Targeting Tim-1 to overcome resistance to transplantation tolerance mediated by CD8 T17 cells

Abstract

The ability to induce durable transplantation tolerance predictably and consistently in the clinic is a highly desired but elusive goal. Progress is hampered by lack of appropriate experimental models in which to study resistance to transplantation tolerance. Here, we demonstrate that T helper 1-associated T box 21 transcription factor (Tbet) KO recipients exhibit allograft tolerance resistance specifically mediated by IL-17-producing CD8 T (T17) cells. Neutralization of IL-17 facilitates long-term cardiac allograft survival with combined T cell co-stimulation (CD28-CD80/86 and CD154-CD40) blockade in Tbet KO recipients. We have used this T17-biased Tbet KO model of allograft tolerance resistance to study the impact of targeting a T cell-co-stimulatory pathway, and demonstrate that targeting T cell Ig and mucin domain-1 (Tim-1) with anti-Tim-1 overcomes this resistance by specifically inhibiting the pathogenic IL-17-producing CD8 T17 cells. These data indicate that in the absence of Th1 immunity, CD8 T17 alloreactivity constitutes a barrier to transplantation tolerance. Targeting TIM-1 provides an approach to overcome resistance to tolerance in clinical transplantation.

Conflict of interest statement

Conflict of interest statement: L.H.G. is on the Board of Directors and holds equity in Bristol Myers Squibb Pharmaceutical Company.

Figures

Fig. 1.

Tbet deficiency accelerates acute cardiac allograft rejection characterized by intense PMN infiltration and T17/Th2 skewing of alloantigen specific cytokine profile. (A) Kaplan-Meier survival curves of fully mismatched (BALB/c into B6) heart allografts in untreated WT and Tbet KO recipients. Each group had 5 to 7 animals. (B) Representative photomicrograph of an H&E-stained sections of cardiac allografts in WT and Tbet KO recipients. Note the dense perivascular PMN (neutrophils and eosinophils) and intramural inflammatory infiltrates in the Tbet KO recipient. (C) Bar graph illustrating Th1, Th2, and T17 proinflammatory cytokine production by the splenocytes of WT and Tbet KO recipients, 7 days after transplantation. Presented are mean ± SD; representative of at least 3 independent experiments.

Fig. 2.

Tbet deficiency prevents induction of transplantation tolerance by combined co-stimulation blockade with persistent T17/Th2 skewing of alloantigen specific cytokine profile, and PMN, CD4, and IL-17-producing CD8 T cell infiltration. (A) Kaplan-Meier survival curves of fully mismatched cardiac allografts in WT and Tbet KO recipients treated with CTLA4Ig+MR1. Each group had 5 to 7 animals. (B) Bar graph illustrating Th1, Th2, and T17 proinflammatory cytokine production by the splenocytes of WT and Tbet KO recipients 14 days after transplantation of heart allograft. Presented are mean ± SD; representative of at least 3 independent experiments. (C) Representative photomicrographs of H&E stained sections of cardiac allografts in WT and Tbet KO recipients treated with CTLA4Ig+MR1. Note the persistent perivascular PMN (including neutrophils and eosinophils) and inflammatory infiltrates in the Tbet KO recipient. (D) Representative photomicrographs of immunofluorescence staining and confocal microscopy of heart allografts harvested 2 weeks after transplantation for IL-17 expression by CD4 and CD8 T cells (400× magnification). Green represents either CD4 or CD8; red represents IL-17; yellow in the merged image represents double staining for IL-17 and CD4 or CD8; Blue represent nuclear staining with DAPI. IL-17 expression by most of the CD8 (Top Right) graft infiltrating T cells in the untreated Tbet KO recipients (Upper) is seen in contrast to WT recipients where only a few of the infiltrating T cells express IL-17. Note the diminished but persistent IL-17-producing CD8 T cell infiltrates (Lower Right) in the Tbet KO recipient treated with CTLA4Ig+MR1 (Lower). Presented are results from 1 experiment and are representative of 3 independent experiments.

Fig. 3.

CD8 but not CD4 T cells mediate resistance to allograft tolerance in Tbet KO recipients. (A) Survival of fully allogeneic cardiac graft in CD4/Tbet or CD8/Tbet DKO recipients treated with CTLA4Ig+MR1 (n = 5 for CD4/Tbet DKO group, and n = 6 for CD8/Tbet DKO and control Tbet KO groups). Survival data presented as Kaplan-Meier plot. (B) Immunofluorescence staining and confocal microscopy of heart allografts harvested 2 weeks after transplantation for IL-17 expression by CD4 and CD8 T cells (400× magnification). CD4 and CD8 T cells infiltrate in the CD8/Tbet DKO and CD4/Tbet DKO, respectively, and only CD8 T cells produce IL-17 (Fig. 3B Upper). Note absence of CD4 T cell infiltration in CD8/Tbet DKO recipients but grafts from CD4/Tbet DKO recipients show reduced but persistent IL-17 producing CD8 T cell infiltration (Fig. 3B Lower). (C) Bar graph illustrating Th1, Th2 and T17 proinflammatory cytokine production by the splenocytes of CD4/Tbet and CD8/Tbet DKO recipients 14 days after transplantation of heart grafts, treated with (Upper) or without CTLA4Ig+MR1 (Lower). Presented are mean ± SD; representative of at least 3 independent experiments.

Fig. 4.

In vivo IL-17 neutralization inhibits rejection and facilitates allograft survival with combined co-stimulation blockade in Tbet KO recipients. (A) Fully mismatched cardiac allograft survival in Tbet KO recipients treated with CTLA4Ig+MR1 and anti-IL-17 mAb (n = 4) or control IgG (n = 6). Survival data presented as a Kaplan-Meier plot. (B) Bar graph illustrating Th1, Th2, and T17 proinflammatory cytokine production by the splenocytes of Tbet KO recipients 14 and 21 days after transplantation of heart graft. Presented are mean ± SD; representative of at least 3 independent experiments.

Fig. 5.

Tim-1, but not ICOS, blockade restores the ability of combined costimulation blockade to induce transplantation tolerance in Tbet KO recipients. (A) Fully mismatched cardiac allograft survival in Tbet KO recipients treated with CTLA4Ig+MR1 and anti-Tim-1 mAb or anti-ICOS mAb (n = 6 for all 3 groups). Survival data presented as a Kaplan-Meier plot. (B) Representative photomicrograph of an H&E-stained section of cardiac allograft, > 100 days after transplantation, in Tbet KO recipients treated with anti-Tim-1 mAb. Note the absence of inflammatory infiltrates and signs of acute or chronic rejection in the Tbet KO recipients treated with anti-Tim-1 mAb. (C) Bar graph illustrating Th1, Th2, and T17 proinflammatory cytokine production by the splenocytes of Tbet KO recipients 14 days after transplantation heart graft. Presented are mean ± SD; representative of at least 3 independent experiments.