Research ArticleIMMUNOLOGY

In situ recruitment of regulatory T cells promotes donor-specific tolerance in vascularized composite allotransplantation

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Science Advances  13 Mar 2020:
Vol. 6, no. 11, eaax8429
DOI: 10.1126/sciadv.aax8429
  • Fig. 1 Treatment with Recruitment-MP (50 mg) prolongs allograft survival indefinitely.

    (A) Hindlimb allograft survival curve showing indefinite survival (>200 days) in six of eight rats treated with Recruitment-MP (50 mg). These results are statistically significant (P < 0.05) when compared to all other groups using a log-rank test. (B) Macroscopically, treatment with 50 mg of Recruitment-MP results in acceptance of the graft with normal hair grown and gross skin appearance, as opposed to controls that exhibit hair loss and skin necrosis. Photo credit: James D. Fisher, University of Pittsburgh.

  • Fig. 2 Recruitment-MP preserves the architectural integrity of intragraft tissues and enhances cutaneous Treg frequency.

    Representative histology from (A) skin and (B) muscle samples from naïve rats, rejecting allografts, and Recruitment-MP–treated allografts at early (POD 33) or late time points [POD > 200; long-term survivor (LTS)]. Tissue was stained with H&E. Scale bars, 100 μm. (C) Immune cell populations in skin of rejecting allografts, Recruitment-MP–treated allografts, and autologous skin from contralateral hindlimbs (POD 29 to 43), as determined by flow cytometry on dissociated skin tissue. The flow cytometry gating strategy is presented in fig. S2. Each dot represents the mean of two 1-cm2 skin biopsies from a single animal (N = 3 for allografts and N = 6 for autologous skin). Teff, effector T cells; NS, not significant. Groups were compared by ANOVA, followed by Tukey’s post hoc tests, and significant differences are indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 Expression of proinflammatory mediators in skin and draining lymph nodes is reduced in Recruitment-MP–treated long-term surviving allografts.

    Relative mRNA expression in (A) skin samples and (B) draining lymph nodes from rejecting hindlimb allografts (N = 9 for skin and N = 11 for draining lymph nodes) or Recruitment-MP–treated long-term surviving allografts (N = 5 for skin and draining lymph nodes). Expression levels are presented as fold changes (2−ΔΔCt) relative to naïve skin (N ≥ 6) or nondraining lymph nodes (N = 7 to 9). Bars represent means ± SD, and dots represent values from individual rats. Groups were compared by Welch’s t test or Mann-Whitney U test, as appropriate, and significant differences are indicated by *P < 0.05, **P < 0.01, or ***P < 0.001.

  • Fig. 4 Functional and phenotypic analysis of CD4+ T cells from animals with long-term surviving hindlimb allografts.

    Flow cytometric analysis of (A) CD4+ FoxP3+ cells (Treg) and (B) CD4+ IFN-γ+ cells (TH1) isolated from allograft draining lymph nodes (DLN) and contralateral nondraining lymph nodes (NDLN) of long-term surviving (LTS) rats treated with 50 mg of Recruitment-MP, and allograft draining lymph nodes from actively rejecting controls (N = 5). Percent Treg and TH1 were normalized to naïve controls. (C) Proliferative capacity of CD4+CD25 Tconv from long-term surviving Recruitment-MP–treated LEW rats with BN allografts, relative to Tconv from naïve LEW rats (N = 3). Tconv were stimulated with BN splenocytes, and percent proliferation normalized to that of naïve LEW Tconv with BN stimulation. (D) CD4+CD25hi Treg isolated from Recruitment-MP–treated animals (N = 4) were more effective on a per-cell basis at suppressing BN-induced proliferation of naïve Tconv than CD4+ CD25hi Treg isolated from naïve LEW rats (N = 3). Groups were compared by ANOVA, followed by Tukey’s post hoc tests (A and B), one-sample t test (C), or Student’s t test (D), and significant differences are indicated by *P < 0.05, **P < 0.01, or ***P < 0.001.

  • Fig. 5 Recruitment-MP confers donor antigen–specific tolerance to animals with long-term surviving grafts.

    (A) CD4+ CD25hi Treg from Recruitment-MP–treated animals (N = 4) were cocultured with CD4+ CD25 Tconv from naïve LEW rats and subject to stimulation with either BN or WF (third-party) splenocytes. Treg isolated from Recruitment-MP–treated animals were more effective at suppressing BN-mediated proliferation than WF-mediated proliferation. Groups were compared by Student’s t test, and a significant difference is indicated by **P < 0.01. (B and C) To test for donor antigen–specific tolerance in vivo, Recruitment-MP–treated animals with long-term surviving BN allografts were challenged with full-thickness nonvascularized skin grafts from LEW (syngeneic control), BN (allogeneic), and WF (allogeneic, third-party) donors. Recruitment-MP–treated animals accepted BN grafts, as evidenced by wound healing and hair growth (C, arrow), but failed to accept third-party WF grafts, as evidenced by contracture and graft necrosis. Additional skin graft images are presented in fig. S3. Photo credit: James D. Fisher, University of Pittsburgh.

  • Fig. 6 Recombinant or synthetic CCL22 induces preferential migration of human Treg to enrich Treg frequency.

    Total CD3+ T cells were isolated from human PBMCs and allowed to migrate through transwell membranes toward different concentrations of recombinant or synthetic CCL22. After 2 hours, migrating cells were collected in the receiver wells and analyzed by flow cytometry (N = 4 wells per group; means ± SD). (A) Frequency of CD4+ CD25+ CD127low Treg among total migrating T cells. Dotted line represents the average Treg frequency among the total CD3+ T cell population before migration (“input”). Groups were compared by two-way independent ANOVA, followed by Dunnett’s test of dose effect. Significant differences compared to the starting population (input) are indicated by *P < 0.05, ***P < 0.001, or ****P < 0.0001. (B) Treg migration index represents the number of CD4+ CD25+ CD127low Treg that migrated toward CCL22, normalized to the average number of Treg that spontaneously migrated in the absence of chemokine. Groups were compared by two-way independent ANOVA, followed by Dunnett’s test of dose effect. Significant differences compared to the no CCL22 control group are indicated by *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001. (C) Representative flow cytometry plots confirming specific FoxP3 expression by CD4+ CD25+ CD127low Treg (blue) and showing greater CCR4 expression by Treg (blue) than conventional CD4+ T cells (red) and CD3+ CD4 T cells (orange).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/11/eaax8429/DC1

    Fig. S1. Characterization of Recruitment-MP and experimental timeline for hindlimb transplantation.

    Fig. S2. Skin flow cytometry gating strategy used for Fig. 2C.

    Fig. S3. Representative images of a hindlimb VCA recipient challenged with LEW, BN, and WF nonvascularized skin grafts.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Characterization of Recruitment-MP and experimental timeline for hindlimb transplantation.
    • Fig. S2. Skin flow cytometry gating strategy used for Fig. 2C.
    • Fig. S3. Representative images of a hindlimb VCA recipient challenged with LEW, BN, and WF nonvascularized skin grafts.

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