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These data demonstrate that a single injection of CD8a:MyD88 T cells delay tumor progression and can prolong mouse survival.

T cell–based immunotherapies are a promising approach for patients with advanced cancers. However, various obstacles limit T-cell efficacy, including suboptimal T-cell receptor (TCR) activation and an immunosuppressive tumor environment. Here, we developed a fusion protein by linking CD8α and MyD88 (CD8α:MyD88) to enhance CD8+ T-cell responses to weakly immunogenic and poorly expressed tumor antigens. CD8α:MyD88–engineered T cells exhibited increased proliferation and expression of effector and costimulatory molecules in a tumor antigen–dependent manner. These effects were accompanied by elevated activation of TCR and Toll-like receptor signaling-related proteins. CD8α:MyD88–expressing T cells improved antitumor responses in mice. Enhanced antitumor activity was associated with a unique tumor cytokine/chemokine signature, improved T-cell infiltration, reduced markers of T-cell exhaustion, elevated levels of proteins associated with antigen presentation, and fewer macrophages with an immunosuppressive phenotype in tumors. Given these observations, CD8α:MyD88 represents a unique and versatile approach to help overcome immunosuppression and enhance T-cell responses to tumor antigens. Cancer Res; 77(24); 7049–58. ©2017 AACR.

Supplementary Figures S1-S11. TLR5L enhances the proliferation of human CD4 T cells (S1); Transduced human T cells coexpressing DMF5 and the TLR5L show significantly increased proliferation, cytokine production, and cytolytic activity than T cells expressing MART-1 TCR alone (S2); Human TLR5 protein expression in Malme-3M and C32 cell lines, and melanoma biopsy specimens (S3); TLR5 on Malme-3M cells enhances the cytokine and chemokine transcript levels (S4); MHC class I and II expression on Malme-3M cells stimulated with supernatants from various experimental conditions (S5); TLR5 engagement on C32 cells enhances the recruitment of various leukocyte subpopulations (S6); Chemokine receptor expression on non-transduced and transduced T cells stimulated with or without TLR5L (S7); Effects of TLR5L on the production of cytokines and chemokines on B16 and/or pmel CD8 T cells (S8); MHC class I and II expression on B16 cells stimulated with supernatants from various experimental conditions (S9); TLR5L stimulation does effect MDSC suppressive capacity or phenotype in vitro (S10); Diagram depicting how intratumoral delivery of TLR5L by CD8 T cells augments anti-tumor responses (S11).

Supplementary Figure Legend. Legends for Supplementary Figures S1-S11.

Chimeric antigen receptor T cells (CAR-Ts) have demonstrated remarkable efficacy in leukemia and lymphomas but limited responses in solid tumors. We conducted a phase I trial (NCT02107963) of GD2 CAR-Ts (GD2-CAR.OX40.28.z.ICD9), demonstrating feasibility and safety of administering GD2 CAR-Ts in children and young adults with neuroblastoma and, for the first time, osteosarcoma. 15 patients aged 8-28 years were enrolled on four dose levels, of which 13 patients were infused. No dose-limiting toxicities were observed, and administration of up to 1x107 GD2-CAR-T/kg was feasible and safe for children and young adults with neuroblastoma and osteosarcoma. At Day 28 following GD2 CAR-T infusion, 23.1% (3/13) of evaluable patients had progressive disease and 76.9% (10/13) had stable disease (SD). 3/10 SD patients remained stable at 60 days post-infusion of GD2 CAR-T, but all patients eventually progressed. Since a major barrier to CAR-T efficacy is inadequate CAR-T expansion, we evaluated CAR-T levels and found that patients stratified into good and poor expander groups, observed across dose levels and associated with pro-inflammatory cytokine signatures in patients. To understand the immune cell contributors to CAR-T expansion, patient pre-treatment apheresis, CAR-T product, and post-infusion samples were evaluated by high-dimensional proteomic (CyTOF), transcriptomic (RNAseq), and epigenetic (ATACseq) analyses. In patient apheresis, good CAR-T expansion associated with more open chromatin and with both proteomic and transcriptomic enrichment of naïve T cells, while poor CAR-T expansion associated with increased levels of T effector memory (TEMRA) cells and enrichment of myeloid derived suppressor cell (MDSC) transcriptomic signatures. CAR-T products across patients, regardless of CAR-T expansion, demonstrated increased T cell activation proteomic signatures, with enhanced exhaustion transcriptomic signatures in poor expanders compared to good. The most robust cellular correlate to good CAR-T expansion was a population of CXCR3-expressing monocytes in pre-treatment apheresis. Interestingly, this CXCR3+ monocyte population reduced in post-infusion timepoints of good expanders, resembling levels found in poor expanders. Our findings were validated in TARGET-OS patient data in The Cancer Genome Atlas, where high CXCR3 expression was found to be associated with survival benefit in osteosarcoma patients. CXCR3 has been extensively studied on T cells, but its function on myeloid populations is yet to be fully explored. These results are the first to demonstrate that the peripheral immune environment prior to CAR-T administration may effectively predict and modulate CAR-T expansion in patients. Citation Format: Tara Murty, Sabina Kaczanowska, Ahmad Alimadadi, Cristina Contreras, Caroline Duault, Priyanka Balasubrahmanyan, Warren Reynolds, Norma Gutierrez, Reema Baskar, Catherine Wu, Franziska Michor, Jennifer Altreuter, Yang Liu, Aashna Jhaveri, Vandon Duong, Hima Anbunathan, Radim Moravec, Joyce Hong, Roshni Biswas, Stephen Van Nostrand, James Lindsay, Mina Pichavant, Elena Sotillo, Bita Sahaf, Sean Bendall, Holden Maecker, Steven Highfill, David Stroncek, Melinda Merchant, John Glod, Catherine Hedrick, Crystal Mackall, Sneha Ramakrishna, Rosandra Kaplan. Immune determinants of CAR-T expansion in solid tumor patients receiving GD2 CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2142.

The pre-metastatic niche is a complex microenvironment formed by the influence of tumor-derived factors on stromal and immune cells at distant sites of disseminated tumor-cell colonization. Signaling through the kinase p38α and regulation of the type I interferon receptor are now linked to formation of the pre-metastatic niche.

Monocytes are innate immune cells recognized for their ability to play both tumor permissive and surveillant roles in cancer. Circulating classical monocytes (CD14+CD16−) can home to the tumor and suppress other immune cells through various mechanisms, including the production of arginase and the release of reactive oxygen species (ROS). Conversely, patrolling nonclassical monocytes (CD14−CD16+) have been shown to employ processes such as phagocytosis and presentation of tumor antigens to prevent metastasis. This heterogeneous monocyte function is influenced by tumor-derived factors that are released during cancer development and progression. Phenotypic and transcriptional alterations in circulating monocytes and other myeloid cells in patients with solid tumors have been reported and associated with poor clinical outcomes. However, perturbations of specific monocyte functions in the setting of solid tumors have not been well explored. Here we present a characterization of monocytes by coupling flow cytometry-based functional assays with sequencing (Func-seq). Healthy donor primary monocytes and monocytic cell lines were used to examine the production of ROS and arginase in response to osteosarcoma-conditioned media and monocyte-mediated phagocytosis of osteosarcoma cells. Bulk RNA-seq and ATAC-seq were performed on FACS-sorted populations to compare differentially expressed genes and establish transcriptomic and epigenetic signatures associated with monocyte-mediated immunosuppression and tumor-cell phagocytosis. The incorporation of functional selection into -omic characterization provides insights into monocyte behavior and potential therapeutic targets to alter their activity in solid tumors. Funding: US National Institutes of Health grants ZIA BC 011332 and ZIA BC 011855 and NCI cancer moonshot.

T cell–based immunotherapies are a promising approach for patients with advanced cancers. However, various obstacles limit T-cell efficacy, including suboptimal T-cell receptor (TCR) activation and an immunosuppressive tumor environment. Here, we developed a fusion protein by linking CD8α and MyD88 (CD8α:MyD88) to enhance CD8+ T-cell responses to weakly immunogenic and poorly expressed tumor antigens. CD8α:MyD88–engineered T cells exhibited increased proliferation and expression of effector and costimulatory molecules in a tumor antigen–dependent manner. These effects were accompanied by elevated activation of TCR and Toll-like receptor signaling-related proteins. CD8α:MyD88–expressing T cells improved antitumor responses in mice. Enhanced antitumor activity was associated with a unique tumor cytokine/chemokine signature, improved T-cell infiltration, reduced markers of T-cell exhaustion, elevated levels of proteins associated with antigen presentation, and fewer macrophages with an immunosuppressive phenotype in tumors. Given these observations, CD8α:MyD88 represents a unique and versatile approach to help overcome immunosuppression and enhance T-cell responses to tumor antigens. Cancer Res; 77(24); 7049–58. ©2017 AACR.

BackgroundImmune suppression is a major hurdle in cancer immunotherapy for solid tumors. Innate myeloid cells are key regulators of the immune system and can dampen the antitumor response against cancer. We have identified that bone marrow-derived myeloid cells play an immunosuppressive role in the metastatic microenvironment, limiting immune surveillance and facilitating the growth of tumor cells. We hypothesized that targeting the myeloid-mediated immune suppression program in the metastatic and primary tumor microenvironment could facilitate antitumor immune activation and be a successful immunotherapeutic approach.MethodsTo take advantage of the unique capability of myeloid cells to home to and infiltrate tumor and metastatic sites, we designed an immunotherapeutic approach in which we generate genetically engineered myeloid cells (GEMys) as a platform to locally deliver modulatory factors into the tumor and metastatic microenvironment.ResultsMice treated with IL-12-secreting GEMys (IL12-GEMys) exhibited a robust IFNγ response associated with increased expression of antigen processing and presentation machinery as well as numbers of T and NK cells expressing markers associated with activation and cytotoxicity. These microenvironmental changes were associated with reduced metastasis, delayed tumor growth, and increased survival. When combined with chemotherapy pre-conditioning, IL12-GEMys cured mice of established tumors and generated long-lived T cell memory, as these mice were immune to subsequent tumor challenge. We are currently working on translating these exciting findings into the human setting.ConclusionsThis work demonstrates that IL12-GEMys can functionally modulate the core program of immune suppression in the pre-metastatic niche to successfully rebalance the dysregulated metastatic microenvironment in cancer. This approach holds promise to limit metastatic progression in patients with high risk and advanced cancers.ReferencesKaczanowska S, Beury DW, Gopalan V, Tycko AK, Qin H, Clements ME, Drake J, Nwanze C, Murgai M, Rae Z, Ju W, Alexander KA, Kline J, Contreras CF, Wessel KM, Patel S, Hannenhalli S, Kelly M, Kaplan RN. Genetically engineered myeloid cells rebalance the core immune suppression program in metastasis. Cell 2021;184:1–20.

Summary Metastasis is the leading cause of cancer-related deaths, and greater knowledge of the metastatic microenvironment is necessary to effectively target this process. Microenvironmental changes occur at distant sites prior to clinically detectable metastatic disease; however, the key niche regulatory signals during metastatic progression remain poorly characterized. Here, we identify a core immune suppression gene signature in pre-metastatic niche formation that is expressed predominantly by myeloid cells. We target this immune suppression program by utilizing genetically engineered myeloid cells (GEMys) to deliver IL-12 to modulate the metastatic microenvironment. Our data demonstrate that IL12-GEMy treatment reverses immune suppression in the pre-metastatic niche by activating antigen presentation and T cell activation, resulting in reduced metastatic and primary tumor burden and improved survival of tumor-bearing mice. We demonstrate that IL12-GEMys can functionally modulate the core program of immune suppression in the pre-metastatic niche to successfully rebalance the dysregulated metastatic microenvironment in cancer.

Tumor metastasis is a critical step in the progression of solid tumors that is associated with patient mortality, yet we have limited knowledge of the metastatic microenvironment to effectively target this process. The pre-metastatic niche is the tumor-promoting microenvironment that is established at distant sites in response to primary tumor growth before detectable metastatic disease. We characterized immune populations in the lungs of tumor-bearing mice by flow cytometry and RNA sequencing approaches. We identified an immune suppression gene signature in pre-metastatic niche formation that is found predominantly within myeloid cells. In addition to the increase of myeloid cells and immunosuppressive pathways, we discovered that T cell populations are reduced in pre-metastatic lungs. We hypothesized that reversing this immunosuppressive environment would restore T cell function and antitumor immunity. We designed a novel approach in which we generated Genetically-Engineered Myeloid cells (GEMys) to deliver IL-12, a potent antitumor molecule, into the pre-metastatic microenvironment. We evaluated the lungs by flow cytometry and observed that IL12-GEMy-treated mice had increased numbers of T cells and enhanced expression of activation markers, resulting in reduced metastasis and increased survival. When combined with chemotherapy pre-conditioning, IL12-GEMys cured mice of established tumors and generated long-lived T cell memory, as these mice were immune to subsequent tumor challenge. These studies demonstrate that IL12-GEMys can functionally modulate the core program of immune suppression in the pre-metastatic niche to successfully rebalance the dysregulated metastatic microenvironment in cancer.

Monocytes are a heterogeneous group of mononuclear innate immune cells that have diverse inflammatory responses. In the context of cancer, monocytes and monocyte-derived cells have been evaluated for their pro- and anti-tumoral effects in the tumor microenvironment. These functions range from induction of tumor cell death to suppression of T cells, promotion of angiogenesis and remodeling of the extracellular matrix. Outside of the primary tumor, monocytes in circulation maintain their dichotomous role in cancer immunosurveillance. Specifically, patrolling non-classical CD14−CD16+ monocytes have been found to play a role in the prevention of metastasis. In contrast, CD14+ monocyte-derived cells from patients with solid tumors have been shown to promote tumor progression. Therefore, understanding this monocytic heterogeneity as well as other unexplored roles (i.e. monocyte-mediated phagocytosis of tumor cells) is crucially important for malignancies with high rates of metastasis. Yet, the phenotypic and functional diversity of circulating monocytes in patients with metastatic solid malignancies is still largely unknown. In this study, we sought to characterize and compare peripheral blood monocytes obtained from healthy donors and patients with advanced stage solid tumors. Through flow cytometric analysis and functional assays, we determined the subpopulation distributions as well as the phagocytic and suppressive activities of the monocytic compartment in patients with advanced cancer and healthy controls. Providing new insights into their cancer-related functions, we highlight the need for consideration of circulating monocytes into immune-targeting approaches in metastatic solid malignancies.

Immune checkpoint inhibitors (ICIs) have improved overall survival for cancer patients, however, optimal duration of ICI therapy has yet to be defined. Given ICIs were first used to treat patients with metastatic melanoma, a condition that at the time was incurable, little attention was initially paid to how much therapy would be needed for a durable response. As the early immunotherapy trials have matured past 10 years, a significant per cent of patients have demonstrated durable responses; it is now time to determine whether patients have been overtreated, and if durable remissions can still be achieved with less therapy, limiting the physical and financial toxicity associated with years of treatment. Well-designed trials are needed to identify optimal duration of therapy, and to define biomarkers to predict who would benefit from shorter courses of immunotherapy. Here, we outline key questions related to health, financial and societal toxicities of over treating with ICI and present four unique clinical trials aimed at exposing criteria for early cessation of ICI. Taken together, there is a serious liability to overtreating patients with ICI and future work is warranted to determine when it is safe to stop ICI.

T cell-based immunotherapies are a promising approach for patients with advanced cancers. However, various obstacles limit T cell efficacy, including suboptimal T cell receptor (TCR) activation and an immunosuppressive tumor environment. Here we developed a fusion protein by linking CD8α and MyD88 (CD8α:MyD88) to enhance CD8+ T cell responses to weakly immunogenic and poorly expressed tumor antigens. CD8α:MyD88-engineered T cells exhibited increased proliferation and expression of effector and co-stimulatory molecules in a tumor antigen-dependent manner. These effects were accompanied by elevated activation of TCR and Toll-like receptor (TLR) signaling-related proteins. CD8α:MyD88-expressing T cells improved anti-tumor responses in mice. Enhanced anti-tumor activity was associated with a unique tumor cytokine/chemokine signature, improved T cell infiltration, reduced markers of T cell exhaustion, elevated levels of proteins associated with antigen presentation, and fewer macrophages with an immunosuppressive phenotype in tumors. Given these observations, CD8α:MyD88 represents a unique and versatile approach to help overcome immunosuppression and enhance T cell responses to tumor antigens.

Cancer metastasis and immune suppression are critical issues in cancer therapy. Here, we show that a β-galactoside–binding lectin [galectin-3 (gal3)] that recognizes the Thomsen-Friedenreich disaccharide (TFD, Galβ1,3GalNAc) present on the surface of most cancer cells is involved in promoting angiogenesis, tumor-endothelial cell adhesion, and metastasis of prostate cancer cells, as well as evading immune surveillance through killing of activated T cells. To block gal3-mediated interactions, we purified a glycopeptide from cod (designated TFD 100 ) that binds gal3 with picomolar affinity. TFD 100 blocks gal3-mediated angiogenesis, tumor-endothelial cell interactions, and metastasis of prostate cancer cells in mice at nanomolar levels. Moreover, apoptosis of activated T cells induced by either recombinant gal3 or prostate cancer patient serum-associated gal3 was inhibited at nanomolar concentration of TFD 100 . Because the gal3–TFD interaction is a key factor driving metastasis in most epithelial cancers, this high-affinity TFD 100 should be a promising antimetastatic agent for the treatment of various cancers, including prostate adenocarcinoma.

A deeper understanding of the metastatic process is required for the development of new therapies that improve patient survival. Metastatic tumor cell growth and survival in distant organs is facilitated by the formation of a pre-metastatic niche that is composed of hematopoietic cells, stromal cells and extracellular matrix (ECM). Perivascular cells, including vascular smooth muscle cells (vSMCs) and pericytes, are involved in new vessel formation and in promoting stem cell maintenance and proliferation. Given the well-described plasticity of perivascular cells, we hypothesized that perivascular cells similarly regulate tumor cell fate at metastatic sites. We used perivascular-cell-specific and pericyte-specific lineage-tracing models to trace the fate of perivascular cells in the pre-metastatic and metastatic microenvironments. We show that perivascular cells lose the expression of traditional vSMC and pericyte markers in response to tumor-secreted factors and exhibit increased proliferation, migration and ECM synthesis. Increased expression of the pluripotency gene Klf4 in these phenotypically switched perivascular cells promoted a less differentiated state, characterized by enhanced ECM production, that established a pro-metastatic fibronectin-rich environment. Genetic inactivation of Klf4 in perivascular cells decreased formation of a pre-metastatic niche and metastasis. Our data revealed a previously unidentified role for perivascular cells in pre-metastatic niche formation and uncovered novel strategies for limiting metastasis.

Osteosarcoma is the most common bone cancer in children and young adults and has a strong propensity to develop lung metastases. E-selectin is known to be involved in the focal adhesion of tumor cells to cytokine exposed endothelial cells and we postulated may play a central role in osteosarcoma progression. Previously we identified that SDF-1, the main ligand for CXCR4, was upregulated in the pre-metastatic niche (Kaplan et al Nature 2005). Many tumor cells express CXCR4 and may use this signaling pathway to direct disseminated tumor cells to pre- and early metastatic sites in the lung. Based on these findings we examined human osteosarcoma cell lines and primary patient derived xenografts (PDXs) for the expression of CXCR4 and E-selectin ligands by flow cytometry. We found robust expression of these ligands in the majority of both the human osteosarcoma cell lines and PDXs examined. We therefore, investigated the impact of targeting these two axes on metastatic progression of orthotopic osteosarcoma using a small molecule, glycomimetic compound with dual inhibitory activity against E-selectin and CXCR4, GMI-1359. Five days post paratibial injection the HOS cell line, female NMRI-nu mice (n=12/group) were treated with saline; GMI-1359 alone (40 mg/kg IP BID x 25 days); doxorubicin (DOX) alone (5 mg/kg IV days 5, 15 and 25), or the combination of GMI-1359 and DOX. All treatments were well tolerated. The % tumor volume in treatment/control on day 27 of mice treated with GMI-1359, DOX or the combination was 35.5, 36.7 and 32.5, respectively. At study conclusion the incidence of lung metastases was approximately 60% and 50% in mice treated with saline or DOX and 15% in mice treated with GMI-1369 alone or in combination with DOX. Moreover, the extent of ectopic bone formation and/or osteolytic lesions was lower in mice treated with GMI-1359 compared to saline and DOX. These results indicate that the E-selectin and CXCR4 axes are important for the progression of osteosarcoma, and further, that inhibition of these two pro-tumor growth components by GMI-1359 has a therapeutic advantage over chemotherapy alone. Furthermore, studies in the adjuvant setting can provide proof of concept of utility of targeting CXCR4 and E- selectin ligands in the metastatic niche as a therapeutic strategy to limit metastatic progression in high risk patients. Citation Format: Wei Ju, Choh L. Yeung, Arnulfo Mendoza, Meera Murgai, Sabina Kaczanowska, Jennifer Zhu, Shil Patel, David A. Stewart, William E. Fogler, John L. Magnani, Rosandra N. Kaplan. Dual E-selectin and CXCR4 inhibition reduces tumor growth and metastatic progression in an orthotopic model of osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5211.

Toll-like receptor (TLR) agonists are potent immunostimulatory agents that have demonstrated great potential for cancer immunotherapy. We have genetically-engineered tumor-specific T cells to deliver and secrete the TLR5 ligand (TLR5L) flagellin to the tumor site to provide costimulation for antitumor immune activity. We found that TLR5L-secreting T cells offered a therapeutic benefit by altering several aspects including augmenting T cell effector function and expansion as well as reshaping the tumor microenvironment toward one that enhances antitumor T cell responses.

The tumor microenvironment counters antitumor T-cell responses, in part, by blunting their activation and infiltration. Ligands that engage Toll-like receptors (TLR) on T cells and antigen-presenting cells can act as potent immune adjuvants. In this study, we show how tumor-reactive T cells engineered to secrete bacterial flagellin, a TLR5 ligand (TLR5L), can engender a costimulatory signal that augments antitumor activity. Human T cells engineered to express TLR5L along with DMF5, a T-cell receptor that recognizes the melanoma antigen MART-127–35 (DMF5TLR5L T cells), displayed increased proliferation, cytokine production, and cytolytic activity against melanoma cells. In a xenogenetic model, adoptive transfer of DMF5TLR5L T cells reduced tumor growth kinetics and prolonged mouse survival. In a syngeneic model, similarly engineered melanoma-reactive T cells (pmelTLR5L) displayed a relative increase in antitumor activity against established tumors, compared with unmodified T cells. In this model, we documented increased T-cell infiltration associated with increased levels of CCR1 and CXCR3 levels on T cells, a reduction in PD-1+Lag3+ T cells and CD11+Gr1+ myeloid-derived suppressor cells, and changes in the chemokine/cytokine profile of tumors. Our findings show how T cell–mediated delivery of a TLR agonist to the tumor site can contribute to antitumor efficacy, in the context of adoptive T-cell immunotherapy. Cancer Res; 75(10); 1959–71. ©2015 AACR.

Chronic inflammation has long been associated with the development of cancer. Amongst the various signaling pathways within cancer cells that can incite the expression of inflammatory molecules are those that activate IL-1 receptor associated kinases (IRAK). The IRAK family comprises of four family members, IRAK-1, IRAK-2, IRAK-3 (also known as IRAK-M), and IRAK-4, which play important roles in both positively and negatively regulating the expression of inflammatory molecules. The wide array of inflammatory molecules that are expressed in response to IRAK signaling within the tumor microenvironment regulate the production of factors that promote tumor growth, metastasis, immune suppression and chemotherapy resistance. Based on published reports and compelling preliminary data, we propose that the dysregulated activation of the IRAK signaling pathway in cancer cells contributes to disease progression by creating a highly inflammatory tumor environment. In this article, we present both theoretical arguments and experimental data in support of this hypothesis.

Review on the ability of different TLR agonists to orchestrate antitumor immune responses, or promote tumor growth, underscoring the impact of choosing among TLR agonists when applying these therapies in the clinic. Various TLR agonists are currently under investigation in clinical trials for their ability to orchestrate antitumor immunity. The antitumor responses are largely attributed to their aptitude to stimulate APCs such as DCs which in turn, activate tumor-specific T cell responses. However, there is a potential for TLR signaling to occur on cells other than professional APCs that could negate antitumor responses or even worse, promote tumor growth. The impetus for this review is twofold. First, there is accumulating data demonstrating that the engagement of TLRs on different T cell subsets and different cancer types could promote tumor growth or conversely, contribute to antitumor responses. Second, the efficacy of TLR agonists as monotherapies to treat cancer patients has been limited. In this review, we discuss how TLR signaling within different T cell subsets and cancer cells can potentially impact the generation of antitumor responses. Based on evidence from preclinical models and clinical trials, we draw attention to several criteria that we believe must be considered when selecting TLR agonists for developing effective immunotherapeutic strategies against cancer.

T cell–based immunotherapies are among the most promising approaches for patients with advanced melanoma and other cancers. The goal of this study is to boost T cell anti-tumor responses by harnessing the costimulatory effects of MyD88 signaling in T cells. We previously demonstrated that Toll-like receptor (TLR) engagement on T cells increases their proliferation, effector function, and survival in a MyD88-dependent manner. However, a challenge to stimulating TLRs on T cells in vivo is inefficiencies in delivering TLR ligands to the tumor site or adverse effects of TLR signaling in cancer cells. We hypothesized that coupling TLR and T cell receptor (TCR) signaling would provide antigen-dependent co-stimulation resulting in enhanced anti-tumor function. We developed a novel synthetic co-receptor consisting of a CD8a and MyD88 fusion protein to induce MyD88 signaling in a TCR–dependent but TLR ligand–independent mechanism. We have compelling data indicating that CD8-MyD88 stimulation improves T cell expansion and function in a tumor antigen–dependent manner. Furthermore, CD8-MyD88 T cells exhibit promising anti-tumor responses in mice with established melanoma tumors. Our strategy represents a universal approach since the CD8-MyD88 is not limited to a specific tumor antigen nor MHC haplotype. CD8-MyD88 also improves T cell responses to low concentrations of antigen, providing the opportunity to expand a large repertoire of tumor-reactive T cells. Furthermore, CD8-MyD88 activates MyD88 signaling in the engineered T cells exclusively, thus eliminating undesirable responses of TLR signaling in tumor cells. These studies represent a novel and versatile therapeutic approach for boosting T cell responses to a variety of cancers.

Toll-like receptors (TLRs) play a significant role in amplifying immune responses against tumors. Our studies demonstrate that activated CD8+ T cells express TLRs and upon stimulation with TLR1-TLR2 agonists, exhibit enhanced T cell survival and proliferation. To define how TLR signaling promotes these processes, we compared gene expression analysis between TLR1-TLR2-stimulated and unstimulated CD8+ and MyD88-/- CD8+ T cells and identified significant increases in the expression levels of five TNF family members. We sought to test the hypothesis that the costimulatory effects of TLR1-TLR2 stimulation on CD8+ T cells were dependent on the function of one of these costimulatory molecules. Blocking CD137 (4-1BB) using blocking antibodies, but not the other TNF family members, dramatically reduced the costimulatory effects of TLR-mediated T cell proliferation. Likewise, 4-1BB -deficient CD8+ T cells did not respond to TLR stimulation. While TLR signals are capable of upregulating antiapoptotic proteins such as BCL2 and BCL-xL, mechanistically, we postulate that 4-1BB activation stabilizes these proteins, hence promoting survival. Adoptive transfer of pmel T cells along with the 4-1BB agonistic antibody, 3H3 and the TLR ligand, Pam3CSK4 was able to reduce tumor growth in a B16 melanoma model. These studies indicate that the costimulatory effects of TLR1-TLR2 signaling in CD8+ T cells mediated by 4-1BB signals are vital for mounting an effective antitumor response.

T cell responses are downregulated by the immunosuppressive tumor microenvironment. On the basis of previous studies demonstrating that Toll-like receptor (TLR) engagement on T cells augments their responses, we hypothesized that T cells engineered to secrete the TLR5 ligand flagellin would enhance T cell costimulation in the tumor environment and augment antitumor activity. Human T cells engineered to express the MART-1 TCR and secrete TLR5-ligand (TLR5-L) responded to lower tumor antigen levels resulting in increased proliferation, cytolytic activity and reversed tumor growth associated with prolonged mouse survival in a xenogeneic model. Treatment with TLR5-L-secreting T cells eradicated tumor in 40% of mice in a syngeneic model. Intratumoral secretion of TLR5-L by T cells reshaped the tumor microenvironment in part by inducing of a distinct chemokine/cytokine profile rendering it more immunogenic and permissive to T cell infiltration. Furthermore, tumor lysis by TLR5-L-producing T cells triggered the priming of endogenous tumor-reactive CD8 and CD4 T cells. Characterization of the cellular composition of the tumor and spleen revealed a reduction in MDSCs in the spleen and increased expression of MHC I in the tumor. This study demonstrates for the first time the ability to use T cells to deliver a TLR agonist to the tumor site and that intratumoral TLR5-L contributes to various processes involved in tumor regression.