T-cell Effector Function Research Articles

GSK-3 Inhibition as a Therapeutic Approach Against SARs CoV2: Dual Benefit of Inhibiting Viral Replication While Potentiating the Immune Response.

The SARS-CoV2 (COVID-19) pandemic and uncertainties in developing a vaccine have created an urgent need for new therapeutic approaches. A key question is whether it is possible to make rational predictions of new therapies based on the presently available scientific and medical information. In this regard, I have noticed an omission in the present analysis in the literature related to the exploitation of glycogen synthase kinase 3 (GSK-3) as a therapeutic approach. This is based on two key observations, that GSK-3 inhibitors can simultaneously block SARs viral replication, while boosting CD8+ adaptive T-cell and innate natural killer (NK) responses. Firstly, it is already clear that GSK-3 phosphorylation of SARs CoV1 N protein on key serine residues is needed for viral replication such that small molecule inhibitors (SMIs) of GSK-3 can inhibit viral replication. In comparing protein sequences, I show here that the key sites in the N protein of SARs CoV1 N for replication are conserved in SARs CoV2. This strongly suggests that GSK-3 SMIs will also inhibit SARs Cov2 replication. Secondly, we and others have previously documented that GSK-3 SMIs markedly enhance CD8+ cytolytic T-cell (CTL) and NK cell anti-viral effector functions leading to a reduction in both acute and chronic viral infections in mice. My hypothesis is that the repurposing of low-cost inhibitors of GSK-3 such as lithium will limit SARS-CoV2 infections by both reducing viral replication and potentiating the immune response against the virus. To date, there has been no mention of this dual connection between GSK-3 and SARs CoV2 in the literature. To my knowledge, no other drugs exist with the potential to simultaneously target both viral replication and immune response against SARs CoV2.

Allogeneic FLT3 CAR T Cells with an Off-Switch Exhibit Potent Activity against AML and Can Be Depleted to Expedite Bone Marrow Recovery

Patients with relapsed or refractory acute myeloid leukemia (AML) have a dismal prognosis and limited treatment options. Chimeric antigen receptor (CAR) Tcells have achieved unprecedented clinical responses in patients with B cell leukemias and lymphomas and could prove highly efficacious in AML. However, a significant number of patients with AML may not receive treatment with an autologous product due to manufacturing failures associated with low lymphocyte counts or rapid disease progression while the therapeutic is being produced. We report the preclinical evaluation of an off-the-shelf CAR Tcell therapy targeting Fms-related tyrosine kinase 3 (FLT3) for the treatment of AML. Single-chain variable fragments (scFvs) targeting various epitopes in the extracellular region of FLT3 were inserted into CAR constructs and tested for their ability to redirect Tcell specificity and effector function to FLT3+ AML cells. A lead CAR, exhibiting minimal tonic signaling and robust activity invitro and invivo, was selected and then modified to incorporate a rituximab-responsive off-switch in cis. We found that allogeneic FLT3 CAR Tcells, generated from healthy-donor Tcells, eliminate primary AML blasts but are also active against mouse and human hematopoietic stem and progenitor cells, indicating risk of myelotoxicity. By employing a surrogate CAR with affinity to murine FLT3, we show that rituximab-mediated depletion of FLT3 CAR Tcells after AML eradication enables bone marrow recovery without compromising leukemia remission. These results support clinical investigation of allogeneic FLT3 CAR Tcells in AML and other FLT3+ hematologic malignancies.

Abstract IA25: Radiation as a way to flip the switch: Resistance to responder in HNSCC

Abstract The de novo response to checkpoint inhibitors in advanced head and neck squamous cell cancer (HNSCC) remains at ~20%. Responders in this small cohort of patients also tend to eventually develop resistance. Efforts at simply combining more checkpoint inhibitors in the recurrent setting to overcome such resistance have also proven inadequate. Mounting evidence suggests that radiation may act as an in situ vaccine, boosting and stimulating antigen presentation, altering the tumor microenvironment (TME), enhancing interferon release, and upregulating PD-L1 expression that leads to enhanced response to checkpoint inhibitors. In patients where resistance has developed, radiation may hit the reset button and reconvert cancers to sensitivity again. The genomic landscape of HNSCC may play a role in looking to rationally combine novel agents already approved in breast and ovarian cancers with DNA damage repair defects, and the tumor mutational burden (TMB) may lend itself to predicting improved response to checkpoint inhibition. However, there is evidence that a high TMB might exist in HNSCC, similar to NSCLC, yet lack a proinflammatory TME that theoretically leads to reduced response to immune-modulating agents. In preclinical animal models of HNSCCs, radiation has been shown to enhance tumor responsiveness to checkpoint inhibitors by fueling the TME. Radiation initiates cell death and the production and release of cytokines and chemokines, such as CXCL9 and CXCL10, into the TME, which leads to infiltration of dendritic cells, macrophages, promotion of antigen-specific T-cell priming and cytotoxic T-cell infiltration and effector function. RT, however, can also modulate the infiltration of an immunosuppressive cell population in a dose-dependent manner. Critical to overcoming resistance or initiating a strong response to immune-enabling drugs may be how and when we deliver the radiation and the type of radiation utilized—hypofractionated or conventionally fractionated doses. High infiltration of Tregs and MDSCCs may contribute to radiation resistance along with resistance to immune-enabling drugs, so altering or depleting this population may lend itself to reversing or preventing resistance from developing. Preclinical evidence suggests that combining radiation with Treg depletion is far more effective than Treg depletion alone for improving response to anti-PD1 or PD-L1 therapy. This is just the beginning of our exploration of radiation’s ability to “flip the switch” and improve response to HNSCC that otherwise would be “cold” to checkpoint inhibition. Breaking down HNSCCs that utilize additional immunosuppressive options such as TGFb may also allow for combinatorial approaches to improve response. This lends itself to paradigm shifts in how we utilize radiation in locally advanced and the metastatic settings. Citation Format: David Raben. Radiation as a way to flip the switch: Resistance to responder in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA25.

Abstract IA15: Suppression of intratumoral regulatory T cells restores radiosensitivity in head and neck cancer: Role for Stat3 inhibition

Abstract Radiation is commonly used in the definitive management of for head and neck cancer. Yet, many patients still fail treatment and develop resistance, especially non-virally driven tumors. Head and neck tumors are highly enriched in regulatory T cells that dampen the response to radiotherapy by creating an immune-inhibitory microenvironment. A key way by which CD4 T cells convert to FOXP3 positive regulatory T cells (Tregs) is through activation of STAT3. We explored mechanisms of Treg signaling and infiltration and assessed their modulation by RT in murine models of head and neck squamous cell carcinoma (HNSCC). Mechanisms of Treg infiltration were investigated in orthotopic murine HNSCC tumors using flow cytometry, multiplex cytokine arrays and targeted therapeutics. Mice were treated with STAT3 ASO, anti-CTLA-4, anti-CD25, and/or anti-PD-L1 alone and in combination with RT. Tumor growth and survival were assessed. Flow cytometry was used to assess phenotypic and functional changes in intratumoral T-cell populations. Multiplex ELISA was performed for assessment of cytokines. Treatment with anti-CD25 concurrently with RT led to tumor eradication, enhanced T-cell cytotoxicity, induced an antitumor antigen-specific memory response, decreased Tregs, and improved survival in orthotopic HNSCC tumor-bearing mice. Treg depletion induced an influx of CD8 and CD4 T cells when combined with RT. In addition, Treg depletion in combination with RT transformed myeloid populations, decreasing M2 macrophages and myeloid-derived suppressor cells (MDSCs) and increasing M1 macrophages. In contrast, RT alone or when combined with anti-CTLA4 and/or anti-PD-L1 did not lead to durable tumor control. To assess whether STAT3 inhibition can target Tregs we employed the STAT3 anti-sense oligonucleotide, AZD5149. STAT3 inhibition in combination with RT, but not as single agent, led to significant tumor growth delay and resulted in significant downregulation of immunosuppressive cytokines including TGF-β1 and IL10 and upregulation of IL2. Analysis of immune populations showed that STAT3 inhibition when combined with RT significantly decreases intratumoral Tregs and enhanced effector T-cell function. Experiments in nude mice demonstrated complete abolishment of the benefit of STAT3 ASO and RT, demonstrating the dependency on T cells for achieving tumor control. These data reveal a critical role for Tregs in mediating resistance to RT. Targeted depletion of Tregs represents an important mechanism of sensitizing tumors to RT. We propose that STAT3 inhibition is a viable and potent therapeutic target against Tregs. Our data support the design of clinical trials integrating STAT3 ASO in the standard of care for cancer patients receiving RT. Citation Format: Sana Karam. Suppression of intratumoral regulatory T cells restores radiosensitivity in head and neck cancer: Role for Stat3 inhibition [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA15.

Anti-VEGF Treatment Enhances CD8+ T-cell Antitumor Activity by Amplifying Hypoxia.

Antiangiogenic therapies that target the VEGF pathway have been used clinically to combat cancer for over a decade. Beyond having a direct impact on blood vessel development and tumor perfusion, accumulating evidence indicates that these agents also affect antitumor immune responses. Numerous clinical trials combining antiangiogenic drugs with immunotherapies for the treatment of cancer are ongoing, but a mechanistic understanding of how disruption of tumor angiogenesis may impact immunity is not fully discerned. Here, we reveal that blockade of VEGF-A with a mAb to VEGF augments activation of CD8+ T cells within tumors and potentiates their capacity to produce cytokines. We demonstrate that this phenomenon relies on the disruption of VEGFR2 signaling in the tumor microenvironment but does not affect CD8+ T cells directly. Instead, the augmented functional capacity of CD8+ T cells stems from increased tumor hypoxia that initiates a hypoxia-inducible factor-1α program within CD8+ T cells that directly enhances cytokine production. Finally, combinatorial administration of anti-VEGF with an immunotherapeutic antibody, anti-OX40, improved antitumor activity over single-agent treatments. Our findings illustrate that anti-VEGF treatment enhances CD8+ T-cell effector function and provides a mechanistic rationale for combining antiangiogenic and immunotherapeutic drugs for cancer treatment.

Cell-Penetrating Anti-Protein Kinase C Theta Antibodies Act Intracellularly to Generate Stable, Highly Suppressive Regulatory T Cells

Regulatory Tcells maintain immunological tolerance and dampen inflammatory responses. Administering regulatory Tcells can prevent the immune-mediated tissue destruction of graft-versus-host disease, which frequently accompanies hematopoietic stem cell transfer. Neutralizing the Tcell-specific kinase, protein kinase C theta, which promotes Tcell effector functions and represses regulatory Tcell differentiation, augments regulatory Tcell immunosuppression and stability. We used a synthetic, cell-penetrating peptide mimic to deliver antibodies recognizing protein kinase C theta into primary human CD4 Tcells. When differentiated exvivo into induced regulatory Tcells, treated cells expressed elevated levels of the regulatory Tcell transcriptional regulator forkhead box P3, the surface-bound immune checkpoint receptor programmed death receptor-1, and pro-inflammatory interferon gamma, previously ascribed to a specific population of stable, highly suppressive human induced regulatory Tcells. The invitro suppressive capacity of these induced regulatory Tcells was 10-fold greater than that of Tcells differentiated without antibody delivery. When administered at the time of graft-versus-host disease induction, using a humanized mouse model, antibody-treated regulatory Tcells were superior to non-treated Tcells in attenuating lethal outcomes. This antibody delivery approach may overcome obstacles currently encountered using patient-derived regulatory Tcells as a cell-based therapy for immune modulation.

Conversion of ATP to adenosine by CD39 and CD73 in multiple myeloma can be successfully targeted together with adenosine receptor A2A blockade

BackgroundPD1/PDL1-directed therapies have been unsuccessful for multiple myeloma (MM), an incurable cancer of plasma cells in the bone marrow (BM). Therefore, other immune checkpoints such as extracellular adenosine and its...

Modulating extracellular TCR-CD3 interaction to identify new immunotherapy targets against cancer

Abstract T cell recognition of antigen and resulting proximal signaling are key steps in the initiation of the adaptive immune response. Previous studies targeting antigen binding site for enhancing T-cell responses to tumor antigens often lead to off-target effects and toxicity. Recently, we used nuclear magnetic resonance (NMR) spectroscopy, mutational analysis and computational docking to derive a 3D structure of the extracellular TCR-CD3 assembly. Further, biomolecular force probe (BFP) measurements allowed us to determine how 2D affinity and force-modulated TCR-pMHC kinetics depend on TCR-CD3 interaction sites and affect transduction of extracellular pMHC-TCR ligation into T cell function. Based on our TCR-CD3 structural model and binding data, we generated TCR libraries for a melanoma-specific TCR (DMF5) using site-specific mutagenesis in the Cbhelix 3 and helix 4-F strand regions of the TCR to optimize the TCR-CD3 interaction and to select for mutants with enhanced T-cell effector function. One Cb helix 4-F strand mutant, NP202203AA showed increased T cell response to antigen and showed enhanced TCR-pMHC bond lifetime (catch-bonds) in BFP assays leading to prolonged T cell signaling. In the future, DMF5 TCR with reengineered CD3 binding regions will be used in tumor rejection in pre-clinical mouse melanoma models for efficacy and toxicity to develop more effective T cell therapies for human targets.

CD6-ALCAM signaling regulates multiple effector/memory T cell functions

Abstract CD6 is a T-cell costimulatory receptor that has been implicated in the pathogenesis of multiple autoimmune and inflammatory diseases. Primarily expressed on CD4 T cells, CD6 promotes immune synapse formation, T-cell activation and T-cell migration via interaction with its ligand activated leukocyte cell adhesion molecule (ALCAM). While the contribution of CD6 to T cell activation has been well described, less is known regarding the role of CD6 on effector and memory T cells (Teff). Thus, to better characterize this, we examined phosphorylation signaling patterns during CD6 co-stimulation together with the impact of CD6 on differentiated Teff functions. Profiling of ~100 phosphorylation targets associated with T-cell receptor signaling revealed that CD6 co-stimulation on T cells activates factors in pathways involved in actin polymerization, motility, integrin activation and T-cell activation. Comparison of Tnaive cells vs. Teff demonstrated differing levels of phosphorylation in response to CD6 stimulation. Furthermore, CD6 signaling on Teff sustained phosphorylation of these pathways at later timepoints compared to CD28 stimulation. Blockade of the CD6 pathway, using the clinically tested anti-CD6 mAb itolizumab during re-stimulation of CD4 Teff cells in the presence of ALCAM, inhibited multiple effector functions including proliferation and changes in blast size. This effect was observed exclusively in the presence of ALCAM, indicating that the effect was specific to blockade of the CD6-ALCAM pathway. These findings demonstrate that the CD6-ALCAM pathway is a key regulator of effector T-cell functions and further support targeting this pathway to directly inhibit both naïve and effector T cell populations.

Tri-functional T-cell engagers target immune checkpoint inhibitors PD-1 and TIGIT to enhance CD8 effector T-cell functions in chronically SHIV-infected rhesus macaques

Abstract Checkpoint inhibitors, such as PD-1 and TIGIT, are crucial dysregulators of CD8 T cell function during chronic HIV/SIV infections. Importantly, these checkpoint inhibitors are highly expressed on the surface of CD4 T cells that harbor latent HIV. We have previously demonstrated that an anti-HIV/anti-CD3 bispecific T-cell engager (BiTE) can be used to redirect follicular CD8 (fCD8) T cells to kill HIV infected cells in vitro. Here, we hypothesize that adding an extra specificity to target PD-1 or TIGIT to the BiTE will further enhance the functional activities of CD8 T cells and simultaneously lower the threshold for reactivation of HIV latently infected cells. By using HIV-infected cell lines and primary CD4 T cells isolated from chronically SHIV-infected lymph nodes (LN) as target cells in in vitro and ex vivo killing assays, we found that trifunctional anti-HIV/anti-CD3-anti-TIGIT or anti-HIV/anti-CD3-anti-PD1 increased (1) T-cell specific activation, (2) antigen-specific CD107a degranulation, levels of granzyme B, cytokine/chemokine release by CD8 T cells, and (3) the killing capability of functionally compromised CD8 T cells when compared to the BiTE. When administered at 100 mcg/kg subcutaneously to chronically SHIV-infected animals, we demonstrated that anti-HIV/anti-CD3-anti-TIGIT could safely be used to augment activation and expansion of CD8 effector T cells that further resulted in a better elimination of the SHIV-infected cells as shown by the reduction in cell-associated SHIV gag DNA and RNA. Together, our results indicate that trifunctional T cell engagers targeting checkpoint inhibitors may serve as novel immunotherapeutic strategies to eliminate infected cells in HIV infected individuals.

Abstract B12: Quantitative high-resolution tissue analysis defines intratumoral hot spots of PMN-MDSC activity in situ

Abstract Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of myeloid cells with T-cell suppressive activity. While the identity and function of circulating MDSC is fairly well established in human cancer patients, the nature of potential MDSC in tissues remains largely enigmatic. However, understanding the biology of MDSC in human tissues is of pivotal importance, because MDSC are believed to regulate the activity of antitumor T cells and may serve as a major resistance mechanism in modern cancer immunotherapy. We developed a whole-mount tissue processing and staining procedure to mark PMN-MDSC subsets and T-cell effector function in situ in an intact 3-dimensional tissue architecture. We then used ultramicroscopy, digital image analysis and 3-D image reconstruction of original data to analyze and quantify the spatial distribution of MDSC and T cells. We identified intratumoral hot spots with high prevalence for both MDSC and T cells. T-cell effector function was reduced in these hot spots. Multicolor immunofluorescence and high-resolution image analysis also revealed the identity and frequency of cellular conjugates between MDSC and T cells. Quantitative spatial analyses indicated that immunosuppressive activity of MDSC requires close proximity of MDSC and T cells. Obtained data were applied to 2-D multicolor immunofluorescence analysis in a larger cohort of head and neck cancer patients with well-defined follow-up. Patients with evidence for strong downregulation of T-cell activity by PMN-MDSC presented with a significantly impaired survival, suggesting a clinical relevance of our findings. Our data suggest that co-targeting of PMN-MDSC and T cells could be beneficial in a subgroup of cancer patients. We also identified parameters useful to select those patients, who are likely to benefit from such immuno-combinations. Citation Format: Yu Si, Anthony Squire, Simon Merz, Kirsten Bruderek, Stephan Lang, Matthias Gunzer, Sven Brandau. Quantitative high-resolution tissue analysis defines intratumoral hot spots of PMN-MDSC activity in situ [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B12.

Abstract A84: B7-H3 promotes antitumor T-cell suppression via MDSCs in colorectal cancer

Abstract One of the mechanisms causing resistance to checkpoint inhibitor treatment is accumulation of suppressive immune cells including myeloid-derived suppressor cells (MDSCs). In CRC patients, accumulation of MDSCs correlates with poor overall survival. Among a subset of CRC patients with high mutation burden, 31.1% respond to checkpoint inhibitor treatment. This implies that targeting resistance mechanisms such as MDSCs in these CRC patients could boost the efficacy of this treatment. B7-H3 (CD279), a member of the B7 family, is expressed on myeloid cells and suppresses T-cell proliferation. Therefore, we hypothesize that B7-H3 on MDSCs could promote CRC tumor growth by suppressing cytotoxic CD8 T cells. We used polyps from ApcMin/+ mice, an intestinal tumor model and a transplantable CRC tumor model such as MC38 colon carcinoma to assess the role of B7-H3. Our findings show that B7-H3 is upregulated in ApcMin/+ polyps and is expressed on in vitro derived monocytic MDSCs (M-MDSCs). The frequency of MDSCs increased with tumor growth, indicating the close correlation between MDSCs and disease burden. Blocking B7-H3 with an antibody in MC38 tumor model significantly reduced tumor volume (control group receiving isotype antibody: 1672.2 ± 121 mm3; B7-H3 antibody treated group: 892.7 ± 36.4 mm3; p<0.05). The frequency of CD4 and CD8 T cells in spleens and tumors of B7-H3 antibody-treated mice was increased, but this was not statistically significant. However, the frequency of CD8 cells producing IFNγ or granzyme B was significantly increased in tumors of B7-H3 antibody-treated mice. This shows that inhibition of B7-H3 in CRC regresses tumor growth and expands cytotoxic CD8 T cells in MC38 tumors. Previously we showed that reduction of intestinal tumors in ApcMin/+Stat6-/- mice correlated with reduced MDSCs and increased CD8 cytotoxic activity, indicating that MDSCs suppressed CD8 cells. Therefore, B7-H3 suppresses cytotoxic CD8 response in colon carcinoma, possibly by mediating the suppressive function of MDSCs. Clinical trials using dual targeting antibodies to B7-H3 and CD3 recruit T cells to kill tumor cells expressing B7-H3. However, impaired T-cell effector function by MDSCs could limit the efficacy of this therapy. Targeting B7-H3 on MDSCs could overcome resistance and boost the efficacy of combination treatments. Citation Format: Asha Jayakumar, Alfred Bothwell. B7-H3 promotes antitumor T-cell suppression via MDSCs in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A84.

Abstract A78: IFNg-activated lymphatic vessels express PD-L1 and suppress effector T-cell function in melanoma

Abstract Peripheral tissues adopt mechanisms of immune suppression that counteract protective immunity to prevent immunopathology and are co-opted by tumors for immune escape. In tumors, cytotoxic, antigen-specific T cells produce IFNg and activate multiple mechanisms of adaptive immune resistance, including upregulation of the T-cell inhibitory molecule programed death ligand 1 (PD-L1). Tumor cell PD-L1 expression does not predict response to anti-PD-L1 immunotherapy, indicating, however, that other sources of PD-L1 in tumor microenvironments may contribute to PD-L1-dependent immune suppression. Lymphatic vessels facilitate antigen and dendritic cell transport to draining lymph nodes and are required for antitumor immunity. Interestingly, lymphatic endothelial cells in lymph nodes are tolerogenic at steady-state and constitutively express PD-L1 to delete self-reactive CD8+ T cells. However, whether lymphatic endothelial cells in tumor microenvironments are also directly immunosuppressive remains unclear. Here we test the hypothesis that peripheral, tumor-associated lymphatic vessels acquire immunosuppressive mechanisms, e.g., PD-L1 expression, that limit effector CD8+ T-cell accumulation and function in melanoma. We demonstrate that host nonhematopoietic, nontumor, PD-L1 limits CD8+ T-cell accumulation in murine melanoma. Further, we show that tumor-associated lymphatic endothelial cells upregulate PD-L1 in response to IFNg produced by tumor-infiltrating, antigen-specific CD8+ T cells. As such, loss of IFNgR by lymphatic endothelial cells improves CD8+ T-cell accumulation in murine melanomas, thereby enhancing tumor control and improved overall survival. Importantly, this robust lymphatic vessel-dependent tumor control was revealed in the context of murine melanomas with significant UVB-induced somatic mutation burden, consistent with a cytotoxic T cell-mediated negative feedback mechanism. Consequently, we present IFNg-mediated activation of tumor-associated lymphatic vessels as a new component of adaptive immune resistance, suggesting that context-dependent lymphatic vessel function may provide novel, targetable mechanisms of immune control in melanoma. Citation Format: Ryan S. Lane, Alec P. Breazeale, Amanda W. Lund. IFNg-activated lymphatic vessels express PD-L1 and suppress effector T-cell function in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A78.

Abstract B63: Immune gene expression profiling of acute myeloid leukemia identifies predictors of survival and actionable targets for treatment

Abstract Background: Acute myeloid leukemia (AML) is characterized by clonal expansion of poorly differentiated myeloid precursors, resulting in impaired hematopoiesis and often bone marrow (BM) failure. The general therapeutic approach in patients with AML has not changed substantially in more than 30 years. Investigation of new strategies, including immunotherapy, remains a priority. Tumor phenotypes are dictated not only by major oncogene drivers, but also by the tumor immunologic microenvironment (TIME) which is inherently immunosuppressive, is equipped to hamper effector T-cell function and includes immune and inflammatory cells, soluble mediators such as interferon (IFN)-gamma and extracellular matrix components. Herein, we profiled the TIME of AML to identify gene signatures that are reflective of general immune status and predictive of antileukemia immune potential. Methods: We used the hybridization-based nCounter® system (NanoString Technologies®, Seattle, WA) and the RNA Pan-Cancer Immune Profiling PanelTM to analyze BM aspirates from 290 adults and 40 children with newly diagnosed, nonpromyelocytic AML. Data were normalized to a set of reference genes and log2-transformed for bioinformatics analysis. The clinical relevance of the association between immune gene signatures and patient outcome was further validated in silico using publicly available cancer transcriptomic datasets (The Cancer Genome Atlas; TCGA). Results: We identified distinct sets of co-expressed genes corresponding to innate immunity, adaptive immunity and IFN-gamma signaling. BM samples with IFN-gamma-dominant gene expression profiles showed up-regulation of actionable immune checkpoints, such as B7-H3, PD-L1 and CTLA-4. A set of 4 differentially expressed immune genes between children and adults with AML (FDR<0.005) was associated with adverse cytogenetic features, leukemia relapse and shorter patient survival, and also stratified survival in the TCGA cohort of adult patients with AML. Finally, all four genes were amplified, deleted or mutated in 7% of 51,175 TCGA solid tumors, with the highest frequency of mutations being detected in primary CNS lymphoma (40%) and diffuse large B-cell lymphoma (25%). Conclusions: Our study identified distinct immune gene programs in the TIME of patients with newly diagnosed AML. From a translational standpoint, immune-enriched and IFN-gamma-dominant AMLs may benefit from immune checkpoint blockade and from new immunotherapy approaches, including dual-affinity T-cell redirecting antibodies targeting CD123. Grant support: John and Lucille van Geest Foundation, UK; Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494). Citation Format: Sergio Rutella, Jayakumar Vadakekolathu, Tressa Hood, Stephen Reeder, Sarah E. Warren, Patrick Danaher, Jan Davidson-Moncada, Alessandra Cesano, Joseph M. Beechem, Sarah K. Tasian, Mark D. Minden. Immune gene expression profiling of acute myeloid leukemia identifies predictors of survival and actionable targets for treatment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B63.

Quantitative Control of Gene-Engineered T-Cell Activity through the Covalent Attachment of Targeting Ligands to a Universal Immune Receptor.

Universal immune receptors represent a rapidly emerging form of adoptive T-cell therapy with the potential to overcome safety and antigen escape challenges faced by conventional chimeric antigen receptor (CAR) T-cell therapy. By decoupling antigen recognition and T-cell signaling domains via bifunctional antigen-specific targeting ligands, universal immune receptors can regulate T-cell effector function and target multiple antigens with a single receptor. Here, we describe the development of the SpyCatcher immune receptor, the first universal immune receptor that allows for the post-translational covalent attachment of targeting ligands at the T-cell surface through the application of SpyCatcher-SpyTag chemistry. The SpyCatcher immune receptor redirected primary human T cells against a variety of tumor antigens via the addition of SpyTag-labeled targeting ligands, both in vitro and in vivo. SpyCatcher T-cell activity relied upon the presence of both target antigen and SpyTag-labeled targeting ligand, allowing for dose-dependent control of function. The mutational disruption of covalent bond formation between the receptor and the targeting ligand still permitted redirected T-cell function but significantly compromised antitumor function. Thus, the SpyCatcher immune receptor allows for rapid antigen-specific receptor assembly, multiantigen targeting, and controllable T-cell activity.

Combining ibrutinib and checkpoint blockade improves CD8+ T-cell function and control of chronic lymphocytic leukemia in Em-TCL1 mice.

Ibrutinib is a Bruton’s tyrosine kinase (BTK) inhibitor approved for the treatment of multiple B-cell malignancies, including chronic lymphocytic leukemia (CLL). In addition to blocking B-cell receptor signaling and chemokine receptor-mediated pathways in CLL cells, that are known drivers of disease, ibrutinib also affects the microenvironment in CLL via targeting BTK in myeloid cells and IL-2–inducible T-cell kinase (ITK) in T cells. These non-BTK effects were suggested to contribute to the success of ibrutinib in CLL. By using the Eμ-TCL1 adoptive transfer mouse model of CLL, we observed that ibrutinib effectively controls leukemia development, but also results in significantly lower numbers of CD8+ effector T cells, with lower expression of activation markers, as well as impaired proliferation and effector function. Using CD8+ T cells from a T-cell receptor (TCR) reporter mouse, we verified that this is due to a direct effect of ibrutinib on TCR activity, and demonstrate that co-stimulation via CD28 overcomes these effects. Most interestingly, combination of ibrutinib with blocking antibodies targeting PD-1/PD-L1 axis in vivo improved CD8+ T-cell effector function and control of CLL. In summary, these data emphasize the strong immunomodulatory effects of ibrutinib and the therapeutic potential of its combination with immune checkpoint blockade in CLL.

Abstract B47: CHIME screening identifies PTPN2 as a novel regulator of antitumor immunity

Abstract Despite the significant clinical responses observed with checkpoint blockade, there is an urgent need to identify new therapeutic targets in immune cells for combination therapies. However, the use of functional genomic approaches in immune cells to discover immunotherapy targets is limited by inefficient vector delivery or perturbation of cell states. To circumvent these limitations, we developed CHIME: CHimeric IMmune Editing, a bone marrow chimeric CRISPR-Cas9 delivery system, to rapidly evaluate gene function in innate and adaptive immune cells in vivo without prior ex vivo manipulation. This approach enables efficient deletion of genes of interest in major immune lineages without altering immune development or function. To discover novel immunotherapy targets we performed an in vivo pooled genetic screen for negative regulators of CD8+ T-cell responses to LCMV Clone 13 viral infection. We found that deletion of the phosphatase Ptpn2 enhances CD8+ T-cell responses to chronic pathogens and cancer. In models of both chronic viral infection and transplantable tumors, Ptpn2 null CD8+ T cells expand more and show increased expression of effector genes compared to wild-type cells. Consistent with this, in the LCMV Clone 13 model, Ptpn2 deletion in CD8+ T cells affects the differentiation and expansion of exhausted subpopulations by increasing IFN-I signaling. Furthermore, deletion of Ptpn2 in the immune system induces a CD8+ T cell-dependent clearance of tumors and synergizes with PD-1 immune checkpoint blockade. Our results suggest that therapeutic inhibition of Ptpn2 in immune cells may enhance CD8+ T-cell effector function and mediate antitumor immunity to improve tumor control. More generally, these findings suggest that this genetic platform can enable rapid target discovery through pooled loss-of-function screening in immune cell lineages in vivo and presents a novel target for potential immune based therapies. Citation Format: Martin LaFleur, Thao Nguyen, Matthew Coxe, Brian Miller, Kathleen Yates, Jacob Gillis, Debattama Sen, John Doench, Nicholas Haining, Arlene Sharpe. CHIME screening identifies PTPN2 as a novel regulator of antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B47.

Gut-Liver Physiomimetics Reveal Paradoxical Modulation of IBD-Related Inflammation by Short-Chain Fatty Acids.

Although the association between the microbiome and IBD and liver diseases is known, the cause and effect remain elusive. By connecting human microphysiological systems of the gut, liver, and circulating Treg and Th17 cells, we created a multi-organ model of ulcerative colitis (UC) exvivo. The approach shows microbiome-derived short-chain fatty acids (SCFAs) to either improve or worsen UC severity, depending on the involvement of effector CD4 Tcells. Using multiomics, we found SCFAs increased production of ketone bodies, glycolysis, and lipogenesis, while markedly reducing innate immune activation of theUC gut. However, during acute Tcell-mediated inflammation, SCFAs exacerbated CD4+ Tcell-effector function, partially through metabolic reprograming, leading to gut barrier disruption and hepatic injury. These paradoxical findings underscore the emerging utility of human physiomimetic technology in combination with systems immunology to study causality and the fundamental entanglement of immunity, metabolism, and tissue homeostasis.

Abstract PR9: Loss of PD-1 promotes antitumor immunity by improving functions of both PD-1+ and PD-1- CD8+ T cells in the tumor microenvironment

Abstract Although PD-1 pathway inhibitors are revolutionizing cancer treatment, the mechanisms by which PD-1 regulates antitumor immunity are not fully understood. To determine PD-1 functions on different cell types, we used PD-1 conditional knockout mice. Subcutaneous transplantation of MC38 adenocarcinoma tumor cells in mice with complete deletion of PD-1 selectively on CD8+ T cells improved CD8+ T functions in the tumor microenvironment (TME). CD8+ T cells were required for the protective effects of PD-1 deletion in this model. To assess whether loss of PD-1 on all cells was necessary for improved antitumor immunity, we restricted PD-1 deletion to only half of the T-cell population. We hypothesized that a cell-intrinsic loss of PD-1 was necessary for improved T-cell fitness and effector functions. Here, deletion of PD-1 indeed led to T cell-intrinsic boosts in function. Unexpectedly, however, there was also a bystander effect that improved functions of PD-1-expressing CD8+ T cells in the TME. These data suggest that complete loss of PD-1 is not necessary for optimal tumor immunity, a finding that has important implications for applying PD-1-based immunotherapies to cancer patients. This abstract is also being presented as Poster B82. Citation Format: Kristen E. Pauken, Vikram R. Juneja, Alison Ringel, Jared H. Rowe, Kelly P. Burke, Peter T. Sage, Martin W. LaFleur, Juhi R. Kuchroo, Noga Ron-Harel, Seth Maleri, Jaclyn M. Long, Gordon J. Freeman, Nicolas Chevrier, Marcia C. Haigis, Arlene H. Sharpe. Loss of PD-1 promotes antitumor immunity by improving functions of both PD-1+ and PD-1- CD8+ T cells in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR9.

Abstract IA04: Impact of tumor-resident DC on antitumor immunity

Abstract Clearance of tumors relies heavily on the production and maintenance of poly-functional CD8+ T-cell responses. Studies of antiviral and antitumor responses highlight that loss of T-cell effector functions, a phenomenon termed T-cell dysfunction or exhaustion, is associated with tumor evasion and is characteristic of a failed immune response. While this correlation is widely appreciated and the T cell-intrinsic mechanisms are well understood, our current understanding of the T cell-extrinsic stimuli that determine T-cell dysfunction versus sustained effector function remains rudimentary. Studies highlight that T-cell dysfunction is induced in tissue microenvironments characterized by high antigen density such as the tumor or the tumor-draining lymph node. Recent studies using mouse models and patient samples suggest that tumor-resident dendritic cells (DC) can have extensive impact on effector T-cell recruitment and function. Based on these observations, it is conceivable that local, nonmigratory DC subsets can engage directly with antigen-specific T cells and impact their effector function by supplying stimulatory or inhibitory signals. We have established model systems of cancer recapitulating the optimal, immunotherapy-sensitive, and immunotherapy-resistant phenotypes. Our data suggest that optimal antitumor immune responses are associated with the presence of a novel subset of DC, which we termed interferon-stimulated gene (ISG)-DC. Our data highlight that ISG-DC are a distinct subset of DC, capable of activating CD8+ T cells and providing a distinct set of costimulatory molecules to re-engage T cells. Mechanistic studies highlight that ISG-DC use an unconventional way of antigen presentation and are sensitized by tumor-derived dsRNA. In sum, we have identified a novel DC subset, which should be further explored for therapeutic engagement. Citation Format: Stefani Spranger. Impact of tumor-resident DC on antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr IA04.