1Department of Pathology, Laboratory Medicine, Transfusion Services and Immunohematology, G. R. Doshi and K. M. Mehta Institute Of Kidney Diseases & Research Centre (IKDRC)- Dr. H.L. Trivedi Institute Of Transplantation Sciences (ITS), AHMEDABAD, India; 2Department of Nephrology and Transplantation Medicine, G. R. Doshi and K. M. Mehta Institute Of Kidney Diseases & Research Centre (IKDRC)- Dr. H.L. Trivedi Institute Of Transplantation Sciences (ITS), Civil Hospital Campus, AHMEDABAD, India
Introduction: Induction of antigen-specific tolerance is critical for the prevention of autoimmunity and maintenance of immune regulation. A subpopulation of suppressive CD4+ T cells, termed regulatory T-cells (Tregs) have recently been recognized as key players in transplantation immunobiology. However there is little knowledge about their source and dose required for tolerance induction. Tregs induction and activation is an attractive approach of immunotherapy for many diseases/ disorders. However Treg therapy may require in vitro expansion since Tregs are present at low frequency in most sites clinically accessible for harvesting. Mesenchymal stem cells (MSC) have promising role in differentiation and preferential activation of Tregs to suppress cytotoxic T-cell activity in transplant recipients. We present in-vitro model of induction of donor-specific Tregs using donor adipose tissue derived MSC (AD-MSC) and recipient peripheral blood mononuclear cells (PBMC).
Material and Methodology: Ten grams subcutaneous adipose tissue was collected from each of 25 renal allograft donors during nephrectomy and cultured in lab using α-minimal essential media. On 10th day, AD-MSC were harvested and cultured in separate culture dishes. PBMC were derived from immunosuppressed recipients and were divided in two parts, one as responder-PBMC (R.PBMC) and other subjected to irradiation served as stimulator-PBMC (S.PBMC). R.PBMC and S.PBMC were layered on AD-MSC for 9 days in culture medium RPMI-1640 supplemented with IL-2 and human albumin. R.PBMC and S.PBMC co-cultured in absence of AD-MSC under similar conditions served as negative controls. Cells were harvested, analysed for viability, suppression assay and phenotypic characterization using FACScan for CD127-/lowCD4+CD25high,CTLA-4+ and infused in thymus of recipients 3 weeks post-transplant.
Results: Mean 3.5 ± 1.3 x103 AD-MSC were generated and co-cultured with mean 3.6 ± 1.9 x104 PBMC. Mean CD127-/lowCD4+CD25high were increased from 2.2 ± 2.3 % to 16.6 ± 12.8% and CTLA-4+ increased from 27.2 ± 11.6 % to 49.8 ± 16.8 % at the end. Interestingly there was rise in CD4+CD8+ cells from mean 2.7 ± 6.8 % to 29.5 ± 27.6 %. Tregs suppressive capacity was maintained in populations layered on AD-MSC for 12 days while control cells lost all suppressive activity after 3 days. Mean quantum of Tregs infused was 2.9 ± 5.3 x 104cells/kg body weight (BW). Tregs infusion was uneventful. Over a mean follow-up of 114.4 ± 48.2 days, all patients are doing well with stable mean serum creatinine of 1.2 mg/dL on immunosuppression of Tacrolimus,0.05 mg/kgBW/day and Prednisone, 10 mg/day without any immune injury.
Conclusion: We have successfully induced T-regs [CD127-/lowCD4+CD25high, CTLA-4+ and CD4+CD8+] using donor AD-MSC from immunosuppressed recipient PBMC. This novel model will open up the gateway to tolerance in autoimmunity and organ transplantation.
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