Altered frequencies of TEMRA and CXCR3-positive cells among autoreactive CD4 T cells associated with recurrent islet autoimmunity in recipients of simultaneous pancreas-kidney transplants

Presenter
Alberto Pugliese, University of Miami

Authors
Helena Reijonen, Isaac Snowhite, Francesco Vendrame, George W. Burke III, Alberto Pugliese

Purpose
Patients with autoimmune, type 1 diabetes (T1D) and end stage renal disease may become recipients of simultaneous pancreas-kidney (SPK) transplants to restore insulin secretion and kidney function. We previously reported that some SPK recipients may develop T1D recurrence (T1DR) on follow-up despite immunosuppression that prevents rejection; in some patients T1DR was confirmed by the demonstration of insulitis and beta cell loss in a pancreas transplant biopsy. Overall, we have observed T1DR in about 5% of SPK recipients on extended follow-up. We previously reported that seroconversion for multiple autoantibodies is a risk factor for T1DR. The aim of this study was to determine whether autoreactive T cells in the circulation of SPK recipients are associated with T1DR and define key phenotypic features of these cells.

Methods
We studied 7 SPK recipients who had developed T1DR and 16 with normal glucose tolerance (NGT), of whom 5 were classified as autoantibody converters at risk for future T1DR (NGT-C) and 11 were autoantibody negative or had stable autoantibody levels from prior to the transplant (stable, NGT-S). We evaluated autoreactive T cells in peripheral blood using a pool of HLA class II tetramers loaded with T1D-associated peptides from multiple autoantigens (GAD65, proinsulin and ZnT8), and a viral antigen (flu) was used as a control. Besides antigen specificity, T cells were analyzed for lineage and phenotype by flow cytometry staining CD4, CD45RA, CD45RO, PD1, CXCR3, CCR7, CCR6 and CCR4. We examined naïve (CD45RA+, CCR7+), effector memory (EM, CD45RO+, CCR7-), central memory (CM, CD45RO+, CCR7+), total memory (EM+CM) and terminally differentially memory (TEMRA, CD45RA+, CCR7-). We measured these phenotypes in the total CD4 T cell compartment and in tetramer positive CD4 T cells. We assessed number of tetramer+ cells/1×106 CD4 T cells and as % of the various subset among the tetramer+ CD4 T cells; Value ranks among patient groups were compared using the non-parametric Mann-Whitney test; two-tailed p values are reported. For some analysis, we used ROC (Receiving Operating Curves).

Summary of Results
T1DR patients had significantly higher numbers of autoreactive CD4 T cells compared to NGT-S patients, and this was observed for naïve, total memory and effector memory compartments. In contrast, T1DR patients had decreased numbers of autoreactive CD4 T cells among TEMRAs compared to NGT-S recipients. For NGT-C patients, numbers of autoreactive CD4 T cells were similar to those of NGT-S patients, except these numbers were higher among naïve T cells.
We then examined the frequency of various subsets among autoreactive CD4 T cells, specifically naive, total memory, effector memory and TEMRA. In T1DR patients there were increased proportions of naïve and total memory autoreactive CD4 T cells compared to NGT-S patients. T1DR patients had very low frequencies of TEMRA autoreactive CD4 T cells compared to NGT-S patients (% mean 1.143 + SEM 0.6335 vs 46.82 + 8.781, respectively, p<0.0026); NGT-C patients also had lower frequency TEMRAs among the autoreactive CD4 T cell (% mean 14.60 + SEM 10.35) compared to NGT-S patients (p= 0.0279). The T1DR and NGT-C patients combined had much lower frequencies of TEMRA autoreactive CD4 T cells compared to NGT-S patients (p<0.00001). Using ROC, the proportions of TEMRA autoreactive CD4 T cells distinguished T1DR and NGT-C from NGT-S patients (AUC= 0.94, p=0.0003, 90% sensitivity, 92% specificity). We did not observe significant differences in the frequencies of naïve and memory subsets among the three patient groups, except that TEMRA CD4 T cells were reduced in T1DR compared to NGT-S patients (% mean 3.286 + SEM 1.358 vs 6.250 + 1.023, respectively, p<0.04).
The frequency autoreactive CD4 T cells expressing PD1, CCR6, or CCR4 did not differ among the three patient groups. However, the frequency of CXCR3-positive CD4 autoreactive T cells was significantly higher in T1DR and NGT-C patients compared to NGT-S patients. The frequency of CXCR3-positive autoreactive CD4 T cells among T1DR and NGT-C patients combined was % mean 25.25 + SEM 6.361 vs % mean 4.455 + SEM 3.431 among NGT-S patients (p= 0.0063). ROC analysis showed that the proportions of CXCR3-positive autoreactive CD4 T cells distinguished T1DR and NGT-C from NGT-S patients (AUC= 0.80, p=0.01, 82% sensitivity, 75% specificity).

Conclusions
Autoreactive CD4 T cells were increased in T1DR compared to NGT-S patients. We show that this applied to both naïve and total memory compartments. NGT-C patients had higher frequencies then NGT-S patients only in the naive compartment. Remarkably, the autoreactive CD4 T cells had very low frequency in the TEMRA compartment of T1DR and NGT-C patients, and this was a distinguishing feature that may be exploited as a biomarker of T1DR. Our results also demonstrate an association of CXCR3-positive autoreactive CD4 T cells with T1DR and autoantibody conversion. The expression of CXCR3 by circulating autoreactive CD4 T cells help identify a stage in which autoreactive T cells may migrate to the pancreas and infiltrate the islets. While our findings are from a relatively small number of patients, given the low frequency of T1DR among SPK recipients, they support CXCR3 as a potential therapeutic target to antagonize recurrent islet autoimmunity.