A Peptide-binding Motif for I-Ag7, the Class II Major Histocompatibility Complex (MHC) Molecule of NOD and Biozzi AB/H Mice

The class II major histocompatibility complex molecule I-Ag7 is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its β chain. To identify the requirements for peptide binding to I-Ag7 and thereby potentially pathogenic T cell epitopes, we analyzed a known I-Ag7-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9–27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-Ag7 M12-Y20/ K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-Ag7 deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-Ag7–restricted T cell epitopes or eluted from I-Ag7. Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM

Weak Proinsulin Peptide–Major Histocompatibility Complexes Are Targeted in Autoimmune Diabetes in Mice

OBJECTIVE—Weak major histocompatibility complex (MHC) binding of self-peptides has been proposed as a mechanism that may contribute to autoimmunity by allowing for escape of autoreactive T-cells from the thymus. We examined the relationship between the MHC-binding characteristics of a β-cell antigen epitope and T-cell autoreactivity in a model of autoimmune diabetes

OCT Signs of Early Atrophy in Age-Related Macular Degeneration: Interreader Agreement: Classification of Atrophy Meetings Report 6.

PURPOSE: To determine the interreader agreement for incomplete retinal pigment epithelium (RPE) and outer retinal atrophy (iRORA) and complete RPE and outer retinal atrophy (cRORA) and their related features in age-related macular degeneration (AMD). DESIGN: Interreader agreement study. PARTICIPANTS: Twelve readers from 6 reading centers. METHODS: After formal training, readers qualitatively assessed 60 OCT B-scans from 60 eyes with AMD for 9 individual features associated with early atrophy and performed 7 different annotations to quantify the spatial extent of OCT features within regions of interest. The qualitative and quantitative features were used to derive the presence of iRORA and cRORA and also in an exploratory analysis to examine if agreement could be improved using different combinations of features to define OCT atrophy. MAIN OUTCOME MEASURES: Interreader agreement based on Gwet's first-order agreement coefficient (AC1) for qualitatively graded OCT features and classification of iRORA and cRORA, and smallest real difference (SRD) for quantitatively graded OCT features. RESULTS: Substantial or better interreader agreement was observed for all qualitatively graded OCT features associated with atrophy (AC1 = 0.63-0.87), except for RPE attenuation (AC1 = 0.46) and disruption (AC1 = 0.26). The lowest SRD for the quantitatively graded horizontal features was observed for the zone of choroidal hypertransmission (± 190.8 μm). Moderate agreement was found for a 3-category classification of no atrophy, iRORA, and cRORA (AC1 = 0.53). Exploratory analyses suggested a significantly higher level of agreement for a 3-category classification using (1) no atrophy; (2) presence of inner nuclear layer and outer plexiform layer subsidence, or a hyporeflective wedge-shaped band, as a less severe atrophic grade; and (3) the latter plus an additional requirement of choroidal hypertransmission of 250 μm or more for a more severe atrophic grade (AC1 = 0.68; P = 0.013). CONCLUSIONS: Assessment of iRORA and cRORA, and most of their associated features, can be performed relatively consistently and robustly. A refined combination of features to define early atrophy could further improve interreader agreement

Germ cell-induced immune suppression in mice. Effect of inoculation of syngeneic spermatozoa on cell-mediated immune responses.

Autologous sperm are immunogenic and can elicit cell-mediated immunity and antibody responses (1-4). Immune responses against spermatozoa do not occur under normal circumstances, due to the fact that germ cells are efficiently sequestered from the immune system by the blood-testis barrier (5). This barrier, which is established before the immune system becomes competent, creates an immunologically privileged site for sperm and the developing spermatozoa. Therefore, immunological tolerance is not generated to autologous male germ cells. Injuries of the blood-testis barrier (e.g., after vasectomy, infection, or biopsy) can result in the production of autoantibodies and T cell reactivity (1-4). Because the immune system is not normally exposed to germ cells, it is possible that sperm and other germ cells exert regulatory infuences on immune potential. In the present study, the influence of inoculation of germ cells on cellular immune reactions was investigated. The results indicate that male C5 7BL/6 mice injected with syngeneic germ cells exhibited: (a) reduced natural killer (NK) 1 cell activity; (b) reduced mixed lymphocyte reactivity (MLR); (c) enhanced auto-proliferation; and (d) decreased potential to generate cytotoxic T lymphocyte (CTL) responses to modified self and allogeneic antigens. The reduction in CTL potential against trinitrophenyl-modified syngeneic spleen cells (TNP-self) was mediated by radiosensitive suppressor T cells. These findings are discussed with respect to possible effects of exposure to male germ cells on the immune system. Materials and Methods Animals. Young adult mice (8-12-wk) were used for all in vitro sensitization studies. The inbred strains C57BI6/J (B6), AKR/J, B10.BR (BR), DBA2/J, and (B6 X DBA/2)F1 (BDFI

Cross-reactive recognition of alloantigens and certain B6 mutant antigens by antigen-specific and polyclonally activated cytotoxic effector cells

Various B6 cytotoxic T lymphocyte (CTL) effector populations were tested for cross-reactive lysis (CRL) of unmodified third-party allogeneic as well as of various K bm and D bm mutant target cells. The effector cells were generated by in vitro stimulation of B6 spleen cells either from unprimed mice against alloantigen, Con A, or a pool of different allogeneic stimulators, or from in vivo hapten-self primed mice against trinitrophenyl (TNP)-self or N-(5-sulfonic-1-naphthyl)ethylenediame (AED)-self antigens. Except for AED-primed effector cells all CTL populations exhibited significant CRL on third-party allogeneic target cells. More importantly, these alloreactive and H-2-restricted effectors also lysed some of the K b mutants, but not others. Thus, unmodified bm1 and bm11 mutants were lysed by all of the B6 wild-type effectors, whereas bm6 and bm9 were only weakly lysed by alloreactive CTL. None of the B6 CTL populations lysed bm13 and bm14 target cells. These data indicate that (a) alloreactive and self-restricted CTL can recognize the same molecular structure of Class I antigens although the epitopes may be different; (b) CTL discriminate between a few amino acid substitutions in certain positions of the H-2 molecule, and (c) the newly generated antigenic determinants of the bm mutants allow them to be divided into three categories in terms of their allogenicity for these cross-reactive CTL populations