Xiufen Chen

PD-1 regulates extrathymic regulatory T-cell differentiation

Treg cells and the programed death‐1/programed death ligand‐1 (PD‐1/PD‐L1) pathway are both critical for maintaining peripheral tolerance to self‐Ags. A significant subset of Treg cells constitutively expresses PD‐1, which prompted an investigation into the role of PD‐1/PD‐L1 interactions in Treg‐cell development, function, and induction in vivo. The phenotype and abundance of Treg cells was not significantly altered in PD‐1‐deficient mice. The thymic development of polyclonal and monospecific Treg cells was not negatively impacted by PD‐1 deficiency. The suppressive function of PD‐1−/− Treg cells was similar to their PD‐1+/+ counterparts both in vitro and in vivo. However, in three different in vivo experimental settings, PD‐1−/− conventional CD4+ T cells demonstrated a strikingly diminished tendency toward differentiation into peripherally induced Treg (pTreg) cells. Our results demonstrate that PD‐1 is dispensable for thymic Treg‐cell development and suppressive function, but is critical for the extrathymic differentiation of pTreg cells in vivo. These data suggest that Ab blockade of the PD‐1/PD‐L1 pathway may augment T‐cell responses by acting directly on conventional T cells, and also by suppressing the differentiation of pTreg cells.

CD40 ligation reverses T cell tolerance in acute myeloid leukemia

Spontaneous antigen-specific T cell responses can be generated in hosts harboring a variety of solid malignancies, but are subverted by immune evasion mechanisms active within the tumor microenvironment. In contrast to solid tumors, the mechanisms that regulate T cell activation versus tolerance to hematological malignancies have been underexplored. A murine acute myeloid leukemia (AML) model was used to investigate antigen-specific T cell responses against AML cells inoculated i.v. versus s.c. Robust antigen-specific T cell responses were generated against AML cells after s.c., but not i.v., inoculation. In fact, i.v. AML cell inoculation prevented functional T cell activation in response to subsequent s.c. AML cell challenge. T cell dysfunction was antigen specific and did not depend on Tregs or myeloid-derived suppressor cells (MDSCs). Antigen-specific TCR-Tg CD8+ T cells proliferated, but failed to accumulate, and expressed low levels of effector cytokines in hosts after i.v. AML induction, consistent with abortive T cell activation and peripheral tolerance. Administration of agonistic anti-CD40 Ab to activate host APCs enhanced accumulation of functional T cells and prolonged survival. Our results suggest that antigen-specific T cell tolerance is a potent immune evasion mechanism in hosts with AML that can be reversed in vivo after CD40 engagement.

Calreticulin promotes immunity and type I interferon-dependent survival in mice with acute myeloid leukemia

ABSTRACT Exposure of cancer cells to particular chemotherapeutic agents or γ-irradiation induces a form of cell death that stimulates an immune response in mice. This “immunogenic cell death” requires calreticulin (CRT) translocation to the plasma membrane, which has been shown to promote cancer cell phagocytosis. However, it remains unclear whether the effect of CRT on cancer cell phagocytosis is alone sufficient to affect tumor immunity. Acute myeloid leukemia (AML) cells expressing cell-surface CRT were generated in order to characterize the mechanism(s) through which CRT activates tumor immune responses. Potent immune-mediated control or rejection of AML was observed in mice with CRT-expressing leukemia. The “CRT effect” was ultimately T-cell dependent, but dendritic cells (DCs), and CD8α+ DCs in particular, were also necessary, indicating that CRT might act directly on these DCs. CRT-expressing AML cells were slightly more susceptible to phagocytosis by DCs in vivo, but this effect was unlikely to explain the potent immunity observed. CRT did not affect classical DC maturation markers, but induced expression of type I interferon (IFN), which was critical for its positive effect on survival. In conclusion, CRT functions as a “danger signal” that promotes a host type I IFN response associated with the induction of potent leukemia-specific T-cell immunity.

Activation of the STING pathway enhances immunity and improves survival in a murine myeloid leukemia model

Type I interferon (IFN) production by innate immune cells is critical to prime spontaneous T cell responses against solid tumors. Emerging pre-clinical data suggests that tumor-derived DNA induces potent IFN-β production by activating a cytosolic DNA sensing receptor called STING (Stimulator of Interferon Genes), ultimately resulting in tumor antigen-specific T cell priming and in some cases, tumor rejection. However, because of their disseminated growth pattern, hematological malignancies, such as acute myeloid leukemia (AML), may not release sufficient quantities of DNA during cell death to activate the STING pathway in innate immune cells, which contributes to the T cell tolerance observed in these hosts. Thus, we hypothesized that STING pathway activation would promote a host type I IFN response sufficient to facilitate leukemia-specific T cell priming and immune-mediated control of AML progression. Murine C1498 AML cells expressing the model SIY antigen (C1498.SIY) were inoculated intravenously into syngeneic C57BL/6 mice which then received a single dose of the murine STING agonist, DMXAA (5,6-dimethylxanthenone-4-acetic acid), or vehicle control 5 days later. DMXAA induced potent IFN-β production by C57BL/6 spleen cells (90-fold induction over vehicle control). STING pathway activation via DMXAA treatment of C1498.SIY-bearing animals resulted in a massive expansion (10-fold over vehicle control) of endogenous SIY antigen-specific T cells as analyzed by SIY/Kb pentamer staining and flow cytometry. Intracellular cytokine staining of SIY-peptide restimulated spleen cells from DMXAA-treated animals revealed that the expanded SIY-specific CD8+ T cells produced high-levels of IFN-γ. This enhanced immune response also translated to a benefit in survival. After C1498.SIY AML cell-challenge, 80% of DMXAA-treated mice survived long-term, while controls succumbed to AML within approximately 3-4 weeks, as expected. DMXAA-treated mice surviving a primary C1498.SIY cell challenge also rejected a secondary challenge with parental C1498 cells (SIY-negative), suggesting DMXAA-induced STING activation led to potent immunological memory against native C1498-expressed antigens. DMXAA treatment of mice following a systemic challenge with parental C1498 cells also led to significantly enhanced survival compared to control-treated animals. This effect was T and/or B cell-dependent, as DMXAA treatment of leukemia-bearing RAG-/- mice had no effect on survival. Collectively, these data suggest that DMXAA-mediated activation of the STING pathway enhances adaptive immunity to AML, culminating in prolonged survival and even disease cure. Human STING agonists are currently in development and, if STING pathway activation demonstrates efficacy in additional pre-clinical leukemia models, it may be of interest to target this pathway in AML patients.

Peripheral T-cell tolerance in hosts with acute myeloid leukemia.

Our laboratory investigates the immune tolerance mechanisms promoted by acute myeloid leukemia (AML). In a murine AML model, we have observed that leukemia antigen-specific T cells are specifically deleted from the host, presumably following interactions with immature host antigen-presenting cells (APCs). Ongoing work focuses on identifying APC subsets that induce T-cell tolerance in AML as well as the precise mechanisms that underlie this phenomenon.

CD8α+ dendritic cells dictate immune responses against murine AML

Spontaneous T cell responses generated against a variety of solid malignancies are often subverted by immune evasion mechanisms active in the tumor microenvironment. In contrast, the mechanisms that regulate T cell activation versus tolerance to hematopoietic malignancies, such as acute myeloid leukemia (AML), have not been well-characterized. Our recent work in a murine AML model has demonstrated that following a systemic introduction of leukemia cells, T cells specific for leukemia-derived antigens underwent abortive proliferation and were deleted from the host. This deletional tolerance in mice with established AML was reversible upon administration of an agonistic anti-CD40 antibody to activate host dendritic cells (DCs), and argued that these cells may play a dominant role in tolerance induction to AML. Investigation of the DCs populations which engulfed AML cells in vivo, and which were likely promoting T cell tolerance, led to the critical observation that AML cells were phagocytosed exclusively by CD11c+CD8α+ DCs (CD8α+ DCs). CD8α+, but not CD8α- DCs purified from mice following an intravenous inoculation of AML cells, were able to cross-present leukemia-derived antigens to T cells in vitro, providing strong evidence that CD8α+ DC generate T cell tolerance to AML. Ongoing work utilizing mice deficient in particular DC subsets is focused on identifying a functional link between CD8α+ DCs and T cell tolerance. Additionally, the receptors expressed selectively on CD8α+ DCs which facilitate phagocytosis and cross-presentation of leukemia derived antigens are under investigation.

Abstract A10: Activation of the STING pathway enhances immunity and improves survival in a murine myeloid leukemia model.

Type I interferon (IFN) production by innate immune cells is critical to prime spontaneous T cell responses against solid tumors. Until recently, the tumor-derived signals which stimulate host type I IFN production have remained elusive. However, emerging data suggest that tumor-derived DNA is sufficient to induce IFN-β production in responding cells through activation of a cytosolic DNA sensing receptor called STING (Stimulator of Interferon Genes). STING-mediated IFN-β production, in turn, leads to functional priming of antigen-specific T cells which mediate tumor rejection in pre-clinical solid tumor models. However, because of their disseminated growth pattern, hematological cancers, such as acute leukemia, may not release sufficient quantities of DNA upon apoptosis to activate the STING pathway in innate immune cells, such as dendritic cells (DCs). The failure of acute leukemias to induce a type I IFN response may contribute to their ability to generate T cell tolerance in the host. Thus, we hypothesized that activating the STING pathway would promote a type I IFN response sufficient to facilitate DC-mediated priming of leukemia-specific T cells and to prolong survival of mice with acute myeloid leukemia (AML). Murine C1498 AML cells engineered to express a model peptide antigen called SIY (C1498.SIY) were inoculated intravenously into syngeneic C57BL/6 mice which were then treated with a selective murine STING agonist, DMXAA (5,6-dimethylxanthenone-4-acetic acid), or vehicle control. Preliminary experiments confirmed that DMXAA administration resulted in robust IFN-β production by C57BL/6 spleen cells (90-fold induction over vehicle control-treated spleen cells). STING pathway activation via DMXAA treatment of C1498.SIY-bearing animals resulted in a massive expansion (10-fold over vehicle control-treated animals) of endogenous SIY antigen-specific T cells as determined by SIY/K b pentamer analysis. Intracellular cytokine staining of SIY-peptide restimulated spleen cells from DMXAA-treated, leukemia-bearing animals, revealed that the expanded SIY-specific CD8+ T cells were functionally active, and produced high-levels of IFN-γ. To determine if this enhanced immune response translated to a benefit in survival, C1498.SIY AML cell-challenged mice were treated with a single dose of DMXAA or vehicle control five days after tumor inoculation. Among mice treated with DMXAA, 80% of mice rejected the leukemia and survived long-term, while control-treated mice succumbed to AML within approximately 3 weeks, as expected. DMXAA-treated mice surviving a primary C1498.SIY cell challenge also rejected a secondary challenge with parental C1498 cells (SIY-negative), suggesting the DMXAA treatment led to potent immunological memory against native C1498-expressed antigens. DMXAA treatment of mice following a systemic challenge with parental C1498 cells also led to enhanced survival compared to control-treated animals. This effect was T and or B cell-dependent, as DMXAA treatment of RAG-/- mice had no effect on survival. Collectively, these data suggest that DMXAA-mediated activation of the STING pathway upstream of type I IFN potently enhances adaptive immunity to AML which culminates in prolonged survival or even disease cure in treated animals. It will be important to determine whether the STING pathway may be successfully manipulated in other murine leukemia models. If so, then STING may be a promising therapeutic target to enhance endogenous immunity in AML patients. Citation Format: Emily K. Curran, Xiufen Chen, Leticia Corrales, Justin Kline. Activation of the STING pathway enhances immunity and improves survival in a murine myeloid leukemia model. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A10.

Calreticulin mediates immune recognition of acute myeloid leukemia cells in vivo

Calreticulin (CRT) is a chaperone protein which normally resides in the endoplasmic reticulum (ER). However, recent studies have demonstrated that pre-apoptotic cancer cells release internalized CRT to their surface prior to death, and this surface exposure of CRT acts as an ‘eat-me’ signal to local phagocytes. Some chemotherapeutic agents and gamma-irradiation have been shown to induce ER stress and promote CRT surface translocation in cancer cells, resulting in their recognition and phagocytosis by innate immune cells, such as macrophages and dendritic cells (DC). In turn, innate immune cells which have phagocytized CRT-expressing tumor cells become capable of priming antigen-specific T cell responses directed against malignant cells. Unfortunately, chemotherapy and irradiation which can induce immunogenic cell death (ICD) through CRT, can also result in local and/or systemic immune suppression in the host. To bypass the requirement of exposing the host to chemotherapy to induce translocation of CRT to the cell surface, C1498 AML cells were engineered to constitutively express cell surface CRT (C1498.CRT). Introduction of C1498.CRT cells subcutaneously (SC) into syngeneic C57BL/6 mice resulted in either significantly delayed tumor outgrowth or complete protection from tumor development compared to parental C1498 cells which universally progressed in vivo. A SC challenge with C1498.CRT cells also protected mice from tumor outgrowth following a subsequent re-challenge with parental C1498 tumor cells, suggesting that C1498.CRT cells promote immunologic memory. Differences in tumor outgrowth between mice inoculated with parental C1498 and C1498.CRT leukemia cells were abrogated in immunodeficient RAG-/- and RAG2-/-γc-/- animals, arguing that the immune-mediated effect of cell-surface CRT expression is dependent upon a functional adaptive immune system. To model a clinically relevant scenario, parental C1498 or C1498.CRT cells were inoculated intravenously (IV) into C57BL/6 mice. Significantly prolonged survival was observed in hosts harboring C1498.CRT versus parental C1498 cells systemically. Systemic inoculation with C1498.CRT cells expressing the model SIYRYYGL (SIY) peptide antigen (C1498.SIY.CRT cells) resulted in enhanced expansion and effector cytokine production by antigen-specific T cells compared to T cells from hosts challenged with control C1498.SIY cells. Our current on-going experiments are focusing on identifying the mechanism(s) through which calreticulin appears to promote anti-tumor immunity.