EH contributed by interpreting the data and revising the manuscript
June 20, 2021
EH contributed by interpreting the data and revising the manuscript. predicted low affinity and low likelihood of cathepsins cleavage were inert controls. Peripheral blood mononuclear cells from these patients were stimulated with the selected idiotope peptides in presence of anti-CD40 for 12 h. T cells were then labeled for activation status with anti-CD154 antibodies and CD3+CD4+ T cells phenotyped as memory (CD45RO+) or na?ve (CD45RO?), with potential for brain migration (CXCR3 and/or CCR6 expression). Anti-CD14 and -CD8 were utilized to exclude monocytes and CD8+ T cells. Unstimulated cells or insulin peptides were unfavorable controls, and EBNA-1 peptides or CD3/CD28 beads were positive controls. The mean proportion of responding memory CD4+ T cells from all nine MS patients was significantly higher for idiotope peptides with predicted high HLA-DR affinity and high likelihood of cathepsin cleavage, than toward predicted inert peptides. Responses were mainly observed toward peptides affiliated with the CDR3 region. Activated memory CD4+ T cells expressed the chemokine receptor CCR6, affiliated with a Th17 phenotype and allowing passage into the central nervous system (CNS). This study suggests that that antigenic properties of BCR idiotopes can be identified using HLA affinity and endosomal processing predictions. It further indicates that MS patients have a memory T cell repertoire capable of recognizing frequent BCR idiotopes found in endogenous CSF, and that these T cells express chemokine receptors allowing them to reach the CSF B cells expressing these idiotopes. models based on these assumptions suggest that nearly half of CSF BCR variable regions from MS patients harbor potential antigenic idiotopes (9). These models included prediction of HLA-DR affinities (25, 26), likelihood of endosomal processing by cysteine cathepsins (27, 28) and modeling of tolerance likelihood based on T cell uncovered motifs (TCEM) (9, 29). Mouse monoclonal to S1 Tag. S1 Tag is an epitope Tag composed of a nineresidue peptide, NANNPDWDF, derived from the hepatitis B virus preS1 region. Epitope Tags consisting of short sequences recognized by wellcharacterizated antibodies have been widely used in the study of protein expression in various systems. It has previously been suggested that frequently occurring TCEM in variable regions (i.e., germline framework motifs) could be tolerogenic, while rare motifs [i.e., complementarity determining region (CDR) 3 or motifs resulting from mutations] potentially could be stimulatory to T cells (10, 29). Thymocytes could be exposed to frequent immunoglobulin heavy chain variable (IGHV) TCEM in the thymus by thymic B cells (30), or by dendritic cells sampling serum immunoglobulins (31, 32). The prediction models used to predict cathepsin cleavage, HLA affinity and TCEM of IGHV have been validated (25C27, 29), and for cathepsin cleavage also using monoclonal FAS-IN-1 antibodies (28). It has however not been verified whether this or any other model actually predicts a repertoire of idiotopes that actually have a corresponding T cell repertoire. As MS is usually a chronic inflammatory disease of the CNS, we expected that relevant blood T cells have a memory phenotype with capacity to migrate into the CNS. The aim of the present study was to examine whether MS patients do have a repertoire of CD4+ T cells that recognize endogenous idiotopes predicted as stimulatory methods can guide identification of T cell stimulatory idiotopes and allow future comparisons between patient groups to establish disease specificity. Methods Patients In this study, we investigated materials collected previously from nine relapsing-remitting MS (RRMS) patients from whom we have immunosequenced the CSF IGHV repertoire (9), and from whom we had collected peripheral blood mononuclear cells (PBMC) in parallel with the FAS-IN-1 CSF cells. Demographic and disease characteristics are described in Supplementary Table 1. The nine patients had on average 1,079 (= 1,213) FAS-IN-1 translated IGHV sequences, which comprised 30C45 amino acids covering part of the framework region 3 (FW3), the entire CDR3 and a part of FW4 (dataset available at http://doi.org/10.6084/m9.figshare.5035703). No material was available to perform renewed sequencing of the full IGHV and/or light chain regions. All participants provided written informed consent before participating. Parameters for Predicting FAS-IN-1 Antigenic Properties of IGHV Idiotopes We utilized.