Mycobacterium tuberculosis-specific CD4+ and CD8+ T cells differ in their capacity to recognize infected macrophages

Abstract

Containment of Mycobacterium tuberculosis (Mtb) infection requires T cell recognition of infected macrophages. Mtb has evolved to tolerate, evade, and subvert host immunity. Despite a vigorous and sustained CD8+ T cell response during Mtb infection, CD8+ T cells make limited contribution to protection. Here, we ask whether the ability of Mtb-specific T cells to restrict Mtb growth is related to their capacity to recognize Mtb-infected macrophages. We derived CD8+ T cell lines that recognized the Mtb immunodominant epitope TB10.44-11 and compared them to CD4+ T cell lines that recognized Ag85b240-254 or ESAT63-17. While the CD4+ T cells recognized Mtb-infected macrophages and inhibited Mtb growth in vitro, the TB10.4-specific CD8+ T cells neither recognized Mtb-infected macrophages nor restricted Mtb growth. TB10.4-specific CD8+ T cells recognized macrophages infected with Listeria monocytogenes expressing TB10.4. However, over-expression of TB10.4 in Mtb did not confer recognition by TB10.4-specific CD8+ T cells. CD8+ T cells recognized macrophages pulsed with irradiated Mtb, indicating that macrophages can efficiently cross-present the TB10.4 protein and raising the possibility that viable bacilli might suppress cross-presentation. Importantly, polyclonal CD8+ T cells specific for Mtb antigens other than TB10.4 recognized Mtb-infected macrophages in a MHC-restricted manner. As TB10.4 elicits a dominant CD8+ T cell response that poorly recognizes Mtb-infected macrophages, we propose that TB10.4 acts as a decoy antigen. Moreover, it appears that this response overshadows subdominant CD8+ T cell response that can recognize Mtb-infected macrophages. The ability of Mtb to subvert the CD8+ T cell response may explain why CD8+ T cells make a disproportionately small contribution to host defense compared to CD4+ T cells. The selection of Mtb antigens for vaccines has focused on antigens that generate immunodominant responses. We propose that establishing whether vaccine-elicited, Mtb-specific T cells recognize Mtb-infected macrophages could be a useful criterion for preclinical vaccine development.

Fig 1. TB10.4-specific CD8⁺ (TB10Rg3) and Ag85b-specific CD4⁺ (P25) T cells both recognize their cognate peptides.

(a) Representative histogram of Nur77 expression in P25 T cells after 2 hours of co-culture with macrophages and (b) time course of Nur77 MFI in P25 T cells. (c) Representative histogram of CD69 in P25 T cells after 2 hours of co-culture with macrophages and (d) time course of CD69 MFI in P25 T cells. (e) Representative histogram of Nur77 in TB10Rg3 T cells after 2 hours of co-culture and (f) time course of Nur77 MFI in TB10Rg3 T cells. (g) Representative histogram of CD69 in TB10Rg3 T cells at 2 hours of co-culture with macrophages and (h) time course of CD69 MFI in TB10Rg3 T cells. (i) CD69 MFI and (j) IFNγ production by P25 T cells after 72 hours of co-culture. (k) CD69 MFI and (l) IFNγ production by TB10Rg3 cells after 72 hours of co-culture. MFI, mean fluorescence intensity; mφ, macrophage.

Fig 2. Ag85b-specific CD4⁺ T cells, but not TB10.4-specific CD8⁺ T cells, inhibit bacterial growth in vitro.

(a) Matched (H-2^b) or mismatched (H-2^k) macrophages were infected with H37Rv for 2 hours, and then, one day post-infection, T cells were added. Separately, TB10.4 or Ag85b peptide was added to Mtb-infected (H-2^b) macrophages, and then T cells were added. CFU were determined 4 days later. (b) C57BL/6 macrophages were infected with H37Rv as described above. The next day, TB10.4₄₋₁₁ peptide and Ag85b_240-254 peptide was added for 1 hour, indicated as “+,” to wells that would later receive TB10.4₄₋₁₁-specific CD8⁺ and P25 CD4⁺ T cells, respectively. Subsequently, unbound peptides were washed away and TB10Rg3, TB10RgP, TB10RgL, TB10RgR, or P25 T cells were added. CFUs were determined 4 days later. Results are representative of at least three experiments. Statistical testing was by one-way ANOVA, using the Dunnett posttest compared to d5. *, #, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.0001.

Fig 3. Ag85b-specific CD4⁺ T cells, but not TB10.4-specific CD8⁺ T cells, recognize Mtb-infected macrophages.

(a-d) T cells were co-cultured with peptide-pulsed, Mtb-infected (at MOI 1, 5, or 10), or uninfected macrophages for 2 hours. (a) Representative histogram of Nur77 expression in P25 T cells and the normalized Nur77 MFI. (b) Representative histogram of CD69 expression in P25 T cells and the normalized CD69 MFI. (c) Representative histogram of Nur77 in TB10Rg3 T cells and the normalized Nur77 MFI. (d) Representative histogram of CD69 expression in TB10Rg3 T cells and the normalized CD69 MFI. (e-f) TB10Rg3 T cells were co-cultured with uninfected, peptide-pulsed, or Mtb-infected macrophages for 2 hours on d1, d3, and d5 post infection. Normalized expression of (e) Nur77 or (f) CD69 by TB10Rg3 T cells. P25 (g) or TB10Rg3 (h) T cells were co-cultured with uninfected, peptide-pulsed, or Mtb-infected macrophages, and IFNγ production was measured after 72 hours. (i) TB10Rg3, TB10RgP, TB10RgL, TB10RgR, or P25 T cells were cultured with uninfected, Mtb-infected, or peptide-pulsed macrophages. After 3 days, IFNγ in the supernatants was measured. Figures are representative of at least 5 (a-d, TB10Rg3), 2 (a-d, P25), 3 (e-h), or 2 (i) experiments. Statistical analysis was done by one-way ANOVA with Dunnett posttest (a-d) or two-way ANOVA with Sidak posttest (g-h). *, p<0.05; **, p<0.01; and ***, p<0.005.

Fig 4. TB10Rg3 CD8⁺ T cells do not recognize lung APCs from infected mice.

(a-d) T cell proliferation after coculture with lung APC from infected mice, with or without cognate peptide, or uninfected TGPM, based on eFluor450 fluorescence dilution after 72 hours. Representative flow plot (a) and quantification (b) of C7 T cell proliferation. Representative flow plot (c) and quantification (d) of TB10Rg3 T cell proliferation. (e) Bacterial burden in the lung APCs during in vitro culture over the course of the experiment in the absence of T cells. Representative of 4 (TB10Rg3) or 2 (C7) experiments.

Fig 5. TB10Rg3 and P25 T cells can recognize macrophages infected with Listeria monocytogenes expressing TB10.4 and Ag85b proteins, respectively.

(a) Representative flow plots showing Nur77 induction by TB10Rg3 T cells after co-culture with macrophages infected with △ActA.TB10 (top row) or △LLO.TB10 (bottom row) Listeria. (b) Analysis of the frequency of Nur77-expressing TB10Rg3 T cells (b, d), or normalized MFI (c, e), after co-culture with △ActA.TB10 (b, c) or △LLO.TB10 (d, e) infected macrophages. (f) Representative flow plots showing Nur77 induction by P25 population after co-culture with macrophages infected with △ActA.TB10 (top row) or △LLO.TB10 (bottom row) Listeria. (g) Analysis of the frequency of Nur77-expressing P25 T cells and (h) normalized MFI of P25 T cells. Representative of at least two experiments. Statistical testing by one-way ANOVA with Dunnett posttest. *, p<0.05; **, p<0.01; and ***, p<0.005.

Fig 6. Probing potential mechanisms for lack of recognition by TB10Rg3 T cells.

(a-c) EsxH (TB10.4) and its partner EsxG were overexpressed together in H37Rv to determine whether increasing TB10.4 abundance would lead to recognition of infected macrophages (esxGH-OE.Mtb). (a) Tetracycline treatment of esxGH-OE.Mtb in broth culture induces protein expression of EsxH (TB10.4) as measured by western blot. A different strain, fbpB-OE.Mtb, in which fbpB (e.g., Ag85b) is induction by tetracycline, does not result in greater EsxH (TB10.4) expression. Protein signal was normalized to that of GroEL2, a chaperonin protein. (b) Tetracycline treatment of esxGH-OE.Mtb in broth culture induces esxG and esxH, but not fbpB (which encodes for Ag85b) and sigA (which encodes for RNA polymerase factor sigma A), mRNA as measured by qPCR. Fold-induction was normalized to baseline (i.e., uninduced). IFNγ production by (c) P25 or (d) TB10Rg3 T cells after co-culture with macrophages infected with uninduced or induced esxGH-OE.Mtb. (e-h) MHC class I and II expression by Mtb infected-macrophages. Representative histograms (e) and fold-change (f) of MHC class I or representative histograms (g) and fold-change (h) of MHC class II expression on infected cells. (i) P25 and (j) TB10Rg3 production of IFNγ after co-culture with macrophages pulsed with titrated amounts of γ-irradiated (non-viable) H37Rv. Data is representative of 3 experiments. Statistical testing by one-way ANOVA with Dunnett posttest. *, p<0.05; **, p<0.01; and ***, p<0.005.

Fig 7. Polyclonal CD8⁺ T cells from the lungs of Mtb-infected mice recognize infected macrophages.

IFNγ production by polyclonal CD4⁺ (a) or CD8⁺ (b) T cells after co-culture with either MHC-matched (H-2^b) or MHC-mismatched (H-2^k), Mtb-infected macrophages. IFNγ production by TB10.4₄₋₁₁-tetramer-depleted (c) or tetramer-enriched (d) polyclonal CD8⁺ T cells after co-culture with either MHC-matched (H-2^b) or MHC-mismatched (H-2^k), Mtb-infected macrophages. Data is representative of at least 2 experiments. Statistical testing by a two-tailed, unpaired Student’s T test. *, p<0.05; **, p<0.01; and ***, p<0.005.