Fibrinogen-like Protein 1 Is a Major Immune Inhibitory Ligand of LAG-3.

Lymphocyte-activation gene 3 (LAG-3) is an immune inhibitory receptor, with major histocompatibility complex class II (MHC-II) as a canonical ligand. However, it remains controversial whether MHC-II is solely responsible for the inhibitory function of LAG-3. Here, we demonstrate that fibrinogen-like protein 1 (FGL1), a liver-secreted protein, is a major LAG-3 functional ligand independent from MHC-II. FGL1 inhibits antigen-specific T cell activation, and ablation of FGL1 in mice promotes T cell immunity. Blockade of the FGL1-LAG-3 interaction by monoclonal antibodies stimulates tumor immunity and is therapeutic against established mouse tumors in a receptor-ligand inter-dependent manner. FGL1 is highly produced by human cancer cells, and elevated FGL1 in the plasma of cancer patients is associated with a poor prognosis and resistance to anti-PD-1/B7-H1 therapy. Our findings reveal an immune evasion mechanism and have implications for the design of cancer immunotherapy.

Immunological impact of cell death signaling driven by radiation on the tumor microenvironment.

Therapeutic irradiation of the tumor microenvironment causes differential activation of pro-survival and pro-death pathways in malignant, stromal, endothelial and immune cells, hence causing a profound cellular and biological reconfiguration via multiple, non-redundant mechanisms. Such mechanisms include the selective elimination of particularly radiosensitive cell types and consequent loss of specific cellular functions, the local release of cytokines and danger signals by dying radiosensitive cells, and altered cytokine secretion by surviving radioresistant cells. Altogether, these processes create chemotactic and immunomodulatory cues for incoming and resident immune cells. Here we discuss how cytoprotective and cytotoxic signaling modules activated by radiation in specific cell populations reshape the immunological tumor microenvironment.

CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity.

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

Cytotoxicity as a form of immunogenic cell death leading to efficient tumor antigen cross-priming.

Antigen cross-priming of CD8+ T cells is a critical process necessary for the effective expansion and activation of CD8+ T cells endowed with the ability to recognize and destroy tumor cells. The cross-presentation of tumor antigens to cross-prime CD8+ T cells is mainly mediated, if not only, by a subset of professional antigen-presenting cells termed type-1 conventional dendritic cells (cDC1). The demise of malignant cells can be immunogenic if it occurs in the context of premortem stress. These ways of dying are termed immunogenic cell death (ICD) and are associated with biochemical features favoring cDC1 for the efficient cross-priming of tumor antigens. Immunosurveillance and the success of immunotherapies heavily rely on the ability of cytotoxic immune cells, primarily CD8+ T cells and NK cells, to detect and eliminate tumor cells through mechanisms collectively known as cytotoxicity. Recent studies have revealed the significance of NK- and CTL-mediated cytotoxicity as a prominent form of immunogenic cell death, resulting in mechanisms that promote and sustain antigen-specific immune responses. This review focuses on the mechanisms underlying the cross-presentation of antigens released during tumor cell killing by cytotoxic immune cells, with an emphasis on the role of cDC1 cells. Indeed, cDC1s are instrumental in the effectiveness of most immunotherapies, underscoring the significance of tumor antigen cross-priming in contexts of immunogenic cell death. The notion of the potent immunogenicity of cell death resulting from NK or cytotoxic T lymphocyte (CTL)-mediated cytotoxicity has far-reaching implications for cancer immunotherapy.

Cancer Immunosurveillance Caught in the Act.

The role of the immune system in the control of cancer formation and progression has been a matter of considerable debate over many years. In this issue of Immunity, Mlecnik et al. (2016) show the importance of immunosurveillance in controlling tumors in a series of microsatellite-instable human colon carcinomas.

Prophylactic TNF blockade uncouples efficacy and toxicity in dual CTLA-4 and PD-1 immunotherapy.

Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1-3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2-/-Il2rg-/- mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.

Antitumor efficacy and reduced toxicity using an anti-CD137 Probody therapeutic.

Costimulation via CD137 (4-1BB) enhances antitumor immunity mediated by cytotoxic T lymphocytes. Anti-CD137 agonist antibodies elicit mild liver inflammation in mice, and the maximum tolerated dose of Urelumab, an anti-human CD137 agonist monoclonal antibody, in the clinic was defined by liver inflammation-related side effects. A protease-activated prodrug form of the anti-mouse CD137 agonist antibody 1D8 (1D8 Probody therapeutic, Pb-Tx) was constructed and found to be selectively activated in the tumor microenvironment. This construct, which encompasses a protease-cleavable linker holding in place a peptide that masks the antigen binding site, exerted antitumor effects comparable to the unmodified antibody but did not result in liver inflammation. Moreover, it efficaciously synergized with both PD-1 blockade and adoptive T-cell therapy. Surprisingly, minimal active Pb-Tx reached tumor-draining lymph nodes, and regional lymphadenectomy did not abrogate antitumor efficacy. By contrast, S1P receptor-dependent recirculation of T cells was absolutely required for efficacy. The preferential cleavage of the anti-CD137 Pb-Tx by tumor proteases offers multiple therapeutic opportunities, including neoadjuvant therapy, as shown by experiments in which the Pb-Tx is given prior to surgery to avoid spontaneous metastases.

The association between the tumor immune microenvironments and clinical outcome in low-grade, early-stage endometrial cancer patients.

Endometrial tumors show substantial heterogeneity in their immune microenvironment. This heterogeneity could be used to improve the accuracy of current outcome prediction tools. We assessed the immune microenvironment of 235 patients diagnosed with low-grade, early-stage endometrial cancer. Multiplex quantitative immunofluorescence was carried out to measure CD8, CD68, FOXP3, PD-1, and PD-L1 markers, as well as cytokeratin (CK), on tissue microarrays. Clustering results revealed five robust immune response patterns, each associated with specific immune populations, cell phenotypes, and cell spatial clustering. Most samples (69%) belonged to the immune-desert subtype, characterized by low immune cell densities. Tumor-infiltrating lymphocyte (TIL)-rich samples (4%) displayed high CD8+ T-cell infiltration, as well as a high percentage of CD8/PD-1+ cells. Immune-exclusion samples (19%) displayed the lowest CD8+ infiltration combined with high PD-L1 expression levels in CK+ tumor cells. In addition, they demonstrated high tumor cell spatial clustering as well as increased spatial proximity of CD8+ /PD-1+ and CK/PD-L1+ cells. FOXP3 and macrophage-rich phenotypes (3% and 4% of total samples) displayed relatively high levels of FOXP3+ regulatory T-cells and CD68+ macrophages, respectively. These phenotypes correlated with clinical outcomes, with immune-exclusion tumors showing an association with tumor relapse. When compared with prediction models built using routine pathological variables, models optimized with immune variables showed increased outcome prediction capacity (AUC = 0.89 versus 0.78) and stratification potential. The improved prediction capacity was independent of mismatch repair protein status and adjuvant radiotherapy treatment. Further, immunofluorescence results could be partially recapitulated using single-marker immunohistochemistry (IHC) performed on whole tissue sections. TIL-rich tumors demonstrated increased CD8+ T-cells by IHC, while immune-exclusion tumors displayed a lack of CD8+ T-cells and frequent expression of PD-L1 in tumor cells. Our results demonstrate the capability of the immune microenvironment to improve standard prediction tools in low-grade, early-stage endometrial carcinomas. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Nivolumab versus sorafenib in advanced hepatocellular carcinoma (CheckMate 459): a randomised, multicentre, open-label, phase 3 trial.

BACKGROUND: Single-agent nivolumab showed durable responses, manageable safety, and promising survival in patients with advanced hepatocellular carcinoma in the phase 1-2 CheckMate 040 study. We aimed to investigate nivolumab monotherapy compared with sorafenib monotherapy in the first-line setting for patients with advanced hepatocellular carcinoma. METHODS: In this randomised, open-label, phase 3 trial done at medical centres across 22 countries and territories in Asia, Australasia, Europe, and North America, patients at least 18 years old with histologically confirmed advanced hepatocellular carcinoma not eligible for, or whose disease had progressed after, surgery or locoregional treatment; with no previous systemic therapy for hepatocellular carcinoma, with Child-Pugh class A and Eastern Cooperative Oncology Group performance status score of 0 or 1, and regardless of viral hepatitis status were randomly assigned (1:1) via an interactive voice response system to receive nivolumab (240 mg intravenously every 2 weeks) or sorafenib (400 mg orally twice daily) until disease progression or unacceptable toxicity. The primary endpoint was overall survival assessed in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of study drug. This completed trial is registered with ClinicalTrials.gov, NCT02576509. FINDINGS: Between Jan 11, 2016, and May 24, 2017, 743 patients were randomly assigned to treatment (nivolumab, n=371; sorafenib, n=372). At the primary analysis, the median follow-up for overall survival was 15·2 months (IQR 5·7-28·0) for the nivolumab group and 13·4 months (5·7-25·9) in the sorafenib group. Median overall survival was 16·4 months (95% CI 13·9-18·4) with nivolumab and 14·7 months (11·9-17·2) with sorafenib (hazard ratio 0·85 [95% CI 0·72-1·02]; p=0·075; minimum follow-up 22·8 months); the protocol-defined significance level of p=0·0419 was not reached. The most common grade 3 or worse treatment-related adverse events were palmar-plantar erythrodysaesthesia (1 [<1%] of 367 patients in the nivolumab group vs 52 [14%] of patients in the sorafenib group), aspartate aminotransferase increase (22 [6%] vs 13 [4%]), and hypertension (0 vs 26 [7%]). Serious treatment-related adverse events were reported in 43 (12%) patients receiving nivolumab and 39 (11%) patients receiving sorafenib. Four deaths in the nivolumab group and one death in the sorafenib group were assessed as treatment related. INTERPRETATION: First-line nivolumab treatment did not significantly improve overall survival compared with sorafenib, but clinical activity and a favourable safety profile were observed in patients with advanced hepatocellular carcinoma. Thus, nivolumab might be considered a therapeutic option for patients in whom tyrosine kinase inhibitors and antiangiogenic drugs are contraindicated or have substantial risks. FUNDING: Bristol Myers Squibb in collaboration with Ono Pharmaceutical.

Differential Interleukin-8 thresholds for chemotaxis and netosis in human neutrophils.

In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.

Perspectives in Immunotherapy: meeting report from the Immunotherapy Bridge, December 1st-2nd, 2021.

Over the past decade, immunotherapy has become an increasingly fundamental modality in the treatment of cancer. The positive impact of immune checkpoint inhibition, especially anti-programmed death (PD)-1/PD-ligand (L)1 blockade, in patients with different cancers has focused attention on the potential for other immunotherapeutic approaches. These include inhibitors of additional immune checkpoints, adoptive cell transfer (ACT), and therapeutic vaccines. Patients with advanced cancers who previously had limited treatment options available may now benefit from immunotherapies that can offer durable responses and improved survival outcomes. However, despite this, a significant proportion of patients fail to respond to immunotherapy, especially those with less immunoresponsive cancer types, and there remains a need for new treatment strategies.The virtual Immunotherapy Bridge (December 1st-2nd, 2021), organized by the Fondazione Melanoma Onlus, Naples, Italy in collaboration with the Society for Immunotherapy of Cancer addressed several areas of current research in immunotherapy, including lessons learned from cell therapies, drivers of immune response, and trends in immunotherapy across different cancers, and these are summarised here.

Heterogenous presence of neutrophil extracellular traps in human solid tumours is partially dependent on IL-8.

Neutrophil extracellular traps (NETs) are webs of extracellular nuclear DNA extruded by dying neutrophils infiltrating tissue. NETs constitute a defence mechanism to entrap and kill fungi and bacteria. Tumours induce the formation of NETs to the advantage of the malignancy via a variety of mechanisms shown in mouse models. Here, we investigated the presence of NETs in a variety of human solid tumours and their association with IL-8 (CXCL8) protein expression and CD8+ T-cell density in the tumour microenvironment. Multiplex immunofluorescence panels were developed to identify NETs in human cancer tissues by co-staining with the granulocyte marker CD15, the neutrophil marker myeloperoxidase and citrullinated histone H3 (H3Cit), as well as IL-8 protein and CD8+ T cells. Three ELISA methods to detect and quantify circulating NETs in serum were optimised and utilised. Whole tumour sections and tissue microarrays from patients with non-small cell lung cancer (NSCLC; n = 14), bladder cancer (n = 14), melanoma (n = 11), breast cancer (n = 31), colorectal cancer (n = 20) and mesothelioma (n = 61) were studied. Also, serum samples collected retrospectively from patients with metastatic melanoma (n = 12) and NSCLC (n = 34) were ELISA assayed to quantify circulating NETs and IL-8. NETs were detected in six different human cancer types with wide individual variation in terms of tissue density and distribution. At least in NSCLC, bladder cancer and metastatic melanoma, NET density positively correlated with IL-8 protein expression and inversely correlated with CD8+ T-cell densities. In a series of serum samples from melanoma and NSCLC patients, a positive correlation between circulating NETs and IL-8 was found. In conclusion, NETs are detectable in formalin-fixed human biopsy samples from solid tumours and in the circulation of cancer patients with a considerable degree of individual variation. NETs show a positive association with IL-8 and a trend towards a negative association with CD8+ tumour-infiltrating lymphocytes. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

CheckMate 040 cohort 5: A phase I/II study of nivolumab in patients with advanced hepatocellular carcinoma and Child-Pugh B cirrhosis.

BACKGROUND & AIMS: Patients with advanced hepatocellular carcinoma (aHCC) and Child-Pugh B liver function are often excluded from clinical trials. In previous studies, overall survival for these patients treated with sorafenib was ∼3-5 months; thus, new treatments are needed. Nivolumab, alone or in combination with ipilimumab, is conditionally approved in the United States to treat patients with aHCC who previously received sorafenib. We describe nivolumab monotherapy outcomes in patients with Child-Pugh B status. METHODS: This phase I/II, open-label, non-comparative, multicentre trial (27 centres) included patients with Child-Pugh B (B7-B8) aHCC. Patients received intravenous nivolumab 240 mg every 2 weeks until unacceptable toxicity or disease progression. Primary endpoints were objective response rate (ORR) by investigator assessment (using Response Evaluation Criteria in Solid Tumors v1.1) and duration of response. Safety was assessed using National Cancer Institute Common Terminology Criteria for Adverse Events v4.0. RESULTS: Twenty-five sorafenib-naive and 24 sorafenib-treated patients began treatment between November 2016 and October 2017 (median follow-up, 16.3 months). Investigator-assessed ORR was 12% (95% CI 5-25%) with 6 patients responding; disease control rate was 55% (95% CI 40-69%). Median time to response was 2.7 months (interquartile range, 1.4-4.2), and median duration of response was 9.9 months (95% CI 9.7-9.9). Treatment-related adverse events (TRAEs) were reported in 25 patients (51%) and led to discontinuation in 2 patients (4%). The most frequent grade 3/4 TRAEs were hypertransaminasemia (n = 2), amylase increase (n = 2), and aspartate aminotransferase increase (n = 2). The safety of nivolumab was comparable to that in patients with Child-Pugh A aHCC. CONCLUSIONS: Nivolumab showed clinical activity and favourable safety with manageable toxicities, suggesting it could be suitable for patients with Child-Pugh B aHCC. LAY SUMMARY: In patients with advanced hepatocellular carcinoma, almost all systemic therapies require very good liver function, i.e. Child-Pugh A status. The evidence from this study suggests that nivolumab shows clinical activity and an acceptable safety profile in patients with hepatocellular carcinoma with Child-Pugh B status who have mild to moderate impairment of liver function or liver decompensation that might rule out other therapies. Further studies are warranted to assess the safety and efficacy of nivolumab in this patient population. CLINICAL TRIAL NUMBER: NCT01658878.

Immunological Mechanisms Responsible for Radiation-Induced Abscopal Effect.

Radiotherapy has been used for more than a hundred years as a local tumor treatment. The occurrence of systemic antitumor effects manifesting as regression of tumors outside of the irradiated field (abscopal effect) was occasionally observed but deemed too rare and unpredictable to be a therapeutic goal. This has changed with the advent of immunotherapy. Remarkable systemic effects have been observed in patients receiving radiotherapy to control tumors that were progressing during immune checkpoint blockade, stimulating interest in using radiation to overcome primary and acquired cancer resistance to immunotherapy. Here, we review the immunological mechanisms that are responsible for the ability of focal radiation to promote antitumor T cell responses that mediate tumor rejection and, in some cases, result in systemic effects.

Cancer immunotherapy resistance based on immune checkpoints inhibitors: Targets, biomarkers, and remedies.

Cancer is one of the main public health problems in the world. Systemic therapies such as chemotherapy and more recently target therapies as well as immunotherapy have improved the prognosis of this large group of complex malignant diseases. However, the frequent emergence of multidrug resistance (MDR) mechanisms is one of the major impediments towards curative treatment of cancer. While several mechanisms of drug chemoresistance are well defined, resistance to immunotherapy is still insufficiently unclear due to the complexity of the immune response and its dependence on the host. Expression and regulation of immune checkpoint molecules (such as PD-1, CD279; PD-L1, CD274; and CTLA-4, CD152) play a key role in the response to immunotherapy. In this regard, immunotherapy based on immune checkpoints inhibitors (ICIs) is a common clinical approach for treatment of patients with poor prognosis when other first-line therapies have failed. Unfortunately, about 70 % of patients are classified as non-responders, or they progress after initial response to these ICIs. Multiple factors can be related to immunotherapy resistance: characteristics of the tumor microenvironment (TME); presence of tumor infiltrating lymphocytes (TILs), such as CD8 + T cells associated with treatment-response; presence of tumor associated macrophages (TAMs); activation of certain regulators (like PIK3γ or PAX4) found present in non-responders; a low percentage of PD-L1 expressing cells; tumor mutational burden (TMB); gain or loss of antigen-presenting molecules; genetic and epigenetic alterations correlated with resistance. This review provides an update on the current state of immunotherapy resistance presenting targets, biomarkers and remedies to overcome such resistance.

Association of inflammatory biomarkers with clinical outcomes in nivolumab-treated patients with advanced hepatocellular carcinoma.

BACKGROUND & AIMS: Nivolumab, a programmed death (PD)-1 (PD-1) inhibitor, led to durable responses, manageable safety, and increased survival in patients with advanced hepatocellular carcinoma (HCC). In our retrospective analysis, we studied the immunobiology and potential associations between biomarkers and outcomes with nivolumab in HCC. METHODS: Fresh and archival tumour samples from dose-escalation and dose-expansion phases of the CheckMate 040 trial were analysed by immunohistochemistry and RNA sequencing to assess several inflammatory gene expression signatures, including CD274 (PD-ligand 1 [PD-L1]), CD8A, LAG3, and STAT1. Biomarkers were assessed for association with clinical outcomes (best overall response by blinded independent central review per RECIST v1.1 and overall survival [OS]). RESULTS: Complete or partial tumour responses were observed in PD-L1-positive and PD-L1-negative patients treated with nivolumab monotherapy. Median OS was 28.1 (95% CI 18.2-n.a.) vs. 16.6 months (95% CI 14.2-20.2) for patients with tumour PD-L1 ≥1% vs. <1% (p = 0.03). Increased CD3 and CD8 showed a non-significant trend towards improved OS (both p = 0.08), and macrophage markers were not associated with OS. Tumour PD-1 and PD-L1 expression were associated with improved OS (p = 0.05 and p = 0.03, respectively). An inflammatory gene signature consisting of 4 genes was associated with improved objective response rate (p = 0.05) and OS (p = 0.01). CONCLUSIONS: PD-1 and PD-L1 expression, biomarkers of inflammation, and inflammatory gene signatures trended with improved survival and response. While further confirmation within a larger phase III trial is needed to evaluate predictive value of these biomarkers, these exploratory analyses suggest that anti-tumour immune response may play a role in the treatment benefit of nivolumab in HCC. LAY SUMMARY: Certain tests may be used to provide a picture of how a tumour is escaping the immune system, allowing it to continue to grow and create more tumours. Therapies such as nivolumab are designed to help the immune system fight the tumour. These tests may be used to determine how effective such therapies will be in the treatment of advanced liver cancer. NCT NUMBER: NCT01658878.

Prognostic value of macrophage polarization markers in epithelial neoplasms and melanoma. A systematic review and meta-analysis.

Macrophage polarization is relevant for tumor biology. M2 polarized macrophages favor tumor growth and survival, while M1 macrophages support tumor destruction and antigen presentation. Markers identifying M1/M2 polarization are a subject of debate. We conducted a systematic review and meta-analysis to investigate the association of proposed macrophage markers with prognosis across epithelial tumors and melanoma. The Medline search engine was used and 195 articles were recovered for full review. Only articles which measured markers using immunohistochemistry or immunofluorescence and had overall survival (OS) as the primary endpoint were included. One hundred and thirteen articles were finally accepted for analysis. CD68 was associated with worse survival across tumors (hazard ratio (HR) = 1.24, 95% CI = 1.11-1.37). Tumor anatomical location influenced this association. Colorectal tumors showed an inverse association between CD68 and OS in contrast to the rest of cancer types (HR = 0.56 vs. 1.34). The approach taken to measure CD68 had an impact on prognosis; when macrophages were measured at the tumor invasion front prognosis was more favorable than when they were measured intratumorally (HR = 0.94 vs. 1.4). CD163, CD204, and CD206 showed a robust association with worse OS (HR = 1.63, 1.95, 1.65, respectively). Tumors arising in the lung and the liver showed a weaker association between CD163 and OS as compared with other locations (ß = -0.5401 for the lung and -0.5940 for the liver compared with other anatomical locations). The counting strategy also had an impact on CD163 association with OS, with hot-spot counting having higher HRs compared with averaging macrophage counts across spots or absolute cell counting (ß = -0.4678). In conclusion, proposed M2 markers are associated with worse survival across epithelial tumors and melanoma. The anatomical origin of tumors influences this association. The compartment where the macrophages were scored and counting strategy influenced the association with OS of CD68 and CD163, respectively.

Diverse immune environments in human lung tuberculosis granulomas assessed by quantitative multiplexed immunofluorescence.

The precise nature of the local immune responses in lung tuberculosis (TB) granulomas requires a comprehensive understanding of their environmental complexities. At its most basic level, a granuloma is a compact, organized immune aggregate of macrophages surrounded by myeloid, B and T cells. We established two complementary multiplex immunolabeling panels to simultaneously evaluate the myeloid and lymphocytic contexture of 14 human lung TB granulomas in formalin-fixed paraffin-embedded tissue samples. We observed diverse CD3+ and CD8+ T-cell and CD20+ B lymphocyte compositions of the granuloma immune environment and a relatively homogeneous distribution of all myeloid cells. We also found significant associations between CD8+ T-cell densities and the myeloid marker CD11b and phagocytic cell marker CD68. In addition, significantly more CD68+ macrophages and CD8+ T cells were found in Mycobacterium tuberculosis-infected granulomas, as detected by Ziehl-Neelsen staining. FOXP3 expression was predominately found in a small subset of CD4+ T cells in different granulomas. As the success or failure of each granuloma is determined by the immune response within that granuloma at a local and not a systemic level, we attempted to identify the presence of reactive T cells based on expression of the T-cell activation marker CD137 (4-1BB) and programmed cell death-1 (PD-1). Only a small fraction of the CD4+ and CD8+ T cells expressed PD-1. CD137 expression was found only in a very small fraction of the CD4+ T cells in two granulomas. Our results also showed that multinucleated giant cells showed strong PD-L1 but not CTLA-4 membrane staining. This study offers new insights into the heterogeneity of immune cell infiltration in lung TB granulomas, suggesting that each TB granuloma represents a unique immune environment that might be independently influenced by the local adaptive immune response, bacterial state, and overall host disease status.

Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies.

4-1BB (CD137, tumor necrosis factor receptor superfamily 9) is an inducible costimulatory receptor expressed on activated T and natural killer (NK) cells. 4-1BB ligation on T cells triggers a signaling cascade that results in upregulation of antiapoptotic molecules, cytokine secretion, and enhanced effector function. In dysfunctional T cells that have a decreased cytotoxic capacity, 4-1BB ligation demonstrates a potent ability to restore effector functions. On NK cells, 4-1BB signaling can increase antibody-dependent cell-mediated cytotoxicity. Agonistic monoclonal antibodies targeting 4-1BB have been developed to harness 4-1BB signaling for cancer immunotherapy. Preclinical results in a variety of induced and spontaneous tumor models suggest that targeting 4-1BB with agonist antibodies can lead to tumor clearance and durable antitumor immunity. Clinical trials of 2 agonist antibodies, urelumab and utomilumab, are ongoing. Despite initial signs of efficacy, clinical development of urelumab has been hampered by inflammatory liver toxicity at doses >1 mg/kg. Utomilumab has a superior safety profile, but is a less potent 4-1BB agonist relative to urelumab. Both antibodies have demonstrated promising results in patients with lymphoma and are being tested in combination therapy trials with other immunomodulatory agents. In an effort to optimally leverage 4-1BB-mediated immune activation, the next generation of 4-1BB targeting strategies attempts to decouple the observed antitumor efficacy from the on-target liver toxicity. Multiple therapeutics that attempt to restrict 4-1BB agonism to the tumor microenvironment and minimize systemic exposure have emerged. 4-1BB is a compelling target for cancer immunotherapy and future agents show great promise for achieving potent immune activation while avoiding limiting immune-related adverse events.

Short-Term Local Expression of a PD-L1 Blocking Antibody from a Self-Replicating RNA Vector Induces Potent Antitumor Responses.

Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFV-aPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment.