Allografts stimulate cross-reactive virus-specific memory CD8 T cells with private specificity

Abstract

Viral infections have been associated with the rejection of transplanted allografts in humans and mice, and the induction of tolerance to allogeneic tissues in mice is abrogated by an ongoing viral infection and inhibited in virus-immune mice. One proposed mechanism for this 'heterologous immunity' is the induction of alloreactive T cell responses that cross-react with virus-derived antigens. These cross-reactive CD8 T cells are generated during acute viral infection and survive into memory, but their ability to partake in the immune response to allografts in vivo is not known. We show here that cross-reactive, virus-specific memory CD8 T cells from mice infected with LCMV proliferated in response to allografts. CD8 T cells specific to several LCMV epitopes proliferated in response to alloantigens, with the magnitude and hierarchy of epitope-specific responses varying with the private specificities of the host memory T cell repertoire, as shown by adoptive transfer studies. Last, we show that purified LCMV-specific CD8 T cells rejected skin allografts in SCID mice. These findings therefore implicate a potential role for heterologous immunity in virus-induced allograft rejection.

Figure 1. Naive and memory phenotype alloreactive CD8 T cells are detectable in LCMV-immune mice

Splenocytes from LCMV-immune B6 mice (A, B and D) or naïve B6 mice (C) were stimulated with peptides or with splenocytes (allogeneic, irradiated LPS-treated cells) that were either syngeneic (H2^b) or allogeneic (BALB/c, H2^d or CBA, H2^k), as described in Materials and Methods. After culture, splenocytes were stained for cell surface markers and for intracellular IFN–γ and TNF. For analysis, samples were gated on CD8⁺ cells, and the values shown represent the percentage of CD8 T cells staining positive for either cytokine. The data are representative of 4 experiments.

Figure 2. H2^d-antigens stimulate LCMV-specific memory CD8 T cells to proliferate in vitro

CFSE-labeled splenocytes from LCMV-immune B6 mice were cultured in vitro for 6 days with P815 cells (H2^d) as described in Materials and Methods. (A) After the in vitro culture, CD8 T cells were evaluated for division by dilution of CFSE, and the values shown represent the percentage of CD8 T cells that are CFSE^lo. (B) Cultured cells were incubated with either syngeneic (H2^b) or allogeneic (H2^d) splenocytes or with a mAb specific for CD3 for 5 hr and then evaluated for the production of IFN–γ̃ (C) Alternatively, cultured cells were incubated with the indicated peptides for 5 hr and then evaluated for the production of IFN–γ. For the intracellular cytokine assays, samples were gated on CD8⁺ cells, and the values shown represent the percentage of either CFSE^lo or CFSE^hi CD8 T cells staining positive for IFN–γ. The data are representative of 5 experiments.

Figure 3. H2^d-skin allografts stimulate LCMV-specific memory CD8 T cells to proliferate in vivo

CFSE-labeled splenocytes (3 × 10⁷) from LCMV-immune B6 mice were adoptively transferred into naïve congenic recipient mice, which were engrafted with either syngeneic (B6, H2^b) skin (A and C) or BALB/c (H2^d) skin (B and D) 1 day later, as described in the Materials and Methods. (A and B) Thirteen days after engraftment, donor CD8 T cells (CD45.2) from the spleens of recipient mice were evaluated for division by dilution of CFSE, and the values represent the percentage of CD8 T cells that are CFSE^lo. Recovered splenocytes were incubated with either syngeneic (H2^b) or allogeneic (H2^d) splenocytes for 5 hr and then evaluated for the production of IFN–γ. (C and D) Alternatively, recovered splenocytes were incubated with the indicated peptides for 5 hr and then evaluated for the production of IFN–γ. For analysis, samples were gated on CD8⁺ cells, and the values shown represent the percentage of either CFSE^lo or CFSE^hi CD8 T cells staining positive for IFN–γ. The data are representative of 4 experiments.

Figure 4. LCMV-immune CD8 T cells from a single immune mouse exhibit similar responses to H2^d-skin allografts in separate recipients

CFSE-labeled splenocytes (3 × 10⁷) from individual LCMV-immune B6 mice (A, B and C) were adoptively transferred into separate congenic recipient mice, which were engrafted with BALB/c (H2^d) skin 1 day later, as described in the Materials and Methods. Thirteen days after engraftment, donor CD8 T cells (CD45.2) from the spleens of recipient mice were incubated with the indicated peptides for 5 hr and then evaluated for the production of IFN–γ. For analysis, samples were gated on CD8⁺ cells, and the values shown represent the percentage of either CFSE^lo or CFSE^hi CD8 T cells staining positive for IFN–γ. The data are representative of 3 experiments.

Figure 5. LCMV-specific CD8 T cells mediate rejection of skin allografts

B6/SJL mice (CD45.1) were infected with LCMV and 8 days later spleens were harvested and pooled. (A) Splenocytes were stained with NP396-MHC tetramers and with mAb to CD8, and double positive cells were purified by sorting. The purity of NP396-tetramer positive cells is shown as the percentage of total cells and as the percentage of CD8 T cells (values in parentheses). (B) Purified NP396-specific CD8 T cells (1–2×10⁶ cells) were then adoptively transferred into B6/SCID mice bearing BALB/c skin grafts. (C) Skin graft survival was monitored over the next 100 days. The data are representative of 2 experiments.