Transplant Tolerance, Not Only Clonal Deletion

Abstract

The quest to understand how allogeneic transplanted tissue is not rejected and how tolerance is induced led to fundamental concepts in immunology. First, we review the research that led to the Clonal Deletion theory in the late 1950s that has since dominated the field of immunology and transplantation. At that time many basic mechanisms of immune response were unknown, including the role of lymphocytes and T cells in rejection. These original observations are reassessed by considering T regulatory cells that are produced by thymus of neonates to prevent autoimmunity. Second, we review "operational tolerance" induced in adult rodents and larger animals such as pigs. This can occur spontaneously especially with liver allografts, but also can develop after short courses of a variety of rejection inhibiting therapies. Over time these animals develop alloantigen specific tolerance to the graft but retain the capacity to reject third-party grafts. These animals have a "split tolerance" as peripheral lymphocytes from these animals respond to donor alloantigen in graft versus host assays and in mixed lymphocyte cultures, indicating there is no clonal deletion. Investigation of this phenomenon excludes many mechanisms, including anti-donor antibody blocking rejection as well as anti-idiotypic responses mediated by antibody or T cells. This split tolerance is transferred to a second immune-depleted host by T cells that retain the capacity to effect rejection of third-party grafts by the same host. Third, we review research on alloantigen specific inhibitory T cells that led to the first identification of the CD4⁺CD25⁺T regulatory cell. The key role of T cell derived cytokines, other than IL-2, in promoting survival and expansion of antigen specific T regulatory cells that mediate transplant tolerance is reviewed. The precise methods for inducing and diagnosing operational tolerance remain to be defined, but antigen specific T regulatory cells are key mediators.

Keywords: chimerism; clonal deletion; graft versus host disease; regulatory T (Treg) cells; transplant tolerance.

Figure 1

Timeline of observations that supported clonal deletion in utero and in neonates. A chronological representation of studies that led to establishment of clonal deletion to explain tolerance.

Figure 2

Discoveries Timeline related to suppressor regulatory T cells. Timeline of major discoveries that led to recognition of role of T regulatory cells in tolerance specially in antigen specific tolerance.

Figure 3

A schematic representation of two subpopulations of CD4⁺T cells produced by the thymus and one of several pathways for their activation by an antigen and cytokines in peripheral lymphoid tissues and sites of inflammation. The activation by an antigen of effector lineage CD4⁺CD25^-CD127⁺CD45RA⁺Foxp3^- cells induces them to produce cytokines that promotes activation of CD25⁺CD127^loCD45RA⁺Foxp3⁺Treg that have been activated by antigen. This figure shows the parallel pathways of activation of effector and regulatory CD4⁺T cells, when producing and being activated by Type-1 cytokines. The cytokines produced by the effector cells are required for the full activation of Treg. Both lineages of cells have been produced by thymus and have migrated to peripheral lymphoid tissue. Their subsequently recirculation from lymphoid tissue to blood and back to lymphoid tissue, is promoted by expression of CD62L and CCR7. This recirculation increases their chances of recognizing antigens. In peripheral lymphoid tissue upon recognition of an antigen, both effector and regulatory CD4⁺T cell populations are activated and proliferate. Effector lineage CD4⁺T cells start producing IL-2 and express IL-2R including CD25 (IL-2Ra chain). Naïve resting Treg expand polyclonally. During an immune response naïve/resting CD4⁺CD25⁺CD127^loCD45RA⁺Foxp3⁺T-bet^-CCR7⁺Treg are activated by an antigen and the IL-2 produced by activated T effector cells and are induced to express the receptor for late Th1 cytokines IL-12 and IFN−γ. Naïve CD4⁺CD25⁺CD127⁺CD45RA⁺Foxp3^-T-bet^-CCR7⁺T cells also acquire CD25, Foxp3 and T-bet expression but no longer express CD45RA. Transient expression of Foxp3 and CD25 on activated effector T cells blurs the distinction between Treg and effector T cells. In the event of ongoing immune response, activated T effector cells, in the presence of IL-2 and IFN-γ get further activated to express the transcription factor t-bet and the chemokine receptor CXCR3. These activated effector CD4⁺T cells produce IFN-γ, which together with IL-12 further activate Treg to Th1-like Treg (CD4⁺CD25^hiCD127^loCD45RA^-Foxp3^hiT-bet⁺IFN-γ⁺ CXCR3⁺). Th1-like Treg express mRNA for Th1 transcription factor T-bet, Th1 cytokine IFN-γ and Th1 chemokine receptor CXCR3. Expression of CXCR3 enables these Treg to migrate to inflamed tissues, where they control immune inflammation as in the graft and promote tolerance. Th-like Treg, such as Th1-like Treg are the mediators of transplant tolerance and are a hundred to a thousand-fold more potent at suppression of rejection than naïve resting Treg. This figure only represents one pathway of activation of Treg and there are others such as Th-2 like Treg promoted by Th2 cells and Type-2 cytokines. The survival of highly activated Treg is dependent on continued antigen stimulation and key cytokines produced by the inflammatory response, IL-2 alone does not sustain these cells and may inhibit them.