GJ performed mRNA expression analyses

GJ performed mRNA expression analyses. for a lack of ML349 central tolerance. However, adoptive EAE was exacerbated in mice lacking PLP in TECs, pointing toward a non-redundant role of the thymus in dominant tolerance to PLP. Our findings reveal multiple layers of tolerance to a central nervous system autoantigen that vary among epitopes and thereby specify disease susceptibility. Understanding how different modalities of tolerance apply to distinct T cell epitopes of a target in autoimmunity has implications for antigen-specific strategies to therapeutically interfere with unwanted immune reactions against self. CD4 T cell response to myelin antigens in classical immunization recall experiments is a robust correlate of disease susceptibility. For instance, PLP-EAE susceptible SJL mice display a vigorous CD4 T cell response upon immunization with PLP protein or particular pools of PLP-peptides, whereas resistant strains such as BL/6, BALB/c, or CBA exhibit a much weaker ML349 response (7, 8). Although none of the strains that are susceptible to EAE induction with a given CNS protein develop spontaneous disease, it is undisputed that the composition and responsiveness of their CD4 T cell compartment is a critical determinant of disease susceptibility. CD4 T cells reactive to MBP or PLP are constituents of the normal human T cell repertoire (12C14). Limitations inherent to human studies so far preclude a conclusive assessment whether this in fact indicates the absence of antigen-specific tolerance or whether these autoreactive cells represent a residual fraction of the repertoire that has escaped tolerance induction. However, a precise understanding of how different modalities of tolerance shape the T cell reactivity to CNS autoantigens and how recessive modes of tolerance, i.e., deletion and anergy, or dominant, i.e., Treg-mediated, tolerance cooperate and/or differentially apply to distinct T cell epitopes of a target in autoimmunity has implications for strategies that aim to therapeutically interfere with unwanted immune reactions against the CNS. Mice lacking particular CNS autoantigens have been used to assess whether the magnitude and quality of the response to a given myelin protein is influenced by antigen-specific tolerance. MOG-specific CD4 T cell responses were found to be identical between prediction of T cell epitopes using the (IEDB) (21, 22). The IEDB algorithm predicts and ranks the relative binding strengths of all 15-mer peptides that can be generated from a given protein. For PLP, the seven 15-mer peptides containing epitope #3 were among the top eight predicted I-Ab binders, and all of the 15-mers harboring epitope #1 were ranked between positions 10 and 20 (Figure S1 in Supplementary Material). Epitope #2-containing 15-mers had the weakest binding scores and ranked between positions 33 and 57. Consistent with this relative ranking, an prediction of ML349 MHC-binding affinities using the SSM-align algorithm (23) yielded mean IC50 values of 168??61?nM for epitope #3-containing peptides and 715??262 or 1,533??498?nM for peptides containing epitopes #1 Rabbit Polyclonal to RBM26 or #2, respectively. Open in a separate window Figure 1 Proteolipid protein (PLP) epitopes and epitope-specific experimental autoimmune encephalomyelitis (EAE) susceptibility in BL/6 mice. (A) with overlapping 25-mers spanning the entire PLP protein. Responses to peptides are shown as proliferation indices. (B) Fine mapping of epitopes with overlapping 12-mer peptides. (C) CD4 T cell recall response of proliferative response to stimulation with titrated amounts of PLP172C183 as cells from TCR-PLP2 stimulation with PLP172C183. Data are from individual mice representative for gene, this resulted in the virtual absence of Foxp3+ cells from thymus and periphery. Importantly, as in with irradiated splenoctyes and peptide PLP9C20 in the presence or absence of titrated numbers of TCR-PLP1+CD25+ CD4 ML349 T cells from TCR-PLP1 into Th1 or Th17 effectors and subsequently transferred into gene in TECs (Foxn1-Cre two distinct, yet mutually not exclusive routes (24). On the one hand, tolerogenic encounter of such antigens by CD4 T cells may depend upon antigen handover and presentation by thymic DCs. On the other hand, mTECs, or TECs in general, may autonomously present endogenously expressed antigen to CD4 T cells unconventional MHC class II-loading pathways (25). Two experimental systems were employed to address this issue in the TCR-PLP1 model. First, we generated TCR-PLP1 (Figure ?(Figure7B),7B), indicating that anergy induction occurred independent of thymic PLP encounter. Consistent with this, the anergy marker FR4 was similarly elevated ML349 on Foxp3CCD25CTCR-PLP1+ cells from both deletional mechanisms and anergy induction rather than Treg conversion..