University of Minnesota Minneapolis, MN, United States
Shawn Mahmud, Thamotharampillai Dileepan, Bryce Binstadt and Marc Jenkins, University of Minnesota, Minneapolis, MN
Background/Purpose: Nearly all patients with mixed connective tissue disease (MCTD) have IgG autoantibodies (autoAb) specific for U170k, a component of the U1-snNRP spliceosomal complex. A genetic link to the MHC II molecule HLA-DRB1*0401 (DR4) and formation of IgG autoAb strongly suggest the importance of CD4+ T cells in MCTD pathogenesis. Defining specific, self-reactive CD4+ T cell populations in MCTD could help drive progress towards specific therapies.
Methods: We used a peptide:MHCII binding prediction algorithm (NetMHCIIpan3.2) to identify U170k peptides likely to bind DR4. We generated fluorophore-labeled HLA-DR4 tetramers loaded with these peptides and subsequently used them to stain and magnetically enrich U170k:DR4-specific CD4+ T cells from naïve and immunized DR4 transgenic (DR4-Tg) mice and from DR4+ healthy human donors.
Results: Three peptides from the RNA binding domain of U170k were predicted to bind DR4: QGDAFKTLFVARVNY, FKTLFVARVNYDTTE, PIKRIHMVYSKRSGK (nonamer cores underlined). Magnetic tetramer enrichment with tetramers loaded with these peptides allowed detection of rare, naïve U170k:DR4-specific CD4+ T cells in HLA-DR4 transgenic mice (DR4-Tg) mice. These cells expanded 5-10 fold and upregulated CD44 after immunization with U170k peptides in Complete Freund's Adjuvant followed by a boost in Incomplete Freund's Adjuvant 30-40 days later (Fig. 1). A single peptide-CFA immunization was insufficient to induce activation of U170k:DR4-specific CD4+ T cells (i.e., a secondary boost with IFA is required; data not shown). Most U170k:DR4-specific CD4+ T cells became T follicular helper (Tfh) cells or T helper type 1 (Th1) cells. Notably, pretreatment of mice with anti-CD20 IgG2c prior to boosting with peptides abrogated the ability to form U170k:DR4-specific Tfh and Tregs (Fig. 2). In healthy human HLA-DR4+ donors, these novel reagents also detected rare, naïve (i.e., not activated) U170k:DR4-specific CD4+ T cells (Fig. 3).
Conclusion: These data demonstrate that novel DR4 tetramers can detect rare, naïve CD4+ T cells specific for U170k:DR4 in DR4-Tg mice and in healthy human DR4+ subjects. U170k:DR4-specific CD4+ T cells expand 5-10-fold and become activated in mice after immunization, chiefly differentiating into Tfh and Th1 cells. Anti-CD20 IgG2c treatment blocked the ability to form U170k:DR4-specific Tfh and Treg cells, suggesting B cells play a role in the activation and differentiation of these antigen-specific CD4+ T cells. We are now using these tetramers in an established DR4-Tg MCTD mouse model that recapitulates some features of human MCTD, specifically development of anti-U170k autoAb and interstitial lung disease (ILD). We aim to determine how the balance of U170k:DR4-specific effector and regulatory CD4+ T cell populations influences autoantibody development and ILD pathogenesis. Moreover, we are preparing to study pediatric and adult patients with anti-U170k antibodies to determine if there are increased numbers of activated U170k:DR4-specific CD4+ T cells in active disease, whether they have altered effector and regulatory phenotypes, and whether this correlates specific disease features such as arthritis, ILD, and/or response to therapy. Figure 1. U170k:DR4-specific CD4+ T cells expand and become activated in DR4-Tg mice immunized with U170k peptides. Adult DR4-Tg mice (n=4 per group, both sexes) were subcutaneously immunized with the predicted DR4-binding U170k peptides emulsified in CFA. A boost with the same peptides in IFA was given at 40 days. Nine days later, APC and PE conjugated DR4 tetramers containing the relevant peptides were used to stain single cell suspensions from lymph nodes and spleen prior to anti-APC and -PE magnetic bead enrichment, surface staining, and flow cytometry. The top panel shows APC- and PE- conjugated tetramer binding cells in representative sham and U170k peptide immunized mice within CD8+ and CD4+ fractions (left). CD44 expression is shown for CD4+Tetramer+ and total CD4+ cells (right). Numbers of tetramer binding cells from individual mice and the proportion of tetramer positive cells expressing CD44 were quantified in the graphs below. Dots represent individual mice and error bars indicate SEM. Statistical significance was determined with Mann-Whitney tests using a cutoff of P < 0.05.
Figure 2. U170k:DR4-specific CD4+ T cells primarily adopt Tfh and Th1 fates. Adult DR4-Tg mice (n=3 per group) were subcutaneously immunized with U170k peptide first in CFA followed by a boost in IFA 30-40 days later. In some mice, anti-CD20 IgG2c (“RTX”) was used to deplete B cells prior to the boost. APC-conjugated U170k:DR4 tetramers were used to magnetically enrich antigen-specific CD4+ T cells prior to surface/intracellular staining and flow cytometry. The top panel shows representative flow cytometry plots from control, peptide-immunized, and peptide-immunized mice that were treated with RTX. Bar graphs in the lower panels show quantitative data with error bars representing SEM. Statistical significance was determined with ANOVA.
Figure 3. Rare U170k:DR4-specific CD4+ T cells are detectable in healthy human blood. Commercially available, de-identified peripheral blood mononuclear cells were isolated from donors expressing at least one copy of DR4. PBMC were incubated with U1-70k:DR4 tetramers, magnetically separated, and stained with antibodies to CD3, CD4, CD8, CD45RA, CCR7, CD25, and CD127. Naïve CD4+ T cells were identified by high expression of CCR7 and CD45RA. Tregs were identified as CD25+ and CD127-low. The top panel shows flow cytometry results from a representative donor. Cumulative results from four donors are in the quantitative figures below. Disclosures: S. Mahmud, None; T. Dileepan, None; B. Binstadt, Pfizer, Pfizer; M. Jenkins, None.