Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here we reveal extensive co-expression of PD-1 and LAG-3 on tumor-infiltrating CD4+ and CD8+ T cells in three distinct transplantable tumors. Dual anti-LAG-3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathological sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3−/−Pdcd1−/− mice demonstrated markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. Additionally, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer.

Regulatory T cells (Tregs) play a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis1. However, they also represent a major barrier to effective anti-tumor immunity and sterilizing immunity to chronic viral infections1. The transcription factor Foxp3 plays a major role in the development and programming of Treg cells2,3. The relative stability of Tregs at inflammatory disease sites has been highly contentious4-6. There is considerable interest in identifying pathways that control Treg stability as many immune-mediated diseases are characterized by either exacerbated or limited Treg function. Here we show that the immune cell-expressed ligand semaphorin-4a (Sema4a) and the Treg-expressed receptor neuropilin-1 (Nrp1) interact to potentiate Treg function and survival in vitro and in inflammatory sites in vivo. Nrp1 is dispensable for suppression of autoimmunity and maintenance of immune homeostasis, but is required by Tregs to limit anti-tumor immune responses and to cure established inflammatory colitis. Sema4a ligation of Nrp1 restrained Akt phosphorylation cellularly and at the immunologic synapse (IS) via phosphatase and tensin homolog (PTEN), which increased nuclear localization of the transcription factor Foxo3a. The Nrp1-induced transcriptome promoted Treg stability by enhancing quiescence/survival factors while inhibiting programs that promote differentiation. Importantly, this Nrp1-dependent molecular program is evident in intratumoral Tregs. Our data support a model in which Treg stability can be subverted in certain inflammatory sites, but is maintained by a Sema4a:Nrp1 axis, highlighting this pathway as a potential therapeutic target that could limit Treg-mediated tumor-induced tolerance without inducing autoimmunity.

SUMMARY Targeting autophagy in cancer cells and in the tumor microenvironment are current goals of cancer therapy. However, components of canonical autophagy play roles in other biological processes, adding complexity to this goal. One such alternative function of autophagy proteins is LC3-associated phagocytosis (LAP), which functions in phagosome maturation and subsequent signaling events. Here we show that impairment of LAP in the myeloid compartment, rather than canonical autophagy, induces control of tumor growth by tumor-associated macrophages (TAM) upon phagocytosis of dying tumor cells. Single cell RNAseq analysis revealed that defects in LAP induce pro-inflammatory gene expression and trigger STING-mediated type I interferon responses in TAM. We found that the anti-tumor effects of LAP impairment require tumor-infiltrating T cells, dependent upon the STING and the type I interferon response. Therefore, autophagy proteins in the myeloid cells of the tumor microenvironment contribute to immune suppression of T lymphocytes by effecting LAP.

Summary Regulatory T (Treg) cells pose a major barrier to effective anti-tumor immunity. Although Treg cell depletion enhances tumor rejection, the ensuing autoimmune sequelae limits its utility in the clinic and highlights the need for limiting Treg cell activity within the tumor microenvironment. Interleukin-35 (IL-35) is a Treg cell-secreted cytokine that inhibits T cell proliferation and function. Using an IL-35 reporter mouse, we observed substantial enrichment of IL-35+ Treg cells in tumors. Neutralization with an IL-35-specific antibody or Treg cell-restricted deletion of IL-35 production limited tumor growth in multiple murine models of human cancer. Limiting intratumoral IL-35 enhanced T cell proliferation, effector function, antigen-specific responses, and long-term T cell memory. Treg cell-derived IL-35 promoted the expression of multiple inhibitory receptors (PD1, TIM3, LAG3), thereby facilitating intratumoral T cell exhaustion. These findings reveal previously unappreciated roles for IL-35 in limiting anti-tumor immunity and contributing to T cell dysfunction in the tumor microenvironment.

Inhibitory receptors expressed on T cells control immune responses while limiting autoimmunity. However, tumors can hijack these ‘checkpoints’ for protection from immune attack. Tumor-specific T cells that exhibit an exhausted, unresponsive phenotype express high levels of inhibitory receptors including CTLA4, PD1 and LAG3, among others. Intratumoral regulatory T cells promote immunosuppression and also express multiple inhibitory receptors. Overcoming this inhibitory receptor-mediated immune tolerance has thus been a major focus of recent cancer immunotherapeutic developments. Here, we review how boosting the host’s immune system by blocking inhibitory receptor signaling with antagonistic monoclonal antibodies restores the capacity of T cells to drive durable antitumor immune responses. Clinical trials targeting the CTLA4 and PD1 pathways have shown durable effects in multiple tumor types. Many combinatorial therapies are currently being investigated with encouraging results that highlight enhanced antitumor immunogenicity and improved patient survival. Finally, we will discuss the ongoing identification and dissection of novel T-cell inhibitory receptor pathways, which could lead to the development of new combinatorial therapeutic approaches.

Dendritic cells are the most potent antigen-presenting cells known; owing to their ability to stimulate antigen-specific cytolytic and memory T-cell responses, their use as cancer vaccines is rapidly increasing. While clinical trials provide evidence that dendritic cells vaccines are safe and elicit immunological responses in most patients, few complete tumor remissions have been reported and further technological advances are required. An effective dendritic cell vaccine must possess and maintain several characteristics: it must migrate to lymph nodes, have a mature, Th1-polarizing phenotype expressed stably after infusion and present antigen for sufficient time to produce a T-cell response capable of eliminating a tumor. While dendritic cells are readily matured ex vivo, their phenotype and fate after infusion are rarely evaluable; therefore, strategies to ensure that dendritic cells access lymphoid tissues and retain an immunostimulatory phenotype are required. In order to best exploit dendritic cells as vaccines, they may require genetic modification and combination with other strategies including adoptive T-cell transfer, inhibition of regulatory T cells or modulation of inflammatory pathways.

Purpose BCR-ABL+ B-ALL leukemic cells are highly dependent on the expression of endogenous anti-apoptotic MCL-1 to promote viability and are resistant to BH3-mimetic agents such as navitoclax (ABT-263) that targets BCL-2, BCL-XL, and BCL-W. However, the survival of most normal blood cells and other cell types are also dependent on Mcl-1. Despite the requirement for MCL-1 in these cell types, initial reports of MCL-1-specific BH3-mimetics have not described any overt toxicities associated with single-agent use, but these agents are still early in clinical development. Therefore, we sought to identify FDA-approved drugs that could sensitize leukemic cells to ABT-263. Experimental Design A screen identified dihydroartemisinin (DHA), a water-soluble metabolite of the anti-malarial artemisinin. Using mouse and human leukemic cell lines, and primary patient-derived xenografts, the effect of DHA on survival was tested and mechanistic studies were carried out to discover how DHA functions. We further tested in vitro and in vivo whether combining DHA with ABT-263 could enhance the response of leukemic cells to combination therapy. Results DHA causes the down-modulation of MCL-1 expression by triggering a cellular stress response that represses translation. The repression of MCL-1 renders leukemic cells highly sensitive to synergistic cell death induced by ABT-263 in a mouse model of BCR-ABL+ B-ALL both in vitro and in vivo. Furthermore, DHA synergizes with ABT-263 in human Ph+ ALL cell lines, and primary patient derived xenografts of Ph+ ALL in culture. Conclusions Our findings suggest that combining DHA with ABT-263 can improve therapeutic response in BCR-ABL+ B-ALL.

To function optimally as vaccines, dendritic cells (DCs) must actively migrate to lymphoid organs and maintain a viable, mature state for sufficient time to effectively present their Ag to cognate T cells. Unfortunately, mature DCs rapidly lose viability and function after injection, and only a minority leaves the vaccine site and migrates to lymph nodes. We show that all of these functions can be enhanced in DCs by removal of IL-1R–associated kinase M (IRAK-M). We found that IRAK-M is induced in DCs by TLR ligation and that its absence from these cells leads to increased activation of the p38-MAPK and NF-κB pathways, which, in turn, improves DC migration to lymph nodes, increases their longevity, and augments their secretion of Th1-skewing cytokines and chemokines. These biological effects have immunological consequences. IRAK-M−/− DCs increase the proliferation and activation of Ag-specific T cells, and a single vaccination with Ag-pulsed, LPS-matured IRAK-M−/− DCs eliminates established tumors and prolongs the survival of EG7 or B16.f10 tumor-bearing mice, without discernible induction of autoimmune disease. Thus, manipulation of IRAK-M levels can increase the potency of DC vaccines by enhancing their Ag-presenting function, migration, and longevity.