From T Cell to Tolerance: Immune Regulation, Apoptosis and Anergy

Angus Thomson, PhD, DSc
Medical Writer: Peggy Keen, RN, PhD, FNP

T-cell activation plays a central role in regulating immune response to antigens. For T-cell activation to occur, two signals are necessary: antigen presentation and costimulation. In the absence of costimulation, anergy occurs and may play a role in development of tolerance. In addition, apoptosis is essential for induction of allograft acceptance.

A series of lectures at the AST Winter Symposium addressed issues of considerable topical importance, including the current understanding of mechanisms underlying transplantation tolerance and strategies for promotion of tolerance induction by costimulation blockade.

APOPTOSIS: A CENTRAL EVENT IN IMMUNE REGULATION?

Lawrence Turka, MD, from the University of Pennsylvania, Philadelphia, gave an overview of the role of apoptosis in tolerance,[1] providing the most current answers to 3 important questions:

What is the effect of calcineurin inhibition on T-cell death and peripheral tolerance at the time of initial T-cell activation?
Is apoptosis from cytokine deprivation required for peripheral tolerance induction by costimulation blockade?
Is activation-induced cell death required for peripheral tolerance induction?

WHAT IS THE EFFECT OF CALCINEURIN INHIBITION ON T-CELL DEATH AND PERIPHERAL TOLERANCE AT THE TIME OF INITIAL T-CELL ACTIVATION?

Calcineurin inhibition antagonizes the effect of costimulation blockade and prevents tolerance induction. In addition to alloantigen recognition and signal transduction via the T-cell receptor (TCR) designated signal 1, an additional costimulatory signal (signal 2) is required for the expression of T-cell growth factors (ie, interleukin-2 [IL-2]) and cell survival genes (ie, Bcl-x) necessary for T-lymphocyte responses that lead to transplantation rejection. Blockade of signal 2 with cytotoxic T-lymphocyte antigen immunoglobulin (CTLA4-Ig) plus monoclonal antibody (mAb) against CD40 ligand induces indefinite survival of major histocompatibility complex (MHC)-mismatched heart allografts in mice. However, the addition of the calcineurin inhibitor cyclosporine that blocks signal 1 to the costimulation-blocking protocol results in rejection.

This concerning ability of calcineurin inhibition to block the tolerizing effects of costimulation blockade has also been reported in nonhuman primates. In recent experimental studies, Terry Strom, MD, and colleagues at Harvard Medical School in Boston, and Larry Turka and investigators at the University of Pennsylvania have shown that this undesired effect reflects the abolition of alloreactive T-cell proliferation in vivo, precluding cell cycle-dependent, activation-induced T-cell apoptosis. Failure to induce apoptosis preserves the pool of alloreactive T cells that retain the potential to mount a rejection response once immunosuppressive therapy is withdrawn.

IS APOPTOSIS FROM CYTOKINE DEPRIVATION REQUIRED FOR PERIPHERAL TOLERANCE INDUCTION BY COSTIMULATION BLOCKADE?

Rapamycin and costimulation blockade are synergistic for tolerance induction. In contrast to the calcineurin inhibitor cyclosporine, rapamycin blocks growth factor IL-2-imparted proliferative signals but does not impair IL-2-mediated priming for activation-induced cell death (AICD). Findings published in November 1999 by the Strom and Turka groups have shown that rapamycin is compatible with costimulation blockade and that in combination, these agents produce tolerance in both heart and more stringent skin allograft models.

The tolerance achieved is associated with massive in vivo apoptosis of alloreactive cells, and thus deletion of the (unusually) large T-cell population that responds to MHC antigens. The important clinical implication of these findings is that global immunosuppression should be avoided to promote deletion of alloreactive T cells. Avoiding global immunosuppression may allow other mechanisms of immune-regulated peripheral tolerance (ie, anergy/immunoregulation) to maintain graft survival.

IS ACTIVATION-INDUCED CELL DEATH REQUIRED FOR PERIPHERAL TOLERANCE INDUCTION?

Deletion of activated T cells through apoptosis is essential for tolerance induction. The requirement of intact T-cell apoptotic pathways for the induction of transplant tolerance to cardiac allografts across MHC barriers has been elegantly demonstrated by Turka and colleagues.

Using mice transgenic for Bcl-xL, in which T cells are resistant to AICD, the researchers showed that these animals are resistant to tolerance induction by transfusion of donor cells in combination with costimulation blockade (CTLA4-Ig or anti-CD40L mAb) and that all eventually reject their transplants. Further studies by the Strom and Turka groups using pancreatic islet allografts in IL-2-deficient mice have provided evidence that IL-2-triggered apoptotic signals are required for tolerance induction.

A ROLE FOR IMMUNE REGULATION IN TRANSPLANT TOLERANCE

Fadi Lakkis, MD, from Emory University in Atlanta, reviewed recent evidence for immune regulatory mechanisms in transplantation tolerance.[2] Although T-cell costimulation and cytokine production are important in generating alloimmune responses, recent data suggest that costimulatory molecules, such as B7-1 and B7-2, and T-cell activating cytokines (interferon-gamma and Il-2) trigger negative feedback mechanisms in T cells that limit alloimmune reactivity. These mechanisms are essential for long-term graft acceptance and in certain situations, transplant tolerance.

EXISTENCE OF REGULATORY CELLS

Our understanding of the role of suppressed/regulatory cells has been unclear in the recent past. But there is now good evidence for the generation of CD4+ suppressor/regulatory T cells in the context of (a) neonatal tolerance to minor and MHC antigens in mice, and (b) in relation to “infectious tolerance” whereby CD4+ cells from skin- or organ-transplanted rodents preconditioned with nondepleting anti-CD4 mAb can transfer tolerance to naive animals that then receive test allografts. In addition, allospecific CD8+ suppressor/regulatory cells (CD8+ , CD28- ) that suppress upregulation of costimulatory molecules on antigen-presenting cells have been described.

EVIDENCE IN CONFLICT WITH THE TH2 PARADIGM

The paradigm of immune deviation, or skewing of the CD4+ T helper cell response toward Th2 predominance and allograft tolerance, has not withstood testing in several animal models, particularly in the use of specific cytokine-neutralizing antibodies or cytokine gene (knockout) mice. In contrast to the Th2 paradigm, interferon-gamma has been shown to facilitate the acceptance of heart allografts in mice treated with donor-specific transfusion plus CTLA4-Ig, whereas tolerance can be induced in IL-4 mice, including neonatal female recipients of male skin grafts.

Another surprising finding from studies of IL-2 knockout or anti-IL-2-treated mice is that IL-2 facilitates tolerance induction. It now appears that, whereas IL-2 and interferon-gamma may play a redundant role during the afferent phase of the alloimmune response, these cytokines may be essential for tolerance-promoting effects, in particular AICD.

A Role for the Common Cytokine Receptor Gamma Chain Regulating IL-2-Dependent Activation-Induced CD8+ T-Cell Death
Interestingly, apoptotic death in specific alloreactive T cells (2C, TCR transgenic) mediated by Fas and tumor necrosis factor receptor (TNFR) death pathways, is enhanced by anti-IL-2R-gamma chain mAb that prevents Bcl-2 induction. It appears that IL-2 prepares CD8+ for AICD by at least two mechanisms: (1) upregulation of a proapoptotic molecule , FasL, and (2) downregulation of a survival molecule, the common cytokine receptor gamma-chain.

COSTIMULATION BLOCKADE: PROGRESS TOWARD CLINICAL APPLICATION

THE CD28 CTLA4-IG PATHWAY

Christian Larsen, MD, DPhil, from Emory University, Atlanta, reviewed progress in mouse and primate models toward clinical application of manipulation of the CD28 pathway by the hybrid molecule CTLA4-Ig and the CD40L-CD40 pathway by the mAb CD40L.[3]

Targeting the CD28 pathway that blocks the B7-CD28 pathway, CTLA4-Ig is a potent inhibitor of T cell responses but unlikely to induce robust tolerance, Dr. Larsen noted. In mouse models, both anti-B7-1 and anti-B7-2 mAbs are required to achieve effects similar to those of CTLA4-Ig. Timing of blockade influences outcome. Thus, delay in administration of CTLA4-Ig (until day 2 posttransplantation) enhances its efficacy. Adjuncts that enhance CTLA4-Ig efficacy include donor-specific transfusion, anti-CD40L mAb and anti-CD4 mAb. Molecular mechanisms underlying its mode of action appear to include preferential CTLA4 signaling and altering the balance of regulators of apoptosis.

In primates, CTLA4-Ig is well tolerated. Alone, it exerts only a modest effect on renal allograft survival but has a large impact on antidonor antibody responses. Its immunosuppressive effects and those of cyclosporine are additive. LEA29Y, a more effective mutant form of CTLA4-IgLEA29Y, is a second-generation fusion protein, developed by Robert Peach, PhD, at Bristol Myers Squibb in Princeton, NJ, that comprises a mutated CTLA4 and human IgG1Fc. It exhibits similar binding to B7-1 but 10-fold higher avidity to B7-2 than parent CTLA4-Ig. LEA29 inhibits mixed leukocyte reactions at 10-fold lower concentrations than CTLA4-Ig.

In rhesus monkeys, LEA29Y monotherapy (at days 0, 4, 14, and weekly thereafter until day 70) prolongs allograft survival much more effectively than CTLA4-Ig, suggesting that increased avidity for B7-2 achieves better results. Excellent long-term renal allograft function (>80 days) is achieved by combining LEA29Y with mycophenolate mofetil and prednisone in a primate model.

TARGETING THE CD40 PATHWAY

CD40 is a member of the TNFR superfamily expressed on antigen-presenting cells that transduce activation signals via TRAF-dependent signaling pathways leading to ND kappa B activation. Its ligand, CD40L, is expressed on activated T cells, endothelial cells, and platelets. Anti-CD40L mAb inhibits B7 and IL-12 induction and is a potent inhibitor of CD4-dependent T-cell responses. But CD8+ responses are not controlled, and the mAb alone does not induce transplant tolerance in mouse models. The mAb may promote immune deviation, and there is evidence that it enhances AICD in alloactivated T cells (see above). Cyclosporine, and possibly corticosteroids, may inhibit its tolerogenic effect whereas these may be enhanced by rapamycin (see above).

In primates (rhesus monkeys), anti-CD40L is effective in inducing the long-term acceptance of renal allografts. There is a trend toward donor-specific unresponsiveness. Notably, surviving grafts contain infiltrating T cells, and there is a persistence of antidonor antibody response.[4] Long-term acceptance, dependent on continued anti-CD40L therapy, is also achieved in rhesus recipients of pancreatic islet allografts. Notably, anti-CD40L can reverse rejection of these grafts.[5] In humans, trials using anti-CD40L mAb in transplantation or autoimmunity have been halted by thromboembolic complications that may reflect (1) prothrombotic contaminants, (2) intrinsic antiplatelet/endothelial cell effects in humans, or (3) epitope-specific effects that may not occur with other anti-CD40L mAbs.

ANTI-CD40 MAB (BMS-224819)

Studies on this mAb to date show that it is effective in inhibiting T-cell dependent antibody responses to sheep red blood cells in primates and that it prolongs renal allograft survival in rhesus monkeys given 6 doses of drug at 20 mg/kg during the first 11 days posttransplant. No synergy with CTLA4-Ig has been observed in relation to graft survival, but a potentially useful inhibitory effect of the two agents on antidonor antibody responses has been observed. Potential interactions with conventional immunosuppressants have yet to be investigated.

REFERENCES

Turka LA. Apoptosis: a central event in immune regulation? In: Program and abstracts of the American Society of Transplantation 4th Annual Winter Symposium; January 13-17, 2000; Fajardo, Puerto Rico.
Lakkis F. Immune regulation revisited: a critical review. In: Program and abstracts of the American Society of Transplantation 4th Annual Winter Symposium; January 13-17, 2000; Fajardo, Puerto Rico.
Larsen C. Interventions in costimulation: the role of cd40 ligand in rejection. In: Program and abstracts of the American Society of Transplantation 4th Annual Winter Symposium; January 13-17, 2000; Fajardo, Puerto Rico.
Kirk AD, Burkly LC, Batty DS, et al. Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nat Med. 1999;5:686-693.
Kenyon NS, Chatzipetrou M, Masetti M, et al. Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc Natl Acad Sci U S A. 1999;96:8132-8137.