MOESM8 of Generation of donor-specific Tr1 cells to be used after kidney transplantation and definition of the timing of their in vivo infusion in the presence of immunosuppression

Additional file 8. Tr1 cell sorting gating strategy

Prolonged, low-dose mATG with a short course of CTLA4-Ig and induction mATG achieves long-term graft survival and favors the emergence of Tregs.

<p><b>A.</b> Kaplan-Meier graph depicting fully MHC-mismatched skin allograft survival following the administration of pld-mATG, i-mATG, i-mATG plus pld-mATG, or no treatment. <b>B.</b> Kaplan-Meier graphs depicting fully MHC-mismatched skin allograft survival in mice administered i-mATG alone, CTLA4-Ig alone,CTLA4-Ig in combination with i-mATG with/without pld-mATG, or no treatment. <b>C.</b> Representative pictures of skin graft from untreated (left panel) and i-mATG plus CTLA4-Ig and pld-mATG-treated mice (right panel). <b>D/E.</b> Bar graphs depicting absolute numbers of CD4⁺ and CD8⁺ cells 7–10 days post-transplantation in mice administered either i-mATG, pld-mATG, CTLA4-Ig or no treatment. <b>F.</b> Bar graphs depicting absolute numbers of CD4⁺CD25⁺FoxP3⁺ cells (Tregs) 7–10 days post-transplantation in mice administered either i-mATG, pld-mATG, CTLA4-Ig or no treatment. Data are representative of ≥3 independent experiments using ≥3 mice per group per timepoint studied.</p

Prolonged graft survival is achieved by inhibiting effector T cells/alloreactive IFNγ secretion and by favoring the emergence of Tregs. A/B.

<p>Graphs demonstrating absolute numbers of CD4⁺ and CD8⁺ T cells with various treatment strategies. <b>C/D.</b> Frequency of Teff cells (CD4⁺/CD8⁺) using calculated absolute numbers at different timepoints in various treatment groups. <b>E.</b> Comparison of donor alloreactive IFN<b>γ</b> production at different timepoints after fully MHC-mismatched skin transplantation in animals administered various treatment protocols. <b>F.</b> Absolute numbers of Tregs at different time points in mice treated with i-mATG, CTLA4-Ig plus pld-mATG. <b>G/H/I.</b> Dot plots representing Tregs/CD4+ ratios in mice treated with pld-mATG alone, i-mATG plus pld-mATGor i-mATG combined with CTLA4-Ig plus pld-mATG during the transplant course. Data are representative of ≥3 independent experiments using ≥3 mice per group per timepoint studied.</p

Treatment with i-mATG+pld-mATG+prolonged CTLA4-Ig induces very-long-term graft survival, an effect primarily due to pld-mATG.

<p><b>A.</b> Skin allograft survival in mice treated with i-mATG, pCTLA4-Ig plus pld-mATG: on day 90 mice were randomized either to cessation of both treatments or to extended treatment with CTLA4-Ig or pld-mATG alone. <b>B/C.</b> Absolute numbers of CD4⁺/CD8⁺ cells at different timepoints post-transplantation in mice treated with i-mATG with pCTLA4-Ig and pld-mATG until day 90 and thereafter randomized to pld-mATG or CTLA4-Ig alone or no further treatment. Data are representative of ≥3 independent experiments using ≥3 mice per group per timepoint studied.</p

Day 90 randomization to pld-mATG enhances graft survival by favoring persistent Treg emergence..

<p><b>A/B.</b> Frequency of CD4⁺/CD8⁺ effector T cells at different timepoints in mice treated with i-mATG, pCTLA4-Ig plus pld-mATG until day 90 and thereafter randomized to extended pld-mATG or CTLA4-Ig or no further treatment. <b>C.</b> Alloreactive IFNγ production at different timepoints in mice treated with i-mATG, pCTLA4-Ig plus pld-mATG until day 90 and thereafter randomized to extended pld-mATG or CTLA4-Ig or no further treatment. <b>D.</b> Absolute numbers of Tregs at different timepoints in skin transplant recipients randomized at day 90 to either extended pld-mATG or CTLA4-Ig or no further treatment. <b>E/F.</b> Treg/CD4⁺ratios at different timepoints after transplantation in mice treated with i-mATG, pCTLA4-Ig plus pld-mATG until day 90, and thereafter randomized to extended pld-mATG or CTLA4-Ig alone. Data are representative of ≥3 independent experiments using ≥3 mice per group per timepoint studied.</p

Skin allograft acceptance requires the expansion of host natural Tregs (nTregs) by prolonged, low-dose mATG. A/B/C.

<p>Kaplan-Meier graphs depicting fully MHC-mismatched skin allograft survival with or without CD25⁺ cell depletion in mice treated with CTLA4-Ig alone (A), pld-mATG alone (B) or i-mATG, CTLA4-Ig plus pld-mATG (C). <b>D.</b> Bar graphs depicting absolute numbers of Thy1.2⁺FoxP3GFP⁺ cells in untreated and mATG-treated groups on day 7. Representative histograms of Thy1.2⁺FoxP3GFP⁺ cells from recipients in each treatment group are shown below the respective bar graphs.</p