Citations

Abstract Introduction Immunotherapy has achieved remarkable success in hematologic malignancies, but its efficacy in solid tumors remains limited due to tumor heterogeneity, immune suppression within the tumor microenvironment, and inefficient T cell infiltration. I hypothesize that enhancing tumor cell sensitivity to cytokine-mediated killing can potentiate paracrine killing, in which T cell—derived cytokines induce tumor cell death even without direct cell—cell contact. This mechanism can overcome antigen heterogeneity and limited T cell penetration, broadening the therapeutic reach of immunotherapy. Methods I propose targeting ADAR1, an RNA-editing enzyme that suppresses innate immune activation, to enhance paracrine killing. Loss of ADAR1 leads to dsRNA accumulation and activation of PKR, MDA5, and RIG-I, sensitizing tumor cells to T cell—derived cytokines such as IFNγ and TNF. To test this, I performed in vitro paracrine killing co-culture assays using T cells from healthy donor PBMCs, antigen-positive tumor targets, and GFP-labeled antigen-negative ADAR1KO tumor targets. GFP signal loss in ADAR1KO cells will indicate enhanced paracrine killing independent of antigen recognition. Results My data show that ADAR1 inhibition sensitizes tumor cells to cytokine-mediated cytotoxicity, though how dsRNA sensor and cytokine receptor signaling intersect remains unclear. Supernatant from antigen-positive T cell—tumor co-cultures induced cell death in ADAR1KO but not WT tumor targets, indicating selective cytokine sensitivity. Consistently, Western blot analysis revealed increased cleaved Caspase-3 and PARP in ADAR1KO cells. The paracrine killing assay is ongoing. Conclusion This study aims to elucidate the interplay between dsRNA sensing and cytokine signaling in the absence of ADAR1 and to define how this crosstalk shapes the efficacy of paracrine killing in solid tumors. These insights could inform new therapeutic strategies to enhance the potency of immunotherapy against resistant and heterogeneous solid tumors. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Introduction It is critical for the immune system to destroy cells harboring intracellular infection to remove pathogen burden. This function is widely attributed to cytotoxic T cells, which can physically engage with their targets through the T cell receptor complex, releasing cytotoxic granules, resulting in a specific death of infected targets. However, effector cytokines tumor necrosis factor-α (TNF) and interferon-γ (IFNγ), secreted by T cells, are themselves cytotoxic in combination to a degree. While TNF has appreciated cell death function through the TNF receptor 1 (TNFR1) signaling pathway, no clear cytotoxic function is attributed to IFNγ. Because of the diffusible nature of these cytokines, it is possible for T cells to elicit cytotoxicity without requiring direct contact with their targets, termed paracrine killing. Whether paracrine killing occurs during viral infection and how it is regulated is unclear. Paracrine killing poses issues of both sensitivity to these cytokines in that high concentrations of these cytokines are needed to affect target cells, as well as specificity in discriminating infected from uninfected host cells. Accounting for both issues, we hypothesize that paracrine killing is elicited through pathogen restriction whereby only infected cells are susceptible to death. Methods Analysis was performed in vitro using the Incucyte live cell imaging system and Western blots with fibroblast targets and a VSV infection model and OT1 T cells were leveraged ex vivo. Results TNF and IFNγ protect cells against the cytopathic effect of the virus. TNF specifically synergizes with VSV resulting in death of the infected cells and the two cytokines have distinct roles that work in concert. We demonstrate that TNF and IFNγ released by activated T cells suppress viral infection of nearby cells in a paracrine manner. Conclusion Here we provide insight and context into how distinct cytokines function in a cooperative manner for immune protection during an infection. Funding Source Mayo Clinic Graduate School of Biomedical Sciences Topic Categories Viral Immunology (VIR)

Cytotoxic T cells produce the cytokine interferon-γ (IFN-γ). We sought to determine whether IFN-γ directly kills target cells and found that T cells used IFN-γ to kill murine melanoma cells lacking the kinases TBK1 and IKKε. In the absence of both kinases, IFN-γ induced the production of TNF receptor 1 (TNFR1) and the sensor Z-DNA binding protein 1 (ZBP1) in the tumor cells to stimulate receptor-interacting protein kinase 1 (RIPK1)–dependent apoptosis in a cell-autonomous manner. IFN-γ also enhanced the activation of nuclear factor κB (NF-κB) signaling in a TNFR1-dependent manner in cells deficient in both TBK1 and IKKε. Because IFN-γ–induced apoptosis occurred in a transcription-dependent manner with slow kinetics, STAT1 and NF-κB cooperated to activate the expression of inflammatory genes in the dying cells. Thus, IFN-γ–induced cell death was accompanied by an inflammatory signature in the absence of TBK1 and IKKε. TBK1 and IKKε not only mediated the induction of type I IFN but also inhibited RIPK1-dependent death and NF-κB–dependent inflammation. Therefore, we propose that these kinases may have gained these intertwined functions so that cells infected by pathogens that produce antagonists of TBK1 and IKKε are eliminated by IFN-γ–secreting T cells in an inflammatory manner to compensate for the inhibition of type I IFN production.

Abstract Multiple Myeloma is the second most common hematologic malignancy among US adults and remains incurable. Immunotherapies, including bispecific T cell engagers (BiTEs) targeting BCMA, have greatly improved outcomes. However, a significant fraction of patients still fail to respond, or develop resistant disease. Conventional models of T cell cytotoxicity focus on delivery of perforin- and granzyme-containing granules following antigen recognition and T cell activation, an inefficient mode of killing that requires effector-to-target cell contact. Activated T cells, however, also secrete cytokines capable of diffusing and acting on distant targets. This raises the possibility of paracrine killing, in which these cytokines induce tumor cell death independently of cell-cell contact.Tumor necrosis factor (TNF) is a key cytokine released during T cell activation and is unique in that it can drive either cell survival or cell death. Although most cells default to cell survival and NFkB-mediated inflammation, several modulators can shift this survival signaling toward cell death. We therefore hypothesized that sensitizing Myeloma cells to TNF could enable T cell paracrine killing and enhance the efficacy of anti-Myeloma T cell immunotherapies.To test this, we first established an in vitro model of clinical response to Teclistamab, a BCMA-directed BiTE, using Multiple Myeloma cell lines that displayed sensitive, intermediate, or resistant responses to T cell killing. Combining these data with assays of cytokine sensitivity and receptor expression, we found that susceptibility to TNF-mediated cell death was a major determinant of Teclistamab sensitivity. This correlation was further supported by reduction or complete abrogation of Teclistamab T cell killing of sensitive Myeloma cell lines when soluble TNF was neutralized from these co-cultures using monoclonal antibodies. Pharmacologic inhibition and CRISPR-mediated deletion of known regulators of TNF survival-signaling further sensitized Myeloma cells to both TNF and T cell killing. In the absence of these regulators, BCMA-negative myeloma targets retained sensitivity to Teclistamab, supporting the idea that the main mechanism of cell death was indeed paracrine killing.Together, these findings establish a direct link between TNF sensitivity and T cell-mediated killing of Myeloma cells, and support TNF-driven paracrine killing as a dominant mechanism of response to Teclistamab. Enhancing this TNF-mediated death signaling may therefore represent a strategy to overcome resistance and improve clinical efficacy of T cell-based immunotherapies in Multiple Myeloma. Citation Format: Allison Carr, Elise Sintim-Aboagye, Adrian Ting, . TNF-driven paracrine killing determines the cytotoxic efficacy of Teclistamab in Myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1636.

ABSTRACT Cytotoxic T lymphocytes (CTLs) are known to eliminate target cells through perforin-mediated, contact-dependent killing - a process limited by the number of effector T cells despite its serial nature. CTLs likely also possess a mass killing mechanism that can eliminate target cells more efficiently. Using CAR-T cells against B7H3 present on B16 tumor targets, we demonstrate that activated T cells can also kill targets in a perforin-independent manner by releasing diffusible TNF and IFNγ capable of killing nearby targets in a paracrine fashion even if they bear no antigen. Against an unperturbed target, paracrine killing is inefficient but is enhanced by the deletion of TNFR1 signaling molecules such as TAK1, HOIP, or TBK1/IKKε. Notably, these molecules are inhibited naturally by pathogen-encoded antagonists. Expression of a microbial-encoded antagonist, such as the Yersinia -encoded YopJ that antagonizes TAK1 or Ebola-encoded VP35 that antagonizes TBK1/IKKε, alters the target cell response to these cytokines from non-lethal to lethal. We propose that target cells are inherently resistant to killing by TNF and IFNγ, but the presence of a microbial factor alters this sensitivity, providing for the selective elimination of the infected cell while minimizing harm to uninfected bystander cells. We propose the term ‘pathogen-restriction’ to describe this discriminatory mechanism. Potentially, any microbial-derived factor that crosstalks with the TNF and IFNγ signaling pathways can function as a pathogen-restriction element. GRAPHICAL ABSTRACT Short Summary T cells use TNF and IFNγ to kill target cells in a paracrine manner where target elimination is restricted by the presence of pathogen-derived factors.

Abstract Description CAR-T cell therapy has shown remarkable success in treating hematologic cancers; however, its efficacy in solid tumors remains limited, partly due to tumor heterogeneity, immune suppression within the tumor microenvironment, and inefficient CAR-T cell infiltration. I hypothesize that increasing the sensitivity of tumor cells to cytokine-mediated killing could enhance the therapeutic impact of CAR-T cells in solid tumors. I propose that targeting Adenosine Deaminases Acting on RNA 1 (ADAR1), an enzyme that edits double-stranded RNA (dsRNA) to prevent immune activation, may be one such strategy. ADAR1 balances inflammation by preventing the overactivation of dsRNA sensors such as PKR, MDA5, and RIG-I. However, in the absence of ADAR1, accumulating dsRNA structures will trigger these dsRNA sensors and alter the tumor cell intrinsic response to T cell-derived cytokines, such as IFNγ and TNF. My data provide evidence that inhibition of ADAR1 in tumor cells sensitize them to killing by cytokines, but it remains unclear how signaling from dsRNA sensors crosstalk with those from cytokine receptors to regulate cell survival or death. My study aims to elucidate the interplay between these different signaling pathways in the absence of ADAR1, and how this interplay determines the efficacy of CAR-T cells against solid tumors. My experimental observations could provide insights into novel therapeutic strategies to improve CAR-T cell potency against resistant tumor types. Funding Sources Mayo Clinic Cancer Center Fellowship Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Multiple myeloma (MM) is the second most common hematologic malignancy among US adults, characterized by high rates of relapse and refractory disease. Anti-Myeloma T cell immunotherapies, such as chimeric antigen receptors (CAR-Ts) and bispecific T cell engagers (BiTEs), have been able to induce deep responses in the clinic and extending survival for many. However, even these responses are not durable and majority of patients still relapse. MM cell signaling in response to cytokines released by these T cells, such as Tumor Necrosis Factor (TNF) Alpha, and Interferon gamma (IFNγ), may offer additional insight into mechanisms of relapse and resistance as well as avenues of therapeutic manipulation. To evaluate the effects of TNF and IFNγ on MM cell death and proliferation, we obtained human MM cell lines (MMCLs) and stably transduced them with a GFP-NLS reporter. We then monitored these cell lines in the presence of TNF, IFNγ, or TNF+IFNγ and tracked their cell death and growth kinetics over time using DiYO-3 incorporation and GFP count, respectively, via IncuCyteS3 live cell imaging. We next utilized Teclistamab, a B cell maturation antigen (BCMA)-directed BiTE, to co-culture MMCLs with healthy donor derived T cells at various effector to target (E:T) ratios and monitored for T cell mediated death again via IncuCyteS3 live cell imaging. While all MMCLs were able to be killed at high E:T ratios (1:1), only cytokine-sensitive lines remained susceptible to T cell killing at lower E:T ratios (1:10). The addition of TNF and/or IFNγ blocking antibodies confirmed that the increased sensitivity of these lines to T cell killing was indeed mediated by the two cytokines. In summary, were able to show an in vitro model of differential MM cell response to Teclistamab mediated T cell killing, and that this difference in response was predicated on the MM cells’ baseline sensitivity to cytokine mediated cell death via TNF and IFNγ. Thus, further dissecting mechanisms underlying resistance or sensitivity of MM cells to cytokine induced death could suggest therapeutic targets with the potential to enhance the efficacy of anti-MM T cell immunotherapies. Citation Format: Allison Carr, Adrian Ting. Role of cytokine-induced cell death on efficacy of teclistamab-mediated T cell killing of multiple myeloma cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1380.

Abstract Chimeric antigen receptor (CAR) T-cells are T-cells engineered with synthetic receptors that directs their specificity and function toward an antigen of choice. This application has been highly successful in treating a variety of blood-borne cancers such as B-cell lymphomas. CAR T-cells against solid tumors have been less efficacious, potentially due to the resistance of tumor cells to T cell-mediated cytotoxicity. Current paradigm suggests that tumor cells are killed by perforin/granzyme-secreting CD8 cytotoxic T cells (CTLs). However, activated CD8 T cells also produce TNF and IFNg, two cytokines that have been reported to have anti-tumor function, but it is not clear whether these cytokines can directly kill tumor targets. It has been reported that defects in kinases in the TNFR1 signaling pathway can sensitize cells to TNF-induced death. Therefore, we hypothesize that by inactivating these kinases, we can sensitize solid tumors to killing by CD8 CAR T-cells producing TNF and IFNg. We intend to conduct proof-of-concept studies to show that sensitizing tumor cells to cytokine-mediated cytotoxicity will enhance the effectiveness of CAR T-cell therapy against solid tumors. Institutional Funding

Abstract There is increasing evidence that immunotherapies make tumor cells more vulnerable to the host’s own immune system that can lead to long-term and durable remission. To date, only a small percentage of cancer patients have benefited from this approach and advances in this field will likely come from a greater understanding of the interaction between immune cells and their tumor targets. T cells are critical to a successful anti-tumor response due to their ability to directly lyse tumor cells by releasing cytolytic granules, and to produce inflammatory cytokines, such as interferon gamma (IFNγ). IFNγ has several functions in an anti-tumor response. On the immune cell side, IFNγ is a major driver of Th1 differentiation that leads to the activation of T cells and NK cells. On the tumor side, IFNγ can enhance MHC class I presentation but also inhibitory receptors such as PD-1. However, it is not as clear whether IFNγ is directly cytotoxic for tumor cells. If that is the case, it is also likely that due to selection pressure exerted by T cells, tumor cells may have acquired mechanisms to protect themselves against this possibility. Thus, I hypothesized that tumor cells are normally resistant to killing by IFNγ but can be sensitized if the death protection mechanisms are disabled. In preliminary studies using the B16F1 murine melanoma model, we found that while wild-type (WT) melanoma cells were completely resistant, tumor cells with knockouts of two kinases (DKO) became highly sensitized to IFNγ-mediated apoptotic death. Furthermore, the DKO tumors were also more easily killed by T cells in an IFNγ-dependent manner. These findings will provide insight into potential targets for therapeutic intervention to enhance immune-mediated destruction of tumors.