Type I interferons are not critical for skin allograft rejection or the generation of donor-specific CD8+ memory T cells

Abstract

Type I interferons (IFN-I) link innate to adaptive immunity in microbial infection, autoimmune disease and tumor immunity. It is not known whether IFN-I have an equally central role in alloimmunity. Here we tested this possibility by studying skin allograft survival and donor-specific CD8+ T-cell responses in mice that lack the IFN-I receptor (IFN-IR-/-). We found that IFN-IR-/- mice reject fully allogeneic wild-type skin grafts at the same rate as wild-type recipients. Similarly, allograft rejection was not delayed if IFN-IR-/- male skin was transplanted to syngeneic IFN-IR-/- female mice. Quantitation of the male (H-Y)-specific CD8+ T-cell response in these mice revealed normal generation of donor-specific CD8+ effector T cells but fourfold reduction in CD8+ memory T cells. Memory CD8+ T cells generated in the absence of IFN-IR had normal phenotype and recall function, assessed by ex vivo cytokine production and the ability of IFN-IR-/- mice to mount second set rejection. Finally, these memory T cells were maintained at a constant number despite their inability to respond to IFN-1. Our findings indicate that IFN-I cytokines are not critical for acute allograft rejection or for the expansion and differentiation of donor-specific CD8+ T cells into long-lived, functional memory T cells.

Figure 1. IFN-I are not required for acute skin allograft rejection

(a) Skin was harvested from BALB/c (H-2^d) mice and grafted onto wt (n = 16) or IFN-1R^−/− (n = 24) 129S6 (H-2^b) mice. Both recipient groups rejected skin grafts with similar kinetics (MST = 15.5 and 18 days, respectively). Control syngeneic skin grafts were not rejected (data not shown). (b) Skin was harvested from wt or IFN-IR^−/− 129S6 male mice and grafted onto wt (n = 33) or IFNI-R^−/− 129S6 female mice (n = 33), respectively. Both recipient groups rejected skin grafts with identical kinetics (MST = 17 days).

Figure 2. Generation of donor-specific CD8⁺ memory T cells in the absence of IFN-I function

Skin grafts were transplanted from wt or IFN-IR^−/− male 129S6 mice onto wt or IFN-IR^−/− female 129S6 mice, respectively. Recipient spleen and lymph nodes were harvested at multiple time points and analyzed for H-Y-specific CD8⁺ T cells by staining with the Uty tetramer. (a) H-Y-specific T cells (CD44^hiUty⁺) were identified after gating on CD8⁺ cells. Plot shown is from an IFN-IR^−/− recipient spleen at 90 days after transplantation. (b) CD8⁺CD44^hiUty⁺ T cells were quantitated in recipient spleen and lymph nodes at the indicated time points (n = 2 – 6 mice/time point). H-Y-specific CD8⁺ T cells expanded similarly in both wt and IFN-IR^−/− recipients (~100 fold), but their number declined four-fold after day 40 in the IFN-IR^−/− group. No difference in the memory maintenance phase was observed between the two groups. (c) wt and IFN-IR^−/− H-Y-specific CD8⁺ memory T cells displayed both central and effector memory phenotypes. Plot shown is from recipient spleens at 90 days after transplantation. Values shown in gates are percentages ± SD (n = 5, p > 0.05).

Figure 3. CD8⁺ memory T cell recall in the absence of IFN-I function

(a) Spleen cells were harvested 90 days after transplanting wt or IFN-IR^−/− 129S6 male skin onto wt or IFN-IR^−/− 129S6 female recipients, respectively, and re-stimulated ex vivo with either male (allogeneic) or female (syngeneic) 129S6 splenocytes for 5 hrs to measure memory recall function. IFNγ and TNFα production were assessed by intracellular staining after gating on CD8⁺Uty⁺ cells. Values shown in allogeneic gates are percentages ± SD (n = 4 – 5, p > 0.05). (b) Second set rejection was measured > 4 weeks after first set rejection in either fully allogeneic (left panel, n = 6 – 8 mice/group) or H-Y mismatched (right panel, n = 9 – 10 mice/group) donor-recipient pairs. Accelerated second set rejection was observed in both wt and IFN-IR^−/− groups.