Fig 2: PTPN2-Deficient KLRG1+ T Cells Can Undergo Robust Secondary Expansion(A) Experimental outline: CD45-congenic C57BL/6J host mice received 104 WT or PTPN2-deficient T cells and 24 h later 1,000 CFUs of Lm-N4. At 7 days post infection, either KLRG1+ effector or CD127+ memory precursor PTPN2-deficient or WT OT-I T cells were sorted and adoptively transferred into new hosts.(B–D) Hosts were sacrificed and OT-I numbers were analyzed 24 h (B, KLRG1+ grafts only) or 3 weeks after the transfer (C) (shown are data for KLRG1+ grafts). (D) In addition, 3 weeks after the transfer, the host mice were infected with 1,000 CFUs Lm-N4 and analyzed 5 days later. Shown are data for KLRG1+ and CD127+ grafts. The data are representative of three independent experiments with four to five mice each. One dot represents one mouse, and the horizontal line the mean in all plots. Statistical analysis: unpaired t test, ∗∗p ≤ 0.001, ∗p ≤ 0.01, nsp ≥ 0.05.

Fig 3: An adoptive transfer model confirms the helped versus helpless CD8 T cell phenotypePurified polyclonal CD8 T cells from C57BL/6J mice were transferred to TCRα KO mice with or without Ag85-specific CD4 T cells (P25) (i.e., helped and helpless, respectively) and then infected with aerosolized Mtb.(A) The survival of helped, helpless, TCRα KO mice that received only P25 cells and TCRα KO mice that did not receive any cells was monitored. Data were combined from three experiments using a total of 17–19 transferred mice per group and 5 TCRα KO mice that received no cells. The difference between the groups was statistically significant (p < 0.0001)as determined using the log-rank test for trend.(B) Determination of synergy between P25 and CD8 T cells as described in the text. The theoretical additive benefit is calculated by the “independent effects” function, while the “synergy” group is the actual survival observed after transfer of P25 and CD8 T cells (i.e., from A). These two scenarios differed statistically (p < 0.0001) by the log-rank test.(C) Lungs from TCRα KO mice that received P25 and CD8 T cells (helped) versus only CD8 T cells (helpless) were analyzed at 4 and 7 wpi by flow cytometry to determine the frequencies of TB10.44-11-specific CD8T cells.(D) The proportion of lung KLRG1+CD127− (SLEC) and KLRG1−CD127+ (MPEC) CD8 T cells was determined 7 wpi. Quadrant numbers represent percentages.(E) The frequencies of lung KLRG1+CD127− (SLEC) among total CD8 T cells was determined 4 and 7 wpi by flow cytometry and analyzed statistically. (C and E) Bars, mean ± SD. Data are representative of three independent experiments, 3–5 mice/group. Statistical significance was analyzed by unpaired t test. *p < 0.01. ns, no significant differences. See also Figures S5-S7.

Fig 4: Mtb-specific CD8 T cell responses are generated in MHCII KO mice(A) Cumulative survival of WT and MHCII KO mice after infection with aerosolized Mtb. Data were compiled from nine experiments with 56 WT and 57 MHCII KO mice. The black ticks represent censored subjects (56 WT and 48 MHCII KO mice) that were analyzed at defined time points. The difference between the strains was statistically significant (p = 0.0002) as determined by the log-rank test.(B–D) The bacterial burden in lungs was determined at 4 and 8 wpi (B). Lung cells from WT and MHCII KO mice were analyzed at 4, 5, and 8 wpi to determine the frequencies (C) and number (D) of TB10.44-11-specific and the frequencies of 32A309-318 -specific CD8 T cells (C).(E and F) The frequencies of KLRG1+CD127− (SLEC) and KLRG1−CD127+ (MPEC) among total CD8 T cells and TB10.44-11-specific CD8 T cells were determined 8 wpi by flow cytometry (E, representative data) and analyzed statistically (F). Representative data of two (B) or three (C, D, and F) independent experiments, 4–5 mice/group. Data represent mean ± SD. Statistical significance was analyzed by unpaired t test (B and F) or multiple t test (C and D). *p < 0.01; ***p < 0.001; ****p < 0.0001. ns, no significant differences.