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, 118 (11), 3619-28

Conferring Indirect Allospecificity on CD4+CD25+ Tregs by TCR Gene Transfer Favors Transplantation Tolerance in Mice

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Conferring Indirect Allospecificity on CD4+CD25+ Tregs by TCR Gene Transfer Favors Transplantation Tolerance in Mice

Julia Yuen-Shan Tsang et al. J Clin Invest.

Abstract

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.

Figures

Figure 1
Figure 1. Expansion of Treg population expressing the transduced TCR α and β chain after retroviral transduction.
Direct allospecific Treg lines were transduced with genes encoding Kd54–68 indirect allospecific TCR (Vα11.2/Vβ13) chains and were restimulated weekly with Kd peptide–pulsed immature BL/6 DCs. To detect surface expression of the transduced TCR genes, the cells were triple stained with anti-mouse Vβ13-FITC, Vα11.1/2-PE, and anti-CD4–APC at different time points. Only CD4+ populations were gated for analysis. The percentage of cells in each quadrant is indicated.
Figure 2
Figure 2. TCR transduction conferred indirect allospecificity to direct allospecific Treg lines.
(A) TCR-transduced Tregs [dTreg(TCR), black bars] were cocultured with mature BL/6 DCs in the presence of different amounts of Kd peptide. T cell proliferation was measured at day 3 by [3H]thymidine incorporation. GFP-transduced Tregs [dTreg(GFP), gray bars] and nontransduced direct allospecific Tregs (dTreg, white bars) were used as controls. Error bars represent mean ± SD of experiments performed in triplicate. (B) CFSE-labeled TCR-transduced Tregs, nontransduced direct allospecific Tregs, and Kd peptide–pulsed DCs expanded indirect allospecific Tregs (DCiTreg) were cocultured with mature BL/6 DCs, Kd peptide–pulsed BL/6 DCs (1 mg/ml), BALB/c DCs, or CBA DCs. The cells were harvested at day 4 of the coculture and stained with anti-CD4–APC and propidium iodide (PI). CFSE dilution of the cells was assessed by flow cytometric analysis. Only CD4+PI populations were gated for analysis. Bracket regions indicated each cell division.
Figure 3
Figure 3. Retroviral transduction did not alter Treg phenotypes and function.
(A) TCR-transduced Tregs or nontransduced Tregs were stained with anti-CD62L–FITC, anti-CD3–FITC, anti-CD25–PE, and anti-FoxP3–APC at the end of weekly stimulation. (B) CD4+ T cells from BL/6 mice were cocultured either with an increasing number of TCR-transduced Tregs (black bars) or nontransduced Tregs with direct allospecificity (gray bars) in the presence of T cell–depleted BL/6 APCs and 1 μg/ml anti-CD3. CD4+ T cells (CD4 only) or Treg lines (CD25+ only) cultured with T cell–depleted splenocytes and anti-CD3 were used as controls. T cell proliferation was measured at day 3 by [3H]thymidine incorporation. Error bars represent mean ± SD of experiments performed in triplicate.
Figure 4
Figure 4. Alloantigen-specific TCR-transduced Tregs can suppress CD4+CD25 responders in an antigen-specific manner.
(A) TCR75 TCR-transgenic CD4+ T cells were either cultured alone (black bars; TCR75) or cocultured with TCR-transduced Tregs [gray bars; TCR75 + dTreg(TCR)] in a 1:1 ratio in the presence of T cell–depleted BL/6 APCs and different amounts of Kd peptides. (B) OT-2 TCR-transgenic CD4+ T cells were cocultured with direct specific Treg lines, GFP-transduced Tregs, or TCR-transduced Tregs in the presence of T cell–depleted BL/6 APCs plus 1.0 μg/ml Kd peptide and 0.5 μg/ml OVA peptide. OT-2 cells cultured without Tregs (white bar) were used as controls. (C) OT-2 TCR-transgenic CD4+ T cells were cocultured with increasing numbers of TCR-transduced Tregs in the presence of T cell–depleted BL/6 APCs plus different amounts of Kd peptides (0, 0.3, 1.0 μg/ml) and 0.5 μg/ml OVA peptide. OT-2 cultured without Tregs (blue bar) were used as controls. (D) DO11.10 TCR-transgenic CD4+ T cells were cocultured with TCR-transduced Tregs in the presence of T cell–depleted BALB/c APCs in the presence of 0.5 μg/ml OVA peptide. DO11.10 culture without Tregs (DO11.10) was used as control. T cell proliferation was measured at day 3 by [3H]thymidine incorporation. Error bars represent mean ± SD of experiments performed in triplicate.
Figure 5
Figure 5. TCR-transduced Tregs can migrate to secondary lymphoid organs and expand in vivo in the presence of specific antigen.
TCR-transduced Tregs (5 × 106) were labeled with 1 μM CFSE and were injected i.v. into either BL/6 or BL/6.Kd mice. Three days later, mice were sacrificed. Lymph nodes and spleens were harvested. Cells from lymph nodes and spleen were stained with anti-CD4–APC. In vivo proliferation of the cells was analyzed by flow cytometric analysis of CFSE dilutions. CFSE-positive cells were gated for analysis. The percentage of CFSE-positive cells in the gated region is indicated.
Figure 6
Figure 6. TCR-transduced Tregs expanded at the site of antigenic challenge.
TCR-transduced Tregs (5 × 106) were labeled with 1 μM CFSE and were injected i.v. into BL/6 mice. One day after transfer, the mice received skin grafts from either BL/6.Kd donors or CBA donors. Mice were sacrificed at day 12 after transplantation. Graft draining lymph nodes, mesenteric lymph nodes, and spleens were harvested. Cells from different lymph nodes and spleens were stained with anti-CD4–APC. In vivo proliferation of Tregs was analyzed by flow cytometric analysis of CFSE dilutions. CFSE-positive cells were gated for analysis.
Figure 7
Figure 7. TCR-transduced Tregs prolonged BL/6.Kd skin graft survival in fully immunocompetent mice.
TCR-transduced Tregs (5 × 106; n = 4), nontransduced Tregs with direct allospecificity (n = 5), or Tregs expanded with autologous BL/6 DCs (AutoTreg; n = 4) were injected i.v. into BL/6 mice. One day after transfer, the mice received skin graft from BL/6.Kd donors. WT BL/6 recipients (n = 4) of grafted BL/6.Kd skin without Treg injection served as controls.
Figure 8
Figure 8. TCR-transduced Tregs with both direct and indirect specificity are required to induce long-term graft survival in fully immunocompetent mice.
(A) B6D2F1 hearts were transplanted into BL/6 recipients. Short-term CD8 T cell depletion was achieved by i.p. injection of 250 mg anti-CD8 at days –1 and 1. TCR-transduced direct allospecific Tregs (5 × 106; n = 4) or nontransduced Tregs with direct allospecificity (n = 6) were injected i.v. at days –1, 7, 14, and 21 into BL/6 mice. WT BL/6 recipients with anti-CD8 without Tregs served as controls (n = 6). (B) Heart graft harvested at day 100 after transplantation were fixed and stained with H&E staining and elastin/van Gieson staining (original magnification, ×400).
Figure 9
Figure 9. TCR-transduced Treg therapy in combination with other treatments can induce tolerance to fully mismatched heart transplant in fully immunocompetent mice.
(A) BALB/c hearts were transplanted into the BL/6 recipients. Short-term CD8+ T cell depletion was achieved by i.p. injection of 250 mg anti-CD8 at days –1 and 1. Rapamycin (Rapa) treatment was given i.p. between days –1 and 14 (1 mg/kg/d). TCR-transduced Tregs (1 × 107; n = 4) and nontransduced direct allospecific Tregs (n = 4) were injected i.v. at days –1, 7, 14, and 21 into BL/6 mice. WT BL/6 recipients with anti-CD8 (n = 3) or rapamycin plus anti-CD8 (n = 3) without Tregs served as controls. (BH) Heart grafts harvested at day 150 after transplantation were fixed and stained with H&E (B and E) and elastin/van Gieson staining (C, D, F, and G) (original magnification, ×400). Arrowheads indicate internal elastic lamina. N, neointima; M, media of arteries. (H) Images were analyzed quantitatively for luminal occlusion, and a total of 123 vessels were analyzed. Data represent the mean value ± SEM. *P < 0.0001 between anti-CD8 + dTreg + Rapa and anti-CD8 + dTreg(TCR) + Rapa groups.

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