A Role for Regulatory Cells in Tolerance Induction in Recipients of Haploidentical Combined Non-Myeloablative Bone Marrow and Kidney Transplantation?.
Authors: Andreola Giovanna , Chittenden Meredith , Shaffer Juanita , Cosimi A. Benedict , Kawai Tatsuo , Cotter Pete , Dey Bimalangshu R. , Tolkoff-Rubin Nina , Preffer Frederic , Saidman Susan , Kattleman Kristin , Spitzer Thomas R. , Sachs David H. , Sykes Megan 
Published: October 2019

Abstract
Following an in vivo T cell depleting non-myeloablative conditioning regimen, 5 patients, aged 22–49, received combined kidney and bone marrow transplantation from a haploidentical related donor. Rituximab was included in the conditioning for patients 4 and 5. All patients developed initial mixed chimerism but lost it by day 21; no patient developed GVHD. Four patients discontinued immunosuppression from 240 to 422 days after BMT and have remained off immunosuppression for 9 to 52 months with no evidence of allograft rejection. Flow cytometry was used to assess lymphocyte subsets recovering after transplant. CD3 counts recovered slowly, exceeding 500 cells/μl at days +271, +365, +640 and +450. While memory CD45RO+ cells were most prevalent among CD4+ cells, naïve-type CD4+CD45RA+ cells, presumably arising from the recipient thymus, ranged from 8% to 56% at the time when total CD4 counts recovered to >100 cells/μl (days +165, +21, +352, +240). Notably, a very high proportion of initially recovering T cells were CD3+CD4+ expressing CD25 in all patients as early as day 7 and persisted over 1 year in 2 patients. At approximately day +120 and +365, we further characterized these cells for CD127, FOXP3, CD45RO, CD45RA, HLA-DR and CD62L expression. At Day +120, all 4 patients showed increased frequencies (10.7±4.6%) of CD25+CD127-FOXP3+ regulatory T cells (Treg) within the CD4 population compared to healthy subjects (3.8±0.4%). Expression of CD45RO, CD45RA, CD62L and HLA-DR was variable. By 1 year post-transplant, frequencies of Treg had decreased to levels similar to those in normal subjects. In vitro assays for CD8 and CD4 T cell-mediated alloreactivity (CML/MLR) showed development of long-lasting donor-specific unresponsiveness by 3 months after transplant in Patients 2, 4 and 5, and by 9 months in Patient 1. Responses to 3rd party recovered in all patients after a period of unresponsiveness. In Patient 1, in whom anti-donor CML reactivity declined gradually to become unresponsive by 9 months, depletion of CD4+CD25+ cells revealed a residual anti-donor CML and MLR response at 1year but not at 18 months. In 2 other patients, depletion of CD4+CD25+ cells did not reveal an anti-donor response at time points analyzed from day +122 to 2 years. In patients in whom renal tubular epithelial cells (RTEC) were cultured from the donor kidney, loss of killing activity against donor RTEC was observed post-transplant. The high percentage of Treg recovering early after transplant suggests that they may play a role in initial tolerance induction. This regulatory mechanism may be followed by later deletion of donor-reactive T cells. The variable ability to detect regulation of anti-donor reactivity may reflect the strength of the initial response, as patients with weak pre-transplant anti-donor responses and rapid post-transplant development of donor unresponsiveness did not reveal anti-donor response when Treg were depleted. In addition, infiltration of Treg at the graft site, not revealed by the assays described, might be responsible for tolerance in these patients.