CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8 HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors.
Human cytomegalovirus (HCMV) is under constant selective pressure from the immune system in vivo. Study of HCMV genes that have been lost in the absence of, or genetically altered by, such selection can focus research toward findings of in vivo significance. We have been particularly interested in the most pronounced change in the highly passaged laboratory strains AD169 and Towne—the deletion of 13–15 kb of sequence (designated the UL/b′ region) that encodes up to 22 canonical genes, UL133-UL150. At least 5 genes have been identified in UL/b′ that inhibit NK cell function. UL135 suppresses formation of the immunological synapse (IS) by remodeling the actin cytoskeleton, thereby illustrating target cell cooperation in IS formation. UL141 inhibits expression of two activating ligands (CD155, CD112) for the activating receptor CD226 (DNAM-1), and two receptors (TRAIL-R1, R2) for the apoptosis-inducing TRAIL. UL142, ectopically expressed in isolation, and UL148A, target specific MICA allotypes that are ligands for NKG2D. UL148 impairs expression of CD58 (LFA-3), the co-stimulatory cell adhesion molecule for CD2 found on T and NK cells. Outside UL/b′, studies on natural variants have shown UL18 mutants change affinity for their inhibitory ligand LIR-1, while mutations in UL40's HLA-E binding peptide differentially drive NKG2C+ NK expansions. Research into HCMV genomic stability and its effect on NK function has provided important insights into virus:host interactions, but future studies will require consideration of genetic variability and the effect of genes expressed in the context of infection to fully understand their in vivo impact.
Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype ‘sheddase’, a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its U L / b’ region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins ( P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.
The treatment of haematologic malignancies with adoptive cell therapy is largely limited to platforms based on patient-derived, autologous αβ T cells. Although successful, this approach comes with challenges including associated toxicities, risk of relapse, high production costs and a requirement to gene edit cells to avoid graft vs host disease (GvHD) risk if the therapy is to be used in an allogeneic setting. In contrast to αβ T cells, human Vδ1 γδ T cells are a subset of T cells defined by expression of heterodimeric T cell receptors (TCRs) composed of a γ chain paired to a Vδ1 chain. Vδ1 γδ T cell function is highly differentiated from αβ T cells, as target cell recognition is not MHC restricted and γδ T cells are not alloreactive. Allogeneic matching of patients is therefore not required for Vδ1 γδ T cell therapeutic approaches. Instead Vδ1 T cells elicit direct anti-tumour responses via activation of diverse receptor repertoires that recognise multiple ligands upregulated on the surface of transformed cells. These key features of Vδ1 T cell biology therefore confer several advantages when generating an allogeneic cell therapy platform. We have developed a good manufacturing practice (GMP) compliant, scalable process to generate αβ T cell depleted, γδ T cell cultures (93.5% ± 3.7 of live), enriched for Vδ1 T cells (67.9% ± 2.2% of live). The cryopreserved therapeutic product shows good recoverability and proliferation after thawing, an activated innate phenotype and produces high levels of IFNγ and chemokines which can stimulate activation of other cells of the immune system. Post-thaw, Vδ1 T cell product has cytotoxic activity against a variety of malignant leukaemia and lymphoma cells in vitro and the cells home to the bone marrow and mediate anti-tumour activity following IV dosing in an in vivo xenograft model. Importantly, Vδ1 T cells have a good safety profile and do not mediate; cytotoxic activity against healthy tissues, mixed lymphocyte reactions or GvHD in in vivo models. Building on our expertise for expanding Vδ1 T cells, we have now developed scalable transient and stable gene engineering platforms for the generation of Vδ1 T cells expressing chimeric antigen receptors (CAR) and other molecules. Genetically engineered cells show good expansion kinetics and recoverability and viability post-cryopreservation. The cells also maintain a favourable activated phenotype post-thaw characterised by high levels of expression of CD27, NCR receptors (DNAM1, NKG2D and NKp30) and stable CAR expression at the cell surface. We have now used the Vδ1 CAR-T platform to develop and screen CAR constructs designed to generate allogeneic CAR-T cell therapies that exploit the unique safety and activity profile of Vδ1 T cells while additionally potentiating on-target tumour cell killing. Using this approach and tool constructs containing CARs targeting CD19, we have demonstrated potent and enhanced killing of a B-cell tumour cell line without eliciting on-target bystander killing of healthy B cells. In addition, the Vδ1 CAR-T anti-tumour activity is not associated with release of cytokines that may potentiate off-target toxicities. These data demonstrate that Vδ1 T cells have the potential to provide a unique CAR-T platform capable of targeting a broader cancer antigen profile than is possible for conventional CAR-T therapies. Citation Format: Istvan Kovacs, Andre Simoes, Tim Recaldin, Elizabeth Reynolds, Katharina Bergerhoff, Mihil Patel, Rebecca Alade, Victoria Hillerdal, Andrew Hutton, Daniel Fowler, Joanna Kawalkowska, Kalle Soderstrom, Valentino Parravicini, Michael Koslowski, Oliver Nussbaumer, Alice Brown. Vδ1γδT-Cells: A unique allogeneic cell therapy platform for the treatment of a broad range of malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 888.
Chimeric antigen receptor (CAR) modification of αβ T cells has revolutionized the field of oncology, driving the focus of attention to the immune system to target and fight malignancies. Currently available αβ T-cell therapies come with many challenges, including alloreactivity, off-target toxicities, cytokine release syndrome, limited survival of infused cells and the necessity to gene edit cells to overcome graft vs. host disease. Whilst some of these limitations can be overcome with complex and costly gene-engineering approaches, utilizing innate immune cells which are inherently non-HMC restricted and able to orchestrate wider immune responses might provide an alternative strategy avoiding many of these obstacles. Many groups developed protocols to grow and modify natural killer cells and invariant natural killer T-cells whilst others, including ours, focused on the development of Vδ1 γδ T-cell-based immunotherapies. Currently, these cell types are evaluated in the clinic in both non-engineered and engineered versions. Most of these engineering principles have been directly translated from the αβ CAR-T field without taking into consideration the biology of innate immune cells: they utilize αβ T-cell specific co-stimulatory molecules and signaling cassettes as well as armoring strategies developed for αβ T-cells. Vδ1 T-cells are tissue resident lymphocytes, which for most of their lifetime remain in epithelia rich tissues. They consistently survey tissues and monitor malignant transformation using a variety of natural cytotoxicity receptors, NK like receptors and the γδ TCR. In contrast to αβ T-cells, Vδ1 T-cells do not follow the ‘two-signal theory’: they must integrate multiple signals from an array of receptors in order to discriminate between healthy and malignant cells. We thus propose that CAR strategies exclusively utilizing CD3ζ activation domains are not using the full potential of innate immune cells for cellular immunotherapy. Another distinguishing feature of Vδ1 T-cells is the absence of traditional autocrine cytokine feedback loops seen in αβ T-cells. Our results demonstrate that traditional cytokine armoring strategies utilizing forced secretion of soluble or membrane-tethered IL-15 are detrimental to the biology of Vδ1 T-cells. By dissecting the IL-15 receptor biology we are now able to create cells that not only survive and grow in the absence of exogenous IL-15 but become more sensitive to endogenous levels. Applying γδ T-cell biology and an adapted understanding of the IL-15 pathway, we have now created novel engineering strategies to tailor specificity, potency, and proliferation of Vδ1 T-cells even in the presence of CAR whilst maintaining the cells’ inherent ability to discriminate between healthy and malignant cells. Preclinical evaluation of these concepts is ongoing with the aim to develop next generation tailored Vδ1T-cell immunotherapies. Citation Format: Jyothi Kumaran, Rajeev Karattil, André Simoes, Rebecca Alade, Mihil Patel, Liz Wood, Gonzalo Mercado Vico, Amy Lane, Sara Tamagno, Sarah Edwards, Andrea Venuso, Sam Illingworth, Alice Brown, Michael Koslowski, Istvan Kovacs, Oliver Nussbaumer. Moving on: Embracing γδ T-cell biology to create truly next generation immunotherapy concepts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2851.
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