B-CELL DEPENDENT T-CELL ALLOREACTIVITY IS ASSOCIATED WITH
FUNCTIONAL OUTCOME & REGULATED BY IL-10 IN CHRONIC ANTIBODYMEDIATED REJECTION
BACKGROUND: Allogeneic renal transplantation is the treatment of choice for end-stage
kidney disease. However, kidney transplants do not last for the natural lifespan of most
recipients. The single biggest cause of renal allograft failure is chronic rejection (CR) (1).
Surprisingly little is known about the cell-mediated interactions that underpin CR. Circulating
antibodies (Ab) against donor human leukocyte antigens (HLA) are thought to be both causative
& important predictors of chronic antibody-mediated rejection (CAMR) (2) but multiple lines of
evidence suggest that cell-mediated immunity is also involved; in CR this is underpinned by
cognate interactions between donor-specific CD4+ T and B lymphocytes. Different subsets of
effector CD4+ T cells, predominantly Th1 cells, which produce interferon-gamma (IFNγ) and
induce macrophage activation, mediate both acute and chronic rejection. Regulation of Th1
cells by IL-10, is essential, as unchecked IFNγ production results in severe tissue damage and
often death (3). We have previously demonstrated heterogeneity and complexity in IFNγ
responses using enzyme-linked immunosorbent (ELISPOT) assay to detect indirect CD4+ T cell
alloresponses in patients with CAMR (4). We investigated the cellular influences on in vitro Tcell alloresponses and we have found that certain ELISPOT patterns are significantly associated
with functional outcome after renal transplantation, establishing the utility of the ELISPOT
assay in CAMR and highlighting new targets for potential therapeutic manipulation.
METHODS: Peripheral blood mononuclear cells were collected & analysed by ELISPOT
assay from two separate patient cohorts at baseline and again 9-12 months later. Of 65 patients
undergoing renal biopsy: the ‘protocol’ cohort consisted of 14 patients with antibody mediated
rejection (AMR) and 5 with no pathology; the ‘for cause’ cohort 38 with AMR and 8 with nonimmunologic damage.
RESULTS: Our findings demonstrate that IFNγ production that is dependent on donor
alloantigen processing and presentation by B cells (“Bdep” pattern) is closely correlated with
later development of kidney transplant dysfunction. In a proportion of patients, IFNγ production
only becomes detectable after B cell (CD19+) depletion (“Breg” pattern); these patients have a
better outcome. In these Breg samples we also found associations with (B cell sources of) IL-10
cytokine production after polyclonal stimulation. These independent associations between
ELISPOT pattern & functional outcome were found to be significant at two separate time points
in both cohorts. IFNγ production also appears differentially regulated by CD25+ cells; in some
patients B cell–dependent allo-specific reactivity was revealed following depletion of CD25+
cells; Surprisingly, these patients appeared to have a worse eGFR trend than other patients with
‘non-regulated’ B-dependent IFNγ production. Furthermore this ELISPOT pattern is also
associated with (a non-B cell source of) IL-10 production after polyclonal stimulation. These
data suggest that patients with T cells that lack the ability to produce regulatory IL-10 appear to
demonstrate poorer long-term outcomes.
CONCLUSIONS: All these data establish the utility of this ELISPOT assay and suggest that
mechanisms that regulate IFNγ production appear to be critical determinants of transplant
success. Moreover the significant associations with regulatory cytokine IL-10 give support to
further investigation of the contribution of the Th1 lifecycle in CR.
REFERENCES
1. Gaston RS et al. Evidence for antibody-mediated injury as a major determinant of late kidney
allograft failure. Transplantation. 2010 Jul 15;90(1):68-74.
2. Colvin RB. Antibody-mediated renal allograft rejection: diagnosis and pathogenesis. J Am
Soc Nephrol 2007; 18: 1046–1056.
3. Cope A, Le Friec G, Cardone J, Kemper C: The Th1 life cycle: molecular control of IFNgnosis and pathogenesis. Trends Immunol 32 278-286 (2011).
4. Shiu KY et al. B-lymphocytes support and regulate indirect T-cell alloreactivity in individual
patients with chronic antibody- mediated rejection Kidney Int. ”in press” (Epub ahead of print)
2015 Apr 1.