Testicular Immune Privilege Promotes Transplantation Tolerance by Altering the Balance between Memory and Regulatory T Cells

Abstract

Immune responses are suppressed in immunologically privileged sites, which may provide a unique opportunity to prolong allograft survival. However, it is unknown whether testicular immune privilege promotes transplantation tolerance. Mechanisms underlying immune privilege are also not well understood. Here we found that islet transplantation in the testis, an immunologically privileged site, generates much less memory CD8+ T cells but induces more Ag-specific CD4+CD25+ regulatory T cells than in a conventional site. These CD4+CD25+ cells exhibited the suppression of alloimmune responses in vivo and in vitro. Despite the immune regulation, intratesticular islet allografts all were rejected within 42 days after transplantation although they survived longer than renal subcapsular islet allografts. However, blocking CD40/CD40L costimulation induced the tolerance of intratesticular, but not renal subcapsular, islet allografts. Tolerance to intratesticular islet allografts spread to skin allografts in the non-privileged sites. Either transfer of memory CD8+ T cells or deletion of CD25+ T cells in vivo broke islet allograft tolerance. Thus, transplantation tolerance requires both costimulatory blockade, which suppresses acute allograft rejection, and a favorable balance between memory and regulatory T cells that could favorably prevent late allograft failure. These findings reveal novel mechanisms of immune privilege and provide direct evidence that testicular immune privilege fosters the induction of transplantation tolerance to allografts in both immunologically privileged and non-privileged sites.

Suppression of Memory CD8 T Cell Generation and Function by Tryptophan Catabolism

“…Seven days later, graft-infiltrating cells were isolated and analyzed for 2C T cell proliferation by the BrdU uptake (25,31). As shown in Fig.…”

“…CD8 ϩ T cell memory phenotype was further confirmed by staining with anti-CD62 ligand (CD62L) allophycocyanin or anti-CD44 allophycocyanin (BD Pharmingen) as previously described (25,35). To purify memory CD8 ϩ T cells for the adoptive transfer experiments or in vitro studies, T CM (1B2 ϩ CD8 ϩ CD44 high CD62L high ) cells were isolated from spleens and lymph nodes by FACSAria cell sorting and T EM (1B2 ϩ CD8 ϩ CD44 high CD62L low ) cells were isolated from livers and kidneys (25,31).…”

“…Islet recipients were 7-to 8-wk-old C57BL/6 female mice. Islets were isolated and transplanted into the subcapsular space of the right kidney of the recipient mice as previously described (31). Recipient mice were rendered diabetic by a single injection of streptozotocin (180 mg/kg; Sigma-Aldrich) 10 -14 days before transplantation.…”

“…Cells were then fixed in 70% ethanol followed by 1% paraformaldehyde and incubated with 50 U/ml DNase I (Sigma-Aldrich). Cells were finally stained with anti-BrdU FITC (BD Biosciences) and analyzed by a FACSCalibur (25,31). To detect apoptosis, graft-infiltrating cells were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton X-100 solution, and labeled with fluorescein-tagged dUTP by the TUNEL method according to the manufacturer's instructions (Roche Applied Science) (25,31).…”

“…Graft-infiltrating cells were isolated as described in our previous publications (25,31). Briefly, the kidneys containing islet allografts were perfused in situ with heparinized 0.9% saline.…”