Allograft dendritic cell p40 homodimers activate donor-reactive memory CD8+ T cells
Hidetoshi Tsuda, Charles A. Su, Toshiaki Tanaka, Katayoun Ayasoufi, Booki Min, Anna Valujskikh, Robert L. Fairchild
Hidetoshi Tsuda, Charles A. Su, Toshiaki Tanaka, Katayoun Ayasoufi, Booki Min, Anna Valujskikh, Robert L. Fairchild
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Research Article Immunology Transplantation

Allograft dendritic cell p40 homodimers activate donor-reactive memory CD8+ T cells

Abstract

Recipient endogenous memory T cells with donor reactivity pose an important barrier to successful transplantation and costimulatory blockade–induced graft tolerance. Longer ischemic storage times prior to organ transplantation increase early posttransplant inflammation and negatively impact early graft function and long-term graft outcome. Little is known about the mechanisms enhancing endogenous memory T cell activation to mediate tissue injury within the increased inflammatory environment of allografts subjected to prolonged cold ischemic storage (CIS). Endogenous memory CD4+ and CD8+ T cell activation is markedly increased within complete MHC-mismatched cardiac allografts subjected to prolonged versus minimal CIS, and the memory CD8+ T cells directly mediate CTLA-4Ig–resistant allograft rejection. Memory CD8+ T cell activation within allografts subjected to prolonged CIS requires memory CD4+ T cell stimulation of graft DCs to produce p40 homodimers, but not IL-12 p40/p35 heterodimers. Targeting p40 abrogates memory CD8+ T cell proliferation within the allografts and their ability to mediate CTLA-4Ig–resistant allograft rejection. These findings indicate a critical role for memory CD4+ T cell–graft DC interactions to increase the intensity of endogenous memory CD8+ T cell activation needed to mediate rejection of higher-risk allografts subjected to increased CIS.

Authors

Hidetoshi Tsuda, Charles A. Su, Toshiaki Tanaka, Katayoun Ayasoufi, Booki Min, Anna Valujskikh, Robert L. Fairchild

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Figure 1

Heart allografts subjected to greater ischemic storage have increased accumulation of endogenous memory CD8+ T cells with donor reactivity at early times after transplant.

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Heart allografts subjected to greater ischemic storage have increased ac...
(A) Memory CD8+ T (CD8+CD44high) cells were purified from the spleens and lymph nodes of CD45.2 C57BL/6 (H-2b) mice 6 weeks after A/J (H-2a) skin transplants, and 2 × 106 aliquots of cells were transferred i.v. to groups of CD45.1 C57BL/6 (n = 5–8/group) mice. Three days later, the CD45.1 C57BL/6 mice received A/J cardiac allografts subjected to either 0.5 or 8 hours of CIS or DBA/1 (H-2q) heart allografts subjected to 8 hours of CIS. Cardiac allograft recipients were sacrificed 12–16 hours after transplant, the allografts were harvested and digested, and aliquots of single cell suspensions were stained with antibody and analyzed by flow cytometry, with examples of gating as shown for each allograft sample to assess and quantitate the infiltration of memory CD8+ memory T cells and the transferred CD45.2 memory CD8+CD44high T cells into the allografts. *P < 0.05, as determined by the Mann-Whitney nonparametric test. (B) A/J hearts subjected to 8 hours of CIS were transplanted to a group of four C57BL/6 mice, and on day 3 after transplant, the graft-infiltrating memory CD8+CD44high T cells were purified, labeled with CFSE, and cultured in media or in a 1:4 mixture with spleen cells from C57BL/6 (Iso), A/J (Allo), and/or DBA/1 (3rd party) mice. After 96 hours, the cultured cells were collected and washed, and the dilution of CFSE by the CD8+ T cells was analyzed by flow cytometry. *P < 0.05, ***P < 0.001, as determined by 1-way ANOVA with Bonferroni’s multiple comparison post-test. (C) A/J hearts subjected to 0.5 or 8 hours of CIS were transplanted to groups of C57BL/6 mice (n = 4–6/group). BrdU was injected i.p. on days 0 and 1. The allografts were harvested after 24, 48, or 72 hours and digested, and aliquots of single cell suspensions were stained with antibody and analyzed by flow cytometry, with examples of gating as shown for each allograft sample. (D) The total number of gated infiltrating memory CD4+ and CD8+ T cells and their incorporation of BrdU was quantitated. *P < 0.05, ** P < 0.01, as determined by the Mann-Whitney nonparametric test.