Analysis of T-cell alloantigen response via a direct pathway in kidney transplant recipients with donor-specific antibodies

Abstract

Donor-specific antibodies (DSAs) are the main cause of graft loss over time. The direct pathway of alloantigen recognition is important in the pathogenesis of acute rejection. Recent studies have suggested that the direct pathway also contributes to the pathogenesis of chronic injury. Nevertheless, there are no reports on T-cell alloantigen response via the direct pathway in kidney recipients with DSAs. We analyzed the T-cell alloantigen response via the direct pathway in kidney recipients with DSAs (DSA+) or without DSAs (DSA-). A mixed lymphocyte reaction assay was implemented to assess the direct pathway response. DSA+ patients showed significantly higher CD8⁺ and CD4⁺ T cell responses to donor cells than DSA- patients. Furthermore, proliferating CD4⁺ T cells showed a marked increase in Th1 and Th17 responses in DSA+ patients than in DSA- patients. In a comparison between anti-donor and third-party responses, the anti-donor CD8⁺ and CD4⁺ T cell response was significantly lower than the anti-third-party response. In contrast, the donor-specific hyporesponsiveness was absent in DSA+ patients. Our study demonstrated that DSA+ recipients have a greater potential for developing immune responses against the donor tissues via the direct alloantigen recognition pathway. These data contribute to an understanding of DSAs pathogenicity during kidney transplantation.

Keywords: direct alloantigen recognition pathway; donor-specific antibody; immune monitoring; kidney transplantation; mixed lymphocyte reaction.

Figure 1

Lymphocyte subsets in patients without donor-specific antibodies (DSA -) and with DSA (DSA+). The number of (A) lymphocytes, (B) T cells (CD3⁺), (C) B cells (CD19⁺), (D) NK cells (CD3^-CD56⁺), and (E) NKT cells (CD3⁺CD56⁺). No significant differences in the number of lymphocytes, T, B, NK, and NKT cells were observed in the peripheral blood between DSA- and DSA+ patients. Data are shown as mean ± SEM. An unpaired t-test was performed.

Figure 2

T and B cell subsets in patients without donor-specific antibodies (DSA-) and with DSA (DSA+). The number of T and B cell subsets was analyzed in the peripheral blood between DSA - and DSA + patients. There were no significant differences in T and B cell subsets between DSA- and DSA+ patients. Data are shown as the mean ± SEM. An unpaired t-test was performed.

Figure 3

Aq1Anti-donor CD8⁺ and CD4⁺ T cell response in patients without donor-specific antibodies (DSA-) and with DSA (DSA+). The CD3⁺ cells isolated from DSA- and DSA+ patients were labeled with CFSE and were cultured with irradiated donor peripheral blood mononuclear cells for 5 days. The cultured cells were stained for CD4 and CD8. Representative flow cytometric data (A: CD8, C: CD4) and cell proliferation rate (B: CD8, D: CD4) are shown. Anti-CD8⁺ and CD4⁺ T-cell hyper-responses were observed in DSA+ patients, and they were significantly higher than those observed in DSA- patients, Data are presented as mean ± SEM. An unpaired t-test was performed. *p<0.01, **p<0.001.

Figure 4

Anti-donor CD4⁺ T cell subset response in patients without donor-specific antibodies negative (DSA-) and with DSA (DSA+). T cells isolated from DSA- and DSA+ patients were labeled with CFSE and were cultured with irradiated donor peripheral blood mononuclear cells for 5 days. The cultured cells were stained for CD4, IFN-γ, IL-4, IL-17 and FOXP3. Representative flow cytometric data (A) and cell proliferation rate (B: CD4⁺IFN- γ ⁺, C: CD4⁺IL-4⁺, D: CD4⁺IL-17⁺, E: CD4⁺FOXP3⁺) are shown. Proliferating CD4⁺ cells showed a marked increase in Th1 (CD4⁺IFN- γ⁺) and Th17 (CD4⁺IL-17⁺) response in DSA+ compared with DSA- those in patients (B, D). However, there were no differences in donor-reactive Th2 (CD4⁺IL-4⁺) or Treg (CD4⁺FOXP3⁺) cells between the groups (C, E). Data are presented as the mean ± SEM. An unpaired t-test was performed. *p<0.01, **p<0.0001.

Figure 5

Analysis of anti-donor CD4⁺ T-cell response in patients with donor-specific antibodies without rejection (DSA+ without rejection). Anti-donor CD4⁺ T cell response was comparable between DSA+ without rejection and DSA- patients (A). However, a significantly higher proportion of the proliferating CD4⁺ T cells showed a Th1 (CD4⁺IFN- γ⁺) and Th17 (CD4⁺IL-17⁺) phenotype in the DSA+ patients without rejection than in DSA- patients (B, C). Data are shown as the mean ± SEM. An unpaired t-test was performed. *p<0.01.

Figure 6

Comparison between anti-donor and third-party response in patients without donor-specific antibodies (DSA-). The isolated T cells from DSA- patients were labeled with CFSE and were cultured with irradiated donor or third-party peripheral blood mononuclear cells for 5 days. The cultured cells were stained for CD8 and CD4. Representative flow cytometric data (A: CD8, D: CD4) and proliferated cells rate (B: CD8, E: CD4) are shown. The response at 1-4, 5-9, and more than 10 (10 years <) years after transplantation is shown (C: CD8, F: CD4). Anti-donor CD8⁺ and CD4⁺ T-cell responses were significantly lower than the anti-third-party response (B, E). This donor-specific T-cell hyporesponsiveness persisted for 10 years after transplantation (C, F). Data are presented as the mean ± SEM. A paired t-test was performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 7

Comparison between anti-donor and third-party responses in patients with donor-specific antibodies (DSA+). T cells isolated from (DSA+) patients were labeled with CFSE and were cultured with irradiated donor or third-party peripheral blood mononuclear cells for 5 days. The cultured cells were stained for CD8 and CD4. Representative flow cytometric data (A: CD8, C: CD4) and proliferated cell rate (B: CD8, D: CD4) are shown. Anti-donor CD8⁺ T-cell response was significantly lower than the third-party response (B). However, CD4⁺ T-cell proliferation was comparable in terms of donor and third-party responses (D). Data are presented as mean ± SEM. A paired t-test was performed. *p<0.05.