Antigen-specific dependence of Tr1-cell therapy in preclinical models of islet transplant

Abstract

Objective: In type 1 diabetes, allogeneic pancreatic islet transplant restores insulin production, but life-threatening immunosuppression is required to avoid graft rejection. Induction of antigen (Ag)-specific tolerance by cell therapy with regulatory T-cells (Tregs) represents an attractive alternative approach but its therapeutic efficacy in islet transplant remains to be determined. Among the different subsets of CD4(+) Tregs, the T inducible regulatory type 1 (Tr1) cells can be generated from naive T-cells in the presence of interleukin-10 (IL-10) and represent one promising therapeutic choice. This study was designed to define the efficacy of Tr1-cell therapy in preclinical models of islet transplant.

Research design and methods: Non-Ag-specific polyclonal Tr1 cells and donor Ag-specific Tr1 cells were transferred, in the absence of any pharmacological treatment, in two distinct mouse models of islet transplant. The two models differed in their therapeutic stringency, based on the mean rejection time of untreated mice that underwent a transplant.

Results: Transfer of polyclonal Tr1 cells engendered graft tolerance only in the nonstringent mouse model. Conversely, cell therapy with Ag-specific Tr1 cells induced an IL-10-dependent tolerance in the stringent mouse model of islet transplant. The therapeutic advantage of Ag-specific Tr1 cells over polyclonal Tr1 cells was due to their donor Ag specificity.

Conclusions: These results demonstrate that Tr1-cell therapy leads to tolerance in settings of islet transplant and that its therapeutic efficacy is highly dependent on the antigen specificity of these cells.

FIG. 1.

A: Naive CD4⁺CD62L⁺ T-cells isolated from the spleens of BALB (left) or B6 (right) mice were activated with αCD3 and αCD28 mAbs in the presence or absence of exogenous IL-10 for 3 weeks. The ability to produce IL-10 and IL-4 was tested by intracellular staining at the end of the culture and upon TPA/ionomycin stimulation. The frequency of Tr1 cells (i.e., IL-10⁺IL-4⁻ cells) differentiated in the presence (+IL-10) or absence of IL-10 is reported in bold. One representative experiment of five (for BALB) and three (for B6) mice is shown. B: Chemically induced diabetic BALB mice received a transplant of islets from B6 mice. The day before transplant, recipient mice were injected with PBS (no cells, n = 7), 2 × 10⁶ of BALB CD4⁺ T-cells enriched in Tr1 cells upon culture in the presence of IL-10 (Tr1 cells, n = 8), or 2 × 10⁶ BALB CD4⁺ T-cells cultured for 3 weeks in the absence of IL-10 (Th0 cells, n = 4). Graft survival was monitored by glycemia levels. A graft was considered rejected when glycemia was >300 mg/dl. At 100 days after transplant, 30 × 10⁶ splenocytes isolated from B6 mice were injected in tolerant mice to boost their immune system. The percentage of graft survival at various time points after transplant is shown (left). Chemically induced diabetic B6 mice received a transplant of islets from BALB mice. The day before transplant, recipient mice were injected with PBS (no cells, n = 8), 2 × 10⁶ B6 CD4⁺ T-cells enriched in Tr1 cells upon culture in the presence of IL-10 (Tr1 cells, n = 6), or 2 × 10⁶ B6 CD4⁺ T-cells cultured for 3 weeks in the absence of IL-10 (Th0 cells, n = 2). Graft survival was monitored by glycemia levels. A graft was considered rejected when glycemia was >300 mg/dl (right).

FIG. 2.

A: Splenic CD4⁺ T-cells isolated from treated B6 mice that underwent a transplant (Tx/treated, n = 4), untreated B6 mice that underwent a transplant (Tx, n = 4), and naive B6 mice (naive, n = 2) were cultured for 1 week in the presence of αCD3 and αCD28 mAbs. The ability to produce IL-10 and IL-4 was tested by intracellular staining at the end of the culture and upon TPA/ionomycin stimulation. The frequency of Tr1 cells (i.e., IL-10⁺IL-4⁻ cells) is reported in bold. One representative plot for each group tested is shown. B: Splenic CD4⁺ T-cells isolated from treated B6 mice that underwent a transplant (Tx/treated, n = 4), untreated B6 mice that underwent a transplant (Tx, n = 4), and naive B6 mice (naive, n = 2) were cultured for 1 week with irradiated APCs isolated from the original donor of the islets (BALB) (upper panels) or from unrelated third-party APCs (C3H) (lower panels). The ability to produce IL-10 and IL-4 was tested by intracellular staining at the end of the culture and upon TPA/ionomycin stimulation. The frequency of Tr1 cells (i.e., IL-10⁺IL-4⁻ cells) is reported in bold. One representative plot for each group tested is shown (left). The ratio between the percentage of Tr1 cells specific for the original donor and the percentage of Tr1 cells specific for a third-party donor are shown (right). C: At the end of 1 week of in vitro Ag-specific stimulation, IL-10 secretion was measured by enzyme-linked immunosorbent assay. The ratio between IL-10 produced specifically in response to APCs from the original donor and IL-10 produced in response to third-party APCs is shown. *P < 0.05.

FIG. 3.

A: Chemically induced diabetic B6 mice received a transplant of islets from BALB mice. The day before transplant, recipient mice were injected with PBS (no cells, n = 7), 2 × 10⁶ CD4⁺CD25⁻ T-cells isolated from the spleen of B6 treated mice that underwent a transplant (Tx/treated, n = 5), or 2 × 10⁶ CD4⁺CD25⁻ T-cells isolated from the spleen of B6 untreated mice that underwent a transplant (Tx mice, n = 2). At 150 days after transplant, tolerant mice were injected with αIL-10R mAb. Graft survival was monitored by glycemia levels. A graft was considered rejected when glycemia was >300 mg/dl. The percentage of graft survival at various time points after transplant is shown. B: Chemically induced diabetic B6 mice received a transplant of islets from C3H mice. The day before transplant, recipient mice were injected with PBS (no cells, n = 6), or 2 × 10⁶ CD4⁺CD25⁻ T-cells isolated from the spleen of B6 mice that received a transplant of BALB islets and were treated (Tx/treated, n = 5). Graft survival was monitored by glycemia levels. A graft was considered rejected when glycemia was >300 mg/dl.