Microenvironment-dependent growth of Sezary cells in humanized IL-15 mice.

Sezary syndrome (SS) is a rare, aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) that lacks adequate therapeutic options and representative small-animal models. Here, we demonstrate that IL-15 is a critical CTCL growth factor. Importantly, an immunodeficient knock-in mouse model genetically engineered to express human IL-15 uniquely supported the growth of SS patient samples relative to conventional immunodeficient mouse strains. SS patient-derived xenograft (PDX) models recapacitated key pathological features of the human disease, including skin infiltration and spread of leukemic cells to the periphery, and maintained the dependence on human IL-15 upon serial in vivo passaging. Detailed molecular characterization of the engrafted cells by single-cell transcriptomic analysis revealed congruent neoplastic gene expression signatures but distinct clonal engraftment patterns. Overall, we document an important dependence of Sezary cell survival and proliferation on IL-15 signaling and the utility of immunodeficient humanized IL-15 mice as hosts for SS - and potentially other T and NK cell-derived hematologic malignancies - PDX model generation. Furthermore, these studies advocate the thorough molecular understanding of the resultant PDX models to maximize their translational impact.

Human CD4 cytotoxic T lymphocytes mediate potent tumor control in humanized immune system mice.

Efficacy of immune checkpoint inhibitors in cancers can be limited by CD8 T cell dysfunction or HLA-I down-regulation. Tumor control mechanisms independent of CD8/HLA-I axis would overcome these limitations. Here, we report potent CD4 T cell-mediated tumor regression and memory responses in humanized immune system (HIS) mice implanted with HT-29 colorectal tumors. The regressing tumors showed increased CD4 cytotoxic T lymphocyte (CTL) infiltration and enhanced tumor HLA-II expression compared to progressing tumors. The intratumoral CD4 T cell subset associated with tumor regression expressed multiple cytotoxic markers and exhibited clonal expansion. Notably, tumor control was abrogated by depletion of CD4 but not CD8 T cells. CD4 T cells derived from tumor-regressing mice exhibited HLA-II-dependent and tumor-specific killing ex vivo. Taken together, our study demonstrates a critical role of human CD4 CTLs in mediating tumor clearance independent of CD8 T cells and provides a platform to study human anti-tumor immunity in vivo.

Spontaneous tumor regression mediated by human T cells in a humanized immune system mouse model.

Immunodeficient mice reconstituted with a human immune system (HIS mice) give rise to human T cells, which make them an attractive system to study human immune responses to tumors. However, such HIS mice typically exhibit sub-optimal responses to immune challenges as well as fail to develop antigen-specific B or T cell memory. Here we report HIS mice mediate spontaneous regression of human B cell lymphoma Raji. Tumor regression was dependent on CD4+ and CD8+ T cell responses and resulted in T cell memory. The T cell memory elicited was mainly Raji-specific, however some level of cross-protection was also elicited to a related B cell lymphoma cell line Ramos. Single-cell RNAseq analysis indicated activation of CD8+ T cells in regressing Raji tumors as well as clonal expansion of specific T cell receptors (TCRs). Cloning of TCRs from Raji-infiltrating T cells into a Jurkat reporter cell line showed reactivity specific for Raji tumor cells. Overall, we report a platform for studying in vivo human T cell tumor immunity by highlighting spontaneous Raji tumor regression, clonal TCR expansion, and T cell memory in HIS mice.

IL-6-GP130 signaling protects human hepatocytes against lipid droplet accumulation in humanized liver models.

Liver steatosis is an increasing health issue with few therapeutic options, partly because of a paucity of experimental models. In humanized liver rodent models, abnormal lipid accumulation in transplanted human hepatocytes occurs spontaneously. Here, we demonstrate that this abnormality is associated with compromised interleukin-6 (IL-6)-glycoprotein 130 (GP130) signaling in human hepatocytes because of incompatibility between host rodent IL-6 and human IL-6 receptor (IL-6R) on donor hepatocytes. Restoration of hepatic IL-6-GP130 signaling, through ectopic expression of rodent IL-6R, constitutive activation of GP130 in human hepatocytes, or humanization of an Il6 allele in recipient mice, substantially reduced hepatosteatosis. Notably, providing human Kupffer cells via hematopoietic stem cell engraftment in humanized liver mice also corrected the abnormality. Our observations suggest an important role of IL-6-GP130 pathway in regulating lipid accumulation in hepatocytes and not only provide a method to improve humanized liver models but also suggest therapeutic potential for manipulating GP130 signaling in human liver steatosis.

Cancer cell-derived type I interferons instruct tumor monocyte polarization.

Monocytes are highly plastic immune cells that modulate antitumor immunity. Therefore, identifying factors that regulate tumor monocyte functions is critical for developing effective immunotherapies. Here, we determine that endogenous cancer cell-derived type I interferons (IFNs) control monocyte functional polarization. Guided by single-cell transcriptomic profiling of human and mouse tumors, we devise a strategy to distinguish and separate immunostimulatory from immunosuppressive tumor monocytes by surface CD88 and Sca-1 expression. Leveraging this approach, we show that cGAS-STING-regulated cancer cell-derived IFNs polarize immunostimulatory monocytes associated with anti-PD-1 immunotherapy response in mice. We also demonstrate that immunosuppressive monocytes convert into immunostimulatory monocytes upon cancer cell-intrinsic cGAS-STING activation. Consistently, we find that human cancer cells can produce type I IFNs that polarize monocytes, and our immunostimulatory monocyte gene signature is enriched in patient tumors that respond to anti-PD-1 immunotherapy. Our work exposes a role for cancer cell-derived IFNs in licensing monocyte functions that influence immunotherapy outcomes.

Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054.

Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.

A single-cell map of dynamic chromatin landscapes of immune cells in renal cell carcinoma.

A complete chart of the chromatin regulatory elements of immune cells in patients with cancer and their dynamic behavior is necessary to understand the developmental fates and guide therapeutic strategies. Here, we map the single-cell chromatin landscape of immune cells from blood, normal tumor-adjacent kidney tissue and malignant tissue from patients with early-stage clear cell renal cell carcinoma (ccRCC). We catalog the T cell states dictated by tissue-specific and developmental-stage-specific chromatin accessibility patterns, infer key chromatin regulators and observe rewiring of regulatory networks in the progression to dysfunction in CD8+ T cells. Unexpectedly, among the transcription factors orchestrating the path to dysfunction, NF-κB is associated with a pro-apoptotic program in late stages of dysfunction in tumor-infiltrating CD8+ T cells. Importantly, this epigenomic profiling stratified ccRCC patients based on a NF-κB-driven pro-apoptotic signature. This study provides a rich resource for understanding the functional states and regulatory dynamics of immune cells in ccRCC.

Humanization of T cell-mediated immunity in mice.

Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαß variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting "VelociT" mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αß T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I­ and MHC-II­restricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.

Flow Cytometric Characterization of Murine B Cell Development.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

Endogenous retroviral proteins provide an immunodominant but not requisite antigen in a murine immunotherapy tumor model.

Clinical observations suggest that responses to cancer immunotherapy are correlated with intra-tumoral T cell receptor (TCR) clonality, tumor mutation burden (TMB) and host HLA genotype, highlighting the importance of host T cell recognition of tumor antigens. However, the dynamic interplay between T cell activation state and changes in TCR repertoire in driving the identification of potential immunodominant antigen(s) remains largely unexplored. Here, we performed single-cell RNA-sequencing on CD8+ tumor-infiltrating T cells (TILs) using the murine colorectal tumor model MC38 to identify unique TCR sequences and validate their tumor reactivity. We found that the majority of clonally expanded TILs are tumor-reactive and their TCR repertoire is unique amongst individual MC38 tumor-bearing mice. Our query identified that multiple expanded TCR clones recognized the retroviral epitope p15E as an immunodominant antigen. In addition, we found that the endogenous retroviral genome encoding for p15E is highly expressed in MC38 tumors, but not in normal tissues, due to epigenetic derepression. Further, we demonstrated that the p15E-specific TILs exhibit an activated phenotype and an increase in frequency upon treatment with anti-41BB and anti-PD-1 combination immunotherapy. Importantly, we showed that although p15E-specific TILs are not required to mount a primary anti-tumor response, they contributed to the development of strong immune memory. Overall our results revealed that endogenous retroviral antigens expressed by tumor cells may represent an important and underappreciated category of tumor antigens that could be readily targeted in the clinic.

Tumor-targeted CD28 bispecific antibodies enhance the antitumor efficacy of PD-1 immunotherapy.

Monoclonal antibodies that block the programmed cell death 1 (PD-1) checkpoint have revolutionized cancer immunotherapy. However, many major tumor types remain unresponsive to anti-PD-1 therapy, and even among responsive tumor types, most of the patients do not develop durable antitumor immunity. It has been shown that bispecific antibodies activate T cells by cross-linking the TCR/CD3 complex with a tumor-specific antigen (TSA). The class of TSAxCD3 bispecific antibodies have generated exciting results in early clinical trials. We have recently described another class of "costimulatory bispecifics" that cross-link a TSA to CD28 (TSAxCD28) and cooperate with TSAxCD3 bispecifics. Here, we demonstrate that these TSAxCD28 bispecifics (one specific for prostate cancer and the other for epithelial tumors) can also synergize with the broader anti-PD-1 approach and endow responsiveness-as well as long-term immune memory-against tumors that otherwise do not respond to anti-PD-1 alone. Unlike CD28 superagonists, which broadly activate T cells and induce cytokine storm, TSAxCD28 bispecifics display little or no toxicity when used alone or in combination with a PD-1 blocker in genetically humanized immunocompetent mouse models or in primates and thus may provide a well-tolerated and "off the shelf" combination approach with PD-1 immunotherapy that can markedly enhance antitumor efficacy.

Kappa-on-Heavy (KoH) bodies are a distinct class of fully-human antibody-like therapeutic agents with antigen-binding properties.

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains.

A novel bispecific antibody platform to direct complement activity for efficient lysis of target cells.

Harnessing complement-mediated cytotoxicity by therapeutic antibodies has been limited because of dependency on size and density of antigen, structural constraints resulting from orientation of antibody binding, and blockade of complement activation by inhibitors expressed on target cells. We developed a modular bispecific antibody platform that directs the complement-initiating protein C1q to target cells, increases local complement deposition and induces cytotoxicity against target antigens with a wide-range of expression. The broad utility of this approach to eliminate both prokaryotic and eukaryotic cells was demonstrated by pairing a unique C1q-recruiting arm with multiple targeting arms specific for Staphylococcus aureus, Pseudomonas aeruginosa, B-cells and T-cells, indicating applicability for diverse indications ranging from infectious diseases to cancer. Generation of C1q humanized mice allowed for demonstration of the efficacy of this approach to clear disease-inducing cells in vivo. In summary, we present a novel, broadly applicable, and versatile therapeutic modality for targeted cell depletion.

Evaluation of the capacities of mouse TCR profiling from short read RNA-seq data.

Profiling T cell receptor (TCR) repertoire via short read transcriptome sequencing (RNA-Seq) has a unique advantage of probing simultaneously TCRs and the genome-wide RNA expression of other genes. However, compared to targeted amplicon approaches, the shorter read length is more prone to mapping error. In addition, only a small percentage of the genome-wide reads may cover the TCR loci and thus the repertoire could be significantly under-sampled. Although this approach has been applied in a few studies, the utility of transcriptome sequencing in probing TCR repertoires has not been evaluated extensively. Here we present a systematic assessment of RNA-Seq in TCR profiling. We evaluate the power of both Fluidigm C1 full-length single cell RNA-Seq and bulk RNA-Seq in characterizing the repertoires of different diversities under either naïve conditions or after immunogenic challenges. Standard read length and sequencing coverage were employed so that the evaluation was conducted in accord with the current RNA-Seq practices. Despite high sequencing depth in bulk RNA-Seq, we encountered difficulty quantifying TCRs with low transcript abundance (<1%). Nevertheless, top enriched TCRs with an abundance of 1-3% or higher can be faithfully detected and quantified. When top TCR sequences are of interest and transcriptome sequencing is available, it is worthwhile to conduct a TCR profiling using the RNA-Seq data.

Combination cancer immunotherapy targeting PD-1 and GITR can rescue CD8+ T cell dysfunction and maintain memory phenotype.

Most patients with cancer do not develop durable antitumor responses after programmed cell death protein 1 (PD-1) or programmed cell death ligand 1(PD-L1) checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing of more than 2000 tumor-infiltrating CD8+ T cells in mice receiving both PD-1 and GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein) antibodies and found that this combination synergistically enhanced the effector function of expanded CD8+ T cells by restoring the balance of key homeostatic regulators CD226 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), leading to a robust survival benefit. Combination therapy decreased CD8+ T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1-Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy.

Humanized mouse model supports development, function, and tissue residency of human natural killer cells.

Immunodeficient mice reconstituted with a human immune system represent a promising tool for translational research as they may allow modeling and therapy of human diseases in vivo. However, insufficient development and function of human natural killer (NK) cells and T cell subsets limit the applicability of humanized mice for studying cancer biology and therapy. Here, we describe a human interleukin 15 (IL15) and human signal regulatory protein alpha (SIRPA) knock-in mouse on a Rag2-/- Il2rg-/- background (SRG-15). Transplantation of human hematopoietic stem and progenitor cells into SRG-15 mice dramatically improved the development and functional maturation of circulating and tissue-resident human NK and CD8+ T cells and promoted the development of tissue-resident innate lymphoid cell (ILC) subsets. Profiling of human NK cell subsets by mass cytometry revealed a highly similar expression pattern of killer inhibitory receptors and other candidate molecules in NK cell subpopulations between SRG-15 mice and humans. In contrast to nonobese diabetic severe combined immunodeficient Il2rg-/- (NSG) mice, human NK cells in SRG-15 mice did not require preactivation but infiltrated a Burkitt's lymphoma xenograft and efficiently inhibited tumor growth following treatment with the therapeutic antibody rituximab. Our humanized mouse model may thus be useful for preclinical testing of novel human NK cell-targeted and combinatory cancer immunotherapies and for studying how they elicit human antitumor immune responses in vivo.

In vivo effector functions of high-affinity mouse IgG receptor FcγRI in disease and therapy models.

Two activating mouse IgG receptors (FcγRs) have the ability to bind monomeric IgG, the high-affinity mouse FcγRI and FcγRIV. Despite high circulating levels of IgG, reports using FcγRI-/- or FcγRIV-/- mice or FcγRIV-blocking antibodies implicate these receptors in IgG-induced disease severity or therapeutic Ab efficacy. From these studies, however, one cannot conclude on the effector capabilities of a given receptor, because different activating FcγRs possess redundant properties in vivo, and cooperation between FcγRs may occur, or priming phenomena. To help resolve these uncertainties, we used mice expressing only FcγRI to determine its intrinsic properties in vivo. FcγRIonly mice were sensitive to IgG-induced autoimmune thrombocytopenia and anti-CD20 and anti-tumour immunotherapy, but resistant to IgG-induced autoimmune arthritis, anaphylaxis and airway inflammation. Our results show that the in vivo roles of FcγRI are more restricted than initially reported using FcγRI-/- mice, but confirm effector capabilities for this high-affinity IgG receptor in vivo.

Cutting edge: FcγRIII (CD16) and FcγRI (CD64) are responsible for anti-glycoprotein 75 monoclonal antibody TA99 therapy for experimental metastatic B16 melanoma.

mAb therapy for experimental metastatic melanoma relies on activating receptors for the Fc portion of IgG (FcγR). Opposing results on the respective contribution of mouse FcγRI, FcγRIII, and FcγRIV have been reported using the gp75-expressing B16 melanoma and the protective anti-gp75 mAb TA99. We analyzed the contribution of FcγRs to this therapy model using bioluminescent measurement of lung metastases loads, novel mouse strains, and anti-FcγR blocking mAbs. We found that the TA99 mAb-mediated effects in a combination therapy using cyclophosphamide relied on activating FcγRs. The combination therapy, however, was not more efficient than mAb therapy alone. We demonstrate that FcγRI and, unexpectedly, FcγRIII contributed to TA99 mAb therapeutic effects, whereas FcγRIV did not. Therefore, FcγRIII and FcγRI are, together, responsible for anti-gp75 mAb therapy of B16 lung metastases. Our finding that mouse FcγRIII contributes to Ab-induced tumor reduction correlates with clinical data on its human functional equivalent human FcγRIIIA (CD16A).

Angiogenic sprouting into neural tissue requires Gpr124, an orphan G protein-coupled receptor.

The vasculature of the CNS is structurally and functionally distinct from that of other organ systems and is particularly prone to developmental abnormalities and hemorrhage. Although other embryonic tissues undergo primary vascularization, the developing nervous system is unique in that it is secondarily vascularized by sprouting angiogenesis from a surrounding perineural plexus. This sprouting angiogenesis requires the TGF-ß and Wnt pathways because ablation of these pathways results in aberrant sprouting and hemorrhage. We have genetically deleted Gpr124, a member of the large family of long N-terminal group B G protein-coupled receptors, few members of which have identified ligands or well-defined biologic functions in mammals. We show that, in the developing CNS, Gpr124 is specifically expressed in the vasculature and is absolutely required for proper angiogenic sprouting into the developing neural tube. Embryos lacking Gpr124 exhibit vascular defects characterized by delayed vascular penetration, formation of pathological glomeruloid tufts within the CNS, and hemorrhage. In addition, they display defects in palate and lung development, two processes in which TGF-ß and/or Wnt pathways also play important roles. We also show that TGF-ß stimulates Gpr124 expression, and ablation of Gpr124 results in perturbed TGF-ß pathway activation, suggesting roles for Gpr124 in modulating TGF-ß signaling. These results represent a unique function attributed to a long N-terminal group B-type G protein-coupled receptor in a mammalian system.

Resistance to diet-induced obesity in mice globally overexpressing OGH/GPB5.

We identified a glycoprotein hormone beta-subunit (OGH, also called GPB5) that, as a heterodimer with the alpha-subunit GPA2, serves as a second ligand for the thyroid-stimulating hormone receptor. Mice in which the OGH gene is deleted (OGH-/-) are indistinguishable from WT littermates in body weight, response to high-fat diet, metabolic parameters, body composition, and insulin tolerance. Mice engineered to transgenically globally overexpress OGH (OGH-TG) develop approximately 2-fold elevations in their basal thyroid levels and weigh slightly less than WT littermates despite increased food intake because of an increase in their metabolic rates. Moreover, when OGH-TG mice are challenged with a high-fat diet, they gain significantly less weight and body fat than their WT littermates. The OGH-TG mice also have reduced blood glucose, insulin, cholesterol, and triglycerides. In contrast to other approaches in which the thyroid axis is activated, OGH-TG mice exhibit only minor changes in heart rate and blood pressure. Our findings suggest that constitutive low-level activation of the thyroid axis (via OGH or other means) may provide a beneficial therapeutic approach for combating diet-induced obesity.