PD-1H/VISTA mediates immune evasion in acute myeloid leukemia.

Acute myeloid leukemia (AML) presents a pressing medical need in that it is largely resistant to standard chemotherapy as well as modern therapeutics, such as targeted therapy and immunotherapy, including anti-programmed cell death protein (anti-PD) therapy. We demonstrate that programmed death-1 homolog (PD-1H), an immune coinhibitory molecule, is highly expressed in blasts from the bone marrow of AML patients, while normal myeloid cell subsets and T cells express PD-1H. In studies employing syngeneic and humanized AML mouse models, overexpression of PD-1H promoted the growth of AML cells, mainly by evading T cell-mediated immune responses. Importantly, ablation of AML cell-surface PD-1H by antibody blockade or genetic knockout significantly inhibited AML progression by promoting T cell activity. In addition, the genetic deletion of PD-1H from host normal myeloid cells inhibited AML progression, and the combination of PD-1H blockade with anti-PD therapy conferred a synergistic antileukemia effect. Our findings provide the basis for PD-1H as a potential therapeutic target for treating human AML.

Immune Inhibitory Molecule PD-1 Homolog (VISTA) Colocalizes with CD11b Myeloid Cells in Melanoma and Is Associated with Poor Outcomes.

Tumors evade immunity through the overexpression of immune inhibitory molecules in the tumor microenvironment such as PD-L1/B7-H1. An immune inhibitory molecule named PD-1 homolog (also known as V-domain Ig-containing suppressor of T cell activation [VISTA]) functions to control both T cells and myeloid cells. Current clinical trials using anti-VISTA-blocking agents for treatment of cancer are ongoing. We sought to determine the extent of VISTA expression in primary cutaneous melanomas (n = 190), identify the critical cell types expressing VISTA, and correlate its expression with PD-L1 expression using multiplexed quantitative immunofluorescence. Within the tumor subcompartments, VISTA is most highly expressed on CD11b myeloid cells, and PD-L1 is most highly expressed on CD68 myeloid cells in our melanoma cohort. There is little correlation between VISTA and PD-L1 expression intensity, suggesting that individual tumors have distinct immunosuppressive tumor microenvironments. High levels of VISTA expression on CD11b myeloid cells but not PD-L1 expression were associated with greater melanoma recurrence and greater all-cause mortality. Our findings suggest that cell-specific VISTA expression may be a negative prognostic biomarker for melanoma and a future potential therapeutic target.

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.

Phase I Study of Single-Agent Anti-Programmed Death-1 (MDX-1106) in Refractory Solid Tumors: Safety, Clinical Activity, Pharmacodynamics, and Immunologic Correlates.

PURPOSE: Programmed death-1 (PD-1), an inhibitory receptor expressed on activated T cells, may suppress antitumor immunity. This phase I study sought to determine the safety and tolerability of anti-PD-1 blockade in patients with treatment-refractory solid tumors and to preliminarily assess antitumor activity, pharmacodynamics, and immunologic correlates. PATIENTS AND METHODS: Thirty-nine patients with advanced metastatic melanoma, colorectal cancer (CRC), castrate-resistant prostate cancer, non-small-cell lung cancer (NSCLC), or renal cell carcinoma (RCC) received a single intravenous infusion of anti-PD-1 (MDX-1106) in dose-escalating six-patient cohorts at 0.3, 1, 3, or 10 mg/kg, followed by a 15-patient expansion cohort at 10 mg/kg. Patients with evidence of clinical benefit at 3 months were eligible for repeated therapy. RESULTS: Anti-PD-1 was well tolerated: one serious adverse event, inflammatory colitis, was observed in a patient with melanoma who received five doses at 1 mg/kg. One durable complete response (CRC) and two partial responses (PRs; melanoma, RCC) were seen. Two additional patients (melanoma, NSCLC) had significant lesional tumor regressions not meeting PR criteria. The serum half-life of anti-PD-1 was 12 to 20 days. However, pharmacodynamics indicated a sustained mean occupancy of > 70% of PD-1 molecules on circulating T cells ≥ 2 months following infusion, regardless of dose. In nine patients examined, tumor cell surface B7-H1 expression appeared to correlate with the likelihood of response to treatment. CONCLUSION: Blocking the PD-1 immune checkpoint with intermittent antibody dosing is well tolerated and associated with evidence of antitumor activity. Exploration of alternative dosing regimens and combinatorial therapies with vaccines, targeted therapies, and/or other checkpoint inhibitors is warranted.

Interaction of glioma-associated microglia/macrophages and anti-PD1 immunotherapy.

Anti-PD-1-based therapy has resulted in a minimal clinical response in malignant gliomas. Gliomas contain numerous glioma-associated microglia/macrophages (GAMs), reported to contribute to an immunosuppressive microenvironment and promote glioma progression. However, whether and how GAMs affect anti-PD-1 immunotherapy in glioma remains unclear. Here, we demonstrated that M1-like GAMs contribute to the anti-PD-1 therapeutic response, while the accumulation of M2-like GAMs is associated with therapeutic resistance. Furthermore, we found that PD-L1 ablation reverses GAMs M2-like phenotype and is beneficial to anti-PD-1 therapy. We also demonstrated that tumor-induced impairment of the antigen-presenting function of GAMs could limit the antitumor immunity of CD4+ T cells in anti-PD-1 therapy. Our study highlights the impact of GAMs activation on anti-PD-1 treatment and provides new insights into the role of GAMs in regulating anti-PD-1 therapy in gliomas.

CD137 (4-1BB)-Based Cancer Immunotherapy on Its 25th Anniversary.

Twenty-five years ago, we reported that agonist anti-CD137 monoclonal antibodies eradicated transplanted mouse tumors because of enhanced CD8+ T-cell antitumor immunity. Mouse models indicated that anti-CD137 agonist antibodies synergized with various other therapies. In the clinic, the agonist antibody urelumab showed evidence for single-agent activity against melanoma and non-Hodgkin lymphoma but caused severe liver inflammation in a fraction of the patients. CD137's signaling domain is included in approved chimeric antigen receptors conferring persistence and efficacy. A new wave of CD137 agonists targeting tumors, mainly based on bispecific constructs, are in early-phase trials and are showing promising safety and clinical activity. SIGNIFICANCE: CD137 (4-1BB) is a costimulatory receptor of T and natural killer lymphocytes whose activity can be exploited in cancer immunotherapy strategies as discovered 25 years ago. Following initial attempts that met unacceptable toxicity, new waves of constructs acting agonistically on CD137 are being developed in patients, offering signs of clinical and pharmacodynamic activity with tolerable safety profiles.

VISTA (PD-1H) Is a Crucial Immune Regulator to Limit Pulmonary Fibrosis.

VISTA (V domain immunoglobulin suppressor of T cell activation, also called PD-1H [programmed death-1 homolog]), a novel immune regulator expressed on myeloid and T lymphocyte lineages, is upregulated in mouse and human idiopathic pulmonary fibrosis (IPF). However, the significance of VISTA and its therapeutic potential in regulating IPF has yet to be defined. To determine the role of VISTA and its therapeutic potential in IPF, the expression profile of VISTA was evaluated from human single-cell RNA sequencing data (IPF Cell Atlas). Inflammatory response and lung fibrosis were assessed in bleomycin-induced experimental pulmonary fibrosis models in VISTA-deficient mice compared with wild-type littermates. In addition, these outcomes were evaluated after VISTA agonistic antibody treatment in the wild-type pulmonary fibrosis mice. VISTA expression was increased in lung tissue-infiltrating monocytes of patients with IPF. VISTA was induced in the myeloid population, mainly circulating monocyte-derived macrophages, during bleomycin-induced pulmonary fibrosis. Genetic ablation of VISTA drastically promoted pulmonary fibrosis, and bleomycin-induced fibroblast activation was dependent on the interaction between VISTA-expressing myeloid cells and fibroblasts. Treatment with VISTA agonistic antibody reduced fibrotic phenotypes accompanied by the suppression of lung innate immune and fibrotic mediators. In conclusion, these results suggest that VISTA upregulation in pulmonary fibrosis may be a compensatory mechanism to limit inflammation and fibrosis, and stimulation of VISTA signaling using VISTA agonists effectively limits the fibrotic innate immune landscape and consequent tissue fibrosis. Further studies are warranted to test VISTA as a novel therapeutic target for the IPF treatment.

Quantitative tissue analysis and role of myeloid cells in non-small cell lung cancer.

BACKGROUND: Despite the prominent role of innate immunity in the antitumor response, little is known about the myeloid composition of human non-small cell lung cancer (NSCLC) with respect to histology and molecular subtype. We used multiplexed quantitative immunofluorescence (QIF) to measure the distribution and clinical significance of major myeloid cell subsets in large retrospective NSCLC collections. METHODS: We established a QIF panel to map major myeloid cell subsets in fixed human NSCLC including 4',6-Diamidino-2-Phenylindole for all cells, pancytokeratin for tumor-epithelial cells, CD68 for M1-like macrophages; and CD11b plus HLA-DR to interrogate mature and immature myeloid cell populations such as myeloid derived suppressor cells (MDSCs). We interrogated 793 NSCLCs represented in four tissue microarray-based cohorts: #1 (Yale, n=379) and #2 (Greece, n=230) with diverse NSCLC subtypes; #3 (Yale, n=138) with molecularly annotated lung adenocarcinomas (ADC); and #4 (Yale, n=46) with patient-matched NSCLC and morphologically-normal lung tissue. We examined associations between marker levels, myeloid cell profiles, clinicopathologic/molecular variables and survival. RESULTS: The levels of CD68+ M1 like macrophages were significantly lower and the fraction of CD11b+/HLA-DR- MDSC-like cells was prominently higher in tumor than in matched non-tumor lung tissues. HLA-DR was consistently higher in myeloid cells from tumors with elevated CD68 expression. Stromal CD11b was significantly higher in squamous cell carcinomas (SCC) than in ADC across the cohorts and EGFR-mutated lung ADCs displayed lower CD11b levels than KRAS-mutant tumors. Increased stromal CD68- and HLA-DR-expressing cells was associated with better survival in ADCs from two independent NSCLC cohorts. In SCC, increased stromal CD11b or HLA-DR expression was associated with a trend towards shorter 5-year survival. CONCLUSIONS: NSCLCs display an unfavorable myeloid immune contexture relative to non-tumor lung and exhibit distinct myeloid-cell profiles across histologies and presence of major oncogenic driver-mutations. Elevated M1-like stromal proinflammatory myeloid cells are prognostic in lung ADC, but not in SCC.

When immunotherapy meets surgery in non-small cell lung cancer.

The results of the most recent Checkmate-816 trial in The New England Journal of Medicine using combination neoadjuvant immunotherapy with platinum-based chemotherapy in resectable non-small cell lung cancer demonstrate the effectiveness of neoadjuvant immunotherapy and provide further support that biology and personalized therapy represent the foundation of lung cancer treatment.

Adaptive immune resistance at the tumour site: mechanisms and therapeutic opportunities.

Tumours employ various tactics to adapt and eventually resist immune attack. These mechanisms are collectively called adaptive immune resistance (AIR). The first defined and therapeutically validated AIR mechanism is the selective induction of programmed cell death 1 ligand 1 (PDL1) by interferon-γ in the tumour. Blockade of PDL1 binding to its receptor PD1 by antibodies (anti-PD therapy) has resulted in remission of a fraction of patients with advanced-stage cancer, especially in solid tumours. However, many clinical trials combining anti-PD therapy with other antitumour drugs conducted without a strong mechanistic rationale have failed to identify a synergistic or additive effect. In this Perspective article, we discuss why defining AIR mechanisms at the tumour site should be a key focus to direct future drug development as well as practical approaches to improve current cancer therapy.

The CD8α-PILRα interaction maintains CD8+ T cell quiescence.

T cell quiescence is essential for maintaining a broad repertoire against a large pool of diverse antigens from microbes and tumors, but the underlying molecular mechanisms remain largely unknown. We show here that CD8α is critical for the maintenance of CD8+ T cells in a physiologically quiescent state in peripheral lymphoid organs. Upon inducible deletion of CD8α, both naïve and memory CD8+ T cells spontaneously acquired activation phenotypes and subsequently died without exposure to specific antigens. PILRα was identified as a ligand for CD8α in both mice and humans, and disruption of this interaction was able to break CD8+ T cell quiescence. Thus, peripheral T cell pool size is actively maintained by the CD8α-PILRα interaction in the absence of antigen exposure.

Resistance Mechanisms to Anti-PD Cancer Immunotherapy.

The transformative success of antibodies targeting the PD-1 (programmed death 1)/B7-H1 (B7 homolog 1) pathway (anti-PD therapy) has revolutionized cancer treatment. However, only a fraction of patients with solid tumors and some hematopoietic malignancies respond to anti-PD therapy, and the reason for failure in other patients is less known. By dissecting the mechanisms underlying this resistance, current studies reveal that the tumor microenvironment is a major location for resistance to occur. Furthermore, the resistance mechanisms appear to be highly heterogeneous. Here, we discuss recent human cancer data identifying mechanisms of resistance to anti-PD therapy. We review evidence for immune-based resistance mechanisms such as loss of neoantigens, defects in antigen presentation and interferon signaling, immune inhibitory molecules, and exclusion of T cells. We also review the clinical evidence for emerging mechanisms of resistance to anti-PD therapy, such as alterations in metabolism, microbiota, and epigenetics. Finally, we discuss strategies to overcome anti-PD therapy resistance and emphasize the need to develop additional immunotherapies based on the concept of normalization cancer immunotherapy.

Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies.

BACKGROUND: On the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved chimeric antigen receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and costimulation of T cells will facilitate clinical development of CD137 agonists in the clinic. METHODS: We used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored on treatment with agonist anti-CD137 monoclonal antibodies (mAbs). Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 mAb (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISA and Luminex. Flow cytometry was used to monitor CD137 surface expression. RESULTS: CD137 costimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 costimulation inside subcutaneously implanted Matrigel plugs. The CD137 signaling domain-containing CAR T cells readily released sCD137 and acquired CD137 surface expression on antigen recognition. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137. CONCLUSION: sCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic costimulatory activity elicited by agonist CD137-targeted agents.

Ablation of T cell-associated PD-1H enhances functionality and promotes adoptive immunotherapy.

Programmed death-1 homolog (PD-1H) is a coinhibitory molecule that negatively regulates T cell-mediated immune responses. In this study, we determined whether ablation of T cell-associated PD-1H could enhance adoptive T cell therapy in experimental tumor models. The expression of PD-1H is upregulated in activated and tumor-infiltrating CD8+ T cells. Activated CD8+ T cells from PD-1H-deficient (PD-1H-KO) mice exhibited increased cell proliferation, cytokine production, and antitumor activity in vitro. Adoptive transfer of PD-1H-KO CD8+ T cells resulted in the regression of established syngeneic mouse tumors. Similar results were obtained when PD-1H was ablated in T cells by CRISPR/Cas9-mediated gene silencing. Furthermore, ablation of PD-1H in CAR-T cells significantly improved their antitumor activity against human xenografts in vivo. Our results indicate that T cell-associated PD-1H could suppress immunity in the tumor microenvironment and that targeting PD-1H may improve T cell adoptive immunotherapy.

An antibody against Siglec-15 promotes bone formation and fracture healing by increasing TRAP+ mononuclear cells and PDGF-BB secretion.

Osteoporosis (OP) is a common age-related disease characterized by a deterioration of bone mass and structure that predisposes patients to fragility fractures. Pharmaceutical therapies that promote anabolic bone formation in OP patients and OP-induced fracture are needed. We investigated whether a neutralizing antibody against Siglec-15 can simultaneously inhibit bone resorption and stimulate bone formation. We found that the multinucleation of osteoclasts was inhibited in SIGLEC-15 conditional knockout mice and mice undergoing Siglec-15 neutralizing antibody treatment. The secretion of platelet-derived growth factor-BB (PDGF-BB), the number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear cells, and bone formation were significantly increased in the SIGLEC-15 conditional knockout mice and antibody-treated mice. The anabolic effect of the Siglec-15 neutralizing antibody on bone formation was blunted in mice with Pdgfb deleted in TRAP+ cells. These findings showed that the anabolic effect of the Siglec-15 neutralizing antibody was mediated by elevating PDGF-BB production of TRAP+ mononuclear cells. To test the therapeutic potential of the Siglec-15 neutralizing antibody, we injected the antibody in an ovariectomy-induced osteoporotic mouse model, which mimics postmenopausal osteoporosis in women, and in two fracture healing models because fracture is the most serious health consequence of osteoporosis. The Siglec-15 neutralizing antibody effectively reduced bone resorption and stimulated bone formation in estrogen deficiency-induced osteoporosis. Of note, the Siglec-15 neutralizing antibody promoted intramembranous and endochondral ossification at the damaged area of cortical bone in fracture healing mouse models. Thus, the Siglec-15 neutralizing antibody shows significant translational potential as a novel therapy for OP and bone fracture.