Sun, N

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 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.

Background Immune checkpoint therapies targeting programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) have been met with limited clinical responses in the treatment of patients of varying cancer types. Current US Food and Drug Administration (FDA)-approved therapies function to prevent PD-1 binding to PD-L1, thereby preventing T-cell suppression. However, these therapies fail to prevent PD-L1 intrinsic signaling and recycling, which can render these treatments ineffective. Moreover, most research primarily focuses on how these antibodies affect tumor cells and their interactions with T cells. Myeloid cells, which also express PD-L1 and influence T-cell responses, are integral to the efficacy of these therapies. However, strategies that can target these cells to boost antitumor responses have not been effective. Methods Our group has developed a new anti-PD-L1 antibody (H1A), which disrupts PD-L1 recycling and redirects it towards degradation. Due to the loss of available PD-L1 protein, H1A effectively prevents PD-L1 intrinsic signaling. Using in vivo humanized PD-1/PD-L1 mouse tumor models, we evaluated the therapeutic efficacy of H1A against current FDA-approved PD-L1 blocking antibodies. Using human-derived immune cell in vitro assays, we pinpoint the biological consequences of H1A-induced PD-L1 degradation in myeloid cells. Results H1A demonstrated improved tumor control and established immunological memory responses in vivo in the treatment of tumors with moderate immunogenicity that are less responsive to current immunotherapies. This was supported by enhanced activation of myeloid cells (major histocompatibility complex (MHC)-II and CD80) and frequencies of effector T-cell populations found intratumorally. Human myeloid cells treated with H1A were also observed to have increased activation (MHC-II and CD80) and cytokine secretion. Similar to our in vivo findings, human peripheral blood lymphocyte cultures demonstrated increased frequencies of effector T-cell populations and greater tumor cell killing. Conclusions This work highlights the importance of targeting PD-L1 recycling and intrinsic signaling, specifically in myeloid cells, to boost antitumor responses. Our studies suggest that H1A can provide a solution to the lack of responses seen with current therapeutics, while also revealing previously unknown intrinsic functions of PD-L1 in myeloid cells.

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.