M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 8 9 4 e1 9 0 3

available at www.sciencedirect.com

ScienceDirect
www.elsevier.com/locate/molonc

Review

Allo-reactive T cells for the treatment of hematological

malignancies5
J.H.F. Falkenburg, I. Jedema*
Department of Hematology, Leiden University Medical Center, Netherlands

A R T I C L E

I N F O

A B S T R A C T

Article history:

Several mechanisms can be responsible for control of hematological tumors by allo-reactive

Received 24 September 2015

T cells. Following allogeneic stem cell transplantation (alloSCT) donor T cells recognizing ge-

Received in revised form

netic disparities presented on recipient cells and not on donor cells are main effectors of tu-

15 October 2015

mor control, but also of the detrimental graft versus host disease (GVHD). Since after

Accepted 16 October 2015

transplantation normal hematopoiesis is of donor origin, any T cell response directed against

Available online 24 October 2015

polymorphic antigens expressed on hematopoietic recipient cells but not on donor cells will
result in an anti-tumor response not affecting normal hematopoiesis. After fully HLA-

Keywords:

matched alloSCT, T cells recognizing polymorphic peptides derived from proteins encoded

Allogeneic stem cell transplantation

by genes selectively expressed in hematopoietic lineages may result in anti-tumor responses

T cells

without GVHD. Due to the high susceptibility of hematopoietic cells for T cell recognition, a

Hematological malignancies

low amplitude of the overall T cell response may also be in favor of the anti-tumor reactivity

Immune therapy

in hematological malignancies. A mismatch between donor and patient for specific HLAalleles can also be exploited to induce a selective T cell response against patient (malignant)
hematopoietic cells. If restricting HLA class II molecules are selectively expressed on hematopoietic cells under non-inflammatory circumstances, allo HLA class-II responses may control
the tumor with limited risk of GVHD. Alternatively, T cells recognizing hematopoiesisrestricted antigens presented in the context of mismatched HLA alleles may be used to treat
patients with hematological cancers. This review discusses various ways to manipulate the
allo-immune response aiming to exploit the powerful ability of allo-reactive T-cells to control
the malignancies without causing severe damage to non-hematopoietic tissues.
2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights
reserved.

1.

Introduction

1.1.

Immune surveillance of hematological cancers

Development of various cancers has been hypothesized to be

the result of the inability of T cells to control the aberrant cell
5

population (Smyth et al., 2001). This hypothesis is based on

the assumption that T cells capable of recognizing the tumor
cells with sufficient avidity to perform immune surveillance
are present in the autologous repertoire (Haanen et al., 2006;
Naito et al., 1998; Zhang et al., 2003). As part of the malignant
transformation certain genes can be induced to be aberrantly

This is a contribution to the special issue edited by Johanna Olweus, Cancer Immunotherapy.
* Corresponding author.
E-mail address: i.jedema@lumc.nl (I. Jedema).
http://dx.doi.org/10.1016/j.molonc.2015.10.014
1574-7891/ 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 8 9 4 e1 9 0 3

expressed or upregulated and may then be a target for a T cell

response. Various antigens from embryonic cancer genes and
other overexpressed tumor associated genes have been proposed to be appropriate targets for such an immune response
(Greiner et al., 2005; Scheibenbogen et al., 2002; van Duin et al.,
2011). Although T cells frequently have been found capable of
specifically recognizing peptides derived from these tumor
associated antigens (TAA), high avidity T cells capable of efficiently eliminating autologous tumor cells have not
frequently been found. This may not be unexpected since
many of these TAA may be presented in the thymus leading
to negative selection of high avidity T cells capable of recognizing the antigens, resulting in deletion of the most efficient
T cell population (Sebzda et al., 1999; Starr et al., 2003). Since
the T cell immune system was developed to recognize non
self-peptides in the context of self HLA molecules, peptides
derived from mutated parts of a tumor associated protein
are more likely to be appropriate targets for T cell mediated
control of the tumor. Recent studies with antibodies modulating checkpoint blockade have illustrated that in some patients T cells capable of controlling the various solid tumors
may be present (Khalil et al., 2015). Whether this is also applicable to hematological tumors is under investigation.

1.2.
Allogeneic stem cell transplantation for cellular
immune therapy
The advantage of selecting a non-self T cell repertoire for
attacking the tumor is attractive, since this T cell repertoire
is not subjected to the negative selection process in the
thymus deleting all relevant high avidity T cells (Sebzda
et al., 1999; Starr et al., 2003). The most broadly applied
example of using the allo-immune repertoire for controlling
hematological cancers is allogeneic hematopoietic stem cell
transplantation (alloSCT). The effectivity of this treatment is
based on genetic differences between patient and donor
allowing recognition of hematological tumors in the patient
by donor T cells. Since after allogeneic stem cell transplantation patient derived hematopoiesis is replaced by donor hematopoiesis, T cell responses from the donor recognizing
non self-antigens on hematopoietic cells of the recipient will
lead to a profound high avidity T cell response capable of eliminating hematopoiesis of recipient origin including the malignant cell population (Barrett, 1997; Falkenburg and Warren,
2011; Horowitz et al., 1990; Kolb, 2008; Riddell et al., 2003).
This graft versus tumor/graft versus leukemia (GVT/GVL)
reactivity following alloSCT has been a well described profound anti-tumor reactivity capable of curing patients with
resistant hematological cancers. However, donor derived
allo-immunity in the patients is frequently coincided by a
similar T cell response against non-hematopoietic normal tissues from the recipient, resulting in organ toxicity (graft
versus host disease (GVHD)) (Ferrara et al., 2009; Markey
et al., 2014; Nash and Storb, 1996). This complication is due
to a comparable immunological phenomenon of donor T cells
capable of recognizing antigens on recipient tissues that are
considered non-self by the donor T cells. After allogeneic
transplantation therefore, a high anti-tumor efficacy can be
obtained, but due to lack of specificity this treatment is
frequently associated with high morbidity and mortality of

1895

GVHD. Thus, the main goal of applying allo-reactivity for control of cancer is to reduce the toxicity and increase the efficacy
by improving the specificity of the T cell response.

2.
Principles of allo-immune control of
hematological cancers after allogeneic stem cell
transplantation
The aim of alloSCT is the induction of an immune response
against patient derived hematopoiesis or specific hematopoietic cell lineages from which the tumor is derived. The aim
could be considered a donor cell mediated inverted immune
rejection of the hematopoietic system of the recipient. The
therapeutic effectivity is not dependent on de-novo changes
in the malignancies as compared to the normal counterpart,
but is based on recognition of polymorphisms that are
anchored in the genome of the recipient. The only requirement of the antigens targeted is that they are polymorphic
and expressed within the cell lineages from which the malignancy originated. This makes this intervention broadly applicable, not restricted to a specific tumor, and not dependent on
mutations within the tumor. Since under these circumstances
multiple non self-antigens are usually targeted at the same
time, a polyclonal response is likely to occur which increases
effectivity since escape variants are less likely to occur. The
establishment of donor hematopoiesis in the patient after
transplantation allows the use of any T cell derived from the
donor that is educated in the donor to be applied for this purpose since there will always be tolerance to donor hematopoiesis after transplantation, irrespective of the diversity of the T
cell repertoire.
The only pre-requisite for a successful application of this
treatment is low or lack of reactivity against normal nonhematopoietic tissues of the recipient. Professional antigen
presenting cells (APC) of recipient origin, like dendritic cells
(DC), are likely to play a dominant role in provoking a donoranti-patient immune response after transplantation. Since
professional APCs like DC are derived from the hematopoietic
system, almost all immune responses against recipient antigens after transplantation are directed against antigens that
are also expressed in hematopoietic cells. Many hematopoietic cells including malignant cells have high expression of HLA
class I and HLA class II, allowing preferential recognition of
hematopoietic cells by the immune system.
Various methods can be used to manipulate the alloimmune response in favor of recognition of hematopoietic
cells after transplantation. This can be obtained by titrated
doses of immune suppression, allowing recognition of hematopoietic cells while preventing the more stringent recognition of non-hematopoietic tissues. Alternatively, the
immune response can be manipulated by first depleting the
stem cell graft of T cells, and subsequently treating the patient
with postponed administration of donor derived T cells after
transplantation as soon as donor hematopoiesis has been
established, the majority of professional APCs have been
replaced by donor APCs, tissue repair has taken place, and
viral infections are under control (Barge et al., 2003). Postponed donor lymphocyte infusion (DLI) may lead to skewing
of the immune response towards hematopoiesis with more

1896

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 8 9 4 e1 9 0 3

limited risk of the development of severe GVHD. Preferential

recognition of hematopoietic cells is also created by the combined expression of HLA class I and HLA class II on many hematopoietic cells and hematopoietic tumors, allowing
concurrent recognition by CD4 and CD8 T cells from the donors. This concurrent expression of HLA class I and HLA class
II less likely occurs in non-hematopoietic cells unless strong
inflammatory circumstances occur.

3.
Minor histocompatibility antigens (MiHA) as
targets for anti-tumor responses after allogeneic SCT
3.1.

Nature of MiHA

T cells are designed to recognize non-self peptides in the

context of self HLA expressed on the cell membrane. Part of
most intracellular proteins are degraded by the proteasomes,
further processed intracellularly to be loaded onto HLA class I
molecules and expressed on the cell membrane. Alternatively,
any protein present in endosomes can be degraded into peptides, which can be presented by HLA class II molecules on
the cell membrane. Thus, HLA molecules present on the cell
membrane are usually mostly loaded with endogenous antigens derived from intracellular proteins. Following alloSCT
with an HLA identical donor, during an allo-immune response
in the patient, donor T cells will recognize patient derived
polymorphic peptides presented in the context of the
matched (self) HLA molecules (Falkenburg and Warren, 2011;
Riddell et al., 2002). Single nucleotide polymorphisms (SNPs)
that differ between donor and recipient are usually responsible for the creation of peptides that are differentially
expressed in HLA molecules of donor and recipient. Polymorphic peptides expressed in self HLA molecules that can be
recognized by T cells from another individual are by definition
called minor histocompatibility antigens (MiHA) (Spierings,
2014). SNPs can be responsible for the creation of MiHA via a
variety of mechanisms. First, non-synonymous SNPs can
lead to proteins carrying amino acid differences between
donor and recipient. If the polymorphic peptides derived
from these proteins are expressed in HLA molecules, this
can lead to development of a strong immune response. In
addition, aberrant expression of proteins in alternative
reading frames that may not create functional proteins, but
that are consistently expressed, are also capable of creating
MiHA. Alternatively, SNPs can be responsible for splice variants or frame shifts resulting in the expression of stretches
of peptides that are different in individuals. These proteins
can be responsible for creating peptides that are present in
some individuals and absent in others. Thus, the repertoire
of MiHA contains peptides that are recognized because of
polymorphic amino acid differences between donor and recipient, or by peptides that are absent in some individuals and
present in others.

3.2.

Development of a MiHA specific immune response

The immune system of the donor has usually not been

exposed to MiHA of recipient origin unless these donors
have been treated with blood transfusions or in case of

pregnancy with fetal-maternal transmission of cells. Thus, T

cells recognizing a specific MiHA are usually present within
the nave T cell repertoire of the donor with a low frequency
(Bleakley et al., 2010; Falkenburg and Warren, 2011).
Since for the development of an appropriate T cell response
triggering by professional APCs is needed, T cells of donor
origin infused into the patient will not always result in the
development of a profound anti recipient immune response.
In most cases after fully HLA identical transplantation an immune response needs to be provoked by professional APC
from patient origin presenting patient derived MiHA (Reddy
et al., 2005). Since professional APCs are derived from the hematopoietic system, such an immune response is likely to be
at least in part directed against MiHAs that are also expressed
in a variety of hematopoietic cells of recipient origin, including
the malignancy, resulting in a frequent GVL reactivity.
If MiHA are derived from genes that are not only expressed
in APCs but also on non-hematopoietic tissues of the recipient, concurrent GVHD may develop. However, if the donor T
cells are directed against MiHA with selective expression on
hematopoietic cells, a specific anti hematopoietic immune
response will occur with destruction of all hematopoietic cells
of the patients, but leaving donor hematopoiesis in the patient
unharmed (de Bueger et al., 1992).
A number of hematopoiesis restricted MiHA have been
described (Spierings, 2014), among which the HA-1 (den
Haan et al., 1998), HA-2 (den Haan et al., 1995), LRH1 (de
Rijke et al., 2005), Bcl2A1 (Akatsuka et al., 2003), and LBARHGDIB-1R (Pont et al., 2015) are most extensively studied.
Although sometimes an immune response selectively targeting MiHA expressed on hematopoietic cells may occur resulting in a specific GVL reactivity, under most circumstances
these T cell responses will be accompanied by immune responses targeting broadly expressed MiHA. Therefore,
without selection of T cells recognizing a restricted set of antigens it is unlikely that unmodified DLI will predictably target
only hematopoietic cells of recipient origin resulting in selective GVL reactivity.

3.3.
GVL

Factors influencing the balance between GVHD and

Gene expression of the targeted antigen is not the only factor

relevant for skewing the immune response towards GVL reactivity. T cells need for appropriate recognition firm attachment to the target cells, as mediated by adhesion molecules
and costimulatory molecules. Many tissues do not highly express these antigens under steady state conditions and therefore do not allow T cells to interact with these tissues resulting
in significant damage. Only in the presence of a danger signal,
resulting in activation of toll-like receptors or by mediation of
activating cytokines, these tissues may be targeted by alloreactive T cells (van der Zouwen et al., 2012). We recently
demonstrated in a cohort of patients that the separation between GVL and GVH reactivity was only in part due to selective
recognition of antigens with restricted tissue expression. Patients who develop an immune response after DLI with a
restricted but not hematopoiesis specific repertoire with a
low amplitude can develop GVL reactivity without significant
GVHD, since cells of hematopoietic origin including malignant

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 8 9 4 e1 9 0 3

cells have a higher likelihood of expressing costimulatory and

adhesion molecules and are more accessible to T cell interaction than many other tissues. These results illustrate that selective GVL reactivity targeting MiHA is in part dependent on
the restricted expression on hematopoietic cells, but is also
influenced by many environmental circumstances including
an inflammatory environment. Skewing the T cell response
specific for MiHA in the direction of GVL may be performed
by selecting for infusion T cells that target MiHA specifically
expressed on hematopoietic cells, or by timing of the immune
response, thus controlling the amplitude and the inflammatory circumstances in the patient. Postponing administration
of unmodified T cells and controlling infections may also
contribute to selective GVL reactivity.

3.4.

HLA class-II restricted MiHA

Since HLA class II molecules are relatively specifically

expressed on hematopoietic cells, adoptive transfer of CD4
T cells recognizing MiHA expressed in HLA class II molecules
may result in a relative skewing towards GVL reactivity
(Griffioen et al., 2008; Oostvogels et al., 2014; Spaapen et al.,
2008, 2009; Stumpf et al., 2009). Since activated APCs highly
express HLA class II molecules, profound CD4 responses
recognizing MiHA expressed in HLA class II molecules
frequently occur. Both CD4 cells recognizing hematopoiesis
restricted MiHA and CD4 T cells recognizing broadly
expressed MiHA may evoke a relatively specific GVL reactivity if the restriction molecule (the HLA class-II molecule)
is selectively expressed on hematopoietic cells of recipient
origin. Under steady state non-inflammatory conditions this
may be the case. However, shortly after transplantation and
under inflammatory circumstances, HLA class II expression
is upregulated on a variety of tissues. Thus, under these circumstances CD4 T cells may also evoke both GVL and GVH
reactivity. Based on these hypotheses administration of purified CD4 cells after transplantation under steady state conditions is now explored to attempt to induce GVL reactivity
with limited coinciding GVHD.

4.
Mismatched HLA molecules as targets for
immune responses after allogeneic stem cell
transplantation
4.1.

The allo-HLA repertoire

From the developing T cell repertoire in the thymus T cells are

depleted that are capable of recognizing self-peptides in the
context of self HLA to prevent the occurrence of auto immune
reactions (Sebzda et al., 1999; Starr et al., 2003). In contrast, T
cell recognition capable of interacting with any peptide presented in the context of non-self (allo) HLA molecules will be
left untouched since there is no reason for deletion. T cells
will normally not encounter peptides in the context of polymorphic non-self HLA-molecules, and no danger of harmful
recognition will occur. Since in evolutional control no rules
are developed in the absence of danger, within the T cell repertoire any T cell with a T cell receptor (TCR) capable of recognizing by chance a peptide presented in the context of non-

1897

self HLA molecules can be preserved. This has resulted in a

high incidence of allo-HLA reactive T cells in the normal T
cell repertoire. Any T cell which is meant to recognize a nonself peptide in the context of self MHC is allowed to crossreact with a non-self HLA molecule presenting any peptide.
In agreement of this observation we have previously illustrated
that virtually any virus specific T cell meant to recognize a viral
peptide in the context of self HLA molecule can be crossreactive with a totally different peptide presented in the
context of an allo-HLA allele (Amir et al., 2010; DOrsogna
et al., 2010; Rist et al., 2009). This does not mean that each individual virus specific T-cell population has a high likelihood of
causing GVHD if infused across one particular HLA barrier. Oligoclonal CMV or EBV specific T cell populations from thirdparty donors have been infused without frequent development
of GVHD (Fuji et al., 2013; Leen et al., 2013; Macesic et al., 2015;
Melenhorst et al., 2010; Vickers et al., 2014). Although this may
be in part due to a relatively rapid rejection of the 3rd party
cells, it is likely that only a relatively broad repertoire of T cells
has a high chance of being cross-reactive against the specific
HLA allele. Because of this random occurring cross-reactivity,
allo-reactive T cells are similarly present in the memory and
nave T cell repertoire. This has major impact for the chance
of allo-HLA reactivity to cause GVHD.

4.2.
Allo-HLA reactive T cells in the nave and memory
compartment
During life, healthy individuals are increasingly exposed to
pathogens leading to the development of the memory T cell
repertoire. Since this memory repertoire contains also allo
cross-reactive T cells targeting allo-HLA molecules, following
alloSCT with HLA mismatched donors, profound alloimmune responses can readily occur. Only in case of a complete nave allo-HLA reactive repertoire as in umbilical cord
blood transplantation, a similar threshold for activation as
in HLA identical transplantation may occur, since also in these
circumstances T cells need to be activated by professional
recipient derived APCs (Farber and Ahmadzadeh, 2002; Ni
and ONeill, 1997). However, in case of transplantation with
stem cell sources or DLI from adult donors, the allo-HLA reactive repertoire can be present in the memory T cell compartment (Macedo et al., 2009; Melenhorst et al., 2011). The high
frequencies, the rapid activation and the highly polyclonal
repertoire of allo-HLA reactive T cells are likely to be the explanation for the rapidly occurring concurrent development of
GVL and GVHD after HLA mismatched transplantation. Better
HLA matching results in a lower chance of developing severe
GVHD allowing to positively influence the balance between
GVL and GVHD. Depletion of the rapidly activating alloreactive memory T cell repertoire shortly after transplantation
can be achieved by treatment of the patient with high-dose
cyclophosphamide resulting in (partial) abrogation of the
allo-HLA response (Holtick et al., 2015). Residual allo-reactive
T cells from the nave repertoire are likely to have different kinetics of activation. Thus, depleting the rapidly activated alloreactive memory T cell repertoire may leave a proportion of
the GVHD and GVL causing T cells unaffected resulting in
preservation of at least some of the GVL reactivity with
acceptable GVHD. Depleting the allo-repertoire from a

1898

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 8 9 4 e1 9 0 3

haplo-identical graft by in vitro depletion of the allo-HLA responses using photo-activation, may also modify the alloimmune response towards the more profitable GVL reactivity
(Bastien et al., 2012; Mielke et al., 2011).

4.3.
Targeting mismatched HLA class-II molecules to
induce GVL reactivity
Whereas HLA class I molecules are expressed on many nucleated cells, HLA class II molecules are preferentially expressed
on cells of hematopoietic origin including hematological tumors. This had led to the assumption that allo-HLA class-II
T cell reactivities may cause a selective GVL response in the
absence of severe GVHD. Indeed several clinical observations
have illustrated that a specific GVL reactivity may be caused
by allo HLA class II reactive T cells (Matsushita et al., 2006;
Rutten et al., 2008, 2013). This phenomenon has been specifically studied for allo-HLA-DP specific T cell reactivities, since
in searching for an HLA matched unrelated donor HLA-DP is
usually not taken into account. 70% of all 10 out of 10 HLA
matched donors are disparate for one or two of the HLA-DP alleles. We previously have illustrated that selective GVL reactivity can occur after donor lymphocyte infusion by specific
allo HLA-DP T cell responses (Rutten et al., 2008, 2013). This
has led to studies investigating the potential of purified CD4
T cells from HLA-DP mismatched individuals to execute a specific GVL reactivity. However, following infusion of these cells
GVHD was still relatively frequently observed. This, again, was
due to the occurrence of inflammatory circumstances leading
to upregulation of HLA class II on GVHD target organs. Since
allo-HLA-DP reactivity may be present in both the naive and
memory T cell repertoire, upregulation of HLA class II molecules on GVH target organs may readily induce allo HLA class
II reactive T cell responses from the memory repertoire resulting is severe GVHD. Therefore, significant modifications are
necessary to recreate the allo-HLA T cell repertoire to treat patients following HLA mismatched transplantation without
harming the patient.

5.
Allo HLA restricted T cell recognition of
hematopoiesis specific targets
5.1.

Peptide recognition by allo-HLA restricted T cells

Allo-immune responses occurring following HLA mismatched

alloSCT are frequently associated with severe GVHD and GVL
reactivity. T cell reactivities against allelic HLA differences between donor and recipient are at least in part specific for the
amino acid polymorphisms present in connection with the
binding groove of the HLA molecules. It has been assumed
that these broad allo-HLA immune responses represented the
dominant repertoire recognizing peptide-HLA complexes
expressed on most cells (Aosai et al., 1991; Rotzschke et al., 1991).
During thymic development T cells will be positively
selected for recognition of peptide-HLA complexes as long as
low avidity interactions take place, whereas only T cells recognizing autologous peptide MHC complexes with high avidity
will be deleted (Viret and Janeway, 1999). This will result in a
large repertoire of T cells that are potentially capable of

recognizing non-self HLA molecules presenting any peptide.

Based on these assumptions it has been hypothesized that
allo-HLA specific T cells recognize the non-self HLA molecule
irrespective of the peptide presented.
However, we and others have illustrated that also in the
allo-HLA repertoire most T cells recognize a specific peptideHLA complex with various avidities (Alexander-Miller et al.,
1993; Amir et al., 2011a; DOrsogna et al., 2010; Munz et al.,
1999; Obst et al., 1998). Some of the TCRs appear to recognize
target cells expressing a non-self HLA molecule with multiple
peptides with low or intermediate avidity, but some T cells
clearly recognize specific peptides presented in non-self HLA
with high specificity and high avidity.

5.2.
Allo-HLA restricted T cells with therapeutic
potential
We and others have illustrated that within the allo-HLA T cell
repertoire T cells can be found with high specificity for HLApeptide complexes of potential therapeutic relevance. High
avidity T cells have been described recognizing peptides
derived from tumor associated antigens (e.g. WT1, PRAME,
NY-ESO) in the context of non-self HLA-A2 or peptides from
B cells specific proteins including CD20, CD79A or the endogenous transcription factor BOB1 in the context of non-self
HLA-A2 or HLA-B7 (Abrahamsen et al., 2010; Amir et al.,
2011b; Jahn et al., 2015; Kronig et al., 2009; Sadovnikova
et al., 1998; Savage et al., 2004). These T cell responses can
be found as rare cell populations within the nave repertoire
of donors who are negative for the targeted allo-HLA allele.
For instance, HLA-A2 or HLA-B7 restricted WT-1, CD20,
CD79A and BOB1 peptide specific T cell responses have
been purified from HLA-A2 and/or HLA-B7 negative healthy
donors via a variety of purification methods. Not only
in vitro, but also in vivo within the detrimental allo-HLA immune responses in patients with GVHD following HLA mismatched transplantation, T cell responses can be found
with potentially beneficial peptide/HLA specificities that
may be used for therapeutic purposes. Recently, we identified
from an HLA-A2 positive patient transplanted with an HLAA2 negative donor allo-reactive donor T cells recognizing
peptides derived from the TAA PRAME restricted by HLA-A2
with high specificity and avidity (Amir et al., 2011b). Such T
cells may be appropriate therapeutic reagents in the context
of HLA mismatched transplantations. Although they are difficult to physically isolate from the T cell repertoire of the
healthy donor, once these T cells have been identified and
the TCR has been cloned, by gene transfer these TCR can be
used to reengineer other immune cells toward highly specific
tumor specific T cell populations of desired specificity.

6.
T cell separations to modify allo-immune
responses after transplantation
6.1.
Postponed T cell infusion to diminish GVHD and
preserve GVL
GVHD after allo-SCT can be prevented or modified by ex vivo T
cell depletion of the graft or by in vivo removal of T cells of

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 8 9 4 e1 9 0 3

donor origin from the recipient using monoclonal antibodies

like alemtuzumab or anti T cell globulins (ATG) infused prior
to or post transplantation (Barge et al., 2006; Kanda et al.,
2011; Storek et al., 2015). Alternatively, activated alloreactive T cells that are rapidly activated shortly after infusion
at the time of transplantation can be depleted by treatment of
the patient shortly after transplantation with high-dose cyclophosphamide (Holtick et al., 2015). Appropriate partial depletion of T cells resulting in significant reduction of GVHD
while preserving part of the allo-immune repertoire may
give rise to a relatively specific GVL reactivity due to the high
immunogenicity of hematopoietic cells, including professional APCs, at the time of the transplant. However, it may
be clear that it is not easy to precisely control these circumstances. Insufficient T cell depletion will give rise to development of GVHD, whereas complete abrogation of the alloimmune T cell responses will also remove the allo-reactive
GVL reactivity. If initially profound T cell depletion is applied
to prevent development of GVHD, the GVL reactivity must be
reintroduced by postponed DLI. Balance between the beneficial GVL reactivity and GVHD is dependent on the dose of
infused T cells and the time interval between transplantation
and DLI and is obviously also dependent on the immunogenetic mismatch between donor and recipient (Barge et al.,
2003; Eefting et al., 2014a, 2014b). At the time of stem cell
transplantation early after conditioning of the patient inflammatory conditions, lymphopenia of the patient, and the high
frequencies of patient derived activated APCs, will lead to profound immune responses which are likely to include clinical
signs of GVHD. Gradually, after transplantation patient APCs
are replaced by donor APCs, lymphopenia is restored, tissue
damage caused by the conditioning regimen will be repaired,
and inflammatory conditions may be more controllable. Postponed DLI allows infusion of higher doses but leaves the patient at risk for recurrence of the disease early after alloSCT.
Whether or not a T cell response directed against the hematopoietic system (including the malignancy) will develop, depends on the presence of APCs of the patient and/or the
immunogenicity of the hematological malignancy. Chronic
myeloid leukemia (CML) has been found to be a very immunogenic malignancy due to its ability to create its own professional APCs, whereas acute lymphoblastic leukemia (ALL) is
far less immunogenic. Thus, early infusion of donor T cells
will lead to GVHD as well as concurrent GVL, appropriate intermediate dose of DLI at an intermediate interval may lead
to less GVHD with hopefully appropriate GVL reactivity,
whereas (too) late infusion of T cells may leave the patient
in an anergic state resulting in no GVHD and no GVL. These responses can be modified to some extent, but it appears to be
difficult to precisely control these clinical circumstances.

6.2.

Separation of T cell populations to decrease GVHD

Several strategies of relatively crude separation of donor T cell

populations before infusion into the patient are being
explored. After HLA identical sibling transplantation initial
removal of nave CD45RA T cells may abrogate GVHD (and
also GVL) keeping the memory CD45RO T cell compartment
intact leading to appropriate control of pathogens after transplantation. In those cases nave T cells may be introduced

1899

later to re-introduce GVL reactivity. This approach may be

less broadly applicable in case of HLA non-matched transplantation since the allo-HLA repertoire is also present in
the memory T cell compartments, as discussed above. This
strategy is now being explored in clinical studies (Bleakley
et al., 2015; Shook et al., 2015; Touzot et al., 2015).
Alternatively, strategies to only deplete alpha-beta T cells
from the graft leaving gammaedelta T cells in the graft are being explored with the assumption that gammaedelta T cells
are capable of controlling certain viral infections in particular
CMV, and may also contribute to an immune response controlling the malignancy. Clinical studies are also undertaken
to validate these hypotheses (Handgretinger, 2012; Locatelli
et al., 2013).
Selection of purified CD4 cells for DLI after transplantation
is also being explored (Meyer et al., 2010; Orti et al., 2009;
Stevanovic et al., 2013). Allo-reactive CD4 T cells may be
capable of controlling infections early after transplantation,
and may induce GVL by eradicating hematopoietic cells that
are HLA class II positive including the malignant cell population. This intervention may bridge patients to more profound
immune intervention with unmodified DLI late after transplantation. Alternatively, approaches are being explored to
select T cell populations with preferential recognition of the
malignant cells of interest. Exposing T cells to activated malignant cells from the patient followed by isolation of these activated T cells is being explored as a method to enrich T cells
with relative specific recognition of (malignant) hematopoietic
cells of recipient origin (Jedema et al., 2007).

7.
Targeting of hematopoietic malignancies by
antigen specific allo-reactive T cells
7.1.
Immunotherapy after alloSCT with in vitro isolated
antigen specific T cells
Hematopoiesis restricted MiHA specific T cells are ideal candidates to execute GVL reactivity in the absence of GVHD. This
treatment can only be performed if the patient is positive for
both the HLA restriction molecule and the MiHA. If the patient
expressed such a MiHA, and the donor is negative for the
MiHA it is likely that the donor has T cells in the repertoire
capable of HLA-restricted recognizing the antigen. Due to
the fact that most donors will not have been exposed to
such MiHAs, selection and purification of MiHA specific T cells
has been found to be challenging.
We have previously explored the possibility to generate
in vitro T cell lines against the hematopoietic MiHA specific
antigen HA-1 to control the malignancy after transplantation
(Meij et al., 2012). Prolonged culture periods were necessary
to generate sufficient relatively pure HA-1 specific T cell lines
from the donor. Thus far such culture methods did not result
in products capable of reproducibly controlling the malignancy in the patient. Two approaches are now being explored
to generate MiHA specific T cell products for clinical application. We are exploring MiHA-HA-1 specific T cells for clinical
application by purification steps using streptamers consisting
of peptide/MHC complexes allowing to process large volumes
of donor lymphocytes for isolation. Although we demonstrate

1900

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 8 9 4 e1 9 0 3

that we can isolate these cells from most HA-1 negative donors, total numbers of antigen specific T cells isolated are still
low, and presently a phase IeII clinical study is being performed to determine whether these numbers are sufficient
to generate a HA-1 specific T cell response after transplantation in vivo.
Amplification of the hematopoietic MiHA specific T cell
population in vivo is also being explored by vaccination of patients after transplantation with dendritic cells (DCs) loaded.
with a variety of hematopoietic MiHA specific peptides. These
investigations have to elucidate whether triggering a hematopoietic restricted MiHA specific T cell response is capable of
executing a GVL response in the absence of GVHD after
transplantation.

7.2.
Immunotherapy using T cells genetically modified
with an allo-antigen specific TCR
T cells can be re-engineered towards new specificities by
transfer of a high affinity TCR from the allo-repertoire (T cell
receptor gene therapy is reviewed elsewhere in this issue). A
high avidity TCR recognizing HA-1 has been isolated from a
patient with a profound HLA- A2 restricted HA-1 specific T
cell response. A clinical grade vector was made to transduce
donor T cells with an HA-1 TCR to create large numbers of
HA-1 specific T cells (van Loenen et al., 2014). We are exploring
a phase IeII clinical study in which patients positive for the
HLA-A2 restriction molecule suffering from a high grade malignancy that is HA-1 positive are treated early after transplantation with virus specific T cells from donor origin transduced
with the HA-1 TCR. In this way a product is created consisting
of virus specific T cells that are also specific for the MiHA HA-1.
This investigation will evaluate whether infusion of the cells
early after transplantation can give rise to expansion of an
HA-1 specific T cell repertoire capable of controlling the disease early after transplantation.
TAA specific T cells recognizing non polymorphic antigens
can also be found in the T cell repertoire of healthy donors. A
variety of WT1, NY-eso, PRAME, Proteinase-3 and other TAAspecific T cells restricted by the autologous HLA-molecules
have been found with various frequencies in the T cell repertoire of healthy donors (Rezvani et al., 2005; van den Ancker
et al., 2013; Weber et al., 2013). A large variety of avidities
have been observed but at present it is unclear whether these
T cells are of sufficient avidity to execute a profound antitumor effect after transplantation. As discussed in paragraph
5 high avidity TAA or tissue specific T cells recognizing a peptide presented in a non-self HLA allele can be found in vitro
and in vivo. Since encouraging clinical responses have been
found by targeting CD19 resulting in complete depletion of B
cells using T cells transduced with a chimeric antigen receptor
(CAR), strategies are being explored how to deplete all B cells
from patients with B cell malignancies using B cell specific
HLA restricted high avidity TCRs. High avidity CD20 or BOB1
specific HLA-A2 or HLA-B07 restricted TCRs capable of
strongly recognizing malignant as well as non-malignant cells
from the B cell compartment including multiple myeloma
have been found. These TCRs have been cloned and characterized and these reagents are now ready to be investigated in
clinical studies. Since TCRs are capable of recognizing

relatively low amounts of peptides in HLA molecules, this

approach may be more sensitive for tumors not expressing
high numbers of target molecules as may be necessary for
appropriate clinical results using CARs.
These clinical investigations will have to be performed to
evaluate whether allo-reactive antigen specific T cells can be
useful to control the malignancy in the absence of GVHD
both after allogeneic SCT as well as in the autologous setting.

R E F E R E N C E S

Abrahamsen, I.W., Stronen, E., Walchli, S., Johansen, J.N.,

Kjellevoll, S., Kumari, S., Komada, M., Gaudernack, G.,
Tjonnfjord, G., Toebes, M., Schumacher, T.N., LundJohansen, F., Olweus, J., 2010. Targeting B cell leukemia with
highly specific allogeneic T cells with a public recognition
motif. Leukemia 24, 1901e1909.
Akatsuka, Y., Nishida, T., Kondo, E., Miyazaki, M., Taji, H., Iida, H.,
Tsujimura, K., Yazaki, M., Naoe, T., Morishima, Y., Kodera, Y.,
Kuzushima, K., Takahashi, T., 2003. Identification of a
polymorphic gene, BCL2A1, encoding two novel hematopoietic
lineage-specific minor histocompatibility antigens. J. Exp.
Med. 197, 1489e1500.
Alexander-Miller, M.A., Burke, K., Koszinowski, U.H.,
Hansen, T.H., Connolly, J.M., 1993. Alloreactive cytotoxic T
lymphocytes generated in the presence of viral-derived
peptides show exquisite peptide and MHC specificity.
J. Immunol. 151, 1e10.
Amir, A.L., DOrsogna, L.J., Roelen, D.L., van Loenen, M.M.,
Hagedoorn, R.S., de, B.R., Van Der Hoorn, M.A., Kester, M.G.,
Doxiadis, I.I., Falkenburg, J.H., Claas, F.H., Heemskerk, M.H.,
2010. Allo-HLA reactivity of virus-specific memory T-cells is
common. Blood 115, 3146e3157.
Amir, A.L., van der Steen, D.M., Hagedoorn, R.S., Kester, M.G., van
Bergen, C.A., Drijfhout, J.W., de Ru, A.H., Falkenburg, J.H., van
Veelen, P.A., Heemskerk, M.H., 2011a. Allo-HLA-reactive T
cells inducing graft-versus-host disease are single peptide
specific. Blood 118, 6733e6742.
Amir, A.L., van der Steen, D.M., van Loenen, M.M.,
Hagedoorn, R.S., de, B.R., Kester, M.D., de Ru, A.H.,
Lugthart, G.J., van, K.C., Hiemstra, P.S., Jedema, I.,
Griffioen, M., van Veelen, P.A., Falkenburg, J.H.,
Heemskerk, M.H., 2011b. PRAME-specific Allo-HLA-restricted
T cells with potent antitumor reactivity useful for
therapeutic T-cell receptor gene transfer. Clin. Cancer Res.
17, 5615e5625.
Aosai, F., Ohlen, C., Ljunggren, H.G., Hoglund, P., Franksson, L.,
Ploegh, H., Townsend, A., Karre, K., Stauss, H.J., 1991. Different
types of allospecific CTL clones identified by their ability to
recognize peptide loading-defective target cells. Eur. J.
Immunol. 21, 2767e2774.
Barge, R.M., Osanto, S., Marijt, W.A., Starrenburg, C.W.,
Fibbe, W.E., Nortier, J.W., Falkenburg, J.H., Willemze, R., 2003.
Minimal GVHD following in-vitro T cell-depleted allogeneic
stem cell transplantation with reduced-intensity conditioning
allowing subsequent infusions of donor lymphocytes in
patients with hematological malignancies and solid tumors.
Exp. Hematol. 31, 865e872.
Barge, R.M., Starrenburg, C.W., Falkenburg, J.H., Fibbe, W.E.,
Marijt, E.W., Willemze, R., 2006. Long-term follow-up of
myeloablative allogeneic stem cell transplantation using
Campath in the bag as T-cell depletion: the Leiden
experience. Bone Marrow Transpl. 37, 1129e1134.
Barrett, A.J., 1997. Mechanisms of the graft-versus-leukemia
reaction. Stem Cells 15, 248e258.

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 8 9 4 e1 9 0 3

Bastien, J.P., Roy, J., Roy, D.C., 2012. Selective T-cell depletion for
haplotype-mismatched allogeneic stem cell transplantation.
Semin. Oncol. 39, 674e682.
Bleakley, M., Heimfeld, S., Loeb, K.R., Jones, L.A., Chaney, C.,
Seropian, S., Gooley, T.A., Sommermeyer, F., Riddell, S.R.,
Shlomchik, W.D., 2015. Outcomes of acute leukemia patients
transplanted with naive T cell-depleted stem cell grafts.
J. Clin. Invest. 125, 2677e2689.
Bleakley, M., Otterud, B.E., Richardt, J.L., Mollerup, A.D.,
Hudecek, M., Nishida, T., Chaney, C.N., Warren, E.H.,
Leppert, M.F., Riddell, S.R., 2010. Leukemia-associated minor
histocompatibility antigen discovery using T-cell clones
isolated by in vitro stimulation of naive CD8 T cells. Blood
115, 4923e4933.
DOrsogna, L.J., Roelen, D.L., Doxiadis, I.I., Claas, F.H., 2010.
Alloreactivity from human viral specific memory T-cells.
Transpl. Immunol. 23, 149e155.
de Bueger, M., Bakker, A., Van Rood, J.J., Van der Woude, F.,
Goulmy, E., 1992. Tissue distribution of human minor
histocompatibility antigens. Ubiquitous versus restricted
tissue distribution indicates heterogeneity among human
cytotoxic T lymphocyte-defined non-MHC antigens.
J. Immunol. 149, 1788e1794.
de Rijke, B., van Horssen-Zoetbrood, A., Beekman, J.M.,
Otterud, B., Maas, F., Woestenenk, R., Kester, M., Leppert, M.,
Schattenberg, A.V., de, W.T., van de Wiel-van Kemenade,
Dolstra, H., 2005. A frameshift polymorphism in P2X5 elicits
an allogeneic cytotoxic T lymphocyte response associated
with remission of chronic myeloid leukemia. J. Clin. Invest.
115, 3506e3516.
den Haan, J.M., Meadows, L.M., Wang, W., Pool, J., Blokland, E.,
Bishop, T.L., Reinhardus, C., Shabanowitz, J., Offringa, R.,
Hunt, D.F., Engelhard, V.H., Goulmy, E., 1998. The minor
histocompatibility antigen HA-1: a diallelic gene with a single
amino acid polymorphism. Science 279, 1054e1057.
den Haan, J.M., Sherman, N.E., Blokland, E., Huczko, E., Koning, F.,
Drijfhout, J.W., Skipper, J., Shabanowitz, J., Hunt, D.F.,
Engelhard, V.H., 1995. Identification of a graft versus host
disease-associated human minor histocompatibility antigen.
Science 268, 1476e1480.
Eefting, M., Halkes, C.J., de Wreede, L.C., van Pelt, C.M.,
Kersting, S., Marijt, E.W., von dem Borne, P.A., Willemze, R.,
Veelken, H., Falkenburg, J.H., 2014a. Myeloablative T celldepleted alloSCT with early sequential prophylactic donor
lymphocyte infusion is an efficient and safe post-remission
treatment for adult ALL. Bone Marrow Transpl. 49, 320.
Eefting, M., von dem Borne, P.A., de Wreede, L.C., Halkes, C.J.,
Kersting, S., Marijt, E.W., Veelken, H., Falkenburg, J.H., 2014b.
Intentional donor lymphocyte induced limited acute graft
versus host disease is essential for long-term survival of
relapsed acute myeloid leukemia after allogeneic stem cell
transplantation. Haematologica 99, 751e758.
Falkenburg, J.H., Warren, E.H., 2011. Graft versus leukemia
reactivity after allogeneic stem cell transplantation. Biol.
Blood Marrow Transpl. 17, S33eS38.
Farber, D.L., Ahmadzadeh, M., 2002. Dissecting the complexity of
the memory T cell response. Immunol. Res. 25, 247e259.
Ferrara, J.L., Levine, J.E., Reddy, P., Holler, E., 2009. Graft-versushost disease. Lancet 373, 1550e1561.
Fuji, S., Kapp, M., Einsele, H., 2013. Alloreactivity of virus-specific
T cells: possible implication of graft-versus-host disease and
graft-versus-leukemia effects. Front Immunol. 4, 330.
Greiner, J., Li, L., Ringhoffer, M., Barth, T.F., Giannopoulos, K.,
Guillaume, P., Ritter, G., Wiesneth, M., Dohner, H., Schmitt, M.,
2005. Identification and characterization of epitopes of the
receptor for hyaluronic acid-mediated motility (RHAMM/
CD168) recognized by CD8 T cells of HLA-A2-positive
patients with acute myeloid leukemia. Blood 106, 938e945.

1901

Griffioen, M., van der Meijden, E.D., Slager, E.H., Honders, M.W.,
Rutten, C.E., van Luxemburg-Heijs, S.A., von dem Borne, P.A.,
Van Rood, J.J., Willemze, R., Falkenburg, J.H., 2008.
Identification of phosphatidylinositol 4-kinase type II beta as
HLA class II-restricted target in graft versus leukemia
reactivity. Proc. Natl. Acad. Sci. U. S. A. 105, 3837e3842.
Haanen, J.B., Baars, A., Gomez, R., Weder, P., Smits, M., de
Gruijl, T.D., von Blomberg, B.M., Bloemena, E., Scheper, R.J.,
van Ham, S.M., Pinedo, H.M., van den Eertwegh, A.J., 2006.
Melanoma-specific tumor-infiltrating lymphocytes but not
circulating melanoma-specific T cells may predict survival in
resected advanced-stage melanoma patients. Cancer
Immunol. Immunother. 55, 451e458.
Handgretinger, R., 2012. Negative depletion of CD3() and
TcRalphabeta() T cells. Curr. Opin. Hematol. 19, 434e439.
Holtick, U., Chemnitz, J.M., Shimabukuro-Vornhagen, A.,
Theurich, S., Chakupurakal, G., Krause, A., Fiedler, A., Luznik, L.,
Hellmich, M., Wolf, D., Hallek, M., von Bergwelt-Baildon, M.,
Scheid, C., 2015. OCTET-CY: a phase II study to investigate the
efficacy of post-transplant cyclophosphamide as sole graftversus-host prophylaxis after allogeneic peripheral blood stem
cell transplantation. Eur. J. Haematol. (in press).
Horowitz, M.M., Gale, R.P., Sondel, P.M., Goldman, J.M., Kersey, J.,
Kolb, H.J., Rimm, A.A., Ringden, O., Rozman, C., Speck, B., 1990.
Graft-versus-leukemia reactions after bone marrow
transplantation. Blood 75, 555e562.
Jahn, L., Hombrink, P., Hassan, C., Kester, M.G., van der
Steen, D.M., Hagedoorn, R.S., Falkenburg, J.H., van
Veelen, P.A., Heemskerk, M.H., 2015. Therapeutic targeting of
the BCR-associated protein CD79b in a TCR-based approach is
hampered by aberrant expression of CD79b. Blood 125,
949e958.
Jedema, I., Meij, P., Steeneveld, E., Hoogendoorn, M.,
Nijmeijer, B.A., van de Meent, M., van Luxemburg-Heijs, S.A.,
Willemze, R., Falkenburg, J.H., 2007. Early detection and rapid
isolation of leukemia-reactive donor T cells for adoptive
transfer using the IFN-gamma secretion assay. Clin. Cancer
Res. 13, 636e643.
Kanda, J., Lopez, R.D., Rizzieri, D.A., 2011. Alemtuzumab for the
prevention and treatment of graft-versus-host disease. Int. J.
Hematol. 93, 586e593.
Khalil, D.N., Budhu, S., Gasmi, B., Zappasodi, R., HirschhornCymerman, D., Plitt, T., De, H.O., Zamarin, D., Holmgaard, R.B.,
Murphy, J.T., Wolchok, J.D., Merghoub, T., 2015. The new era of
cancer immunotherapy: manipulating T-cell activity to
overcome malignancy. Adv. Cancer Res. 128, 1e68.
Kolb, H.J., 2008. Graft-versus-leukemia effects of transplantation
and donor lymphocytes. Blood 112, 4371e4383.
Kronig, H., Hofer, K., Conrad, H., Guilaume, P., Muller, J.,
Schiemann, M., Lennerz, V., Cosma, A., Peschel, C.,
Busch, D.H., Romero, P., Bernhard, H., 2009. Allorestricted T
lymphocytes with a high avidity T-cell receptor towards NYESO-1 have potent anti-tumor activity. Int. J. Cancer 125,
649e655.
Leen, A.M., Bollard, C.M., Mendizabal, A.M., Shpall, E.J.,
Szabolcs, P., Antin, J.H., Kapoor, N., Pai, S.Y., Rowley, S.D.,
Kebriaei, P., Dey, B.R., Grilley, B.J., Gee, A.P., Brenner, M.K.,
Rooney, C.M., Heslop, H.E., 2013. Multicenter study of banked
third-party virus-specific T cells to treat severe viral infections
after hematopoietic stem cell transplantation. Blood 121,
5113e5123.
Locatelli, F., Bauquet, A., Palumbo, G., Moretta, F., Bertaina, A.,
2013. Negative depletion of alpha/beta T cells and of CD19 B
lymphocytes: a novel frontier to optimize the effect of innate
immunity in HLA-mismatched hematopoietic stem cell
transplantation. Immunol. Lett. 155, 21e23.
Macedo, C., Orkis, E.A., Popescu, I., Elinoff, B.D., Zeevi, A.,
Shapiro, R., Lakkis, F.G., Metes, D., 2009. Contribution of naive

1902

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 8 9 4 e1 9 0 3

and memory T-cell populations to the human alloimmune

response. Am. J. Transpl. 9, 2057e2066.
Macesic, N., Langsford, D., Nicholls, K., Hughes, P., Gottlieb, D.J.,
Clancy, L., Blyth, E., Micklethwaite, K., Withers, B.,
Majumdar, S., Fleming, S., Sasadeusz, J., 2015. Adoptive T cell
immunotherapy for treatment of ganciclovir-resistant
cytomegalovirus disease in a renal transplant recipient. Am. J.
Transpl. 15, 827e832.
Markey, K.A., MacDonald, K.P., Hill, G.R., 2014. The biology of
graft-versus-host disease: experimental systems instructing
clinical practice. Blood 124, 354e362.
Matsushita, M., Yamazaki, R., Ikeda, H., Mori, T., Sumimoto, H.,
Fujita, T., Okamoto, S., Ikeda, Y., Kawakami, Y., 2006. Possible
involvement of allogeneic antigens recognised by donorderived CD4 cytotoxic T cells in selective GVL effects after
stem cell transplantation of patients with haematological
malignancy. Br. J. Haematol. 132, 56e65.
Meij, P., Jedema, I., Van Der Hoorn, M.A., Bongaerts, R., Cox, L.,
Wafelman, A.R., Marijt, E.W., Willemze, R., Falkenburg, J.H.,
2012. Generation and administration of HA-1-specific T-cell
lines for the treatment of patients with relapsed leukemia
after allogeneic stem cell transplantation: a pilot study.
Haematologica 97, 1205e1208.
Melenhorst, J.J., Leen, A.M., Bollard, C.M., Quigley, M.F.,
Price, D.A., Rooney, C.M., Brenner, M.K., Barrett, A.J.,
Heslop, H.E., 2010. Allogeneic virus-specific T cells with HLA
alloreactivity do not produce GVHD in human subjects. Blood
116, 4700e4702.
Melenhorst, J.J., Scheinberg, P., Williams, A., Ambrozak, D.R.,
Keyvanfar, K., Smith, M., McCoy Jr., J.P., Hensel, N.F.,
Douek, D.C., Barrett, A.J., 2011. Alloreactivity across HLA
barriers is mediated by both naive and antigen-experienced T
cells. Biol. Blood Marrow Transpl. 17, 800e809.
Meyer, R.G., Wagner, E.M., Konur, A., Bender, K., Schmitt, T.,
Hemmerling, J., Wehler, D., Hartwig, U.F., Roosnek, E.,
Huber, C., Kolbe, K., Herr, W., 2010. Donor CD4 T cells convert
mixed to full donor T-cell chimerism and replenish the CD52positive T-cell pool after alemtuzumab-based T-cell-depleted
allo-transplantation. Bone Marrow Transpl. 45, 668e674.
Mielke, S., McIver, Z.A., Shenoy, A., Fellowes, V., Khuu, H.,
Stroncek, D.F., Leitman, S.F., Childs, R., Battiwalla, M.,
Koklanaris, E., Haggerty, J., Savani, B.N., Rezvani, K.,
Barrett, A.J., 2011. Selectively T cell-depleted allografts from
HLA-matched sibling donors followed by low-dose
posttransplantation immunosuppression to improve
transplantation outcome in patients with hematologic
malignancies. Biol. Blood Marrow Transpl. 17, 1855e1861.
Munz, C., Obst, R., Osen, W., Stevanovic, S., Rammensee, H.G.,
1999. Alloreactivity as a source of high avidity peptide-specific
human CTL. J. Immunol. 162, 25e34.
Naito, Y., Saito, K., Shiiba, K., Ohuchi, A., Saigenji, K., Nagura, H.,
Ohtani, H., 1998. CD8 T cells infiltrated within cancer cell
nests as a prognostic factor in human colorectal cancer.
Cancer Res. 58, 3491e3494.
Nash, R.A., Storb, R., 1996. Graft-versus-host effect after
allogeneic hematopoietic stem cell transplantation: GVHD and
GVL. Curr. Opin. Immunol. 8, 674e680.
Ni, K., ONeill, H.C., 1997. The role of dendritic cells in T cell
activation. Immunol. Cell Biol. 75, 223e230.
Obst, R., Munz, C., Stevanovic, S., Rammensee, H.G., 1998. Alloand self-restricted cytotoxic T lymphocytes against a peptide
library: evidence for a functionally diverse allorestricted T cell
repertoire. Eur. J. Immunol. 28, 2432e2443.
Oostvogels, R., Lokhorst, H.M., Minnema, M.C., van, E.M., van
den Oudenalder, K., Spierings, E., Mutis, T., Spaapen, R.M.,
2014. Identification of minor histocompatibility antigens
based on the 1000 genomes project. Haematologica 99,
1854e1859.

Orti, G., Lowdell, M., Fielding, A., Samuel, E., Pang, K.,
Kottaridis, P., Morris, E., Thomson, K., Peggs, K.,
Mackinnon, S., Chakraverty, R., 2009. Phase I study of highstringency CD8 depletion of donor leukocyte infusions after
allogeneic hematopoietic stem cell transplantation.
Transplantation 88, 1312e1318.
Pont, M.J., Hobo, W., Honders, M.W., van Luxemburg-Heijs, S.A.,
Kester, M.G., van Oeveren-Rietdijk, A.M., Schaap, N., de
Boer, H.C., van Bergen, C.A., Dolstra, H., Falkenburg, J.H.,
Griffioen, M., 2015. LB-ARHGDIB-1R as a novel minor
histocompatibility antigen for therapeutic application.
Haematologica 100, e419ee422 (in press).
Reddy, P., Maeda, Y., Liu, C., Krijanovski, O.I., Korngold, R.,
Ferrara, J.L., 2005. A crucial role for antigen-presenting cells
and alloantigen expression in graft-versus-leukemia
responses. Nat. Med. 11, 1244e1249.
Rezvani, K., Brenchley, J.M., Price, D.A., Kilical, Y., Gostick, E.,
Sewell, A.K., Li, J., Mielke, S., Douek, D.C., Barrett, A.J., 2005. Tcell responses directed against multiple HLA-A*0201-restricted
epitopes derived from Wilms tumor 1 protein in patients with
leukemia and healthy donors: identification, quantification,
and characterization. Clin. Cancer Res. 11, 8799e8807.
Riddell, S.R., Berger, C., Murata, M., Randolph, S., Warren, E.H.,
2003. The graft versus leukemia response after allogeneic
hematopoietic stem cell transplantation. Blood Rev. 17,
153e162.
Riddell, S.R., Murata, M., Bryant, S., Warren, E.H., 2002. Minor
histocompatibility antigensetargets of graft versus leukemia
responses. Int. J. Hematol. 76 (Suppl. 2), 155e161.
Rist, M., Smith, C., Bell, M.J., Burrows, S.R., Khanna, R., 2009.
Cross-recognition of HLA DR4 alloantigen by virus-specific
CD8 T cells: a new paradigm for self-/nonself-recognition.
Blood 114, 2244e2253.
Rotzschke, O., Falk, K., Faath, S., Rammensee, H.G., 1991. On the
nature of peptides involved in T cell alloreactivity. J. Exp. Med.
174, 1059e1071.
Rutten, C.E., van Luxemburg-Heijs, S.A., Griffioen, M.,
Marijt, E.W., Jedema, I., Heemskerk, M.H., Posthuma, E.F.,
Willemze, R., Falkenburg, J.H., 2008. HLA-DP as specific target
for cellular immunotherapy in HLA class II-expressing B-cell
leukemia. Leukemia 22, 1387e1394.
Rutten, C.E., van Luxemburg-Heijs, S.A., Halkes, C.J., van
Bergen, C.A., Marijt, E.W., Oudshoorn, M., Griffioen, M.,
Falkenburg, J.H., 2013. Patient HLA-DP-specific CD4 T cells
from HLA-DPB1-mismatched donor lymphocyte infusion can
induce graft-versus-leukemia reactivity in the presence or
absence of graft-versus-host disease. Biol. Blood Marrow
Transpl. 19, 40e48.
Sadovnikova, E., Jopling, L.A., Soo, K.S., Stauss, H.J., 1998.
Generation of human tumor-reactive cytotoxic T cells against
peptides presented by non-self HLA class I molecules. Eur. J.
Immunol. 28, 193e200.
Savage, P., Gao, L., Vento, K., Cowburn, P., Man, S., Steven, N.,
Ogg, G., McMichael, A., Epenetos, A., Goulmy, E., Stauss, H.J.,
2004. Use of B cell-bound HLA-A2 class I monomers to
generate high-avidity, allo-restricted CTLs against the
leukemia-associated protein Wilms tumor antigen. Blood 103,
4613e4615.
Scheibenbogen, C., Letsch, A., Thiel, E., Schmittel, A.,
Mailaender, V., Baerwolf, S., Nagorsen, D., Keilholz, U., 2002.
CD8 T-cell responses to Wilms tumor gene product WT1 and
proteinase 3 in patients with acute myeloid leukemia. Blood
100, 2132e2137.
Sebzda, E., Mariathasan, S., Ohteki, T., Jones, R., Bachmann, M.F.,
Ohashi, P.S., 1999. Selection of the T cell repertoire. Annu. Rev.
Immunol. 17, 829e874.
Shook, D.R., Triplett, B.M., Eldridge, P.W., Kang, G., Srinivasan, A.,
Leung, W., 2015. Haploidentical stem cell transplantation

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 8 9 4 e1 9 0 3

augmented by CD45RA negative lymphocytes provides rapid

engraftment and excellent tolerability. Pediatr. Blood Cancer
62, 666e673.
Smyth, M.J., Godfrey, D.I., Trapani, J.A., 2001. A fresh look at
tumor immunosurveillance and immunotherapy. Nat.
Immunol. 2, 293e299.
Spaapen, R.M., de Kort, R.A., van den Oudenalder, K., van, E.M.,
Bloem, A.C., Lokhorst, H.M., Mutis, T., 2009. Rapid
identification of clinical relevant minor histocompatibility
antigens via genome-wide zygosity-genotype correlation
analysis. Clin. Cancer Res. 15, 7137e7143.
Spaapen, R.M., Lokhorst, H.M., van den Oudenalder, K.,
Otterud, B.E., Dolstra, H., Leppert, M.F., Minnema, M.C.,
Bloem, A.C., Mutis, T., 2008. Toward targeting B cell cancers
with CD4 CTLs: identification of a CD19-encoded minor
histocompatibility antigen using a novel genome-wide
analysis. J. Exp. Med. 205, 2863e2872.
Spierings, E., 2014. Minor histocompatibility antigens: past,
present, and future. Tissue Antigens 84, 347e360.
Starr, T.K., Jameson, S.C., Hogquist, K.A., 2003. Positive and
negative selection of T cells. Annu. Rev. Immunol. 21, 139e176.
Stevanovic, S., van Bergen, C.A., van Luxemburg-Heijs, S.A., van
der Zouwen, B., Jordanova, E.S., Kruisselbrink, A.B., van de
Meent, M., Harskamp, J.C., Claas, F.H., Marijt, E.W.,
Zwaginga, J.J., Halkes, C.J., Jedema, I., Griffioen, M.,
Falkenburg, J.H., 2013. HLA class II upregulation during viral
infection leads to HLA-DP-directed graft-versus-host disease
after CD4 donor lymphocyte infusion. Blood 122, 1963e1973.
Storek, J., Mohty, M., Boelens, J.J., 2015. Rabbit anti-T cell globulin
in allogeneic hematopoietic cell transplantation. Biol. Blood
Marrow Transpl. 21, 959e970.
Stumpf, A.N., van der Meijden, E.D., van Bergen, C.A.,
Willemze, R., Falkenburg, J.H., Griffioen, M., 2009.
Identification of 4 new HLA-DR-restricted minor
histocompatibility antigens as hematopoietic targets in
antitumor immunity. Blood 114, 3684e3692.
Touzot, F., Neven, B., Dal-Cortivo, L., Gabrion, A., Moshous, D.,
Cros, G., Chomton, M., Luby, J.M., Terniaux, B., Magalon, J.,
Picard, C., Blanche, S., Fischer, A., Cavazzana, M., 2015.
CD45RA depletion in HLA-mismatched allogeneic
hematopoietic stem cell transplantation for primary
combined immunodeficiency: a preliminary study. J. Allergy
Clin. Immunol. 135, 1303e1309.
van den Ancker, W., Ruben, J.M., Westers, T.M., Wulandari, D.,
Bontkes, H.J., Hooijberg, E., Stam, A.G., Santegoets, S.J.,

1903

Ossenkoppele, G.J., de, G.T., van de Loosdrecht, A., 2013.

Priming of PRAME- and WT1-specific CD8 T cells in healthy
donors but not in AML patients in complete remission:
implications for immunotherapy. Oncoimmunology 2,
e23971.
van der Zouwen, B., Kruisselbrink, A.B., Jordanova, E.S.,
Rutten, C.E., von dem Borne, P.A., Falkenburg, J.H., Jedema, I.,
2012. Alloreactive effector T-cells require the local formation
of a proinflammatory environment to allow crosstalk and
high avidity interaction with non-hematopoietic tissues to
induce GvHD reactivity. Biol. Blood Marrow Transpl. 18,
1353e1367.
van Loenen, M.M., de, B.R., van, L.E., Meij, P., Jedema, I.,
Falkenburg, J.H., Heemskerk, M.H., 2014. A good
manufacturing practice procedure to engineer donor virusspecific T-cells into potent anti-leukemic effector cells.
Haematologica 99, 759e768.
van Duin, M., Broyl, A., de, K.Y., Goldschmidt, H.,
Richardson, P.G., Hop, W.C., van der Holt, B., JosephPietras, D., Mulligan, G., Neuwirth, R., Sahota, S.S.,
Sonneveld, P., 2011. Cancer testis antigens in newly diagnosed
and relapse multiple myeloma: prognostic markers and
potential targets for immunotherapy. Haematologica 96,
1662e1669.
Vickers, M.A., Wilkie, G.M., Robinson, N., Rivera, N., Haque, T.,
Crawford, D.H., Barry, J., Fraser, N., Turner, D.M.,
Robertson, V., Dyer, P., Flanagan, P., Newlands, H.R.,
Campbell, J., Turner, M.L., 2014. Establishment and operation
of a good manufacturing practice-compliant allogeneic
Epstein-Barr virus (EBV)-specific cytotoxic cell bank for the
treatment of EBV-associated lymphoproliferative disease. Br. J.
Haematol. 167, 402e410.
Viret, C., Janeway Jr., C.A., 1999. MHC and T cell development.
Rev. Immunogenet. 1, 91e104.
Weber, G., Gerdemann, U., Caruana, I., Savoldo, B., Hensel, N.F.,
Rabin, K.R., Shpall, E.J., Melenhorst, J.J., Leen, A.M.,
Barrett, A.J., Bollard, C.M., 2013. Generation of multi-leukemia
antigen-specific T cells to enhance the graft-versus-leukemia
effect after allogeneic stem cell transplant. Leukemia 27,
1538e1547.
Zhang, L., Conejo-Garcia, J.R., Katsaros, D., Gimotty, P.A.,
Massobrio, M., Regnani, G., Makrigiannakis, A., Gray, H.,
Schlienger, K., Liebman, M.N., Rubin, S.C., Coukos, G., 2003.
Intratumoral T cells, recurrence, and survival in epithelial
ovarian cancer. N. Engl. J. Med. 348, 203e213.