Long-Term Cardiac Allograft Survival across an MHC Mismatch after “Pruning” of Alloreactive CD4 T Cells

Abstract

Specific tolerance to allografts has been achieved by a variety of means. We have previously shown that ex vivo removal of dividing CD4+ T cells from an MLR or “pruning” delays skin allograft rejection. We tested pruning of alloreactive T cells as a strategy for retaining a broad T cell repertoire while removing alloreactive T cells in a model of cardiac allograft transplant. Using CFSE staining of responder BALB/c cells with stimulator C57BL/6 cells in an MLR, SCID mice were reconstituted with either dividing (D) or nondividing (ND) CD4+ T cells derived from an MLR and then challenged with heterotopic cardiac allografts. Mice reconstituted with D CD4+ T cells rejected cardiac allografts from the stimulator strain with a median survival time (MST) of 29 days, while mice reconstituted with ND CD4+ T cells maintained allografts from the stimulator strain (MST of >100 days) while rejecting third-party allografts (B10.BR) (MST = 11 days). ELISPOT assays demonstrate donor-specific hyporesponsiveness of the ND CD4+ T cells. TCR β-chain V region (TRBV) repertoire analysis demonstrates clonal expansion within both rejecting D cardiac allografts and ND cardiac allografts surviving for the long-term. Histology showed greater allograft infiltration by the D CD4+ T cells. The surviving ND cardiac allografts demonstrated reduced cellular infiltration and reduced incidence of allograft vasculopathy, but with the development of chronic fibrosis. Thus, pruning of alloreactive T cells allows long-term-specific cardiac allograft survival while retaining the ability to reject third-party allografts.

Infiltrating Foxp3+ Regulatory T Cells From Spontaneously Tolerant Kidney Allografts Demonstrate Donor-Specific Tolerance

“…Kidney and skin grafts were fixed in 10% formalin and embedded in paraffin before staining with periodic acid-Schiff (PAS) or hematoxylin and eosin (H&E) (22,26). PAS staining was performed on 5-mm paraffin kidney sections to assess tubulitis, glomerulosclerosis and interstitial fibrosis (22).…”

“…The expression of IL-10, TGF-b, IFN-g, transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) and chemokine receptor 3 (Cxcr3) on sorted K-Tregs from Days 7-14 and !69 PTx mice was determined by TaqMan R Gene Expression Assay (Applied Biosystems, Invitrogen, Life Technologies, Mulgrave, Victoria, Australia) as described (26,29).…”

Extreme lymphoproliferative disease and fatal autoimmune thrombocytopenia in FasL and TRAIL double-deficient mice

“…TCR CDR3 gene usage was determined by RT-PCR, using V␤-specific PCR, and CDR3 spectratyping was performed with a second round of PCR using 1 L of PCR product from each TCRV␤, with FAM-labeled universal constant region primer, as described previously. 17 …”

Approaching the Promise of Operational Tolerance in Clinical Transplantation

“…Furthermore, its creation of a "window" during the first month after transplantation, where there are low numbers of T or B cells also open possibilities for therapies that could take advantage of the low T-cell precursor frequency in the recipient. For example, it would be possible to reintroduce recipient T cells, harvested before transplant and cultured with donor antigen-presenting cells, to create donor-specific Tregs (35), or to "prune" alloreactive T cells to eliminate donor-reactive T cells (36). Alemtuzumab is now used widely in transplantation, and approximately 10% of all transplant patients in the United States receive it for induction therapy (37).…”