The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8+ T Cells

Abstract

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+)and CD8(+)T cells secreting gamma interferon (IFN-γ) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.

FIG 1

Percentages of volunteers whose PBMC showed proliferative responses to individual 20-mer peptides derived from Rv1860 among 28 latently M. tuberculosis-infected healthy volunteers (HV) (A) and 20 TB patients (PAT) (B). The three most immunodominant peptides, 1803, 1821, and 1826, are highlighted in panel A.

FIG 2

Proliferation and cytokine secretions by PBMC in response to immunodominant peptides from Rv1860. (A) Peptide 1803; (B) peptide 1821; (C) peptide 1826. Twenty-eight HV and 20 PAT were compared. The P values for significant differences between the two groups were computed using Mann-Whitney tests; the P values that did not remain significant after Bonferroni's correction are indicated as ns (not significant).

FIG 3

Polychromatic flow cytometry analysis of cytokine production profiles of Rv1860-derived peptide-specific T cells from latently M. tuberculosis infected volunteers (HV). Whole-blood ICC samples stimulated with a mixture of peptides 1803, 1821, and 1826 were stained and analyzed as described in Materials and Methods. Percentages of CD4⁺ (left bar in each pair) were compared with those of CD8⁺ (right bar in each pair) T cells secreting the different combinations of cytokines IFN-γ, IL-2, TNF-α, and MIP-1β indicated below the pairs of bars as + or − signs. The boxes represent the 25th and 75th percentile values, and the bars denote medians. Dots represent individual values. ^#P values computed by the nonparametric Wilcoxon test for significant differences between CD4⁺ and CD8⁺; *significant P values below the Bonferroni-corrected critical value of 0.05/15 = 0.0033.

FIG 4

Polychromatic flow cytometry analysis of cytokine production profiles of Rv1860-derived peptide-specific T cells in TB patients (PAT). Whole-blood ICC samples stimulated with a mixture of peptides 1803, 1821, and 1826 were stained and analyzed as described in Materials and Methods. Percentages of CD4⁺ (left bar in each pair) were compared with those of CD8⁺ (right bar in each pair) T cells secreting the different combinations of cytokines IFN-γ, IL-2, TNF-α, and MIP-1β indicated below the pairs of bars as + or −. The boxes represent the 25th and 75th percentile values, and the bars denote medians. Dots represent individual values. ^#P values computed by the nonparametric Wilcoxon test for significant differences between CD4⁺ and CD8⁺; *significant P values below the Bonferroni-corrected critical value of 0.05/15 = 0.0033.

FIG 5

Polyfunctional human T cell response to secreted antigen ESAT-6. The plots show comparisons of frequencies of CD4⁺ (left bar in each pair) with CD8⁺ (right bar in each pair) T cells in latently infected HV (top panel) and PAT (bottom panel) secreting different combinations of the cytokines IFN-γ, IL-2, TNF-α, and MIP-1β indicated below the pairs of bars as + or −. ^#P values for significant differences between HV and PAT computed by the nonparametric Wilcoxon test available within SPICE. None of the values were significant following Bonferroni's correction for multiple comparisons.