Fig 2: CD4 T cells that have trogocytosed and display MHC-II present antigen and stimulate other cognate naive T cells(A) Experimental setup.(B) Proliferation of naive OT2 and AND Tresp cells upon 3 days of co-culture with purified OT2 or AND Tpres cells previously exposed to BMDCs loaded with OVAp or MCCp, respectively. Fluorescence-activated cell sorting (FACS) plots show representative images of CTV-labeled OT2 and AND Tresp proliferation upon co-culture with or without OT2 Tpres cells. Bar graph shows quantification of AND and OT2 Tresp proliferation upon incubation with AND or OT2 Tpres cells (means ± SEMs of quadruplicates; ****p < 0.0001 [2-tailed unpaired Student’s t test]).(C) Proliferation of OT2 Tresp upon 3 days of co-culture with purified OT2 Tpres cells, previously exposed to k/b BMDCs loaded with OVAp, MCCp, or no peptide (no-Ag). The number of cell divisions was calculated according to CTV dilution. The bar plot shows the means ± SEMs of triplicates.**p < 0.01; ***p < 0.001; *****p < 0.00001 (2-tailed unpaired Student’s t test).(D) Cumulative number of cell divisions of CTV-labeled Tpres and Tresp AND T cells co-cultured for 3 and 5 days. The graph shows the means ± SEMs of quadruplicates. ***p < 0.001; *****p < 0.00001 (2-tailed unpaired Student’s t test).(E) Induction of ERK phosphorylation upon co-incubation of Tpres and Tresp AND T cells for the indicated time points. Line plots represent means ± SEMs (n = 3). *p < 0.05 (2-tailed unpaired Student’s t test).(F) Two-color contour plots showing the expression of CD25 and PD1 activation markers by CD4 AND Tpres and Tresp cells after co-incubation for the indicated time periods. Tpres cells were previously incubated overnight with MCCp-loaded BMDCs. Time point 0 h shows marker expression before co-incubation.(G) Quantification of data shown in (F). Bar plots represent means ± SEMs (n = 3). ***p < 0.001; *****p < 0.00001 (2-tailed unpaired Student’s t test).

Fig 3: Abundance of DCs and antigen determines Treg versus Th17 response in vivo(A) Experimental setup of the immunization protocol with different numbers of DCs.(B) OT2 CD4 response to antigen was evaluated by measuring the expression of the CD44 activation marker as a function of the dose of OVAp-loaded DCs. Bar graphs show the means ± SEMs (n = 3 mice per condition; unpaired 2-tailed Student’s t test; **p < 0.01).(C) Two-color contour plots showing percentages of Foxp3+CD25+ Treg cells and IL-17A+CCR6+ Th17 cells within the CD45.2+CD4+ OT2 cell population in the function of the dose of DC APCs. Bar plots show the means ± SEMs (n = 3 mice per condition; unpaired 2-tailed Student’s t test; *p < 0.05; ***p < 0.001).(D) Experimental setup of intraperitoneal (i.p.) infection with increasing number of plaque-forming units (PFUs) of MVA-OVA.(E) CD8 T cell response to the virus was evaluated by measuring the percentage of I-Ab-OVAp tetramer+ cells within the CD8+ T cell population. Graph shows the means ± SEMs (n = 2–4 mice per condition; unpaired 2-tailed Student’s t test; **p < 0.01).(F) Quantification of the percentage of Foxp3+CD25+ Treg and IL-17A+CCR6+ Th17 within the splenic endogenous CD4 T cell population. Graphs show the means ± SEMs (n = 2–4 mice per condition; unpaired 2-tailed Student’s t test; *p < 0.05; **p < 0.01; ***p < 0.001).

Fig 4: Tpres cells are enriched in Treg and Tresp cells in Th17(A) Experimental setup. After purification by cell sorting, AND Tpres cells are incubated for 3–6 days in the absence or presence of naive AND Tresp cells.(B) ELISA measurement of IL-2 and TNF-a concentrations in supernatants of Tpres cells cultured for 3 days in the presence or absence of Tresp cells. The bar graphs show the means ± SEMs of triplicates.**p < 0.01 (2-tailed unpaired Student’s t test).(C) Expression of CCR6, CD25, and Foxp3 by Tpres and Tresp cells from the experiment in (B), measured by flow cytometry. Bar graphs represent means ± SEMs (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 (2-tailed unpaired Student’s t test).(D) Generation of FoxP3+CD25+ T cells within AND Tpres and Tresp cell populations. After overnight incubation with MCCp-loaded BMDCs and purification, Tpres were cultured alone (Tpres alone) or together with naive AND Tresp cells (Tpres+Tresp) for 6 days and then analyzed by flow cytometry for Foxp3 and CD25 expression. In parallel, AND T cells were cultured uninterrupted for 6 days with MCC-loaded BMDCs (DC+Tpres). Data represent the means ± SEMs of biological triplicates. **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA test).(E) Tpres and Tresp cells were stained with surface CD25 and intracellular IL-17A, Foxp3, ROR?t, or IFN?. Two-color contour plots are on the left and quantification in the bar plots to the right. **p < 0.01; ****p < 0.0001 (2-tailed unpaired Student’s t test).(F) Quantification via qRT-PCR of mRNA expression of sorted Tpres, Tresp cells, and naive AND T cells. Data are presented as the means ± SEMs of n = 2–4 biological replicates normalized to the mean values of naive cells (set as 1). *p < 0.05; **p < 0.01 (2-tailed unpaired Student’s t test).

Fig 5: Deficiency in T-T antigen presentation leads to reduced Th17/Treg ratios and resistance to EAE(A) Evolution of neurological symptoms (score) and body weight in MOG-immunized WT and Rhog-/- mice. Neurological scores were according to Borroto et al. (2016) Graphs represent the means ± SEMs (n = 8–9 mice per group; paired 2-tailed Student’s t test [weight] and non-parametric matched-pairs signed rank Wilcoxon 2-tailed test [score]). **p < 0.01; ****p < 0.0001.(B) Two-color contour plots showing expression of Foxp3, CD25, IL-17A, and CCR6 in WT and Rhog-/- mice sacrificed at day 22 (A). Bar graphs show the percentages of Foxp3+CD25+ and IL17A+CCR6+ T cells (means ± SEMs; n = 8–9 mice per group; unpaired 2-tailed Student’s t test; *p < 0.05; ***p < 0.001).(C) WT and Rhog-/- mice were immunized with MOG, and the draining popliteal lymph nodes were collected 7 days later. The presence of MOG-reactive T cells was analyzed by flow cytometry on the CD4+CD44+ activated population by incubation with I-Ab OVA329-337 tetramer. Bar plot shows the means ± SEMs (n = 4–5 mice per group; unpaired 2-tailed Student’s t test. ns, not significant).(D) Experimental setup of the bone-marrow adoptive transfer experiment.(E) BM reconstitution was tested in the blood of chimeric mice before immunization with MOG. Bar plots show the percentage (means ± SEMs) of CD4 T, CD8 T, and B cells within the white blood cell population in bone marrow chimeras and, as reference, in Cd3e-/- and WT C57BL/6 mice.(F) Score and body weight evolution in BM chimeras reconstituted with either WT or Rhog-/- T cells. Graphs represent the means ± SEMs (n = 6–8 mice per group; 2-tailed Student’s t test [weight] and non-parametric matched-pairs signed rank Wilcoxon 2-tailed test [score]; **p < 0.01; ***p < 0.001).(G) Two-color contour plots showing the expression and the percentages of Foxp3 and CD25 Treg markers and of the IL-17A and ROR?t Th17 markers in WT and Rhog-/- BM chimeras sacrificed at day 14 (F). Bar plots show the means ± SEMs (n = 6–8 mice per group; unpaired 2-tailed Student’s t test; *p < 0.05; **p < 0.01).