The scientific objectives of the ITN are:

To conduct clinical trials at all phases to determine the safety, toxicity and efficacy of promising tolerogenic strategies in islet, kidney and liver transplantation, autoimmune diseases and allergy & asthma

To investigate the basic mechanisms of immune tolerance in these diseases as an integral part of clinical trials

To develop and/or refine and validate, immune and surrogate marker assays to monitor the induction, maintenance and loss of tolerance in these diseases

Research supported by the ITN comes from a year-round, open call for proposals from academic and industry researchers from around the world. Since October 1999, the ITN has approved more than 15 clinical trials and 10 tolerance assays or core facilities for further development. A number of trials are now underway and core facilities, including a clinical sample repository are operational and analyzing samples from ITN trials.

Targets for Clinical Tolerance Induction

Trials implemented within the ITN generally target the normal biological processes generally thought to regulate tolerance, which are based on four mechanisms: clonal deletion, clonal inactivation, cytokine-dependent immune deviation and suppression (1). Each operates to varying degrees in the generation and maintenance of tolerance, although their relative contribution may vary depending on the nature of the antigen and the location in which "tolerization" occurs.

The primary mediators of immune reactivity in autoimmune, allergy, asthma, and transplant settings are T and B lymphocytes. T lymphocytes have antigen-specific receptors that recognize foreign and self antigens, and B lymphocytes produce antibodies that are reactive against foreign and self tissues. Accessory cells known as antigen-presenting cells (APC) activate naïve T cells by presentation of the antigen together with major histocompatibility complex (MHC) antigens, the primary targets for allorecognition. However, as illustrated in Figure 1, the activation of T and B cells requires signaling not only through the T and B cell receptors (TCR/BCR; Signal 1) but also through co-stimulatory pathways (Signal 2) (2). After activation, a number of cell surface and soluble molecules are known to regulate further the immune response. The number of cell surface molecules that are known to regulate T-cell activation is growing, and more complete descriptions of the molecular actions of proapoptotic molecules such as Fas, Trance, and tumor necrosis factor (TNF) have been compiled (1). Each of these mechanisms provides potential targets for therapeutic interventions, many of which are rapidly nearing clinical application.

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Figure 1. T-cell activation requires signaling through the T-cell receptor (TCR) (Signal 1) and co-stimulatory pathways (Signal 2). For example, antigen/MHC binding to the TCR and CD28 interaction with B7.1 or B7.2 expressed on the surface of the antigen presenting cells (APC). Both of these signal pathways allow opportunity for intervention in immune-mediated diseases.

Signal 1: An increasing knowledge of the various steps in T-cell activation through the T-cell receptor (TCR) has provided a number of opportunities for intervention. It is now known that the process requires binding of the antigen/MHC complex to the TCR/CD3 complex. This event initiates a cascade of signaling that begins with the activation of several cytoplasmic protein tyrosine kinases. Recruitment of the CD4 (or CD8) co-receptor and its associated tyrosine kinase, Lck, into the vicinity of the TCR complex is believed to induce phosphorylation of CD3 proteins, which ultimately leads to downstream signal progression. In vitro studies have suggested that interruption of this signaling pathway at a number of points may lead to tolerance. Thus, mAb that are directed at the TCR and co-receptor molecules, altered TCR ligands, and MHC-derived peptides present novel approaches to tolerance induction. A number of these are ready to be applied clinically. Promising drugs include nonmitogenic anti-CD3 mAb, anti-CD4 mAb, systemic and oral peptide therapies (Copaxone and MHC peptides), Campath-1H (anti-CD52), and DNA vaccination for allergy therapy, e.g., ragweed. One of the most successful strategies for tolerance induction in nonhuman primate renal transplantation has been depletion of immune cells at the time of the transplantation. Gradual repopulation of the immune cells occurs after the organ transplantation, often in the absence of inflammation and associated rejection. Pilot studies have been initiated in transplant recipients to test these new drugs in humans. As an example, nonmitogenic anti-CD3 mAb have been used in several phase I kidney transplant trials with similar efficacy as the original mitogenic OKT3 mAb without the severe associated side effects (3, 4). Similar early clinical studies have been performed with Campath 1 (5). Although it is early in the study, the results are promising and may represent an alternative approach to the development of tolerance.

Signal 2: The additional co-stimulatory signal required for complete T-cell activation, cell cycle progression, survival, and maximum effector function presents a number of additional therapeutic targets. Candidate co-stimulatory molecules are under investigation for tolerance induction. Some of these are soluble factors, such as interleukin-2 (IL-2) and IL-12, and many are T-cell surface receptors, such as CD28, lymphocyte function-associated antigen-1, 4-1BB, CD2, CD30, CD44, and CD40 ligand (CD40L). Each has the ability to augment the T-cell proliferative response to antigenic stimuli. It is likely that each of these acts through different mechanisms, some delivering co-stimulatory biochemical signals to the T cell, some enhancing adhesion to antigen-presenting cells, and still others mediating homing to target tissues. Specifically, blockade of T-cell co-stimulatory targets CD28 and CD40L during TCR engagement has been shown to induce a state of antigen-specific tolerance (6). Prevention of CD28/B7 and CD40L/CD40 interactions with anti-CD40L and CTLA4Ig during kidney transplantation in monkeys results in greatly prolonged allograft survival without the need for other immunosuppressive drugs (7). Additional approaches may include alloantigen pretreatment, e.g., donor-specific transfusion, under the cover of short-term immunotherapy with anti-CD154, CTLA4Ig (anti-B7 mAb), or nondepleting anti-CD4 mAb. Furthermore, the CD2 pathway has repeatedly been found to exert a major role in human immune responses, out of proportion to its effects in rodents (which lack lymphocyte function-associated antigen-3 and only engage CD2 with CD48). Finally, combined targeting of Signal 1 and Signal 2, e.g., nonmitogenic anti-CD3 plus B7 or CD40L blockade, should result in a synergistic effect on tolerance induction. These therapies are some of the most promising and exciting candidates for clinical tolerance induction in transplantation and autoimmune diseases.

Targeting Clonal Inactivation/Deletion: It is clear that the least pathogenic T cell is a dead T cell. Thus, multiple therapeutics have been developed to promote clonal deletion either centrally within the thymus or in the periphery. One approach that can be applied clinically is the use of autologous or allogeneic bone marrow transplantation to "re-educate" the immune response and promote deletion of autoreactive cells in the thymus. In addition, mixed chimerism with nonmyeloablative conditioning holds promise for the induction of transplant tolerance. This approach has been shown to achieve reliable and robust tolerance in rodents, and recent studies in large animals and humans show that mixed chimerism can also be induced with tolerable, nonmyeloablative conditioning (8). Regimens that combine extrathymic T-cell deletion (using mAb) followed by central T-cell tolerance induced by donor bone marrow under the cover of combined co-stimulatory blockade and low-dose total body irradiation are in the planning stage. Another, distinct approach uses proapoptotic therapies that take advantage of the Fas, TNF, and Trance pathways to promote activation-induced programmed cell death to eliminate antigen-stimulated T cells. These therapies can be combined with other T-cell inhibitors, such as anti-CTLA-4 agonists, to shut down the ongoing immune responses and promote tolerance. In more direct approaches, cytokines, such as transforming growth factor-ß, IL-10, and others, have been shown broadly to suppress pathogenic T cells, whereas IL-4 and interferon- can alter the balance of Th1 and Th2 responses (immune deviation) that can be used to regulate asthma, allergy, transplantation, and autoimmunity (9).

References

1. van Parijs L, Perez VL, Abbas AK: Mechanisms of peripheral T cell tolerance. Novartis Found Symp 215 : 5-20, 33-40,1998.

2. Lenschow DJ, Walunas TL, Bluestone JA: CD28/B7 system of T cell co-stimulation. Annu Rev Immunol 14 : 233-258,1996 [Full Text]

3. Woodle ES, Xu D, Zivin RA, Auger J, Charette J, O'Laughlin R, Peace D, Jollife LK, Haverty T, Bluestone JA, Thistlethwaite JR Jr: Phase I trial of a humanized, Fc receptor nonbinding OKT3 antibody, huOKT3 l(Ala-Ala) in the treatment of acute renal allograft rejection. Transplantation 68:608 -616, 1999. [Full Text] (pdf)

4. Friend PJ, Hale G, Chatenoud L, Rebello P, Bradley J, Thiru S, Phillips JM, Waldmann H: Phase I study of an engineered aglycosylated humanized CD3 antibody in renal transplant rejection. Transplantation 68:1632 -1637, 1999. [Full Text] (pdf)

5. Calne R, Moffatt SD, Friend PJ, Jamieson NV, Bradley JA, Hale J, Firth J, Bradley J, Smith KG, Waldmann H: Proper tolerance with induction using Campath 1H and low-dose cyclosporin monotherapy in 31 cadaveric renal allograft recipients. Nippon Geka Gakkai Zasshi 101 : 301-306, 2000.

6. Larsen CP, Elwood ET, Alexander DZ, Ritchie SC, Hendrix R, Tucker-Burden C, Cho HR, Aruffo A, Hollenbaugh D, Linsley PS, Winn KJ, Pearson TC: Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381:434 -438, 1996

7. Kirk AD, Harlan DM, Armstrong NN, Davis TA, Dong Y, Gray GS, Hong X, Thomas D, Fechner JH Jr, Knechtle SJ: CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci USA 94:8789 -8794, 1997 [Full Text]

8. Huang CA, Fuchimoto Y, Scheier-Dolberg R, Murphy MC, Neville DM Jr, Sachs DH: Stable mixed chimerism and tolerance using a non-myeloablative preparative regimen in a large-animal model. J Clin Invest 105:173 -181, 2000 [Full Text]

9. Singh VK, Mehrotra S, Agarwal SS: The paradigm of Th1 and Th2 cytokines: Its relevance to autoimmunity and allergy. Immunol Res 20: 147-161,1999.