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. 2016 Oct;16(10):2854-2864.
doi: 10.1111/ajt.13814. Epub 2016 May 5.

Tracking of TCR-Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice

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Free PMC article

Tracking of TCR-Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice

M L Miller et al. Am J Transplant. .
Free PMC article

Abstract

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long-lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25+ regulatory T cells (Tregs), blocking programmed death ligand-1 (PD-L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD-1/PD-L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.

Keywords: T cell biology; animal models: murine; basic (laboratory) research/science; heart transplantation/cardiology; immunobiology; tolerance: costimulation blockade; tolerance: mechanisms.

Conflict of interest statement

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Figures

Figure 1:
Figure 1:. Alloreactive T cells seeded in tolerant mice are not completely deleted and are not ignorant of the graft, but are maintained at low numbers
A. Experimental design. B. The percentages and total numbers of CD4+ TCR75 cells recovered from the spleen and peripheral lymph nodes 60 days post adoptive transfer into naïve untransplanted C57BL/6 mice, mice receiving BALB/c donor splenocytes (DST) and mice receiving BALB/c donor splenocytes, anti-CD154, and a heart transplant (Tol). n=3 mice per cell dose (103, 104, 105) per group. Different cell doses are represented by different symbols. C. Representative flow plots of percentages of CD44hi cells amongst TCR75 cells and their quantification. Different cell doses were pooled. Mean values were compared using one-way ANOVA with Bonferroni correction for pairwise comparisons ns=not significant, ***p<0.001.
Figure 2:
Figure 2:. Transfer of a high dose (HD) but not low dose (LD) of alloreactive T cells is sufficient to break established tolerance
A. Experimental design. B. Graft survival in tolerant mice that were adoptively transferred at least 60 days post-transplantation with 105 TCR75 cells (low dose, LD, n=9) or 2–4 × 106 cells (high dose, HD, n=13), p<0.05 by log-rank test.
Figure 3:
Figure 3:. Low dose (LD) alloreactive T cells seeded during the maintenance phase of tolerance are well controlled
A. Experimental design. B. Representative plots of CFSE dilution of 2.5 × 105 TCR75 cells adoptively transferred into naïve mice (Naïve+LD, n=3), or into mice undergoing acute rejection (AR+LD, n=3), or into tolerant mice (Tol+LD, n=3), at four days post-transfer in the spleen. C. Percentages of TCR75 cells expressing CD44hi in the spleen and heart graft 8 days post adoptive transfer. Percentages (D) and total numbers (E) of TCR75 cells recovered in the spleen and graft 8 days post adoptive transfer. Percentages of TCR75 cells expressing IFNγ and TNFα upon PMA/ionomycin restimulation (F) and PD-1 without restimulation (G) 8 days after adoptive transfer. C-G. Results were pooled from two or more independent experiments. Mean values were compared using one-way ANOVA with Bonferroni correction for pairwise comparisons or Student’s t test where appropriate. ns=not significant, *p<0.05, **p<0.01, ***p<0.001.
Figure 4:
Figure 4:. HD alloreactive T cells are controlled in the periphery of tolerant mice but not in the graft
A-C. TCR75 cells injected at high doses (2–4 × 106 cells, HD) into naïve mice or tolerant mice 60 days post transplantation and were recovered from spleens 5, 10, or 14 days later (n=2–4 mice per group per time point). A. Total numbers of TCR75 cells in the spleen. Percentages of splenic TCR75 cells expressing IFNγ and TNFα upon PMA/ionomycin restimulation (B) and PD-1 without restimulation (C) on the indicated days post adoptive transfer. Mean values for each time point were compared using two-way ANOVA with Bonferroni correction for pairwise comparisons ###p<0.001, between tolerant and naïve groups ***p<0.001, between tolerant and acute rejection groups. D-F. Graft-infiltrating cells were analyzed 7–14 days post adoptive transfer into tolerant mice (n=10–12 mice per group). D. Percentages and total numbers of Foxp3+ cells within TCR75 and endogenous CD4+ T cells in the graft. E. Total numbers of graft-infiltrating TCR75 cells and endogenous CD4+ and CD8+ T cells. F. Total numbers of IFNγ-producing graft-infiltrating TCR75 cells and endogenous CD4+ and CD8+ T cells. Results were pooled from three or more independent experiments. Mean values of total graft T cells were compared using one-way ANOVA with Bonferroni correction for pairwise comparisons *p<0.05, **p<0.01, ***p<0.001.
Figure 5:
Figure 5:. Maintenance of tolerance of primary grafts is dependent on controlling the size of the alloreactive T cell pool through regulatory T cells and signals from PD-L1
A. C57BL/6 mice were transplanted with BALB/c hearts and treated with anti-CD154+DST. On day >45 post-transplantation, tolerant recipients were treated with individual or combined treatments to block pathways of peripheral tolerance. B. Graft survival following transfer of naïve TCR75 T cell (105), blockade of PD-L1, or administration of anti-CD25 alone or in combination. Graft rejection was significantly induced only when all 3 therapies were combined (triangles, p<0.05).

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