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Stem-like CD8 T cells are regulated by the transcription factor TCF1 and are key players in the response to immune checkpoint blockade (ICB). However, recent findings indicate that the dependance on TCF1+ stem-like T cells for ICB efficacy may not be equal across patients or in different tumor contexts. Here we leveraged TCF1 conditional knock-out (TCF1 cKO) mice to investigate how TCF1 instructs the early fate and functions of CD8 T cells upon ICB therapy in tumors that differ for immunogenicity and levels of tumor antigen expression. Strikingly, we discovered that TCF1 expression in CD8 T cells is required for ICB efficacy in poorly immunogenic B16OVA melanomas but is dispensable in highly immunogenic MC38OVA colorectal tumors. Single-cell RNA sequencing and immunophenotyping in the tumor draining lymph node (TDLN) revealed defective priming and expansion of tumor-specific TCF1 cKO T cells in B16OVA- but not MC38OVA-bearing mice treated with ICB. Conversely, ICB therapy efficiently expanded tumor-specific TCF1 WT T cells in the TDLN of both tumor models. In vitro, we found defective proliferation, reduced PD1 and CD28 up-regulation and reduced phosphorylation of key molecules downstream the T cell receptor pathway in TCF1 cKO T cells stimulated with low but not high TCR signals. These data indicate that TCF1 poises T cells for optimal responsiveness in suboptimal priming conditions such as those found in low antigen expressing tumors. Furthermore, in the absence of TCF1 we found the accumulation in the TDLN of a subset of tumor-specific naïve T cells poised to give rise to short-lived effectors, which are less suited to sustain anti-tumor responses in poorly immunogenic tumors where expansion of T cells retaining memory potential is required for durable responses. Single-cell RNA sequencing and immunophenotyping within the tumor microenvironment showed that in MC38OVA tumors both WT and TCF1 cKO mice expanded a CD8 subset sharing a signature with transitory effectors, which were shown to mediate ICB efficacy in chronic viral infection models. Expansion of cytotoxic CD8 T cells likely accounted for the strong anti-tumor response observed in both WT and TCF1 cKO mice. Conversely, B16OVA tumors failed to expand transitory effectors and accumulated Tox+ dysfunctional CD8 T cells. Importantly, in the absence of TCF1 dysfunctional T cells became destabilized, failed to persist and shared features with CD8 T cells found in patients that fail ICB. Altogether, the reduced priming of stem-like T cells in the TDLN combined with a destabilized dysfunctional T cell state in the tumor contributed to the failure of TCF1 cKO mice to sustain effective ICB responses in poorly immunogenic tumors. Our study highlights a role for TCF1 during the priming and early stages of the anti-tumor CD8 T cell response with important implications for guiding optimal therapeutic interventions in cancers with low frequency of TCF1+ CD8 T cells and low neoantigen expression. Citation Format: Giulia Escobar, Katherine Tooley, Joan Pages Oliveras, Linglin Huang, Hanning Cheng, Chang Xue, Davide Mangani, Natanael Hazel, Carola Rutigliani, Luca Biasco, Ana C. Anderson. Tumor context dictates reliance on TCF1 for response to immunotherapy. [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 4157.

Stem-like CD8 T cells that are regulated by the transcription factor TCF1 are key players in the response to immune checkpoint blockade (ICB). However, recent findings indicate that the dependance on TCF1+ stem-like T cells for ICB efficacy may not be equal across patients or in different tumor contexts and raises the question of what determines reliance on TCF1 for effective responses. Here we leveraged TCF1 conditional knock-out (TCF1-cKO) mice to investigate how TCF1 instructs the early fate and functions of CD8 cells upon ICB therapy in tumors that differ for immunogenicity and levels of tumor antigens expression. Strikingly, we discovered that TCF1 expression in CD8 T cells is required for ICB efficacy in poorly immunogenic B16OVA melanomas but is dispensable in highly immunogenic MC38OVA colorectal tumors. Single-cell-RNA sequencing and immunophenotyping revealed defective priming and expansion of tumor-specific TCF1-cKO T cells in the tumor draining lymph-node (TDLN) of B16OVA- but not MC38OVA-bearing mice treated with ICB. In vitro, we found defective proliferation, reduced PD-1 and CD28 up-regulation and reduced phosphorylation of key molecules downstream the T cell receptor pathway when TCF1-cKO T cells were stimulated with low doses of antigens but not when stimulated with strong TCR signals. These data indicate that TCF1 poises T cells for optimal antigen responsiveness. Furthermore, transcriptional profiling of T cells in the TDLN revealed the accumulation of a subset of tumor-specific naïve T cells poised to give rise to short-lived effectors in TCF1-cKO mice and thus is less suited to sustain anti-tumor responses in poorly immunogenic tumors where expansion of T cells retaining stem-like potential is required for durable anti-tumor responses. In the MC38OVA tumor, single-cell-RNA sequencing and immunophenotyping showed that both WT and TCF1-cKO mice displayed an expanded CD8 subset sharing a signature with transitory effector cells that mediate ICB efficacy in chronic viral infection models. These cells likely accounted for the strong anti-tumor response observed in both WT and TCF1-cKO mice. Conversely, B16-OVA tumors retained a higher frequency of stem-like cells, failed to expand transitory effectors and accumulated Tox+ CD8 T cells, sharing a signature with CD8 cells expanded in non-responders to ICB. Importantly, loss of TCF1 was associated with reduced maintenance and proliferation of stem-like precursors and reduced expression of the Tox gene which was required for the survival of the dysfunctional T cells. Altogether, these data indicate that the reduced priming and maintenance of stem-like T cells combined with a destabilized dysfunctional T cell state in the tumor microenvironment contributed to the failure of TCF1-cKO mice to sustain effective ICB responses in poorly immunogenic tumors. Our study highlights a role for TCF1 in the early stages of the CD8 T cell response with important implications for guiding the choice of optimal therapeutic interventions in tumors expressing low neoantigen levels. Citation Format: Giulia Escobar, Katherine Tooley, Joan Pagès Oliveras, Cheng Hanning, Xue Chang, Davide Mangani, Hazel Natanael, Carola Rutigliani, Luca Biasco, Ana Carrizosa Anderson. Tumor context dictates reliance on TCF1 for response to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B35.

Unleashing cytotoxic CD8+ T cells for effective cancer treatment requires understanding T cell states across different tumor microenvironments. Here, we developed an algorithm to recover both shared and tumor type specific programs and used it to analyze a scRNA-seq compendium of 38,852 CD8+ T cells from 141 patients spanning nine different human cancers. We uncovered a pan-cancer T cell dysfunction program that was predictive of clinical responses to immunotherapy and highlighted CXCR6 as a pan-cancer marker of dysfunctional T cells. In mouse models, CXCR6 increased following checkpoint blockade and was repressed by TCF1. Its ligand, CXCL16, was expressed primarily by myeloid cells, and was co-regulated with antigen presentation genes. CXCR6 deletion decreased Tox, CX3CR1, and Bcl2 expression, predisposing dysfunctional PD-1+Tim3+CD8+ T cells to apoptosis, and compromising tumor growth control. Our approach discovered a TCF1:CXCR6-CXCL16 regulatory axis essential for effective anti-tumor immunity, revealing a new perspective on T cell dysfunction and new opportunities for modulating this cell state.

Recent advances have redefined a role for T cell factor 1 (TCF1) that goes beyond T cell development and T memory formation and encompasses new functions in the regulation of T cell biology. Here, we discuss the multifaceted and context-dependent role of TCF1 in peripheral T cells, particularly during disease-induced inflammatory states such as autoimmunity, cancer, and chronic infections. Understanding how TCF1 fine-tunes peripheral T cell biology holds the potential to tailor improved immune-targeted therapies.

Summary Identifying signals in the tumor microenvironment (TME) that shape CD8+ T cell phenotype can inform novel therapeutic approaches for cancer. Here, we identified a gradient of increasing glucocorticoid receptor (GR) expression and signaling from naive to dysfunctional CD8+ tumor-infiltrating lymphocytes (TILs). Conditional deletion of the GR in CD8+ TILs improved effector differentiation, reduced expression of the transcription factor TCF-1, and inhibited the dysfunctional phenotype, culminating in tumor growth inhibition. GR signaling transactivated the expression of multiple checkpoint receptors and promoted the induction of dysfunction-associated genes upon T cell activation. In the TME, monocyte-macrophage lineage cells produced glucocorticoids and genetic ablation of steroidogenesis in these cells as well as localized pharmacologic inhibition of glucocorticoid biosynthesis improved tumor growth control. Active glucocorticoid signaling associated with failure to respond to checkpoint blockade in both preclinical models and melanoma patients. Thus, endogenous steroid hormone signaling in CD8+ TILs promotes dysfunction, with important implications for cancer immunotherapy.

Immunotherapy is emerging as a new pillar of cancer treatment with potential to cure. However, many patients still fail to respond to these therapies. Among the underlying factors, an immunosuppressive tumor microenvironment (TME) plays a major role. Here we show that monocyte-mediated gene delivery of IFNα inhibits leukemia in a mouse model. IFN gene therapy counteracts leukemia-induced expansion of immunosuppressive myeloid cells and imposes an immunostimulatory program to the TME, as shown by bulk and single-cell transcriptome analyses. This reprogramming promotes T-cell priming and effector function against multiple surrogate tumor-specific antigens, inhibiting leukemia growth in our experimental model. Durable responses are observed in a fraction of mice and are further increased combining gene therapy with checkpoint blockers. Furthermore, IFN gene therapy strongly enhances anti-tumor activity of adoptively transferred T cells engineered with tumor-specific TCR or CAR, overcoming suppressive signals in the leukemia TME. These findings warrant further investigations on the potential development of our gene therapy strategy towards clinical testing., An immune suppressive tumor microenvironment (TME) is a limitation for immunotherapy. Here the authors show that, in a B cell acute lymphoblastic leukemia mouse model, gene-based delivery of IFNα reprograms the leukemia-induced immunosuppressive TME into immunostimulatory and enhances T-cell responses.

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases.

An improved understanding of the anti-tumor CD8(+) T cell response after checkpoint blockade would enable more informed and effective therapeutic strategies. Here we examined the dynamics of the effector response of CD8(+) tumor-infiltrating lymphocytes (TILs) after checkpoint blockade therapy. Bulk and single-cell RNA profiles of CD8(+) TILs following combined Tim-3+PD-1 blockade in preclinical models revealed significant changes in the transcriptional profile of PD-1(−) TILs. These cells could be divided into subsets bearing characterstics of naïve-, effector-, and memory-precursor-like cells. Effector- and memory-precursor-like TILs contained tumor-antigen specific cells, exhibited proliferative and effector capacity and expanded in response to different checkpoint blockade therapies across different tumor models. The memory-precursor-like subset shared features with CD8(+) T cells associated with response to checkpoint blockade in patients, and was compromised in the absence of Tcf7. Expression of Tcf7/Tcf1 was requisite for the efficacy of diverse immunotherapies, highlighting the importance of this transcriptional regulator in the development of effective CD8(+) T cell responses upon immunotherapy.

The establishment of an immunosuppressive microenvironment is one of the hallmarks of cancer and the major impediment to the successful application of cancer immunotherapy. By exploiting the tumor homing ability of a subpopulation of monocytes we turned them into an efficient vehicle for the tumor-targeted delivery of a potent immune-stimulatory molecule: interferon-alpha (IFNα);. We are now combining the ability of IFNα to reverse the immunosuppressive tumor microenvironment with established and new cancer immunotherapies for the treatment of advanced breast cancer, including disseminated lung and bone metastasis. We identified and characterized a specific subpopulation of pro-tumoral macrophages characterized by the expression of the Angiopoietin receptor TIE2 (TEMs: TIE2-expressing monocytes/macrophages) and endowed with pro-angiogenic and immunosuppressive activities. As a strategy aimed at reversing the immunosuppressive microenvironment, we exploited the tumor homing ability and selective expression of the TIE2 receptor to turn TEMs into IFNα delivery vehicles by engineering hematopoietic stem cells (HSCs) to express an IFNα transgene under the control of the Tie2 promoter. To progress this strategy to clinical application we recently developed a humanized vector expressing a human IFNα gene, and have studied the feasibility and safety of engineering human HSCs for its expression in their TEM progeny. Moreover, to demonstrate the efficacy of our human IFNα delivery platform, we challenged human hematochimeric mice with a human breast cancer cell line. Type I interferons (IFNs) are pleiotropic cytokines involved in innate and adaptive immunity that have been shown to promote anti-tumor immune responses. The broad anti-tumor biological activities of type I IFNs have provided the rationale for testing administration of exogenous IFNα as an anti-cancer treatment, which has proven effective against several solid and hematological malignancies. However, clinical use of IFNα has since declined because of the substantial toxicity associated with systemic administration and the limited efficacy at the maximal tolerated doses, thus calling for safer and more effective delivery strategies. Our cell- and gene-based IFNα delivery strategy strongly inhibited primary breast cancer tumors and breast cancer lung metastasis in mouse and human hematochimeric models by promoting the recruitment and activation of both innate and adaptive immune cells with no evident signs of toxicity. Moreover, our studies validated the feasibility, safety and therapeutic potential of a new HSC-based gene therapy strategy to treat established tumors, and could, therefore, revive interest in testing high-dose chemotherapy followed by autologous HSC transplantation in advanced breast cancer patients. Importantly, we think that the multiple activities of type I IFNs in the complex network of cell interactions that lead to activation and deployment of immune responses may represent a valid strategy to promote and improve the outcome of cancer immunotherapy for the treatment of advanced breast cancer including lung and bone metastasis. Preliminary data on new therapeutic strategies combining TEM-mediated delivery of IFNα with monoclonal antibodies against inhibitory checkpoint molecules such as PD1 and CTLA4, will be presented. Citation Format: Giulia Escobar, Davide Moi, Ryan Galea, Moustafa Sherif, Anna Ranghetti, Luigi Naldini, Roberta Mazzieri. Genetic engineering of tumor-infiltrating monocytes to inhibit primary and metastatic breast cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B181.

An immunosuppressive tumor microenvironment is a cancer hallmark and a major impediment to successful immunotherapy. We engineered hematopoietic progenitors to target expression of an interferon-α (IFNα) transgene specifically to their monocytic progeny, including tumor-infiltrating macrophages. Mice chimeric for these IFNα-expressing macrophages showed activation of innate and adaptive immune cells against breast cancer and inhibited disease progression.

Colorectal cancer (CRC) metastatic dissemination to the liver is one of the most life-threatening malignancies in humans and represents the leading cause of CRC-related mortality. Herein, we adopted a gene transfer strategy into mouse hematopoietic stem/progenitor cells to generate immune-competent mice in which TEMsa subset of Tie2(+) monocytes/macrophages found at peritumoral sitesexpress interferon-alpha (IFN), a pleiotropic cytokine with anti-tumor effects. Utilizing this strategy in mouse models of CRC liver metastasis, we show that TEMs accumulate in the proximity of hepatic metastatic areas and that TEM-mediated delivery of IFN inhibits tumor growth when administered prior to metastasis challenge as well as on established hepatic lesions, improving overall survival. Further analyses unveiled that local delivery of IFN does not inhibit homing but limits the early phases of hepatic CRC cell expansion by acting on the radio-resistant hepatic microenvironment. TEM-mediated IFN expression was not associated with systemic side effects, hematopoietic toxicity, or inability to respond to a virus challenge. Along with the notion that TEMs were detected in the proximity of CRC metastases in human livers, these results raise the possibility to employ similar gene/cell therapies as tumor site-specific drug-delivery strategies in patients withCRC. Colorectal cancer (CRC) metastatic dissemination to the liver is one of the most life-threatening malignancies in humans and represents the leading cause of CRC-related mortality. Herein, we adopted a gene transfer strategy into mouse hematopoietic stem/progenitor cells to generate immune-competent mice in which TEMs-a subset of Tie2+ monocytes/macrophages found at peritumoral sites-express interferon-alpha (IFNα), a pleiotropic cytokine with anti-tumor effects. Utilizing this strategy in mouse models of CRC liver metastasis, we show that TEMs accumulate in the proximity of hepatic metastatic areas and that TEM-mediated delivery of IFNα inhibits tumor growth when administered prior to metastasis challenge as well as on established hepatic lesions, improving overall survival. Further analyses unveiled that local delivery of IFNα does not inhibit homing but limits the early phases of hepatic CRC cell expansion by acting on the radio-resistant hepatic microenvironment. TEM-mediated IFNα expression was not associated with systemic side effects, hematopoietic toxicity, or inability to respond to a virus challenge. Along with the notion that TEMs were detected in the proximity of CRC metastases in human livers, these results raise the possibility to employ similar gene/cell therapies as tumor site-specific drug-delivery strategies in patients with CRC.