Background/Purpose: Mixed connective tissue disease (MCTD) is a systemic autoimmune disorder marked by a U1-small nuclear ribonucleoprotein (U1-snRNP) autoantibody. The majority of U1-snRNP antibodies in MCTD target the 70 kD subunit of the protein complex (U170). How self-reactive T and B cells cooperate to drive autoantibody production in MCTD is unknown. Here, we use novel tetramers of U170 peptides bound to HLA-DRB1*0401 (U170:DR4) to detect self-reactive CD4+ T cells in a previously published mouse model of MCTD and diseased human patients. Our goals are to quantify and better characterize self-reactive CD4+ T cells in MCTD, which may eventually drive development of antigen-specific immunotherapy.

Methods: Because MCTD is linked to DR4, we use commercially available DR4 transgenic mice as previously published. We generated fluorophore-labeled DR4 tetramers with peptides bioinformatically predicted to bind DR4. We used these reagents to stain and magnetically enrich U170:DR4-specific CD4+ T cells from mice immunized with human U170 and separately from human peripheral blood mononuclear cells (PBMCs) from diseased patients. In mice, U170-specific antibodies and blood cells were quantified with a commercially available ELISA and flow cytometry, respectively.

Results: Our novel U170:DR4 tetramers identified mouse CD4+ T cells expanding and upregulating CD44 and differentiating into Th1, Th17, and Tfh (Fig. 1). U170-specific antibody production in DR4 Tg mice accelerates until six weeks and plateaus (Fig. 2). Mice also develop a mild thrombocytopenia 8-10 weeks after immunization. Lastly, U170:DR4-specific tetramers detected rare CD45RA low (memory) cells in a DR4 positive MCTD patient (Fig. 3).

Conclusion: DR4 Tg mice produce autoantibodies in response to U170 immunization, and cognate U170-specific CD44+ T cells were detected alongside them using novel tetramers. U170-specific cells in mice were Th1, Th17, and Tfh. Mild thrombocytopenia, which is seen in MCTD, was also present at late time points in mice. Preliminary human data indicate these novel tetramers will be useful tools in longitudinal studies of humans with MCTD undergoing therapy and for development of specific treatments, such as chimeric antigen receptor (CAR) regulatory T cells.

Expanding and activated mouse U170-specific CD4+ T cells primarily become Th1, Tfh, and Th17 cells in DR4-Tg mice immunized with recombinant U170 protein. DR4 Tg mice (both sexes, n=6 each group) were subcutaneously immunized with human recombinant U170 protein emulsified in CFA, and secondary lymphoid organs stained with U170:DR4 tetramers and surface markers as displayed in (a), and quantified in (b) (mean ±SEM, Mann-Whitney). (c) Analysis of phenotypes of bulk (top row) and tetramer+ cells (bottom row) with intracellular staining.

DR4-Tg mice immunized with U170 develop U170-specific antibodies and mild thrombocytopenia. (a) DR4-Tg mice (n=14 each group at 2-8 weeks, n=5 each group at 16 weeks) were immunized with U170 protein. U170-specific antibodies were measured over time with ELISA modified to detect mouse IgG (mean ±95% CI). (b) Flow cytometry-based enumeration of platelets, red blood cells (RBC), leukocytes (WBC, neutrophils, lymphocytes) (n=9, 8-10 weeks post-immunization, mean ±SEM, Mann-Whitney).

Detection of U170-specific T cells in healthy humans and MCTD patients. PBMCs were isolated and stained with U170:DR4 tetramers and surface markers as shown in (a) and quantified in (b) (n=3 healthy control, 1 patient; mean, Mann-Whitney).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/detecting-autoreactive-cd4-t-cells-relevant-to-mixed-connective-tissue-disease/