Post on 10-Jan-2017
The influence of Mycobacterium tuberculosis on B cells during disease and treatment responseJACQUES DU PLESSISSUPERVISORS: PROF G. WALZL & DR A. LOXTON
But why B cells, TB immunity is T cell driven?
B cells are diverse cells that contribute to a broad range of functions in immunity, including:◦ They respond to TLR activation and is involved in antigen presentation◦ Sole producers of antibodies (humoral immunity)
Growing body of evidence implicating B cells in atypical functions:◦ B cells function as either effectors/regulators of immunity via cytokine production
◦ B cells are now well known for producing IL-10 in regulatory roles◦ B cells produce pro-inflammatory cytokines in effector roles ◦ Rauch et al. (2012) identified B cells as the primary producers of GM-CSF – from innate
response activator (IRA) B cells
Whilst TB infection induces a wide variety of T cell lineages (Th1, Th2, Th17, Treg), which is well documented, the influence of the infection on B cells during disease and treatment response is largely unknown – warranting further research.
Hypothesis / Aims We hypothesise that B cells play a role during M.tb infection and TB disease, and that treatment has an influence on its frequency, maturation and role as effectors/regulators.
We aim to explore the influence of M.tb on B cells by:◦ Identifying specific B cell phenotype frequencies (immunophenotyping) and,◦ To assess specific B cell functional capacity during M.tb infection/exposure
B cell Immunophenotyping – Methods
Cohorts:◦ Healthy community controls (n = 20)◦ Other-lung disease (OLD) patients (n = 24)◦ Active TB disease patients (n = 52)
Time points:◦ Diagnosis◦ Day 2 on treatment◦ Day 7 on treatment◦ Week 24 (End of treatment, TB samples
only)
Flow cytometry panel:◦ CD3◦ CD4 T cell markers◦ CD8◦ CD19: B cell marker, present on all subtypes◦ CD23: Activation marker◦ CD27: Memory marker◦ CD138: Plasma cell marker◦ IgM: Antibody – present in immature, mature
and memory B cells
Immunophenotyping – Gating Strategy
B cell phenotypes:• Mature B cells• Activated B cells• Naïve B cells• Marginal zone B cells• Memory B cells• Memoryhigh B cells• Total plasma B cells• Memory-Plasma B
cells• Memoryhigh-plasma B
cells
Immunophenotyping – Results p= .02880
Dx W 24-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Mem
ory
(IgM
+CD
27++
)
p= .04680
Dx W 246
8
10
12
14
16
18
20
22
24
26
28
Mar
gina
l Zon
e(I
gM+C
D27
+CD
23-)
p= .00389
Dx W 242
4
6
8
10
12
14
16
18
20
22
Mem
ory-
Pla
sma
(IgM
+CD
138+
CD
27+)
B cell phenotypes that are significantly different between TB diagnosis and end of treatment
Immunophenotyping – Results (Continued)
p= .02092
TB OLD5
10
15
20
25
30
35
40
45
50
Mar
ginal
Zone
(IgM
+CD2
7+CD
23-)
Marginal Zone B cells are significantly different between TB and OLD at diagnosis.
Functional B cell work – Workflow
Frozen PBMCs
Select B cells with MACS beads(Negative selection)
Stimulate with antigen(16 hours)
Stimulate with antigen(16 hours)
Flow cytometry (Functional panel)
Supernatant
MSD multiplex analysis
Fresh whole blood (~70ml/patient)
Functional B cell work – Methods One group:
◦ IGRA+ healthy community individuals (LTBI), n = 11
Six stimulating conditions:◦ Unstimulated: Negative control◦ PHA: Positive control◦ PPD ◦ BCG◦ LPS: TLR-4◦ TLR9-agonist: B cell activation control
Flow cytometry panel : Phenotype and ICS◦ CD3: General T cell marker◦ CD19: General B cell marker◦ CD27: Memory marker◦ CD138: Plasma cell marker◦ IL-10: Anti-inflammatory cytokine◦ IL-17: Delayed response pro-inflammatory cytokine◦ IL-21: B cell recruitment and maturation cytokine◦ TNF-α: Pro-inflammatory cytokine
MSD kits used: Secreted proteins◦ Pro-inflammatory panel 1 (humans)
◦ IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α◦ Human MMP 3-Plex Ultra-Sensitive Kit
◦ MMP-1, MMP-3, MMP-9
TB specific
Functional B cell work – Results (Secreted Prots)
Qlucore omics explorer: Unbiased hierarchical analysis
Functional B cell work – Phenotyping and ICS Gating Strategy
PHA PPD TLR9-a LPS BCG
IL-10
IL-17
CD138+
CD27-CD138-
CD27+
CD27+CD138+
CD27+CD138+
CD27+CD138+
CD27+CD138+
CD27+CD138+
CD27+
CD27+
CD27+
CD27+
CD27-CD138-
CD27-CD138-
CD27-CD138-
CD27-CD138-
CD138+
CD138+
CD138+
CD138+
CD138+
CD27-CD138-
CD27+
CD27+CD138+
TNF-α
IL-21
PHA PPD TLR9-a
CD138+
CD138+
CD138+
CD138+
CD27-CD138-
CD27-CD138-
CD27-CD138-
CD27-CD138-
CD27+
CD27+
CD27+
CD27+
CD27+CD138+
CD27+CD138+
CD27+CD138+
CD27+CD138+LPS BCG
Plasma-memory B cells may be key B cells subsets during innate recruitment of B cells during tuberculous challenge.
CD138+
CD138+
CD27-CD138-
CD27-CD138-
CD27+
CD27+
CD27+CD138+
CD27+CD138+
Conclusions – Primary Findings◦ Memory-based B cell phenotypes and marginal zone (MZ) B cells can distinguish
between TB at diagnosis and end of treatment (week 24)
◦ Marginal zone B cell frequencies are distinguishable at diagnosis between tuberculosis and individuals diagnosed with other-lung diseases (OLD).
◦ Mature B cells best distinguish between all three groups (TB, OLD and healthy community controls) at diagnosis
◦ B cells readily and differentially produce pro-inflammatory cytokines following antigenic challenge.
Conclusions – Continued ◦ BCG stimulation results in the significantly higher production of IL-1β
compared to the other stimulations.
◦ TLR-based stimulations resulted in the greatest cytokine yields from B cells. From the Ingenuity Pathway Analysis (IPA), we learned that the production of B cell derived IL-1β primarily facilitates pathways implicated in intra-cellular communication.
◦ Plasma-memory B cells (CD19+CD27+CD138+) are the primary contributors of cytokine (IL-10, IL-21 and TNF-α), except for IL-17 which seems to be mainly produced by plasma B cells (CD19+CD138+).
Study Limitations◦ Small study groups
◦ Functional work was on latent M.tb infection
◦ Ideally one would want to expand this research to other groups and samples such as active TB disease, TST-/IGRA- or BAL (bronchoalveolar lavage)
Degree Outputs
◦ Brief Report: Unique peripheral B cell populations during M. tuberculosis infection and disease (Journal of Immunology Research)
◦ The Functional Response of B cells to Antigenic Stimulation during Latent Tuberculosis (PLOS ONE)
Acknowledgements◦ Prof Gerhard Walzl◦ Dr Andre Loxton◦ Dr Nelita du Plessis◦ Dr Leanie Kleynhans◦ Dr Katharina Ronacher◦ Dr Novel Chegou◦ Belinda Kriel and everyone in the lab◦ All of my fellow students in the group◦ The whole SUN-IRG group◦ NRF for my bursary
Questions
Frequency distribution of B cell subset
3+7+ = CD23+CD27+ (Naïve cells)7+3- = CD27+CD23- (MZ Memory)7-3+ = CD27-CD23+ (Activated B cells)7-3- = CD27-CD23- (Naïve B cells)
TB
HC
IL-2 IL-10 IL-12p70
IL-1β IL-6 TNF-α
B cells produce pro-inflammatory cytokines differentially based on stimulation.
US PHA PPD TLR9a LPS BCG