Differential requirement of CD27 costimulatory signaling for naïve versus alloantigen-primed effector/memory CD8+ T cells

Abstract

CD8(+) memory T cells endanger allograft survival by causing acute and chronic rejection and prevent tolerance induction. We explored the role of CD27:CD70 T-cell costimulatory pathway in alloreactive CD8(+)/CD4(+) T-cell activation. CD27-deficient (CD27(-/-)) and wild-type (WT) B6 mice rejected BALB/c cardiac allografts at similar tempo, with or without depletion of CD4(+) or CD8(+) T cells, suggesting that CD27 is not essential during primary T-cell alloimmune responses. To dissect the role of CD27 in primed effector and memory alloreactive T cells, CD27(-/-) or WT mice were challenged with BALB/c hearts either 10 or 40 days after sensitization with donor-type skin grafts. Compared to WT controls, allograft survival was prolonged in day 40- but not day 10-sensitized CD27(-/-) recipients. Improved allograft survival was accompanied by diminished secondary responsiveness of memory CD8(+) T cells, which resulted from deficiency in memory formation rather than their lack of secondary expansion. Chronic allograft vasculopathy and fibrosis were diminished in CD27(-/-) recipients of class I- but not class II-mismatched hearts as compared to WT controls. These data establish a novel role for CD27 as an important costimulatory molecule for alloreactive CD8(+) memory T cells in acute and chronic allograft rejection.

Figure 1. BALB/c cardiac allograft survival in CD27^−/− and wild-type (WT) B6 recipients

(A) Both CD27^−/− (n = 5) and WT recipients (n = 5) rejected allografts at a similar tempo. (B) Depletion of CD4⁺ cells in both CD27^−/− (n = 5) and WT recipients (n = 5) significantly prolonged allograft survival, but there was no difference in allograft survival between the two groups. (C) Depletion of CD8⁺ cells had no effect on allograft survival in both CD27^−/− (n = 5) and WT recipients (n = 5).

Figure 2. Frequency of IFN-γ(Th1)- and IL-4 (Th2)-producing alloreactive T cells, as assessed by ELISPOT (A, B) and percentage of alloreactive CD4⁺ and CD8⁺ effector-memory cells, as measured by flow cytometry, in CD27^−/− and wild-type (WT) B6 recipients 7 days after transplantation of BALB/c hearts

(A, B) The frequency of alloreactive IFN-γ- and IL-4-producing splenocytes was significantly increased in CD27^−/− recipients compared to WT recipients. (C, D) While the frequency of alloreactive CD4⁺ effector-memory cells did not differ between CD27^−/− and WT recipients, the frequency of CD8⁺ effector-memory T cells was significantly higher in CD27^−/− recipients compared to WT recipients. Data and density plots are representative of two independent experiments (n = 3/3 each).

Figure 3. BALB/c cardiac allograft survival in CD27^−/− and wild-type (WT) B6 recipients presensitized with donor-type skin transplantation 40 days prior to cardiac transplantation

(A) Allografts survived significantly longer in CD27^−/− recipients (n = 5) compared to WT recipients (n = 5). (B) Improved allo-graft survival in CD27^−/− recipients was preserved and further enhanced after depletion of CD4⁺ T cells (n = 5). (C) Allograft survival did not differ between CD27^−/− (n = 5) and WT (n = 5) recipients after CD8⁺ T-cell depletion.

Figure 4. Donor-specific antibodies (DSA) production in day 40 skin-sensitized CD27^−/− and wild-type B6 recipients 7 days after cardiac transplantation, assessed by flow cytometry

(A) CD27 deficiency did not affect production of IgG1-type DSA. (B) CD27 deficiency did not affect production of IgG2a-type DSA. Data are shown as percentage of IgG1- or IgG2a-loaded splenocytes, and are representative of two independent experiments (n = 3/3 each).

Figure 5. Frequency of IFN-γ(Th1)- and IL-4 (Th2)-producing alloreactive T cells in day 40 skin-sensitized CD27^−/− and wild-type (WT) B6 recipients prior to and 4 and 7 days after cardiac transplantation, assessed by ELISPOT

(A) The frequency of alloreactive IFN-γ-producing splenocytes was significantly decreased in CD27^−/− recipients compared to WT recipients on day 40 after skin sensitization and 4 and 7 days after challenge with donor-type cardiac transplantation. (B) The frequency of alloreactive IL-4-producing splenocytes did not differ between CD27^−/− and WT recipients at any of above time points. (C) The frequency of IFN-γ-producing CD4⁺ T cells purified from splenocytes was lower in CD27^−/− animals than in WT controls on day 40 after skin transplantation; however, the decline in frequency of IFN-γ-producing CD8⁺ memory T cells purified from same splenocytes was markedly more enhanced in CD27^−/− mice as compared to WT controls. (D) The frequency of donor-specific IL-4-producing cells among purified CD4⁺ or CD8⁺ T cells derived from day 40-sensitized splenocytes did not differ between CD27^−/− and WT mice. Data are representative of three independent experiments (n = 3/3 each) and indicate the mean of quintuplicate results in each experiment. *p < 0.05; ***p < 0.001.

Figure 6. Frequency of alloreactive CD4⁺ and CD8⁺ effector-memory T cells in day 40-sensitized CD27^−/− and wild-type (WT) recipients prior to and 4 and 7 days after the cardiac transplantation

(A) and (B) The frequency of CD8⁺ effector-memory T cells was significantly decreased in CD27^−/− recipients compared to WT recipients at any time point examined. (C) and (D) The frequency of CD4⁺ effector-memory T cells did not significantly differ between CD27^−/− and WT recipients over the studied time course. The density plots shown are representative of at least three separate experiments (n = 3/3 each). *p < 0.05; ***p < 0.001.

Figure 7. Histological findings of single MHC class I (bm1)- or class II (bm12)-mismatched allografts in B6 CD27^−/− and wild-type (WT) recipients

(A) Elastica-van Gieson’s staining of bm1 allografts shows essentially normal vessels in CD27^−/−recipients compared to advanced arteriosclerosis in WT recipients (× 200). (B) Masson’s trichrome stainings of bm1 allografts show only mild epicardial fibrosis in CD27^−/− recipients, but severe epicardial and in-tramyocardial fibrosis in WT recipients. (C) Vasculopathy score. Bm1 but not bm12 allografts display a lower degree of graft arteriosclerosis in CD27^−/− recipients compared to WT recipients. (D) Fibrosis rate. Bm1 but not bm12 allografts display a lower degree of fibrosis in CD27^−/−recipients compared to WT recipients.

Figure 8. Frequencies of alloreactive IFN-γ-producing splenocytes and CD8⁺ memory T cells in CD27^−/− and wild-type (WT) B6 recipients of bm1 allografts

(A) The frequency of alloreactive IFN-γ-producing splenocytes was significantly decreased in CD27^−/−recipients compared to WT recipients. (B) Alloreactive CD8⁺ T cells are the main source of IFN-γproduction. Frequency of CD8⁺ IFN-γ-producing splenocytes is significantly decreased in CD27^−/− animals. (C) The frequency of CD8⁺ effector-memory and CD8⁺ central-memory cells as well as the overall CD8⁺ memory T-cell pool was significantly decreased in CD27^−/− animals when compared to WT animals. Data are representative of two independent experiments (n = 3/3 each). ELISPOT data are shown as the mean of quintuplicate results in each experiment. *p < 0.05; **p < 0.01.

Figure 9. Frequencies of alloreactive IFN-γ-producing splenocytes and CD4⁺ memory T cells in CD27^−/− and wild-type (WT) B6 recipients of bm12 allografts

(A) The frequency of alloreactive IFN-γ-producing splenocytes was similar in CD27^−/− and WT recipients. (B) Alloreactive CD4⁺ T cells are the main source of IFN-γproduction. Frequency of CD4⁺ IFN-γ-producing splenocytes are similar in CD27^−/− and WT animals. (C) The frequency of CD4⁺ effector-memory cells is similar in CD27^−/− and WT animals. Data are representative of two independent experiments (n = 3/3 each). ELISPOT data are shown as the mean of quintuplicate results in each experiment.