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Human Regulatory T Cells With Alloantigen Specificity Are More Potent Inhibitors of Alloimmune Skin Graft Damage Than Polyclonal Regulatory T Cells

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Human Regulatory T Cells With Alloantigen Specificity Are More Potent Inhibitors of Alloimmune Skin Graft Damage Than Polyclonal Regulatory T Cells

Pervinder Sagoo et al. Sci Transl Med.

Abstract

Graft rejection by the immune system is a major cause of transplant failure. Lifelong immunosuppression decreases the incidence of graft rejection; however, nonspecific immunosuppression results in increased susceptibly to infection and cancer. Regulatory T cells (T(regs)), which suppress the activation of the immune system and induce tolerance, are currently under evaluation for use in clinical transplantation. Ex vivo expanded polyclonal T(regs) that are introduced into transplant recipients alter the balance of T effector cells to T(regs); however, experimental data suggest that alloantigen-specific T(regs) would be more effective at preventing graft rejection. We have developed a method to enrich alloantigen-specific human T(regs) based on the coexpression of activation markers, CD69 and CD71. These T(regs) could be readily expanded in vitro and demonstrated potent antigen-specific suppression. In a humanized mouse model of alloimmune-mediated injury of human skin grafts, alloantigen-specific T(regs) resulted in a significant reduction in clinically relevant indicators of dermal tissue injury when compared with polyclonal T(regs), restoring a histology comparable to healthy skin. This method of human allospecific T(reg) selection should be scalable to the clinic. The improved in vivo efficacy of alloantigen-specific T(regs) over polyclonal T(regs) shown here suggests that generating "customized" T(regs) with defined anti-donor allospecificities may improve current practice in clinical immunotherapy.

Figures

Fig. 1
Fig. 1
Screening of human Treg activation markers by flow cytometry. (A) Human Tregs were isolated from whole CD4+ T cells from peripheral blood by magnetic bead selection (CD4+CD25+) or cell sorting (CD4+CD25+CD127lo/−) and enriched to purities of ~90% and 98% by each method, respectively. Data are representative of nine independent experiments. (B) Phenotype of freshly isolated Tregs and autologous Teffs (CD4+CD25) was studied by flow cytometry, analyzing typical Treg markers and basal expression of activation markers under investigation. (C) Data are summarized, plotting means ± SD (n = 6). (D) Expression of T cell activation markers by purified Tregs and autologous Teffs after 1 to 5 days of polyclonal stimulation with CD3CD28 beads. Data are representative of six experiments.
Fig. 2
Fig. 2
Analysis of Treg activation marker modulation in response to direct pathway allogeneic stimulation. (A) Tregs and autologous Teffs were cultured with mature allogeneic CD1c+ DCs for 1 to 5 days. Expression of activation markers was analyzed by flow cytometry and is expressed as the change (Δ) in percentage of activation marker–positive cells after in vitro stimulation, relative to unstimulated cells (Fig. 1B). Data plotted show means ± SD (n = 7). (B) Polyclonal and allogeneic stimulation results in coexpression of CD69 and CD71 by Tregs. Data are representative of 12 independent experiments. (C) CD69 and CD71 up-regulation induced by allogeneic stimulation was inhibited by addition of αHLA-DRβ1–blocking antibody to cultures but did not affect expression in polyclonally stimulated cultures. Data are representative of four experiments. (D) Detailed analysis of CD69+CD71+ Treg phenotype after allogeneic stimulation (4 days). Inset images show that the activated Treg phenotype is reflected in cell cultures by activated cell clusters (n = 4).
Fig. 3
Fig. 3
CD69+CD71+ enrichment of potent alloantigen-specific Tregs. (A) Suppressive function of alloantigen (donor) or polyclonal (CD3CD28) activated (CD69+CD71+) Tregs was tested by in vitro CFSE dilution assays after 5 days (upper panels). Flow cytometry data are summarized in line plots (lower panels) as percentage suppression of proliferation mediated by Tregs on autologous CD4+CD25 Teffs stimulated with Donor or HLA-disparate 3rdParty DCs. (B) Summary of five independent experiments showing suppressive capacity of enriched alloantigen-activated CD69+CD71+ Tregs and nonactivated CD69CD71 Tregs against the primary allostimulator or 3rdParty allostimulator. (C) Number of activated Tregs isolated by cell sorting and final cell number expanded in vitro after 4 to 6 weeks for several Treg lines. (D) Maintenance of Treg marker expression by two different antigen-specific Treg lines (t1 and t2) after in vitro expansion, and expression of IFN-γ and IL-17 upon stimulation (phorbol 12-myristate 13-acetate/ionomycin) of Tregs. High FoxP3 expression is maintained by expanded Tregs, compared to polyclonally activated Teffs, which express intermediate FoxP3. (E) Alloantigen-specific Tregs enriched with CD69+CD71+ maintained antigen-specific suppressive function after in vitro expansion for 6 weeks. Percentage suppression of autologous Teff proliferation by donor alloantigen-specific Tregs (AgS-Tregs), in response to primary allostimulator or 3rdParty simulation, is highlighted within histograms. Data are representative of three independent assays. Statistical significance was calculated with paired Student’s t tests. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 4
Fig. 4
Assessment of enriched donor alloantigen-specific and polyclonal Treg function with a humanized mouse model of skin allograft injury. (A) Strategy for generation of human skin donor alloantigen–specific (AgS-Tregs) and polyclonal (Pc-Tregs) Tregs and in vivo assessment of their protective function with a humanized mouse model of allogeneic CD4+ T effector-mediated damage of allografted tissue in NOD/scid/IL-2Rγ−/− (NSG) and BALB/cRag2−/−γc−/− strains. (B) Histological assessment of donor-allografted human skin 4 weeks after animals received either PBS (Nil) or 5 × 106 allogeneic human T effectors (Teffs). Human CD45 staining shows an intense Teff infiltrate, with focal localization at dermo-epidermal junctions (arrows). Cells across epidermal basal lamina are positive for Ki67, indicating keratinocyte hyperproliferation. Loss of involucrin expression shows epidermal barrier dysfunction in Teff-treated animals.
Fig. 5
Fig. 5
More potent protection of skin allografts from alloimmune injury by antigen-specific Tregs than polyclonal Tregs. (A) Human skin grafts from mice receiving PBS only (Nil), 5 × 106 T effectors alone (Teffs), or in combination with 1 × 106 donor-specific (AgS) or polyclonal (Pc) Tregs were analyzed after 4 weeks by staining skin allografts for human CD45, Ki67, and involucrin. (B to E) More detailed characterization of skin allografts, examining (B) epidermal keratinocyte expression of Ki67, (C) TUNEL+ nuclei in dermal infiltrates, (D) human CD31 dermal microvasculature, and (E) detection of CD3+FoxP3+ Tregs in perivascular dermal infiltrates was performed. Representative immunofluorescent images from each treatment group are shown (inset images at higher magnification). (F to I) Quantitative histological analysis of three to five independent visual fields per allograft, where plotted lines represent the mean (n = 6 animals per treatment group, pooled data from two independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001, Kruskal-Wallis test. Quantification of CD3+FoxP3+ cells is expressed as a ratio of total CD3+ cells quantified per visual field and was statistically compared by one-way ANOVA.

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