The 2004 FASEB Summer Research Conference on Transplant Immunology: Closer to the Goal of Transplant-Specific Tolerance

T and APC interaction

T cell activation requires interaction of T cell receptors (TCR) and major histocompatibility complex (MHC)-peptide complexes in the nanometer scale between T lymphocytes and antigen presenting cells (APCs). In addition to antigen receptor, there are essential interactions of adhesion, costimulatory and inhibitory molecules that are organized within the immunological synapse (IS) (1). On the basis of their functional outcome, co-signaling molecules can be divided into co-stimulators and co-inhibitors, which promote and suppress T cell activation, respectively, by positively and negatively controlling priming, growth, differentiation, and functional maturation of a T cell response. The IS has proven to be a provocative concept, particularly in stimulating discussion on similarities among intercellular communication mechanisms controlling disparate biological processes, including the choice between immunity and tolerance. Recent studies have clarified some of the underlying molecular mechanisms and functions of the IS.

In light of this, a major concept emerging from this FASEB meeting was that the state of T cell activation (i.e., effector versus anergic) as well the type of APC (i.e., tolerogenic versus immunogenic) can profoundly condition the IS and the outcome of an immune response. Work presented from Mike Dustin's lab (New York University School of Medicine, NY) showed that anergic T cells exhibit impaired calcium mobilization after TCR triggering and are unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Another major player of the IS is a professional APC often represented by a dendritic cell (DC), and certain DC subsets appear to possess the unique ability to cross-present exogenous antigens. Gabrielle Belz (University of Melbourne, Australia) presented results indicating that CD8α⁺ murine bone marrow-derived DCs can uniquely cross-present antigens, although other signals provided to the T cells by DCs are crucial in determining the type (tolerogenic or immunogenic) and quality of primary and memory T cell responses.

Regulation of immune responses by dendritic cell subsets

DC subsets are characterized by high plasticity and can perform various functions depending on environmental factors (2). An emerging theme is that professional APCs are not only involved in the activation of T cell responses, but also act in maintaining immune homeostasis. There is a large body of evidence indicating that DCs exert regulatory functions in the process of tolerance induction. Immaturity of DCs, bone marrow lineage, treatment with specific molecules, location after transplantation and environment seem to be the prerequisites for displaying their tolerogenic activity. Robert Lechler (Imperial College, London, UK) described a novel approach to inducing indirect pathway regulatory T cells in a rat model. DCs co-expressing donor and recipient MHC molecules and induced to become tolerogenic by treatment with dexamethasone (DEX) were shown to render recipient T cells unresponsive to indirectly presented alloantigens. This led to indefinite survival of kidney grafts after a short course of cyclosporine used to inhibit the early direct pathway response in a mouse model. Activation of immature DCs in the presence of DEX results in alternatively matured DCs that present antigen in the absence of a proper co-stimulatory context (3).

There is increasing evidence that DCs display opposite functions in the immune system, as they may induce immunity or tolerance depending on intrinsic and environmental factors. In particular, Francesca Fallarino (University of Perugia, Italy) showed data that indicate that reverse signaling through B7 molecules as well CD200R engagement in DCs can induce the expression of high levels of the enzyme indoleamine 2,3-dioxygenase (IDO) in DC subsets. Through localized tryptophan deficiency combined with the release of proapoptotic catabolites, DCs appear to exert an IDO-dependent homeostatic control over the proliferation and survival of peripheral T cells and promote antigen-specific tolerance (Andrew Mellor, Medical College of Georgia, Augusta, GA).

Regulation of immune responses by T cells

It is now increasingly appreciated that tolerance is a highly active process, with tolerogenic DCs and also heterogeneous regulatory T cells (Tregs, Tr1, and NKT cells) being the major players. DCs can induce the differentiation of specific regulatory T cell subsets and meanwhile the same regulatory T cell can modulate DC functions. CD4⁺CD25⁺ (Tregs) are a distinct population of T lymphocytes that have the capacity to dominantly suppress the proliferation of responder T cells in vitro and inhibit autoimmune disease in vivo (5). Despite expression of all three subunits of the high-affinity IL-2R, CD4⁺CD25⁺ T cells remain hypoproliferative in response to IL-2 alone, suggesting a distinct IL-2R signaling pattern. Larry Turka (University of Pennsylvania, PA) showed data to that effect, demonstrating that engagement of the IL-2R on Tregs activates JAK/STAT signaling, as expected, but fails to activate downstream targets of the PI3K (phosphatidylinositol 3-kinase) signaling pathway, such as Akt or p70^s6kinase. This inability was correlated with the expression of the lipid phosphatase and tensin homologue deleted on chromosome 10, PTEN.

In several murine models but not yet in humans it has been shown that Tregs are crucial in regulating organ-specific autoimmune diseases and inducing tolerance to allogeneic organ transplants. However, the role of Tregs in human health and diseases is yet to be clarified. In addition, in other preclinical models it has been reported that high numbers of Tregs can also modulate graft-versus-host disease (GVHD) if administered in conjunction with allogeneic hematopoietic stem cell transplantation in mice. In a clinical setting, it may prove difficult to obtain sufficient numbers of Tregs to achieve a therapeutic effect, motivating a search for methods for their ex vivo expansion. Along these lines, Bruce Blazar (University of Minnesota, MN) described conditions for stimulating Tregs using cell-sized beads coated with anti-CD3 and anti-CD28 monoclonal antibodies along with CD4⁺ feeder cells and IL-2. In this system, it was possible to obtain marked CD4⁺/CD25⁺ cell expansion, in the range of 100-fold, with retention and enhancement of regulatory function. Suppressive activity was associated with L-selectin expression by the Tregs. Importantly, at this time there is no clear answer to whether or not in vitro expanded Tregs may need to be specific for alloantigens in order to suppress.

Other subsets of regulatory T cells retain their suppressive activity after in vitro culture. In this regard, Li Zhang (University of Toronto, Canada) showed that in vitro propagated TCR⁺CD3⁺CD4⁻CD8⁻ T cell clones that were expanded after donor lymphocyte infusion (DLI) were able to suppress allogeneic immune responses by specifically killing donor-reactive CD8⁺ T cells in vitro and to prolong cardiac allograft survival by injection in vivo. In addition to the well-known CD4⁺ regulatory T cell subset characterized by the expression of the CD25 molecule, other T cells have been associated with immunoregulatory functions. Among them, CD1d-restricted natural killer T (NKT) cells exhibiting a limited TCR repertoire can act not only in the defense against pathogens, but also regulate adaptive immune responses. Joan Stein-Streilein (Schepens Eyes Research Institute, Boston, MA) showed that NKT cells stimulated by F4/80⁺ APCs are essential for both anterior chamber associated immune deviation (ACAID) and corneal graft survival. In vivo activation of NKT cell populations can be achieved by α-galactosyl ceramide KRN 7000 with strong agonist properties for the TCR of NKT cells. Activated CD1-restricted T cells carry out effector, helper, and adjuvant-like functions and interact with other cell types including macrophages, DCs, NK, T, and B cells, thereby contributing to both innate and adaptive immune responses (4). Therefore it will be of interest to identify selective NKT cell ligands capable of activating specific NKT functions such as Th1 or Th2 responses. Interestingly, Steve Porcelli (Albert Einstein College of Medicine, Bronx, NY) showed several compounds containing altered fatty acid chains that stimulate strong release of Th2-associated but not Th1 cytokines that appear to be superior to KRN7000 at blocking the development of insulitis in NOD mice.

T and B cell memory responses

Special attention has been focused on the mechanism governing the development of memory T cells, which aid in rapid protection from previously encountered pathogens. Ironically, the features of memory T cells beneficial in the setting of a pathogen may instead be deleterious in the setting of organ transplantation. Memory T cells have lower activation thresholds and costimulatory requirements than naive T cells and, therefore, memory T cells are likely to be resistant to the effects of costimulatory blockade (6). Peter S. Heeger (The Cleveland Clinic Foundation, OH) communicated interesting data suggesting that alloreactive CD4⁺ T cells resistant to costimulatory blockade and with a memory phenotype do not directly mediate graft destruction, but indirectly induce anti-donor CD8⁺ T effector cells and antibody responses. The specific interference with both alloreactive memory CD4⁺ and CD8⁺ T cells has indeed important implications for designing approaches to induce tolerance in human transplant recipients. A fundamental, as yet unanswered question is what determines which effector T cells will survive to become long-lived memory cells, and which will die. Susan M. Kaech (Yale University, New Haven, CT) suggested that a potential marker for the ‘memory-destined’ effector CD8⁺ T cells is in fact increased IL-7 receptor α-chain (IL-7Rα). Her results suggest that specific growth factors may be required to maintain a memory T cell phenotype and this could be a general principle applying to B cells as well. In this regard, Rudolf A. Manz (Humbold University, Berlin, Germany) reported that long-lived plasma cells producing autoantibodies can survive only in specific niches in the bone marrow and these cells are relatively resistant to immunosuppressive treatment including cyclophosphamide. The number of niches increases in patients with autoimmune diseases.

B  cell  tolerance

Graft-reactive antibodies directed against ABO-incompatible carbohydrate antigens or classical alloantigens contribute to allograft rejection and therefore approaches capable of inducing B cell tolerance to both classes of antigens might be of use to improve allograft survival. Anita Chong (University of Chicago, IL) showed that transient anti-CD40L administration associated with the injection of donor derived intact active bone (IAB) induces a vigorous tolerance, resulting in suppression to both alloantibodies as well anti-Gal antibodies. This effect did not require the depletion of pre-existing T cells in a cardiac allograft model. These data provide experimental evidence that peripheral mechanisms can effectively inhibit donor-reactive B cells. Another promising tolerogenic approach is mixed hematopoietic chimerism, which can simultaneously tolerize both xenoreactive T cells and, by different mechanisms, both pre-existing and newly developing B-1 cells that recognize Gal and other xenodeterminants (Megan Sykes, Massachusetts General Hospital, Harvard Medical School, Boston). Further, xenogeneic tissues are subject to robust immune rejection, and so tolerance induction is essential to the success of clinical xenotransplantation. Sykes and her colleagues showed that in porcine hematopoietic chimeras, human thymus grafts are populated with porcine class II (high) cells in addition to human cells, and human T cells are rendered tolerant of the porcine hematopoietic donor. This study proves the principle that porcine chimerism is able to induce tolerance of xenoreactive human T cells. An expansion of this approach can be the use of organs from cloned genetically modified animals. Recently, pigs deficient in the gene for α 1,3 galactosyltransferase have been generated (8). Interestingly, Yifan Dai (University of Pittsburgh, PA) reported that no hyperacute rejection was observed when organs from those pigs were transplanted into non-human primates. However an acute vascular rejection started five days after transplantation, suggesting that additional genetic modifications are needed to overcome the potential of chronic rejection.

Manipulating vascular inflammation

These latter data clearly emphasize that the interference with vascular lesions that can cause tissue hypoxia, injury and local inflammation can result in increased graft survival. Interestingly, Miguel Soares (Gulbenkian Institute of Science Inflammation, Oeiras, Purtugal) showed that expression of heme oxygenase (HO-1), a stress responsive gene that catabolizes heme into iron, carbon monoxide (CO) and biliverdin in the graft vasculature, can suppress the pathogenesis of chronic graft rejection by a direct interference with the inflammatory response (CO) and T cell activation (biliverdin). Another way to modulate graft vascular lesions can be represented by the use of anticoagulants. By generating different transgenic mice expressing anticoagulants specifically on the platelets, endothelial cells (EC) or leukocytes, Anthony Dorling (Imperial College, London) reported on the effects of selective expression of anticoagulants on EC and not at the other sites. The effect was the unexpected inhibition of even chronic rejection. Interestingly, along the same lines, Jonathan Bromberg (Mount Sinai School of Medicine, New York, NY) reported that the effects of the immunosuppressive activity of the drug FTY720 may be now interpreted as a result of inhibiting T cell migration, through agonistic interaction with the sphingosine-1- phosphate receptor-1 (S1P1). Additionally, a new action on endothelial cells was described in which FTY720 preserves vascular integrity by enhancing adherens junction assembly and endothelial barrier functions.

Bone marrow transplantation

Allogeneic hematopoietic cell transplantation (HCT) has emerged as an important and increasingly utilized treatment modality for hematologic malignancies (9). Interestingly, the use of relatively non-toxic, nonmyeloablative total body irradiation (TBI)-based conditioning regimens results in donor engraftment leading to the development of graft-versus-tumor (GVT) effect after allogeneic HCT. Preliminary results from 600 patients receiving HCT from HLA-matched related or unrelated donors indicate that this approach is potentially curative for susceptible pathologies with a reduced risk of treatment-related morbidity and mortality (Brenda Sandmaier, Fred Hutchinson Cancer Research, Seattle, WA).

Impaired immune reconstitution has significant problems limiting allogeneic bone marrow transplantation (BMT), and therapies to enhance immune reconstitution would be important. Interleukin 7 (IL-7) is the most potent thymopoietic cytokine identified thus far. Christal Mackall (National Cancer Institute of Health, Bethesda, MD) reported that IL-7 lowers the threshold for the development of clinically significant GVHD in mice, increases the rate of GVHD lethality for a given T-cell dose, and induces more pronounced tissue inflammation in GVHD target organs. These results must be considered carefully as preparations for the clinical development of this agent in allogeneic BMT proceed. In addition, based on the double effect of the cytokine she pointed to its possible use as immune adjuvant in the design of antitumor immunotherapy approaches.

Donor T cells in the allograft are vital for promoting engraftment, eradicating malignant cells (the graft-versus-leukemia GVL effect), and reconstituting immunity. Unfortunately, they mediate GVHD, which is an attack on recipient tissues. T cell depletion prevents GVHD but increases the risk of graft rejection and leukemic relapse. In human transplants, Andrea Velardi (University of Perugia, Italy) showed that donor-versus-recipient natural killer (NK)-cell alloreactivity could reduce leukemia relapse and graft rejection without inducing GVHD. Interestingly, in mice the pretransplant infusion of alloreactive NK cells obviates the need for high-intensity conditioning and reduces GVHD. NK cell alloreactivity may thus provide a powerful tool for enhancing the efficacy and safety of allogeneic hematopoietic transplantation.

New technologies for immune monitoring

Profiles of global gene and protein expression have recently begun to demonstrate significance in many aspects of biological research, including pathway mapping, better understanding of gene regulation and assessment of extracellular stimuli on gene/protein expression. Microarrays or gene chips are exciting investigative tools for analyzing expression changes across thousands of genes in concert in tissues and cells of interest. Interestingly, Minnie Sarwal (Stanford University School of Medicine, Stanford, CA) reported data on the recent application of microarrays to transplant research, especially for unraveling the staging of rejection, stratifying patients towards more individualized treatment regimes, and discovering noninvasive biomarkers for monitoring of intragraft events.

The gene profiles have to be confirmed by protein expression and here several technologies are needed to integrate all these pieces of information. Related to this issue, Vicki Seyfert (Immune Tolerance Network, Bethesda, MD) described that the Immune Tolerance Network (ITN http://www.immunetolerance.org) has embarked on a strategy to utilize techniques such as real time PCR, macroarrays, ELISPOT analysis, or tetramers, together with new complex computational modeling algorithms to assess immune cell functions following administration of tolerogenic drugs or following activation of tolerogenic pathways in the transplant setting. The enormous amount of information coming from such these studies should help us to detect new molecules involved in early transplant rejection and to define specific markers of tolerance initiation, maintenance or breakdown.