T cell responses to Mycobacterium indicus pranii immunotherapy and adjunctive glucocorticoid therapy in tuberculous pericarditis

Figures

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  Fig. 1. Consort diagram of 2-by-2 factorial design of the immunogenicity sub-study of the IMPI trial. A total of 64 tuberculous pericarditis (TBP) patients were randomly assigned to receive either 5 injections of Mycobacterium indicus pranii (MIP) or saline. The MIP or placebo groups were further subdivided by randomization to receive either 6 weeks of oral prednisolone or placebo. Blood samples were collected at baseline, 2, 6 and 24 weeks after treatment initiation.

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  Fig. 2. Flow cytometry gating strategy to identify cytokine expressing CD4 and CD8 T cells. Doublets were excluded by forward scatter (FSC) area versus height gating. T cells were identified as CD3 positive and side scatter (SSC)-low cells and further subdivided into CD4 or CD8 positive cells (A). Interferon-gamma (IFN-γ), Interleukin-2 (IL-2), Interleukin-22 (IL-22) and tumor necrosis factor (TNF) expression in unstimulated (UNS) samples or after PPD and MIP stimulation in CD4 (B) and CD8 (C) T cells.

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  Fig. 3. Heterogenous cytokine-expressing CD4 T cell responses between the four groups at study baseline, prior to treatment initiation. Frequencies of cytokine co-expressing CD4 T cells in peripheral blood of tuberculous pericarditis patients, detected after PPD stimulation at baseline in placebo (orange), prednisolone (blue), MIP (green) and MIP + prednisolone (red) treatment groups. Frequencies observed in unstimulated blood were subtracted from those in PPD stimulated blood. Horizontal lines within the boxes depict medians, boxes the IQR and whiskers the range (Tukey). Mann-Whitney test was used to compare responses between all four treatment groups. We adjusted p-values using the Benjamini-Hochberg method (false discovery rate (FDR) adjustment within each cytokine combination across all treatment group). Only p-values < 0.1 are shown (for all p-values refer to Suppl Tab. 2).

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  Fig. 4. Antigen-specific CD4 T cell responses increase during follow-up. Frequencies of cytokine co-expressing CD4 T cells in peripheral blood of tuberculous pericarditis patients after PPD stimulation in placebo (a) adjunctive prednisolone (b) MIP immunotherapy (c) or combinatorial prednisolone and MIP therapy (d). Frequencies observed in unstimulated blood were subtracted from those in PPD stimulated blood. Horizontal lines of graphs depict the median, boxes the IQR and whiskers the range (Tukey). Wilcoxon comparison of responses at 2 weeks (pink), 6 weeks (royal blue) and 24 weeks (purple) post treatment time points to baseline (light blue). We used the Bonferroni method to account for multiple testing and a p-value below 0.00238 (21 comparisons) was considered significant and highlighted in bold. Only p-values < 0.1 are shown.

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  Fig. 5. Comparison of changes in antigen-specific CD4 T cell responses relative to baseline between the treatment groups after PPD stimulation. Relative differences in frequencies of cytokine co-expressing CD4 T cells in peripheral blood after PPD stimulation between baseline (0 weeks) and 2 weeks (top), 6 weeks (middle) or 24 weeks (bottom), in the placebo (orange), prednisolone (blue), MIP (green) and MIP + prednisolone (red) treatment groups. Horizontal lines depict the median, boxes the IQR and whiskers the range (Tukey). Mann-Whitney was used to compare placebo (orange), prednisolone (blue) and MIP (green) cytokine values to MIP + prednisolone (red) treatment. We adjusted p-values using the Benjamini-Hochberg method (FDR adjustment within each cytokine combination across all treatment group). Only p-values < 0.1 are indicated (all p and q values in Suppl Tab 3) and p-values with associated q-values below 0.05 were considered significant and are highlighted in bold.