VSIG4 interaction with heparan sulfates inhibits VSIG4-complement binding.

V-set and immunoglobulin domain-containing 4 (VSIG4) is a complement receptor of the immunoglobulin superfamily that is specifically expressed on tissue resident macrophages, and its many reported functions and binding partners suggest a complex role in immune function. VSIG4 is reported to have a role in immune surveillance as well as in modulating diverse disease phenotypes such as infections, autoimmune conditions, and cancer. However, the mechanism(s) governing VSIG4's complex, context-dependent role in immune regulation remains elusive. Here, we identify cell surface and soluble glycosaminoglycans, specifically heparan sulfates, as novel binding partners of VSIG4. We demonstrate that genetic deletion of heparan sulfate synthesis enzymes or cleavage of cell-surface heparan sulfates reduced VSIG4 binding to the cell surface. Furthermore, binding studies demonstrate that VSIG4 interacts directly with heparan sulfates, with a preference for highly sulfated moieties and longer glycosaminoglycan chains. To assess the impact on VSIG4 biology, we show that heparan sulfates compete with known VSIG4 binding partners C3b and iC3b. Furthermore, mutagenesis studies indicate that this competition occurs through overlapping binding epitopes for heparan sulfates and complement on VSIG4. Together these data suggest a novel role for heparan sulfates in VSIG4-dependent immune modulation.

Multifunctional Alleles: A novel method for the generation of "All-In-One" null and conditional alleles.

The engineering of conditional alleles has evolved from simple floxing of regions of genes to more elaborate methods. Previously, we developed Conditional by Inversion (COIN), an allele design that utilizes an exon-splitting intron and an invertible genetrap-like module (COIN module) to create null alleles upon Cre-mediated inversion. Here we build upon COINs by generating a new Multifunctional Allele (MFA), that utilizes a single gene-targeting step and three site-specific recombination systems, to generate four allelic states: 1. The initial MFA (generated upon targeting) functions as a null with reporter (plus drug selection cassette) allele, wherein the gene of interest is inactivated by both inversion of a critical region of its coding sequence and simultaneous insertion of a reporter gene. MFAs can also be used as 'reverse-conditional' alleles as they are functionally wild type when they are converted to COIN alleles. 2. Null with reporter (minus drug selection cassette), wherein the selection cassette, the inverted critical region, and the COIN module are removed. 3. COIN-based conditional-null via removal of the selection cassette and reporter and simultaneous re-inversion of the critical region of the target. 4. Inverted COIN allele, wherein the COIN allele in turn is reconverted to a null allele by taking advantage of the COIN module's gene trap while simultaneously deleting the critical region.

Development and migration of plasma cells in the mouse lymph node.

In this study, we imaged the differentiation and migratory behavior of nascent plasma cells (PCs) in mouse lymph nodes by intravital microscopy. Pre-PCs exhibited a unique migration pattern characterized by long, linear paths that were randomly oriented. Although chemotaxis via Galphai coupled-receptors has been implicated in PC migration, treatment with Pertussis toxin (Ptx), which ablates these signals, did not prevent movement of pre-PCs while it arrested other lymphocytes. In vitro, pre-PCs displayed processive amoeboid locomotion on surfaces coated with integrin ligand, whereas fully differentiated PCs moved slowly or were arrested. Both PC arrest and differentiation occurred in the medullary cords. Ptx treatment before PC differentiation blocked their accumulation in the medullary cords but pre-PCs still differentiated in other lymph node regions. Taken together, we suggest pre-PCs undergo a persistent random walk to find the medullary cords, where localized chemokines help retain these cells until they undergo differentiation and arrest in situ.

Conditionals by inversion provide a universal method for the generation of conditional alleles.

Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.

Designing meaningful continuous representations of T cell receptor sequences with deep generative models.

T Cell Receptor (TCR) antigen binding underlies a key mechanism of the adaptive immune response yet the vast diversity of TCRs and the complexity of protein interactions limits our ability to build useful low dimensional representations of TCRs. To address the current limitations in TCR analysis we develop a capacity-controlled disentangling variational autoencoder trained using a dataset of approximately 100 million TCR sequences, that we name TCR-VALID. We design TCR-VALID such that the model representations are low-dimensional, continuous, disentangled, and sufficiently informative to provide high-quality TCR sequence de novo generation. We thoroughly quantify these properties of the representations, providing a framework for future protein representation learning in low dimensions. The continuity of TCR-VALID representations allows fast and accurate TCR clustering and is benchmarked against other state-of-the-art TCR clustering tools and pre-trained language models.

CD8- DCs induce IL-12-independent Th1 differentiation through Delta 4 Notch-like ligand in response to bacterial LPS.

Toll-like receptor (TLR) ligation is believed to skew T cell responses toward T helper (Th)1 differentiation by inducing interleukin (IL)-12 secretion by CD8(+) dendritic cells (DCs). However, TLR-dependent Th1 responses occur in the absence of IL-12. To determine how DCs induce Th1 differentiation in the absence of IL-12, we examined the response of IL-12-deficient DCs to bacterial lipopolysaccharide (LPS). We find that LPS activates MyD88-dependent Delta 4 Notch-like ligand expression by CD8(-) DCs, and that these cells direct Th1 differentiation by an IL-12-independent and Notch-dependent mechanism in vitro and in vivo. Thus, activation of the two DC subsets by TLR4 leads to Th1 responses by two distinct MyD88-dependent pathways.

In vivo imaging of germinal centres reveals a dynamic open structure.

Germinal centres are specialized structures wherein B lymphocytes undergo clonal expansion, class switch recombination, antibody gene diversification and affinity maturation. Three to four antigen-specific B cells colonize a follicle to establish a germinal centre and become rapidly dividing germinal-centre centroblasts that give rise to dark zones. Centroblasts produce non-proliferating centrocytes that are thought to migrate to the light zone of the germinal centre, which is rich in antigen-trapping follicular dendritic cells and CD4+ T cells. It has been proposed that centrocytes are selected in the light zone on the basis of their ability to bind cognate antigen. However, there have been no studies of germinal-centre dynamics or the migratory behaviour of germinal-centre cells in vivo. Here we report the direct visualization of B cells in lymph node germinal centres by two-photon laser-scanning microscopy in mice. Nearly all antigen-specific B cells participating in a germinal-centre reaction were motile and physically restricted to the germinal centre but migrated bi-directionally between dark and light zones. Notably, follicular B cells were frequent visitors to the germinal-centre compartment, suggesting that all B cells scan antigen trapped in germinal centres. Consistent with this observation, we found that high-affinity antigen-specific B cells can be recruited to an ongoing germinal-centre reaction. We conclude that the open structure of germinal centres enhances competition and ensures that rare high-affinity B cells can participate in antibody responses.

Ongoing Dll4-Notch signaling is required for T-cell homeostasis in the adult thymus.

The essential role of the Delta-like ligand 4 (Dll4)-Notch signaling pathway in T-lymphocyte development is well established. It has been shown that specific inactivation of Dll4 on thymic stromal cells during early post-natal development leads to a deregulation in T-cell differentiation. However, whether ongoing Dll4-Notch signaling is required for T-cell development in the adult thymus is unknown. The use of anti-Dll4 Abs allowed us to confirm and expand previous studies by examining the kinetics and the reversibility of Dll4-Notch signaling blockade in T-cell development in adult mice. We found that anti-Dll4 treatment reduced thymic cellularity after 7 days, as a consequence of a developmental delay in T-cell maturation at the pro-T-cell double negative 1 (CD4(-) CD8(-) c-kit(+) CD44(+) CD25(-) ) stage, leading to decreased numbers of immature double-positive (CD4(+) CD8(+) ) T cells without affecting the frequency of mature single positive CD4(+) and CD8(+) thymocytes, while promoting alternative thymic B-cell expansion. This cellular phenotype was similarly observed in both young adult and aged mice (>1.5 years), extending our understanding of the ongoing role for Dll4-Notch signaling during T-cell development in the adult thymus. Finally, after cessation of Dll4 Ab treatment, thymic cellularity and thymocyte subset ratios returned to normal levels, indicating reversibility of this phenotype in both adult and aged mice, which has important implications for potential clinical use of Dll4-Notch inhibitors.

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.

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.

CD22-targeted CD28 bispecific antibody enhances antitumor efficacy of odronextamab in refractory diffuse large B cell lymphoma models.

Although many patients with diffuse large B cell lymphoma (DLBCL) may achieve a complete response to frontline chemoimmunotherapy, patients with relapsed/refractory disease typically have poor outcomes. Odronextamab, a CD20xCD3 bispecific antibody that provides "signal 1" through the activation of the T cell receptor/CD3 complex, has exhibited early, promising activity for patients with highly refractory DLBCL in phase 1 trials. However, not all patients achieve complete responses, and many relapse, thus representing a high unmet medical need. Here, we investigated whether adding a costimulatory "signal 2" by engaging CD28 receptors on T cells could augment odronextamab activity. We demonstrate that REGN5837, a bispecific antibody that cross-links CD22-expressing tumor cells with CD28-expressing T cells, enhances odronextamab by potentiating T cell activation and cytolytic function. In preclinical DLBCL studies using human immune system-reconstituted animals, REGN5837 promotes the antitumor activity of odronextamab and induces intratumoral expansion of reprogrammable T cells while skewing away from a dysfunctional state. Although REGN5837 monotherapy shows limited activity and no toxicity in primate studies, it augments T cell activation when dosed in combination with odronextamab. In addition, analysis of non-Hodgkin lymphoma clinical samples reveals an increase in CD28+CD8+ T cells after odronextamab treatment, demonstrating the presence of a population that could potentially be targeted by REGN5837. Collectively, our data demonstrate that REGN5837 can markedly enhance the antitumor activity of odronextamab in preclinical NHL models, and the combination of these two bispecific antibodies may provide a chemotherapy-free approach for the treatment of DLBCL.

Sequence of αPD-1 relative to local tumor irradiation determines the induction of abscopal antitumor immune responses.

Although radiotherapy has been used for over a century to locally control tumor growth, alone it rarely induces an abscopal response or systemic antitumor immunity capable of inhibiting distal tumors outside of the irradiation field. Results from recent studies suggest that combining immune checkpoint blockades to radiotherapy may enhance abscopal activity. However, the treatment conditions and underlying immune mechanisms that consistently drive an abscopal response during radiation therapy combinations remain unknown. Here, we analyzed the antitumor responses at primary and distal tumor sites, demonstrating that the timing of αPD-1 antibody administration relative to radiotherapy determined the potency of the induced abscopal response. Blockade of the PD-1 pathway after local tumor irradiation resulted in the expansion of polyfunctional intratumoral CD8+ T cells, a decrease in intratumoral dysfunctional CD8+ T cells, expansion of reprogrammable CD8+ T cells, and induction of potent abscopal responses. However, administration of αPD-1 before irradiation almost completely abrogated systemic immunity, which associated with increased radiosensitivity and death of CD8+ T cells. The subsequent reduction of polyfunctional effector CD8+ T cells at the irradiated tumor site generated a suboptimal systemic antitumor response and the loss of abscopal responses. Therefore, this report maximizes the potential synergy between radiotherapy and αPD-1 immunotherapy, information that will benefit clinical combinations of radiotherapy and immune checkpoint blockade.

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.

A class of costimulatory CD28-bispecific antibodies that enhance the antitumor activity of CD3-bispecific antibodies.

T cell activation is initiated upon binding of the T cell receptor (TCR)/CD3 complex to peptide-major histocompatibility complexes ("signal 1"); activation is enhanced by engagement of a second "costimulatory" receptor, such as the CD28 receptor on T cells binding to its cognate ligand(s) on the target cell ("signal 2"). CD3-based bispecific antibodies act by replacing conventional signal 1, linking T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific and bridging to TCR/CD3 with the other. Although some of these so-called TSAxCD3 bispecifics have demonstrated promising antitumor efficacy in patients with cancer, their activity remains to be optimized. Here, we introduce a class of bispecific antibodies that mimic signal 2 by bridging TSA to the costimulatory CD28 receptor on T cells. We term these TSAxCD28 bispecifics and describe two such bispecific antibodies: one specific for ovarian and the other for prostate cancer antigens. Unlike CD28 superagonists, which broadly activate T cells and resulted in profound toxicity in early clinical trials, these TSAxCD28 bispecifics show limited activity and no toxicity when used alone in genetically humanized immunocompetent mouse models or in primates. However, when combined with TSAxCD3 bispecifics, they enhance the artificial synapse between a T cell and its target cell, potentiate T cell activation, and markedly improve antitumor activity of CD3 bispecifics in a variety of xenogeneic and syngeneic tumor models. Combining this class of CD28-costimulatory bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide well-tolerated, off-the-shelf antibody therapies with robust antitumor efficacy.

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.

CD8+ T cells in beige adipogenesis and energy homeostasis.

Although accumulation of lymphocytes in the white adipose tissue (WAT) in obesity is linked to insulin resistance, it remains unclear whether lymphocytes also participate in the regulation of energy homeostasis in the WAT. Here, we demonstrate enhanced energy dissipation in Rag1-/- mice, increased catecholaminergic input to subcutaneous WAT, and significant beige adipogenesis. Adoptive transfer experiments demonstrated that CD8+ T cell deficiency accounts for the enhanced beige adipogenesis in Rag1-/- mice. Consistently, we identified that CD8-/- mice also presented with enhanced beige adipogenesis. The inhibitory effect of CD8+ T cells on beige adipogenesis was reversed by blockade of IFN-γ. All together, our findings identify an effect of CD8+ T cells in regulating energy dissipation in lean WAT, mediated by IFN-γ modulation of the abundance of resident immune cells and of local catecholaminergic activity. Our results provide a plausible explanation for the clinical signs of metabolic dysfunction in diseases characterized by altered CD8+ T cell abundance and suggest targeting of CD8+ T cells as a promising therapeutic approach for obesity and other diseases with altered energy homeostasis.

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.

Delta-like Ligand-4-Notch Signaling Inhibition Regulates Pancreatic Islet Function and Insulin Secretion.

Although Notch signaling has been proposed as a therapeutic target for type-2 diabetes, liver steatosis, and atherosclerosis, its direct effect on pancreatic islets remains unknown. Here, we demonstrated a function of Dll4-Notch signaling inhibition on the biology of insulin-producing cells. We confirmed enhanced expression of key Notch signaling genes in purified pancreatic islets from diabetic NOD mice and showed that treatment with anti-Dll4 antibody specifically abolished Notch signaling pathway activation. Furthermore, we showed that Notch inhibition could drive proliferation of ß-islet cells and confer protection from the development of STZ-induced diabetes. Importantly, inhibition of the Dll4 pathway in WT mice increased insulin secretion by inducing the differentiation of pancreatic ß-islet cell progenitors, as well as the proliferation of insulin-secreting cells. These findings reveal a direct effect of Dll4-blockade on pancreatic islets that, in conjunction with its immunomodulatory effects, could be used for unmet medical needs hallmarked by inefficient insulin action.

Characterization of the Anti-PD-1 Antibody REGN2810 and Its Antitumor Activity in Human PD-1 Knock-In Mice.

The Programmed Death-1 (PD-1) receptor delivers inhibitory checkpoint signals to activated T cells upon binding to its ligands PD-L1 and PD-L2 expressed on antigen-presenting cells and cancer cells, resulting in suppression of T-cell effector function and tumor immune evasion. Clinical antibodies blocking the interaction between PD-1 and PD-L1 restore the cytotoxic function of tumor antigen-specific T cells, yielding durable objective responses in multiple cancers. This report describes the preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4(S228P) high-affinity anti-PD-1 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. REGN2810 was characterized in a series of binding, blocking, and functional cell-based assays, and preclinical in vivo studies in mice and monkeys. In cell-based assays, REGN2810 reverses PD-1-dependent attenuation of T-cell receptor signaling in engineered T cells and enhances responses of human primary T cells. To test the in vivo activity of REGN2810, which does not cross-react with murine PD-1, knock-in mice were generated to express a hybrid protein containing the extracellular domain of human PD-1, and transmembrane and intracellular domains of mouse PD-1. In these mice, REGN2810 binds the humanized PD-1 receptor and inhibits growth of MC38 murine tumors. As REGN2810 binds to cynomolgus monkey PD-1 with high affinity, pharmacokinetic and toxicologic assessment of REGN2810 was performed in cynomolgus monkeys. High doses of REGN2810 were well tolerated, without adverse immune-related effects. These preclinical studies validate REGN2810 as a potent and promising candidate for cancer immunotherapy. Mol Cancer Ther; 16(5); 861-70. ©2017 AACR.

Immunoregulatory properties of mast cell-derived exosomes.

Transmission of information from mast cells to neighboring or distant cells must be established continuously in order to ensure homeostasis or to initiate immune and inflammatory responses. Owing to their strategic location in peripheral tissues and their prompt response to various stimuli, mast cells can be considered as the cell prototype to fulfill such a sentinel function. There are several ways for mast cells to communicate with other cells including cell-cell interactions via membrane-associated receptors, cytokines and other soluble mediators, and a newly described messenger which consists of membrane vesicles called exosomes carrying a number of immunoregulatory molecules.