Adoptive cellular therapy with T cells expressing the dendritic cell growth factor Flt3L drives epitope spreading and antitumor immunity.

Adoptive cell therapies using genetically engineered T cell receptor or chimeric antigen receptor T cells are emerging forms of immunotherapy that redirect T cells to specifically target cancer. However, tumor antigen heterogeneity remains a key challenge limiting their efficacy against solid cancers. Here, we engineered T cells to secrete the dendritic cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L-secreting T cells expanded intratumoral conventional type 1 DCs and substantially increased host DC and T cell activation when combined with immune agonists poly (I:C) and anti-4-1BB. Importantly, combination therapy led to enhanced inhibition of tumor growth and the induction of epitope spreading towards antigens beyond those recognized by adoptively transferred T cells in solid tumor models of T cell receptor and chimeric antigen receptor T cell therapy. Our data suggest that augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative tumor escape following adoptive cell therapy.

PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours.

Although adoptive T-cell therapy has shown remarkable clinical efficacy in haematological malignancies, its success in combating solid tumours has been limited. Here, we report that PTPN2 deletion in T cells enhances cancer immunosurveillance and the efficacy of adoptively transferred tumour-specific T cells. T-cell-specific PTPN2 deficiency prevented tumours forming in aged mice heterozygous for the tumour suppressor p53. Adoptive transfer of PTPN2-deficient CD8+ T cells markedly repressed tumour formation in mice bearing mammary tumours. Moreover, PTPN2 deletion in T cells expressing a chimeric antigen receptor (CAR) specific for the oncoprotein HER-2 increased the activation of the Src family kinase LCK and cytokine-induced STAT-5 signalling, thereby enhancing both CAR T-cell activation and homing to CXCL9/10-expressing tumours to eradicate HER-2+ mammary tumours in vivo. Our findings define PTPN2 as a target for bolstering T-cell-mediated anti-tumour immunity and CAR T-cell therapy against solid tumours.

CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity.

Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR-Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.

Distinct roles of two eIF4E isoforms in the germline of Caenorhabditis elegans.

Germ cells use both positive and negative mRNA translational control to regulate gene expression that drives their differentiation into gametes. mRNA translational control is mediated by RNA-binding proteins, miRNAs and translation initiation factors. We have uncovered the discrete roles of two translation initiation factor eIF4E isoforms (IFE-1, IFE-3) that bind 7-methylguanosine (m7G) mRNA caps during Caenorhabditiselegans germline development. IFE-3 plays important roles in germline sex determination (GSD), where it promotes oocyte cell fate and is dispensable for spermatogenesis. IFE-3 is expressed throughout the germline and localizes to germ granules, but is distinct from IFE-1 and PGL-1, and facilitates oocyte growth and viability. This contrasts with the robust expression in spermatocytes of IFE-1, the isoform that resides within P granules in spermatocytes and oocytes, and promotes late spermatogenesis. Each eIF4E is localized by its cognate eIF4E-binding protein (IFE-1:PGL-1 and IFE-3:IFET-1). IFE-3 and IFET-1 regulate translation of several GSD mRNAs, but not those under control of IFE-1. Distinct mutant phenotypes, in vivo localization and differential mRNA translation suggest independent dormant and active periods for each eIF4E isoform in the germline.

IL-15 Preconditioning Augments CAR T Cell Responses to Checkpoint Blockade for Improved Treatment of Solid Tumors.

Chimeric antigen receptor (CAR) T cell therapy has been highly successful in hematological malignancies leading to their US Food and Drug Administration (FDA) approval. However, the efficacy of CAR T cells in solid tumors is limited by tumor-induced immunosuppression, leading to the development of combination approaches, such as adjuvant programmed cell death 1 (PD-1) blockade. Current FDA-approved methods for generating CAR T cells utilize either anti-CD3 and interleukin (IL)-2 or anti-CD3/CD28 beads, which can generate a T cell product with an effector/exhausted phenotype. Whereas different cytokine preconditioning milieu, such as IL-7/IL-15, have been shown to promote T cell engraftment, the impact of this approach on CAR T cell responses to adjuvant immune-checkpoint blockade has not been assessed. In the current study, we reveal that the preconditioning of CAR T cells with IL-7/IL-15 increased CAR T cell responses to anti-PD-1 adjuvant therapy. This was associated with the emergence of an intratumoral CD8+CD62L+TCF7+IRF4- population that was highly responsive to anti-PD-1 therapy and mediated the vast majority of transcriptional and epigenetic changes in vivo following PD-1 blockade. Our data indicate that preservation of CAR T cells in a TCF7+ phenotype is crucial for their responsiveness to adjuvant immunotherapy approaches and should be a key consideration when designing clinical protocols.

Spatial and temporal translational control of germ cell mRNAs mediated by the eIF4E isoform IFE-1.

Regulated mRNA translation is vital for germ cells to produce new proteins in the spatial and temporal patterns that drive gamete development. Translational control involves the de-repression of stored mRNAs and their recruitment by eukaryotic initiation factors (eIFs) to ribosomes. C. elegans expresses five eIF4Es (IFE-1-IFE-5); several have been shown to selectively recruit unique pools of mRNA. Individual IFE knockouts yield unique phenotypes due to inefficient translation of certain mRNAs. Here, we identified mRNAs preferentially translated through the germline-specific eIF4E isoform IFE-1. Differential polysome microarray analysis identified 77 mRNAs recruited by IFE-1. Among the IFE-1-dependent mRNAs are several required for late germ cell differentiation and maturation. Polysome association of gld-1, vab-1, vpr-1, rab-7 and rnp-3 mRNAs relies on IFE-1. Live animal imaging showed IFE-1-dependent selectivity in spatial and temporal translation of germline mRNAs. Altered MAPK activation in oocytes suggests dual roles for IFE-1, both promoting and suppressing oocyte maturation at different stages. This single eIF4E isoform exerts positive, selective translational control during germ cell differentiation.

Generating CAR T cells from tumor-infiltrating lymphocytes.

Background: Tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T-cell therapies have demonstrated promising, though limited, efficacy against melanoma. Methods: We designed a model system to explore the efficacy of dual specific T cells derived from melanoma patient TILs by transduction with a Her2-specific CAR. Results: Metastatic melanoma cells in our biobank constitutively expressed Her2 antigen. CAR-TIL produced greater amounts of IFN compared with parental TIL, when co-cultured with Her2 expressing tumor lines, including autologous melanoma tumor lines, although no consistent increase in cytotoxicity by TIL was afforded by expression of a CAR. Results of an in vivo study in NSG mice demonstrated tumor shrinkage when CAR-TILs were used in an adoptive cell therapy protocol. Conclusion: Potential limitations of transduced TIL in our study included limited proliferative potential and a terminally differentiated phenotype, which would need addressing in further work before consideration of clinical translation.

CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy.

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.

MAIT cells regulate NK cell-mediated tumor immunity.

The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.

Macrophage-Derived CXCL9 and CXCL10 Are Required for Antitumor Immune Responses Following Immune Checkpoint Blockade.

PURPOSE: Response rates to immune checkpoint blockade (ICB; anti-PD-1/anti-CTLA-4) correlate with the extent of tumor immune infiltrate, but the mechanisms underlying the recruitment of T cells following therapy are poorly characterized. A greater understanding of these processes may see the development of therapeutic interventions that enhance T-cell recruitment and, consequently, improved patient outcomes. We therefore investigated the chemokines essential for immune cell recruitment and subsequent therapeutic efficacy of these immunotherapies. EXPERIMENTAL DESIGN: The chemokines upregulated by dual PD-1/CTLA-4 blockade were assessed using NanoString-based analysis with results confirmed at the protein level by flow cytometry and cytometric bead array. Blocking/neutralizing antibodies confirmed the requirement for key chemokines/cytokines and immune effector cells. Results were confirmed in patients treated with immune checkpoint inhibitors using single-cell RNA-sequencing (RNA-seq) and paired survival analyses. RESULTS: The CXCR3 ligands, CXCL9 and CXCL10, were significantly upregulated following dual PD-1/CTLA-4 blockade and both CD8+ T-cell infiltration and therapeutic efficacy were CXCR3 dependent. In both murine models and patients undergoing immunotherapy, macrophages were the predominant source of CXCL9 and their depletion abrogated CD8+ T-cell infiltration and the therapeutic efficacy of dual ICB. Single-cell RNA-seq analysis of patient tumor-infiltrating lymphocytes (TIL) revealed that CXCL9/10/11 was predominantly expressed by macrophages following ICB and we identified a distinct macrophage signature that was associated with positive responses to ICB. CONCLUSIONS: These data underline the fundamental importance of macrophage-derived CXCR3 ligands for the therapeutic efficacy of ICB and highlight the potential of manipulating this axis to enhance patient responses.

Dual PD-1 and CTLA-4 Checkpoint Blockade Promotes Antitumor Immune Responses through CD4+Foxp3- Cell-Mediated Modulation of CD103+ Dendritic Cells.

Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4+Foxp3- cells activated by dual PD-1/CTLA-4 blockade modulated the myeloid compartment, including activation of conventional CD103+ dendritic cells (DC) and expansion of a myeloid subset that produces TNFα and iNOS (TIP-DCs). CD4+Foxp3- T cell-mediated activation of CD103+ DCs resulted in enhanced IL12 production by these cells and IL12 enhanced the therapeutic effect of dual PD-1/CTLA-4 blockade. Given the importance of these myeloid subsets in the antitumor immune response, our data point to a previously underappreciated role of CD4+Foxp3- cells in modulating this arm of the antitumor immune response. Cancer Immunol Res; 6(9); 1069-81. ©2018 AACR.

Healthy by Design: Using a Gender Focus to Influence Complete Streets Policy.

BACKGROUND: Public health leaders in Yellowstone County, Montana, formed an alliance to address community-wide issues. One such issue is Complete Streets, with its vision of safe streets for all. This case study focuses on development and adoption of a Complete Streets policy. It examines how a community coalition, Healthy By Design, infused a gender focus into the policymaking process. METHODS: An incremental and nonlinear policymaking process was aided by a focus on gender and health equity. The focus on a large constituency helped to frame advocacy in terms of a broad population's needs, not just special interests. RESULTS: The city council unanimously adopted a Complete Streets resolution, informed by a gender lens. Healthy By Design further used gender information to successfully mobilize the community in response to threats of repeal of the policy, and then influenced the adoption of a revised policy. CONCLUSIONS: Policies developed with a focus on equity, including gender equity, may have broader impact on the community. Such policies may pave the way for future policies that seek to transform gender norms toward building a healthier community for all residents.

Agonist immunotherapy restores T cell function following MEK inhibition improving efficacy in breast cancer.

The presence of tumor-infiltrating lymphocytes in triple-negative breast cancers is correlated with improved outcomes. Ras/MAPK pathway activation is associated with significantly lower levels of tumor-infiltrating lymphocytes in triple-negative breast cancers and while MEK inhibition can promote recruitment of tumor-infiltrating lymphocytes to the tumor, here we show that MEK inhibition adversely affects early onset T-cell effector function. We show that α-4-1BB and α-OX-40 T-cell agonist antibodies can rescue the adverse effects of MEK inhibition on T cells in both mouse and human T cells, which results in augmented anti-tumor effects in vivo. This effect is dependent upon increased downstream p38/JNK pathway activation. Taken together, our data suggest that although Ras/MAPK pathway inhibition can increase tumor immunogenicity, the negative impact on T-cell activity is functionally important. This undesirable impact is effectively prevented by combination with T-cell immune agonist immunotherapies resulting in superior therapeutic efficacy.MEK inhibition in breast cancer is associated with increased tumour infiltrating lymphocytes (TILs), however, MAPK activity is required for T cells function. Here the authors show that TILs activity following MEK inhibition can be enhanced by agonist immunotherapy resulting in synergic therapeutic effects.

Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy.

Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types.

A Multifunctional Role for Adjuvant Anti-4-1BB Therapy in Augmenting Antitumor Response by Chimeric Antigen Receptor T Cells.

Adoptive immunotherapy utilizing chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematologic cancers, but results against solid malignancies have been limited to date, due in part to the immunosuppressive tumor microenvironment. Activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong costimulatory signals for augmenting and diversifying T-cell responses. We therefore hypothesized that a combination of α-4-1BB and CAR T-cell therapy would result in improved antitumor responses. Using a human-Her2 self-antigen mouse model, we report here that α-4-1BB significantly enhanced CAR T-cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Treatment also increased the expression of IFNγ and the proliferation marker Ki67 in tumor-infiltrating CAR T cells when combined with α-4-1BB. Strikingly, α-4-1BB significantly reduced host immunosuppressive cells at the tumor site, including regulatory T cells and myeloid-derived suppressor cells, correlating with an increased therapeutic response. We conclude that α-4-1BB has a multifunctional role for enhancing CAR T-cell responses and that this combination therapy has high translational potential, given current phase I/II clinical trials with α-4-1BB against various types of cancer. Cancer Res; 77(6); 1296-309. ©2017 AACR.

CD73: A potential biomarker for anti-PD-1 therapy.

In our recent study, we show that tumoral CD73 expression limits the efficacy of anti-PD-1 therapy, and this is rescued by concomitant A2A blockade. Since CD73 is known to be overexpressed in several human cancers and A2A antagonists have undergone clinical trials for Parkinson's Disease, this combination warrants further investigation.

Adenosine Receptor 2A Blockade Increases the Efficacy of Anti-PD-1 through Enhanced Antitumor T-cell Responses.

Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent clinical trials with anti-CTLA-4 and anti-PD-1/PD-L1 antibodies (mAbs) suggest that targeting multiple immunosuppressive pathways may significantly improve patient survival. The generation of adenosine by CD73 also suppresses antitumor immune responses through the activation of A2A receptors on T cells and natural killer (NK) cells. We sought to determine whether blockade of A2A receptors could enhance the efficacy of anti-PD-1 mAb. The expression of CD73 by tumor cells limited the efficacy of anti-PD-1 mAb in two tumor models, and this was alleviated with concomitant treatment with an A2A adenosine receptor antagonist. The blockade of PD-1 enhanced A2A receptor expression on tumor-infiltrating CD8(+) T cells, making them more susceptible to A2A-mediated suppression. Thus, dual blockade of PD-1 and A2A significantly enhanced the expression of IFNγ and Granzyme B by tumor-infiltrating CD8(+) T cells and, accordingly, increased growth inhibition of CD73(+) tumors and survival of mice. The results of our study indicate that CD73 expression may constitute a potential biomarker for the efficacy of anti-PD-1 mAb in patients with cancer and that the efficacy of anti-PD-1 mAb can be significantly enhanced by A2A antagonists. We have therefore revealed a potentially novel biomarker for the efficacy of anti-PD-1 that warrants further investigation in patients. Because our studies used SYN-115, a drug that has already undergone phase IIb testing in Parkinson disease, our findings have immediate translational relevance for patients with cancer.

Induction of cap-independent BiP (hsp-3) and Bcl-2 (ced-9) translation in response to eIF4G (IFG-1) depletion in C. elegans.

During apoptosis, activated caspases cleave the translation initiation factor eIF4G. This cleavage disrupts cap-dependent mRNA translation initiation within the cell. However, a specific subset of mRNAs can still be recruited for protein synthesis in a cap-independent manner by the residual initiation machinery. Many of these mRNAs, including cell death related mRNAs, contain internal ribosome entry sites (IRESes) that promote their enhanced translation during apoptosis. Still other mRNAs have little dependence on the cap recognition mechanism. The expression of the encoded proteins, both anti- and pro-apoptotic, allows for an initial period of attempted cell survival, then commitment to cell death when damage is extensive. In this study we address the translational regulation of the stress and apoptosis-related mRNAs in C. elegans: BiP (hsp-3) (hsp-4), Hif-1 (hif-1), p53 (cep-1), Bcl-2 (ced-9) and Apaf-1 (ced-4). Altered translational efficiency of these messages was observed upon depletion of cap-dependent translation and induction of apoptosis within the C. elegans gonad. Our findings suggest a physiological link between the cap-independent mechanism and the enhanced translation of hsp-3 and ced-9. This increase in the efficiency of translation may be integral to the stress response during the induction of physiological apoptosis.

Chimeric antigen receptor-redirected T cells display multifunctional capacity and enhanced tumor-specific cytokine secretion upon secondary ligation of chimeric receptor.

AIM: The aim of the current study was to fully elucidate the functions of T cells genetically modified with an erbB2-specific chimeric antigen receptor (CAR). MATERIAL & METHODS: In this study, key functional parameters of CAR T cells were examined following antigen-specific stimulation of the chimeric anti-erbB2 receptor. RESULTS: Gene-modified T cells produced the cytokines IFN-γ, IL-2, IL-4, IL-10, TNF-α and IL-17, and the chemokine RANTES upon CAR ligation. A multifunctional capacity of these CAR T cells was also demonstrated, where 13.7% of cells were found to simultaneously express IFN-γ and CD107a, indicative of cytolytic granule release. In addition, the CAR T cells were able to respond to a greater degree on the second ligation of CAR, which has not been previously shown. IFN-γ secretion levels were significantly higher on second ligation than those secreted following initial ligation. CAR-expressing T cells were also demonstrated to lyze erbB2-expressing tumor cells in the absence of activity against bystander cells not expressing the erbB2 antigen, thereby demonstrating exquisite specificity. CONCLUSION: This study demonstrates the specificity of CAR gene-engineered T cells and their capacity to deliver a wide range of functions against tumor cells with an enhanced response capability after initial receptor engagement.

A germline-specific isoform of eIF4E (IFE-1) is required for efficient translation of stored mRNAs and maturation of both oocytes and sperm.

Fertility and embryonic viability are measures of efficient germ cell growth and development. During oogenesis and spermatogenesis, new proteins are required for both mitotic expansion and differentiation. Qualitative and quantitative changes in protein synthesis occur by translational control of mRNAs, mediated in part by eIF4E, which binds the mRNAs 5' cap. IFE-1 is one of five eIF4E isoforms identified in C. elegans. IFE-1 is expressed primarily in the germ line and associates with P granules, large mRNPs that store mRNAs. We isolated a strain that lacks IFE-1 [ife-1(bn127)] and demonstrated that the translation of several maternal mRNAs (pos-1, pal-1, mex-1 and oma-1) was inefficient relative to that in wild-type worms. At 25 degrees C, ife-1(bn127) spermatocytes failed in cytokinesis, prematurely expressed the pro-apoptotic protein CED-4/Apaf-1, and accumulated as multinucleate cells unable to mature to spermatids. A modest defect in oocyte development was also observed. Oocytes progressed normally through mitosis and meiosis, but subsequent production of competent oocytes became limiting, even in the presence of wild-type sperm. Combined gametogenesis defects decreased worm fertility by 80% at 20 degrees C; ife-1 worms were completely sterile at 25 degrees C. Thus, IFE-1 plays independent roles in late oogenesis and spermatogenesis through selective translation of germline-specific mRNAs.