Extended Data Fig. 8: Generation of MHC II deficient SJL mice and analysis of T cell compartment following RR-EAE challenge.

(a) Schematic of CRISPR/Cas9-based H2-Ab1^s targeting strategy. (b) Schematic showing the WT and mutant H2-Ab1^s locus with details of the respective 247 bp deletion. (c) Genomic PCR analysis of H2-Ab1^{s −/−} SJL mouse (631 bp) and WT control (~900 bp). Representative out of infinite number of experiments. (d) Flow cytometric analysis of I-A^s on B220⁺ B cells in blood of WT C57BL6, WT SJL and H2-Ab1^s hetero- or homozygote SJL mice establishing MHC-II deficiency of H2-Ab1^{s −/−} SJL mouse. (e) Flow cytometric analysis of MHCII surface expression (I-A^b) on microglia of Mg^ΔMHCII mice and controls (diseased brains). N = 20 individual mice per group, pooled from 3 independent experiments. Lines represent mean. Significance was calculated using two tailed t-test. ****p < 0.0001. (f) Flow cytometric analysis of FoRBY2 CD4⁺ T cells extracted from spleen and lymph nodes of 2D2 donor animals post MACS isolation, prior to adoptive transfer. (g) Flow cytometric analysis of blood of Mg^ΔMHCII mice and control recipient mice at day 6 post immunization showing detection of engrafted 2D2 cells as CD45.1^- TCRβ11⁺ cells, gated from GR1⁻CD45^hi CD11b⁻ CD4⁺ TCRb⁺ T cells. N = 11 individual mice per group, line represent mean, data are pooled from 3 independent experiments. Significance was calculated using two tailed t-test. (h) Flow cytometric analysis of Gitr, Foxp3 and CD25 expression on CD4⁺ T cells, showing that Foxp3⁺ or CD25⁺ Tregs can be defined by Gitr. (i) Gating strategy for detection of grafted cells and GITR Tregs. Related to Fig. 7d, e. j Normalized MFI of Gitr and Klrg1 on Gitr^hi Treg population in control and Mg^ΔMHCII mice. n = 20 individual mouse per group, lines represent mean, data pooled from 3 independent experiments. Significance was calculated using two tailed t-test.

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