K Kildey1,2, AJ Kassianos1,2,3,4, B LAW1,2,3, X Wang1,2, E See5, GT John2, R Wilkinson1,2,3,4, H Healy1,2
1Conjoint Kidney Research Laboratory, Pathology Queensland, Brisbane, Australia; 2Kidney Health Service, Royal Brisbane and Women’s Hospital, Brisbane, Australia; 3Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia; 4School of Medicine, University of Queensland, Brisbane, Australia. 5Department of Nephrology, Princess Alexandra Hospital, Brisbane, Australia.
Aim: To identify, enumerate and phenotype the human lymphocyte subsets in kidney allograft rejection.
Background: Lymphocytes are powerful effectors in kidney transplant rejection. However, the respective contributions of different lymphocyte subsets in episodes of allograft rejection remain uncertain, with previous studies restricted to basic immunohistochemical methods. This study uses a multi-colour flow cytometric-based approach to unequivocally define and evaluate the lymphocyte subsets in human allograft rejection.
Methods: We extracted renal lymphocytes from healthy control kidney tissue and transplant biopsies stratified based on the histopathological diagnosis of (a) non-rejection, (b) T cell-mediated rejection (TCMR), (c) antibody-mediated rejection (ABMR) and (d) mixed (TCMR+ABMR) rejection. Lymphocyte subsets were characterised by fourteen-colour flow cytometry.
Results: We detected significantly elevated numbers of total T cells (CD45+CD3+) in TCMR and mixed transplant biopsies compared with healthy kidney tissue. Within this T cell compartment, numbers of T helper cells (CD3+CD4+), cytotoxic T cells (CD3+CD8+), γ/δ T cells (CD3+γ/δ+) and natural killer (NK)-T cells (CD3+CD16+) were significantly elevated in TCMR and mixed transplant biopsies compared with healthy kidney tissue. Of CD3– lymphoid cells, total NK cells (CD3–CD56+) were significantly elevated in TCMR and mixed transplant biopsies compared with healthy kidney tissue – in particular, the CD56bright NK cell subset in TCMR biopsies and the CD56dim NK cell subset in mixed rejection biopsies.
Conclusions: Collectively, our data demonstrate the feasibility of flow cytometry to characterise the immunophenotypes in kidney transplant tissue. Our results indicate that lymphocyte subsets, in particular NK cell subsets, are differentially recruited during different types of rejection. Further functional dissection of these subsets may provide useful diagnostic and prognostic information for patients experiencing allograft rejection.