When the antifungal compound rapamycin was discovered in 1972 within bacteria isolated from soil samples acquired on the South Pacific island of Rapa Nui, its future use in preventing transplant rejection could not have been predicted. As advancing research unearthed the evolutionarily conserved signal transduction pathway mTOR (mechanistic target of rapamycin),1 an understanding of the role of rapamycin in abrogating T-cell activation developed. There has since been much interest in detailing the potential of this class of immunosuppressant in modulating or enhancing regulatory T (Treg) cell function.2,3 An additional interest has been the anticancer potential of mTOR inhibitors, although these effects are not entirely clear or consistent.4-7 During the late 1980s and early 1990s, another receptor-signaling pathway, exclusive to resting Treg cells and activated T cells bearing the OX40 (CD134) receptor was also discovered.8 OX40 is enriched at sites of autoimmune inflammation9,10 and is found on the surface of circulating lymphocytes in systemic autoimmune diseases. Once ligated, OX40 signaling promotes the development of a cytokine-producing effector T (Teff) cells and prolongs the survival of memory T (Tmem) cells. Blockade or deficiency of OX40 has been shown to effectively attenuate autoimmune pathology.11,12 In vivo studies have demonstrated that OX40 ligand (OX40L) blockade can abrogate autoimmunity,11 potentially representing a more effective target than its receptor.13 In transplantation, blockade of OX40 effectively targets both CD4+ and CD8+ T cells to prevent rejection.14
Leslie Kean’s group has contributed extensively to the field of transplant tolerance, with a specific focus on the summative benefits of combinatorial therapies targeting T cell costimulatory and coinhibitory pathways. Having previously published promising results in their nonhuman primate (NHP) transplant model,15-21 the group is currently involved in clinical trials assessing the efficacy of CTLA4-Ig therapy to prevent severe graft versus host disease (GVHD); moreover, current trials include an anti-CD28 Fab22 for the treatment of rheumatoid arthritis. There is certainly a strong argument for using combinatorial therapies to manipulate lymphocyte signaling pathways to simultaneously tilt the immune balance toward allograft tolerance in both Treg cell and Teff cell compartments.22
In their recent study published in Science Translational Medicine,23 the authors aimed to identify an immunoprophylactic therapy that can synergize with the protolerogenic capabilities of sirolimus and in doing so highlighted the ability of OX40-OX40L blockade to control Tmem and Teff function while preserving the homeostasis and function of Treg. Using an NHP model of GVHD, they demonstrated increased expression of OX40L on lymph node-derived myeloid dendritic cells after allotransplantation in the absence of immunoprophylaxis. Moreover, they observed the OX40-encoding transcript TNFRSF4 in T cells after allotransplantation when using sirolimus. Based on those observations, they assessed the effects of OX40-OX40L blockade using a monoclonal anti-OX40L antibody (KY1005) with or without sirolimus in their high-risk NHP allogeneic GVHD model. KY1005 monotherapy reduced the rates of proliferating T cells and OX40+ CD4+ conventional T cells (predominantly central memory [TCM]) without impacting the proportion of Treg while modestly extended recipient survival (+11.5 days compared with untreated controls). Anti-OX40L treatment also reduced CD4+ T-cell expression of IL-17A, the hallmark cytokine of the Th17 profile typically associated with breakthrough acute GVHD (aGVHD).
The response to combined anti-OX40L and mTOR inhibition was much more effective: GVHD-free survival lasted the full 100-day experimental period with negligible clinical disease scores recorded.
Additionally, all recipients demonstrated effective donor engraftment with robust T-cell chimerism. Allospecific T-cell activation was controlled with significantly reduced CD4+ and CD8+ proliferation, decreased proportions (however, not numbers) of CD4+ TCM cells, and enhanced reconstitution of Treg and naive CD4+ T cells. Recipients that received the combinatorial treatment had distinct immunological transcriptome profiles, suggesting a unique synergistic effect of the combined therapies.
This study impressively demonstrates how the simultaneous blockade of synergistic inflammatory pathways can enhance efficacy. The mTOR pathway is known to affect T-cell function in several ways: (i) impacting T-cell proliferation in response to antigen stimulation (via IL-2); (ii) switching energy generation (known as the Warburg effect24) required during the transition of T cells from a resting state to a state of activation, differentiation, or proliferation25; and (iii) as a critical regulator of FoxP3 expression and Treg development.26 Blocking these effects while concurrently antagonizing a costimulatory signal essential for full T-cell activation has provided a therapy capable of delivering long-term allograft tolerance while preventing breakthrough aGVHD. This approach provided a significant advantage compared with other costimulation modulators, such as belatacept,27 while avoiding side effects notoriously associated with first-line calcineurin inhibitors.
Indeed, enhanced reconstitution of Tregs concurrent with reduced donor-specific Teffs is a highly attractive therapy based on the outcome of this study. In addition to being of relevance in preventing aGVHD, an enriched Treg pool has been frequently documented as key to providing long term protection against chronic allograft rejection in animal models.28-30 It will be interesting to test the durability of Treg reconstitution with a combinatorial treatment of KY1005 and mTOR inhibition as OX40 signaling is said to maintain T-cell survival.31
The complex relationship between OX40 signaling and Treg function has been comprehensively documented, yet there is no clear consensus on whether OX40 enhances or inhibits suppressive capacity. Studies have shown that OX40-deficient Tregs display an impaired ability to control inflammation in vivo.32 In addition, within the “right” cytokine milieu, OX40 engagement expands Tregs33,34 while inducing long-term allograft survival.35 Conversely, OX40 stimulation has been shown to profoundly inhibit FoxP3 expression.36 Moreover, antibody-mediated activation of OX40 has been shown to deplete Tregs while enhancing the antitumor immune response.37 In a model of skin transplantation, OX40 blockade at the time of exposure to alloantigen attenuated graft rejection.38 This effect had been linked to both enhanced suppressive potency and prolonged survival of Tregs upon blockade of OX40-OX40L signaling, findings that are in accordance with the present study (Figure 1). Thus, supported by the Science Translational Medicine publication and by the work of others, OX40L blockade may augment tolerance.27
Transduction of reverse signals from OX40 to OX40L has been shown to produce enhanced maturation and cytokine production in human dendritic cells39 as well as proliferation and IgG secretion by activated B cells.40,41 In turn, OX40L blockade has recently demonstrated suppressive effects mediated in part by inhibiting reverse-signaling on activated APCs and mesenchymal cells42 (Figure 1). Considering the importance of the cytokine milieu for OX40+ Tregs, and to obtain a greater understanding of the mechanisms underlying the findings reported, it would be intriguing to identify how far the effects conferred by OX40L blockade are linked with reverse signaling to APC’s and subsequent alterations of their phenotype and cytokine profiles. It should be noted that because there is no leukemia model in NHPs, evaluation of the graft-versus-leukemia response after hematopoeitic stem cell transplantation has not been possible. It will therefore be critically important for clinical trials to have robust cessation criteria to safeguard against the risk of relapse.
The exciting findings of this innovative study address several problems with current established immunosuppressive therapies and provide a strong basis for clinical translation. The reported results are promising because the simultaneous targeting of costimulatory and mTOR pathways target Teff cells and Treg cells differentially, with the potential to tilt the balance toward allograft tolerance.
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