Viral and bacterial infections interfere with peripheral tolerance induction and activate CD8+ T cells to cause immunopathology

Abstract

We studied the impact of various infectious and proinflammatory agents on the induction of peripheral T cell tolerance. Adoptive transfer of CD8+ T cells from lymphocytic choriomeningitis virus (LCMV) T cell receptor transgenic mice into LCMV antigen transgenic mice expressing the LCMV glycoprotein epitope (gp) 33-41 under control of a major histocompatibility complex class I promoter led to efficient induction of peripheral tolerance after a period of transient activation. If, however, the recipient mice were challenged with viral or bacterial infections or proinflammatory agents (lipopolysaccharide or Poly:IC) early after cell transfer, tolerance induction was prevented and instead, CD8+ T cell activation leading to vigorous expansion and generation of cytolytic activity ensued. This became manifest in significant immunopathology mainly involving destruction of the splenic architecture and lysis of antigen-expressing lymphocyte and macrophage populations. Important parameters involved in the activation of host-reactive T cells by nonspecific infectious agents included the presence, localization, and quantity of the specific transgene-encoded self-antigen; in contrast, CD4+ T cells were not required. In mice surviving the acute phase, the transferred CD8+ T cells persisted at high levels in an anergic state; they were unable to generate cytolytic activity in vitro or to control LCMV infection in vivo. These results impinge on our understanding of the role of infectious agents in graft verus host reactions towards minor histocompatibility antigens.

Figure 1

Functional presentation of gp 33–41 in H8 mice. (a) The transgene construct: a fragment of the LCMV glycoprotein sequence including the gp 33–41 CTL epitope was cloned into the genomic sequence of H-2K^b. (b and c) Functional expression of gp 33–41 in peripheral lymphoid tissue. (b) Spleen cells from day 8 immune C57BL/6 mice were used as effector cells against Con A blast target cells obtained from H8 or C57BL/6 mice; as a control, C57BL/6 Con A blast target cells pulsed with gp 33–41 were used. Spontaneous release was <28%. (c) H8-, C57BL/6-, and gp 33–41-pulsed C57BL/6 spleen cells were used as stimulators for TCR318 responder spleen cells in a [³H]thymidin incorporation assay. (d) Functional gp 33 expression on fibroblasts. Embryonal fibroblasts from H8 and C57BL/6 mice were left unmanipulated or were pulsed with gp 33–41 and used as targets for effector cells obtained from LCMV-WE–infected C57BL/6 mice in a 5-h ⁵¹Cr–release assay; spontaneous release was <30%.

Figure 2

Peripheral tolerance induction after transient activation of transferred gp 33–specific CD8⁺ T cells in H8 mice. (a–d) H8 and littermate contol mice were transfused with 5 × 10⁷ CFSE-labeled spleen cells from RAG1-deficient mice transgenic for a gp 33–41-specific T cell receptor (TCR×RAG). 40 h after transfer, mesenteric lymph node cells were analyzed by flow cytometry. CFSE staining of CD8⁺ cells from littermate (B6; a) or H8 recipient mice (b) is shown. CFSE⁺ CD8⁺ lymph node cells from both recipients were compared for their expression of CD62L (c) and CD44 (d). (e) Spleen cells of H8 mice transfused with 5 × 10⁷ spleen cells from TCR318 mice were analyzed at the indicated times after transfer for gp 33–specific cytotoxicity on EL-4 target cells in a 15-h ⁵¹Cr–release assay. Spleen cells from C57BL/6 mice immunized with LCMV-WE 50 d earlier (B6 memo) were used as control. Spontaneous release was <25%. (   f   ) H8 mice and littermate control (B6) mice were transfused with the indicated number of spleen cells from TCR318 mice. The percentage of CD8⁺ PBLs expressing the transgenic Vα and Vβ chains was monitored at the indicated time points before and after challenge infection with 200 PFU LCMV.

Figure 3

Virus-induced host-specific CD8⁺ T cells mediate GVH-like immunopathology. H8 and littermate control mice were transfused with 10⁶ spleen cells from TCR318 mice and infected with 200 PFU LCMV 1 d later. At different time points after infection, tissue sections of spleen (d35), gut, and liver (d50, separate experiment) were stained with hematoxilin–eosin (HE) or with Abs of the desired specificity. B220, CD45R⁺ B cells; CD4, CD4⁺ T cells (YTS-191); CD8, CD8⁺ T cells (YTS-169); MOMA, marginal metallophilic macrophages; 4C11, FDCs.

Figure 4

Kinetics of splenic immunopathology mediated by virus-induced host-specific CD8⁺ T cells. H8 and littermate control (B6) mice were transfused with 10⁶ spleen cells from TCR318 mice and infected with 200 PFU LCMV 1 d later. At the indicated time points after transfer, mice were killed and the absolute number of B cells per spleen was determined by flow cytometry.

Figure 5

Virus infection interferes with peripheral tolerance induction and activates host-specific CD8⁺ T cells. H8 and littermate control (B6) mice were transfused with 10⁶ spleen cells from TCR318 mice. 3 d later, the recipient mice were infected with 200 PFU LCMV-WE and the percentage of CD8⁺ PBLs expressing the transgenic Vα and Vβ chains was monitored by flow cytometric analysis (a). 8 d after challenge infection, spleen cells from some recipient mice were tested for gp 33–specific cytotoxicity on EL-4 target cells in a 5-h ⁵¹Cr–release assay; spontaneous release was <18% (b). Data from one of four similar experiments are shown.

Figure 6

Anergy of persisting virus-induced host-specific CD8⁺ T cells. (a–d) Phenotypical analysis of TCR318 T cells: H8 and littermate control (B6) mice were transfused with 10⁶ spleen cells from TCR318 mice and infected with 200 PFU LCMV-WE 3 d later. 35 d after infection, CD8⁺ spleen cells of recipient mice were analyzed for expression of the transgenic Vα and Vβ chains (a and b). Forward light scatter of Vα2⁺Vβ8⁺CD8⁺ TCR318 T cells (c) and CD49d expression of Vα2⁺ CD8⁺ T cells (d) were compared between H8 (bold lines) and littermate mice (dashed lines). (e–g) Functional analysis of TCR318 T cells. 35 d after LCMV infection, spleen cells of H8 and littermate control (B6) mice were (e) tested for gp 33–specific cytotoxicity after 5 d of restimulation on LCMV-infected macrophages in the presence of 5% Con A supernatant and (   f   ) used as responder cells in a [³H]thymidine incorporation assay stimulated by C57BL/6 spleen cells pulsed with gp 33–41. In experiment g, the LCMV virus titer was analyzed in the indicated organs of the recipient mice. Data from one of two to four similar experiments are shown.

Figure 7

Antigen presentation by bone marrow–derived versus epithelial cells for activation of host-specific CTLs. 6 wk after bone marrow reconstitution, H8–B6 and B6–H8 bone marrow chimeras were transfused with 10⁶ spleen cells from TCR318 mice. 3 d later, the recipient mice were infected with 200 PFU LCMV-WE and the percentage of CD8⁺ PBLs expressing the transgenic Vα and Vβ chains was monitored by flow cytometric analysis (a). 8 d after challenge infection, spleen cells from some recipient mice were tested for gp 33–specific cytotoxicity on EL-4 target cells in a 5-h ⁵¹Cr–release assay; spontaneous release was <18% (b). ^† Deaths.

Figure 8

Various infectious and inflammatory stimuli can interfere with tolerance induction and activate host-specific CD8⁺ T cells. H8 (closed symbols) and littermate control mice (open symbols) were transfused with 10⁷ spleen cells from TCR318 mice and 1 d later injected with 500 μg Poly:IC, 10 μg LPS, 2 × 10⁶ PFU VSV-Indiana, 2 × 10⁶ PFU vaccinia WR, 200 PFU LCMV-WE 8.7, or 200 PFU LCMV-WE. The percentage of CD8⁺ PBLs expressing the transgenic Vα and Vβ chains was monitored by flow cytometry (a, b, e, and f   ). 8 d after challenge infection, spleen cells from some recipient mice were tested for gp 33–specific cytotoxicity on EL-4 target cells in a 5-h ⁵¹Cr–release assay (c). 20 d after infection, mice were killed and the absolute number of B cells per spleen was determined by flow cytometry (d ). In experiment e, mice were depleted of CD4⁺ T cells 3 and 1 d before cell transfer. In experiment f, the number of transferred TCR318 spleen cells was varied as indicated and mice were infected either with LCMV-WE (closed symbols) or with LCMV-WE 8.7 (open symbols).