Self-peptides prolong survival in murine autoimmunity via reduced IL-2/IL-7-mediated STAT5 signaling, CD8 coreceptor, and V alpha 2 down-regulation

Abstract

Although the pathogenic role of B cells and CD4 T cells has been studied extensively, less is known about the role of CD8 T cells in autoimmunity and self-tolerance. To evaluate the role of CD8 T cells in autoimmunity and its modulation using self-peptides, we used mice expressing soluble OVA (sOVA) under control of the keratin-14 promoter. Spontaneous autoimmunity occurred when sOVA mice were crossed with OT-I mice, whose CD8 T cells carry a Valpha2/Vbeta5-transgenic TCR with specificity for the OVA(257-264) peptide. Eighty-three percent of OVA/OT-I mice died during the first 2 wk of life due to multiple organ inflammation. In contrast, preventive or therapeutic OVA(257-264) peptide injections induced a dose-dependent increase in survival. Healthy survivors exhibited reductions in peripheral CD8 T cells, CD8 coreceptor, and Valpha2 expression. Furthermore, CD8 T cells from healthy mice were anergic and could not be activated by exogenous IL-2. A block in IL-2/IL-7 signaling via the STAT5 pathway provided the basis for low surface expression of the CD8 coreceptor and failure of IL-2 to break CD8 T cell anergy. Thus, the soluble TCR ligand triggered multiple tolerance mechanisms in these sOVA/OT-I mice, making this treatment approach a potential paradigm for modulating human autoimmune diseases.

Figure 1

sOVA mice express OVA mRNA in stratified epithelia and are susceptible to OT-I cells. (A) The expression of the OVA transgene in indicated tissues of sOVA mice and C57BL/6 controls was quantified by real-time PCR. Error is given as standard deviation of triplicate measurements. (B) The presence of sOVA protein in tongue epithelial lysates was confirmed by Western blot using rabbit anti-chicken ovalbumin IgG. M: marker, 1: C57BL/6 tongue, 2: sOVA tongue, 3: non-transfected 293 T cells, 4: OVA-transfected 293 T cells. (C) Dose-response titration of disease induction by i.v. transfer of OVA-specific OT-I cells. Adoptive transfer of at least 1×10⁵ CD8 T cells led to weight loss and death of sOVA mice. 1×10⁶ OT-I cells did not induce disease in C57BL/6 mice. Numbers in brackets depict the fraction of surviving mice at given time points. One representative experiment out of 3 is displayed (N=5/group). (D) H&E sections (20×) obtained 4 days after adoptive transfer of 5 × 10⁶ OT-I cells, showing extensive lymphocytic infiltration in tongue and esophageal sections from sOVA mice but not in C57BL/6 controls.

Figure 2

83% of sOVA/OT-I mice die from CD8-mediated inflammation within 3 weeks of life. (A) Survival curve of sOVA/OT-I mice (N=28) and OT-I heterozygous littermates (N=32), showing one representative experiment out of 3. (B) Daily weight gain of sOVA/OT-I pups and OT-I littermates showing an arrest of weight gain on day 9, which precedes death within the next days. (C) H&E sections (20×) obtained from the same mice as in Figure 2B showing inflammatory infiltrates in tongue, esophagus, skin, liver and lung of sOVA/OT-I (a-e) but not OT-I littermate controls (f-j). (D) In vivo depletion of CD8⁺ T cells with YTS mAB 169.4 CD8⁺ (50 μg/day, i.p.; N=25) on days 5, 7 and 9 of life extends the life of sOVA/OT-I mice by three more weeks, compared to PBS-treated solvent controls (N=14). (E) FACS analysis of skin draining lymph nodes from sOVA/OT-I mice revealed increasing percentages of CD8 T cells correlating with advancing disease stages (0 healthy, 1 sluggish weight gain, 2 hunched appearance, 3 moribund). P < 0.05. (F) FACS analysis of CD8 T cells in skin draining lymph nodes of sick sOVA/OT-I pups showing increased expression of the activation markers CD25 and CD69 together with a downregulation of CD62L.

Figure 3

Self-peptide treatment delays death of sOVA/OT-I mice. (A) Survival curve of sOVA/OT-I mice injected with OVAp showing a dose dependent survival benefit induced by repeated OVAp injections on day 5 and 9 of life, while 100 μg VSV NP52-59 control peptide led inevitably to death. Displayed is one representative experiment out of 3 (18 mice/ group). (B) Survival curve of sick pups (reduced weight gain and motility, hunched appearance) injected on day 10 after birth. 100 μg of OVAp rescued 60% of sick pups by day 21 and 40% by day 42 (N=21), while lower doses of 10 μg (injected on day 9 of life, N=12) and solvent (PBS, day 10 of life) had no beneficial effect (N=18). (C) 10 day old sOVA/OT-I pups have a 20% lower body weight. N=34 mice/group. Data is given as mean +/- SEM (P=0.003).

Figure 4

CD8 T cells in healthy sOVA/OT-I mice are functionally disabled proximally at the T cell receptor with CD8-coreceptor downregulation and lack of Vα2-chain expression. (A) FACS analysis of CD8-coreceptor and Vα2 expression in skin draining lymph nodes in sOVA/OT-I mice from different disease states and OT-I littermates. CD8 T cells of healthy spontaneous or healthy peptide-treated survivors exhibited either reduced expression of the CD8-coreceptor or the TCR Vα2-chain. In contrast, CD8 T cells in acutely sick pups and OT-I littermates showed strong CD8 and Vα2-expression. (B) Pregating on CD8⁺ cells for comparison of the Vα2/Vβ5 expression pattern revealed that only Vα2, but not Vβ5 was downregulated in sOVA/OT-I mice. (C) CFSE dilution assay showing a reduced proliferative response of Vα2⁺ /Vβ5⁺ CD8 T cells from skin draining lymph nodes in healthy spontaneous survivors, while CD8 T cells from sick pups and OT-I controls proliferated vigorously. In contrast, bypass of antigen-specific T cell receptor ligation with anti-CD3 or complete bypass of the TCR using PMA/ionomycin induced proliferation also by tolerized OT-I cells from the healthy sOVA/OT-I survivors.

Figure 5

The T cell receptor of CD8 cells from healthy spontaneous- and peptide treated survivors exhibited reduced ligand sensitivity. Flow cytometric analysis of TCR-peptide sensitivity using OVA_257-264-pentamers for staining of CD8 T cells from skin draining lymph nodes. Pregating on CD8+ Vβ5+ OT-I cells showed reduced OVA_257-264-pentamer binding by CD8 T cells from sOVA-OT-I survivors (spontaneous and peptide-treated) compared to naive OT-I mice or sick pups.

Figure 6

No restoration of CD8 and reduced STAT5 signaling in response to IL-2- or IL-7 in healthy spontaneous survivors. (A/B) CD8 expression was analyzed on CD8+ Vα2+ T cells after 16h incubation ex vivo with indicated concentrations of IL-7 (A) or IL-2 (B). One representative experiment out of 5 is depicted. (C/D) STAT5-phosphorylation in CD8⁺ cells was analyzed after 30 minutes of ex vivo stimulation with indicated concentrations of IL-7 (C) or IL-2 (D). One representative experiment out of 3 is depicted.

Figure 7

CD8 T cells from healthy 1× treated sOVA/OT-I survivors (100μg OVAp on day 10 of life) show similar characteristics as spontaneous survivors or prophylactically treated sOVA/OT-I mice. Total cell counts of Vα2⁺ or Vα2^- CD8 T cells in skin draining lymph nodes are diminished in surviving sOVA/OT-I mice treated with a single dose of 100μg OVA on day 10 (A). FACS analysis of skin draining lymph nodes revealed a reduction of CD8-coreceptor (B) and the TCR Vα2-chain (pregated on CD8⁺ cells, 7C). CD8 expression was analyzed on CD8⁺ Vα2⁺ T cells after 16h incubation ex vivo with indicated concentrations of IL-7 (D) or IL-2 (E)