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: H83, a fusion protein of immunodominant antigens, confer robust long-term protection. Female CB6F1 mice were vaccinated with 10 µg of either MPT70, ESAT-6, Rv3020c, Rv3019c, or Tris-HCL buffer three times s.c. at the base of the tail. (A) Immunogenicity of single antigens were assessed two weeks after 3rd immunization by stimulating splenocytes in vitro with individual antigens or media. Figures represent percentage of cytokine producing (TNFa and/or IFN-y and/or IL-2) CD4 T cells after intracellular cytokine staining (ICS) (n = 4). The gating strategy for antigen-specific CD4 T cells is shown in Supplementary Figure 1 . Paired t-test, two-tailed, t-values (6.84, 5.52, 4.71, 3.51, df = 3). (B) Vaccine-mediated protection were determined by counting the bacterial numbers in the lungs of MPT70-, ESAT-6-, Rv3020c-, and Rv3019c-vaccinated mice at week 4 post Mtb Erdman infection (n = 7–8). Colony forming units (CFUs) for one saline mouse was too numerus to count. One-way ANOVA with Tukey’s multiple comparison test, F-value (5, 41) = 41.59. (C) The fusion protein, hybrid 83 (H83), was composed of MPT70, ESAT-6, Rv3020c, and Rv3019c in the mentioned order. (D) Percentage of cytokine-producing CD4 T cells after H83 vaccination measured by ICS. Splenocytes were restimulated with H83, MPT70, ESAT-6, Rv3020c, Rv3019c, or media two weeks after 3rd immunization (n = 4). Brown-Forsythe and Welch ANOVA tests with Dunnett’s multiple comparison test, F-value (5.000, 6.606) = 4.351, W-value (5.000, 7.664) =5.975. (E) CB6F1 mice were vaccinated with 2 µg H83 three times or once with 2x105 CFU BCG Danish. Six weeks after 3rd subunit vaccination or ten weeks after BCG immunization, mice were challenged with Mtb. Bacterial numbers were determined in the lungs of H83- and BCG-vaccinated mice in two individual experiments at week 6 and one experiment at week 17 post Mtb infection (n = 8). One-way ANOVA with Tukey’s multiple comparison test was performed, and the exact p-values indicated, F-values (3, 28) = 44.02, 23.07, 21.26. Data shows box plots with whiskers indicating minimum and maximum values.

Fig 4: Effects of Z. morio hemolymph on peripheral blood parameters in E. coli-induced mastitis. (A) The total number of peripheral blood leukocytes. (B) The percentage of lymphocytes on blood leukocytes. (C) The total number of peripheral blood platelet. (D–F) The percentage of CD4+, CD8+ and TNF-a+ T cells among CD3+ T cells determined in peripheral blood lymphocytes of mice in different groups by flow cytometric analysis. The representative flow cytometry dot plot shows the gating strategy for CD4, CD8 and TNF-a expression in peripheral CD3+ T cells. (G) The percentage of IL-2+ T cells among CD3+CD4+ T cells determined in peripheral blood lymphocytes of mice in different groups by flow cytometric analysis. The representative flow cytometry dot plot shows the gating strategy for IL-2 expression in peripheral CD3+ T cells. XTRC beetle stands for Z. morio hemolymph-treatment. The data from each group are presented as the mean ± SEM (n = 8 per group). * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 5: Tumor-Reactive CD4+ T Cells Acquire Cytotoxic Phenotype following Lymphopenia-Induced Expansion(A–C) B16-tumor-bearing mice were left untreated or treated with Trp1 cells (Trp1 ctrl), Trp1 + GVAX + aCTLA-4, or RT + Trp1 + aCTLA-4 as per Figure S1A. (A) Tumor growth and survival (N = 5/group). (B) T-bet and IFN-? expression by Trp1 TILs (N = 10–11/group in 2 experiments) and (N = 5–6/group in two experiments), respectively. (C) Representative plot and quantification of GzmB expression by Trp1 TILs (N = 13–17/group in four experiments).(D and E) WT and Ifngr1-/- mice bearing B16 tumors were left untreated or treated with Trp1 or Trp1xPrf-1-/- cells alone or in combination with RT + aCTLA-4. (E) Tumor growth and survival (N = 5/group).(F–H) Foxp3- Trp1 cells were sorted from B16 tumors from mice treated with GVAX + aCTLA-4 or RT + aCTLA-4 as per Figure S1D. (G) Total differential gene expression between Th Trp1 cells and Th-ctx Trp1 cells (p = 0.01) and differentially expressed transcription factors between Trp1 Th-ctx cells and Trp1 Th cells (p = 0.01). (H) Reactome pathway enrichment analysis of immune-system-related pathways and cytokine signaling pathways (red) (NES > 2, p < 0.05). Right panel: gene set enrichment analysis of IL-2-dependent genes (Castro et al., 2012; GEO: GSE39110).All quantification plots show mean ± SEM (one-way ANOVA).