17Jun14

Mycobacterium Tuberculosisspecific
lunginnateandadaptiveimmunityin
closecontactsofTBindexcases
LiezelSmithPhD
LunginfectionandImmunityunit,
UniversityofCapeTown

Acknowledgements

ProfKeertanDheda
Dr.MalikaDavids
Dr.AnkeBinder
Mr.RichardMeldau
LungInfectionandImmunityUnit
Participants

17Jun14

References

Introduction
Despite effective drugs and improved diagnostics, TB
remains out of control
What we need effective vaccine!
Can only occur if we understand the mechanisms
and what constitutes protective immunity
Well documented: ~50% of close contacts of smear
positive cases in Africa remain TST ve
p
Morrison J, Lancet Inf Dis, 2008
Even higher number remain IGRA ve (no evidence of
T cell activation defined by RD1 antigens despite
heavy recent contact)

17Jun14

NonInfected

LTBI

Studyhypothesis
Close contacts of smearpositive TB cases who fail to convert
their immunodiagnostic tests (Non infected [NI]i.e. Both TST and
IGRA negative) have different Immunological biomarker
signatures (measurable differences in the levels of biomarkers
and cellular phenotypes) compared to contacts that convert
their tests (LTBIs i.e. Both TST and IGRA positive).

17Jun14

Aim
TToelucidatethedifferencesinthefrequencyand
l id t th diff
i th f
d
profileofinnateimmunepathways

Participants&Procedures
Inclusion criteria
HIV uninfected
Age>18 years
Lived for at least 3 months
in same room as Smear
positive TB case or contact
score>200
Exclusion criteria
Symptoms,
examination
and/or CXR suggests active
disease
Pregnancy
smoker

Bronchoscopy
Earlymorning(810am)
100200mlBALfluid
collectedfollowing
instillationof230300mls
saline
Mean(Range)BALcellconc:
1.3x105/ml(0.36 4.8)
Mean(Range)BALcelltotal:
23x106 (6.872)
Phlebotomy
~50mlsofbloodtakenat
timeofbronchoscopy

17Jun14

StudyParticipants
30 Samples

Noninfected/non
converters

LTBI/converters
TST+ve ;IGRA
+ve
15
Blood
samples

TSTve ;IGRAve

15
BALsamples

15

15

Blood
samples

BAL
samples

TST/IGRA
NIvs.LTBI
PeripheralBlood
Cell types
Celltypes

BAL

LTBI
(%counts)

NI
(%counts)

Pvalue

LTBI
(%counts)

NI
(%counts)

Pvalue

Lymphocytes
(CD3+)

66.65

67.72

0.89

5.22

7.18

0.25

Macrophages
(CD14+)

22.02

16.67

0.09

60.13

66.37

0.17

Natural killer cells

Naturalkillercells
(CD56+)

14 3
14.3

10 68
10.68

0 73
0.73

6 55
6.55

14 8
14.8

0 23
0.23

Dendriticcells
(CD3CD33+)

8.72

10.68

0.18

8.27

11.42

0.62

Nodifferencesnotedincellfractionsineithercompartment

17Jun14

AutophagymarkerinCD4Tcells
CD3+CD4+ coexpressing LC3B
are significantly higher in NI vs LTBI
for unstimulated cells from both
Blood and BAL when compared to
PPDstimulated cells.

ExpressionofTLR9inMacrophages

The frequency of macrophages expressing TLR9 is

significantly higher in NI vs LTBI in both PPD
stimulated and unstimulated cells from BAL.

17Jun14

ExpressionofCathelicidininDendritic
cells

CD3CD33+ coexpressing CAP

Indicate a trend of being higher in
NI vs LTBI for both PPDstimulated
and unstimulated cells from Blood
and BAL.

ExpressionofPerforininNaturalKiller
cells

The frequency of natural killer cells

expressing
Perforin
was
significantly higher in LTBI vs NI in
PPDstimulated and unstimulated
cells from BAL. No significant
difference seen in Blood.

17Jun14

Patternrecognitionreceptors
Marker

Blood
LTBI

Pvalue
NI

BAL
LTBI

Pvalue
NI

CD14+Mannosereceptor

31.50

21.55

ns

86.61

80.30

ns

CD14+TLR2

32.86

36.20

ns

17.98

22.15

ns

CD14+TLR4

49.53

52.67

ns

68.56

72.51

ns

No differences between LTBI and NI noted in either compartment,

unstimulated or PPD stimulated, for Macrophage Mannose Receptor, TLR2,
TLR4 and DCSIGN

MicroarrayAnalysis

Pathwayy and cluster analysis

y
indicate clear differences in the
overall transcriptional profiles
between LTBI and NI participants
with several genes being down
regulated.

17Jun14

Summary
Compartment specific differences between blood and BAL cells noted as
expected.
LTBI showed a trend for decreased LC3B,
LC3B TLR9,Cathlecidin
TLR 9 Cathlecidin and increased
expression of Perforin when compared to cells from NI participants.
There seems to be differences between LTBI and NI, however small patient
numbers mean results are preliminary and need confirmation.
Pathway cluster analysis indicate clear differences in the overall
transcriptional profiles between LTBI and NI participants with several
genes being downregulated in NI participants.

These findings may aid in the identification of new biomarkers of protective

immunity that are urgently needed for the development of new and
improvement of current vaccines.

Futurework
Increasesamplesize
Explorespecificinnatepathwaysthatdiffer
betweenLTBIandNI

17Jun14

Thankyou
Save the Date:
11 12 October 2014

MDR- and XDR-TB Conference

Lung infection and Immunity Unit
Cape Town

Thee Long
o g walk
a to TB
Freedom
Contact: liezel.smith@uct.ac.za

Alveolar
pneumocytes

Innate immunity
No detectable T cell priming (IGRA neg., TST neg.)
NK-cells
Neutrophils

Clearance of
Infection

1.Close contacts
inhale M.tb

NK-T cells
Macrophages
-defensin

T-cells

Cathelicidin

Adaptive immunity
Evidence of T cell priming (IGRA pos., TST pos.)$
Cytokines

Th1,Th2 & Th17

CD4+ T cells

T cell
CD4+ T cells

Macrophage

M.tb

3. The remainder of exposed persons have

conversion of TST or IGRA and a
proportion have presumed infection**

CD4 Naive
Tcell

CD1
lipid
TLR2
TLR4
TLR9

2. ~50% or more of exposed

persons have no immuno
evidence of M.tb
diagnostic
g
sensitisation and may
remain uninfected through
sterilizing immunity#

MHCII

Treg

MHCI

CD8 Naive
T cell

CD8+ T cells

7. Reinfection

6.ReversionofTSTorIGRA
(Acuteorchronicresolving
infection)

In
I ~ 95 %
containment

~5%

~ 2 to 5 %

4. LTBI**

5. Clinically detectable
active or subclinical
disease

Schwander and Dheda, AJRCCM, 2011

10