Dàlia Raïch-Regué

Murine dendritic cell rapamycin-resistant and rictor-independent mTOR controls IL-10, B7-H1, and regulatory T-cell induction

Mammalian target of rapamycin (mTOR) is an important, yet poorly understood integrative kinase that regulates immune cell function. mTOR functions in 2 independent complexes: mTOR complex (mTORC) 1 and 2. The immunosuppressant rapamycin (RAPA) inhibits mTORC1 but not mTORC2 and causes a paradoxical reduction in anti-inflammatory interleukin (IL) 10 and B7-homolog 1 (B7-H1) expression by dendritic cells (DCs). Using catalytic mTOR inhibitors and DCs lacking mTORC2, we show that restraint of signal transducer and activator of transcription 3-mediated IL-10 and B7-H1 expression during DC maturation involves a RAPA-insensitive and mTORC2-independent mTOR mechanism. Relatedly, catalytic mTOR inhibition promotes B7-H1-dependent and IL-1β-dependent DC induction of regulatory T cells (Tregs). Thus, we define an immunoregulatory pathway in which RAPA-sensitive mTORC1 in DCs promotes effector T-cell expansion and RAPA-insensitive mTORC1 restrains T(reg) induction. These findings identify the first known RAPA-insensitive mTOR pathway that is not mediated solely by mTORC2 and have implications for the use of catalytic mTOR inhibitors in inflammatory disease settings.

Regulatory Myeloid Cells in Transplantation

Regulatory myeloid cells (RMC) are emerging as novel targets for immunosuppressive (IS) agents and hold considerable promise as cellular therapeutic agents. Herein, we discuss the ability of regulatory macrophages, regulatory dendritic cells, and myeloid-derived suppressor cells to regulate alloimmunity, their potential as cellular therapeutic agents, and the IS agents that target their function. We consider protocols for the generation of RMC and the selection of donor- or recipient-derived cells for adoptive cell therapy. Additionally, the issues of cell trafficking and antigen (Ag) specificity after RMC transfer are discussed. Improved understanding of the immunobiology of these cells has increased the possibility of moving RMC into the clinic to reduce the burden of current IS agents and to promote Ag-specific tolerance. In the second half of this review, we discuss the influence of established and experimental IS agents on myeloid cell populations. IS agents believed historically to act primarily on T cell activation and proliferation are emerging as important regulators of RMC function. Better insights into the influence of IS agents on RMC will enhance our ability to develop cell therapy protocols to promote the function of these cells. Moreover, novel IS agents may be designed to target RMC in situ to promote Ag-specific immune regulation in transplantation and to usher in a new era of immune modulation exploiting cells of myeloid origin.

mTORC2 Deficiency in Myeloid Dendritic Cells Enhances Their Allogeneic Th1 and Th17 Stimulatory Ability after TLR4 Ligation In Vitro and In Vivo

The mammalian/mechanistic target of rapamycin (mTOR) is a key integrative kinase that functions in two independent complexes, mTOR complex (mTORC) 1 and mTORC2. In contrast to the well-defined role of mTORC1 in dendritic cells (DC), little is known about the function of mTORC2. In this study, to our knowledge, we demonstrate for the first time an enhanced ability of mTORC2-deficient myeloid DC to stimulate and polarize allogeneic T cells. We show that activated bone marrow–derived DC from conditional Rictor−/− mice exhibit lower coinhibitory B7-H1 molecule expression independently of the stimulus and enhanced IL-6, TNF-α, IL-12p70, and IL-23 production following TLR4 ligation. Accordingly, TLR4-activated Rictor−/− DC display augmented allogeneic T cell stimulatory ability, expanding IFN-γ+ and IL-17+, but not IL-10+ or CD4+Foxp3+ regulatory T cells in vitro. A similar DC profile was obtained by stimulating Dectin-1 (C-type lectin family member) on Rictor−/− DC. Using novel CD11c-specific Rictor−/− mice, we confirm the alloreactive Th1 and Th17 cell-polarizing ability of endogenous mTORC2-deficient DC after TLR4 ligation in vivo. Furthermore, we demonstrate that proinflammatory cytokines produced by Rictor−/− DC after LPS stimulation are key in promoting Th1/Th17 responses. These data establish that mTORC2 activity restrains conventional DC proinflammatory capacity and their ability to polarize T cells following TLR and non-TLR stimulation. Our findings provide new insight into the role of mTORC2 in regulating DC function and may have implications for emerging therapeutic strategies that target mTOR in cancer, infectious diseases, and transplantation.

Adenosine Triphosphate-Competitive mTOR Inhibitors: A New Class of Immunosuppressive Agents That Inhibit Allograft Rejection

The mechanistic/mammalian target of rapamycin (mTOR) is inhibited clinically to suppress T cell function and prevent allograft rejection. mTOR is the kinase subunit of two mTOR‐containing complexes, mTOR complex (mTORC) 1 and 2. Although mTORC1 is inhibited by the macrolide immunosuppressant rapamycin (RAPA), its efficacy may be limited by its inability to block mTORC1 completely and its limited effect on mTORC2. Adenosine triphosphate (ATP)‐competitive mTOR inhibitors are an emerging class of mTOR inhibitors that compete with ATP at the mTOR active site and inhibit any mTOR‐containing complex. Since this class of compounds has not been investigated for their immunosuppressive potential, our goal was to determine the influence of a prototypic ATP‐competitive mTOR inhibitor on allograft survival. AZD8055 proved to be a potent suppressor of T cell proliferation. Moreover, a short, 10‐day course of the agent successfully prolonged murine MHC‐mismatched, vascularized heart transplant survival. This therapeutic effect was associated with increased graft‐infiltrating regulatory T cells and reduced CD4+ and CD8+ T cell interferon‐γ production. These studies establish for the first time, that ATP‐competitive mTOR inhibition can prolong organ allograft survival and warrant further investigation of this next generation mTOR inhibitors.

Intratumoral delivery of mTORC2-deficient dendritic cells inhibits B16 melanoma growth by promoting CD8+ effector T cell responses

ABSTRACT Dendritic cells (DC) play a pivotal role in the induction and regulation of immune responses. In cancer, DC-based vaccines have proven to be safe and to elicit protective and therapeutic immunological responses. Recently, we showed that specific mTORC2 (mechanistic target of rapamycin complex 2) deficiency in DC enhances their ability to promote Th1 and Th17 responses after LPS stimulation. In the present study, bone marrow-derived mTORC2-deficient (Rictor−/−) DC were evaluated as a therapeutic modality in the murine B16 melanoma model. Consistent with their pro-inflammatory profile (enhanced IL-12p70 production and low PD-L1 expression versus control DC), intratumoral (i.t.) injection of LPS-activated Rictor−/− DC slowed B16 melanoma growth markedly in WT C57BL/6 recipient mice. This antitumor effect was abrogated when Rictor−/− DC were injected i.t. into B16-bearing Rag−/− mice, and also after selective CD8+ T cell depletion in wild-type hosts in vivo, indicating that CD8+ T cells were the principal regulators of tumor growth after Rictor−/− DC injection. I.t. administration of Rictor−/− DC also reduced the frequency of myeloid-derived suppressor cells within tumors, and enhanced numbers of IFNγ+ and granzyme-B+ cytotoxic CD8+ T cells both in the spleens and tumors of treated animals. These data suggest that selective inhibition of mTORC2 activity in activated DC augments their pro-inflammatory and T cell stimulatory profile, in association with their enhanced capacity to promote protective CD8+ T cell responses in vivo, leading to slowed B16 melanoma progression. These novel findings may contribute to the design of more effective DC-based vaccines for cancer immunotherapy.

Cutting Edge: Flt3 Ligand Mediates STAT3-Independent Expansion but STAT3-Dependent Activation of Myeloid-Derived Suppressor Cells

The Flt3–Flt3 ligand (Flt3L) pathway is critically involved in the differentiation and homeostasis of myeloid cells, including dendritic cells (DC); however, its role in the expansion and function of myeloid-derived suppressor cells (MDSC) has not been determined. In this article, we describe the ability of Flt3L to expand and activate murine MDSC capable of suppressing allograft rejection upon adoptive transfer. Although Flt3L expands and augments the stimulatory capacity of myeloid DC, MDSC expanded by Flt3L have increased suppressive activity. Although STAT3 is considered the central transcription factor for MDSC expansion, inhibition and genetic ablation of STAT3 did not block, but rather augmented, Flt3L-mediated MDSC expansion. MDSC suppressive function, preserved when STAT3 inhibition was removed, was reduced by genetic STAT3 deletion. Both STAT3 inhibition and deletion reduced Flt3L-mediated DC expansion, signifying that STAT3 had reciprocal effects on suppressive MDSC and immunostimulatory DC expansion. Together, these findings enhance our understanding of the immunomodulatory properties of Flt3L.

Renal Allograft Survival in Nonhuman Primates Infused with Donor Antigen-Pulsed Autologous Regulatory Dendritic Cells

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte‐derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg‐pulsed autologous DCreg (1.4–3.6 × 106/kg) were administered intravenously, 1 day before MHC‐mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag‐pulsed DCreg (n = 5) was associated with evidence of modulated host CD4+ and CD8+ T cell responses to donor Ag and attenuation of systemic IL‐17 production. Circulating anti‐donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag‐pulsed DCreg rejected its graft in association with progressively elevated anti‐donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag‐pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen.