Immune Escape Mechanisms Research Articles

EV-Mediated Chemoresistance in the Tumor Microenvironment: Is NF-κB a Player?

Drug resistance is a major impediment to patient survival and remains the primary cause of unsuccessful cancer therapy. Drug resistance occurs in many tumors and is frequently induced by chemotherapy which triggers a defensive response both in cancerous and cancer-associated cells that constitute the tumor microenvironment (TME). Cell to cell communication within the TME is often mediated by extracellular vesicles (EVs) which carry specific tumor-promoting factors able to activate survival pathways and immune escape mechanisms, thus sustaining tumor progression and therapy resistance. NF-κB has been recognized as a crucial player in this context. NF-κB activation is involved in EVs release and EVs, in turn, can trigger NF-κB pathway activation in specific contexts, based on secreting cytotype and their specific delivered cargo. In this review, we discuss the role of NF-κB/EVs interplay that sustain chemoresistance in the TME by focusing on the molecular mechanisms that underlie inflammation, EVs release, and acquired drug resistance.

Simplexviruses Successfully Adapt to Their Host by Fine-Tuning Immune Responses.

Primate herpes simplex viruses are species-specific and relatively harmless to their natural hosts. However, cross-species transmission is often associated with severe disease, as exemplified by the virulence of macacine herpesvirus 1 (B virus) in humans. We performed a genome-wide scan for signals of adaptation of simplexviruses to their hominin hosts. Among core genes, we found evidence of episodic positive selection in three glycoproteins, with several selected sites located in antigenic determinants. Positively selected noncore genes were found to be involved in different immune-escape mechanisms. The herpes simplex virus (HSV)-1/HSV-2 encoded product (ICP47) of one of these genes is known to down-modulate major histocompatibility complex class I expression. This feature is not shared with B virus, which instead up-regulates Human Leukocyte Antigen (HLA)-G, an immunomodulatory molecule. By in vitro expression of different ICP47 mutants, we functionally characterized the selection signals. Results indicated that the selected sites do not represent the sole determinants of binding to the transporter associated with antigen processing (TAP). Conversely, the amino acid status at these sites was sufficient to determine HLA-G up-regulation. In fact, both HSV-1 and HSV-2 ICP47 induced HLA-G when mutated to recapitulate residues in B virus, whereas the mutated version of B virus ICP47 failed to determine HLA-G expression. These differences might contribute to the severity of B virus infection in humans. Importantly, they indicate that the evolution of ICP47 in HSV-1/HSV-2 led to the loss of an immunosuppressive effect. Thus, related simplexviruses finely tune the balance between immunosuppressive and immunostimulatory pathways to promote successful co-existence with their primate hosts.

African swine fever virus MGF505-3R inhibits cGAS-STING-mediated IFN-β pathway activation by degrading TBK1

African swine fever virus (ASFV) is an important pathogen causing acute infectious disease in domestic pigs and wild boars that seriously endangers the global swine industry. As ASFV is structurally complex and encodes a large number of functional proteins, no effective vaccine has been developed to date. Thus, dissecting the mechanisms of immune escape induced by ASFV proteins is crucial. A previous study showed that the ASFV-encoded protein is an important factor in host immunity. In this study, we identified a negative regulator, MGF505-3R, that significantly downregulated cGAS/STING- and poly (dG:dC)-mediated IFN-β and interferon stimulation response element (ISRE) reporter activity and suppressed IFNB1 and IFIT2 mRNA levels. In addition, TBK1, IRF3 and IκBα phosphorylation levels were also inhibited. Mechanistically, MGF505-3R interacted with cGAS/TBK1/IRF3 and targeted TBK1 for degradation, thereby disrupting the cGAS-STING-mediated IFN-β signaling pathway, which appears to be highly correlated with autophagy. Knockdown MGF505-3R expression enhanced IFN-β and IL-1β production. Taken together, our study revealed a negative regulatory mechanism involving the MGF505-3R-cGAS-STING axis and provided insights into an evasion strategy employed by ASFV that involves autophagy and innate signaling pathways.

Abstract 1378: CD8 T cell mediated killing of MHC class 1 negative tumors requires antigen presenting myeloid cells and interferon gamma

Abstract Major Histocompatibility Complex (MHC) Class I downregulation is a well described mechanism of tumor immune escape, posing a challenge for T cell based immunotherapies including immune checkpoint blockade (ICB). Recent studies, however, have demonstrated mixed roles of MHC Class 1 and the critical component beta-2-microglobulin (β2m) expression in cancer progression and ICB response, with some studies showing inactivation of antigen presentation to be associated with resistance to ICB and others showing low β2m expression to be associated with favorable prognosis. Glioblastoma (GBM) in particular expresses little or no MHC Class 1 and patients remain unresponsive to ICB. We thus sought to evaluate the role of MHC Class 1 in ICB, given that we have previously demonstrated that combination ICB with anti-PD-1 and co-stimulation with 4-1BB agonism has marked efficacy against intracranial murine glioma tumors in a CD8 T cell dependent manner. Surprisingly, in a CT2A murine glioma tumor line expressing the antigen TRP2 and lacking cell surface MHC I (CT2A-TRP2-β2mKO), the efficacy of combination 4-1BB and PD-1 therapy (ICB) was re-demonstrated in a CD8 dependent manner, independent of NK cells, CD4 T cells, and B cells. Furthermore, the efficacy of immunotherapy against intracranial CT2A-TRP2-β2mKO was demonstrated to be antigen dependent, with an adoptive lymphocyte transfer (ALT) of TRP2 TCR transduced T cells (TRP2 T cells) into a CD8KO mouse sufficient to eliminate CT2A-TRP2-β2mKO in the setting of ICB. Additionally, an ALT of TRP2 T cells did not kill CT2A-β2mKO tumors in the setting of ICB, while OT-1 mice whose CD8+ T cells primarily recognize OVA peptide with CT2A-TRP2-β2mKO tumors did not respond to ICB. In vitro studies revealed that TRP2 T cells demonstrated anti-tumor cytotoxicity against MHC Class I negative CT2A-TRP2-β2mKO tumor cells in the presence of TRP2 loaded bone marrow derived macrophages (TRP2 Mφ), but neither cell type was individually sufficient to induce tumor cell death, while the combination of TRP2 T cells and TRP2 Mφ demonstrated no cytotoxicity against CT2A-β2mKO tumors. Transwell experiments in which TRP2 Mφ and CT2A-TRP2-β2mKO tumor cells were separated by a 0.5µm membrane permeable to T cells but not Mφ or tumor cells revealed that contact between TRP2 Mφ and tumor cells was not necessary to induce T cell dependent killing. Indeed, tumor-bearing β2mKO bone marrow chimeras lacking MHC class 1 on hematopoeitically derived cells did not respond to ICB, highlighting the importance of antigen presentation from myeloid cells. The mechanism of killing was found to be dependent on interferon gamma (IFNγ), as IFNγKO mice did not respond to ICB. These findings demonstrate that tumors with low MHC Class 1 expression may still be targeted by T cell dependent immunotherapies such as ICB when antigen presentation can occur from myeloid cells. Citation Format: Emily Lerner, Vincent D'Anniballe, William Tomaszewski, Jonathan Perera, Xiuyu Cui, Jessica Waibl-Polania, Daniel Wilkinson, Michael D. Gunn, Peter E. Fecci, Karolina Woroniecka. CD8 T cell mediated killing of MHC class 1 negative tumors requires antigen presenting myeloid cells and interferon gamma [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 1378.

Abstract 3609: Defining extrinsic and intrinsic mechanisms of immune evasion in TRACERx using imaging mass cytometry

Abstract Introduction: The relationship between the evolving cancer genome and its tumor microenvironment (TME) is poorly understood. TRACERx has examined the intrinsic mechanisms of immune escape in non-small cell lung cancer. Here, we applied imaging mass cytometry (IMC) to address the contribution of extrinsic mechanisms in the context of intrinsic mechanisms and genomic features such as neoantigen burden. Methods: We applied two 35-antibody IMC panels to the treatment naïve multiregion TRACERx cohort (n=81 patients, 221 regions), and developed an IMC analysis pipeline to study T cell differentiation states, cancer associated fibroblasts, vasculature, and innate and adaptive immunity. Identities and functional states were assigned to over 4 million cells and their positions were mapped within tumor and stromal compartments. Results: We identified four distinct TMEs according to the local densities of lymphoid and myeloid cells: high infiltrate, stroma TIL enriched, myeloid enriched, and low infiltrate. We confirmed frequent intratumor TME heterogeneity, previously inferred from RNA sequencing. In high and stroma TIL enriched TMEs, we found the balance of effector and suppressor cells either favoured an exhausted (exhausted T cells and Tregs, n=16, 26.7%), suppressed (effector T cells and suppressive macrophages, n=31, 51.6%) or effector phenotype (high M1:M2, n=13, 21.6%). Tregs within exhausted TMEs neighboured effector and exhausted T cells, whereas effector T cells were nearest tumor cells within effector TMEs. Cell-type specific checkpoint molecule expression favoured specific compartments and TMEs. For example, the highest density of intratumoral PDL1+ M2-macrophages was observed within high infiltrate regions. Complementary intrinsic and extrinsic immune evasion mechanisms were identified, including immune avoiding architectures and stromal barriers as spatial mechanisms of evasion. Tumor cells were found to express lactate transporter, MCT4, in high infiltrate environments. Neoantigen burden associated with immune cell densities in a histology-specific manner. In lung adenocarcinoma, neoantigen burden was associated with a high infiltrate TME and suppressive myeloid populations such as M2-macrophages. In squamous cell carcinoma, we observed significant associations with CD8+ T cells, including exhausted populations, specifically in tumors lacking HLALOH. Conclusions: IMC allowed for the identification of spatial mechanisms of immune evasion that, when combined with intrinsic mechanisms, provided a more complete understanding of how lung tumors escape immune predation. By visualising single cells in their native context, we observed various immunosuppressive TMEs that defined high infiltrate tumors. Histology-specific relationships between immune cells and neoantigen burden point to different immune pressures, which may influence tumor evolution. Citation Format: Katey Enfield, Emma Colliver, Mihaela Angelova, Alastair Magness, Claudia Lee, Kristiana Grigoriadis, Oriol Pich, Clare Puttick, Dina Levi, Philip Hobson, David Moore, Takahiro Karasaki, Selvaraju Veeriah, TRACERx Consortium, Mariam Jamal-Hanjani, Nicholas McGranahan, Erik Sahai, Julian Downward, Crispin Hiley, Charles Swanton. Defining extrinsic and intrinsic mechanisms of immune evasion in TRACERx using imaging mass cytometry [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 3609.

Abstract 124: PRDX4 links ER-specific redox vulnerability to innate immunity in pancreatic cancer

Abstract The prognosis of pancreatic ductal carcinoma (PDAC) remains dismal, with an overall 5-year survival rate of 7%. Immunotherapy (IT) has provided new hope for other hard-to-treat cancers, but PDAC has shown striking resistance to IT by orchestrating mechanisms of immune escape. This spurs an interest to identify novel strategies to improve PDAC sensitivity to IT. We recently unveiled that perturbating redox homeostasis by targeting the antioxidant protein peroxiredoxin 4 (PRDX4) render pancreatic cancer cells highly vulnerable. PRDX4 supports redox homeostasis by metabolizing H2O2 in the endoplasmic reticulum (ER), and its loss leads to oxidative stress and toxicity. Interestingly, cell death induced by PRDX4 depletion is accompanied by DNA damage and accumulation of cytosolic DNA, a potent primer for immune therapy. In line with that, PRDX4 knockdown results in the transcriptional upregulation of inflammatory genes, including type I interferons (IFNs), primary cytokines and interferon-stimulated genes (ISGs). Importantly, our data indicate that this induction is dependent on the critical regulator of the innate immune responses, STING, as treatment with a STING inhibitor, or a STING knockdown, substantially blunts their upregulation. It is known that following DNA damage detection, the ER-localized STING translocates to the Golgi, where it associates with the kinase TBK1 and mediates the induction of inflammatory target genes, depending on IRF3 and NF-κB transcription factors. We observe an activation of downstream STING effectors upon PRDX4 loss, and co-depletion with NFκB significantly rescues the upregulation of those genes. As such, we propose that PRDX4 links ER-specific redox vulnerability to innate immunity signaling in PDAC. Since PRDX4 loss causes increased DNA damage and inflammatory signaling, our preliminary data suggest that PRDX4 could be a novel therapeutic target that may act synergistically with immune checkpoint inhibitors. Citation Format: Lucie Malbeteau, Pallavi Jain, Shane Harding, Bradley Wouters, Marianne Koritzinsky. PRDX4 links ER-specific redox vulnerability to innate immunity in pancreatic cancer [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 124.

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.

Abstract 1259: PD-L1 expression by RNA-sequencing and survival from pembrolizumab in non-small cell lung cancer (NSCLC)

Abstract PURPOSE: The immunohistochemistry companion diagnostic test for pembrolizumab (IHC 22C3 pharmDx) lacks sensitivity, challenging immunotherapy selection for NSCLC patients with lower levels of expression. Unlike IHC 22C3, which restricts assessment of PD-L1 expression to viable tumor cells as a tumor proportion score (% TPS), mRNA next generation sequencing (RNA-seq) measures PD-L1 expression in the tumor microenvironment for both tumor and inflammatory background cells. RNA-seq previously demonstrated concordance with IHC and may be a robust alternative testing method for multiple tumor types. Here, we sought to optimize PD-L1 RNA-seq cutoff values in NSCLC to improve clinical sensitivity. PROCEDURE: NSCLC patients included in the study (n=3,465) were tested for PD-L1 expression by IHC 22C3 and clinically validated RNA-seq, measured as % rank (0-100) relative to a reference population based on normalized reads per million (nRPM). Patients were divided into an RNA-seq cut-off discovery cohort (n=3,168), and a test cohort pembrolizumab treated patients. Principal components analysis (PCA) was used to classify patients based on test results and explore cut-off values in the discovery cohort. Kaplan Meier curves and a Cox proportional hazards regression models assessed overall survival (OS) hazard ratios (HR) for RNA-seq versus standard of care IHC cut-offs in the test cohort. RESULTS: Unsupervised PCA clustering identified three distinct PD-L1 groups separated by combinations of significant over- and under-representation of RNA-seq and IHC result measures from prior testing. The groups were labeled as “low” (rank ≤40), “moderate” (rank 41-73), and “high” (rank ≥74), based on the median RNA-seq rank for each group (+/- 1SD for low and high). Both the low and moderate groups were overrepresented by patients in the PD-L1 IHC low and negative groups. The moderate group was overrepresented by patients with moderately high PD-L1 RNA-seq ranks (median=70), while the low group was overrepresented by patients that were not PD-L1 high by RNA-seq. The high group was overrepresented by patients high for PD-L1 by both IHC and RNA-seq. OS HRs were better for RNA-seq high versus moderate (HR=0.05, CI 0.00-0.63, p=.02), and RNA-seq high versus low (HR=0.16, CI 0.03-0.86, p=.03) groups compared to standard of care IHC 22C3 high versus low groups, (HR=0.21, CI 0.04-1.07, p=.06). Findings were non-significant for the RNA-seq moderate versus low groups, likely due to the limited and disproportionately high number of patients with poor performance status in these groups. CONCLUSIONS: PD-L1 expression by RNA-seq demonstrated improved clinical sensitivity in predicting OS versus standard of care PD-LI IHC in a pembrolizumab treated NSCLC patient cohort. Additional studies are needed to further define cut-offs in the context of performance status, and better understand immune escape mechanisms in the moderate group. Citation Format: Mary K. Nesline, Sarabjot Pabla, Yong Hee Lee, Paul DePietro, Amy Early, Roger Klein, Shengle Zhang, Jeffrey Conroy. PD-L1 expression by RNA-sequencing and survival from pembrolizumab in non-small cell lung cancer (NSCLC) [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 1259.

Metastasis Related Epithelial-Mesenchymal Transition Signature Predicts Prognosis and Response to Immunotherapy in Gastric Cancer.

BackgroundIncreasing evidence has revealed the effect of epithelial-mesenchymal transition (EMT) on tumor microenvironment and cancer treatment. However, an EMT-based signature to predict the prognosis and therapeutic effect in gastric cancer (GC) has rarely been established.MethodsDifferentially expressed genes (DEGs) between paired primary gastric and ovarian metastatic tumors were identified through comparative RNA-seq analysis, followed by the construction of metastasis-related EMT signature (MEMTS) based on DEGs and EMT gene set. Then, both The Cancer Genome Atlas (TCGA) cohort and the Asian Cancer Research Group (ACRG) cohort were analyzed to explore the potential association between MEMTS and prognosis in GC. Samsung Medical Center (SMC) cohort and two individual immunotherapy treatment cohorts, including Kim cohort and Hugo cohort, were utilized to evaluate the predictive value of MEMTS on the response to adjuvant therapy and immunotherapy, respectively. Finally, the potential association of MEMTS with tumor environment and immune escape mechanisms was investigated.ResultsHigh MEMTS predicted a poor prognosis in patients with GC. Patients with low MEMTS potentially gained more benefits from adjuvant chemoradiotherapy than those with high MEMTS. MEMTS reliably predicted the response to immunotherapy in GC (area under the curve = 0.896). MEMTS was significantly associated with cancer-associated fibroblasts and stromal score in the aspect of the tumor microenvironment.ConclusionMEMTS serves as a potential biomarker to predict the prognosis and response to adjuvant therapy and immunotherapy in GC. MEMTS-based evaluation of individual tumors enables personalized treatment for GC patients in the future.

IMMU-15. The immunologic context of pediatric central nervous system malignancies and identification of pan-histology immunomodulatory targets

Abstract Central nervous system tumors account for the most childhood cancer mortality. Immunotherapies have made major contributions to treat adult cancers, but application of immunotherapy for childhood brain tumors has been limited, in part due to the unique CNS microenvironment and mechanisms of immune escape in this context. To investigate the immunologic context, we query the transcriptomic profile of ~700 primary brain tumors released by the Children’s Brain Tumor Network. An immune subtype classification from The Cancer Genome Atlas project reveals that 81% of high-grade tumors across molecular subtypes are characterized by an immunosuppressive phenotype (C4) while an inflammatory phenotype (C3) is more common in low-grade lesions (p<0.001). Adjusting for histologic grade and extent of resection, C4 associates with worse overall (OS) and progression-free survival (PFS) in this cohort (HR 3.1, p=0.008 and 1.6, p=0.03 respectively). Deconvolution of the transcriptome reveals that relative to C3, C4 tumors have decreased T-cell signature, OR 0.4 (0.2–0.8), and increased macrophage and tumor-proliferation signature (OR 2.0, 1.3–3.3, and 3.1, 2.3–4.2, respectively). In contrast to C3 tumors, T-cell signature in C4 tumors adversely impacts survival and correlates with multiple immunosuppressive genes and cytokines. Among them, the immune checkpoint CD276 has the highest associated impact on survival in C4 tumors (HR of log increase is 1.9, p<0.001). Additionally, high-grade lesions have suppressed expression of antigen-presenting genes. EZH2 is implicated in downregulating antigen presentation and is found to be significantly upregulated in all high-grade lesions in this cohort. Treatment with the EZH1/2 inhibitor valemetostat resulted in upregulation of antigen-presenting genes and tissue differentiation pathways across three murine syngeneic models, one modeling diffuse midline glioma and two embryonal models. Future in-vivo studies with genetic and chemical modification of immunomodulatory genes of interest aim to identify immunotherapeutic targets with potential for broad applicability in pediatric neuro-oncology.

A Novel Exosome-Relevant Molecular Classification Uncovers Distinct Immune Escape Mechanisms and Genomic Alterations in Gastric Cancer.

Objective: Gastric cancer (GC) is a highly heterogeneous malignant carcinoma. This study aimed to conduct an exosome-based classification for assisting personalized therapy for GC. Methods: Based on the expression profiling of prognostic exosome-related genes, GC patients in The Cancer Genome Atlas (TCGA) cohort were classified using the unsupervised consensus clustering approach, and the reproducibility of this classification was confirmed in the GSE84437 cohort. An exosome-based gene signature was developed via Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis. Immunological features, responses to immune checkpoint inhibitors, and genetic alterations were evaluated via computational methods. Results: Two exosome-relevant phenotypes (A and B) were clustered, and this classification was independent of immune subtypes and TCGA subtypes. Exosome-relevant phenotype B had a poorer prognosis and an inflamed tumor microenvironment (TME) relative to phenotype A. Patients with phenotype B presented higher responses to the anti-CTLA4 inhibitor. Moreover, phenotype B occurred at a higher frequency of genetic mutation than phenotype A. The exosome-based gene signature (GPX3, RGS2, MATN3, SLC7A2, and SNCG) could independently and accurately predict GC prognosis, which was linked to stromal activation and immunosuppression. Conclusion: Our findings offer a conceptual frame to further comprehend the roles of exosomes in immune escape mechanisms and genomic alterations of GC. More work is required to evaluate the reference value of exosome-relevant phenotypes for designing immunotherapeutic regimens.

Natural killer cell activity as a biomarker for the diagnosis of lung cancer in high-risk patients

ObjectiveNatural killer (NK) cells play an essential role in the immune response against cancer. However, immune escape mechanisms may cause inferior NK cell activity (NKA) in patients with cancer. This prospective study examined the relationship between NKA and lung cancer in a high-risk cohort.MethodsIn a cohort study, 250 participants referred by their general practitioner for suspicion of lung cancer were included. Before clinical investigation, blood was collected into NK Vue tubes, and the level of interferon gamma after 24 hours served as a surrogate marker for NKA.ResultsAmong 250 patients, 79 were diagnosed with lung cancer. No difference in NKA was found between patients with lung cancer and control participants in which lung cancer was ruled out (median 226 pg/mL vs. 450 pg/mL). However, there was a significant difference in NKA between patients with late-stage lung cancer and controls (median 161 pg/mL vs. 450 pg/mL). A linear regression model showed that NKA was not influenced by age, sex or smoking status.ConclusionsThe significantly lower NKA in patients with late-stage lung cancer warrants further investigation combining NKA with other biomarkers and examining the potential role of NKA as a marker of disseminated disease.

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]

Systematic analysis the genomic and microenvironment characteristics with the status of human leukocyte antigen (HLA) and the integration of HLA LOH, TMB and PD-L1 could further distinguish the responders to immunotherapy.

e20529 Background: Although studies have shown that loss of heterozygosity (LOH) at the human leukocyte antigen (HLA) locus is one of the mechanisms of immune escape, the effect of HLA LOH on the immune checkpoint inhibitors (ICIs) treatment is still need to be further investigated, especially for the patients with positive programmed cell death-Ligand 1 (PD-L1) expression and high tumor mutation burden (TMB). Methods: 425 sample of Chinese lung cancer patients who received PD-L1 immunotherapy were performed with targeted panel sequencing to analyzed the genomic characteristics with HLA status. 994 non-small cell lung cancer (NSCLC) sample from The Cancer Genome Atlas (TCGA) were used to analyze the impact of HLA LOH on the tumor microenvironment. In addition, 89 samples from previous study were used to analyze the association of HLA status with the ICIs treatment in NSCLC. Results: The genomic characteristics from 425 sample of Chinese lung cancer patients who received PD-L1 immunotherapy were analyzed. We found that mutation frequency of driver genes and TMB in the HLA LOH high group were significantly increased. Transcriptome analysis revealed HLA LOH played a critical role in the tumor immune microenvironment in PD-L1+/TMB-H population and the impact on lung adenocarcinoma was greater than that on lung squamous cell carcinoma. Progression-free survival of PD-L1+/TMB-H population was significantly decreased in HLA LOH group which suggest that comprehensive considering HLA LOH as a biomarker may improve clinical response to immunotherapy. Conclusions: This work showed the genomic and immune microenvironment characteristics of HLA LOH and demonstrated HLA LOH as a significant negative predictor of outcomes after ICIs treatment, especially in PD-L1+/TMB-H population. These data suggested that comprehensive considering HLA LOH as a biomarker may improve clinical response to immunotherapy.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

Addition of tyrosine kinase inhibitors (TKIs) in patients (pts) with unresectable hepatocellular carcinoma (HCC) who progress on first-line immunotherapy (IO).

e16193 Background: Atezolizumab plus bevacizumab is now standard first-line therapy for advanced HCC. Early phase trials in HCC and phase III trials in other solid tumors testing IO and TKI have shown greater antitumor activity with the combination than with either agent alone. Cancer and vascular endothelial cells release VEGF, which not only increases angiogenesis, promoting tumor growth, but also induces an immunosuppressive tumor microenvironment (TME). Preclinical studies suggest that TKIs with anti-VEGF activity have immunomodulatory potential, enabling the infiltration and activation of effector T cells to the TME. Experimental models of HCC with combination of IO plus TKI also reverted immune escape mechanisms mediated by β-catenin, Wnt, and TGFβ activation and by Treg or neutrophil infiltration. At our institution, pts who were treated with IO alone in the front line often had a TKI added on progression of disease (PD). We aimed to evaluate whether the addition of a TKI could rescue IO failure. Methods: We conducted an IRB-approved retrospective chart review of pts with unresectable HCC at Mount Sinai Health System who received IO from 1/2017 to 6/2021 and who subsequently received a TKI on PD. We assessed the objective response rate (ORR), median progression-free survival (mPFS) and overall survival (mOS). Follow-up CT/MRI imaging studies were conducted every 2-3 months after addition of TKI. Results: Of 277 pts who received first-line IO during the study period, 46 evaluable pts subsequently had TKIs added to IO upon PD. Pts were predominantly male (37/46), with median age 63 years, and 38 had cirrhosis. Etiologies of HCC were as follows: 9 HBV, 22 HCV, 15 alcohol-related, 7 NASH-associated. All pts received first-line nivolumab. TKIs added were lenvatinib (44/46) and sorafenib (2). At the start of IO, most pts (31/46) had Child Pugh class A liver disease and performance status ECOG 0 (31/46). TKIs were added upon PD and after a median of 5.8 months (range 2.1-28.1 months) from IO initiation. ORR of IO plus TKI was 15% (7/46; 2 complete responses and 5 partial responses). 16 pts had stable disease, and 13 had PD. Among the 11/46 pts who initially responded to first-line IO, 2 achieved a response with TKI. There were 5 responses among the 35/46 non-responders to first-line IO. TKI-related grade 3-4 adverse events, including liver toxicity, rash, and encephalopathy, led to discontinuation in 7/46 pts. mOS after adding TKI was 9.5 months (95% CI: 6.1-13.7) and mPFS was 5.4 months (95% CI: 3.6-9.7). The mOS and mPFS of adding TKI after PD did not differ between IO initial responders and non-responders. Overall, mOS from IO initiation was 17.6 months (95% CI: 14.5-24.4). Conclusions: Adding a TKI after PD on single agent IO in pts with advanced HCC did not add a signal of clinical benefit compared with reported outcomes from second-line TKIs alone.

Immune Microenvironment of Muscular-Invasive Urothelial Carcinoma: The Link to Tumor Immune Cycle and Prognosis.

In this study, we investigated the relationship between the tumor immune microenvironment (TIME), histological differentiation and hypoxia in patients with muscular-invasive urothelial carcinomas (MIUC) after radical cystectomy. Forty-two cases of pT2-3N0M0 MIUCs underwent clinical, histological and immunohistochemical evaluation by counting CD8+, FOXP3+, CD68+, CD163+ cells and polymorphonuclear leukocytes (PMN) in intra-tumoral and peritumoral areas, assessing PD-L1 and GLUT1 expression for defining the impact of tumor immune contexture on patients’ outcomes. Five-year survival rates and overall survival were calculated. Most of the MIUCs demonstrated the immune-desert or immune-excluded TIME, reflecting altered mechanisms of T-cells’ activation or traffic into tumors. Tumor immune contexture was closely related to histological differentiation. CD8+ cells were scant in MIUCs with papillary and squamous differentiation, while basal-like or mesenchymal-like histological differentiation was associated with increased density of CD8+ cells. A high rate of PD-L1 expression (47.6%) was not related to immune cell infiltration. M2-macrophages predominated under CD8+ lymphocytes. The abundance of PMN and CD163+ macrophages in MIUCs was associated with high GLUT1 expression. CD8+, CD68+, FOXP3+ cells and PD-L1 status did not affect patients’ outcomes, while high CD163+ density and PMN infiltration were associated with the unfavorable outcome of patients with MIUC. These data drive the hypothesis that in MIUC, immune escape mechanisms are shifted towards the role of the innate immunity cells rather than CD8+ lymphocytes’ functioning.

Host-virus interactions in PK-15 cells infected with Pseudorabies virus Becker strain based on RNA-seq

Pseudorabies is a highly contagious viral disease caused by the pseudorabies virus (PRV), and it is one of the most devastating diseases for the swine industry worldwide. However, the host-virus interaction and virus-related host factors at the mRNA level in virus natural host (pig) cells, are not fully understood. Here, we performed time-course RNA sequencing of the PK-15 cells infected with a recombinant strain PRV-Becker-GFP to study the dynamic competition between the host and the virus. At early stage of infection (3 hpi), our results suggested that the activation of cytosolic DNA-sensing pathway and NOD-like receptor signaling pathway might play a role in recognition of PRV, and the activation of NF-kappa B signaling pathway and TNF signaling pathway might be involved in immune response against the virus. However, all these pathways were subsequently inhibited by PRV. Additionally, our data indicated the fatty acid degradation pathway was significantly downregulated during late stage of infection (9 hpi), which was likely to accumulate fatty acids for viral envelope synthesis. Moreover, we verified the expression of 5 representative genes (ALDH1B1, ACAA2, ACSL3, ADH5, and EHHADH) related to fatty acid degradation pathway by RT-qPCR. Overall, our findings provide valuable information to better understand host-virus interactions and the immune escape mechanism of PRV-Becker as a virulent strain, offering novel targets for porcine anti-PRV breeding research and potential clinical treatment.

HIV-1 mutants that escape the cytotoxic T-lymphocytes are defective in viral DNA integration.

HIV-1 replication is durably controlled without antiretroviral therapy (ART) in certain infected individuals called elite controllers (ECs). These individuals express specific human leukocyte antigens (HLA) that tag HIV-infected cells for elimination by presenting viral epitopes to CD8+ cytotoxic T-lymphocytes (CTL). In HIV-infected individuals expressing HLA-B27, CTLs primarily target the viral capsid protein (CA)-derived KK10 epitope. While selection of CA mutation R264K helps HIV-1 escape this potent CTL response, the accompanying fitness cost severely diminishes virus infectivity. Interestingly, selection of a compensatory CA mutation S173A restores HIV-1 replication. However, the molecular mechanism(s) underlying HIV-1 escape from this ART-free virus control by CTLs is not fully understood. Here, we report that the R264K mutation-associated infectivity defect arises primarily from impaired HIV-1 DNA integration, which is restored by the S173A mutation. Unexpectedly, the integration defect of the R264K variant was also restored upon depletion of the host cyclophilin A. These findings reveal a nuclear crosstalk between CA and HIV-1 integration as well as identify a previously unknown role of cyclophilin A in viral DNA integration. Finally, our study identifies a novel immune escape mechanism of an HIV-1 variant escaping a CA-directed CTL response.

Nano Drug Delivery System for Tumor Immunotherapy: Next-Generation Therapeutics.

Tumor immunotherapy is an artificial stimulation of the immune system to enhance anti-cancer response. It has become a powerful clinical strategy for treating cancer. The number of immunotherapy drug approvals has been increasing in recent years, and many treatments are in clinical and preclinical stages. Despite this progress, the special tumor heterogeneity and immunosuppressive microenvironment of solid tumors made immunotherapy in the majority of cancer cases difficult. Therefore, understanding how to improve the intratumoral enrichment degree and the response rate of various immunotherapy drugs is key to improve efficacy and control adverse reactions. With the development of materials science and nanotechnology, advanced biomaterials such as nanoparticle and drug delivery systems like T-cell delivery therapy can improve effectiveness of immunotherapy while reducing the toxic side effects on non-target cells, which offers innovative ideas for improving immunity therapeutic effectiveness. In this review, we discuss the mechanism of tumor cell immune escape and focus on current immunotherapy (such as cytokine immunotherapy, therapeutic monoclonal antibody immunotherapy, PD-1/PD-L1 therapy, CAR-T therapy, tumor vaccine, oncolytic virus, and other new types of immunity) and its challenges as well as the latest nanotechnology (such as bionic nanoparticles, self-assembled nanoparticles, deformable nanoparticles, photothermal effect nanoparticles, stimuli-responsive nanoparticles, and other types) applications in cancer immunotherapy.