Leukocyte-associated immunoglobulin-like receptor-1 blockade in combination with programmed death-ligand 1 targeting therapy mediates increased tumour control in mice.

Collagen expression and structure in the tumour microenvironment are associated with tumour development and therapy response. Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a widely expressed inhibitory collagen receptor. LAIR-2 is a soluble homologue of LAIR-1 that competes for collagen binding. Multiple studies in mice implicate blockade of LAIR-1:collagen interaction in cancer as a promising therapeutic strategy. Here, we investigated the role of LAIR-1 in anti-tumour responses. We show that although LAIR-1 inhibits activation, proliferation, and cytokine production of mouse T cells in vitro, tumour outgrowth in LAIR-1-deficient mice did not differ from wild type mice in several in vivo tumour models. Furthermore, treatment with NC410, a LAIR-2-Fc fusion protein, did not result in increased tumour clearance in tested immunocompetent mice, which contrasts with previous data in humanized mouse models. This discrepancy may be explained by our finding that NC410 blocks human LAIR-1:collagen interaction more effectively than mouse LAIR-1:collagen interaction. Despite the lack of therapeutic impact of NC410 monotherapy, mice treated with a combination of NC410 and anti-programmed death-ligand 1 did show reduced tumour burden and increased survival. Using LAIR-1-deficient mice, we showed that this effect seemed to be dependent on the presence of LAIR-1. Taken together, our data demonstrate that the absence of LAIR-1 signalling alone is not sufficient to control tumour growth in multiple immunocompetent mouse models. However, combined targeting of LAIR-1 and PD-L1 results in increased tumour control. Thus, additional targeting of the LAIR-1:collagen pathway with NC410 is a promising approach to treating tumours where conventional immunotherapy is ineffective.

B7-H4 Modulates Regulatory CD4+ T Cell Induction and Function via Ligation of a Semaphorin 3a/Plexin A4/Neuropilin-1 Complex.

The potent immune regulatory function of an agonistic B7-H4-Ig fusion protein (B7-H4Ig) has been demonstrated in multiple experimental autoimmune models; however, the identity of a functional B7-H4 receptor remained unknown. The biological activity of B7-H4 is associated with decreased inflammatory CD4+ T cell responses as supported by a correlation between B7-H4-expressing tumor-associated macrophages and Foxp3+ T cells within the tumor microenvironment. Recent data indicate that members of the semaphorin (Sema)/plexin/neuropilin (Nrp) family of proteins both positively and negatively modulate immune cell function. In this study, we show that B7-H4 binds the soluble Sema family member Sema3a. Additionally, B7-H4Ig-induced inhibition of inflammatory CD4+ T cell responses is lost in both Sema3a functional mutant mice and mice lacking Nrp-1 expression in Foxp3+ T cells. These findings indicate that B7-H4Ig binds to Sema3a, which acts as a functional bridge to stimulate an Nrp-1/Plexin A4 heterodimer to form a functional immunoregulatory receptor complex resulting in increased levels of phosphorylated PTEN and enhanced regulatory CD4+ T cell number and function.

BCAM (basal cell adhesion molecule) protein expression in different tumor populations.

Basal Cell Adhesion Molecule (BCAM), a receptor for laminin subunit α5, plays a crucial role in the pathogenesis of various malignancies. Notably, evidence of hypermethylation at multiple immune checkpoints in patients with low BCAM expression suggests these individuals may respond favorably to immunotherapy using ICIs (immune checkpoint inhibitors). This finding lays the foundation for the hypothesis that BCAM may serve as an important biomarker in cancer patients. To investigate this potential, we evaluated BCAM expression patterns in 3114 patients from both discovery and validation cohorts, spanning seven cancer types, using quantitative immunofluorescence (QIF). We also explored the correlation between BCAM and PD-L1 expressions within these cohorts, aiming to establish its potential predictive value for immunotherapy response. Our findings indicate that BCAM was highly expressed in ovarian (79.2%) and lung (78.5%) tumors, with lower yet significant expression in breast (37.7%), head and neck (31.3%), and bladder-urothelial tumors (27.6%). Notably, high BCAM expression was associated with better OS in NSCLC. More importantly, BCAM expression did not correlate with PD-L1 protein expression in any of these tumors, highlighting its independent predictive potential. The widespread expression of BCAM across multiple tumor types, coupled with its lack of correlation with PD-L1 expression, highlights its potential as a predictive novel biomarker across various cancer types.

The FLRT3-UNC5B checkpoint pathway inhibits T cell-based cancer immunotherapies.

Cancers exploit coinhibitory receptors on T cells to escape tumor immunity, and targeting such mechanisms has shown remarkable clinical benefit, but in a limited subset of patients. We hypothesized that cancer cells mimic noncanonical mechanisms of early development such as axon guidance pathways to evade T cell immunity. Using gain-of-function genetic screens, we profiled axon guidance proteins on human T cells and their cognate ligands and identified fibronectin leucine-rich transmembrane protein 3 (FLRT3) as a ligand that inhibits T cell activity. We demonstrated that FLRT3 inhibits T cells through UNC5B, an axon guidance receptor that is up-regulated on activated human T cells. FLRT3 expressed in human cancers favored tumor growth and inhibited CAR-T and BiTE + T cell killing and infiltration in humanized cancer models. An FLRT3 monoclonal antibody that blocked FLRT3-UNC5B interactions reversed these effects in an immune-dependent manner. This study supports the concept that axon guidance proteins mimic T cell checkpoints and can be targeted for cancer immunotherapy.

Up-regulated PLA2G10 in cancer impairs T cell infiltration to dampen immunity.

T cells are often absent from human cancer tissues during both spontaneously induced immunity and therapeutic immunotherapy, even in the presence of a functional T cell-recruiting chemokine system, suggesting the existence of T cell exclusion mechanisms that impair infiltration. Using a genome-wide in vitro screening platform, we identified a role for phospholipase A2 group 10 (PLA2G10) protein in T cell exclusion. PLA2G10 up-regulation is widespread in human cancers and is associated with poor T cell infiltration in tumor tissues. PLA2G10 overexpression in immunogenic mouse tumors excluded T cells from infiltration, resulting in resistance to anti-PD-1 immunotherapy. PLA2G10 can hydrolyze phospholipids into small lipid metabolites, thus inhibiting chemokine-mediated T cell mobility. Ablation of PLA2G10's enzymatic activity enhanced T cell infiltration and sensitized PLA2G10-overexpressing tumors to immunotherapies. Our study implicates a role for PLA2G10 in T cell exclusion from tumors and suggests a potential target for cancer immunotherapy.

B7-H4Ig inhibits mouse and human T-cell function and treats EAE via IL-10/Treg-dependent mechanisms.

We evaluated the therapeutic efficacy and mechanisms of action of both mouse and human B7-H4 Immunoglobulin fusion proteins (mB7-H4Ig; hB7-H4Ig) in treating EAE. The present data show that mB7-H4Ig both directly and indirectly (via increasing Treg function) inhibited CD4⁺ T-cell proliferation and differentiation in both Th1- and Th17-cell promoting conditions while inducing production of IL-10. B7-H4Ig treatment effectively ameliorated progression of both relapsing (R-EAE) and chronic EAE correlating with decreased numbers of activated CD4⁺ T-cells within the CNS and spleen, and a concurrent increase in number and function of Tregs. The functional requirement for Treg activation in treating EAE was demonstrated by a loss of therapeutic efficacy of hB7-H4Ig in R-EAE following inactivation of Treg function either by anti-CD25 treatment or blockade of IL-10. Significant to the eventual translation of this treatment into clinical practice, hB7-H4Ig similarly inhibited the in vitro differentiation of naïve human CD4⁺ T-cells in both Th1- and Th17-promoting conditions, while promoting the production of IL-10. B7-H4Ig thus regulates pro-inflammatory T-cell responses by a unique dual mechanism of action and demonstrates significant promise as a therapeutic for autoimmune diseases, including MS.

Regulation of tumor immunity and immunotherapy by the tumor collagen extracellular matrix.

It has been known for decades that the tumor extracellular matrix (ECM) is dysfunctional leading to loss of tissue architecture and promotion of tumor growth. The altered ECM and tumor fibrogenesis leads to tissue stiffness that act as a physical barrier to immune cell infiltration into the tumor microenvironment (TME). It is becoming increasingly clear that the ECM plays important roles in tumor immune responses. A growing body of data now indicates that ECM components also play a more active role in immune regulation when dysregulated ECM components act as ligands to interact with receptors on immune cells to inhibit immune cell subpopulations in the TME. In addition, immunotherapies such as checkpoint inhibitors that are approved to treat cancer are often hindered by ECM changes. In this review we highlight the ways by which ECM alterations affect and regulate immunity in cancer. More specifically, how collagens and major ECM components, suppress immunity in the complex TME. Finally, we will review how our increased understanding of immune and immunotherapy regulation by the ECM is leading towards novel disruptive strategies to overcome immune suppression.

Quantitative, Spatially Defined Expression of Leukocyte-associated Immunoglobulin-like Receptor in Non-small Cell Lung Cancer.

Targeting the interaction of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) and its ligands has been shown to reinstate antitumor immunity. In addition, the introduction of the LAIR-1 decoy protein, LAIR-2, sensitizes previously resistant lung tumors to programmed death-1 (PD-1) blockade, indicating the potential of LAIR-1 as an alternative marker for anti-PD-1 resistance in lung cancer. Here, we assessed LAIR-1 as compared with programmed death-ligand 1 (PD-L1) expression in various tumors, with a focus on non-small cell lung cancer (NSCLC) and its histologic subtypes using multiplexed quantitative immunofluorescence (mQIF) in 287 (discovery cohort) and 144 (validation cohort) patients with NSCLC. In addition, using multispectral imaging technology on mQIF images, we evaluated the localization of LAIR-1 on various cell types. We observed that CD14+, CD68+, and CD163+ monocytes and CK+ tumor cells predominantly expressed LAIR-1 more than other cell types. Furthermore, LAIR-1 expression in the tumor compartment was significantly higher in patients with lung adenocarcinoma (LUAD) than those with lung squamous cell carcinoma subtype (**, P = 0.003). Our results indicated that high tumor LAIR-1 expression in patients with LUAD is negatively associated with OS (overall survival, HR = 2.4; *, P = 0.02) highlighting its prognostic value in LUAD but not in other subtypes. The Pearson correlation between LAIR-1 and PD-L1 is 0.31; however, mutual exclusive staining pattern (i.e., several cases were positive for LAIR-1 and negative for PD-L1) was observed. Altogether, our data suggest that the combination therapy of anti-PD-1/PD-L1 with anti-LAIR-1 or the anti-LAIR-1 monotherapy alone may be promising cancer immunotherapeutic strategies. Significance: The spatial, quantitative assessment of LAIR-1 in NSCLC shows positive association of OS with high LAIR-1+/CD68+ cell densities and negative association of OS with high LAIR-1 expression in LUAD tumor subtype.

Anti-Siglec-15 Antibody Prevents Marked Bone Loss after Acute Spinal Cord Injury-Induced Immobilization in Rats.

Rapid and extensive sublesional bone loss after spinal cord injury (SCI) is a difficult medical problem that has been refractory to available interventions except the antiresorptive agent denosumab (DMAB). While DMAB has shown some efficacy in inhibiting bone loss, its concurrent inhibition of bone formation limits its use. Sialic acid-binding immunoglobulin-like lectin (Siglec)-15 is expressed on the cell surface of mature osteoclasts. Anti-Siglec-15 antibody (Ab) has been shown to inhibit osteoclast maturation and bone resorption while maintaining osteoblast activity, which is distinct from current antiresorptive agents that inhibit the activity of both osteoclasts and osteoblasts. The goal of the present study is to test a Siglec-15 Ab (NP159) as a new treatment option to prevent bone loss in an acute SCI model. To this end, 4-month-old male Wistar rats underwent complete spinal cord transection and were treated with either vehicle or NP159 at 20 mg/kg once every 2 weeks for 8 weeks. SCI results in significant decreases in bone mineral density (BMD, -18.7%), trabecular bone volume (-43.1%), trabecular connectivity (-59.7%), and bone stiffness (-76.3%) at the distal femur. Treatment with NP159 almost completely prevents the aforementioned deterioration of bone after SCI. Blood and histomorphometric analyses revealed that NP159 is able to greatly inhibit bone resorption while maintaining bone formation after acute SCI. In ex vivo cultures of bone marrow cells, NP159 reduces osteoclastogenesis while increasing osteoblastogenesis. In summary, treatment with NP159 almost fully prevents sublesional loss of BMD and metaphysis trabecular bone volume and preserves bone strength in a rat model of acute SCI. Because of its unique ability to reduce osteoclastogenesis and bone resorption while promoting osteoblastogenesis to maintain bone formation, Siglec-15 Ab may hold greater promise as a therapeutic agent, compared with the exclusively antiresorptive or anabolic agents that are currently used, in mitigating the striking bone loss that occurs after SCI or other conditions associated with severe immobilization. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Siglec-15 Promotes Evasion of Adaptive Immunity in B-cell Acute Lymphoblastic Leukemia.

Siglec-15 (Sig15) has been implicated as an immune checkpoint expressed in solid tumor-infiltrating macrophages and is being targeted in clinical trials with mAbs to normalize the tumor immune microenvironment and stimulate antitumor immunity. However, the role of Sig15 in hematologic malignancies remains undefined. Sig15 mRNA and protein expression levels in hematologic malignancies were determined from publicly available databases, cell lines, and primary patient samples. Human B-cell acute lymphoblastic leukemia (B-ALL) cell lines were used to identify signaling pathways involved in the regulation of Sig15 expression. Secreted/soluble Sig15 and cytokine levels were measured from the plasma of children with leukemia and healthy controls. Knockdown and knockout of Siglec15 in a murine model of B-ALL was used to evaluate the effect of leukemia-derived Sig15 on the immune response to leukemia. We observed pathologic overexpression of Sig15 in a variety of hematologic malignancies, including primary B-ALL samples. This overexpression was driven by NFκB activation, which also increased the surface localization of Sig15. Secreted/soluble Sig15 was found to circulate at elevated levels in the plasma of children with B-ALL and correlated with an immune-suppressive cytokine milieu. Genetic inhibition of Sig15 in murine B-ALL promoted clearance of the leukemia by the immune system and a marked reversal of the immune-privileged leukemia bone marrow niche, including expanded early effector CD8+ T cells and reduction of immunosuppressive cytokines. Thus, Sig15 is a novel, potent immunosuppressive molecule active in leukemia that may be targeted therapeutically to activate T lymphocytes against leukemia cells. Significance: We demonstrate that Sig15 is overexpressed in hematologic malignancies driven by NFκB, is required for immune evasion in a mouse model of leukemia, and, for the first time, that it circulates at high levels in the plasma of children with leukemia.