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