Risk prediction of de novo donor specific antibody (DSA) would be very important for long term graft outcome after organ transplantation. The purpose of this study was to elucidate the association of eplet mismatches and predicted indirectly recognizable HLA epitopes (PIRCHE) scores with de novo DSA production. Our retrospective cohort study enrolled 691 living donor kidney transplantations. HLA-A, B, DRB and DQB eplet mismatches and PIRCHE scores (4 digit of HLA-A, B, DR, and DQ) were determined by HLA matchmaker (ver 2.1) and PIRCHE-II Matching Service, respectively. Weak correlation between eplet mismatches and PIRCHE scores was identified, although both measurements were associated with classical HLA mismatches. Class II (DRB+DQB) eplet mismatches were significantly correlated with the incidence of de novo class II (DR/DQ) DSA production [8/235 (3.4%) in eplet mismatch ≤ 13 vs. 92/456 (20.2%) in eplet mismatch ≥ 14, p < 0.001]. PIRCHE scores were also significantly correlated with de novo class II DSA production [26/318 (8.2%) in PIRCHE ≤ 175 vs. 74/373 (19.8%) in PIRCHE ≥ 176, p < 0.001]. Patients with low levels of both class II eplet mismatches and PIRCHE scores developed de novo class II DSA only in 4/179 (2.2%). Analysis of T cell and B cell epitopes can provide a beneficial information on the design of individualized immunosuppression regimens for prevention of de novo DSA production after kidney transplantation.

In pre-sensitizing events, immunological memory is mainly created via indirect allorecognition where CD4+ T cells recognize foreign peptides in the context of self-HLA class II (pHLA) presented on antigen-presenting cells. This recognition makes it possible for naive CD4+ T-helper cells to differentiate into memory cells, resulting in the creation of further antibody memory. These responses contribute to effective secretion of donor-specific anti-HLA antibodies (DSA) after second encounters with the same peptide. Preformed donor-reactive CD4+ memory T cells may induce early immune responses after transplantation; however, the tools to evaluate them are limited. This study evaluated shared T cell epitopes (TEs) between the pre-sensitizing and donor HLA using an in silico assay, an alternative to estimate donor-reactive CD4+ memory T cells before transplantation. In 578 living donor kidney transplants without preformed DSA, 69 patients had anti-HLA antibodies before transplantation. Of them, 40 had shared TEs and were estimated to have donor-reactive CD4+ memory T cells. De novo DSA formation in the early phase was significantly higher in the shared TE-positive group than in the anti-HLA antibody- and shared TE-negative groups (p=0.001 and p=0.02, respectively). In conclusion, evaluation of shared TEs for estimating preformed donor-reactive CD4+ memory T cells may help predict the risk of early de novo DSA formation after kidney transplantation.

Background. Generation of donor-specific human leukocyte antigen antibody (DSA) via indirect allorecognition is detrimental to long-term survival of transplant organs. The detection of such immune responses would make it possible to define patients with high risk of sensitization. In this study, we established a novel method for evaluating indirect allorecognition to assess sensitization in kidney transplant recipients. Methods. Recipient CD14 + monocytes were mixed with donor peripheral blood mononuclear cells; cultured in the presence of IL-4, GM-CSF, IL-1β, and TNFα; and used as pulsed dendritic cells (DCs). Cell proliferation and cytokine production were evaluated by carboxyfluorescein diacetate succinimidyl ester-based T cell proliferation assay and Enzyme-Linked ImmunoSpot assay, respectively. Results. CD4 + T cell proliferation was strongly observed in following coculture with allogeneic antigen-pulsed DC leading to interferon-γ and IL-21 production. About 1% of CD4 + T cells exhibited Tfh-like phenotype (PD-1 high CXCR5 + ICOS + CD40L + ). Recipient DC pulsed with donor peripheral blood mononuclear cells was cocultured with recipient CD45RA+CD4+ and CD45RA-CD4+ (generally defined as naive and memory in humans, respectively) T cells. Irrespective of preformed or de novo DSA status, CD45RA + CD4 + T cells constantly produced IL-21. In contrast, IL-21-produced CD45RA − CD4 + T cells were significantly higher in preformed DSA-positive patients than those in negative patients (80.8 ± 51.2 versus 14.8 ± 20.4, P < 0.001). In de novo DSA-positive patients, IL-21produced CD45RA − CD4 + T cells were significantly increased after transplantation compared with before transplantation (9.23 ± 9.08 versus 43.9 ± 29.1, P < 0.001). Conclusions. Assessment of indirect pathway CD4 + T cell response could provide new insights into the underlying mechanism of de novo DSA production, leading to the development of effective strategies against antibody-mediated rejection.

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