TBK1 and IKKε protect target cells from IFNγ-mediated T cell killing via an inflammatory apoptotic mechanism.

Cytotoxic T cells produce interferon gamma (IFNγ), which plays a critical role in anti-microbial and anti-tumor responses. However, it is not clear whether T cell-derived IFNγ directly kills infected and tumor target cells, and how this may be regulated. Here, we report that target cell expression of the kinases TBK1 and IKKε regulate IFNγ cytotoxicity by suppressing the ability of T cell-derived IFNγ to kill target cells. In tumor targets lacking TBK1 and IKKε, IFNγ induces expression of TNFR1 and the Z-nucleic acid sensor, ZBP1, to trigger RIPK1-dependent apoptosis, largely in a target cell-autonomous manner. Unexpectedly, IFNγ, which is not known to signal to NFκB, induces hyperactivation of NFκB in TBK1 and IKKε double-deficient cells. TBK1 and IKKε suppress IKKα/ß activity and in their absence, IFNγ induces elevated NFκB-dependent expression of inflammatory chemokines and cytokines. Apoptosis is thought to be non-inflammatory, but our observations demonstrate that IFNγ can induce an inflammatory form of apoptosis, and this is suppressed by TBK1 and IKKε. The two kinases provide a critical connection between innate and adaptive immunological responses by regulating three key responses: (1) phosphorylation of IRF3/7 to induce type I IFN; (2) inhibition of RIPK1-dependent death; and (3) inhibition of NFκB-dependent inflammation. We propose that these kinases evolved these functions such that their inhibition by pathogens attempting to block type I IFN expression would enable IFNγ to trigger apoptosis accompanied by an alternative inflammatory response. Our findings show that loss of TBK1 and IKKε in target cells sensitizes them to inflammatory apoptosis induced by T cell-derived IFNγ.

Salvage therapy expands highly cytotoxic and metabolically fit resilient CD8+ T cells via ME1 up-regulation.

Patients with advanced cancers who either do not experience initial response to or progress while on immune checkpoint inhibitors (ICIs) receive salvage radiotherapy to reduce tumor burden and tumor-related symptoms. Occasionally, some patients experience substantial global tumor regression with a rebound of cytotoxic CD8+ T cells. We have termed the rebound of cytotoxic CD8+ T cells in response to salvage therapy as T cell resilience and examined the underlying mechanisms of resilience. Resilient T cells are enriched for CX3CR1+ CD8+ T cells with low mitochondrial membrane potential, accumulate less reactive oxygen species (ROS), and express more malic enzyme 1 (ME1). ME1 overexpression enhanced the cytotoxicity and expansion of effector CD8+ T cells partially via the type I interferon pathway. ME1 also increased mitochondrial respiration while maintaining the redox state balance. ME1 increased the cytotoxicity of peripheral lymphocytes from patients with advanced cancers. Thus, preserved resilient T cells in patients rebound after salvage therapy and ME1 enhances their resiliency.

A Novel PD-L1 Antibody Promotes Antitumor Function of Peripheral Cytotoxic Lymphocytes after Radical Nephrectomy in Patients with Renal Cell Carcinoma.

The intrinsic and acquired resistance to PD-1/PD-L1 immune checkpoint blockade is an important challenge for patients and clinicians because no reliable tool has been developed to predict individualized response to immunotherapy. In this study, we demonstrate the translational relevance of an ex vivo functional assay that measures the tumor cell killing ability of patient-derived CD8 T and NK cells (referred to as "cytotoxic lymphocytes," or CLs) isolated from the peripheral blood of patients with renal cell carcinoma. Patient-derived PBMCs were isolated before and after nephrectomy from patients with renal cell carcinoma. We compared the efficacy of U.S. Food and Drug Administration (FDA)-approved PD-1/PD-L1 inhibitors (pembrolizumab, nivolumab, atezolizumab) and a newly developed PD-L1 inhibitor (H1A Ab) in eliciting cytotoxic function. CL activity was improved at 3 mo after radical nephrectomy compared with baseline, and it was associated with higher circulating levels of tumor-reactive effector CD8 T cells (CD11ahighCX3CR1+GZMB+). Treatment of PBMCs with FDA-approved PD-1/PD-L1 inhibitors enhanced tumor cell killing activity of CLs, but a differential response was observed at the individual-patient level. H1A demonstrated superior efficacy in promoting CL activity compared with FDA-approved PD-1/PD-L1 inhibitors. PBMC immunophenotyping by mass cytometry revealed enrichment of effector CD8 T and NK cells in H1A-treated PBMCs and immunosuppressive regulatory T cells in atezolizumab-treated samples. Our study lays the ground for future investigation of the therapeutic value of H1A as a next-generation immune checkpoint inhibitor and the potential of measuring CTL activity in PBMCs as a tool to predict individual response to immune checkpoint inhibitors in patients with advanced renal cell carcinoma.

Cancer-targeted photoimmunotherapy induces antitumor immunity and can be augmented by anti-PD-1 therapy for durable anticancer responses in an immunologically active murine tumor model.

The complex immunosuppressive nature of solid tumor microenvironments poses a significant challenge to generating efficacious and durable anticancer responses. Photoimmunotherapy is a cancer treatment strategy by which an antibody is conjugated with a non-toxic light-activatable dye. Following administration of the conjugate and binding to the target tumor, subsequent local laser illumination activates the dye, resulting in highly specific target cell membrane disruption. Here we demonstrate that photoimmunotherapy treatment elicited tumor necrosis, thus inducing immunogenic cell death characterized by the release of damage-associated molecular patterns (DAMPs). Photoimmunotherapy-killed tumor cells activated dendritic cells (DC), leading to the production of proinflammatory cytokines, T cell stimulation, priming antigen-specific T cells, and durable memory T cell responses, which led complete responder mice to effectively reject new tumors upon rechallenge. PD-1 blockade in combination with photoimmunotherapy enhanced overall anticancer efficacy, including against anti-PD-1-resistant tumors. The combination treatment also elicited abscopal anticancer activity, as observed by reduction of distal, non-illuminated tumors, further demonstrating the ability of photoimmunotherapy to harness local and peripheral T cell responses. With this work we therefore delineate the immune mechanisms of action for photoimmunotherapy and demonstrate the potential for cancer-targeted photoimmunotherapy to be combined with other immunotherapy approaches for augmented, durable anticancer efficacy. Moreover, we demonstrate responses utilizing various immunocompetent mouse models, as well as in vitro data from human cells, suggesting broad translational potential.