Estimation of Sensitization Status in Renal Transplant Recipients by Assessing Indirect Pathway CD4+ T Cell Response to Donor Cell-pulsed Dendritic Cell

Abstract

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-1highCXCR5+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-21–produced 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.