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Liver regeneration is under metabolic and immune regulation. Despite increasing recognition of the involvement of neutrophils in regeneration, it is unclear how the liver signals to the bone marrow to release neutrophils after injury and how reparative neutrophils signal to hepatocytes to reenter the cell cycle. Here we report that loss of the liver tumour suppressor Lifr in mouse hepatocytes impairs, whereas overexpression of leukaemia inhibitory factor receptor (LIFR) promotes liver repair and regeneration after partial hepatectomy or toxic injury. In response to physical or chemical damage to the liver, LIFR from hepatocytes promotes the secretion of cholesterol and CXCL1 in a STAT3-dependent manner, leading to the efflux of bone marrow neutrophils to the circulation and damaged liver. Cholesterol, via its receptor ERRα, stimulates neutrophils to secrete hepatocyte growth factor to accelerate hepatocyte proliferation. Altogether, our findings reveal a LIFR-STAT3-CXCL1-CXCR2 axis and a LIFR-STAT3-cholesterol-ERRα-hepatocyte growth factor axis that form bidirectional hepatocyte-neutrophil cross-talk to repair and regenerate the liver.

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Introduction: Combination immunotherapy, exemplified by atezolizumab plus bevacizumab, has become the standard of care for inoperable hepatocellular carcinoma (HCC). However, the lack of predictive biomarkers and limited understanding of response mechanisms remain a challenge. Methods: Using data from the IMbrave150plus cohort, we applied an immune signature score (ISS) predictor to stratify HCC patients treated with atezolizumab plus bevacizumab or with sorafenib alone into potential high and low response groups. By applying multiple statistical approaches including a Bayesian covariate prediction algorithm, we refined the signature to 10 key genes (ISS10) for clinical use while maintaining similar predictive power to the full model. We further validated ISS10 in an independent HCC cohort treated with nivolumab plus ipilimumab. Results: The study identified a significant association between the ISS and treatment response. Among patients classified as high responders, those treated with the atezolizumab plus bevacizumab combination exhibited improved overall and progression-free survival as well as better objective response rate compared to those treated with sorafenib. We also observed a significant correlation between ISS10 and response to nivolumab plus ipilimumab treatment. Analysis of immune cell subpopulations revealed distinct characteristics associated with ISS subtypes. In particular, the ISS10 high subtype displayed a more favorable immune environment with higher proportions of anti-tumor macrophages and activated T-cells, potentially explaining its better response. Conclusions: Our study suggests that ISS and ISS10 are promising predictive biomarkers for enhanced therapeutic outcomes in HCC patients undergoing combination immunotherapy. These markers are crucial for refining patient stratification and personalized treatment approaches to advance the effectiveness of standard-of-care regimens.

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STING agonists can reprogram the tumor microenvironment to induce immunological clearance within the central nervous system. Using multiplexed sequential immunofluorescence (SeqIF) and the Ivy Glioblastoma Atlas, STING expression was found in myeloid populations and in the perivascular space. The STING agonist 8803 increased median survival in multiple preclinical models of glioblastoma, including QPP8, an immune checkpoint blockade-resistant model, where 100% of mice were cured. Ex vivo flow cytometry profiling during the therapeutic window demonstrated increases in myeloid tumor trafficking and activation, alongside enhancement of CD8+ T cell and NK effector responses. Treatment with 8803 reprogrammed microglia to express costimulatory CD80/CD86 and iNOS, while decreasing immunosuppressive CD206 and arginase. In humanized mice, where tumor cell STING is epigenetically silenced, 8803 therapeutic activity was maintained, further attesting to myeloid dependency and reprogramming. Although the combination with a STAT3 inhibitor did not further enhance STING agonist activity, the addition of anti-PD-1 antibodies to 8803 treatment enhanced survival in an immune checkpoint blockade-responsive glioma model. In summary, 8803 as a monotherapy demonstrates marked in vivo therapeutic activity, meriting consideration for clinical translation.

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Lack of robust activation of Stimulator of Interferon Genes (STING) pathway and subsequent induction of type I IFN responses is considered a barrier to antitumor immunity in acute myeloid leukemia (AML). Using common human AML cell lines as in vitro tools to evaluate the efficacy of novel STING agonists, we found most AML lines to be poor producers of IFNs upon exposure to extremely potent agonists, suggesting cell-intrinsic suppression of STING signaling may occur. We observed unexpected patterns of response that did not correlate with levels of STING pathway components or of known enzymes associated with resistance. To identify a genetic basis for these observations, we cloned and sequenced STING from the cDNA of human AML cell lines and found both frequent mutations and deviations from normal RNA splicing. We identified two novel spliced isoforms of STING in these lines and validated their expression in primary human AML samples. When transduced into reporter cells, these novel STING isoforms exhibited complete insensitivity to agonist stimulation. These observations identify alternative splicing as a mechanism of STING pathway suppression and suggest that most AML silences the STING pathway through direct modification rather than through engagement of external inhibitory factors. SIGNIFICANCE: We find that AML acquires resistance to innate immune activation via the STING pathway through aberrant splicing of the STING transcript including two novel forms described herein that act as dominant negatives. These data broaden understanding of how cancers evolve STING resistance, and suggest that the AML tumor microenvironment, not the cancer cell, should be the target of therapeutic interventions to activate STING.

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We previously showed that ablation of tumor hypoxia can sensitize tumors to immune checkpoint blockade (ICB). Here, we used a Kras+/G12D TP53+/R172H Pdx1-Cre-derived (KPC-derived) model of pancreatic adenocarcinoma to examine the tumor response and adaptive resistance mechanisms involved in response to 2 established methods of hypoxia-reducing therapy: the hypoxia-activated prodrug TH-302 and vascular endothelial growth factor receptor 2 (VEGFR-2) blockade. The combination of both modalities normalized tumor vasculature, increased DNA damage and cell death, and delayed tumor growth. In contrast with prior cancer models, the combination did not alleviate overall tissue hypoxia or sensitize these KPC tumors to ICB therapy despite qualitative improvements to the CD8+ T cell response. Bulk tumor RNA sequencing, flow cytometry, and adoptive myeloid cell transfer suggested that treated tumor cells increased their capacity to recruit granulocytic myeloid-derived suppressor cells (G-MDSCs) through CCL9 secretion. Blockade of the CCL9/CCR1 axis could limit G-MDSC migration, and depletion of Ly6G-positive cells could sensitize tumors to the combination of TH-302, anti-VEGFR-2, and ICB. Together, these data suggest that pancreatic tumors modulate G-MDSC migration as an adaptive response to vascular normalization and that these immunosuppressive myeloid cells act in a setting of persistent hypoxia to maintain adaptive immune resistance.

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Immunotherapy, in the form of hematopoietic stem cell transplantation (HSCT), has been part of the standard of care in the treatment of acute leukemia for over 40 years. Trials evaluating novel immunotherapeutic approaches, such as targeting the programmed death-1 (PD-1) pathway, have unfortunately not yielded comparable results to those seen in solid tumors. Major histocompatibility complex (MHC) proteins are cell surface proteins essential for the adaptive immune system to recognize self versus non-self. MHC typing is used to determine donor compatibility when evaluating patients for HSCT. Recently, loss of MHC class II (MHC II) was shown to be a mechanism of immune escape in patients with acute myeloid leukemia after HSCT. Here we report that treatment with the tyrosine kinase inhibitor, dasatinib, and an anti-PD-1 antibody in preclinical models of Philadelphia chromosome positive B-cell acute lymphoblastic leukemia is highly active. The dasatinib and anti-PD-1 combination reduces tumor burden, is efficacious, and extends survival. Mechanistically, we found that treatment with dasatinib significantly increased MHC II expression on the surface of antigen-presenting cells (APC) in a tumor microenvironment-independent fashion and caused influx of APC cells into the leukemic bone marrow. Finally, the induction of MHC II may potentiate immune memory by impairing leukemic engraftment in mice previously cured with dasatinib, after re-inoculation of leukemia cells. In summary, our data suggests that anti-PD-1 therapy may enhance the killing ability of dasatinib via dasatinib driven APC growth and expansion and upregulation of MHC II expression, leading to antileukemic immune rewiring.

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Obesity, in which the functional importance of small nucleolar RNAs (snoRNAs) remains elusive, correlates with risk for many cancer types. Here, we identify that the serum copies of adipocyte-expressed SNORD46 correlate with body mass index (BMI), and serum SNORD46 antagonizes interleukin-15 (IL-15) signaling. Mechanically, SNORD46 binds IL-15 via G11, and G11A (a mutation that significantly enhances binding affinity) knockin drives obesity in mice. Functionally, SNORD46 blocks IL-15-induced, FER kinase-dependent phosphorylation of platelet glycoprotein 4 (CD36) and monoglyceride lipase (MGLL) in adipocytes, leading to inhibited lipolysis and browning. In natural killer (NK) cells, SNORD46 suppresses the IL-15-dependent autophagy, leading to reduced viability of obese NK. SNORD46 power inhibitors exhibit anti-obesity effects, concurring with improved viability of obese NK and anti-tumor immunity of CAR-NK cell therapy. Hence, our findings demonstrate the functional importance of snoRNAs in obesity and the utility of snoRNA power inhibitors for antagonizing obesity-associated immune resistance.

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One of the major obstacles to treating pancreatic ductal adenocarcinoma (PDAC) is its immunoresistant microenvironment. The functional importance and molecular mechanisms of Schwann cells in PDAC remains largely elusive. We characterized the gene signature of tumor-associated nonmyelinating Schwann cells (TASc) in PDAC and indicated that the abundance of TASc was correlated with immune suppressive tumor microenvironment and the unfavorable outcome of patients with PDAC. Depletion of pancreatic-specific TASc promoted the tumorigenesis of PDAC tumors. TASc-expressed long noncoding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) was triggered by the tumor cell-produced interleukin-6. Mechanistically, PVT1 modulated RAF proto-oncogene serine/threonine protein kinase-mediated phosphorylation of tryptophan 2,3-dioxygenase in TASc, facilitating its enzymatic activities in catalysis of tryptophan to kynurenine. Depletion of TASc-expressed PVT1 suppressed PDAC tumor growth. Furthermore, depletion of TASc using a small-molecule inhibitor effectively sensitized PDAC to immunotherapy, signifying the important roles of TASc in PDAC immune resistance.

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BACKGROUND: The lack of murine glioblastoma models that mimic the immunobiology of human disease has impeded basic and translational immunology research. We, therefore, developed murine glioblastoma stem cell lines derived from Nestin-CreERT2QkL/L; Trp53L/L; PtenL/L (QPP) mice driven by clinically relevant genetic mutations common in human glioblastoma. This study aims to determine the immune sensitivities of these QPP lines in immunocompetent hosts and their underlying mechanisms. METHODS: The differential responsiveness of QPP lines was assessed in the brain and flank in untreated, anti-PD-1, or anti-CTLA-4 treated mice. The impact of genomic landscape on the responsiveness of each tumor was measured through whole exome sequencing. The immune microenvironments of sensitive (QPP7) versus resistant (QPP8) lines were compared in the brain using flow cytometry. Drivers of flank sensitivity versus brain resistance were also measured for QPP8. RESULTS: QPP lines are syngeneic to C57BL/6J mice and demonstrate varied sensitivities to T cell immune checkpoint blockade ranging from curative responses to complete resistance. Infiltrating tumor immune analysis of QPP8 reveals improved T cell fitness and augmented effector-to-suppressor ratios when implanted subcutaneously (sensitive), which are absent on implantation in the brain (resistant). Upregulation of PD-L1 across the myeloid stroma acts to establish this state of immune privilege in the brain. In contrast, QPP7 responds to checkpoint immunotherapy even in the brain likely resulting from its elevated neoantigen burden. CONCLUSIONS: These syngeneic QPP models of glioblastoma demonstrate clinically relevant profiles of immunotherapeutic sensitivity and potential utility for both mechanistic discovery and evaluation of immune therapies.

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Median survival of patients with glioblastoma (GBM) treated with standard of care which consists of maximal safe resection of the contrast-enhancing portion of the tumor followed by radiation therapy with concomitant adjuvant temozolomide (TMZ) remains 15 months. The tumor microenvironment (TME) is known to contain immune suppressive myeloid cells with minimal effector T cell infiltration. Stimulator of interferon genes (STING) is an important activator of immune response and results in production of Type 1 interferon and antigen presentation by myeloid cells. This review will discuss important developments in STING agonists, potential biomarkers for STING response, and new combinatorial therapeutic approaches in gliomas.

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Immune checkpoint inhibitors (ICIs) have been increasingly used in combination for cancer treatment but are associated with myocarditis. Here, we report that tumor-bearing mice exhibited response to treatment with combinatorial anti-programmed cell death 1 and anti-cytotoxic T lymphocyte antigen-4 antibodies but also presented with cardiovascular toxicities observed clinically with ICI therapy, including myocarditis and arrhythmia. Female mice were preferentially affected with myocarditis compared to male mice, consistent with a previously described genetic model of ICI myocarditis and emerging clinical data. Mechanistically, myocardial tissue from ICI-treated mice, the genetic mouse model, and human heart tissue from affected patients with ICI myocarditis all exhibited down-regulation of MANF (mesencephalic astrocyte-derived neurotrophic factor) and HSPA5 (heat shock 70-kDa protein 5) in the heart; this down-regulation was particularly notable in female mice. ICI myocarditis was amplified by heart-specific genetic deletion of mouse Manf and was attenuated by administration of recombinant MANF protein, suggesting a causal role. Ironically, both MANF and HSPA5 were transcriptionally induced by liganded estrogen receptor ß and inhibited by androgen receptor. However, ICI treatment reduced serum estradiol concentration to a greater extent in female compared to male mice. Treatment with an estrogen receptor ß-specific agonist and androgen depletion therapy attenuated ICI-associated cardiac effects. Together, our data suggest that ICI treatment inhibits estradiol-dependent expression of MANF/HSPA5 in the heart, curtailing the cardiomyocyte response to immune injury. This endocrine-cardiac-immune pathway offers new insights into the mechanisms of sex differences in cardiac disease and may offer treatment strategies for ICI myocarditis.

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CD47-SIRPα interaction acts as a "don't eat me" signal and is exploited by cancer to downregulate innate and adaptive immune surveillance. There has been intense interest to develop a mechanism of blockade, and we aimed to analyze the emerging data from early clinical trials. We performed a systematic review and meta-analysis of relevant databases and conference abstracts including clinical trials using CD47 and/or SIRPα inhibitors in cancer treatment. Nonlinear mixed models were applied for comparison of response and toxicity. We retrieved 317 articles, 24 of which were eligible. These included 771 response-evaluable patients with hematologic (47.1%) and solid tumors (52.9%). Of these, 6.4% experienced complete response, 10.4% partial response, and 26.1% stable disease for a 16.7% objective response rate (ORR), 42.8% disease control rate, and 4.8-month median duration of response. ORR was significantly higher for hematologic cancers (25.3%) than solid cancers (9.1%, p=0.042). Comparing by mechanism, seven CD47 monoclonal antibodies (mAbs) and six selective SIRPα blockers were given alone or combined with checkpoint inhibitors, targeted therapy, and/or chemotherapy. In solid cancers, selective SIRPα blockade showed a higher ORR (16.2%) than anti-CD47 mAbs (2.8%, p=0.079), which was significant for combination therapies (ORR 28.3% vs 3.0%, respectively, p=0.010). Responses were seen in head and neck, colorectal, endometrial, ovarian, hepatocellular, non-small cell lung, and HER2+gastroesophageal cancers. Dose-limiting toxicity (DLT) was seen in 3.3% of patients (5.4% anti-CD47 mAbs, 1.4% selective SIRPα blockers; p=0.01). The frequency of treatment-related adverse events (TRAEs) ≥grade 3 was 18.0%, similar between the two groups (p=0.082), and mostly laboratory abnormalities. For anti-CD47 mAbs, the most common toxicities included grade 1-2 fatigue (27.2%), headache (21.0%), and anemia (20.5%). For selective SIRPα blockers, these included grade 1-2 infusion reaction (23.1%) and fatigue (15.8%). Anti-CD47 mAbs were significantly more likely than selective SIRPα blockers to cause grade 1-2 fever, chills, nausea/vomiting, headache, and anemia. In conclusion, combination therapies using selective SIRPα blockade had higher response rates in solid tumors than anti-CD47 mAb combinations. Hematologic changes were the main TRAEs, and selective SIRPα blockers seemed to have a better grade 1-2 toxicity profile. Treatment was well-tolerated with minimal DLTs.

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Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited significantly elevated postirradiation CD47 levels. The combination of RT, anti-SIRPα, and anti-PD-1 reversed adaptive immune resistance and drove efficient TAA cross-presentation, resulting in robust TAA-specific CD8 T cell priming, functional activation of T effectors, and increased T cell clonality and clonal diversity. We observed significantly higher complete response rates to RT/anti-SIRPα/anti-PD-1 in both irradiated and abscopal tumors and prolonged survival in three distinct murine CRC models, including a cecal orthotopic model. The efficacy of triple combination therapy was STING dependent as knockout animals lost most benefit of adding anti-SIRPα and anti-PD-1 to RT. Despite activation across the myeloid stroma, the enhanced dendritic cell function accounts for most improvements in CD8 T cell priming. These data suggest ATR-mediated CD47 and PD-L1 up-regulation as a key mechanism restraining radiation-induced immune priming. RT combined with SIRPα and PD-1 blockade promotes robust antitumor immune priming, leading to systemic tumor regressions.

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Therapeutic monoclonal antibodies directed against PD-L1 (e.g., atezolizumab) disrupt PD-L1:PD-1 signaling and reactivate exhausted cytotoxic T-cells in the tumor compartment. Although anti-PD-L1 antibodies are successful as immune checkpoint inhibitor (ICI) therapeutics, there is still a pressing need to develop high-affinity, low-molecular-weight ligands for molecular imaging and diagnostic applications. Affibodies are small polypeptides (∼60 amino acids) that provide a stable molecular scaffold from which to evolve high-affinity ligands. Despite its proven utility in the development of imaging probes, this scaffold has never been optimized for use in mRNA display, a powerful in vitro selection platform incorporating high library diversity, unnatural amino acids, and chemical modification. In this manuscript, we describe the selection of a PD-L1-binding affibody by mRNA display. Following randomization of the 13 amino acids that define the binding interface of the well-described Her2 affibody, the resulting library was selected against recombinant human PD-L1 (hPD-L1). After four rounds, the enriched library was split and selected against either hPD-L1 or the mouse ortholog (mPD-L1). The dual target selection resulted in the identification of a human/mouse cross-reactive PD-L1 affibody (M1) with low nanomolar affinity for both targets. The M1 affibody bound with similar affinity to mPD-L1 and hPD-L1 expressed on the cell surface and inhibited signaling through the PD-L1:PD-1 axis at low micromolar concentrations in a cell-based functional assay. In vivo optical imaging with M1-Cy5 in an immune-competent mouse model of lymphoma revealed significant tumor uptake relative to a Cy5-conjugated Her2 affibody.

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BACKGROUND: The combination of ISA101, a human papilloma virus (HPV) 16 peptide vaccine, and nivolumab showed a promising response rate of 33% in patients with incurable HPV-16+ cancer. Here we report long-term clinical outcomes and immune correlates of response. METHODS: Patients with advanced HPV-16+ cancer and less than two prior regimens for recurrence were enrolled to receive ISA101 (100 µg/peptide) on days 1, 22, and 50 and nivolumab 3 mg/kg every 2 weeks beginning day 8 for up to 1 year. Baseline tumor samples were stained with multiplex immunofluorescence for programmed death-ligand 1 (PD-L1), programmed cell death protein-1 (PD-1), CD3, CD8, CD68, and pan-cytokeratin in a single panel and scanned with the Vectra 3.0 multispectral microscope. Whole transcriptome analysis of baseline tumors was performed with Affymetrix Clariom D arrays. Differential gene expression analysis was performed on responders versus non-responders. RESULTS: Twenty-four patients were followed for a median of 46.5 months (95% CI, 46.0 months to not reached (NR)). The median duration of response was 11.2 months (95% CI, 8.51 months to NR); three out of eight (38%) patients with objective response were without progression at 3 years. The median and 3-year overall survival were 15.3 months (95% CI, 10.6 months to 27.2 months) and 12.5% (95% CI, 4.3% to 36%), respectively. The scores for activated T cells ((CD3+PD-1+)+(CD3+CD8+PD-1+)), activated cytotoxic T cells (CD3+CD8+PD-1+), and total macrophage ((CD68+PD-L1-)+(CD68+PD-L1+)) in tumor were directly correlated with clinical response (p<0.05) and depth of response with the two complete response patients having the highest degree of CD8+ T cells. Gene expression analysis revealed differential regulation of 357 genes (≥1.25 fold) in non-responders versus responders (p<0.05). Higher expression of immune response, inflammatory response and interferon-signaling pathway genes were correlated with clinical response (p<0.05). CONCLUSIONS: Efficacy of ISA101 and nivolumab remains promising in long-term follow-up. Increased infiltration by PD-1+ T cells and macrophages was predictive of response. Enrichment in gene sets associated with interferon-γ response and immune infiltration strongly predicted response to therapy. A randomized trial is ongoing to test this strategy and to further explore correlates of immune response with combined nivolumab and ISA101, versus nivolumab alone. TRIAL REGISTRATION NUMBER: NCT02426892.

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In order to fuel their relentless expansion, cancers must expand their vasculature to augment delivery of oxygen and essential nutrients. The disordered web of irregular vessels that results, however, leaves gaps in oxygen delivery that foster tumor hypoxia. At the same time, tumor cells increase their oxidative metabolism to cope with the energetic demands of proliferation, which further worsens hypoxia due to heightened oxygen consumption. In these hypoxic, nutrient-deprived environments, tumors and suppressive stroma evolve to flourish while antitumor immunity collapses due to a combination of energetic deprivation, toxic metabolites, acidification, and other suppressive signals. Reversal of cancer hypoxia thus has the potential to increase the survival and effector function of tumor-infiltrating T cells, as well as to resensitize tumors to immunotherapy. Early clinical trials combining hypoxia reduction with immune checkpoint blockade have shown promising results in treating patients with advanced, metastatic, and therapeutically refractory cancers.

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Hepatocellular carcinoma (HCC) is the most common liver cancer and one of the leading causes of cancer-related deaths in the world. Multiple immunotherapeutic approaches have been investigated to date, and immunotherapy has become the new standard of care therapy in HCC. However, the current role of immunotherapy in HCC remains non-curative. Given this context, a high priority for oncology is understanding the biomarkers that predict clinical response to immunotherapy, have the potential to improve patient selection to maximize the clinical benefit, and spare unnecessary toxicity. In this review, we summarize the key predictive and prognostic biomarkers investigated in immunotherapy clinical trials in HCC and the emerging biomarkers to serve as a roadmap for future clinical trials. Biomarkers from tumoral tissues including PDL-1 expression, tissue infiltrating lymphocytes, tumor mutational burden (TMB) and specific immune signatures, and from peripheral blood including neutrophil-to-lymphocytes ratio, platelet-to-lymphocytes ratio, circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and specific cytokines, along with gut microbiota are among the studied biomarkers to date in the HCC era. More integrative approaches, including mathematical biomarkers to predict immunotherapy outcomes, are yet to be studied in HCC.

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Immune checkpoint blocking antibodies are a cornerstone in cancer treatment; however, they benefit only a subset of patients and biomarkers to guide immune checkpoint blockade (ICB) treatment choices are lacking. We designed this study to identify blood-based correlates of clinical outcome in ICB-treated patients. We performed immune profiling of 188 ICB-treated patients with melanoma using multiparametric flow cytometry to characterize immune cells in pretreatment peripheral blood. A supervised statistical learning approach was applied to a discovery cohort to classify phenotypes and determine their association with survival and treatment response. We identified three distinct immune phenotypes (immunotypes), defined in part by the presence of a LAG-3+CD8+ T cell population. Patients with melanoma with a LAG+ immunotype had poorer outcomes after ICB with a median survival of 22.2 months compared to 75.8 months for those with the LAG- immunotype (P = 0.031). An independent cohort of 94 ICB-treated patients with urothelial carcinoma was used for validation where LAG+ immunotype was significantly associated with response (P = 0.007), survival (P < 0.001), and progression-free survival (P = 0.004). Multivariate Cox regression and stratified analyses further showed that the LAG+ immunotype was an independent marker of outcome when compared to known clinical prognostic markers and previously described markers for the clinical activity of ICB, PD-L1, and tumor mutation burden. The pretreatment peripheral blood LAG+ immunotype detects patients who are less likely to benefit from ICB and suggests a strategy for identifying actionable immune targets for further investigation.

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