Increased activated memory B-cells in the peripheral blood of patients with erythema nodosum leprosum reactions - Fig 1

<p>Gating strategy for B-cell sub populations; (1A) gating B-lymphocytes by FSCA versus CD19, (1B) gating mature B-cells (CD19⁺CD10^-), (1C) memory B-cells obtained by gating mature B-cells for CD27 versus CD21.</p

Correlations among paired antigen-specific PB and MBC responses detected in the community controls and TB patients.

<p>Correlations among paired antigen-specific PB and MBC responses detected in the community controls and TB patients.</p

<i>Mycobacteria</i>-specific PB and MBC responses in the active TB patients and community controls.

<p>Frequencies of antigen-specific PBs and MBCs were determined by 6-hour ex-vivo and 6-day in-vitro ELISPOTs respectively. Frequencies of antigen-specific PBs and MBCs are presented as antibody secreting cells per million PBMCs. (<b>A</b>) Frequencies of mycobacteria-specific PBs in the active TB patients (filled circles), community controls responding to ESAT-6/CFP-10 (half filled triangles) and healthy community controls lacking ESAT-6 and CFP-10 responses (open circles) (<b>B</b>) Frequencies of mycobacteria-specific MBCs in the active TB patients (filled circles), community controls responding to ESAT-6/CFP-10 (half filled triangles) those without ESAT-6 and CFP-10 responses (open circles). **** P<0.0001, ***, P<0.001; **, P<0.01.</p

Paired <i>mycobacteria</i>-specific PB and MBC responses in healthy community controls.

<p>Matched frequencies of antigen-specific PBs and MBCs were determined by 6-hour ex-vivo and 6-day in-vitro ELISPOTs respectively. Matched frequencies of PPD- (<b>A</b>), ESAT-6- (<b>B</b>), CFP-10- (<b>C</b>), Ag85A- (<b>D</b>) and Ag85B- (<b>E</b>) specific PBs and MBCs in healthy community controls are shown. Open square across all panels represents an interesting donor who presented with a higher PB:MBC ratio on various antigens tested. Frequencies of antigen-specific PBs and MBCs are presented as antibody secreting cells per million PBMCs.</p

Modulation of PD-1 and GrzB protein co-expression during active TB disease and chemotherapy.

<p>Frozen PBMCs from Pakistan were thawed and PD-1 and GrzB co-expression was investigated by flow cytometry. (A) The proportion of PD-1 cells which are also GzB positive in the CD3⁺CD8⁺ lymphocyte gate, CD3⁺CD8^- lymphocyte gate and CD3^- lymphocyte (NK cell) gate at diagnosis in TB patients (P: n = 18), latently infected contacts (C+: n = 7) and uninfected contacts (C-: n = 4). P values are derived from the Kruskal-Wallis test with Dunn’s multiple testing correction (B) Modulation of PD-1/GzB co-expression with the intensive phase of chemotherapy on CD3⁺CD8⁺ T cells, CD3⁺CD8^- lymphocytes and CD3^- lymphocytes (NK cells), analysed with the Wilcoxon signrank test. The bars show medians and the interquartile range.</p

Study subjects:clinical and demographic information.

<p>Study subjects:clinical and demographic information.</p

<i>Ex vivo</i> gene expression of PD-1 and its ligands in active TB disease.

<p>RNA was extracted from whole blood samples added to Tempus tubes and expression investigated by qRT-PCR. (A) PD-1 expression in <i>ex vivo</i> venous blood from untreated active TB cases at diagnosis (n = 23), in IGRA+ (C+: n = 11) and in IGRA- contacts (C-: n = 8). (B) PD-L1 expression in <i>ex vivo</i> venous blood from untreated active TB cases at diagnosis (n = 24), in latently-infected household contacts (C+: n = 12) and in uninfected contacts (C-: n = 11). (C) PD-L2 expression in <i>ex vivo</i> venous blood from untreated active TB cases at diagnosis (n = 24), in latently-infected household contacts (C+: n = 12) and in uninfected contacts (C-: n = 12). The lines in the centre of each group represent medians. (D) ROC comparisons were conducted between TB patients and household contacts (C+ and C- combined) to determine whether PD-1, PD-L1 or PD-L2 expression could distinguish active TB patients from healthy people. PD-1/PD-L1/PD-L2 mRNA expression was determined by qRT-PCR, with results shown normalised against the housekeeping gene Cyclophilin A. P values in A) to C) were derived from Kruskal-Wallis test with Dunn’s multiple testing correction. AUC = Area Under the Curve.</p

PD-1 protein expression during active TB disease and chemotherapy.

<p>Frozen PBMCs from Pakistan were thawed and stained to analyse PD-1 protein expression. (A) PD-1 protein expression on CD3⁺CD8⁺ T cells, CD3⁺CD8^- T cells and CD3^- lymphocytes at diagnosis in 18 TB patients (moderate severity) (P), 7 latently infected contacts (C+) and 5 uninfected contacts (C-). P values are derived from the Kruskal-Wallis test with Dunn’s multiple testing correction. (B) Modulation of PD-1 protein expression on CD3⁺CD8⁺ T cells, CD3⁺CD8^- T cells and CD3^- lymphocytes with the intensive phase of chemotherapy in patients who were sputum smear negative after 2 months of treatment (n = 11), analysed with the Wilcoxon signrank test. The bars show medians and the interquartile range.</p

The median percentage of total B-lymphocytes (CD19⁺), mature B-cells (CD19⁺CD10^-) and naïve B-lymphocytes (CD19⁺CD10^-CD27^-CD21⁺) in the PBMCs of) patients with ENL and LL controls before, during and after treatment respectively (2A,2B,2C); within ENL patients before, during and after treatment respectively (2D,2E,2F).

<p>Statistical test: Mann-Whitney unpaired test (U) (2A, 2B, 2C) and Wilcoxon matched-pairs signed rank test (2D, 2E, 2F). * <i>P≤ 0</i>.<i>05; *</i>* <i>P≤ 0</i>.<i>005;</i> *** <i>P≤ 0</i>.<i>001</i>. Box and whiskers show median ± interquartile range.</p

The median percentage of resting memory B-cells (CD19⁺CD10^-CD27⁺CD21⁺), activated memory B-cells (CD19⁺CD10^-CD27⁺CD21^-) and tissue-like memory B-cells (CD19⁺CD10^-CD27^-CD21^-) in the PBMCs of patients with ENL and LL controls before, during and after treatment respectively (3A,3B,3C); within ENL patients before, during and after treatment respectively (3D,3E,3F).

<p>Statistical test: Mann-Whitney unpaired test (U) (3A,3B, 3C)) and Wilcoxon matched-pairs signed rank test (3D, 3E, 3F)B). * <i>P≤ 0</i>.<i>05; *</i>* <i>P≤ 0</i>.<i>005;</i> *** <i>P≤ 0</i>.<i>001</i>. Box and whiskers show median ± interquartile range.</p