Citations

In the host immune system, cytotoxic T cells (CTLs) play a critical role in mounting an effective attack against tumorigenic cells. CTLs can drive this immunity through two mechanisms: direct lysis of tumor cells via cytolytic granule release and secretion of inflammatory cytokines such as interferon-γ (IFN-γ). While IFN-γ is well known for its role in upregulating major histocompatibility complex (MHC) class I presentation and differentiating naïve T cells into T helper 1 cells, its role in direct killing of tumor cells is less understood. In a recent study, we found that tumor cells lacking the signaling kinases TANK-binding kinase 1 (TBK1) and inhibitor of κB kinase ε (IKK-ε) were more easily killed by IFN-γ-producing T cells than their wild-type counterparts. In the absence of both kinases, IFN-γ upregulated the expression of both tumor necrosis factor receptor 1 (TNFR1) and the left-handed nuclei acid sensor Z-DNA-binding protein 1 (ZBP1) to trigger cell-autonomous apoptosis. Loss of TBK1 and IKK-ε also heightened nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation downstream of IFN-γ, along with increased production of inflammatory chemokines and cytokines. Thus, IFN-γ-induced death was paired with an inflammatory environment when the two kinases were absent. Our study illustrates that TBK1 and IKK-ε not only have a role in mediating a type I IFN response but also in regulating IFN-γ-induced death and NF-κB-dependent inflammation. Herein, we briefly highlight how the wiring of these kinases may have evolved over time to combat opportunistic pathogens and how this may shape our approach to cancer immunotherapy.

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.

Abstract Background Apolipoprotein E (APOE), the major genetic risk factor for Alzheimer's disease (AD), has three common alleles (2, 3 and 4), giving rise to genotypes that are associated with six levels of AD risk: (2/2<2/3<3/3<2/4<3/4<4/4). In our new Arizona APOE Cohort Study, we have begun to establish a longitudinal cohort of more than 300 cognitively unimpaired persons (50‐90 year‐old) stratified by APOE genotype and age decile, matched by age and sex, and have an extensive battery of cognitive, clinical, amyloid (NAV4694) and tau (MK6240) PET, MRI, cerebrospinal fluid (CSF), plasma, and DNA. The study is intended to clarify the impact of APOE4 and APOE2 allelic dose on the predisposition to and protection from AD and inform the design of secondary and primary prevention therapies. Here, we describe the study and the resource of data and samples that are now available to researchers. Methods Participants were recruited from across the United States using GeneMatch and other sources. Comprehensive clinical assessments, brain images, and lumbar punctures are performed every 2 years, 50mL blood samples are acquired annually, and whole genome sequencing is in progress. Results As of January 2025, baseline assessments were completed in 303 participants, 68±9 years of age, including 205 females and 98 males. Sixteen percent are from underrepresented groups. Baseline CSF samples were obtained in 89% of the participants. The complete cohort consists of 17, 63, 41, 65, 66, and 51 persons with the APOE 2/2, 2/3, 3/3, 2/4, 3/4, and 4/4 genotype, respectively. In the first 287 participants with available amyloid PET scans 6% of 2/2, 5% of 2/3, 14% of 3/3, 26% of 2/4, 29% of 3/4, and 52% of 4/4 participants had a positive amyloid PET (≥20 centiloids). Conclusions Building up on our decades of experience in APOE research, we launched the new Arizona APOE Cohort which promises to provide a valuable resource of data and samples to advance the study of APOE, its variants and interactions with other risk and protective factors, and the development, treatment, and prevention of AD. Baseline data are now available to interested and eligible research collaborators.

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.