Destabilization of peptide:MHC interaction induces IL-2 resistant anergy in diabetogenic T cells

Abstract

Autoreactive T cells are responsible for inducing several autoimmune diseases, including type 1 diabetes. We have developed a strategy to induce unresponsiveness in these cells by destabilizing the peptide:MHC ligand recognized by the T cell receptor. By introducing amino acid substitutions into the immunogenic peptide at residues that bind to the MHC, the half life of the peptide:MHC complex is severely reduced, thereby resulting in abortive T cell activation and anergy. By treating a monoclonal diabetogenic T cell population with an MHC variant peptide, the cells are rendered unresponsive to the wild type ligand, as measured by both proliferation and IL-2 production. Stimulation of T cells with MHC variant peptides results in minimal Erk1/2 phosphorylation or cell division. Variant peptide stimulation effectively initiates a signaling program dominated by sustained tyrosine phosphatase activity, including elevated SHP-1 activity. These negative signaling events result in an anergic phenotype in which the T cells are not competent to signal through the IL-2 receptor, as evidenced by a lack of phospho-Stat5 upregulation and proliferation, despite high expression of the IL-2 receptor. This unique negative signaling profile provides a novel means to shut down the anti-self response.

Keywords: I-A(g7); Signaling; T cell anergy; Type 1 diabetes.

Figure 1. BDC2.5 T cells do not proliferate in response to variant peptide

A. Sequence of the mimotope (parent) and variant peptides. B. BDC2.5 splenocytes were stimulated with a range of concentrations of both parent and variant peptides. Proliferation was assessed by [³H] thymidine incorporation as described in the methods. Data are representative of at least three independent experiments. Points represent means +/− SEM. All variant peptides differ significantly from the parent mimotope (p<0.05) as determined by One-way ANOVA with Dunnett post test.

Figure 2. Variant peptide treatment induces anergy

A. BDC2.5 cells were activated for 14d with 1µM mimotope, restimulated with either mimotope or variant (YPDV) for an additional 14d and subsequently challenged with the indicated concentrations of mimotope peptide. Proliferation was assessed by [³H] thymidine incorporation. Data represents average of four independent experiments performed in duplicate. Points indicate mean values +/− SEM. p=0.0015 as determined by Student’s t test. B. Anergy was induced as in part A. Mimotope or variant treated BDC2.5 T cells were restimulated with 10µM mimotope peptide. Supernatants were harvested after 24h, and IL-2 production was determined by ELISA. Samples were assessed in triplicate and data are representative of three independent experiments. Error bars indicate SEM. P=0.0310 by Student’s t test.

Figure 3. MHC anchor substitutions decrease half life of peptide: MHC complexes

Decay curves (A) and half life (B) of mimotope and variant peptide as determined by HPSEC. Decay curves are representative of five independent repeats. Half life values represent the average of five replicates. Y-axis values represent the natural log of the normalized peak height at each time point divided by the baseline peak height. Half life values are calculated as the natural log 2 of the slope of the decay curve. p=0.0067 for differences in half life as determined by Student’s t test.

Figure 4. Variant stimulation induces no detectable Erk1/2 phosphorylation

A. BDC2.5 splenocytes were stimulated with mimotope peptide for 14d. Live cells were stimulated with an I-A^g7 expressing cell line and either 10µM mimotope (black bars, top row) or 10µM variant (YPDV, white bars, bottom row) for the indicated times. Cells were fixed, permeabilized and Erk1/2 phosphorylation was determined by flow cytometry. Y-axis represents percent of CD4+ T cells that are pErk1/2+. Right side displays representative plots of pErk1/2 vs. SSC at time=0 (no stimulation), 30 min (peak), and 5h (post-peak). Flow plots are gated on CD4+ lymphocytes. Data compiled from three independent experiments, p=0.0031 by Student’s t test, error bars represent SEM. B. c-Jun phosphorylation. Cells were stimulated as in A. Y-axis represents percent of CD4+ T cells that are p-c-Jun+. Right panel displays representative histograms of c-Jun phosphorylation, with shaded histograms representing mimotope stimulated cells and open histograms representing variant stimulation for the indicated time points. Data compiled from two independent experiments, p=0.0007 by Student’s t test, error bars represent SEM.

Figure 5. Variant stimulated cells exhibit increased total tyrosine phosphatase and specific SHP-1 phosphatase activity

A. BDC2.5 T cells were stimulated as in Figure 4. p-nitrophenylphosphate solution was added to whole lysates and allowed to develop overnight at 37°C. Colorimetric change was assessed at 405nm. Data graphed are the averages of three independent experiments and represent mean +/− SEM. p=0.0086 by Student’s t test. B. Cells were stimulated as in Figure 4. After cell lysis, SHP-1 was immunoprecipitated and incubated with a pTyr containing substrate peptide. Malachite green was added and amount of free phosphate was determined by colorimetric change at 620nm. Data graphed are representative of three independent experiments.

Figure 6. Addition of exogenous IL-2 does not rescue proliferation of anergized T cells

BDC2.5 T cells were stimulated for 14d with 1µM mimotope peptide. Live cells were restimulated for an additional 14d with either 1µM mimotope of 10µM variant peptide plus irradiated antigen presenting cells. Live cells were restimulated with irradiated antigen presenting cells and indicated doses of mimotope without the addition of IL-2 (Mimotope vs. variant, p=0.05) or with IL-2 (mimotope+IL-2 vs. variant+IL-2 p=0.001). Data represent mean +/− SEM, and p values were calculated by a one way ANOVA with Bonferroni post test.

Figure 7. MHC variant peptide anergized cells are resistant to signaling through the IL-2 receptor

A. Expression of CD25 is slightly elevated in variant anergized cells (open histogram) as compared to mimotope-cultured cells (filled, dark grey histogram). Light grey tinted histogram represents no stain control. B. 30 minutes following stimulation with 100ng/ml recombinant IL-2 mimotope cultured cells exhibit significant Stat5 phosphorylation (open histogram) compared with unstimulated cells (filled histogram). Anergized cells stimulated under the same conditions exhibit no Stat5 phosphorylation above background (open histogram: IL-2 stimulated; filled histogram: no stimulation). Data are representative of two independent experiments. C. Average of percent maximum pStat5 geometric mean fluorescence intensity at various times following IL-2 stimulation. Data represents average of two independent experiments. Points indicate averages of percent maximum fluorescence intensity +/− SEM. * p<0.02.