Acta Biotheor (2013) 61(2):223-258 DOI 10.

1007/s10441-013-9180-x REGULAR ARTICLE

Can an Engineer Fix an Immune System?Rethinking theoretical biology

Claudio Mattiussi

Received: 21 October 2012 / Accepted: 20 February 2013 / Published online: 03 March 2013 Springer Science+Business Media Dordrecht 2013

Abstract In an instant classic paper (Lazebnik, in Cancer Cell 2(3); 2002: 179182) biologist Yuri Lazebnik deplores the poor effectiveness of the approach adopted by biologists to understand and x biological systems. Lazebnik suggests that to remedy this state of things biologist should take inspiration from the approach used by engineers to design, understand, and troubleshoot technological systems. In the present paper I substantiate Lazebniks analysis by concretely showing how to apply the engineering approach to biological problems. I use an actual example of electronic circuit troubleshooting to ground the thesis that, in engineering, the crucial phases of any non-trivial troubleshooting process are aimed at generating a mechanistic explanation of the functioning of the system, which makes extensive recourse to problem-driven qualitative reasoning possibly based on cognitive artifacts applied to systems that are known to have been designed for function. To show how to translate these ndings into biological practice I consider a concrete example of biological model building and troubleshooting, aimed at the identication of a x for the human immune system in presence of progressing cancer, autoimmune disease, and transplant rejection. The result is a novel immune system modelthe danger model with regulatory cells and new, original hypotheses concerning the development, prophylaxis, and therapy of these unwanted biological processes. Based on the manifest efcacy of the proposed approach, I suggest a refocusing of the activity of theoretical biologists along the engineering-inspired lines illustrated in the paper. Keywords Qualitative reasoning Mechanistic explanation Biological modeling Danger model Regulatory cells Cancer immune therapy Autoimmunity Diabetes Multiple sclerosis Transplantation

C. Mattiussi (&) Via Antonio Ban 13, 20871 Vimercate, MB, Italy e-mail: claudio.mattiussi@gmail.com

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1 Introduction In an instant classic paper (Lazebnik 2002) biologist Yuri Lazebnik comments on what he perceives as serious shortcomings of the approaches currently adopted by biologists in their attempt to understand and x biological systems. To identify the origins of these limitations and suggest ways to overcome them Lazebnik compares the effectiveness of the biologists approach to biological systems to the effectiveness of the engineers approach to technological systems. Lazebnik argues for a greater effectiveness of the latter and attributes at least in part this fact to the engineers use of formal languages endowed with two key properties: that of being unambiguous and that of being amenable to quantitative analysis. In his paper Lazebnik adopts the point of view of a professional biologist and amateur electronic engineer. In the present paper I complement Lazebniks illuminating analysis from the perspective given by my experience as professional electronic engineer and amateur biologist, albeit with some qualied experience at the interface between the elds (Floreano and Mattiussi 2008; Mattiussi et al. 2008). To anticipate the main points of my argument, I start by arguing that the formal languages used by engineers in their approach to the troubleshooting of technological systems are rst and foremost used to support qualitative reasoning aimed at explaining the workings of the system. The generation of a mechanistic explanation is greatly facilitated by the fact that the troubleshooting process is problem-driven and concerns a system that is known to have been designed for function. I thus reason that these aspects are crucial in the attainment of the effectiveness noticed by Lazebnik in the engineers discourse and must be kept in mind while attempting to realize in biology a comparable level of effectiveness. 1.1 Outline of the Paper Mimicking Lazebniks paper, I start in Sect. 2 by describing and commenting in the rst person on an experience of troubleshooting of an electronic circuit: in particular, Sect. 2.1 discusses how trying to x a system helps to assess the level of understanding of its functionality; Sects. 2.2, 2.3, and 2.4 illustrate the role of formal description languages and the increasing level of understanding attained during the successive phases of troubleshooting process, and Sect. 2.5 shows how system understanding is turned in a successful circuit repair. Section 3 distils the general epistemological lessons that can be drawn from the circuit troubleshooting example; Sect. 3.1 stresses the crucial role that qualitative mechanistic explanations play in system understanding; Sect. 3.2 denes and discusses the concept of model used in science and engineering, and Sect. 3.3 emphasizes the cognition-extending potential of such models and of formal model description techniques. Section 4 shows how to transfer to biology the lessons learned in circuit troubleshooting: Sect. 4.1 is devoted to the denition of a model for the human immune system (IS), with Sects. 4.1.1 and 4.1.2 dedicated to the description of existing IS models and the discussion of their shortcomings, and Sect. 4.1.3 to the denition of a brand new IS model built upon the older models. In Sect. 4.2 I show how the new IS model leads to a new, original scenario for cancer development, and in Sect. 4.3 I list a few hitherto puzzling

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cancer-related observations that, on the contrary, are predicted by the new model; in Sect. 4.4 I formulate new proposals for cancer prevention and cure that follow from the new model, and in Sect. 4.5 I extend the discussion to the prevention and cure of transplant rejection and of autoimmune diseases such as type 1 diabetes and multiple sclerosis. Finally, Sects. 5 and 6 are devoted to the discussion of the relationship between qualitative and quantitative models (with special emphasis on the role of numerical simulations in Sect. 5.2 and the role of formal description languages in Sect. 5.4), and to the formulation of some claims about the degree of applicability and generality of the proposed approach and about its potential impact on theoretical biology.

2 Troubleshooting an Electronic Circuit One of the best ways to learn electronics is to try to understand the workings of electronic circuits designed by capable engineers. The fundamental tool used by electronic engineers to represent a circuit is the circuit schematic1 (Fig. 1). In his paper Lazebnik uses the schematic representation of electronic circuits as the primary example of the unambiguous and quantitative formal tools used by engineers. 2.1 The Importance of Fixing Unfortunately, by just pondering on a circuit schematic it is only too easy to fall into the trap of believing you have understood the workings of the circuit when in fact you have not. An excellent way to reduce the probability of this happening is to use the knowledge gained from the schematic to try troubleshooting a broken exemplar of the circuit. Given the proper equipment, the inability to x the circuit is a proof that you have not understood how it works (Williams 1995). Note this must not be construed as a claim that the process of troubleshooting is essential to circuit or, more generally, to system analysis and understanding but only that, when it comes into play, it can act as a guiding target during the analysis, and its outcome is a useful test of the level of understanding eventually attained. Many years ago I was asked by a friend to troubleshoot the electronic circuit controlling the electrically actuated brakes of a newly bought car trailer. At the time, the electronic braking circuit (from now on simply the braking circuit) was a recent innovation substituting the cumbersome overrun brake, a mechanical system based on the inertia of the trailer pushing on the decelerating tow car (Oertle 1969). At rst the braking circuit was able to elicit from the trailer brakes a smooth contribution to the deceleration effort, which felt to the driver of the tow vehicle as roughly proportional to the pressure exerted on the brake pedal of the car. Unfortunately, the original specimen of the braking circuit had stopped working after a few weeks, a rst replacement suffered a similar fate, and a second one
1 Despite the presence of a few circuit schematics no knowledge of electronics is required to understand the gist of the argument developed in the paper.

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Fig. 1 Unstructured schematic. Tentative reconstruction of the schematic drawn while tearing apart one of the broken exemplars of the braking circuit. The circuit was connected to the car and trailer electric network via the terminals drawn outside of the dotted rectangle

followed suit shortly thereafter, suggesting a recurring failure caused by a basic aw in the design of the circuit. The three exemplars of the braking circuit exhibited the same failure behavior, namely, a slight pressure on the brake pedalsufcient to engage the switch that activates the brake lights of the carproduced the maximal activation and the consequent lock of the trailer brakes. 2.2 Drawing the Schematic Since the schematic of the braking circuit was not available, the rst task was to draw it out of the broken specimens. The component side of the printed circuit board was covered by a hard and opaque epoxy resin completely hiding the components. Removal of the resin in order to identify the components resulted in the destruction of one the circuit specimens but permitted to draw a circuit schematic like that shown in Fig. 1. In fact, after so many years I was unable to unearth the actual original schematic incrementally drawn by hand while removing of the resin. The schematic of Fig. 1 is intended as a tentative replacement of that original schematic, which almost certainly must have looked even more tangled than the one shown here. The unavoidable messiness of this rst draft is a consequence of the fact that while you are tearing apart the circuit to identify and annotate the devices and their connectivity you have no idea of how the circuit functionality is realized by what you are unraveling. Therefore, except for very simple circuits, you are typically unable to arrange the devices in a way that reects their role in the circuit operation, and the resulting schematic is to some extent functionally opaque. The second step consisted thus in analyzing this rst schematic in order to explain the workings of the circuit and then draw a new schematic exhibiting in its structure the newly acquired understanding. The information available for this purpose was the known overall function of the circuit, i.e., the control of the trailer brakes according to the deceleration of the towing car, and the nature of the signals brought to the circuit connector as shown in the schematic of Fig. 1.

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2.3 Structuring the Schematic Let me summarize the steps that led to the unraveling of the circuit workings. The signal coming from the brake pedal switch of the towing car conveys the information that the driver has pressed the pedal. However, this signal has a binary nature and thus carries no information about the magnitude of the desired braking contribution. Conversely, I knew that the functioning circuit could produce a braking contribution from the trailer brakes which somewhat reected this intended magnitude. Since none of the signals brought to the braking circuit trough its external connector carried this information I concluded that the circuit was capable of generating in some way a second signal expressing this magnitude information. Inspection of the components constituting the circuit revealed a peculiar assembly of three elements (indicated by TOR, LAMP and PR in the schematic of Fig. 1) that constituted an (ingenuous, albeit, by today standards, rudimentary) accelerometer converting the amount of deceleration experienced by the braking circuit into a resistance value. Starting from this observation it was possible to untangle the schematic of Fig. 1 and progressively transform it into the structured schematic of Fig. 2. 2.4 Simplifying the Schematic Besides explaining the workings of the functioning circuit it is also necessary to verify that the hypothesized operational mechanism is compatible with the observed failure behavior. If successful, this step will also produce a series of hypotheses about the cause of the malfunction. In executing this step it is preferable to start by considering a simplied version of the schematic, which accounts only for the main functionality of the circuit, and proceed to include in the analysis additional

Fig. 2 Structured schematic. The structured version of the schematic shown in Fig. 1. Contrary to Fig. 1, now the disposition of the components reects their role in the functioning of the circuit, roughly identied by the labeled braces at the bottom. Consequently, using this schematic the circuit can be easily understood and mentally manipulated by anyone familiar with electronic circuits and their schematic representation

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components only if the simplied schematic is unable to account for the behavior observed in the real circuit. The elements that generate the main functionality of the braking circuit are a small subset of the components that appear in the circuit. Figure 3 is an example of a possible simplied circuit schematic capable of conveying the essence of the braking circuit functionality (note that this simplied schematic was never actually drawn but just imagined while looking at the complete schematic of Fig. 2). The simplied schematic disregards or abstracts as black boxes the circuitry that is devoted to auxiliary functions. Disregarding that additional circuitry does not imply that it is considered useless. On the contrary, it implies taking for granted that it is correctly performing its designed function. 2.5 Fixing the Circuit As a rst hypothesis, I tentatively attributed the observed failure to the burning of the accelerometer light bulb denoted in the schematics by LAMP, seen as the most fragile component of the assembly. Using the schematic of Fig. 3 one can indeed verify that a burned light bulb is compatible with the observed failure mechanism. However, an additional prediction concerning the signals at the input of the amplier U1 went unfullled. Thus, the malfunctioning of the braking circuit could not be attributed to the burning of the light bulb. A second suspect for the observed failure was a broken amplier U1, which could have been damaged, for example, by an over-voltage over its power line, an event far from unusual in automotive electrical circuits. This hypothesis is also compatible with the observed failure mechanism. Moreover, it is also compatible with the signals that I could measure at the input and output terminals of U1. To further test

Fig. 3 Simplied structured schematic. A simplied version of the schematic shown in Fig. 2, showing only the components and sub-circuits that are essential to the production of the main functionality of the braking circuit

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this hypothesis I substituted in one of the broken specimens the original operational amplier U1 with a new one and added an over-voltage suppression device. A few measurements revealed that the intervention had indeed restored the circuit to its original functionality. The braking circuit thus modied has been working awlessly ever since, which corresponds to more than two decades of smooth operation.

3 Deconstructing Troubleshooting Let me try to identify in what I have described the most important features of the troubleshooting process of an electronic circuit in view of their application to a biological case. Note once again that what is important here is the role of troubleshooting as a guiding target for the reverse engineering process and as a nonnegotiable measure of the level of understanding of the operation of the system eventually attained, not the troubleshooting process per se. Thus, the obvious observation that the braking circuit was either poorly designed or was designed having in mind the wrong set of specications is immaterial for the application to biology of the lessons learned in this example of troubleshooting of an engineered system. This point is important because biological systems are the result of a long evolutionary process and we would not expect them to be poorly designed or badly specied like the braking circuit. 3.1 Qualitative Mechanistic Explanations The starting point in the deconstruction of troubleshooting is the observation that the braking circuit is a system designed for function (rather than, say, for appearance (French 1994)) and that, at least for a while, it has been correctly performing its intended function. This observation justies the assumption that the designer has realized a functional behavior that is robust relatively to a reasonable degree of variation of the value of the system parameters. In turn, this permits the exclusive use of qualitative reasoning in the preliminary analysis of the circuit, as testied by the absence of parameter values in the schematics of Figs. 1, 2, and 3. The goal of this analysis is the production of a qualitative mechanistic explanation of the circuit working, i.e., a description of how the components that compose the system are organized to produce the overall functionality (Craver 2007). This denition of mechanistic explanation implies the presence of multiple levels of description of the system under analysis (Darden 2007). The description tops off at the whole system level: in this case, the braking circuit with its overall functionality. From the point of view of a pragmatic engineer, the need to descend at least one level in the analysis of the system exists only if the substitution of the whole system with a working one of equivalent (or improved) functionality is impractical. In turn, assuming that the malfunction is due to the failure of one of the components at that level of analysis, the troubleshooter can either proceed to the substitution of that component or analyze the working of that component as a system on its own. This recursive analysis bottoms out at the level where the

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troubleshooter deems expedient to intervene to restore the functionality of the component under analysis and, with it, that of the whole system. This means that the analysis of the system functionality is a problem-driven process where the nature of the problem and the nature of the available troubleshooting tools dictate the levels at which the description tops off and bottoms out. In the braking circuit example the strategy initially adopted was indeed the substitution of the whole circuit considered as a black box of known functionality. Having ascertained the long term futility of this approach, an analysis and explanation of the circuit working at the component level was undertaken. At the component level, the malfunctioning device U1 was substituted with a new one and this xed the system and stopped the descent to further levels of description. In this case the option of descending the level of description for the amplier U1 was out of question because U1 was an integrated circuit which, besides being very cheap, cannot be practically xed at its component level. However, in the 1960s an operational amplier with similar functionality would have been an expensive discrete circuit and the choice would have certainly be to descend at the level of description of its components in order to x it. In general we can thus expect that technological advances inuence the choice of the level at which to intervene. Note that the strategy described above is adopted more generally when one is not satised with the current behavior of the system and wants to modify it, the case of a broken system being a particular case of unsatisfying behavior. 3.2 Models and Theories Taking inspiration from Russo (2004), I call model an abstract structure such that some of its elements are put in correspondence by a set of correspondence rules with some observable property of a real system and whose elements can be manipulated to produce qualitative or quantitative predictions about the behavior of the real system. The elements of the abstract system are said to represent the elements of the real system with which they are put in correspondence. It is understood that the correspondence holds only within a certain modeling envelope2, i.e., a range of values for the system and environmental parameters. When the predictions produced by the model are quantitative, it is understood that even within the modeling envelope the correspondence between represented elements and their abstract representation holds only up to a certain degree of approximation. In sciences such as physics, the ensemble constituted by the abstract system with its prediction rules, by the correspondence rules, and by the limitations specied by the modeling envelope and by the degree of approximation specied for the quantitative correspondence constitute a scientic theory of the concrete system that is modeled. From this denition follows that producing a mechanistic explanation of the braking circuit corresponds to building a theory about the circuit workings. Now, it may sound strange to say that explaining the workings of an engineered system corresponds to building a theory about the system and its functionality. However,
I propose the term modeling envelope for the range of conditions where the correspondence rules hold with the stipulated accuracy, taking inspiration from the term operational envelope used in electronic engineering to refer to the range of conditions where a circuit complies with the stipulated specications.
2

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we must keep in mind that engineering, much like medicine and biology, belongs to what philosopher R.G. Collingwood called the practical sciences of nature, i.e., the sciences of nature whose primary aim is not achieve theoretical knowledge about nature but to enable man to enlarge his control of nature (Collingwood 2002, pp. 286287). In other words, the theories of engineering and biology are not timeless truths about nature but historically contingent tools created to understand, manipulate, and control particular systems that are the transient result of a process of articial or natural evolution (Craver 2007). Nonetheless, we can continue to use in engineering and biology the term model to refer to what according to the given denition should be called theory, provided we keep in mind that the correspondence rules, the modeling envelope, and the degree of approximation must be considered part of the lot. In physics and in engineering a system model is built with a practical or a theoretical goal in mind. For example, in the braking circuit case, the goal was the recovery of the intended circuit functionality and the prevention of further faults. In building the model the physicist and the engineer will try to abstract from the real system only the aspects that are relevant to the intended goal. Thus, a given real system admits in general a plurality of models dictated by a multiplicity of modeling goals. There is nothing contradictory in the idea of a multiplicity of models corresponding to a single real system because a model has a purely epistemic status, not an ontic one (Moss and Nicholson 2012). Note that in drawing a schematic like that of Fig. 2 we are implicitly assuming that each component entering the schematic is accompanied by a specication of the model we are adopting for that component. 3.3 Cognitive Artifacts and Computational Models Circuit schematics like those of Figs. 1 and 2 might be construed as mere records of the components that compose the braking circuit along with their connectivity. However, from that point of view the schematic of Fig. 1 and that of Fig. 2 are equivalent, whereas I have argued above that for the expert engineer they are quite different in terms of expressiveness. This fact suggests the schematic-as-a-mererecord interpretation is too restrictive. A suitably drawn schematic is better construed as a cognitive artifact (Norman 1991), i.e., an artifact thatif properly usedguides, supports, enhances, or extends the cognitive functions of the human mind (Clark 2008). By revealing to the expert engineer the causal structure of the circuit functionality, a schematic like that of Fig. 2 facilitates the emergence of the mechanical explanation of the circuit behavior and permits the (typically qualitative) model-based mental simulation of circuit operation. Note that, on the one hand, an artifact such as the well-structured schematic of Fig. 2 is not by itself a cognitive artifact; it becomes one only in the context of the extended cognitive system composed by both the mind of the trained electronic engineer and the schematic (Nersessian 2008). On the other hand, a poorly drawn schematic like that of Fig. 1 does not turn into a cognitive artifact by the mere virtue of being examined by an expert engineer. Summing up, when properly used the discipline of circuit

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schematics plays not only the role of unambiguous formal description language supporting quantitative analysis signaled by Lazebnik (2002), but plays also the role of a powerful tool affording model-based mental simulation and qualitative reasoning. In the context of troubleshooting, a model must rst and foremost support the generation of qualitative predictions about the normal and faulty behavior of the system that are functional to the formulation of a repair strategy. The nal phases of the troubleshooting process typically require a modicum of quantitative analysis. If the x consists only in the substitution of a broken component the quantitative aspect is essentially a check of the compatibility of the substituted components with the original ones. The amount of quantitative analysis increases when what is required is a change of functionality or an improvement in some specication, and can become substantial when a circuit is designed from scratch. Nonetheless, the amount of quantitative analysis is still surprisingly small in the initial phases of a new design, when the engineer typically combines a few backof-the-envelope calculations with a robust qualitative appreciation of the components and standard sub-circuits needed to produce the intended overall functionality (Gilbert 1991). It is only in the nal phases of a design that the analysis becomes frankly quantitative and makes use of computers and numerical simulation, for example, in order to optimize the values of the parameters of the circuit elements according to some predened goal. Interestingly, it was observed that even in systems composed by elements that operate in parallel, model-based mental simulation proceeds sequentially along the chain of direct causation revealed by the model (Nersessian 2008).3 On the contrary, computer-based simulation of systems is typically based on the numerical integration of systems of algebraic and differential equations and proceeds in parallel for all the components of the system. Note also that the unstructured schematic of Fig. 1 and the structured one of Fig. 2 are equivalent when it comes to set up a numerical simulation of the circuit. These observations suggest that a computational model, no matter how detailed and accurate, is unable by itself to contribute much to the explanation of the circuit behavior. Richard Hamming notoriously observed that: The purpose of computing is insight, not numbers (Hamming 1962). What precedes suggests that for all but the simplest systems, insight from computation can be attained only via a preliminary model-based qualitative reasoning, possibly buttressed by suitable cognitive artifacts. Summing up, contrary to the laymans lore according to which engineers are mainly focused on numbers and quantities, in engineering qualitative reasoning always precedes, nourishes, and authorizes quantitative analysis. Moreover, apart from very simple or familiar systems, the power of the unaided mind is often
3 Some linguists consider language as a cognitive artifact that, by imposing sequentiality, fosters the appearance of a more articulate, analytic, and hierarchical form of thought relatively to non-linguistic forms. For example, taking inspiration form the 18th century philosopher Condillac, linguist Raffaele Simone distinguishes a simultaneous and a sequential form of intelligence. In his words, signs are responsible for a real cognitive revolution in man, for they permit the transition from a merely simultaneous thinking to a sequential representation of ideas. _ The thus gained successivity of ideas enhances the development of all the operations of mind and introduces a quite new principle of thinking (Simone 1987, p.68)

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inadequate to perform the required qualitative reasoning. The creation of qualitative models and their embodiment as cognitive artifacts mediated by suitable formal languages is in these cases essential for the understanding and troubleshooting of complex technological systems.

4 Troubleshooting the Immune System Having distilled the principles that govern the understanding and troubleshooting of technological artifacts it is now time to address the question if the same or very similar principles also apply to biological systems. Some authors have rmly maintained that this is indeed the case (e.g., Bernard 1865; Platt 1964) whereas other authors have argued against this view (e.g., Madar et al. 2009). As this disagreement cannot be reconciled merely by crying louder, the best strategy to support the rst view is to show how these principles can be fruitfully applied to some non-trivial examples of biological system understanding and troubleshooting. The biological system that I will consider is the human immune system (IS). According to the strategy used in the troubleshooting of the braking circuit, the starting point is the denition of the overall function realized by this biological system. The IS has in fact at least two main related functions. The rst is to protect its ownerthe hostfrom exploitation and damage inicted by rogue cells and by organismsthe pathogensthat, in virtue of their small size, have the potential to evolve at a much faster pace than the host. The second function of the IS is the management of the operations of tissue healing and regeneration that are required to repair the damage provoked by a trauma, by a pathogen, or by the IS itself while ghting a pathogen. Note that if the overall function of the system was only partially known, we could start with a guess and then rene the guess according to the observations. The rst example of apparent malfunction that I will consider is the presence of progressing cancer, that is, of uncontrolled cellular proliferation that damages the host but nevertheless does not seem to be restrained by the IS. The rst step of the troubleshooting process is the identication of the level of organization at which the intervention will take place. At the topmost level there is the IS; thus, we must ask if the substitution of the whole IS is possible and desirable. Indeed, the execution of a bone marrow transplant after total body irradiation can be seen as an attempt at realizing this substitution. However, the complications associated with this procedure make it the preferred choice only in a very limited number of cases. This means that it is preferable to descend at least at the next lower level to examine the possibility of intervention. This implies dening a model for the IS at its component level. 4.1 Adopting a Model for the Immune System In describing models for the IS in the presence of cancer I will take for granted a knowledge of the elements that compose the human IS and of their nomenclature as can be derived from a modern textbook such as (Abbas et al. 2012). The IS elements

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will be named according to their functional rather than phenotypic characterization. For example, I will refer to a regulatory T cell rather than to, say, a CD25?FOX3?T cell. Note that the elements of the models described below must not be confused with the real thing; for example, a T cell in one of the models is an abstraction that disregards many properties of the biological elements it represents. A common assumption of all the models discussed below is the existence of tumor-specic antigens (TSA) (Schreiber et al. 2011) and the presentation of their epitopes on major histocompatibility complex (MHC) molecules. In other words it is assumed that the tumor cells cannot manage to become invisible to the IS. I will briey discuss below what happens if this hypothesis is dropped. 4.1.1 The Self/Nonself Model (SNM) To warm up to biological system modeling let us begin with a simple and admittedly outdated model of the IS: the self/nonself model (SNM). The SNM was one of the rst models proposed for the IS and is based on the assumption that early in life the IS gets rid of all the immune effectors that recognize antigens present in the thymus, which dene the self (Medzhitov et al. 2002). According to the SNM, once this deletion phase is terminated the IS is licensed to attack any element that it recognizes, on the hypothesis that it must necessarily be nonself. To verify the adequacy of this theory for the troubleshooting task at hand we need to compare its predictions with a set of relevant observations. Matzinger (2002) made this comparison not only for the original SNM but also for its more recent versions and found that the SNM is indeed inadequate as its predictions disagree with the observations in many crucial respects. From our perspective the main shortcoming is that the SNM is incompatible with the presence of cancer in an immunocompetent individual. The reason is that cancerous cells present on their surface TSA that are the result of recent genome mutations and consequently could not have been present during the early deletion phase. 4.1.2 The Danger Model (DM) To obviate to the shortcomings of the SNM, Matzinger and Fuchs proposed a new IS model: the danger model (DM) (Fuchs and Matzinger 1996). The DM is built on top of the SNM but differs from it in a crucial aspect: it assumes that the activation of the immune effectors requires the presence of danger signals. These signals can be either molecular patterns associated with known pathogens, chemicals produced by stressed host cells, or molecular patterns associated with the non-programmed death of host cells (Matzinger and Kamala 2011; Gallucci and Matzinger 2001). To implement this policy, the IS has a set of professional antigen presenting cells (APC)in particular dendritic cells and, when antigen is limiting, B cellsthat possess surface receptors capable of sensing a vast repertoire of danger signals. In the absence of danger signals (a situation called the steady state) an APC remains in an inactive state and patrols the host tissues, engulfs at a leisurely pace antigens in its surrounding medium, processes and displays the captured antigens on MHC-II molecules, and homes periodically to the draining lymph nodes to present the

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captured antigens to helper T cells (Th). If a T cell recognizes with its T cell receptor the antigen presented by an inactive APC it is either instructed by the APC to die by apoptosis or is anergized. This is done on the presupposition that this T cell is potentially reactive against host tissues and commensal organisms (Lathrop et al. 2011). This deletion of T cells performed outside of the thymus realizes a peripheral tolerance that complements the central tolerance realized in the thymus, a mechanism that the DM inherits from the SNM. When an APC senses a danger signal it switches to an active state, increases is rate of capture of antigens present locally, processes and displays the captured antigen on MHC-II molecules, and homes to the draining lymph node to present the captured antigens to Th cells. According to the DM, if a Th cell recognizes with its T cell receptor the antigen presented by an active APC it is activated by a costimulatory signal delivered by the APC. Beyond danger signals, an APC is also able to sense and process additional cues produced by the host tissues in order to establish the kind of immune response that is best suited in the context of that tissue (Matzinger and Kamala 2011). If a humoral immune response is called for, the Th cell is instructed to activate the B cells that display on their MHC-II molecules its cognate antigen. If a cellular immune response is to be preferred, the Th licenses the APC to cross-present the captured antigens on MHC-I molecules and activate cytotoxic T cells (Tc) that recognize the presented antigens with their T cell receptor (Kurts et al. 2010). A mix of cellular and humoral immunity activation can also be imagined. Much more detail about the DM can be found in Matzingers original papers (e.g., Matzinger 2002; Matzinger and Kamala 2011) andpresented exactly from the engineering viewpoint adopted in this paperin chapter 5 of Floreano and Mattiussi (2008). In particular, Matzinger explains the presence of progressing cancer as follows. When the tumor starts to grow it does not produce danger signals. Thus, it has the opportunity to tolerize the IS to the TSA by deletion or anergy of the TSA-specic T cells (I denote them as TSAT cells). At a certain point the growing tumor will start to produce danger signals caused, for example, by the hypoxia of tumor cells that are too distant from the capillaries to obtain oxygen by diffusion. These signals would nally activate the APC, but these would not nd any T cells to activate since they would have been deleted or anergized in the previous phase. Given this prediction following from the DM, one would not expect to nd in the tumor mass any non-anergized TSAT cell. On the contrary, a population of TSAT cells is typically observed among the tumor-inltrating lymphocytes and these cells, far from being anergized, exert a denite immunosuppressive action (Fridman et al. 2012). 4.1.3 The Danger Model with Regulatory Cells (DMR) The T cells with suppressive activity that are observed among the tumor-inltrating lymphocytes are called regulatory T cells (Treg) (Sakaguchi et al. 2008). Their existence and function was hypothesized several decades ago, on the basis of the observation that T cells could not only enhance but also attenuate the immune response. However, the actual existence of what was then tentatively called

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suppressor T cells remained controversial for lack of reliable phenotypic markers and for the experimental demise of the putative molecular mechanisms of action (for details, see Sakaguchi 2011, and references therein) until, quite recently, they could be eventually identied phenotypically (Sakaguchi 2005). Treg with the phenotypic markers of Th, Tc, and NKT cells have been observed, as well as double negative (DN) Treg (which do not express Th, Tc, or NKT markers) (Wood et al. 2012; Juvet and Zhang 2012); in what follows I will simply refer to Treg relying on the context or on the assumed cell function if disambiguation is required. Since the DM makes several correct predictions concerning the behavior of the human IS, I will proceed to build incrementally on the DM a new IS modelthe danger model with regulatory cells (DMR)rather than dene a new IS model from scratch. The DMR will include as a new component the Treg. Thus, to specify the new model I need to specify: (1) how the Treg are generated; (2) how they behave in relation to the other elements of the new model inherited from the DM, and (3) if they are plastic, that is, if they ever cease to behave as regulatory cells and revert to a non-regulatory state. First, a model for the generation of Treg can be dened simply by extending the tolerization mechanisms dened in the DM. In the DM the central and peripheral tolerization is based on the deletion or anergization of lymphocytes that recognize an antigen presented by an inactive APC. In the DMR I assume that a third outcome of this kind of interaction is possible, namely, the transformation of the T cell into a Treg. Since the DMR is intended as a qualitative model of the IS there is no need to specify the details of the mechanism leading to the choice between deletion, anergization, and conversion to a regulatory phenotype. It is instead sufcient to assume that the conversion of an non-regulatory T cell to a Treg happens often enough to make of the Treg a non negligible agent of immune tolerance. Considering now Treg behavior, in vitro and in vivo observations suggest that Treg exert an antigen-specic down-regulation of the immune response that implies an interaction with the APC (Sakaguchi 2005). However, the exact nature of the interaction is still unknown. In specifying the behavior of the Treg in the DMR one must take into account the following observation. The Treg appear as an additional tolerogenic mechanism on top of the one realized by deletion and anergy. This mechanism requires additional resources and it is easily imagined that if it were currently not useful in evolutionary terms it would have been inactivated by some mutation. Consequently, we can expect to nd in the mechanism that implements tolerance by Treg some improvement on the deletion and anergy mechanism of the DM. In the DM there is indeed at least one aspect of the mechanism of lymphocyte activation by APC that could be improved. When an APC is activated by danger signals, before homing to the draining lymph node it takes a sample of the antigens present at the activation place. Besides pathogen-associated antigens this sample can include antigens associated with healthy host tissues. This means that according to the DM the APC might subsequently activate an autoreactive lymphocyte that has fortuitously escaped deletion and anergization. This activated lymphocyte could then proceed to attack healthy host tissue, although its hypothesized short activation time would limit this effect to a few cell killings (Matzinger 2002).

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Thanks to the presence of this new componentthe TregI can devise and specify into the DMR a mechanism that avoids even this moderate autoimmune effect. Let me assume that, before being allowed to interact with effector T cells, an activated APC must be inspected by the local Treg pool, and that this inspection would result in the removal of all the peptide-MHC complexes (pMHC) recognized by the Treg pool. This hypothetical two-phase mechanism of T cell access to the APC is corroborated by the in vivo observation of initial crowding of Treg around active APC entering lymph nodes, followed by a second phase of APC interaction with non-regulatory T cells (Bousso 2008). A possible mechanism of pMHC removal from APC is the stripping by trogocytosis of the pMHC by the Treg (Davis 2007; Juvet and Zhang 2012). This process of APC stripping would confer to the DMR at least four advantages over the original DM.4 The rst advantage is the absence of the autoimmune phenomenon described above, due to autoantigens captured and presented by active APC. The second advantage is the possibility for the Treg to use the acquired pMHC to behave as a regulatory APC and realize an additional antigen-specic tolerization mechanism by killing lymphocytes that recognize the acquired pMHC (Zhou et al. 2011). The third advantage is the speedup of the activation of adaptive immunity in presence of novel pathogens. This speedup is realized because the T cells that will later interact with the APC to establish if their cognate antigen is displayed on the APC surface will be confronted only with antigens that have not been stripped by the Treg. By the mechanism of generation of the Treg hypothesized above the antigens that can be found on the surface of the active APC after Treg inspection are antigens that have not been previously encountered in the steady state. This correspond to a focusing of the later inspection of the stripped APC by effector T cells on the subset of captured antigens that are specically associated with the danger signal that activated the APC. The speedup factor is the ratio of the number of all the antigens captured by the APC to the number of antigens that remain on the APC surface after Treg stripping. This speedup factor might prove vital in presence of a pathogen whose antigens are recognized only by rare circulating T cells. The fourth advantage deserves a particular emphasis because it concerns a step often ignored in establishing an IS model, namely, the transition from the phase of immune pathogen attack to the equally important phase of tissue regeneration and healing (Nathan 2002). The possibility to model in the DMR an informed transition between the two phases is based on the possibility that an active APC be stripped by the Treg of all its pMHC issuing externally acquired antigens. This is the situation that prevails, for example, when a pathogen infection has been successfully cleared by the IS but the concerned tissue is still stressed due to the damages inicted during the ght phase. Thus, an active APC stripped by Treg of all these pMHC is an indicator of the opportunity that the IS switches from pathogen eradication to tissue regeneration and healing. Note, however, that the absence of pathogen antigens in the sample engulfed by any particular APC might be due to a stochastic sampling effect. Thus, in the DMR the licensing of the healing process will not be entrusted to
4 The physical transfer (rather than, say, simple inactivation or resorption) of the pMHC from APC to Treg is essential only to realize the second advantage described in the text.

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a single completely stripped APC. Rather, APC that have been stripped of all their pMHC displaying externally acquired antigens will produce anti-inammatory, regenerative signals, whereas APC that after Treg inspection still have some of these pMHC will produce pro-inammatory, immunogenic signals (Sozzani et al. 2007). The outcome will thus be the product of a decision mechanism similar to the quorum sensing observed in colonies of bacteria (Almeida et al. 2012). To complete the denition of the DMR I must nally dene the fate of the Treg. One can imagine two scenarios. In the rst scenario the Treg are not plastic, i.e., once an inactive T cell is transformed into a Treg it remains a regulatory cell for all its remaining lifetime. This mechanism has the disadvantage that a pathogen or a tumor could gain long term protection by rst behaving well and avoiding the generation of danger signals, generate in this way Treg specic for its specic antigens, and then proliferate under the umbrella of the established Treg protection. In the second scenario the Treg are plastic, i.e., there exists a mechanism of reconversion of a Treg into a non-regulatory T cell (Bailey-Bucktrout and Bluestone 2011). For example, one could assume that the execution of one or a few operations of pMHC stripping on an APC would result in the (possibly, suitably delayed) reversion of the Treg to its original non-regulatory state. In this way a steady-state tissue would produce Treg specic for tissue-associated antigens whereas an inamed tissue would consume those same Treg. To this end one must just add to the DMR the assumption that Treg are attracted into inamed sites at early phases of infection (Mailloux and Young 2010). This second, plastic Treg scenario also has a drawback, namely, that if a tissue expresses a tissue-restricted antigen (TRA)5 and becomes inamed, it will stop to produce TRATreg and will start to consume them. Since by denition those TRA are not expressed elsewhere by the host, if the inammation state persists it will eventually lead to the exhaustion of the existing stock of TRATreg and to the consequent autoimmune attack against the tissue. However, contrary to the unwanted consequence of the non-plastic Treg scenario, the unwanted consequence of the plastic Treg scenario can be easily prevented. It is sufcient to ensure that no TRA exists in the host tissues, for example, by ensuring that antigens that are associated with molecules that are functionally required only in a single tissue be also (typically, non-functionally) expressed in at least one additional distinct tissue of the host. In fact, it is known that humans possess a gene called autoimmune regulator (AIRE) whose function is the ectopic expression of antigens (Anderson et al. 2002). The AIRE gene is expressed in the thymus and possibly in other regions of the lymphatic system (Eldershaw et al. 2011) and exerts its function by encoding a transcription factor that provokes the transcription of a multitude of proteins that otherwise would be tissue-restricted. Based on this evidence, I will adopt for the DMR the plastic Treg scenario, which contemplates the production of Treg by tissues in steady state and their consumption in the presence of danger signals.

5 Tissue-restricted rather than the equally plausible tissue-specic to avoid a conict of acronyms with tumor-specic antigen.

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4.2 Using the Model to Understand Cancer Development Now that I have dened the IS model I can proceed to verify its compatibility with the observed malfunction of the system, that is, the presence of progressing cancer. The currently mainstream model of cancer development is the somatic mutation theory (SMT) (Soto and Sonnenschein 2011). It posits that the process starts with a single cell that, for unspecied causes, accumulates in its genome a certain number of mutations and as a consequence starts to proliferate uncontrollably. This produces a growing primary tumor wherein some cells can subsequently acquire further mutations and start to metastasize. In this scenario, in the phase that goes from the single originator cell to a microtumor of a few millimeters in diameter, the cells of the growing tumor receive oxygen and nutrients by diffusion from the nearest existing blood vessels. This is called the avascular phase of tumor growth (Wicki and Christofori 2008). In this phase the tumor cells do not produce danger signals and, according to the DMR, their interaction with the local APC leads to the generation of TSATreg. If the growth continues beyond the microtumor size, however, some cells of the growing tumor come to be too distant from the blood vessels to receive an adequate supply of oxygen and nutrients by diffusion (Valastyan and Weinberg 2011). As a consequence, starving and hypoxic tumor cells start to produce danger signals. According to the tenets of the DMR, by activating the local APC, the presence of danger signals stops the production of TSATreg and starts a phase of TSATreg consumption. Since in this phase, by denition, the TSA exist only in the growing tumor, there are no other places where TSATreg are produced. Thus, the stock of TSA Treg is inexorably eroded. As soon as the stock is exhausted, the tumor loses its TSA Treg protection and is attacked and cleared by the IS. I have thus arrived at the surprising conclusion that the scenario proposed by the mainstream model of cancer development is incompatible with the DMR. Thankfully, the SMT is not a fact established beyond doubt by multiple direct observations but only a hypothesis based on indirect observations and still open to debate (e.g., Soto and Sonnenschein 2011). Thus, I am not forced to discard outright the DMR for incompatibility with the observed failure mode. On the contrary, I can proceed and follow the model wherever it leads. To this end it is expedient to adopt an evolutionary Darwinian point of view (Valastyan and Weinberg 2011; Greaves and Maley 2012) and say that a cell clone that continues to grow beyond the avascular phase is nonviable in the environment provided by the host with a functioning IS. However, as Wicki and Christofori observe: Avascular tumors can reach a steady state, where tumor cell proliferation and apoptosis are in balance and a net increase in tumor volume does not occur (Wicki and Christofori 2008, p.68). This kind of tumor, which I will call steady-state (nonmetastatic) microtumor (SSM), does not generate danger signals and is thus viable in the context of a functioning IS as described by the DMR. Note that the steady-state in the SSM name evokes both its immunological status (absence of danger signals) and the dynamical equilibrium of its size. The dynamical nature of the equilibrium is essential because it ensures that the Darwinian process continues to operate in the SSM despite its unchanging size. In particular, this evolutionary process can lead to the generation of a mutant cell clone

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possessing the ability to metastasize. This fact leads to a dissemination in the host of a progeny of what I will call steady-state metastatic microtumors (SMM), each capable of further SMM seeding. Being in steady state, each SMM will generate a stock of TSATreg whose size depends on the local trafc of APC and T cells. This local trafc is particularly intense in special tissues and organs of the host such as the thymus, the bone marrow, the spleen, the lymphatic nodes, and possibly the liver and the appendix (Mailloux and Young 2010; Chen et al. 2004). This means that if a colony of SMM happens to settle in one of these special sites the rate of generation of TSATreg can become substantial. The same result is obtained in a less localized way when a substantial number of SMM is disseminated in the body. The presence in the host of a colony of SMM has the effect of gradually changing the evolutionary scenario experienced by each SMM. While initially the outgrowth of a microtumor beyond the avascular phase is stopped by the IS and is thus evolutionarily nonviable, the growing stock and increased potential of production of TSA Treg due to the presence of the colony of SMM makes their outgrowth evolutionary viable. Now a mutation in one of the SMM cells leading to its uncontrolled proliferation would lead to the growth of one of the microtumors beyond the avascular size. As before, this would generate danger signals, which in turn would activate the APC, attract TSATreg and consume them. Contrary to what happened in the absence of other SMM, however, now the local consumption would be compensated by the inux of TSATreg produced by the other SMM, which are still in the steady-state. Thus, the TSATreg stock will not be exhausted and the active APC will continue to be completely stripped of their pMHC. The only condition for this to happen is that the mutation that led to the uncontrolled proliferation of one of the SMM did not generate TSA that were not present in the original SMM. This is not difcult to imagine considering that the uncontrolled proliferation can be due to a mutation that might have just disabled the expression of a tumor suppressing gene. Holding this condition, the APC, instead of generating immunogenic signals and activating IS effectors against the growing microtumor, would produce healing and regenerative factors (Curiel et al. 2004). In this sense, rather than a wound that does not heal (Dvorak 1986) the primary tumor might be characterized as a wound that does not stop healing (Schafer and Werner 2008). In particular, given the hypoxic status of the growing tumor, the fully-stripped active APC would produce pro-angiogenic factors that would promote the vascularization of the growing tumor (corresponding to the activation of the socalled angiogenic switch) (Coukos and Benencia 2009). This growing tumor would become what in the context of the SMT appears to be the primary tumor. Note that in the DMRcontrary to the scenario hypothesized by the SMTbesides the potential of uncontrolled growth the cells of the primary tumor possess from the beginning the ability to metastasize6, and only the typical inefciency of the metastatic spread (Valastyan and Weinberg 2011) can lead to a signicant delay before cells shed by the primary tumor settle and start to proliferate.

Unless this capacity has been disabled by the mutations that led to uncontrolled proliferation.

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4.3 Further Predictions About Cancer Following from the Model Let me consider a few additional predictions that follow from the DMR scenario of cancer development and are actually observed in vivo, but appear puzzling in the context of the SMT. A rst obvious implication of the DMR scenario is the presence of a colony of SMM in the body of a patient with overt cancer. The DMR predicts indeed that colonies of SMM are to be found also in individuals who do not harbor a growing primary tumor. In fact it was observed that (Folkman and Kalluri 2004): Many of us may have tiny tumours without knowing it. In fact, autopsies of individuals who died of trauma often reveal microscopic colonies of cancer cells (my emphasis), also known as in situ tumours. It has been estimated that more than one-third of women aged 4050, who did not have cancer-related disease in their life-time, were found at autopsy with in situ tumours in their breast. But breast cancer is diagnosed in only 1 % of women in this age range. Similar observations are also reported for prostate cancer in men. Virtually all autopsied individuals aged 5070 have in situ carcinomas in their thyroid gland, whereas only 0.1 % of individuals in this age group are diagnosed with thyroid cancer during this period of their life. The existence of these in situ or dormant microtumors has been puzzling biologists for a long time (Uhr and Pantel 2011). In the scenario offered by the SMT, these quiescent microtumors must necessarily be interpreted as metastases of the primary tumor. However, this interpretation raises the question of why some metastases grow and other become quiescent. Moreover, according to SMT one would expect that the cells of dormant microtumors harbor more mutations than the primary tumor and more similar to the ones observed in growing metastases than to those observed in the primary tumor, whereas the contrary is actually observed (Klein 2009). Conversely, all these observations make perfect sense in the light of the DMR, since the dormant microtumors of the old model correspond to the SMM of the new model and, in it, their formation precedes that of the primary tumor rather than following it as predicted by the SMT. The DMR also explains the related observation that some of the neoplastic growths that in the light of SMT are considered metastases appear to be disseminated from the beginning of the formation of the primary tumor, being observed in patients whose primary tumor is detected in its early stages (Valastyan and Weinberg 2011). This is puzzling in the light of the SMT, which posits that the acquisition of metastatic potential requires a series of additional mutations besides the one that led to the uncontrolled proliferation. In fact, within the SMT the very observation of the high frequency of metastatic dissemination is puzzling. After all, shedding cells to distant sites should put a cell clone at a selective disadvantage in the Darwinian struggle between clones taking place within the primary tumor (Aranda-Anzaldo 2001; Merlo et al. 2006). Conversely, in the light of the DMR the early spread of metastases from the primary tumor is obvious since the metastatic competence is a prerequisite for the establishment of the conditions that allow the switch to the phase of uncontrolled growth.

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Remark If we drop the hypothesis that the tumor cell must necessarily present some TSA on its MHC molecules, the DMR scenario of tumor development from the hypothetical rst mutated cell changes as follows. When the tumor reaches the size limits of the avascular stage, the APC are activated by the danger signals, but upon presentation of the engulfed antigens to Treg they are completely stripped and consequently enter the healing state and start to produce pro-angiogenic and regenerative factors. Thus, as above, the tumor can grow not only undisturbed but actually actively supported by the immune system. However, contrary to the scenario with obligatory TSA presentation, in this scenario the primary tumor can grow directly from this rst cell clone and there is no compulsory metastatic phase. Indeed, as noted above, the acquisition of metastatic properties by a clone of the growing tumor is evolutionarily penalized. Consequently, the acquisition of metastatic potential by the growing tumor would be a rare event and these hypothetical TSA-free tumors would almost always be benign. Similar conclusions hold for the establishment of tumor protection by SSM production and secretion into the circulation of free TSA (McKhann and Yarlott 1975). Another signicant prediction of the new model follows from the fact that the mechanism of tolerance based on Treg requires a certain algebraic relationship between Treg production and consumption. In particular, it might happen that when the primary tumor reaches a certain size the rate of Treg production is limiting for further grow of both the primary tumor and of its metastases. Consequently, the DMR predicts that, sometimes, when the primary tumor is surgically removed, its metastases will be observed to resume their formerly arrested growth. This phenomenon is indeed observed in cancer patients (Carmona Bayonas 2011). Another observation that appears puzzling from the SMT perspective is the existence of a double peak in the delay of recurrence of treated tumors (Retsky et al. 2008). In the light of the SMT, if a malignant tumor is completely eradicated before it can shed metastases, one would not expect any recurrence. If, instead, the tumor is incompletely treated, leaving behind some cells of the primary tumor, or if it is treated after the shedding of metastases, one would expect a temporal distribution of recurrences with a single peak. The DMR predicts instead that if the primary tumor is completely eradicated before it can shed viable metastases, only the colony of SMM will remain in the patient. Otherwise, after the treatment there will be, besides the colony of SMM, some cells of the primary tumor or some of its metastases. The double peak of recurrences is predicted on the basis of the expectation that the time required for the mutation leading to the outgrowth of another SMM be different from the time required for the regrowth of the cells seeded by the primary tumor. Many other predictions can be made based on the tenets of the DMR. Rather than exploring them one by one, the formulation and the comparison with the observations reported in the literature of the predicted effect of, for example, the complete ablation of the Treg population (Colombo and Piconese 2007), or that of the APC population (Gilboa 2004), or that of the tumor-inltrating Treg population (Yu et al. 2005), the inhibition of Treg migration to the tumor (Byrne et al. 2011), the effect of the inoculation of activated APC loaded with tumor associated antigens (Tacken et al. 2007), the inoculation of activated APC completely stripped of their

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pMHC and that of activated Tc (Mapara and Sykes 2004) is left as an exercise for the reader.7 4.4 Fixing the System: Cancer Prophylaxis and Therapy The scenario of cancer development predicted by the DMR contains three main transitions: that from a normal cell to a cell clone characterized by the controlled proliferation which generates the SSM; the transition from the latter to a cell clone with controlled proliferation and metastatic character which constitutes the SMM; and the transition from the latter to the clone with uncontrolled proliferation and metastatic character that forms the primary tumor and its metastases. The DMR does not include a mechanistic model of these three crucial transitions and just assumes that they can happen with a certain probability. Due to the qualitative character of the DMR the probabilities need not even be specied, granted that they all are positive. Nonetheless, an intervention capable of blocking any of these transitions would obviously prevent the development of cancer. Thus, in the search for cancer prophylaxes and therapies the development of an adequate mechanistic model for these transitions would likely provide a level of control over cancel development even greater than that provided by an adequate mechanistic model of the IS. Note that the effect of chronic inammation has been cited as a possible facilitating factor for cancer development (Smithers 1962). In fact, according to the DMR the presence of inammation in the absence of infection would result in the APC producing healing and growth factors. This would lead to an increased proliferation and turnover of cells, with the accompanying increased accumulation of mutations favoring the emergence of the cell clones forming the SSM and SMM. In this light, a strategy of cancer prevention based on the prevention of chronic inammation could be envisaged (Dalgleish and Haefner 2006). Nonetheless, in the absence of an unequivocal model for the initiating factors we are forced to settle for interventions which assume that one or more of the transitions to overt cancer have already been crossed. Let me assume that, in the context of the DMR, a primary tumor with disseminated metastases has already developed. This state of things implies and requires the presence of a colony of SMM that produce the TSATreg consumed by the primary tumor and by its metastases. Thus, if we could nd a way to remove all the SMM, the supply of TSATreg would stop and the primary tumor along with its metastases would eventually be cleared by the immune system. The DMR tells us that there is a simple strategy to achieve that result, which does not imply surgery, cytocidal irradiation, or cytotoxic chemotherapy. From the DMR follows that the continued production of the TSATreg by the SMM and the protection of the SMM from immune attack requires the permanence of the SMM in steady state, that is, the absence of danger signals in their surroundings. This means that simply by creating and maintaining danger signals (and the
7 This little provocation was inspired by the following statement by Russo (Russo 2004, p. 18): A simple criterion to verify whether a theory is scientic is to check whether one can compile an exercise manual; if that is not possible, its certainly not a scientic theory.

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coincidental inammation) in the SMM surroundings we can stop the production of TSA Treg. Consequently, the SMM, the primary tumor, and its metastases will all be consuming the existing stock of TSATreg, which will be no longer replenished. Once the stock of is exhausted, both the primary tumor with its metastases and the SMM, being all seats of danger signals, will be cleared by the IS.8 Note that despite the non-specicity of the required danger signals, this kind of cancer therapy is not only antigen-specic; it is also patient-specic by default. Its application implies obviously the availability of techniques for the localization of the SMM and for the creation and maintenance of generic danger signals in correspondence of the SMM locations. As noted above, the most productive and thus more dangerous SMM colonies will presumably be found in special sites of the lymphatic system such as the spleen and the bone marrow (Zhao et al. 2012). The required danger signals could be produced, for example, by mild localized irradiation, by adjuvants (Gallucci et al. 1999), and even by controlled pathogenic or non-pathogenic microorganism spread. In fact, we could conjecture that some cases of spontaneous regression of cancer in the presence of infection (Hoption Cann et al. 2002) and the regressions observed following the injection of Coleys rin (Terry 1975) are the consetoxin (Starnes 1992) and bacillus Calmette-Gue quence of the ensuing coincidental generation of danger signals at the SMM sites. Note also that the localization of existing SMM and SSM, and the generation and maintenance of danger signals at their location before the development of a primary tumor constitutes a primary prophylaxis of cancer. In the light of the DMR we can instead maintain that the complete ablation of the TSA Treg does not constitute a resolutive cancer therapy. The ablation of the TSATreg would indeed expose the primary tumor and its metastases to the attack of the IS. However, for lack of danger signals the SMM will not be attacked and will continue to produce TSATreg.9 On the one hand this implies that there is the possibility that the ablation of the TSATreg must be repeated several times before the primary tumor and its metastases are cleared. Moreover, even after tumor eradication the colony of SMM will persist and might thus lead to a recurrence of the disease. Another strategy that could be effective in producing an immune attack of the tumor but presents the problem of SMM permanence is the drug-induced temporary inhibition of all Treg production. This strategy has also the drawback of possible autoimmune side-effects. Incidentally, in the light of the DMR an antigen-specic Treg ablation strategy could instead be effective in the case of chronic infections that follow from the manipulation of the IS by a pathogen (Belkaid 2007) or from a host-pathogen compromise (Rouse and Suvas 2004), provided the chronicization is not based on the establishment of a protected source of pathogen-specic Treg. For example, Das et al. (2013) report that Mycobacterium tuberculosis (Mtb) may maintain
8 Note that if the sites where SMM are located are also sites of ectopic expression of host TRA, the corresponding tissues must be protected from inammation during the therapy.

In this description I am not considering the role of humoral immune response, whose model the DMR inherits from the DM. Taking it into account would lead to the possible production of antibodies for the TSA, which might opsonize the cells of the SMM and lead to their demise even without prior inammation.

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long-term intracellular viability in a human bone marrow. In the light of the DMR, these protective cellular niches where Mtb persists and which are highly trafcked by lymphocytes can act just as this protected source of pathogen-specic Treg. However, even excluding this case (for which, taking inspiration from the case of cancer, one can apply the strategy of generating and maintaining danger signals in the niches where the pathogens persist), the direct ablation of the existing pathogen-specic Treg population appears inexpedient. Being antigen-specic, the ablationconsidered as a pattern-recognition taskappears almost as difcult as the direct antigen-specic ablation of the pathogen. However, one could imagine applying the following technique to indirectly produce a pathogen-specic Treg ablation without any pattern-recognition effort. This technique is inspired by the system restore strategy adopted by some computer operating systems (OS). An unexpected side-effect of a software installation or OS update can render a computer unusable. To avoid a complete reinstallation of the system some OS generate a restore point at predened intervals and before every major software update. In this way, if the computer becomes unstable after the operation, the user has the option of executing a roll-back to a previous working system conguration. The application of this strategy could be imagined in the context of the DMR. An IS restore point would be represented by a stored sample of the Treg population, and might be executed at regular intervals. In the presence of a chronic infection that is maintained by an existing and not renewing population of pathogen-specic Treg, one could ablate all the Treg population and reconstitute it with a population generated from the most recent infection-free sample. An interesting question raised by this hypothetical therapy is the (possible rejuvenating) effect of the roll-back on cells and tissues that are not infected but are inamed at the time of the reinoculation and have changed their antigen repertoire as a consequence of normal aging. 4.5 More Mending: Autoimmune Diseases and Transplant Rejection The denition of the DMR was the result of a problem-driven process where the problem is the progression of cancer in immunocompetent individuals. The model of cancer development that ensued revealed that, after all, cancer is not a malfunction of the IS. It is instead the consequence of the normal working of the human IS, combined with an evolutionary process that takes place in the host and results in the development of a Treg protection for tissues that, for the well-being of the host, should not be protected. In this perspective, autoimmune diseases represent a problem complementary to cancer because they are due to the lack of Treg protection for tissues that should be protected. Let me consider as a rst example type 1 diabetes (T1D). As in the case of cancer, the initiating factor is of somewhat undened and probably multiple origin (Knip and Simell 2012). In T1D the initiating factor leads to an inammation of the pancreas. According to the DMR, this inammation will activate the local APC, which will then migrate to the draining lymph nodes and present the engulfed antigens to the Treg pool. Normally, this process would result in the stripping of APC of all the pMHC with the exception of those carrying pathogen antigens, if

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any. Thus, in the presence of pathogens the reaction would be an immune attack towards the pathogen. In the absence of pathogens the APC would produce regenerative signals that might possibly lead to a reorganization of the tissue that alleviates the existing stress. It may happen, however, that some of the antigens that characterize the insulin producing b-cells of the pancreas are TRA (tissue restricted antigens). This means that these antigens are not expressed elsewhere in the body. Consequently, the existing inammation would consume the existing stock of TRATreg and, if the inammation persists long enough, this would lead to the immune attack and destruction of the b-cells. Repeated rounds of inammation would progressively erode the existing population of b-cells and lead to the manifestation of the insulinrelated symptoms of T1D. The possibility of this kind of effect was predicted while introducing the DMR. According to the DMR any tissue harboring TRA is at risk of immune attack as a consequence of protracted inammation. This is a tradeoff that is related to the decision by evolution to adopt plastic Treg. The surprising thing, then, is the very existence of TRA, since evolution should have operated so as to prevent it. Indeed, there is evidence that the activation of the AIRE gene mentioned above leads also to the expression of the b-cell related proteins that appear as TRA in the autoimmune attack leading to T1D (Anderson et al. 2002). It has been hypothesized that the post-translational modications that are performed in the b-cells of the pancreas differ from those performed in the tissues that express the AIRE gene (Diez et al. 2001). Consequently, the proteins produced in the pancreas are slightly different from those produced ectopically. If the differences are singled out in the processing of these proteins for presentation on MHC molecules, the Treg produced ectopically would not protect the pancreas cells. This further condition for the emergence of the autoimmune attack explains the inuence on the disease of the genes encoding the MHC molecules (Noble and Erlich 2012). There is nally a dependence of T1D on environmental factors that led to the formulation of the so-called hygiene hypothesis, according to which the lack of childhood contact with certain pathogens, gut bacteria, and foods favors the later emergence of autoimmune diseases such as T1D (Bach and Chatenoud 2012). In the light of the DMR the protection should be due to protective10 molecular mimicry, that is, the similarity of some epitopes of the pathogen and bacteria antigens with the TRA of the tissues that should be protected by Treg but are not (Lathrop et al. 2011). Of course, the generation of the TRATreg requires the absence of inammation and might be due to an asymptomatic colonization of the host (on the other hand, a recrudescence of the pathogen infection would inhibit the Treg production and could lead to a relapse (Christen and von Herrath 2005), although for reasons opposite to the ones typically invoked). An endemic colonization of this kind in an evolutionarily signicant past could have led to a reduction of the selection pressure against

10 Typically, molecular mimicry is considered as possible source of autoimmune attack rather than as a protective element (Fasano and Shea-Donohue 2005).

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post-translational modication that introduces differences between the functionally expressed and the AIRE-expressed antigens (Embry 2004). To formulate a T1D therapy in the light of DMR one can take inspiration from the strategy pursued by cancer in the establishment of its immune protection. This means identifying the TRA that are the objective of immune attack in T1D and establishing colonies of cells carrying these TRA (or some technological surrogate such as, e.g., coated beads (Zhang et al. 2007)). These colonies should be preferentially established in locations of high lymphocyte trafc (e.g., the bonemarrow) so as to permit the production of a number of TRATreg sufcient for the protection, in this case, of the pancreas b-cells. Note that once a supply of TRATreg is ensured, the presence of danger signals and of the consequent inammation in the pancreas (possibly even therapeutically induced) would result, by the principles of the DMR, in the production of healing signals that might lead to the regeneration of the lost or exhausted b-cells. Finally, also in this case we can observe that the in vitro production and successive inoculation of TRATreg is an inferior therapeutic option with respect to the establishment of a colony of TRATreg producing agents, because aging and inammations of the b-cells would deplete the stock of TRATreg and require periodicTRATreg reinoculations. Alternatively one could envisage a prophylaxis for individuals at risk based on the disabling of the plasticity of Treg. For example, this could be realized by targeting an element of the hypothetical signaling pathway that in the DMR is responsible for the return of Treg to a non-regulatory state after an encounter with an active APC issuing the cognate antigen. This strategy would prevent or at least assuming a limited lifetime for Tregdelay the exhaustion of the existing pool ofTRATreg following tissue inammation. However, if the disabling of the plasticity of Treg is not antigen-specic, it carries the risk of immune exploitation (by, e.g., opportunistic infections and malignancies) inherent in non-plastic Treg that was mentioned above while dening the DMR (Sect. 4.1.3). On the other hand, an antigen-specic disabling of Treg considered, as above, as a pattern-recognition task appears of difcult technical implementation. The prevention of other autoimmune diseases can proceed along the lines expounded for T1D, provided one takes into account, in particular, the possible different nature of the initiating factor. For example, there is evidence that in multiple sclerosis (MS) (Goverman 2009) the still poorly understood and probably manifold initiating factors lead to an initial death of oligodendrocytes, the cells that myelinate the neuron axons in the central nervous system (CNS) (Coyle 2011). The subsequent autoimmune attack is apparently directed against myelin epitopes and, like in T1D, seems to be due to slight differences between the myelin synthesized in the CNS and that expressed under AIRE control in the lymphatic system (Anderson et al. 2002). The confounding factor is that the actual progression of the disease appears typically to be composed of a mix of oligodendrocyte destruction and regeneration. In the light of the DMR this could be tentatively explained as follows. First, there is evidence that once the oligodendrocytes are killed by the initiating factor they are efciently scavenged by macrophages that clear all the myelin debris. Thus, in the CNS regions of oligodendrocyte death the local APC (mainly microglia (Tambuyzer

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et al. 2009)) activated by the danger signals will not engulf myelin debris and, in the absence of pathogens, would be completely stripped by local Treg. This would promote oligodendrocyte regeneration and axon remyelination. In fact, in this scenario, the maintenance of the inammation for a small time interval beyond the vanishing of the initiating factor could lead to more encompassing regeneration (Foote and Blakemore 2005). However, in the absence of myelin-specic Treg protection hypothesized for MS, the oligodendrocytes of adjacent inamed regions will be attacked by immune effectors. This mix of oligodendrocyte death and regeneration would proceed until the initiating stress factor abates, leaving probably a substantial total damage. One can imagine that even in the presence of adequate myelin-specic Treg protection, each instance of activation of the initiating factor would still lead to a small net demyelination whose effects, contrary to the case of MS, would become apparent only in old age. This would lead to the long run convergence of CNS damage in long-term MS patients and a class of MS-free aging individuals (Coyle 2011). Summing up, even if protection against MS overt damage could be obtained just like in the case of T1D by establishing sites of ectopic myelin-specic Treg production, the MS initiating factor should also be identied in order to obtain a truly encompassing CNS protection. Finally, let me consider the problem of rejection in allogenic tissue transplantation. After an initial phase of the so-called direct immune attack provoked by donor APC, an indirect allorecognition phase ensues where the cells carrying donorspecic antigens are attacked by the immune system of the transplant recipient (Wood et al. 2012). This latter phase can be assimilated to an autoimmune response where the donor-specic antigens play the role of TRA. Thus, also in this case a strategy of establishment of a colony of sites of ectopic donor-specic Treg production can be envisaged. One could even conjecture that a concurrent organ and bone-marrow transplant is an attempt at establishing just this kind of situation, assuming that the bone marrow was such an ectopic Treg production site in the donor (Pham et al. 2000). In the case of nonliving donors, one could imagine an initial phase of effector (but not regulator) immunosuppression covering the period required to implant the ectopic colony of donor-specic Treg producers and establish an adequate stock of transplant-protecting Treg. Note that on the one hand one could say that in the presence of an ectopic colony of donor-specic Treg producers the transplanted tissue is protected from the consequences of inammation just like any other recipient tissue. Conversely, one could say that a non-transplanted tissue that following inammation is the object of autoimmune attack for lack of Treg protection is seen by the IS just like a transplanted tissue. Note also that the infusion of donor Treg concomitant with transplantation would promote transplantation tolerance but would not ensure the much more signicant operational tolerance (i.e., the long-term allograft survival in the absence of continuous drug-based immunosuppression (Wood et al. 2012, p.417)) because the transplanted tissue, owing to its donor-specic TRA, would still be subject to TRATreg depletion, with the consequent immune attack following longterm inammation.

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5 Discussion 5.1 Quality Considering the complexity of the cancer phenomenon, the DMR might appear naively simple as a candidate for understanding and curing cancer. For example, the DMR does not even mention the plethora of cytokines and immunosuppressive agents that are known to prevail in the tumor environment (Baronzio and Freitas 2008). The point is that simplication is at the heart of modeling and that identifying the elements that can be omitted from a model is as important as identifying those that must be included (Bredeweg and Struss 2004). Like the components of the braking circuit missing in the simplied circuit of Fig. 3, the immune components not mentioned in the DMR are not considered irrelevant but, on the contrary, they are assumed to correctly realize the function they are designed by evolution to perform. It is only when the predictions based on a model go unfullled that we need to consider its modication. And it is only when predictions are denite that they can go unfullled. Typically, when a model produces a signicant number of correct predictions it is modied incrementally rather than discarded and rebuilt from scratch. Moreover, the unfullled predictions often point the way to the model improvement and to the experiments that must be performed to establish how it must be changed. For these reasons, Lazebniks quote (2002, p.179) according to which in biology [a] eld reaches a stage at which models, that seemed so complete, fall apart, predictions that were considered so obvious are found to be wrong appears puzzling to an engineer. Paraphrasing Gall (1986, p.65), an engineer typically expects that an adequate complex model be invariably found to have evolved from an adequate simple model and, thus, that it cannot suddenly crumble into dust. Note that in pursuing a solution to the cancer problem we found at the cellular level a troubleshooting strategy that looked technically feasible. So, in our quest we were not forced to descend to the next lower level of organization. However, it might well be that an even better strategy exists at a lower level of organization, for example at the intracellular level of gene expression and signal transduction where most cancer research is currently focused. More generally, the problem-oriented engineering perspective on the understanding of system function is not incompatible with the quest for a level of understanding that goes beyond the current technical limits of system manipulability and repairability. In particular, the adoption of the engineering perspective does not necessarily imply the adoption of the if aint broke dont x it attitude, which is obviously incompatible with fundamental scientic thinking in biology just like in any other domain of science. In addition to its roles in grounding system understanding and grouping under one heading seemingly unrelated phenomena, the mechanistic nature of the model permits to make specic predictions about the system behavior. These predictions include the answer to the crucial experimental question what did you expect? (Collingwood 1978, p.140). Moreover, it makes possible the generation of what if, counterfactual predictions, which are essential for the interventions on the system aimed at changing its behavior. On the contrary, the correlative

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(or covariational) approach used in biology and expressed in the kind of arrow diagrams stigmatized by Lazebnik is unable to produce satisfying predictions. In Lazebniks own words (Lazebnik 2002, p.181): Even if a[n arrow] diagram makes overall sense it is usually useless for a quantitative analysis, which limits its predictive or investigative value to a very narrow range. The language used by biologists for verbal communications is not better and is not unlike that used by stock market analysts. Both are vague and avoid clear predictions. The problem of the correlative approach stems from the use of averages and the ensuing possibility of reasoning in terms of an average behavior which may not correspond to any actual individual behavior. As Paul Thagard put it (2000, p.145) Explanation is not statistical. For example, consider the case of Treg in the DMR. Should we consider them tolerogenic or immunogenic? At rst sight their function is exclusively tolerogenic. However, Treg also focus and thus enhance the immune response against the dangerous elements by stripping the active APC of their non dangerous pMHC. In this sense they are immunogenic. Thus, in general, Treg are both tolerogenic and immunogenic and no average behavior captures this duality. A similar observation can be made about the manipulation of chemokine signaling. Let us assume that, in the context of the DMR, the initial meeting of activated APC with Treg is due to a hypothetical chemokine X emitted by one of the parties and chemotactically sensed by the other. A depletion of X or an interference with the production or sensing of X would prevent the action of the Treg. However, locally ooding the host with X would also disrupt chemotaxis and would produce an effect very similar to that of the depletion. These are only two examples of how observations made on a biological system appear puzzling or contradictory in the absence of an underlying mechanistic model. Most importantly, the same holds for observations obtained from animal models of diseases. If one does not ensure that the human disease and its putative animal model admit a structurally similar mechanistic model, there is the real risk that the results of observations and manipulations performed on the animal model not only do not transfer to humans but be positively misleading in their role of models. Finally, let me emphasize the fact that biological models, like any other model, have an epistemic rather than ontic status. This observation answers the objection to Lazebnik suggestions put forward by Madar and coworkers. In their own words (Madar et al. 2009, p. 6379) Should we adopt the formal description approach [advocated by Lazebnik]? In our opinion the answer is no. We should always take into account that the denition of each component in a system might be temporal and partial and that questioning it, despite the confusion it might cause, is our scientic obligation. From the epistemic point of view advocated in this paper it is obvious that the denition of each component in a system (is) temporal and partial. Madar and coworkers objection is valid only if one holds the assumption that modelers should strive for unique, true models and that any reliance on temporary and partial models must be condemned. But true model is an oxymoron: by denition, models cannot be true; at most, they are adequate for the solution of the problem that prompted their creation (where the desire to attain a

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given level of understanding or to produce a model with a given predictive power can be also construed as examples of a such a problem).11 5.2 Quantity I have shown above that in the design and troubleshooting of electronic circuits there comes a point where some qualitative analysis is required. The same is true when these activities are performed on biological systems. For example, assuming that the DMR is adequate for describing tumor development and therapy, one would probably like to estimate the time interval between the establishment of inammation at the SMM locations and the depletion of the TSATreg stock that starts the process of immune tumor clearance. In the electronic circuit case, the rst step in the transition to quantitative analysis is the specication of the numerical values of the parameters that characterize the component models (e.g., resistance of resistors, gain of transistors, etc.). There is however a caveat in applying this strategy to biological models. Take for example the case of the sophisticated computer model of the T7 bacteriophage virus built by Drew Endy and John Yin. Endy and Yins model simulated mathematically how all 56 of the viruss genes were translated into 59 proteins, how those proteins subverted the host cell and even how the viruses would evolve resistance to various RNA-based drugs. That seems impressive. But peek inside the equations, Endy says, and youll nd that despite including measurements from 15 years of laborious experiments, there are still a tremendous number of degrees of freedom. The equations can be tweaked to produce almost any behavior. A useful model must suggest a hypothesis that forces the model builder to do an experiment, Endy says. This one didnt (Gibbs 2001, p.54, my emphasis). This problem, however, is not specic of quantitative biology. In the case of physics it is illustrated by an anecdote concerning physicists Enrico Fermi and Dyson (2004). In 1953, after several years of toil, Dyson and his collaborators had just obtained the results of a complex series of calculations concerning the interactions of some elementary particles. The computational results were in good agreement with the results of Fermi measurements. SoDyson narratesI made an appointment to meet with Fermi and show him our results. Proudly, I rode the Greyhound bus from Ithaca to Chicago with a package of our theoretical graphs to show to Fermi but he hardly glanced at them. Then he delivered his verdict in a quiet, even voice. There are two ways of doing calculations in theoretical physics, he said. One way, and this is the way I prefer, is to have a clear physical picture of the process that you are calculating. The other way is to have a precise and selfconsistent mathematical formalism. You have neither. In desperation I asked Fermi whether he was not impressed by the agreement between our calculated numbers and his measured numbers. He replied, How many arbitrary parameters did you use for your calculations? I thought for a moment about our cut-off procedures and said, Four. He said, I remember my friend Johnny von Neumann
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Of course, and most importantly, the choice of the problem must remain a primary ethical concern of the scientist.

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used to say, with four parameters I can t an elephant, and with ve I can make him wiggle his trunk. With that, the conversation was over (Dyson 2004, p.297, my emphasis). Fermi, one of the pioneers of computational physics, did not condemn computational models as such. He just reminded Dyson that, as Bredweg and Struss admirably synthesize (Bredeweg and Struss 2004, p.14) the existence or creation of a conceptual and qualitative model of a situation for a certain task to be solved is a prerequisite for a scientic quantitative model, not its result, as sometimes suggested. This is true for physics, for engineering, and for biology and medicine. In the words of Bernard (1865, p.227), in biology, medicine, as in all tude qualitative des phe nome ` nes [doit] ne cessairement experimental sciences le ce der leur e tude quantitative.12 In the absence of these previous, founding pre qualitative models, the parameters of the quantitative models correspond to the arbitrary parameters deprecated by Fermi and von Neumann. Note that the structured schematic of Fig. 2 contains more than 30 components and thus its quantitative specication requires at least the same number of parameters. Yet, it can be used to produce reliable computational predictions without incurring in von Neumanns tting-the-elephant effect. The reason lies in the fact that the parameters refer to quantities that are dened at the right level of organization and posses a well dened qualitative physical meaning in the circuit model. This makes the quantitative and the computational models robust relatively to the parameter values. The same robustness can be found in biology. Indeed, as Wimsatt reminds us (2007, p.111) If you nd a property that appears to be biologically important, and it is not robust, be suspicious of your model or the assumed parameter values. Incidentally, note that in general the quantitative analysis of nonlinear systems is problematic, whereas qualitative analysis is much less conditioned by nonlinearity (De Kleer 1984, p.263), as suggested by the fact that nonlinearity never surfaced as a problem in all the above discussions about qualitative models of circuits and biological systems that certainly contain some nonlinearity. Finally, let me add a note about the question of quantitative data in biology. The purported recent abundance of data in biology has almost become a truism. Still, as Sorger reminds us (2005, p.10) far from being data-rich as often claimed, biology is extremely data-poorparticularly in terms of quantitative, time-dependent and spatially resolved measurements. Conversely, an engineer using a simple oscilloscope to perform measurements on a circuit can easily capture thousands of high-resolution time-dependent data points in a single sweep of the oscilloscope. The scarcity of quantitative high-resolution time-dependent measurement data in biology can result in the observation that even soundly based quantitative biological models may happen to be insufciently constrained for the estimation of parameters from data. Even in this case, the availability of a qualitative mechanistic model can make a difference because data can be collected with a theoretical question in mind and be relevant to that question. On the contrary, data collected free of any theoretical burden (Krohs and Callebaut 2007, p.184) are typically found to have reduced relevance and explanatory value.
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The qualitative study of phenomena [must] necessarily precede their quantitative study.

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5.3 Structure So far I have taken for granted the existence of an adequate qualitative mechanistic model for the system and problem of interest. It is now time to ask if this assumption holds in general, that is if every biological and technological system admits a qualitative model. Put in the afrmative the question seems difcult to tackle. On the other hand, a single counterexample would be sufcient to prove that the answer is negative. I take the counterexample from the description given by Barrie Gilberta distinguished designer of analog electronic circuitsof his youthful attempts to understand a circuit. For a few years after World War II it was possible to buy at a very low price pieces of ex-government equipmentreal serious electronic systems (Gilbert 1991, p.178). Gilbert was very eager to probe the secrets of each apparatus he bought. As he recounts (Gilbert 1991, p.178, my emphasis) Most enigmatic was an IFF (Identication Friend or Foe) receiver: two back-to-back steel chassis_ Between these was a space into which a detonator tted; clearly, the secret of the IFF circuitry was something to protect at all costs, and that made it all the more interesting to trace it out. Alas, try as I might, it made no sense at all. Everything seemed to be connected to everything else, forming an impenetrable electronic labyrinth. To this day, I wonder how that stirring electronic mind made its crucial decision between friend and foe. The rst clue coming from this example is the existence of systems that presumably do not admit a satisfying qualitative explanation of their functioning. The second clue is the fact that systems where everything is connected to everything have a good probability of belonging to this category of undecipherable systems. On the other hand, several authors have pointed out that both technological and biological systems do not have this kind of structure. On the contrary, for reasons of biological evolvability and technical designability, biological and technological systems typically have a hierarchical and modular structure (Simon 1996). Obviously, this observation does not constitute a proof that all properly structured biological and technological systems and subsystems can be given an adequate qualitative mechanistic explanation and model. However, recalling the denition given above of a mechanistic explanation as a description of how the components that compose the system are organized to produce the overall functionality, the existence of a hierarchical organization of modules suggests that biological and technological systems are typically eligible for an analysis in terms of interacting components. Indeed, the biological system that served as example in this paperthe immune systemhas proved to be particularly amenable to mechanistic explanation and modeling. 5.4 Language All the biological models discussed in this paper were described only verbally. These models, being simple and qualitative can be kept in mind, explained, and mentally manipulated without the assistance of cognitive artifacts. However, there certainly comes a point when the complexity of the system of interest requires some

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kind of formal language to convey unequivocally the model and mentally manipulate it. Presumably, different system and different levels of organization of the same system require distinct formal languages. Therefore, it is difcult to offer detailed guidelines for formal language denition that hold in general. Nonetheless, I will try to list a few high-level principles derived from the examples of modeling presented above. First of all, a formal language for system modeling must support qualitative and mechanistic reasoning. Second, it must be able to behave as a cognitive artifact when operated by an individual suitably trained in the use of the language, especially when the system under consideration is complex. Third, it must support robust quantitative analysis (e.g., robust parametrization). Fourth, it must give an unequivocal representation of the model when used in the quantitative mode. Fifth, it must force the modeler to make explicit his or her assumptions, that is, to specify the model behavior for each component of the model, and for the interactions between the components contemplated by the model. The fact that the user and the model expressed in terms of the formal language become an extended cognitive system only when the user has been properly trained in the use of the language creates a bootstrap problem in the invention of new formal languages. When a language has just been created, not even its inventors can benet from it, even if the language is good in the sense that it has the potential to support the production of cognitive artifacts. Thus, initially, good languages and bad languages do not appear different in their support to modeling. This will be a problem especially in biology, where many new languages would presumably be invented, given the peculiarities of biological system that make unsuitable many of the formal languages developed for technological systems (of course, exceptions exist; for example, in Floreano and Mattiussi (2008) gure 5.9 on page 362 shows how the life cycle of a Th cell can be described using Grafcet, a formalism z developed for the description of the operation of industrial control systems (Hru and Zhou 2007)). Note that, on the contrary, a representation tool much used in biology such as cartoons does not require a special training to be understood (at least by people exposed to a western milieu) but its expressive potential is very limited.

6 Conclusion In a recent editorial (Moore 2012), BioEssays editor Andrew Moore wrote: I have a vision of a future in which [biologists] who think more about others results than producing their own are valued just as much. _[O]ne could call [this kind of biologist] a professional integrator: someone who gains higher level insights by critically analyzing and integrating the discrete ndings of others to create a new model that is more than the sum of its parts._Arguably there are so many results around that we need more dedicated people who explicitly dont produce new results, but rather distill out higher level insights. We could complement Moores recommendation to his wished professional integrator with the exhortation to also aim at creating models that make new predictions, which can be veried and, indeed, connect what was previously seen as not connected.

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In physics, this kind of scientist already exists and is called theoretical physicist, as opposed to experimental physicist. What is needed in biology is a new kind of theoretical biologist, one who does not rush to build a quantitative, mathematical, or computational model but starts by creating the equivalent in biology of Fermis clear physical picture (Dyson 2004). However, we must not forget that in physics there is no notion of system function nor that of malfunction, which are instead essential to biological thinking (Knight 2002). Thus, in the endeavor of realizing the equivalent in biology of the gure of theoretical physicist it is necessary to take into account also the point of view of the engineer, who builds models not only aspiring to a certain predictive power or intuitive physical clarity, but also aiming at the understanding of system functions and the troubleshooting of their malfunctions. The potential of such an engineering-inspired, qualitative, problem-driven, mechanistic approach to model building for the attainment of the goals of biology and medicine is signicant. The quantity and quality of original hypotheses, predictions, experimental questions, and proposed prophylaxes and therapies ensuing from the new immune system model built in this paper following this approach, for elds as complex and crowded as cancer, autoimmune disease, and transplantation research should be convincing enough for any biologist leaning toward the dryer side of research (Moore 2012).
Acknowledgments Many thanks to Daniel Marbach for bringing to my attention Lazebniks paper, to Enrica Cantamessa and to Maurizio and Raffaele Persello for their help in reconstructing the history of the braking circuit, and to Delia Giallonardo, Roberta Maestri, Marco Palombi, Maurizio Persello, Markus Waibel, and to an anonymous reviewer for their precious feedback on the manuscript.

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