HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis.

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4+ T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4+ T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4+ T cells in MS.

Autoantigen-specific CD4+ T cells acquire an exhausted phenotype and persist in human antigen-specific autoimmune diseases.

Pro-inflammatory autoantigen-specific CD4+ T helper (auto-Th) cells are central orchestrators of autoimmune diseases (AIDs). We aimed to characterize these cells in human AIDs with defined autoantigens by combining human leukocyte antigen (HLA)-tetramer-based and activation-based multidimensional ex vivo analyses. In aquaporin4-antibody-positive neuromyelitis optica spectrum disorder (AQP4-NMOSD) patients, auto-Th cells expressed CD154, but proliferative capacity and pro-inflammatory cytokines were strongly reduced. Instead, exhaustion-associated co-inhibitory receptors were expressed together with FOXP3, the canonical regulatory T cell (Treg) transcription factor. Auto-Th cells responded in vitro to checkpoint inhibition and provided potent B cell help. Cells with the same exhaustion-like (ThEx) phenotype were identified in soluble liver antigen (SLA)-antibody-autoimmune hepatitis and BP180-antibody-positive bullous pemphigoid, AIDs of the liver and skin, respectively. While originally described in cancer and chronic infection, our data point to T cell exhaustion as a common mechanism of adaptation to chronic (self-)stimulation across AID types and link exhausted CD4+ T cells to humoral autoimmune responses, with implications for therapeutic targeting.

Microbial peptides activate tumour-infiltrating lymphocytes in glioblastoma.

Microbial organisms have key roles in numerous physiological processes in the human body and have recently been shown to modify the response to immune checkpoint inhibitors1,2. Here we aim to address the role of microbial organisms and their potential role in immune reactivity against glioblastoma. We demonstrate that HLA molecules of both glioblastoma tissues and tumour cell lines present bacteria-specific peptides. This finding prompted us to examine whether tumour-infiltrating lymphocytes (TILs) recognize tumour-derived bacterial peptides. Bacterial peptides eluted from HLA class II molecules are recognized by TILs, albeit very weakly. Using an unbiased antigen discovery approach to probe the specificity of a TIL CD4+ T cell clone, we show that it recognizes a broad spectrum of peptides from pathogenic bacteria, commensal gut microbiota and also glioblastoma-related tumour antigens. These peptides were also strongly stimulatory for bulk TILs and peripheral blood memory cells, which then respond to tumour-derived target peptides. Our data hint at how bacterial pathogens and bacterial gut microbiota can be involved in specific immune recognition of tumour antigens. The unbiased identification of microbial target antigens for TILs holds promise for future personalized tumour vaccination approaches.

Attenuated immune control of Epstein-Barr virus in humanized mice is associated with the multiple sclerosis risk factor HLA-DR15.

Immune responses to Epstein-Barr virus (EBV) infection synergize with the main genetic risk factor HLA-DRB1*15:01 (HLA-DR15) to increase the likelihood to develop the autoimmune disease multiple sclerosis (MS) at least sevenfold. In order to gain insights into this synergy, we investigated HLA-DR15 positive human immune compartments after reconstitution in immune-compromised mice (humanized mice) with and without EBV infection. We detected elevated activation of both CD4+ and CD8+ T cells in HLA-DR15 donor-reconstituted humanized mice at steady state, even when compared to immune compartments carrying HLA-DRB1*04:01 (HLA-DR4), which is associated with other autoimmune diseases. Increased CD8+ T cell expansion and activation was also observed in HLA-DR15 donor-reconstituted humanized mice after EBV infection. Despite this higher immune activation, EBV viral loads were less well controlled in the context of HLA-DR15. Indeed, HLA-DR15-restricted CD4+ T cell clones recognized EBV-transformed B cell lines less efficiently and demonstrated cross-reactivity toward allogeneic target cells and one MS autoantigen. These findings suggest that EBV as one of the main environmental risk factors and HLA-DR15 as the main genetic risk factor for MS synergize by priming hyperreactive T-cell compartments, which then control the viral infection less efficiently and contain cross-reactive CD4+ T cell clones.

miR-27a and miR-214 exert opposite regulatory roles in Th17 differentiation via mediating different signaling pathways in peripheral blood CD4+ T lymphocytes of patients with relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is one of the most prevalent autoimmune diseases, which involves the central nervous system. In this illness, Treg/Th17 cell imbalance causes the defect. Several studies revealed that T helper 17 (Th17) cells play a crucial role in pathogenesis, inflammation, and autoimmunity of several autoimmune diseases such as MS. In the present study, we assessed transcript levels of miR-27a and miR-214, in purified CD4+ T cells of MS patients, during relapsing and remitting phases in inducing differentiation of T naïve cells to Th17 cells. Forty RR-MS patient samples including those in relapsing (n=20) and remitting (n=20) phases were participated in this study. In addition, transcript levels of IL-17A, RORγt, IL-23R, Foxp3, and TGF-ß in purified CD4+ T cells of patients in relapsing and remitting phases of RRMS patients were compared to healthy controls. Expression levels of miR-27a and miR-214 were measured by RT-qPCR and compared to healthy control group (n=10). Data indicated upregulation of miR27a in relapsing phase of multiple sclerosis compared to remitting phase and healthy volunteers while miR-214 downregulated in relapsing phase of MS compared to remitting phase and healthy volunteers. In silico studies demonstrated pathways which miR-27a and miR-214 could effect on CD4+ T cell lineage fate including TGF-ß and mTOR signaling, respectively. Our data suggest that miR-27a may probably inhibit negative regulators of Th17 cell differentiation, thus promoting its differentiation while miR-214 has an adverse effect.

Dynamics of T cell repertoire renewal following autologous hematopoietic stem cell transplantation in multiple sclerosis.

Autologous hematopoietic stem cell transplantation (aHSCT) is a highly effective treatment of multiple sclerosis (MS). It depletes autoreactive cells and subsequently renews adaptive immune cells. The possible proinflammatory potential of surviving T cells early after aHSCT has not been studied. Here, we examined the dynamics of new and surviving T cells in 27 patients after aHSCT by multidimensional flow cytometry, T cell receptor (TCR) sequencing, specificity testing, telomere length profiling, and HLA genotyping. Early after aHSCT, naïve T cells are barely detectable, whereas effector memory (EM) T cells quickly reconstitute to pre-aHSCT values. EM CD4+ T cells early after aHSCT have shorter telomeres, have higher expression of senescence and exhaustion markers, and proliferate less than those before aHSCT. We find a median TCR repertoire overlap of 26% between the early post-aHSCT EM CD4+ T cells and pre-aHSCT, indicating persistence of EM CD4+ T cells early after transplantation. The EM CD4+ TCR repertoire overlap declines to 15% at 12 months after aHSCT, whereas the naïve TCR repertoire entirely renews. HLA-DR-associated EM CD4+ T cell reactivity toward MS-related antigens decreased after aHSCT, whereas reactivity toward EBV increased. Our data show substantial survival of pre-aHSCT EM CD4+ T cells early after transplantation but complete renewal of the T cell repertoire by nascent T cells later.

Vaccination with Designed Neopeptides Induces Intratumoral, Cross-reactive CD4+ T-cell Responses in Glioblastoma.

PURPOSE: The low mutational load of some cancers is considered one reason for the difficulty to develop effective tumor vaccines. To overcome this problem, we developed a strategy to design neopeptides through single amino acid mutations to enhance their immunogenicity. EXPERIMENTAL DESIGN: Exome and RNA sequencing as well as in silico HLA-binding predictions to autologous HLA molecules were used to identify candidate neopeptides. Subsequently, in silico HLA-anchor placements were used to deduce putative T-cell receptor (TCR) contacts of peptides. Single amino acids of TCR contacting residues were then mutated by amino acid replacements. Overall, 175 peptides were synthesized and sets of 25 each containing both peptides designed to bind to HLA class I and II molecules applied in the vaccination. Upon development of a tumor recurrence, the tumor-infiltrating lymphocytes (TIL) were characterized in detail both at the bulk and clonal level. RESULTS: The immune response of peripheral blood T cells to vaccine peptides, including natural peptides and designed neopeptides, gradually increased with repetitive vaccination, but remained low. In contrast, at the time of tumor recurrence, CD8+ TILs and CD4+ TILs responded to 45% and 100%, respectively, of the vaccine peptides. Furthermore, TIL-derived CD4+ T-cell clones showed strong responses and tumor cell lysis not only against the designed neopeptide but also against the unmutated natural peptides of the tumor. CONCLUSIONS: Turning tumor self-peptides into foreign antigens by introduction of designed mutations is a promising strategy to induce strong intratumoral CD4+ T-cell responses in a cold tumor like glioblastoma.

Integrative computational mRNA-miRNA interaction analyses of the autoimmune-deregulated miRNAs and well-known Th17 differentiation regulators: An attempt to discover new potential miRNAs involved in Th17 differentiation.

Th17 cells are a lineage of CD4(+) T helper cells in immune system which differentiate from naïve CD4(+) T cells and have demonstrated to play a critical role in the pathogenesis of different autoimmune disorders. miRNAs are a novel group of non-coding RNAs which participate in post-transcriptional regulation of gene expression mostly by pairing with 3'UTR of their mRNA targets and inhibition of its translation. It has been demonstrated that miRNAs function in various cellular processes such as differentiation, proliferation, and apoptosis. By now, several signaling pathways and their downstream positive and negative regulators involve in Th17 differentiation have been discovered. Several studies have reported the aberrant miRNA expression profile in patients with autoimmune disease called autoimmune-deregulated miRNAs. Here, using integrative miRwalk database which assembles the data gathered from ten different bioinformatics databases designed to predict miRNA-target interaction, we analyzed possible targeting effect of "autoimmune-deregulated miRNAs" on prominent positive and negative regulators of Th17 differentiation. Our resulting mRNA-miRNA network simply nominated several miRNAs with strong possibility which probably may have inducing (miR-27b, miR-27a, miR-30c, miR-1, and miR-141) or inhibitory (miR-20b, miR-93, miR-20a, miR-152, miR-21, and miR-106a) role in Th17 differentiation by targeting negative or positive regulators of Th17 differentiation, respectively.

miR-141 and miR-200a, Revelation of New Possible Players in Modulation of Th17/Treg Differentiation and Pathogenesis of Multiple Sclerosis.

BACKGROUND: One of the main issues in pathogenesis of MS is Th17/Treg imbalance. There are growing interests in nominating miRNAs involved in Th17 cell differentiation, suggesting them as new therapeutic agents that may reduce progression of different autoimmune diseases specially MS. OBJECTIVES: We assessed transcript levels of miR-141 and miR-200a in MS patients, during relapsing and remitting phases. We also investigated possible role of miR-141, miR-200a in inducing differentiation to Th17 cells. MATERIALS AND METHODS: Forty RR-MS patient samples including relapsing (n=20) and remitting (n=20) phases were chosen. Expression level of miR-141 and miR-200a were measured by RT-q PCR and compared to healthy control group (n=10). In-silico analyses on miR-141 and miR-200a targetome showed involvement of both miRNAs in T helper cell differentiation pathways including TGF-ß, mTOR and JAK/STAT. RESULTS: We observed that percentage of RORγt+ CD4+ T cells increase in relapsing phase while FOXP3+ CD4+ increase in remitting phase of MS patients. Furthermore, both miR-141 and miR-200a show up-regulation in relapsing phase of MS patients compared to remitting and control groups. Interestingly, expression level of target genes of miR-141 and miR-200a, which were assessed through in-silico methods, show down-regulation in relapsing phase of MS patients. CONCLUSIONS: According to our results, miR-141 and miR-200a may be key miRNAs in progression of symptoms of MS through inducing differentiation of Th17 cells and inhibiting differentiation to Treg cells. Our data suggest that these miRNAs may probably inhibit negative regulators of Th17 cell differentiation, thus promoting its differentiation.