Extended Data Fig. 10: Functional analysis of 8F10 T cells developed in NOD or B16A hosts.

a–f, The bone marrow chimaera was constructed as in Fig. 4a, and T cells were examined after 6 (a–c) or 9 (d–f) weeks. a, b, d, e, Bulk CD4⁺ T cells were purified from iLNs of individual NOD or B16A mice (three per group) by two rounds of MACS negative selection. To examine cytokine repertoire (a, d), half of the individual T cell samples were combined. The remainder were kept as individual samples, labelled with CFSE (1.5 µM), and used to measure cell proliferation (b, e). In either case, T cells were mixed with NOD.Rag1^–/– splenocytes (1:2 ratio) and stimulated with B:9–23 for 16 (a, d) or 72 (b, e) hours. a, Representative FACS plots showing intracellular cytokine staining of the 8F10 T cells from NOD-iLN or B16A-iLN, after stimulation with B:9–23 for 16 h (brefeldin A was added for the last 4 h). Production of IL-4, IL-17A, IL-5 and IL-10 was not detected. Data are representative of two independent experiments with 3 mice combined per experiment. b, Representative FACS plots (top) showing CFSE dilution of the 8F10 T cells stimulated by B:9–23 or the control HEL11–25 peptide for 72 h. The results of 6 individual mice from two independent experiments are summarized in the box plots (bottom). Box plots show the median, box edges represent the first and third quartiles, and the whiskers extend from the minimum to the maximum. **P < 0.01, two-tailed unpaired Student’s t-test. c, Representative FACS plots showing ex vivo surface staining of FR4 and CD73 as well as CD39 and TIGIT on endogenous CD4⁺ or 8F10 T cells in the iLNs of NOD or B16A mice. Data are representative of three mice analysed in two independent experiments. d–f, Experiments were performed in week 9 following the procedures described in a–c. The data in d–f are from a single experiment.

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