Fig 2: Responses of Autoreactive CD4+ TCCs to RASGRP2 and HLA-DR-SPs(A) Dose-response curves of TCC14 to RASGRP2(78–87) and HLA-DR-SPs.(B) Irradiated BLS-DR2b cells were incubated with peptides for 12 h and then co-cultured with TCC14. Shown are expression levels of CD69 and CD25 on TCC14 at different time points after co-culture. ?MFI indicates the mean fluorescence intensity (MFI) value above the no-peptide control.(C–E) CD45RA- PBMCs from 3 HLA-DR15+ RRMS_NAT patients were stimulated with RASGRP2(78–87) to generate TCCs. Proliferation of memory CD4+ T cells was analyzed on day 12 (C). Acquired TCCs were co-cultured with irradiated autologous PBMCs as APCs and stimulated with RASGRP2(78–87) or DRB1(57–70), and proliferation was detected on day 3. The TCCs responding to RASGRP2(78–87) and DRB1(57–70) are highlighted in blue (D). Five new 1159AG_TCCs that responded to RASGRP2(78–87) and DRB1(57–70) were generated from RRMS_NAT-2. Their corresponding TCRVß sequence and functional phenotype are shown (E).(F) Restriction of the RASGRP2(78–87)-specific 1159AG_TCCs was tested with irradiated BLS-DR2b cells and stimulation with RASGRP2(78–87).(G) Th1/Th2/Th17-related cytokines in supernatants of 1159AG_TCCs after co-culture with irradiated BLS-DR2b cells and stimulation with RASGRP2(78–87) for 3 days.(H and I) 1159AG_TCCs were co-cultured with irradiated BLS-DR2b cells and stimulated with RASGRP2(78–87) or DRB1/5(184–199) for 3 days. Shown is proliferation of 1159AG_TCCs (H) and IFN-? in supernatants (I).Data are expressed as mean ± SEM, and p values were determined by unpaired t test. See also Figures S4 and S5 and Table S1.

Fig 3: Elution of DR2a- and DR2b-Presented Peptides from Primary B Cells and Monocytes of HLA-DR15+ MS Patients Using Allele-Specific Monoclonal Abs(A) Genomic organization of HLA-DR loci of the HLA-DR15 haplotype with one HLA-DRA gene (DRA*01:01P) and two HLA-DRB genes (DRB1*15:01 and DRB5*01:01). These genes encode two HLA-DR heterodimers (serotypes) DR2a and DR2b.(B and C) HLA-DR genotyping results of MS patients (n = 1,017). Proportions of DRB1*15:01+ (DR2b+) and DRB1*15:01- (DR2b-) MS patients (B). Frequencies of DRB1*15:01+ (DR2b+) or DRB1*15:01- (DR2b-) MS patients also carrying the DRB5*01:01 (DR2a) (C).(D) Cross-reactivity between DR2a and DR2b allele-specific monoclonal Abs was tested using BLS transfectants expressing DR2a (BLS-DR2a) or DR2b (BLS-DR2b) (scale bar, 100 µm).(E) Binding affinities of DR2a or DR2b allele-specific monoclonal Abs. The x axis indicates fluorescence intensity.(F and G) 9 amino acid binding motifs of DR2a-presented (F) and DR2b-presented peptides (G) on B cells and monocytes based on NetMHCII 2.3 analysis and visualization by iceLogo. Putative peptide anchor positions for DR2a and DR2b are highlighted in gray.(H) Overlap of the DR2a- or DR2b-presented unique peptides between B cells and monocytes at the group level (HDs, n = 3; RRMS, n = 3; RRMS_NAT, n = 3).(I) Overlap of the source proteins of DR2a- or DR2b-presented peptides between B cells and monocytes at the group level (HDs, n = 3; RRMS, n = 3; RRMS_NAT, n = 3).See also Figures S1 and S2 and Tables S1, S2, and S3.