Reduced self-reactivity of an autoreactive T cell after activation with cross-reactive non-self-ligand

Abstract

Autoreactive CD4(+) T lymphocytes are critical to the induction of autoimmune disease, but because of the degenerate nature of T cell receptor (TCR) activation such receptors also respond to other ligands. Interaction of autoreactive T cells with other non-self-ligands has been shown to activate and expand self-reactive cells and induce autoimmunity. To understand the effect on the autoreactivity of naive cross-reactive T cells of activation with a potent nonself ligand, we have generated a TCR transgenic mouse which expresses a TCR with a broad cross-reactivity to a number of ligands including self-antigen. The activation of naive transgenic recombination activating gene (Rag)2(-)(/)(-) T cells with a potent non-self-ligand did not result in a enhancement of reactivity to self, but made these T cells nonresponsive to the self-ligand and anti-CD3, although they retained a degree of responsiveness to the non-self-ligand. These desensitized cells had many characteristics of anergic T cells. Interleukin (IL)-2 production was selectively reduced compared with interferon (IFN)-gamma. p21(ras) activity was reduced and p38 mitogen-activated protein kinase (MAPK) was relatively spared, consistent with known biochemical characteristics of anergy. Surprisingly, calcium fluxes were also affected and the anergic phenotype could not be reversed by exogenous IL-2. Therefore, activation with a hyperstimulating non-self-ligand changes functional specificity of an autoreactive T cell without altering the TCR. This mechanism may preserve the useful reactivity of peripheral T cells to foreign antigen while eliminating responses to self.

Figure 1.

Phenotype of naive ex-vivo transgenic 1B6 T cells. The 1B6 TCR transgenic T cells retain the same hierarchy of response to peptide ligands as the Q1.1B6 T cell clone. LNCs from a TCR transgenic mouse were stimulated with different peptides and proliferation was assessed by measuring the incorporation of [³H]thymidine added after 48 h of culture.

Figure 2.

Culture with the superagonist peptide L144 affects the functional response of transgenic 1B6 T cells. Lymph node CD4⁺ T cells from 1B6 TCR transgenic Rag2 ^{− / −} mice were stimulated three times in vitro with 50 μg/ml Q144, 50 μg/ml L144, or 0.005 μg/ml L144. 2 wk after the third stimulation they were incubated with SJL/J splenocytes as APCs and the indicated concentrations of (A) L144, Q144, W144 peptides, or anti-CD3 and APCs, or (B) PMA and ionomycin (c1 = 100 ng/ml PMA, 2 μg/ml ionomycin; subsequent concentrations diluted 1:5) shown compared with activation by anti-CD3 (c1 = 10 μg/ml, subsequent concentrations diluted 1:5). Proliferation was assessed by pulsing the cultures with [³H]thymidine after 48 h.

Figure 3.

Selective loss of IL-2 but not IFN-γ production from desensitized 1B6 T cells. CD4⁺ 1B6 Rag2 ^{− / −} T cells were cultured as described in Fig. 1 and activated with L144 peptide and APC or with PMA/ionomycin. Supernatants were harvested after 40 h and analyzed for IL-2 and IFN-γ concentration by ELISA. Activation with antigen, but not PMA/ionomycin fails to induce IL-2 secretion from the desensitized cells.

Figure 4.

AICD and receptor/coreceptor expression are comparable. (A) Naive CD4⁺ 1B6 Rag2 ^{− / −} T cells were cultured with different antigens for 48 h and then assessed for AICD by annexin and 7-AAD staining. The fraction of CD4⁺ cells undergoing AICD was similar in all antigen-treated groups. (B) CD4⁺ 1B6 Rag2 ^{− / −} T cell lines were assessed for expression of antigen receptor and CD4 before they were used in the assays shown in Figs. 1 and 2. The level of receptor expression was equivalent in the different populations.

Figure 5.

1B6 T cells are hyporesponsive but not deleted in vivo after immunization with L144. Naive female SJL mice were reconstituted with transgenic 1B6 T cells or LNCs from littermate controls and immunized with L144 (50 μg/ml) or Q144 (50 μg/ml). 10 d after immunization, LNCs were assessed for the number of surviving 1B6-clonotype–positive CD4⁺ cells by flow cytometry (A) and for their functional response by proliferation (B). 1B6 T cells expanded well in mice immunized with L144, but were hyporesponsive compared with cells obtained from mice immunized with Q144 when they were tested in a proliferation assay ex vivo.

Figure 6.

Impaired activity of p21^ras, p44,42 MAPK, and JNK in the desensitized cells. (A) T cells of the responsive (cultured with L144 0.005 μg/ml) and desensitized (cultured with L144 50 μg/ml) cell lines were stimulated by cross-linking anti-CD3. At the indicated time points the cells were lysed and the active, GTP-bound form of p21^ras was immunoprecipitated with Raf-1 RBD agarose beads. The immunoprecipitated proteins were analyzed by SDS-PAGE and Western blot for p21^ras. As a control for equal content of total p21^ras protein in the different samples before immunoprecipitation, an aliquot of the cell lysate (corresponding to 2 × 10⁶ cells) was separately analyzed by Western blot for p21^ras (bottom half of the panel). (B) T cells from the responsive and desensitized cell lines were stimulated by cross-linking anti-CD3 as described for panel A. At the indicated time points the cells were lysed and aliquots of the cell lysates (corresponding to 2 × 10⁶ cells) were analyzed by SDS-PAGE and Western blot with phosphorylation-state–specific mAbs for the phosphorylated forms of p44,42 MAPK, JNK, and p38 MAPK. To control for equal loading, parallel Western Blots with mAbs for each kinase are shown below the respective blots for the phospho-form.

Figure 7.

Impairment in sustained calcium influx in the desensitized cells. T cells of the responsive and desensitized cell lines were loaded with the indicator dye Indo-1-AM. Calcium mobilization of the cells was monitored continuously by flow cytometry upon stimulation of the T cells with ionomycin, cross-linking anti-CD3, or with I-A^s/B7 expressing DAS cells, that had been loaded with the indicated concentrations of L144 peptide (start of stimulation indicated by arrow). Calcium mobilization in antibody and antigen stimulated cells was measured and compared with activation with ionomycin, by comparing the number of gated events above a baseline in the total time after stimulation (converted to arbitrary units and taking flux induced with ionomycin as 1.0).

Figure 8.

Impaired tyrosine phosphorylation of TCRζ, CD3ɛ, and ZAP-70 in desensitized cells. (A) T cells of the responsive and desensitized cell lines (2 × 10⁷ cells for each time point) were stimulated with I-A^s/B7 expressing DAS cells (loaded with 100 μg/ml of L144 peptide) as APCs. At the indicated times, the cells were lysed and the lysates subjected to immunoprecipitation with anti-CD3ɛ mAb. The immunoprecipitated proteins were analyzed by Western blot with an anti-phosphotyrosine–specific mAb. The relative ratio of p23 and p21 were measured as indicated by image analysis using the Scion Image package. We were unable to determine the total loading of CD3ɛ by stripping and reprobing. (B) The responsive and tolerant cells were activated with L144 peptide and I-A^s/B7 expressing DAS cells. After lysis and immunoprecipitation with anti-ZAP-70 antibody, Western blotting with anti-phosphotyrosine-specific mAb was performed and showed reduced ZAP-70 phosphorylation in the desensitized cells. After stripping the blot was reprobed with anti-ZAP-70 to control for loading.