Donor-reactive CD8 memory T cells infiltrate cardiac allografts within 24-h posttransplant in naive recipients

Abstract

Normal immune responses stimulated by pathogenic and environmental antigens generate memory T cells that react with donor antigens and no currently used immunosuppressive drug completely inhibits memory T-cell function. While donor-reactive memory T cells clearly compromise graft outcomes, mechanisms utilized by memory T cells to promote rejection are largely unknown. In this study, we investigated how early endogenous memory cells infiltrate and express effector function in cardiac allografts. Endogenous CD8 memory T cells in nonsensitized recipients distinguish syngeneic versus allogeneic cardiac allografts within 24 h of reperfusion. CD8-dependent production of IFN-gamma and CXCL9/Mig was observed 24 to 72 h posttransplant in allografts but not isografts. CXCL9 was produced by donor cells in response to IFN-gamma made by recipient CD8 T cells reactive to donor class I major histocompatibility complex (MHC) molecules. Activated CD8 T cells were detected in allografts at least 3 days before donor-specific effector T cells producing IFN-gamma were detected in the recipient spleen. Early inflammation mediated by donor-reactive CD8 memory T cells greatly enhanced primed effector T-cell infiltration into allografts. These results suggest that strategies for optimal inhibition of alloimmunity should include neutralization of infiltrating CD8 memory T cells within a very narrow window after transplantation.

Figure 1

Early induction of IFN-γ and CXCL9 in allografts is CD8 T cell dependent. A. C57BL/6 mice received syngeneic or complete MHC mismatched A/J cardiac allografts. Groups of 4 iso- (—) and allografts ( - - ) were harvested 24, 48, and 72 hours post-transplant, RNA was isolated from total graft homogenates, and relative real-time PCR was used to measure cytokine and chemokine expression. Data are normalized relative to isograft expression levels at 24 hours post-transplant. ( = p ≤ 0.05) B. Wild-type C57BL/6, B6.CD4^−/−, B6.CD8^−/− or B6.Rag1^−/− mice received syngeneic or A/J cardiac allografts. Groups of 5 grafts were harvested 72 hours post-transplant and intragraft levels of CXCL9 were measured by ELISA. ( = p ≤ 0.0001, = p ≤ 0.002)

Figure 2

Early CXCL9 production requires IFN-γ produced by class I MHC alloreactive CD8 T cells. A. L^d-reactive 2C TCR-transgenic CD8 cells or IFN-γ^−/− 2C cells were adoptively transferred into Rag1^−/− mice. After 10 weeks BALB/C (L^d-expressing) or DBA/1 (L^d null) hearts were transplanted into groups (n = 4) of reconstituted Rag1^−/− recipients and graft CXCL9 proteins levels were measured by ELISA 72 hours post-transplant. ( = p ≤ 0.05 vs. BALB/C ≤ Rag1^−/− + 2C) B. (C57BL/10xDBA/1)F1 mice received syngeneic, class II MHC-disparate bq1, or complete MHC-disparate C3H cardiac allografts. Protein levels of CXCL9 were measured in groups of 5 grafts harvested 72 hours after transplantation. ( = p ≤ 0.005 vs. complete MHC disparity)

Figure 3

Donor cells are the major source of CXCL9 produced in the graft early post-transplant. Wild-type, B6.CXCL9^−/−, or B6.IFN-γR^−/− mice received wild-type syngeneic, wild-type A/J, or A/J.CXCL9^−/− cardiac allografts. Protein levels of CXCL9 were measured in groups of 5 grafts harvested 72 hours after transplantation. ( = p ≤ 0.01)

Figure 4

Activated CD8 T cells infiltrate allografts before donor-specific T cell priming is detected in the spleen. A. Flow cytometry was used to detect C57BL/6 CD8 T cells infiltrating A/J cardiac allografts 24, 48, and 72 hours post-transplant. Representative data from 3 independent experiments are shown. B. Cardiac grafts and spleens were removed from C57BL/6 recipients of A/J allografts 48 hours after transplantation. Equal numbers of splenic and graft-infiltrating CD8 cells were isolated by flow sorting and stained for further analysis. Representative data are shown. C. Spleens were harvested from non-transplanted mice or C57BL/6 recipients of A/J cardiac allografts on days 1–5 post-transplant. Splenocytes were mixed with A/J (■) or third party DBA/1 (□) stimulator cells in 24-hour IFN-γ ELISPOT assays. (n = 3/group, = p ≤ 0.05, = p ≤ 0.005)

Figure 5

Adoptive transfer of CD8⁺CD44^hi memory cells restores early CXCL9 production in CD8^−/− allograft recipients. A/J skin grafts were placed on C57BL/6 mice and 8–10 weeks later naïve (CD8⁺CD44^lo) and memory (CD8⁺CD44^hi) cell populations were flow-sort purified from spleens and 5 × 10⁶ cells were adoptively transferred to B6.CD8^−/− mice which then received either A/J or DBA/1 cardiac allografts. For adoptive transfer of primed T cells, CD8 T cells were isolated from spleens of C57BL/6 mice actively rejecting A/J cardiac allografts at day 7 post-transplant. A. Flow cytometric detection of adoptively transferred CD8 T cells infiltrating allografts in CD8^−/− recipients 72 hours post-transplant. Representative data are shown. B. Quantification of the adoptively transferred CD8 T cell populations within spleens and allografts 72 hours post-transplant (n = 4/group, = p ≤ 0.05). C. Quantification of IFN-γ and CXCL9 mRNA within allografts 72 hours post-transplant and normalized to expression levels in the no-transfer control group (n = 4/group, = p ≤ 0.05). D. Using the experimental design described above, 2 × 10⁵ central memory (CD8⁺CD44^hiCD62L^hi) or effector memory (CD8+CD44^hiCD62L^lo) cells were flow-sort purified and adoptively transferred into CD8^−/− recipients of A/J cardiac allografts. Quantification of graft-infiltration by the adoptively transferred CD8 T cells 72 hours after transplantation is shown (n = 4/group).

Figure 6

Early CD8 T cell alloreactivity is not inhibited by α-CD40L mAb or CTLA4-Ig. C57BL/6 recipients of A/J cardiac allografts were treated with control Ig or α-CD40L mAb (0.4 mg on day 0, +1), CTLA4-Ig (0.35 mg on day 0, +1, +2), or α-IFN-γ (0.2 mg 8 hours before and after transplantation). Protein levels of CXCL9 were measured in grafts harvested 72 hours after transplantation (n ≥ 3 group and the experiment was performed twice with identical results, = p ≤ 0.05 vs. IgG).

Figure 7

Early CD8 memory T cell alloreactivity enhances recruitment of primed effector T cells to the allograft. Wild-type C57BL/6 mice were treated with control Ig or CD8 depleting antibodies (0.2 mg on days −3, −2, −1, +1). On day 0 mice received A/J cardiac allografts. On day +2, 8 × 10⁶ purified CD90.1 CD4⁺ cells from spleens of wild-type CD90.1 C57BL/6 mice rejecting A/J cardiac allografts on day 7 post-transplant were adoptively transferred and 48 hours later allografts were harvested and graft-infiltrating CD90.1 CD4⁺ T cells were detected using flow cytometry. A. Representative data illustrating the gating strategy used to identify the graft-infiltrating CD4⁺ CD90.1 test population. B. Quantification of the total number of graft-infiltrating CD4⁺ CD90.1 cells (n = 6/group, = p ≤ 0.05). C–D. Relative mRNA quantification of IFN-γ and CXCL9 in grafts harvested from IgG and γ-CD8 treated recipients 4 days after transplantation and 48 hours after adoptive transfer. Data are normalized to a sample randomly chosen from the γ-CD8 treated group (n = 6/group, = p ≤ 0.05).