Hēth R. Turnquist

Immunoregulatory Functions of mTOR Inhibition

The potent immunosuppressive action of rapamycin is commonly ascribed to inhibition of growth factor-induced T cell proliferation. However, it is now evident that the serine/threonine protein kinase mammalian target of rapamycin (mTOR) has an important role in the modulation of both innate and adaptive immune responses. mTOR regulates diverse functions of professional antigen-presenting cells, such as dendritic cells (DCs), and has important roles in the activation of effector T cells and the function and proliferation of regulatory T cells. In this Review, we discuss our current understanding of the mTOR pathway and the consequences of mTOR inhibition, both in DCs and T cells, including new data on the regulation of forkhead box P3 expression.

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival.

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4+ Foxp3+ regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b+ cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4+ Foxp3+ Tregs, including an ST2L+ population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8+ IFN-γ+ cells. Also, despite reducing overall CD3+ cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3+ cells. Whereas control graft recipients displayed increases in systemic CD11b+ Gr-1hi cells, IL-33–treated recipients exhibited increased CD11b+ Gr-1int cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4+ Foxp3+ Tregs that underlie IL-33–mediated cardiac allograft survival.

IL-33 synergizes with TCR and IL-12 signaling to promote the effector function of CD8+ T cells.

The effector functions of CD8+ T cells are influenced by tissue inflammatory microenvironments. IL‐33, a member of the IL‐1 family, acts as a danger signal after its release during cell necrosis. The IL‐33/ST2 axis has been implicated in various Th2 responses. Its role in CD8+ T‐cell‐mediated immune response is, however, not known. Here we find that type 1 cytotoxic T (Tc1) cells cultured in vitro unexpectedly express high levels of the IL‐33 receptor ST2. Interestingly, the expression of ST2 in Tc1 cells is dependent on T‐bet, a master Th1/Tc1 transcription factor. In addition, IL‐33 enhances TCR‐triggered IFN‐γ production. IL‐33 together with IL‐12 can stimulate IFN‐γ production in Tc1 cells. Moreover, IL‐33 synergizes with IL‐12 to promote CD8+ T‐cell effector function. The synergistic effect of IL‐33 and IL‐12 is partly mediated by Gadd45b. Together, these in vitro data establish a novel role of IL‐33 in promoting effector type 1 adaptive immune responses.

IL-33 Is an Unconventional Alarmin That Stimulates IL-2 Secretion by Dendritic Cells To Selectively Expand IL-33R/ST2+ Regulatory T Cells

IL-33 is a recently characterized IL-1 family member that is proposed to function as an alarmin, or endogenous signal of cellular damage, as well as act as a pleiotropic cytokine. The ability of IL-33 to potentiate both Th1 and Th2 immunity supports its role in pathogen clearance and disease immunopathology. Yet, IL-33 restrains experimental colitis and transplant rejection by expanding regulatory T cells (Treg) via an undefined mechanism. We sought to determine the influence of IL-33 on hematopoietic cells that drives Treg expansion and underlies the therapeutic benefit of IL-33 administration. In this study, we identify a feedback loop in which conventional mouse CD11c+ dendritic cells (DC) stimulated by IL-33 secrete IL-2 to selectively expand IL-33R(ST2+)– suppressive CD4+Foxp3+ Treg. Interestingly, this occurs in the absence of classical DC maturation, and DC-derived (innate) IL-2 increases ST2 expression on both DC and interacting Treg. ST2+ Treg represent an activated subset of Foxp3+ cells, demonstrated to be ICOShighCD44high compared with their ST2− counterparts. Furthermore, although studies have shown that IL-33–exposed DC promote Th2 responses, we reveal that ST2+ DC are required for IL-33–mediated in vitro and in vivo Treg expansion. Thus, we have uncovered a relationship between IL-33 and innate IL-2 that promotes the selective expansion of ST2+ Treg over non-Treg. These findings identify a novel regulatory pathway driven by IL-33 in immune cells that may be harnessed for therapeutic benefit or for robust expansion of Treg in vitro and in vivo.

IL-1β-Driven ST2L Expression Promotes Maturation Resistance in Rapamycin-Conditioned Dendritic Cells

Maturation resistance and tolerogenic properties can be conferred on human and murine dendritic cells (DC), crucial regulators of T cell responses, by exposure to rapamycin (RAPA), a “tolerance-sparing” immunosuppressive agent. Mechanisms underlying this acquired unresponsiveness, typified by diminished functional responses to TLR or CD40 ligation, have not been identified. We report that in vitro and in vivo conditioning of murine myeloid DC with RAPA elicits the de novo production of IL-1β by otherwise phenotypically immature DC. Interestingly, IL-1β production promotes overexpression of the transmembrane form of the IL-1R family member, IL-1R-like 1, also know as ST2 on RAPA-conditioned DC (RAPA-DC). ST2 is the recently identified receptor for IL-33, a cytokine favoring Th2 responses. In addition, transmembrane ST2, or ST2L, has been implicated as a potent negative regulator of TLR signaling. RAPA-DC generated from ST2−/− mice exhibited higher levels of costimulatory molecules (CD86) than wild-type RAPA-DC. Consistent with its regulatory function, IL-1β-induced ST2L expression suppressed the responsiveness of RAPA-DC to TLR or CD40 ligation. Thus, as a result of their de novo production of IL-1β, RAPA-DC up-regulate ST2L and become refractory to proinflammatory, maturation-inducing stimuli. This work identifies a novel mechanism through which a clinically important immunosuppressant impedes the capacity of DC to mature and consequently stimulate effector/adaptive T cell responses.

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.

Histone deacetylase inhibition facilitates GM-CSF-mediated expansion of myeloid-derived suppressor cells in vitro and in vivo.

Chromatin‐modifying HDACi exhibit anti‐inflammatory properties that reflect their ability to suppress DC function and enhance regulatory T cells. The influence of HDACi on MDSCs, an emerging regulatory leukocyte population that potently inhibits T cell proliferation, has not been examined. Exposure of GM‐CSF‐stimulated murine BM cells to HDACi led to a robust expansion of monocytic MDSC (CD11b+Ly6C+F4/80intCD115+), which suppressed allogeneic T cell proliferation in a NOS‐ and HO‐1‐dependent manner with similar potency to control MDSCs. The increased yield of MDSCs correlated with blocked differentiation of BM cells and an overall increase in HSPCs (Lin–Sca‐1+c‐Kit+). In vivo, TSA enhanced the mobilization of splenic HSPCs following GM‐CSF administration and increased the number of CD11b+Gr1+ cells in BM and spleen. Increased numbers of Gr1+ cells, which suppressed T cell proliferation, were recovered from spleens of TSA‐treated mice. Overall, HDACi enhance MDSC expansion in vitro and in vivo, suggesting that acetylation regulates myeloid cell differentiation. These findings establish a clinically applicable approach to augment this rare and potent suppressive immune cell population and support a novel mechanism underlying the anti‐inflammatory action of HDACi.

New dog and new tricks: evolving roles for IL-33 in type 2 immunity

IL‐33 is a more recently identified member of the IL‐1 cytokine family, expressed in the nucleus of epithelial cells and released into the extracellular space following tissue damage. The impact of IL‐33 as a regulator of the adaptive immune response has been studied extensively, with an understood role for IL‐33 in the effector functions of CD4+ Th2 cells. IL‐33, however, is now being shown to initiate the Th2‐polarizing function of DCs, and stimulate the secretion of the type 2‐associated cytokines, IL‐4, IL‐5, and IL‐13, from tissue‐resident innate‐immune cells, especially ILCs and MCs. IL‐33 also initiates and perpetuates local inflammatory responses through the recruitment and activation of type 2‐ and inflammatory‐associated effectors, such as eosinophils, basophils, and neutrophils. As such, IL‐33 drives and amplifies type 2‐dependent immunity, as well as type 2‐dependent tissue destruction and inflammation. It is also becoming apparent that IL‐33 supports the reparative capacity of macrophage and ILCs, but these functions may also contribute to chronic fibrotic diseases. Herein, we review new developments in the understanding of IL‐33 as it functions in Th2 cells and type 2 immunity. This includes a discussion of our evolving understanding of how IL‐33 directly and indirectly promotes type 2 immune responses through action on innate cells in immunity and the pathogenesis of atopic and fibrotic diseases.

Tolerogenic dendritic cell-regulatory T-cell interaction and the promotion of transplant tolerance.

Evidence has accumulated that targeting of donor alloantigen to quiescent dendritic cells (DC) in situ or adoptive DC therapy is associated with the expansion or induction of regulatory T cells (Treg) in experimental organ transplantation. These Treg can mediate suppression of antidonor T-effector cell responses and promote allograft tolerance. In addition, Treg can exert reciprocal, inhibitory effects on DC that maintain their tolerogenic properties. Several groups have exploited DC to expand allo-Ag-specific Treg in vitro, followed by adoptive transfer of the Treg to graft recipients, an approach that holds promise for tolerogenic cell therapy in clinical cell and organ transplantation.

Peri-alloHCT IL-33 administration expands recipient T-regulatory cells that protect mice against acute GVHD

During allogeneic hematopoietic cell transplantation (alloHCT), nonhematopoietic cell interleukin-33 (IL-33) is augmented and released by recipient conditioning to promote type 1 alloimmunity and lethal acute graft-versus-host disease (GVHD). Yet, IL-33 is highly pleiotropic and exhibits potent immunoregulatory properties in the absence of coincident proinflammatory stimuli. We tested whether peri-alloHCT IL-33 delivery can protect against development of GVHD by augmenting IL-33-associated regulatory mechanisms. IL-33 administration augmented the frequency of regulatory T cells (Tregs) expressing the IL-33 receptor, suppression of tumorigenicity-2 (ST2), which persist following total body irradiation. ST2 expression is not exclusive to Tregs and IL-33 expands innate immune cells with regulatory or reparative properties. However, selective depletion of recipient Foxp3(+) cells concurrent with peri-alloHCT IL-33 administration accelerated acute GVHD lethality. IL-33-expanded Tregs protected recipients from GVHD by controlling macrophage activation and preventing accumulation of effector T cells in GVHD-target tissue. IL-33 stimulation of ST2 on Tregs activates p38 MAPK, which drives expansion of the ST2(+) Treg subset. Associated mechanistic studies revealed that proliferating Tregs exhibit IL-33-independent upregulation of ST2 and the adoptive transfer of st2(+) but not st2(-) Tregs mediated GVHD protection. In total, these data demonstrate the protective capacity of peri-alloHCT administration of IL-33 and IL-33-responsive Tregs in mouse models of acute GVHD. These findings provide strong support that the immunoregulatory relationship between IL-33 and Tregs can be harnessed therapeutically to prevent GVHD after alloHCT for treatment of malignancy or as a means for tolerance induction in solid organ transplantation.