Background/Purpose: CD4+ T cells play a key role in autoantibody production by B cells. Autoantibodies targeting the 70kD subunit of U1-small nuclear ribonucleoprotein (U170) are found in mixed connective tissue disease (MCTD) patients and many with systemic lupus erythematosus (SLE). Identifying the specific autoreactive CD4+ T cells could provide insights into MCTD/SLE pathogenesis and provide opportunities for developing antigen-specific therapies. Here, we use tetramers of U170 peptides bound by HLA-DRB1*0401 (DR4) molecules, and separately, activation-induced marker (AIM) assays to detect and characterize U170-specific CD4+ T cells in mice and humans.

Methods: For tetramer enrichment, peptides from U170 predicted to bind DR4 were used to generate fluorophore-conjugated U170:DR4 tetramers.  DR4 transgenic mice were immunized with full length U170 and secondary lymphoid organs (SLO) harvested for U170:DR4 tetramer enrichment and phenotyping by flow cytometry. For AIM assays, SLOs from immunized mice were harvested and CD4+ T cells were magnetically separated and plated 1:1 with antigen-presenting cells derived from irradiated splenocytes from unimmunized mice. Cells were stimulated with full-length U170 or the U170 U1-RNA recognition motif (RRM) and incubated overnight prior to staining and flow cytometry. Human PBMC were obtained from healthy controls from commercial vendors and MCTD/SLE patients from the UMN Pediatric Rheumatology clinic. Human PBMC used in tetramer analysis were HLA typed for selection of DR4+ donors.

Results: In mice, U170:DR4 tetramers identified a clear population of activated, CD4+ T cells that expand in immunized animals (Fig. 1A) and adopted primarily T-helper type 1 (Th1) and T follicular helper (Tfh) fates (Fig 1C). Using separate AIM assays from immunized mice, we found a population of CD40L+ and OX40+ CD4+ T cells that emerged following antigen restimulation (Fig. 1B).  CD40L+ and OX40+ cells emerging after antigen restimulation with U170 were primarily Th1 and Tfh (Fig. 1C). In two human donors with well-controlled MCTD on medications, rare U170:DR4 tetramer binding CD4+ T cells were detected bearing expression of CD45RA but lacking CCR7, suggesting chronic antigen stimulation. (Fig 2A). AIM assays conducted on human cells from three donors with well controlled SLE on immune suppression showed variable detection of an CD40L+ and OX40+ subset in response to U170 stimulation (Fig 2B).

Conclusion: U170-specific CD4+ T cells are detectable in U170-immunized mice using U170:DR4 tetramers and also AIM assays, which produce similar results. Detection of U170-specific cells in human donors has thus far proven difficult on the basis of the low abundance of DR4+ donors in our patient population as well as the confounding effects of immune suppression. We are currently expanding our program by incorporating novel tetramers built with other MCTD and SLE-associated MHCII molecules and seeking to enroll MCTD/SLE donors who are treatment ‘naïve’. Our data identify peptide:MHCII tetramer enrichment and AIM assays as promising tools further probe how U170 autoreactivity relates to disease pathogenesis and responses to treatments in MCTD and SLE.

Figure 1: Autoreactive CD4+ T cells in mice detected using U170:DR4 tetramers and AIM assays. (A) SLOs from naïve (top row) and U170 immunized (bottom row) DR4-transgenic mice were processed and stained with U170:DR4 tetramers, magnetically enriched, and stained with surface antibodies prior to flow cytometry. (B) CD4+ T cells isolated from U170 immunized mice and restimulated in vitro with irradiated splenocytes and full-length U170, RRM, or staphylococcal enterotoxin B (SEB). After incubation, cells were stained with fluorophore-conjugated antibodies to detect CD40-L and OX40 upregulation. SEB and an unstimulated control were used as positive and negative controls, respectively. Cells are gated as CD4+, CD44-hi. (C) U170:DR4+ (from tetramer assays, left columns) and CD40-L/OX40+ cells (from AIM assays, right columns) were phenotyped with intracellular staining for the indicated lineage markers.

Figure 2: Detection of rare U170-specific CD4+ T cells in humans with U170:DR4 tetramers or AIM assays. PBMCs from DR4+ controls and MCTD/SLE patients were stained with U170:DR4 tetramers and fluorophore-conjugated antibodies. TEMRA frequency within U170:DR4+ cells is quantified and mean represented by line (A). (B) AIM assays with PBMCs from DR4- samples were stimulated in vitro with U170 protein and represented as fold change of OX40+ and CD40L+ cells over the average of healthy controls in the corresponding column graph. Upstream gates of all plots in AIM assays are CD4+, CD45RA-.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/profiling-u170-specific-self-reactive-cd4-t-cells-in-mctd-and-sle/