David H. Raulet

I was pleased to receive an invitation to write a historical perspective on my career. As I understand it, these perspectives aim to illustrate the often-twisted paths to discoveries, the fortuitous events that often enable them, and the pitfalls along the way. At the same time, they can be expected to illustrate the influence of the particular “tastes” and interests of a scientist in the choices that are made along the way and that there are many ways to position oneself in a field of study. This article describes my initial interest in molecular biology, the early events that led me to undertake graduate studies in cellular immunology with a focus on MHC restriction of T cell specificity and T cell activation, and the serendipitous events that led to my eventual transition to a focus on natural killer cells and cancer immunotherapy. I hope that this piece provides some of the illustrations mentioned and will therefore be of interest to a range of readers with an interest in immunology research.

Summary: Natural Killer (NK) cell desensitization induced by persistent stimulation limits durable antitumor immunity, yet the molecular mechanisms governing this dysfunctional state remain poorly defined. To identify conserved regulators of NK cell desensitization, we performed comparative transcriptomic analyses across multiple murine models of persistent activation and dysfunction. This approach defined a shared transcriptional program of desensitization and identified Itpkb to be upregulated across multiple distinct contexts. Genetic and pharmacological inhibition of ITPKB enhanced degranulation, cytokine production, and cytotoxicity in both murine and human NK cells under multiple desensitization settings. Mechanistically, ITPKB regulated signaling downstream of persistent activation through the IP ₃ /IP ₄ axis, limiting calcium mobilization and NFAT-dependent transcriptional responses in desensitized NK cells. Furthermore, inhibition or deletion of ITPKB enhanced NK-cell mediated tumor control in vivo and improved the efficacy of adoptively transferred CAR-NK cells, underscoring the translational potential of targeting this pathway. Together, these findings identify ITPKB as a cell-intrinsic regulator of NK cell desensitization and support targeting the IP ₃ /IP ₄ signaling axis to enhance NK cell-mediated antitumor immunity.

Therapies to activate the STING immune response pathway represent promising potential anticancer treatments. However, the native STING activating molecule, 2',3'-cGAMP, is a poor drug candidate due to its susceptibility to nuclease degradation and its relatively poor cell uptake. In this study, we present a nanoscale delivery vehicle based on the bacteriophage MS2 virus-like particle that can both protect cGAMP and deliver it into cells to access and bind cytosolic STING. MS2-delivered cGAMP achieved greatly increased STING activation potency relative to both free cGAMP and a nuclease-resistant synthetic cGAMP analog. In an in vivo murine colon carcinoma model, MS2-cGAMP elicited significant and prolonged antitumor activity in a STING-dependent manner at 50-fold lower concentrations relative to free cGAMP and synthetic analogs. These results demonstrate that MS2 delivery of cGAMP can yield a highly potent STING agonist immunotherapy with in vivo anticancer activity.

Cytokines dimerize two receptor chains to activate Janus kinases and signal transducer and activator of transcription (STAT) transcription factors that regulate immune cells, but they have therapeutic liabilities. We engineered “Trikines” to compel cis formation of three-chain cytokine receptor complexes at the cell surface that induce bespoke STAT transcriptional signaling programs. Trikines coactivated phosphorylation of STAT5 (pSTAT5) and pSTAT3 signatures distinct from natural cytokines by assembling trimeric combinations of interleukin-2 (IL-2), IL-10, and IL-21 receptors. In preclinical models, an IL-2–based Trikine restrained terminal differentiation of T cells, promoted stemness, and enhanced durability of tumor control without observable toxicity. An IL-10–based Trikine induced immune infiltration into poorly immunogenic tumors, showing efficacy in preclinical models of small cell lung cancer and pancreatic cancer. Trikines obviate the need for cell engineering to customize STAT signatures and may hold potential for immunotherapy.

Cancer cells frequently lose major histocompatibility complex class I (MHC I) to evade CD8 T cell recognition. Natural killer (NK) cells are poised to target MHC I–deficient cancer cells, but MHC I loss alone is often insufficient to unleash fully effective NK cell responses. Here, we show that selective intratumoral (IT) ablation of regulatory T cells (T _reg cells) elicited potent antitumor NK cell responses that controlled MHC I–deficient and even MHC I ⁺ cancers that expressed NKG2D ligands. T _reg cells controlled the activation, maturation, and antitumor cytotoxic activity of NK cells within the tumor microenvironment. Mechanistically, depletion of IT-T _reg cells relieved the inhibition of cDC2-dependent induction of IL-2 production by conventional CD4 T cells that was necessary for NK cell activation. Systemically administered antibodies that selectively depleted IT-T _reg cells similarly empowered NK cell–dependent tumor control. These findings expand the breadth of T _reg cell–mediated cancer immunosuppression to encompass antitumor NK cells and suggest that therapeutic targeting of T _reg cells in tumors can control CD8 T cell–resistant cancers.

Cancer cells frequently lose MHC I to evade CD8 ⁺ T cell recognition. While Natural Killer (NK) cells are poised to target MHC I-deficient cancer cells, MHC I loss alone is often insufficient to unleash fully effective NK cell responses. Here we show that selective intra-tumoral (IT) ablation of regulatory T (Treg) cells elicited potent antitumor NK cell responses that controlled MHC I-deficient and even MHC I ⁺ cancers. Tregs controlled the activation, maturation, and anti-tumor cytotoxic activity of NK cells within the tumor microenvironment. Mechanistically, IT Tregs prevented the cDC2-dependent induction of IL-2 production by CD4 ⁺ Tconv cells that was necessary for NK cell activation. Systemically administered antibodies that selectively depleted IT Tregs similarly empowered NK- dependent tumor control. These findings expand the breadth of Treg-mediated cancer immunosuppression to encompass antitumor NK cells and suggest that targeting Tregs in tumors can control CD8 ⁺ T cell-resistant cancers. One Sentence Summary: IT Treg ablation drives NK cell tumor control via CD4 ⁺ Tconv-derived IL-2, eliminating MHC I+/MHC I- cancers without systemic toxicity.

Background: While chimeric antigen receptor (CAR) T-cells are promising, there is a rapidly growing interest in developing other CAR-expressing immune cells. However, to date, no reported studies evaluated these cells side-by-side in immune-competent glioma models. Methods: We developed a novel C57BL/6-background transgenic mouse strain with all hematopoietic cells carrying the anti-epidermal growth factor receptor (EGFR)vIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. Crossing with mice transgenic for Vav-Cre allowed the expression of anti-EGFRvIII CAR in all hematopoietic cells. In particular, we evaluated CAR-T, CAR-NKT, CAR-NK-cells, and CAR-macrophages in a syngeneic mouse SB28EGFRVIII glioma model. Results: CAR-NK and CAR-NKT-cells demonstrated anti-tumor effects comparable to CAR-T cells in vitro. A single intratumoral administration of CAR-T and CAR-NKT cells in combination mediated superior therapeutic efficacy compared to CAR-T cells or CAR-NKT-cells alone. A single intravenous infusion of CAR-NK cells following lymphodepletion failed to mediate significant anti-glioma effects. Additionally, intratumoral injection of CAR-NK cells did not confer therapeutic benefit. Contrary to previous reports using human macrophages, CAR-macrophages did not demonstrate enhanced antigen-presentation activity against glioma cells compared to non-CAR macrophages. Intratumorally administered CAR-macrophages failed to demonstrate local persistence or anti-tumor effects in vivo. Conclusions: These data provide a valuable basis as to which immune cells can mediate effective anti-glioma response in an immuno-competent glioma environment. Our data also suggest that a combination of CAR-T and CAR-NKT-cells may represent a promising therapeutic strategy.

Type I Interferons (Type I IFNs) are critical for host defense against a wide range of viral infections but can enhance the pathogenicity of several bacterial pathogens, including Mycobacterium tuberculosis (Mtb) ^1–4 . Given the significance of Type I IFN signaling in determining Mtb infection outcomes we sought to uncover new molecular mechanisms controlling Type I IFN activation during mycobacterial infection. We performed a genome-wide CRISPR interference screen in human macrophages to identify genes that regulate IFN-β, during infection with M. marinum (Mm) . In addition to known regulators of Type I IFN, we identified UFL1, which encodes the E3 ligase for the ubiquitin-like protein UFM1, as a major regulator of IFN-β during mycobacterial infection. Depletion of other components of the UFMylation pathway, DDRGK1 and UFM1, also resulted in increased IFN-β, indicating that UFMylation activity is required for Type I IFN regulation. Deficiency in UFMylation resulted in increased production and secretion of IFN-β during macrophage infection with both Mm and Mtb compared to control cells. Additionally, silencing UFL1 resulted in increased expression of several interferon stimulated genes and other pro-inflammatory genes, including TNF and IL-6, at both the mRNA and protein level. Transcriptional profiling revealed a surprisingly broad increase in pro-inflammatory responses of UFL1-deficient cells during Mtb infection, particularly for interferon-stimulated genes. Our data reveals an unexpected role of the UFMylation pathway in suppressing Type I IFN, a pro-inflammatory immune pathway with detrimental effects on Mtb infection outcome.

Activation of the endoplasmic reticulum (ER)–resident adaptor protein STING, a component of a cytosolic DNA–sensing pathway, induces the transcription of genes encoding type I interferons (IFNs) and other proinflammatory factors. Because STING is activated at the Golgi apparatus, control of the localization and activation of STING is important in stimulating antiviral and antitumor immune responses. Through a genome-wide CRISPR interference screen, we found that STING activation required the Golgi-resident protein ACBD3, which promotes the generation of phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network, as well as other PI4P-associated proteins. Appropriate localization and activation of STING at the Golgi apparatus required ACBD3 and the PI4P-generating kinase PI4KB. In contrast, STING activation was enhanced when the lipid-shuttling protein OSBP, which removes PI4P from the Golgi apparatus, was inhibited by the US Food and Drug Administration–approved antifungal itraconazole. The increase in the abundance of STING-activating phospholipids at the trans-Golgi network resulted in the increased production of IFN-β and other cytokines in THP-1 cells. Furthermore, a mutant STING that could not bind to PI4P failed to traffic from the ER to the Golgi apparatus in response to a STING agonist, whereas forced relocalization of STING to PI4P-enriched areas elicited STING activation in the absence of stimulation with a STING agonist. Thus, PI4P is critical for STING activation, and manipulating PI4P abundance may therapeutically modulate STING-dependent immune responses.

Natural killer (NK) cells are a major subset of innate immune cells that are essential for host defense against pathogens and cancer. Two main classes of inhibitory NK receptors (NKR), KIR and CD94/NKG2A, play a key role in suppressing NK activity upon engagement with tumor cells or virus-infected cells, limiting their antitumor and antiviral activities. Here, we find that single-chain NKR antagonists linked to a VHH that binds the cell surface phosphatase CD45 potentiate NK and T activities to a greater extent than NKR blocking antibodies alone in vitro. We also uncovered crosstalk between NKG2A and Ly49 that collectively inhibit NK cell activation, such that CD45-NKG2A and CD45-Ly49 bispecific molecules show synergistic effects in their ability to enhance NK cell activation. The basis of the activity enhancement by CD45 ligation may reflect greater antagonism of inhibitory signaling from engagement of MHC I on target cells, combined with other mechanisms, including avidity effects, tonic signaling, antagonism of weak inhibition from engagement of MHC I on non-target cells, and possible CD45 segregation within the NK cell-target cell synapse. These results uncover a strategy for enhancing the activity of NK and T cells that may improve cancer immunotherapies.