Fig 2: MPT70-specific CD4 T cells maintain a low differentiation state compared to ESAT-6. (a) The functional differentiation score (FDS) of MPT70- and ESAT-6-specific CD4 T cells over the course of Mtb Erdman infection (n = 4). The FDS is defined as the ratio of all IFN-?-producing CD4 T cell subsets divided by subsets producing other cytokines (IL-2, TNF-a), but not IFN-? (high FDS = high IFN-? production). Multiple t tests with correction for multiple testing using the Holm-Sidak method were used to assess statistical differences. Values shown are means ± SEM. Flow cytometry gating is shown as depicted in Fig. S1a in the supplemental material, using the antibodies shown in Table 4. (b) Frequencies of KLRG1-expressing MPT70- and ESAT-6-specific CD4 T cells throughout infection (n = 4). Values shown are means ± SEM. Multiple t tests with correction for multiple testing using the Holm-Sidak method were used to assess statistical differences. (c) Frequency of CD45-labeled MPT70- and ESAT-6-specific CD4 T cells in the lung-associated vasculature (CD45+) 20 weeks postinfection (p.i.) with Mtb (n = 4). A two-tailed, paired t test was used. (d, top) Schematic representation of custom-made I-Ab:MPT7038-52 MHC-II tetramer. (d, bottom) Representative concatenated FACS plots showing frequencies of I-Ab:MPT7038-52 and I-Ab:ESAT-64-17 tetramer (Tet)-positive CD4 T cells or corresponding hClip tetramer-positive CD4 T cells in lungs of mice 12 weeks after Mtb infection (n = 4). (e) Frequency of I-Ab:MPT7038-52 and I-Ab:ESAT-64-17 CD4 T cells 12 to 16 weeks after Mtb infection expressing CXCR3, KLRG1, and T-bet. A parametric, two-tailed, paired t test was used to assess statistical differences (n = 12). Flow cytometry gating is shown as depicted in Fig. S3 using the antibodies shown in Table 3. (f) Concatenated FACS plot of CX3CR1+ KLRG1+ coexpressing ESAT-64-17 CD4 T cells (n = 4).

Fig 3: Ena/VASP deficiency reduces T cell accumulation in response to Listeria Monocytogenes challenge. Congenically distinct naïve WT CD45.1/.2 OT-I and EVL/VASP dKO CD45.2/.2 OT-I T cells were isolated from donor mice and transferred at a 1:1 ratio I.V. into WT CD45.1/.1 recipient mice. (A) Graphical schematic of the experimental method. (B) Representative flow cytometry gating scheme used to detect transferred T cells. (C) WT and EVL/VASP dKO naïve T cells have similar persistence in vivo. 1×106 WT and EVL/VASP dKO naïve OT-I CD8+ T cells, were co-transferred I.V. into uninfected WT CD45.1/.1 recipient mice and spleens were harvested 6 days later to determine a persistence baseline. (D–F) EVL/VASP dKO T cells have reduced proliferation and accumulation in response to LM challenge. 10,000 of each naïve WT CD45.1/.2 OT-I and EVL/VASP dKO CD45.2/.2 OT-I T cells were co-transferred I.V. into WT CD45.1/.1 recipient mice one day before infection with LM-OVA (2×103 PFUs). Spleens from recipient mice were then harvested 3 or 6 days later for analysis by flow cytometry. (D) T cell numbers recovered from the spleen at day 6 post LM infection (left) and ratios of EVL/VASP dKO/WT CD8+ T cells (right). EVL/VASP dKO T cell numbers were normalized to the ratio of EVL/VASP dKO/WT T cells recovered from spleens of uninfected recipient mice at day 6 from the same experiment [i.e., homing ratio in (C)]. (E) Example CTV proliferation dye dilution curves and quantification of the average number of divided cells, determined by CTV dilution, from spleens on day 3 post LM-OVA infection. (F) Representative histograms of KLRG1 and CD127 expression, and flow cytometry contour plots showing the gates used to identify SLECs (KLRG1hi CD127lo). The number (normalized to uninfected mice) and percent of WT and EVL/VASP dKO SLEC T cells are plotted. Data are from =9 experiments each with =2 mice per group/experiment, except for (E), (F), which are from 3 independent experiments with =2 mice per group/experiment. Significance was defined by paired t tests (for T cell numbers) and one sample t tests compared to a hypothetical value of 1.0 (for the EVL/VASP dKO/WT ratios); ns is not significant, * is p < 0.05, ** is p < 0.01, and **** is p < 0.0001.

Fig 4: MPT70-specific CD4 T cells maintain a low differentiation state compared to ESAT-6.(a) The functional differentiation score (FDS) of MPT70 and ESAT-6-specific CD4 T cells over the course of Mtb Erdman infection (n=4). The FDS is defined as the ratio of all IFN-? producing CD4 T cell subsets divided by subsets producing other cytokines (IL-2, TNF-a), but not IFN-? (high FDS = high IFN-? production). Multiple t-tests with correction for multiple testing using the Holm-Sidak method. Shown as average mean ± SEM. Flow Cytometry gating as depicted in Figure S1, using antibody panel 2. (b) Frequencies of KLRG1 expressing MPT70 and ESAT-6-specific CD4 T cells throughout infection (n=4). Shown as average mean ± SEM. Multiple t-tests with correction for multiple testing using the Holm-Sidak method. (c) Frequency of CD45-labelled MPT70 and ESAT-6 specific CD4 T cells in the lung-associated vasculature (CD45+) 20 weeks post-infection (p.i.) with Mtb (n=4). Paired t-test, two-tailed. (d, upper) Schematic representation of custom-made I-Ab:MPT7038–52 MHC-II tetramer. (d, lower) Representative concatenated FACS plots showing frequencies of I-Ab:MPT7038–52 and I-Ab:ESAT-64–17 tetramer+ CD4 T cells or corresponding hClip tetramer+ CD4 T cells in lungs of mice 12 weeks post Mtb infection (n=4). (e) Frequency of I-Ab:MPT7038–52 and I-Ab:ESAT-64-17 CD4 T cells 12–16 weeks post Mtb infection expressing CXCR3, KLRG1, and T-bet. Parametric, paired t-test, two-tailed (n=12). Flow Cytometry gating as depicted in Figure S3 using antibody panel 1. (f) Concatenated FACS plot of CX3CR1+KLRG1+ co-expressing ESAT-64-17 CD4 T cells (n=4).

Fig 5: Overexpression of MPT70 accelerates T cell differentiation and increases bacterial susceptibility to MPT70 vaccination. (a) In vitro fold gene expression of sigK, rskA, and MPT70 in H37Rv::mpt70high compared to WT H37Rv. All genes were tested in technical duplicates and normalized to esxA expression using primers in Table 1. (b) In vivo fold gene expression of MPT70 and ESAT-6 in the lungs of H37Rv::mpt70high-infected mice compared to WT H37Rv-infected mice 3 weeks after Mtb challenge (n = 4). Genes were analyzed in technical duplicates using primers and probes in Table 2, normalized to 16S rRNA expression, and shown as fold increase from WT H37Rv. A two-tailed, unpaired t test was used to assess statistical differences. (c) Bacterial burden in the lungs of mice injected with PBS at day 1, week 3, week 12, and week 22 after infection with either H37Rv:: mpt70high or WT H37Rv infection (n = 5). Values shown are means ± SEM. Multiple t tests with correction for multiple testing using the Holm-Sidak method were used to assess statistical differences. (d) Frequency of lung MPT70- and ESAT-6-specific CD4 T cells 4 weeks after Mtb infection (n = 10). Box plots with whiskers indicating the minimum and maximum values are shown. The mean values are indicated with + symbols. A unpaired, two-tailed t test was used to assess statistical differences. (e) Frequency of lung MPT70-specific CD4 T cells 3 weeks after Mtb infection in mice injected with PBS (white boxes) and mice vaccinated with MPT70 (blue boxes) (n = 5). A unpaired, two-tailed t test was used to assess statistical differences. (f) Representative concatenated FACS plots (n = 10) showing the expression of CX3CR1, CXCR3, KLRG1, or CD45 on MPT70-specific CD4 T cells 4 weeks after H37Rv:: mpt70high infection (blue) or H37Rv infection (gray). Flow cytometry gating is shown as depicted in Fig. S1a, using antibodies shown in Table 5. (g) Bacterial numbers were determined in the lungs of mice vaccinated with PBS or mice vaccinated with MPT70 and ESAT-6 at day 1, week 3, week 12, and week 22 after WT H37Rv infection (left) or H37Rv::mpt70high infection (right) (n = 4 to 5). One mouse was excluded from the week 12 time point (H37Rv::mpt70high, MPT70 vaccinated), as the mouse was very sick, had high weight loss, and met the study’s predefined humane endpoints (P value = 0.67, if included). Values shown are means ± SEM. Statistical differences were assessed using one-way ANOVA with Tukey’s multiple-comparison test.