List of 145 differentially-expressed genes in PVRIG-knockout vs wild-type CD8 TILs (day 18 post-implantation).

Fusobacterium nucleatum is an oral pathogen associated with periodontal disease, preterm births and the exacerbation of colorectal, esophageal, pancreatic, and breast cancers. F. nucleatum was previously found to inhibit killing of cancer cells by natural killer (NK) cells and tumor infiltrating T cells by inducing the NK cells killing-suppressing receptors TIGIT and CEACAM1. In this study, we show that the incubation of F. nucleatum with primary human natural killer (NK) cells causes the cleavage of the activating receptors CD16, NKp44 and NKp46 by fusolisin. Fusolisin is a fusobacterial outer-membrane, auto-transporter, serine protease and to date the only functional protease found in F. nucleatum. We previously showed that fusolisin is essential for fusobacterial growth in culture. High expression of a functional recombinant fusolisin in E. coli was enabled using codon optimization and replacement of the fusolisin's signal peptide with that of the E. coli OmpA. Recombinant fusolisin was found to cleave the same NK-activating receptors degraded by F. nucleatum. Cleavage of these activating receptors by fusolisin inhibited NK cells activity including killing of tumor cells in-vitro and in-vivo. Our results provide a new bacterial protease-dependent mechanism in which tumors colonized by F. nucleatum are protected from NK cells attack by exploiting fusolisin proteolytic activity. Importantly, our previous and current results suggest that fusolisin might serve to target for tumor-colonized fusobacteria. Citation Format: Amjad Shhadeh, Liat Dassa, Jamal Fahoum, Nicole Haj, Reuven Wiener, Ofer Mandelboim, Gilad Bachrach. Suppression of anti-tumor immunity by the Fusobacterium nucleatum protease fusolisin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5893.

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The GPI-anchoring pathway plays important roles in normal development and immune modulation. MHC Class I Polypeptide-related Sequence A (MICA) is a stress-induced ligand, downregulated by human cytomegalovirus (HCMV) to escape immune recognition. Its most prevalent allele, MICA*008, is GPI-anchored via an uncharacterized pathway. Here, we identify cleft lip and palate transmembrane protein 1-like protein (CLPTM1L) as a GPI-anchoring pathway component and show that during infection, the HCMV protein US9 downregulates MICA*008 via CLPTM1L. We show that the expression of some GPI-anchored proteins (CD109, CD59, and MELTF)-but not others (ULBP2, ULBP3)-is CLPTM1L-dependent, and further show that like MICA*008, MELTF is downregulated by US9 via CLPTM1L during infection. Mechanistically, we suggest that CLPTM1L's function depends on its interaction with a free form of PIG-T, normally a part of the GPI transamidase complex. We suggest that US9 inhibits this interaction and thereby downregulates the expression of CLPTM1L-dependent proteins. Altogether, we report on a new GPI-anchoring pathway component that is targeted by HCMV.

Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells without prior sensitization. One pivotal activating NK receptor is NKG2D, which binds a family of eight ligands, including the major histocompatibility complex (MHC) class I-related chain A (MICA). Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus causing morbidity and mortality in immunosuppressed patients and congenitally infected infants. HCMV encodes multiple antagonists of NK cell activation, including many mechanisms targeting MICA. However, only one of these mechanisms, the HCMV protein US9, counters the most prevalent MICA allele, MICA*008. Here, we discover that a hitherto uncharacterized HCMV protein, UL147A, specifically downregulates MICA*008. UL147A primarily induces MICA*008 maturation arrest, and additionally targets it to proteasomal degradation, acting additively with US9 during HCMV infection. Thus, UL147A hinders NKG2D-mediated elimination of HCMV-infected cells by NK cells. Mechanistic analyses disclose that the non-canonical GPI anchoring pathway of immature MICA*008 constitutes the determinant of UL147A specificity for this MICA allele. These findings advance our understanding of the complex and rapidly evolving HCMV immune evasion mechanisms, which may facilitate the development of antiviral drugs and vaccines.

Stress can induce cell surface expression of MHC-like ligands, including MICA, that activate NK cells. Human cytomegalovirus (HCMV) glycoprotein US9 downregulates the activating immune ligand MICA*008 to avoid NK cell activation, but the underlying mechanism remains unclear. Here, we show that the N-terminal signal peptide is the major US9 functional domain targeting MICA*008 to proteasomal degradation. The US9 signal peptide is cleaved with unusually slow kinetics and this transiently retained signal peptide arrests MICA*008 maturation in the endoplasmic reticulum (ER), and indirectly induces its degradation via the ER quality control system and the SEL1L-HRD1 complex. We further identify an accessory, signal peptide-independent US9 mechanism that directly binds MICA*008 and SEL1L. Collectively, we describe a dual-targeting immunoevasin, demonstrating that signal peptides can function as protein-integral effector domains.

AbstractAlthough checkpoint inhibitors that block CTLA-4 and PD-1 have improved cancer immunotherapies, targeting additional checkpoint receptors may be required to broaden patient response to immunotherapy. PVRIG is a coinhibitory receptor of the DNAM/TIGIT/CD96 nectin family that binds to PVRL2. We report that antagonism of PVRIG and TIGIT, but not CD96, increased CD8+ T-cell cytokine production and cytotoxic activity. The inhibitory effect of PVRL2 was mediated by PVRIG and not TIGIT, demonstrating that the PVRIG–PVRL2 pathway is a nonredundant signaling node. A combination of PVRIG blockade with TIGIT or PD-1 blockade further increased T-cell activation. In human tumors, PVRIG expression on T cells was increased relative to normal tissue and trended with TIGIT and PD-1 expression. Tumor cells coexpressing PVR and PVRL2 were observed in multiple tumor types, with highest coexpression in endometrial cancers. Tumor cells expressing either PVR or PVRL2 were also present in numbers that varied with the cancer type, with ovarian cancers having the highest percentage of PVR−PVRL2+ tumor cells and colorectal cancers having the highest percentage of PVR+PVRL2− cells. To demonstrate a role of PVRIG and TIGIT on tumor-derived T cells, we examined the effect of PVRIG and TIGIT blockade on human tumor-infiltrating lymphocytes. For some donors, blockade of PVRIG increased T-cell function, an effect enhanced by combination with TIGIT or PD-1 blockade. In summary, we demonstrate that PVRIG and PVRL2 are expressed in human cancers and the PVRIG–PVRL2 and TIGIT–PVR pathways are nonredundant inhibitory signaling pathways.See related article on p. 244

AbstractA limitation to antitumor immunity is the dysfunction of T cells in the tumor microenvironment, in part due to upregulation of coinhibitory receptors such as PD-1. Here, we describe that poliovirus receptor–related immunoglobulin domain protein (PVRIG) acts as a coinhibitory receptor in mice. Murine PVRIG interacted weakly with poliovirus receptor (PVR) but bound poliovirus receptor–like 2 (PVRL2) strongly, making the latter its principal ligand. As in humans, murine NK and NKT cells constitutively expressed PVRIG. However, when compared with humans, less PVRIG transcript and surface protein was detected in murine CD8+ T cells ex vivo. However, activated CD8+ T cells upregulated PVRIG expression. In the mouse tumor microenvironment, infiltrating CD8+ T cells expressed PVRIG whereas its ligand, PVRL2, was detected predominantly on myeloid cells and tumor cells, mirroring the expression pattern in human tumors. PVRIG-deficient mouse CD8+ T cells mounted a stronger antigen-specific effector response compared with wild-type CD8+ T cells during acute Listeria monocytogenes infection. Furthermore, enhanced CD8+ T-cell effector function inhibited tumor growth in PVRIG−/− mice compared with wild-type mice and PD-L1 blockade conferred a synergistic antitumor response in PVRIG−/− mice. Therapeutic intervention with antagonistic anti-PVRIG in combination with anti–PD-L1 reduced tumor growth. Taken together, our results suggest PVRIG is an inducible checkpoint receptor and that targeting PVRIG–PVRL2 interactions results in increased CD8+ T-cell function and reduced tumor growth.See related article on p. 257

Natural killer (NK) cells are lymphocytes of the innate immune system capable of killing hazardous cells, including virally infected cells. NK cell-mediated killing is triggered by activating receptors. Prominent among these is the activating receptor NKG2D, which binds several stress-induced ligands, among them major histocompatibility complex (MHC) class I-related chain A (MICA). Most of the human population is persistently infected with human cytomegalovirus (HCMV), a virus which employs multiple immune evasion mechanisms, many of which target NK cell responses. HCMV infection is mostly asymptomatic, but in congenitally infected neonates and in immunosuppressed patients it can lead to serious complications and mortality. Here we discovered that an HCMV protein named UL148A whose role was hitherto unknown is required for evasion of NK cells. We demonstrate that UL148A-deficient HCMV strains are impaired in their ability to downregulate MICA expression. We further show that when expressed by itself, UL148A is not sufficient for MICA targeting, but rather acts in concert with an unknown viral factor. Using inhibitors of different cellular degradation pathways, we show that UL148A targets MICA for lysosomal degradation. Finally, we show that UL148A-mediated MICA downregulation hampers NK cell-mediated killing of HCMV-infected cells. Discovering the full repertoire of HCMV immune evasion mechanisms will lead to a better understanding of the ability of HCMV to persist in the host and may also promote the development of new vaccines and drugs against HCMV.IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen which is usually asymptomatic but that can cause serious complications and mortality in congenital infections and in immunosuppressed patients. One of the difficulties in developing novel vaccines and treatments for HCMV is its remarkable ability to evade our immune system. In particular, HCMV directs significant efforts to thwarting cells of the innate immune system known as natural killer (NK) cells. These cells are crucial for successful control of HCMV infection, and yet our understanding of the mechanisms which HCMV utilizes to elude NK cells is partial at best. In the present study, we discovered that a protein encoded by HCMV which had no known function is important for preventing NK cells from killing HCMV-infected cells. This knowledge can be used in the future for designing more-efficient HCMV vaccines and for formulating novel therapies targeting this virus.

The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like domain-containing receptor (ILDR) 2 as a novel B7-like protein with robust T cell inhibitory activity, expressed in immune cells and in immune-privileged and inflamed tissues. A fusion protein, consisting of ILDR2 extracellular domain with an Fc fragment, that binds to a putative counterpart on activated T cells showed a beneficial effect in the collagen-induced arthritis model and abrogated the production of proinflammatory cytokines and chemokines in autologous synovial-like cocultures of macrophages and cytokine-stimulated T cells. Collectively, these findings point to ILDR2 as a novel negative regulator for T cells, with potential roles in the development of immune-related diseases, including autoimmunity and cancer.

3074 Background: While inhibitors of CTLA4 and PD1 have emerged as effective cancer therapies, the majority of treated patients do not derive long term benefit. Employing our computational discovery platform, we discovered PVRIG as an immune suppressive molecule expressed on T and NK cells and identified COM701, an antibody (Ab) targeting human PVRIG that enhances T cell function and anti-tumor responses. Methods: Anti-human PVRIG Ab COM701 was identified as an antagonistic Ab that enhanced T cell function in multiple assays. Antagonistic anti-mouse PVRIG Abs and PVRIG deficient (PVRIG-/-) mice were generated and characterized using syngeneic tumor models. Results: PVRIG was induced upon T cell activation, with long term activation leading to the highest expression. PVRL2 was identified as the ligand for PVRIG, placing PVRIG in the DNAM/TIGIT immunoreceptor axis. Compared to normal adjacent tissues, PVRIG and PVRL2 were both induced in the tumor microenvironment of several human cancers. To target PVRIG for therapeutic intervention, we identified COM701, a high affinity Ab that disrupts the interaction of PVRIG with PVRL2. COM701 enhanced CD8 T cell proliferation and IFN-g production in vitro and had an additive or synergistic effect on T cell activation when further combined with an anti-PD1 or anti-TIGIT Ab. Consistent with a checkpoint function for human PVRIG, mouse PVRIG-/- T cells showed increased function compared to wild type T cells. A surrogate antagonistic anti-mPVRIG Ab reduced growth of CT26 and B16 tumors when combined with an anti-PDL1 Ab in vivo. MC38 tumors also grew slower in PVRIG-/- mice compared to wild type mice and ex vivo analysis pointed to functional differences in anti-cancer immunity. Conclusions: We demonstrated that targeting PVRIG with COM701, a high affinity antagonistic Ab, increased human T cell function. We further showed that PVRIG was induced in the tumor microenvironment and that disruption of PVRIG/PVRL2 interaction resulted in reduced tumor growth in preclinical models. These data demonstrate that PVRIG is a promising target for the treatment of cancer and provide the rationale for COM701 as a potential cancer immunotherapy.

Abstract Background: While blockade of the CTLA4 and PD1 pathways has emerged as an effective treatment of cancer, the majority of patients do not derive long term benefit. This provides a rationale for identifying and targeting additional checkpoints. Employing our unique computational algorithms, we identified PVRIG, a new member of the B7/CD28 family. We report here the expression pattern, functional characterization, and anti-tumor activity of blocking antibodies targeting PVRIG as well as characterization of PVRIG KO mice. Materials and Methods: PVRIG is expressed by T and NK cells within the tumor microenvironment. We identified PVRL2 as its counterpart and characterized the PVRIG-PVRL2 interaction. Antibody discovery was carried out with phage display and hybridoma platforms and antibodies against the human protein were screened for their ability to enhance T-cell activity in vitro, while surrogate antibodies targeting the mouse protein were assessed in syngeneic models for effects on tumor growth. PVRIG -/- KO mice were generated and characterized including phenotyping and anti-tumor immune response. Results: PVRIG is expressed on different T cell subsets and on NK, NKT and γδ T-cells. Within T cells, memory subsets possess the highest level of PVRIG and its expression is induced upon long term activation with different stimuli. Within tumor microenvironment, PVRIG was found to be expressed on NK and CD8+ T cells in multiple cancers. A high affinity lead Ab was selected, COM701, for further clinical development and demonstrated blockade of the interaction of PVRIG with PVRL2 as well as enhancement of activation of both primary and tumor-derived effector immune cells through a PVRL2-dependent mechanism. Moreover, COM-701 showed notable enhancement of T cell function in-vitro when combined with PD1 or TIGIT Ab blockade. The lead antibody, COM-701, is currently in preclinical development. A surrogate antibody, that blocks PVRIG-PVRL2 interaction, was shown to inhibit growth of colon carcinoma and melanoma in syngeneic models upon combined treatment with anti-PDL1 antibody. Comparative analysis of PVRIG KO versus WT derived T cells revealed enhanced reactivity of PVRIG null T cells upon polyclonal activation in presence of PVRL2-Ig. Accordingly, MC38 tumors grew slower in PVRIG KO than in WT mice and ex vivo analysis pointed to the quantitative and functional differences in anticancer immunity developed in these mice. Conclusion: We describe the identification of PVRIG as a novel T cell immune checkpoint. We further demonstrate that antibody blockade of the PVRIG-PVRL2 interaction has the potential to be efficiently combined with PD1 or TIGIT blockade for enhancing anti-tumor immunity. COM-701 is a high affinity antagonistic antibody that is currently in preclinical development. Taken together, these data demonstrate the utility of targeting PVRIG in addition to other B7 family checkpoints for the treatment of cancer. Citation Format: Ofer Levy, Chris Chan, Gady Cojocaru, Spencer Liang, Eran Ophir, Sudipto Ganguly, Maya Kotturi, Tal Friedman, Benjamin Murter, Liat Dassa, Ling Leung, Shirley Greenwald, Meir Azulay, Sandeep Kumar, Zoya Alteber, Xiaoyu Pan, Andy Drake, Ran Salomon, Arthur Machlenkin, John Hunter, Zurit Levine, Drew Pardoll, Mark White. Discovery and development of COM701, a therapeutic antibody targeting the novel immune checkpoint PVRIG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 581. doi:10.1158/1538-7445.AM2017-581

Senescent cells, formed in response to physiological and oncogenic stresses, facilitate protection from tumourigenesis and aid in tissue repair. However, accumulation of such cells in tissues contributes to age-related pathologies. Resistance of senescent cells to apoptotic stimuli may contribute to their accumulation, yet the molecular mechanisms allowing their prolonged viability are poorly characterized. Here we show that senescent cells upregulate the anti-apoptotic proteins BCL-W and BCL-XL. Joint inhibition of BCL-W and BCL-XL by siRNAs or the small-molecule ABT-737 specifically induces apoptosis in senescent cells. Notably, treatment of mice with ABT-737 efficiently eliminates senescent cells induced by DNA damage in the lungs as well as senescent cells formed in the epidermis by activation of p53 through transgenic p14 ARF . Elimination of senescent cells from the epidermis leads to an increase in hair-follicle stem cell proliferation. The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies.

Natural killer (NK) cells form an important arm of the innate immune system and function to combat a wide range of invading pathogens, ranging from viruses to bacteria. However, the means by which NK cells accomplish recognition of pathogens with a limited repertoire of receptors remain largely unknown. In the current study, we describe the recognition of an emerging fungal pathogen, Candida glabrata, by the human NK cytotoxic receptor NKp46 and its mouse ortholog, NCR1. Using NCR1 knockout mice, we observed that this receptor-mediated recognition was crucial for controlling C. glabrata infection in vitro and in vivo. Finally, we delineated the fungal ligands to be the C. glabrata adhesins Epa1, Epa6, and Epa7 and demonstrated that clearance of systemic C. glabrata infections in vivo depends on their recognition by NCR1. As NKp46 and NCR1 have been previously shown to bind viral adhesion receptors, we speculate that NKp46/NCR1 may be a novel type of pattern recognition receptor.

Meeting abstracts The past few years have witnessed a renaissance in the field of immuno-oncology largely due to the clinical success in targeting the immune checkpoints CTLA-4 and PD-1. Towards identification of novel immune checkpoint drug targets we developed a dedicated predictive discovery