Fig 2: Largely normal B cell development and composition of B cell compartments in the bone marrow, spleen, and peritoneal cavity of MIM-/- mice. (A) Equal numbers of WT and MIM-KO splenic B cells were lysed and analyzed by immunoblotting with MTSS1 antibodies [4385S (Ab #1), 4386S (Ab #2), Cell Signaling Technology]. Arrowhead indicates the position of the band corresponding to MIM. (B) Bone marrow, spleen, and peritoneal cavity cell populations from WT and MIM-/- mice were extracted for analysis by flow cytometry (C–G) using the gating strategy shown in Supplementary Figures S1, S2. Data of three to six independent experiments are shown as mean ± SEM. (C) Percentages of total CD19+, CD19+ IgM+, and CD19- CD117+ cells in the bone marrow. (D) Percentages of B cell precursor and mature recirculating B cell populations in the bone marrow. Progression through consecutive B cell developmental stages was analyzed based on the major surface phenotypic markers shown in the schematic below. Percentage of corresponding bone marrow populations are shown as mean ± SEM. (E) Percentages of CD19+ B cells and total (upper panel) as well as CD4+ and CD8+ T cell populations (lower panel) in the spleen. (F) Percentages of major B cell subsets in the spleen. T1–3 (transitional 1–3), Fo (follicular), MZ (marginal zone) B cells are analyzed. (G) Percentages of CD23-CD5+ (B1a), CD23-CD5- (B1b), and CD23+CD5- (B2) B cells in the peritoneal cavity. Mean ± SEM is shown. *p < 0.05, **p < 0.01.

Fig 3: Generation of bone marrow chimeras of the clones in thymoproteasome deficient mice. WT or Psmb11-/- mice were sub-lethally irradiated and transplanted with hematopoietic stem cells transgenic for the indicated clones. Eight weeks later, thymi and LNs were analyzed by flow cytometry. n = 4-8 mice per group from three independent experiments. (A) Representative FACS plots showing thymic mature SP8 for all of the clones in WT or Psmb11-/- mice. (B) Numbers of donor mature SP8 in thymi of WT or Psmb11-/- mice. Mean. n = 4-5 mice per group from two independent experiments. (C) Numbers of donor CD8+ T cells isolated from thymi of WT or Psmb11-/- mice depicted as a ratio between WT and Psmb11-/- (KO). Median. The statistical analysis was performed using the two-tailed Mann Whitney U test. (D) A linear fit between the WT : KO ratio of mature SP8 cells and CD5 gMFI levels. (E) Numbers of donor CD8+ T cells isolated from LNs of WT or Psmb11-/- mice. n=4-8 mice per group from three independent experiments. The statistical analysis was performed using Kruskal–Wallis test with Dunn’s post-tests. (F) Numbers of donor CD8+ T cells isolated from LNs of WT or Psmb11-/- mice depicted as a ratio between WT and Psmb11-/- (KO). Median. The statistical analysis was performed using the two-tailed Mann Whitney U test. ** p<0.01, ns (not significant) p>0.05.

Fig 4: The self-reactivity does not dictate the magnitude of immune response of naïve CD8+ T cells. (A-C) 104 CD8+ T cells from the indicated clone were transferred into RIP.OVA mice. The next day, mice were infected with Lm-OVA. Urine glucose levels were monitored on a daily basis on days 5-14 post infection. (A) Scheme of the experiment. (B) Percentage of the diabetic mice is shown. Number of the diabetic mice and total number of mice per group is indicated on top of each column. Three (C1, C2, C8, C17) or four (C7, C12) independent experiments. For the incidence of diabetes over time, see Figure S3B . (C) Glucose concentration in blood on day 7 post-infection. Mean. Clone 1: n=11, Clone 2: n= 11, Clone 7: n=15, Clone 8: n=13, Clone 12: n=22, Clone 17: n=12. (D–F) Phenotypic analysis of two additional monoclonal TCRs reactive to OVA, TCR8 and TCR28. Splenocytes and LN cells were isolated from sub-lethally irradiated Ly5.1 mice transplanted with the indicated clones and CD8+ T cells were analyzed by flow cytometry. (D) Expression of CD5 in CD8+ LN cells. A representative experiment out of three in total. (E) Percentage of AIMT (CD44+ CD49-) T cells among CD8+ splenocytes. Mean. n = 5 mice per group from two independent experiments. (F) Staining of monoclonal LN cells with fluorescently labeled Kb-OVA tetramer. A representative experiment out of three in total. (G) Monoclonal naïve (CD44-) CD8+ T cells were sorted from LN cells and adoptively transferred to polyclonal Ly5.1 host mice (10 000 cells/mouse), which were infected one day later with transgenic Lm-OVA. Five days after the infection, the percentage of adoptively transferred GFP+ T cells among all CD8+ T cells was determined by flow cytometry. Mean. n = 6-12 per group from three independent experiments. The statistical significance was calculated using the Kruskall-Wallis test. (H, I) A linear fit between average expansion and (H) CD5 gMFI or (I) EC20 of the tetramer staining. The Pearson correlation coefficient and p value is shown.

Fig 5: CD5 levels on CD4+ T lymphocytes correlate with their capacity to upregulate p40 and differentiate into T-bethigh cells.(a–f) CD5 levels on TCR-Tg CD4+ T lymphocytes are positively associated with the extent of MP differentiation and DC1 p40 expression but not with CD40L expression or naïve cell levels. a CD5 levels on CD4+ T cells from P25, OT-II, and Marilyn TCR-Tg and WT mice. b CD44 and CD62L expression in CD4+ T lymphocytes from indicated animals. c MFI of CD5 expression obtained in (a) versus the frequency and the number of MP as well as naïve cells among CD4+ T cells obtained in (b) plotted against each other. d The representative histograms show CD40L expression on MP lymphocytes from each TCR-Tg animal while the scatter plot indicates the relationship between ?MFI of CD40L and MFI of CD5. e The contour plots depict expression of T-bet and CXCR3 in MP cells from each TCR-Tg animal and the scatter plot displays the relationship between the frequency of T-bethighCXCR3+ cells among the MP population and the MFI of CD5. f The contour plots display p40 expression in CD8a+ DCs from the indicated animals while the bar graph shows the frequency (mean ± SD) of p40+ cells among the same DC subset. Data are pooled from two independent experiments (Marilyn n = 4 mice; OT-II n = 5 mice; P25 n = 3 mice). (g–i) CD5 levels in CD4+ T lymphocytes positively correlate with their capacity to augment DC1 p40 expression. Naïve CD4+ T lymphocytes sorted from P25, OT-II, and Marilyn TCR-Tg mice were transferred into Rag KO animals and donor-derived CD4+ T cells and host-derived CD8a+ DCs analyzed 3 weeks later. g and h MFI of CD5 plotted against (g) the frequency and the number of CD44highCD62Llow as well as CD44lowCD62Lhigh donor cells and (h) ?MFI of CD40L in the different transgenic T-cell populations following adaptive transfer. i Representative contour plots showing p40 expression in host-derived CD8a+ DCs from the indicated groups together with a scatter plot depicting the relationship between MFI of CD5 and the frequency of p40+CD8a+ DCs from each group. Results from the analysis of untransferred WT and Rag KO mice are also included. Data are pooled from two independent experiments (Marilyn n = 3 mice; OT-II n = 4 mice; P25 n = 4 mice). Red, green, and blue lines/dots show P25, OT-II, and Marilyn TCR-Tg T cells, respectively. A two-sided t-test was performed to assess significance. r, Pearson’s correlation coefficient. Source data are provided as a Source Data file.