Potential Immune Escape Mechanism Research Articles

Anti-PD-L1 blockade facilitates antitumor effects of radiofrequency ablation by improving tumor immune microenvironment in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a complex disease with advanced presentation that significantly affects survival rates. Therefore, novel therapeutic strategies are needed. In this study, we investigate the tumor microenvironment (TME) in HCC by analyzing 13 HCC samples at single cell level. We identified key cell populations, including CD8 + T cells, Tregs, M1/M2 macrophages, and CD4 + memory T cells, and explored their roles and interactions. Our research revealed an early enrichment of CD8 + T cells, which could potentially lead to their exhaustion and facilitate tumor progression. We also investigated the impact of percutaneous radiofrequency ablation (RFA) on the immune microenvironment. Using a dual tumor mouse model, we demonstrated that RFA induces necrosis, enhancing antigen presentation and altering immune responses. Our results indicate that RFA increases PD-L1 expression in residual liver tissue, suggesting potential immune escape mechanisms. Furthermore, the combination of RFA and anti-PD-L1 therapy in the mouse model resulted in significant improvements in immune modulation. This included increased CD8 + T cell efficacy and decreased Treg infiltration. This combination shows promise as an approach to counteract HCC progression by altering the immune landscape. This study highlights the critical interaction within the TME of HCC and suggests the possibility of improving patient outcomes by targeting immune evasion mechanisms through combined therapeutic strategies.

LPA suppresses HLA-DR expression in human melanoma cells: a potential immune escape mechanism involving LPAR1 and DR6-mediated release of IL-10.

While immune checkpoint inhibitors (ICIs) are promising in the treatment of metastatic melanoma, about half of patients do not respond well to them. Low levels of human leukocyte antigen-DR (HLA-DR) in tumors have been shown to negatively influence prognosis and response to ICIs. Lysophosphatidic acid (LPA) is produced in large amounts by melanoma and is abundantly present in the tumor microenvironment. LPA induces the release of various cytokines and chemokines from tumor cells, which affect cancer development, metastasis, and tumor immunity. In the present study, we investigated the role of LPA-induced IL-10 release in regulating HLA-DR expression and the underlying mechanisms in human melanoma cells. We showed that LPA (0.001-10 μM) dose-dependently increased DR6 transcript levels through activating LPAR1 in HEK293T cells. Knockdown of NF-κB1 abrogated the LPA-increased DR6 expression without affecting basal DR6 expression in both A2058 and A375 melanoma cell lines. LPA (10 µM) significantly increased IL-10 transcripts in A2058 and A375 melanoma cells, the effect was abolished by pharmacological inhibition of LPAR1 or knockdown of DR6. We found a statistically significant correlation between the expression of LPAR1, DR6 and IL-10 in human melanoma tissue and an association between increased expression of LPAR1 and reduced effectiveness of ICI therapy. We demonstrated that LPA (10 µM) markedly suppressed HLA-DR expression in both A375 and A2058 melanoma cells via activating the LPAR1-DR6-IL-10 pathway. These data suggest that the LPAR1-DR6-IL-10 autocrine loop could constitute a novel mechanism used by tumor cells to evade immunosurveillance by decreasing HLA-DR expression.

A potential mechanism of tumor immune escape: Regulation and application of soluble natural killer group 2 member D ligands (Review).

The immune system is integral to the surveillance and eradication of tumor cells. Interactions between the natural killer group 2 member D (NKG2D) receptor and its ligands (NKG2DLs) are vital for activating NKG2D receptor‑positive immune cells, such as natural killer cells. This activation enables these cells to identify and destroy tumor cells presenting with NKG2DLs, which is an essential aspect of tumor immunity. However, tumor immune escape is facilitated by soluble NKG2DL (sNKG2DL) shed from the surface of tumor cells. The production of sNKG2DL is predominantly regulated by metalloproteinases [a disintegrin and metalloproteinases (ADAM) and matrix metalloproteinase (MMP) families] and exosomes. sNKG2DL not only diminish immune recognition on the tumor cell surface but also suppress the function of immune cells, such as NK cells, and reduce the expression of the NKG2D receptor. This process promotes immune evasion, progression, and metastasis of tumors. In this review, an in‑depth summary of the mechanisms and factors that influence sNKG2DL production and their contribution to immune suppression within the tumor microenvironment are provided. Furthermore, due to the significant link between sNKG2DLs and tumor progression and metastasis, they have great potential as novel biomarkers. Detectable via liquid biopsies, sNKG2DLs could assess tumor malignancy and prognosis, and act as pivotal targets for immunotherapy. This could lead to the discovery of new drugs or the enhancement of existing treatments. Thus, the application of sNKG2DLs in clinical oncology was explored, offering substantial theoretical support for the development of innovative immunotherapeutic strategies for sNKG2DLs.

Multiplexed Spatial Profiling of Hodgkin Reed-Sternberg Cell Neighborhoods in Classic Hodgkin Lymphoma.

Classic Hodgkin lymphoma (cHL) is a B-cell lymphoma that occurs primarily in young adults and, less frequently, in elderly individuals. A hallmark of cHL is the exceptional scarcity (1%-5%) of the malignant Hodgkin Reed-Sternberg (HRS) cells within a network of nonmalignant immune cells. Molecular determinants governing the relationship between HRS cells and their proximal microenvironment remain largely unknown. We performed spatially resolved multiplexed protein imaging and transcriptomic sequencing to characterize HRS cell states, cellular neighborhoods, and gene expression signatures of 23.6 million cells from 36 newly diagnosed Epstein-Barr virus (EBV)-positive and EBV-negative cHL tumors. We show that MHC-I expression on HRS cells is associated with immune-inflamed neighborhoods containing CD8+ T cells, MHC-II+ macrophages, and immune checkpoint expression (i.e., PD1 and VISTA). We identified spatial clustering of HRS cells, consistent with the syncytial variant of cHL, and its association with T-cell-excluded neighborhoods in a subset of EBV-negative tumors. Finally, a subset of both EBV-positive and EBV-negative tumors contained regulatory T-cell-high neighborhoods harboring HRS cells with augmented proliferative capacity. Our study links HRS cell properties with distinct immunophenotypes and potential immune escape mechanisms in cHL.

Pervasive loss of antigen presentation in solid tumors driven by loss of HLA class I expression.

e14654 Background: Human Leukocyte Antigens (HLA) play a key role in inducing responses to immune checkpoint inhibitor (ICI) therapy. HLA class I (HLA-I) expression is essential for antigen presentation in cancer cells and its loss leads to immune evasion. Expression of HLA-II shows presence of adaptive immune response via antigen presenting cells. To investigate the complex interplay of HLA class I/II in solid tumors, we study the landscape and association of several HLA phenotypes with biomarkers of tumor response and resistance to ICIs in a large pan-cancer cohort. Methods: A real-world cohort of 24,186 solid tumors was assessed by an immune expression profiling assay of 395 genes that were background subtracted, normalized, and ranked yielding a percentile rank value [0-100]. HLA Class I and class II expressions were estimated as average expression of HLA-A, HLA-B, HLA-C genes, and HLA HLA-DP, HLA-DQ, HLA-DR genes, respectively. Both HLA classes were labelled as high (≥66), moderate (≥33 & <66) or low (<33). We studied the distribution of HLA I/II phenotype defined as Ihi/IIhi comprised hot tumors with no loss of HLA-I and Ilo/IIhi defines immune evasion via loss of HLA-I in hot tumors. Ihi/IIlo phenotype comprises less inflamed tumors with high HLA-I expression and Ilo/IIlo comprises of cold tumors. We also performed differential expression of ICI targets including PD-1, PD-L1, LAG-3, TIM3 and expression-based signatures of inflammation and cell proliferation. Results: Loss of HLA-I expression was observed in 44.1% (n= 10669) of all solid tumors with most frequent loss observed in prostate (55%, n=450) cancer. Similarly, loss of HLA-II expression was observed in 34.7% (n=8396) of solid tumors with highest loss observed in neuroendocrine (59%; n=345) cancers. 49% of all tumors presented with four HLA-phenotypes, where 17.9% (n=4332) were characterized by Ilo/IIlo phenotype. Ihi/IIlo was present in 13% (n=3127) of all tumors. Both Ihi/IIhi and Ilo/IIhi phenotypes were significantly (p<0.001) associated with increased tumor inflammation. Other ICI targets such as PD-L1, TIM3, CTLA4 and LAG3 were significantly (p<0.001) overexpressed in this immune evasion phenotype compared to other phenotypes. Conversely, Ilo/IIlo phenotype was significantly associated with cold tumors (p<0.001), hinting at lack of adaptive immune response in these tumors. Conclusions: This study offers insight into pervasive loss of antigen presentation via HLA expression. Concurrent loss of HLA Class I expression with increased class II expression and additional ICI target co-expression is a potential immune escape mechanism in immunologically active solid tumors. Distinct and significant association of these MHC phenotypes with tumor inflammation, checkpoint markers and PD-L1 highlight the potential use of comprehensive immune profiling for further studying combination strategies to correct soft loss of HLA-I and ICI therapies.

Lessons from COVID‐19: Aspects of prevention, therapeutics, and diagnostics against SARS‐CoV‐2 with special focus on JN.1 and XBB sublineages

AbstractThe end of second decade in the 21st century witnessed a prominent disease outbreak caused by the novel coronavirus SARS‐CoV‐2 (including most diverse omicron subvariants), where the death toll crossed the boundary of 6.9 million across the globe by December 19, 2023. All spheres of central dogma of molecular biology and host‒pathogen interaction was explored to find ways in diagnostics, isolation, curtailment, and therapy. Above all, diagnostics and therapeutics against COVID‐19 took an enormous jump, which needs to be evaluated for accuracy and feasibility and requires serious compilation for current and future generations. With the same objective, this review encompasses the diverse ways including prevention practiced and proposed during the handling of this pandemic across the globe. It involves the role of mutations in viruses and subsequent epitope mapping with potential immune escape mechanisms of SARS‐CoV‐2 variants including the conservancy of T‐cell epitopes has also been highlighted. The efficacy in antigen/antibody‐based diagnostics, RT‒PCR‐ and NGS‐based confirmation of pathogen presence, and imaging (X‐ray/CT‐scan) for symptoms and damage assessment has been thoroughly filtered. The possibility of errors in diagnostics and their cause and consequences have also been presented for the ease of readers and further improvisers.

Potential immune evasion of the severe acute respiratory syndrome coronavirus 2 Omicron variants.

Coronavirus disease 2019 (COVID-19), which is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic. The Omicron variant (B.1.1.529) was first discovered in November 2021 in specimens collected from Botswana, South Africa. Omicron has become the dominant variant worldwide, and several sublineages or subvariants have been identified recently. Compared to those of other mutants, the Omicron variant has the most highly expressed amino acid mutations, with almost 60 mutations throughout the genome, most of which are in the spike (S) protein, especially in the receptor-binding domain (RBD). These mutations increase the binding affinity of Omicron variants for the ACE2 receptor, and Omicron variants may also lead to immune escape. Despite causing milder symptoms, epidemiological evidence suggests that Omicron variants have exceptionally higher transmissibility, higher rates of reinfection and greater spread than the prototype strain as well as other preceding variants. Additionally, overwhelming amounts of data suggest that the levels of specific neutralization antibodies against Omicron variants decrease in most vaccinated populations, although CD4+ and CD8+ T-cell responses are maintained. Therefore, the mechanisms underlying Omicron variant evasion are still unclear. In this review, we surveyed the current epidemic status and potential immune escape mechanisms of Omicron variants. Especially, we focused on the potential roles of viral epitope mutations, antigenic drift, hybrid immunity, and "original antigenic sin" in mediating immune evasion. These insights might supply more valuable concise information for us to understand the spreading of Omicron variants.

Immune response of silver pomfret (Pampus argenteus) to Photobacterium damselae subsp. Damselae: Virulence factors might induce immune escape by damaging phagosome

Photobacterium damselae subsp. damselae (PDD) is a dangerous bacterial disease that affects silver pomfret (Pampus argenteus) and its main virulence factors are pore-forming toxins. In this study, a highly pathogenic strain was obtained from naturally infected silver pomfret and was introduced into healthy fish for experimentation. Liver, gill, kidney and spleen samples were collected at 0, 24, 48, 72 and 96 h. Histopathological evaluations of these tissues, via HE staining, showed multi-organ bleeding, inflammatory infiltration and histopathocyte vacuolation as the main pathological changes. The levels of AKP, ACP, LZM, SOD, GSH and CAT in serum also displayed an increase following infection. Transcriptome sequencing of liver, kidney and spleen was performed using samples 96 h post-infection. The analysis of the resulting 770,878,120 clean reads collectively assembled into 89,310 unigenes. The WGCNA analysis demonstrated enrichment of phagosome and cytokine-cytokine receptor interaction in the liver, drug metabolism-cytochrome P450 in the kidney, and MAPK signaling pathway and other signaling pathways in the spleen. DEGs related to immune and immune-related pathways, such as Phagosome, Autophagy-animal and Glycerolipid metabolism, were significantly enriched. Selected key genes from these KEGG pathways were examined using RT-qPCR. The expression level of autophagy related genes (pi3k, bcl2) increased after infection, while the gene associated with MAPK and apoptosis (Caspase 9, mapk4) significantly increased after 72 h infection. The expression of phagosome related genes (atp6v1f, ctsl) increased initially, but later decreased. The genes related to innate immunity (ccl4, il15) were up-regulated, while those related to acquired immunity (mhc2a, mhc2b) showed a downward trend. Lysosome-related genes (gilta, giltb, cftr, c-type lectin, m6pra and m6prb) were up-regulated in liver and spleen and down-regulated in kidney and gill. The data obtained illustrated that PDD activated the immune defenses of the fish, increasing metabolic expenditure, but that the pore-forming toxins released by PDD could destroy the phagosome membrane and help the bacteria escape, which could disturb antigen presentation and inhibit the activation of the acquired immunity. This study reveals the potential immune escape mechanism of PDD in the host for the first time and has the potential to aid in the development of anti-PDD drugs.

LMAP-07 POTENTIAL MECHANISM OF IMMUNE ESCAPE RELATED TO TUMOR-ASSOCIATED MACROPHAGE IN GLIOBLASTOMA AFTER COMBINED THERAPY WITH RADIOTHERAPY AND IMMUNE CHECKPOINT BLOCKER

Abstract Immune checkpoint blockers (ICBs) have revolutionized the treatment of cancer. Radiotherapy (RT) has been demonstrated to increase the survival benefit of ICBs in some cancers through its local and systemic immunomodulation, although its efficacy in glioblastoma (GBM) is very limited. With a thorough investigation of the immune responses to single and dual treatment utilizing single-cell RNA-seq analyses on the synergy of RT and anti-PD-L1 antibody (aPD-L1), we studied the potential mechanism of immune escape in GBM even when RT and ICB are combined. Our findings demonstrated a statistically significant longer survival of mice treated with RT and aPD-L1 combined than either single modality of treatment in an orthotopic syngeneic GBM model achieving a 70% cure rate. In the tumor tissues collected from the mouse brain that failed dual treatment, i.e., immune escaped, immune cell profiling still demonstrated much lower T regulatory cells and higher CD8+ cytotoxic T cells infiltrated to tumor, and elevated CD8+/CD4+ and CD8+/Treg ratios in the dual therapy group compared to single therapies, as expected with synergistic effects of dual treatment. RT with aPD-L1 markedly reduced CD8+ and CD4+ T exhaustion cells and elevated T-memory cells in comparison to single modality groups. Nevertheless, compared to the RT alone or no treatment control group, dual therapy failed to decrease the proportion of Arginase1-positive glioma-associated macrophages (GAM) in macrophage/monocytes in tumor microenvironment, which were considerably increased if treated with aPD-L1 only. Moreover, the combination of RT with aPD-L1 did not alter immunosuppressive gene expression in the macrophage/monocyte groups. In conclusion, our research demonstrated that RT and aPD-L1 together may elicit a potent and durable antitumor immune response in the GBM mouse model through regulating lymphocytes but not macrophages. Our findings point to the urgent need for research into GAM-modulating drug and their potential to prevent immunological escape.

Spatial immunophenotypes predict clinical outcome in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (iCCA) is a severe malignant tumour that shows only modest responses to immunotherapy. We aimed to identify the spatial immunophenotypes of iCCA and delineate potential immune escape mechanisms. Multiplex immunohistochemistry (mIHC) was performed to quantitatively evaluate the distribution of 16 immune cell subsets in intratumour, invasive margin and peritumour areas in a cohort of 192 treatment-naïve patients with iCCA. Multiregion unsupervised clustering was used to determine three spatial immunophenotypes, and multiomics analyses were carried out to explore functional differences.Results: iCCA displayed a region-specific distribution of immune cell subsets with abundant CD15+ neutrophil infiltration in intratumour areas. Three spatial immunophenotypes encompassing inflamed (35%), excluded (35%) and ignored (30%) phenotypes were identified. The inflamed phenotype showed characteristics of abundant immune cell infiltration in intratumour areas, increased PD-L1 expression and relatively favourable overall survival. The excluded phenotype with a moderate prognosis was characterized by immune cell infiltration restricted to the invasive margin or peritumour areas and upregulation of activated hepatic stellate cells, extracellular matrix and Notch signalling pathways. The ignored phenotype, with scarce immune cell infiltration across all subregions, was associated with MAPK signalling pathway elevation and a poor prognosis. The excluded and ignored phenotypes, constituting non-inflamed phenotypes, shared features of an increased angiogenesis score, TGF-β and Wnt-β catenin pathway upregulation and were enriched for BAP1 mutations and FGFR2 fusions. We identified three spatial immunophenotypes with different overall prognoses in iCCA. Tailored therapies based on the distinct immune evasion mechanisms of the spatial immunophenotypes are needed. The contribution of immune cell infiltration in the invasive margin and peritumour areas has been proved. We explored the multiregional immune contexture of 192 patients to identify three spatial immunophenotypes in intrahepatic cholangiocarcinoma (iCCA). By integrating genomic and transcriptomic data, phenotype-specific biological behaviours and potential immune escape mechanisms were analysed. Our findings provide a rationale to develop personalized therapies for iCCA.

Abstract 5282: Mitotane induces GDF-15 expression in vitro and in vivo: A potential therapy-induced mechanism of immune escape in adrenocortical carcinoma

Abstract Context: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with poor prognosis and limited treatment options. The only approved therapy is mitotane, an adrenal toxicant, alone or in combination with cytotoxic chemotherapy. Response rate is only ~20%. Results of later line immune checkpoint inhibition (ICI) in ACC are disappointing, most likely due to an immunologically cold, immunosuppressive microenvironment. Among the factors induced in mitotane-treated ACC cell lines, we previously found growth/differentiation factor 15 (GDF-15), an anti-inflammatory cytokine physiologically expressed during pregnancy. Aim: As GDF-15 is an emerging mediator of tumor immune escape, we investigated changes in GDF-15 secretion upon mitotane treatment in ACC in vitro and in vivo, and explored a potential correlation with response to therapy and survival. Methods: Four different human ACC cell lines (NCI-H295R, CU-ACC1, CU-ACC2 and JIL-2266) were treated with mitotane. GDF-15 secretion and expression were determined by ELISA and immunoblot. GDF-15 levels in sera of 142 ACC patients (94 prior and 48 during mitotane) were analyzed by ELISA and correlated with survival from the time point of blood collection. GDF-15 serum levels were quantified in three responders and seven non-responders to ICI treatment. Results: In vitro, 24h treatment with 25 µM mitotane-induced GDF-15 secretion and expression in all cell lines by up to one order of magnitude. In 32 patient samples, GDF-15 serum concentrations increased from 0.6±1 ng/l prior mitotane to 2.1 ± 2.7 ng/l after mitotane initiation, p<0.0001.Importantly, patients with low serum GDF-15 levels before and during mitotane treatment had a significantly longer overall survival compared to patients with higher GDF-15 serum levels. This association retained statistical significance after adjustment for the known prognostic factors Ki67 and ENSAT tumour stage in the mitotane treated group (95% CI 1.15-.1.73, HR1.41, p=0.001). In an exploratory cohort of three responders and seven non-responders to ICI we found a trend for higher GDF-15 serum concentrations in the non-responders. Conclusion: Mitotane induces GDF-15 both in vivo and in vitro and potentially contributes to the poor response rates to ICI in ACC. Citation Format: Isabel Weigand, Tanja Anderlik, Jochen Schreiner, Hanna Remde, Otilia Kimpel, Laura-Sophie Landwehr, Eva Hoster, Florian Wedekink, Alexandra S. Triebig, Tanja Maier, Jörg Wischhusen, Martin Reincke, Martin Fassnacht, Matthias Kroiss. Mitotane induces GDF-15 expression in vitro and in vivo: A potential therapy-induced mechanism of immune escape in adrenocortical carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5282.

Pembrolizumab for the treatment of disease relapse after allogeneic hematopoietic stem cell transplantation.

A failed graft-versus-tumor (GVT) effect is a common mechanism of relapse after allogeneic hematopoietic cell transplantation (alloHCT). Although targeting the PD-1/PD-L1 axis may restore GVT effects, PD-1 blockade exacerbates graft-versus-host disease (GVHD) in murine models, and severe GVHD can occur in patients treated with anti-PD-1 therapy after alloHCT. Therefore, we developed a prospective study to assess the safety and efficacy of pembrolizumab in patients relapsing after alloHCT. Eligible patients received pembrolizumab (200 mg every 3 weeks) for up to 2 years. Twelve patients were enrolled (8patients with acute myeloid leukemia, 1 patient with myelodysplastic syndrome, 1 patient with classical Hodgkin lymphoma, and 2 patients with diffuse large B-cell lymphoma [DLBCL]). All participants received reduced-intensity preparative regimens with invivo T-cell depletion. The median time from alloHCT to enrollment was 587 days (range, 101-4211). Three participants (25%) experienced grade 3 to 4 immune-related adverse events (irAE) (pneumonitis, 2 patients; hyperthyroidism, 1 patient), all occurring after 1 to 2 cycles, and resolving after pembrolizumab discontinuation and corticosteroid treatment. irAEs of any grade occurred in 5 patients (42%). No treatment-emergent GVHD was observed. Overall and complete response (CR) rates were 22% (2/9). Both patients achieving CRs hadPD-L1 gene-amplified lymphomas and diffuse PD-L1 expression on pretreatment biopsies. An acquired EZH2 mutation was identified at relapse in a patient with DLBCL who achieved an initial CR to pembrolizumab, which was associated with downregulated HLAexpression on malignant B cells, implicating EZH2 mutations as a potential immune escape mechanism after PD-1-blockade therapy. In conclusion, after alloHCT, treatment with pembrolizumab is feasible and associated with objective responses in relapsed lymphoid malignancies but can induce severe irAEs, requiring vigilant monitoring. Thistrial was registered at www.clinicaltrials.gov as #NCT02981914.

Double-edged roles of IFNγ in tumor elimination and immune escape

Interferon-gamma (IFNγ) is a pleiotropic cytokine implicated in tumor immune surveillance, with its antiproliferative, pro-apoptotic, and immune-provoking effects. Regarding the antitumor effects of IFNγ, IFNγ-dependent therapies have been proposed and have undergone many clinical trials for various cancer types but the outcomes were not satisfactory. Recent studies have suggested that cancer cells develop immune evasion strategies to escape from IFNγ-dependent immunosurveillance by various mechanisms. In this review, we summarize recent advances in the effects and molecular mechanisms of IFNγ on target cells, as well as potential immune escape mechanisms of tumor cells. Furthermore, we discuss how to target IFNγ signaling and overcome immune evasion to provide promising therapeutic strategies for the treatment of patients with cancer.

Wnt/β-Catenin Signaling and Resistance to Immune Checkpoint Inhibitors: From Non-Small-Cell Lung Cancer to Other Cancers.

Lung cancer is the leading cause of cancer-related deaths worldwide. The standard of care for advanced non-small-cell lung cancer (NSCLC) without driver-gene mutations is a combination of an anti-PD-1/PD-L1 antibody and chemotherapy, or an anti-PD-1/PD-L1 antibody and an anti-CTLA-4 antibody with or without chemotherapy. Although there were fewer cases of disease progression in the early stages of combination treatment than with anti-PD-1/PD-L1 antibodies alone, only approximately half of the patients had a long-term response. Therefore, it is necessary to elucidate the mechanisms of resistance to immune checkpoint inhibitors. Recent reports of such mechanisms include reduced cancer-cell immunogenicity, loss of major histocompatibility complex, dysfunctional tumor-intrinsic interferon-γ signaling, and oncogenic signaling leading to immunoediting. Among these, the Wnt/β-catenin pathway is a notable potential mechanism of immune escape and resistance to immune checkpoint inhibitors. In this review, we will summarize findings on these resistance mechanisms in NSCLC and other cancers, focusing on Wnt/β-catenin signaling. First, we will review the molecular biology of Wnt/β-catenin signaling, then discuss how it can induce immunoediting and resistance to immune checkpoint inhibitors. We will also describe other various mechanisms of immune-checkpoint-inhibitor resistance. Finally, we will propose therapeutic approaches to overcome these mechanisms.

Characterizing the tumor microenvironment at the single-cell level reveals a novel immune evasion mechanism in osteosarcoma

The immune microenvironment extensively participates in tumorigenesis as well as progression in osteosarcoma (OS). However, the landscape and dynamics of immune cells in OS are poorly characterized. By analyzing single-cell RNA sequencing (scRNA-seq) data, which characterize the transcription state at single-cell resolution, we produced an atlas of the immune microenvironment in OS. The results suggested that a cluster of regulatory dendritic cells (DCs) might shape the immunosuppressive microenvironment in OS by recruiting regulatory T cells. We also found that major histocompatibility complex class I (MHC-I) molecules were downregulated in cancer cells. The findings indicated a reduction in tumor immunogenicity in OS, which can be a potential mechanism of tumor immune escape. Of note, CD24 was identified as a novel “don’t eat me” signal that contributed to the immune evasion of OS cells. Altogether, our findings provide insights into the immune landscape of OS, suggesting that myeloid-targeted immunotherapy could be a promising approach to treat OS.

TIE-2 Signaling Activation by Angiopoietin 2 On Myeloid-Derived Suppressor Cells Promotes Melanoma-Specific T-cell Inhibition.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immune suppressive cells detected in several human cancers. In this study, we investigated the features and immune suppressive function of a novel subset of monocytic MDSC overexpressing TIE-2 (TIE-2+ M-MDSC), the receptor for the pro-angiogenic factor angiopoietin 2 (ANGPT2). We showed that patients with melanoma exhibited a higher circulating rate of TIE-2+ M-MDSCs, especially in advanced stages, as compared to healthy donors. The distribution of the TIE-2+ M-MDSC rate toward the melanoma stage correlated with the serum level of ANGPT2. TIE-2+ M-MDSC from melanoma patients overexpressed immune suppressive molecules such as PD-L1, CD73, TGF-β, and IL-10, suggesting a highly immunosuppressive phenotype. The exposition of these cells to ANGPT2 increased the expression of most of these molecules, mainly Arginase 1. Hence, we observed a profound impairment of melanoma-specific T-cell responses in patients harboring high levels of TIE-2+ M-MDSC along with ANGPT2. This was confirmed by in vitro experiments indicating that the addition of ANGPT2 increased the ability of TIE-2+ M-MDSC to suppress antitumor T-cell function. Furthermore, by using TIE-2 kinase-specific inhibitors such as regorafenib or rebastinib, we demonstrated that an active TIE-2 signaling was required for optimal suppressive activity of these cells after ANGPT2 exposition. Collectively, these results support that TIE-2+ M-MDSC/ANGPT2 axis represents a potential immune escape mechanism in melanoma.

Immune surveillance in glioblastoma: Role of the NKG2D system and novel cell-based therapeutic approaches.

Glioblastoma, formerly known as glioblastoma multiforme (GBM), is the most frequent and most aggressive brain tumour in adults. The brain is an immunopriviledged organ, and the blood-brain barrier shields the brain from immune surveillance. In this review, we discuss the composition of the immunosuppressive tumour micromilieu and potential immune escape mechanisms in GBM. In this respect, we focus on the role of the NKG2D receptor/ligand system. NKG2D ligands are frequently expressed on GBM tumour cells and can activate NKG2D-expressing killer cells including NK cells and γδ T cells. Soluble NKG2D ligands, however, contribute to tumour escape from immunological attack. We also discuss the current immunotherapeutic strategies to improve the survival of GBM patients. Such approaches include the modulation of the NKG2D receptor/ligand system, the application of checkpoint inhibitors, the adoptive transfer of ex vivo expanded and/or modified immune cells or the application of antibodies and antibody constructs to target cytotoxic effector cells in vivo. In view of the multitude of pursued strategies, there is hope for improved overall survival of GBM patients in the future.

Novel hypoxia features with appealing implications in discriminating the prognosis, immune escape and drug responses of 947 hepatocellular carcinoma patients.

BackgroundHypoxia has a profound impact on the development and progression of hepatocellular carcinoma (HCC). This study aimed to explore and elucidate how hypoxia affect prognosis, immune escape and drug responses in HCC.MethodsHCC-specific hypoxia signatures were identified based on the intersect of differentially expressed genes (DEGs) of GSE41666 and GSE15366. The hypoxia score was calculated using the gene set variation analysis (GSVA) function and validated on GSE18494. We collected five cohorts [The Cancer Genome Atlas (TCGA), GSE14520, GSE39791, GSE36376, GSE57957] for further analysis. First, we analyzed the effect of the hypoxia score on prognosis. Next, we systematically analyzed the potential hypoxia-related immune escape mechanisms and the effect of hypoxia upon immunotherapy. Then, we predicted and screened potential sensitive drugs for HCC patients with high hypoxia levels using machine learning and docking.ResultsWe constructed a novel HCC-specific hypoxia score and undertook further analysis in five cohorts (TCGA, GSE14520, GSE39791, GSE36376, GSE57957). We observed that patients with high hypoxia scores exhibited worse overall survival (OS) in TCGA and GSE14520. We also constructed a hypoxia-related nomogram that had good performance in predicting HCC patients’ prognosis. Furthermore, patients with lower hypoxia scores had a lower risk of immune escape and thus may benefit from immunotherapy. Finally, we predicted and screened VLX600 as the candidate drug for HCC patients with high hypoxia scores. We further explored and elucidated why VLX600 was more sensitive in HCC patients with high hypoxia than with low hypoxia HCC patients using weighted gene co-expression network analysis (WGCNA).ConclusionsThis study provides further evidence of the link between hypoxia and prognosis and immune escape in HCC patients. Moreover, our research screened VLX600 as a potential drug for HCC patients with high hypoxia levels and elucidated the potential mechanism.

Abstract 253: Analyses of tumor-specific T cell dynamics and tumor immune contexture in microsatellite instability-high cancers in response to first-line therapy

Abstract Microsatellite instability-high (MSI-H) phenotype arises from defective DNA repair mechanisms and is found in many malignancies, including colorectal and endometrial cancers. Due to high neoantigen loads derived from tumor-specific mutations, evidence of tumor-infiltrating T cells and high response rates to checkpoint blockade (30-60%), MSI-H cancers can be leveraged to study the features of neoantigen-induced anti-tumor immunity in response to therapy and disease outcome. Previously we identified a set of recurrent frameshift (fs) mutations in MSI-H cancers that yielded immunogenic polyepitope neoantigens (neoAgs) eliciting CD8+ T cell responses. Here we investigate whether these shared fs-neoAgs engendered spontaneous T cell responses in MSI-H colorectal and endometrial cancer patients. Using peripheral blood mononuclear cells (PBMCs), we evaluated the frequency of the fs-neoAg-specific T cells by ex vivo IFN-γ ELISPOT upon fs-peptide stimulation. Additionally, we isolated naïve and memory T cells from MSI-H patient PBMCs and expanded fs-neoAg-specific T cells within each subset. Fs-neoAg-induced effector cytokine (IFN-γ, TNF-α) production was measured by flow cytometry and was detected only in the memory subset, supporting the hypothesis that shared fs-neoAgs prime CD8+ T cells in vivo to generate clonally-expanded memory T cells in MSI-H patients. To further evaluate the clonal expansion of fs-neoAg-specific T cells in MSI-H cancer patients, we identified fs-neoAg-specific T cell receptors (TCRs). T cell lines enriched for fs-neoAg-specific T cells were generated from patient PBMCs and sequenced for variable β chains of TCRs. These fs-neoAg-specific TCRs were monitored in the blood and tumor tissues, including those from primary and recurrent sites. Finally, to determine the spatial relationships between the infiltrating immune cells and the malignant cells at single cell resolution, we performed multiplexed immunohistochemistry using CODEX technology. A panel of 35 markers was used to evaluate the myeloid (HLA-DR, CD11c, CD14, CD68, CD206) and T (CD4, CD8, Foxp3) cell subsets, the activation (CD107a, CD69, Ki67) and exhaustion (PD-1, TIM3, LAG3) status of T cells, T cell states (TCF-1, TOX, PD-1) and the presence of tertiary lymphoid structures/antigen presenting cell niches (CD19, CD20, HLA-DR) in the tumor microenvironment of primary or recurrent MS-stable and MSI-H tumors from endometrial cancer patients. We also investigated potential immune escape mechanisms in MSI-H tumors, such as the loss of B2M and MHC-I expression. This study will provide insights into the quality and quantity of shared anti-tumor T cells within the periphery and tumor tissues and will help identify the immune correlates of response or resistance to therapy in MSI-H cancer patients. Citation Format: Cansu Cimen Bozkus, Mona Saleh, Rachel Brody, Stephanie V. Blank, Nina Bhardwaj. Analyses of tumor-specific T cell dynamics and tumor immune contexture in microsatellite instability-high cancers in response to first-line therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 253.

Association of LAG-3 expression in circulating T cells and response to combination temozolomide (TMZ) and nivolumab (NIVO) in advanced neuroendocrine neoplasms (NENs): Results from an investigator-initiated phase 2 trial.

4123 Background: LAG-3 is an immune checkpoint present on NK cells, activated T cells and myeloid cells that inhibit T cell responses. Recent evidence demonstrating the safety and efficacy of LAG-3 inhibition has increased interest in this pathway for the treatment of multiple malignancies but the role in NEN is unclear. We present results from correlative peripheral blood mass cytometry (CyTOF) performed in a phase 2 trial (NCT03728361) of the combination of NIVO and TMZ in pts with advanced NEN. Methods: Patients (pts) with progressive NEN of any grade or primary location and any line of therapy were eligible. Small cell lung cancer was excluded. Clinical results from NCT03728361 will be presented in a separate abstract. Study treatment consisted of NIVO 480 mg IV every 4 weeks and TMZ 150 mg/m2 for 5 consecutive days out of a 28-day cycle. Peripheral blood mononuclear cells (PBMCs) were available from 16 out of 28 patients at screening (baseline) and cycle 1, day 15 (C1D15) and analyzed via CyTOF. Antibody labelling was performed using a 37 marker Maxpar Direct Immune Profiling Assay (Fluidigm). Immune cell populations were compared using two sample t-tests between pts with partial response (PR) and non-partial response (non-PR). Results: At screening, no differences were observed in PD-1, TIM3, or KLRG1 positive T-cell populations between pts with PR or non-PR. Patients with a PR had a significantly lower % of LAG-3 expressing T cells (p=0.029). There was a trend towards a lower % CD8+LAG-3+ T cells in pts with PR (p=0.086). At C1D15: The % of CD8+ LAG-3+ T cells were significantly higher in PRs vs. non-PR (p = 0.015). In matched samples comparing T cell populations at screening to C1D15, LAG-3+ CD8+ T cells increased significantly in PRs when compared to non-PRs (p=0.021). Conclusions: The % of LAG-3+ T cell population at baseline associates with non-response to TMZ/NIVO in NENs. Among responders, there was a significant increase in CD8+ LAG-3+ T cells by Day 15 compared to baseline indicating a potential mechanism of immune escape and eventual resistance. Clinical trial information: NCT03728361. [Table: see text]