CTL-mediated Killing Research Articles

Hippo-signaling-controlled MHC class I antigen processing and presentation pathway potentiates antitumor immunity

The major histocompatibility complex class I (MHC class I)-mediated tumor antigen processing and presentation (APP) pathway is essential for the recruitment and activation of cytotoxic CD8+ T lymphocytes (CD8+ CTLs). However, this pathway is frequently dysregulated in many cancers, thus leading to a failure of immunotherapy. Here, we report that activation of the tumor-intrinsic Hippo pathway positively correlates with the expression of MHC class I APP genes and the abundance of CD8+ CTLs in mouse tumors and patients. Blocking the Hippo pathway effector Yes-associated protein/transcriptional enhanced associate domain (YAP/TEAD) potently improves antitumor immunity. Mechanistically, the YAP/TEAD complex cooperates with the nucleosome remodeling and deacetylase complex to repress NLRC5 transcription. The upregulation of NLRC5 by YAP/TEAD depletion or pharmacological inhibition increases the expression of MHC class I APP genes and enhances CD8+ CTL-mediated killing of cancer cells. Collectively, our results suggest a crucial tumor-promoting function of YAP depending on NLRC5 to impair the MHC class I APP pathway and provide a rationale for inhibiting YAP activity in immunotherapy for cancer.

Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells.

Cytotoxic CD8 +T lymphocytes (CTLs) are key players of adaptive anti-tumor immunity based on their ability to specifically recognize and destroy tumor cells. Many cancer immunotherapies rely on unleashing CTL function. However, tumors can evade killing through strategies which are not yet fully elucidated. To provide deeper insight into tumor evasion mechanisms in an antigen-dependent manner, we established a human co-culture system composed of tumor and primary immune cells. Using this system, we systematically investigated intrinsic regulators of tumor resistance by conducting a complementary CRISPR screen approach. By harnessing CRISPR activation (CRISPRa) and CRISPR knockout (KO) technology in parallel, we investigated gene gain-of-function as well as loss-of-function across genes with annotated function in a colon carcinoma cell line. CRISPRa and CRISPR KO screens uncovered 187 and 704 hits, respectively, with 60 gene hits overlapping between both. These data confirmed the role of interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α) and autophagy pathways and uncovered novel genes implicated in tumor resistance to killing. Notably, we discovered that ILKAP encoding the integrin-linked kinase-associated serine/threonine phosphatase 2 C, a gene previously unknown to play a role in antigen specific CTL-mediated killing, mediate tumor resistance independently from regulating antigen presentation, IFN-γ or TNF-α responsiveness. Moreover, our work describes the contrasting role of soluble and membrane-bound ICAM-1 in regulating tumor cell killing. The deficiency of membrane-bound ICAM-1 (mICAM-1) or the overexpression of soluble ICAM-1 (sICAM-1) induced resistance to CTL killing, whereas PD-L1 overexpression had no impact. These results highlight the essential role of ICAM-1 at the immunological synapse between tumor and CTL and the antagonist function of sICAM-1.

Genome-wide CRISPR screens define determinants of epithelial-mesenchymal transition mediated immune evasion by pancreatic cancer cells.

The genetic circuits that allow cancer cells to evade immune killing via epithelial mesenchymal plasticity remain poorly understood. Here, we showed that mesenchymal-like (Mes) KPC3 pancreatic cancer cells were more resistant to cytotoxic T lymphocyte (CTL)-mediated killing than the parental epithelial-like (Epi) cells and used parallel genome-wide CRISPR screens to assess the molecular underpinnings of this difference. Core CTL-evasion genes (such as IFN-γ pathway components) were clearly evident in both types. Moreover, we identified and validated multiple Mes-specific regulators of cytotoxicity, such as Egfr and Mfge8. Both genes were significantly higher expressed in Mes cancer cells, and their depletion sensitized Mes cancer cells to CTL-mediated killing. Notably, Mes cancer cells secreted more Mfge8 to inhibit proliferation of CD8+ T cells and production of IFN-γ and TNFα. Clinically, increased Egfr and Mfge8 expression was correlated with a worse prognosis. Thus, Mes cancer cells use Egfr-mediated intrinsic and Mfge8-mediated extrinsic mechanisms to facilitate immune escape from CD8+ T cells.

Arming a killer: mitochondrial regulation of CD8+ T cell cytotoxicity.

While once regarded as ATP factories, mitochondria have taken the spotlight as important regulators of cellular homeostasis. The past two decades have witnessed an intensifying interest in the study of mitochondria in cells of the immune system, with many new and unexpected roles for mitochondria emerging. Immune cells offer intriguing insights as mitochondria appear to play different roles at different stages of T cell development, matching the changing functions of the cells. Here we briefly review the multifaceted roles of mitochondria during T cell differentiation, focusing on CD8+ cytotoxic T lymphocytes (CTLs) and we consider how mitochondrial dysfunction can contribute to CTL exhaustion. In addition, we highlight a newly appreciated role for mitochondria as homeostatic regulators of CTL-mediated killing and explore the emerging literature describing mechanisms linking cytosolic and mitochondrial protein synthesis.

MicroRNAs affecting the susceptibility of melanoma cells to CD8+ T cell-mediated cytolysis.

The regulatory functions of microRNAs (miRNAs) in anti-tumour immunity have been mainly described in immune effector cells. Since little is known about miRNA effects on the susceptibility of target cells during T cell-target cell interaction, this study focused on the identification of miRNAs expressed in tumour cells controlling their susceptibility to CD8+ T cell-mediated cytotoxicity. Luciferase expressing B16F10 melanoma (B16F10 Luci+ ) cells transfected with individual miRNAs covering a comprehensive murine miRNA library were screened for their susceptibility to lysis by an established cytotoxic T lymphocyte (CTL) line (5a, clone Nβ) specific for the melanoma-associated antigen tyrosinase-related protein 2. miRNAs with the most pronounced effects on T cell-mediated lysis were validated and stably expressed in B16F10 cells. In silico analyses identified common targets of miRNA sets determined by the screen, which were further confirmed by small interfering RNA (siRNA)-mediated silencing experiments modulating immune surveillance. The Ingenuity Pathway Analysis (IPA) software and RNA sequencing (RNA-seq) data from miRNA-overexpressing cell lines were applied to investigate the underlying mechanisms. The Cancer Genome Atlas (TCGA)-derived miRNA sequencing data were used to assess the correlation of miRNA expression with melanoma patients' survival. The miRNA screen resulted in the selection of seven miRNAs enhancing CTL-mediated melanoma cell killing in vitro. Upon stable overexpression of selected miRNAs, hsa-miR-320a-3p, mmu-miR-7037-5p and mmu-miR-666-3p were determined as most effective in enhancing susceptibility to CTL lysis. In silico analyses and subsequent siRNA-mediated silencing experiments identified Psmc3 and Ndufa1 as common miRNA targets possibly involved in the functional effects observed. The analyses of RNA-seq data with IPA showed pathways, networks, biological functions and key molecules potentially involved in the miRNA-mediated functional effects. Finally, based on TCGA data analysis, a positive correlation of the conserved miRNAs among the panel of the seven identified miRNAs with overall survival of melanoma patients was determined. For the first time, this study uncovered miRNA species that affect the susceptibility of melanoma cells to T cell-mediated killing. These miRNAs might represent attractive candidates for novel therapy approaches against melanoma and other tumour entities.

SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly.

CTL-mediated killing of virally infected or malignant cells is orchestrated at the immune synapse (IS). We hypothesized that SARS-CoV-2 may target lytic IS assembly to escape elimination. We show that human CD8+ T cells upregulate the expression of ACE2, the Spike receptor, during differentiation to CTLs. CTL preincubation with the Wuhan or Omicron Spike variants inhibits IS assembly and function, as shown by defective synaptic accumulation of TCRs and tyrosine phosphoproteins as well as defective centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing and cytokine production. These defects were reversed by anti-Spike antibodies interfering with ACE2 binding and reproduced by ACE2 engagement by angiotensin II or anti-ACE2 antibodies, but not by the ACE2 product Ang (1-7). IS defects were also observed ex vivo in CTLs from COVID-19 patients. These results highlight a new strategy of immune evasion by SARS-CoV-2 based on the Spike-dependent, ACE2-mediated targeting of the lytic IS to prevent elimination of infected cells.

ICAM-1 on Breast Cancer Cells Suppresses Lung Metastasis but Is Dispensable for Tumor Growth and Killing by Cytotoxic T Cells.

Breast tumors and their derived circulating cancer cells express the leukocyte β2 integrin ligand Intercellular adhesion molecule 1 (ICAM-1). We found that elevated ICAM-1 expression in breast cancer cells results in a favorable outcome and prolonged survival of breast cancer patients. We therefore assessed the direct in vivo contribution of ICAM-1 expressed by breast cancer cells to breast tumorigenesis and lung metastasis in syngeneic immunocompetent mice hosts using spontaneous and experimental models of the lung metastasis of the C57BL/6-derived E0771 cell line, a luminal B breast cancer subtype. Notably, the presence of ICAM-1 on E0771 did not alter tumor growth or the leukocyte composition in the tumor microenvironment. Interestingly, the elimination of Tregs led to the rapid killing of primary tumor cells independently of tumor ICAM-1 expression. The in vivo elimination of a primary E0771 tumor expressing the ovalbumin (OVA) model neoantigen by the OVA-specific OVA-tcr-I mice (OT-I) transgenic cytotoxic T lymphocytes (CTLs) also took place normally in the absence of ICAM-1 expression by E0771 breast cancer target cells. The whole lung imaging of these cells by light sheet microscopy (LSM) revealed that both Wild type (WT)- and ICAM-1-deficient E0771 cells were equally disseminated from resected tumors and accumulated inside the lung vasculature at similar magnitudes. ICAM-1-deficient breast cancer cells developed, however, much larger metastatic lesions than their control counterparts. Strikingly, the vast majority of these cells gave rise to intravascular tumor colonies both in spontaneous and experimental metastasis models. In the latter model, ICAM-1 expressing E0771- but not their ICAM-1-deficient counterparts were highly susceptible to elimination by neutrophils adoptively transferred from E0771 tumor-bearing donor mice. Ex vivo, neutrophils derived from tumor-bearing mice also killed cultured E0771 cells via ICAM-1-dependent interactions. Collectively, our results are a first indication that ICAM-1 expressed by metastatic breast cancer cells that expand inside the lung vasculature is involved in innate rather than in adaptive cancer cell killing. This is also a first indication that the breast tumor expression of ICAM-1 is not required for CTL-mediated killing but can function as a suppressor of intravascular breast cancer metastasis to lungs.

A Sampling of Highlights from the Literature

What We're Reading| June 03 2022 A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors Author & Article Information Online Issn: 2326-6074 Print Issn: 2326-6066 ©2022 American Association for Cancer Research2022American Association for Cancer Research Cancer Immunol Res (2022) 10 (6): 671. https://doi.org/10.1158/2326-6066.CIR-10-6-WWR Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record June 3 2022 Citation A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors. Cancer Immunol Res 1 June 2022; 10 (6): 671. https://doi.org/10.1158/2326-6066.CIR-10-6-WWR Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Perforin pores can be repaired in cancer cells by ESCRT proteins (from Fig. 8 of Rudd-Schmidt et al. Nature Communications 2019) CTLs can destroy cancer cells through polarized release of perforins, which puncture the cancer cell's plasma membrane, and granzymes, which enter through the holes created by the perforins, initiating apoptosis. Using multiple high-resolution imaging techniques to visualize the cancer cell–CTL synapse over time, Ritter et al. find that endosomal sorting complexes required for transport (ESCRT) proteins are recruited within cancer cells to the synapse upon CTL release of perforin. Cancer-cell susceptibility to CTL-mediated killing is increased if ESCRT function in the cancer cells is blocked. The data identify ESCRT-mediated repair of perforin-induced pores as a potential mechanism by which cancer cells resist CTL-mediated killing. Ritter AT, …, Mellman I. Science 2022 April 21;376:377–382. IFNγ signaling increases CAR T-cell cytotoxicity against solid tumors (from LeCornichon via Wikimedia Commons) Chimeric-antigen... You do not currently have access to this content.

ESCRT-mediated membrane repair protects tumor-derived cells against T cell attack.

Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds. We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release. Inhibition of ESCRT machinery in cancer-derived cells enhanced their susceptibility to CTL-mediated killing. Thus, repair of perforin pores by ESCRT machinery limits granzyme entry into the cytosol, potentially enabling target cells to resist cytolytic attack.

The Expanding Arsenal of Cytotoxic T Cells.

Cytotoxic T cells (CTLs) are the main cellular mediators of the adaptive immune defenses against intracellular pathogens and malignant cells. Upon recognition of specific antigen on their cellular target, CTLs assemble an immunological synapse where they mobilise their killing machinery that is released into the synaptic cleft to orchestrate the demise of their cell target. The arsenal of CTLs is stored in lysosome-like organelles that undergo exocytosis in response to signals triggered by the T cell antigen receptor following antigen recognition. These organelles include lytic granules carrying a cargo of cytotoxic proteins packed on a proteoglycan scaffold, multivesicular bodies carrying the death receptor ligand FasL, and the recently discovered supramolecular attack particles that carry a core of cytotoxic proteins encased in a non-membranous glycoprotein shell. Here we will briefly review the main features of these killing entities and discuss their interrelationship and interplay in CTL-mediated killing.

Targeting the NANOG/HDAC1 axis reverses resistance to PD-1 blockade by reinvigorating the antitumor immunity cycle

Immune checkpoint blockade (ICB) therapy has shifted the paradigm for cancer treatment. However, the majority of patients lack effective responses because of the emergence of immune-refractory tumors that disrupt the amplification of antitumor immunity. Therefore, the identification of clinically available targets that restrict antitumor immunity is required to develop potential combination therapies. Here, using transcriptomic data on patients with cancer treated with programmed cell death protein 1 (PD-1) therapy and newly established mouse preclinical anti–PD-1 therapy–refractory models, we identified NANOG as a factor restricting the amplification of the antitumor immunity cycle, thereby contributing to the immune-refractory feature of the tumor microenvironment (TME). Mechanistically, NANOG induced insufficient T cell infiltration and resistance to CTL-mediated killing via the histone deacetylase 1–dependent (HDAC1-dependent) regulation of CXCL10 and MCL1, respectively. Importantly, HDAC1 inhibition using an actionable agent sensitized NANOGhi immune-refractory tumors to PD-1 blockade by reinvigorating the antitumor immunity cycle. Thus, our findings implicate the NANOG/HDAC1 axis as a central molecular target for controlling immune-refractory tumors and provide a rationale for combining HDAC inhibitors to reverse the refractoriness of tumors to ICB therapy.

Tnfrsf4-expressing regulatory T cells promote immune escape of chronic myeloid leukemia stem cells.

Leukemia stem cells (LSCs) promote the disease and seem resistant to therapy and immune control. Why LSCs are selectively resistant against elimination by CD8+ cytotoxic T cells (CTLs) is still unknown. In this study, we demonstrate that LSCs in chronic myeloid leukemia (CML) can be recognized and killed by CD8+ CTLs in vitro. However, Tregs, which preferentially localized close to CD8+ CTLs in CML BM, protected LSCs from MHC class I–dependent CD8+ CTL–mediated elimination in vivo. BM Tregs in CML were characterized by the selective expression of tumor necrosis factor receptor 4 (Tnfrsf4). Stimulation of Tnfrsf4 signaling did not deplete Tregs but reduced the capacity of Tregs to protect LSCs from CD8+ CTL–mediated killing. In the BM of newly diagnosed CML patients, TNFRSF4 mRNA levels were significantly increased and correlated with the expression of the Treg-restricted transcription factor FOXP3. Overall, these results identify Tregs as key regulators of immune escape of LSCs and TNFRSF4 as a potential target to reduce the function of Tregs and boost antileukemic immunity in CML.

Cell Softness Prevents Cytolytic T-cell Killing of Tumor-Repopulating Cells.

Biomechanics is a fundamental feature of a cell. However, the manner by which actomysin tension affects tumor immune evasion remains unclear. Here we show that although cytotoxic T lymphocytes (CTL) can effectively destroy stiff differentiated tumor cells, they fail to kill soft tumor-repopulating cells (TRC). TRC softness prevented membrane pore formation caused by CTL-released perforin. Perforin interacting with nonmuscle myosin heavy-chain 9 transmitted forces to less F-actins in soft TRC, thus generating an inadequate contractile force for perforin pore formation. Stiffening TRC allowed perforin the ability to drill through the membrane, leading to CTL-mediated killing of TRC. Importantly, overcoming mechanical softness in human TRC also enhanced TRC cell death caused by human CTL, potentiating a mechanics-based immunotherapeutic strategy. These findings reveal a mechanics-mediated tumor immune evasion, thus potentially providing an alternative approach for tumor immunotherapy. SIGNIFICANCE: Tumor-repopulating cells evade CD8+ cytolytic T-cell killing through a mechanical softness mechanism, underlying the impediment of perforin pore formation at the immune synapse site.

Abstract 5543: TAK1 deficiency in tumor cells enhances sensitivity to CTL-mediated killing via TNF-α

Abstract The clinical successes achieved by different immunotherapies have resulted in a paradigm shift in treatment modalities. Despite these significant advances, not all patients benefit from the use of these therapies, creating a need to develop additional approaches to enhance and broaden their clinical application. To identify genes whose products can increase or decrease the sensitivity of tumor cells to the immune system, we used a CTL assay to screen a whole genomic CRISPR library. We co-culture a mouse cell line, ID8, expressing a model antigen (Ova) with transgenic CD8 T cells (OT-I) recognizing this antigen. A set of controls that enhance or decrease CTL activity behaved as expected. Comparison of the CRISPR score identified several hits that increased or decreased the sensitivity of the tumor cells to CTL killing. Subsets of these hits belong to two pathways involved in CTL-mediated killing: the IFN-γ and the TNF-α signaling pathways. We evaluated which of these hits would be amenable to therapeutic modulation, and decided to focus on the kinase TAK1 for confirmation and validation studies. A TAK1 deficient cell line was more sensitive to CTL killing, which was prevented by expression of TAK1, confirming the role of TAK1 in this process. A TAK1 gene carrying an inactivation mutation K63W did not rescue the effects of TAK1 KO, indicating that TAK1 enzymatic activity was necessary. Several pathways mediate CTL killing: Perforin/Granzyme B, IFN-γ, TNF-α, Fas & TRAIL pathways. To determine TAK1 MOA, we studied the effects of a Perforin/Granzyme B inhibitor CMA. CMA inhibited CTL activity in a dose-dependent manner on WT cells, but did not inhibit CTL activity on TAK1 deficient cells, indicating TAK1 effects are independent of this pathway. We then tested the sensitivity of TAK1 KO cells to TNF-α. TAK1 KO cells were more sensitive to TNF-α mediated killing, and similar results were observed with several additional cell lines (MC38, EMT6, KP). TNF-α can activate the JNK, p38, and NF-κB pathways, and the apoptosis extrinsic pathway to regulate cell growth and cell death. Kinetics studies monitoring pathway activity upon TNF-α stimulation showed that TAK1 KO cell lines induced cFLIP degradation before observing PARP cleavage, and that the NF-κB pathway, which has been observed to mediate cFLIP synthesis, was not activated. We proceeded to evaluate the effects of TAK1 deficiency in a mouse syngeneic model. TAK1 deficiency resulted in reduced growth and increased survival in the MC38 in vivo model. In summary, by screening a CRISPR library against a CTL assay, we identified TAK1 as a novel potential target for immunotherapies. TAK1 deficiency enhances CTL killing and results in decreased tumor growth and increased survival in vivo. This results support the development of TAK1 inhibitors to enhance the anti-tumor action of the immune system. Citation Format: Juan J. Miret, Troy A. Luster, Patrick Lizotte, Min Wu, Sarah Nzikoba, Luke Taus J. Taus, Prafulla C. Gokhale, Paul Kirschmeier, David Barbie, Cloud P. Paweletz. TAK1 deficiency in tumor cells enhances sensitivity to CTL-mediated killing via TNF-α [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5543.

Investigation of Cytotoxic T Lymphocyte Function during Allorejection in the Anterior Chamber of the Eye.

Cytotoxic T lymphocytes (CTL) are an essential part of our immune system by killing infected and malignant cells. To fully understand this process, it is necessary to study CTL function in the physiological setting of a living organism to account for their interplay with other immune cells like CD4+ T helper cells and macrophages. The anterior chamber of the eye (ACE), originally developed for diabetes research, is ideally suited for non-invasive and longitudinal in vivo imaging. We take advantage of the ACE window to observe immune responses, particularly allorejection of islets of Langerhans cells by CTLs. We follow the onset of the rejection after vascularization on islets until the end of the rejection process for about a month by repetitive two-photon microscopy. We find that CTLs show reduced migration on allogeneic islets in vivo compared to in vitro data, indicating CTL activation. Interestingly, the temporal infiltration pattern of T cells during rejection is precisely regulated, showing enrichment of CD4+ T helper cells on the islets before arrival of CD8+ CTLs. The adaptation of the ACE to immune responses enables the examination of the mechanism and regulation of CTL-mediated killing in vivo and to further investigate the killing in gene-deficient mice that resemble severe human immune diseases.

Microenvironment-Dependent Gradient of CTL Exhaustion in the AE17sOVA Murine Mesothelioma Tumor Model.

The immune system, and in particular, cytotoxic CD8+ T cells (CTLs), plays a vital part in the prevention and elimination of tumors. In many patients, however, CTL-mediated tumor killing ultimately fails in the clearance of cancer cells resulting in disease progression, in large part due to the progression of effector CTL into exhausted CTL. While there have been major breakthroughs in the development of CTL-mediated “reinvigoration”-driven immunotherapies such as checkpoint blockade therapy, there remains a need to better understand the drivers behind the development of T cell exhaustion. Our study highlights the unique differences in T cell exhaustion development in tumor-specific CTL which arises over time in a mouse model of mesothelioma. Importantly, we also show that peripheral tumor-specific T cells have a unique expression profile compared to exhausted tumor-infiltrating CTL at a late-stage of tumor progression in mice. Together, these data suggest that greater emphasis should be placed on understanding contributions of individual microenvironments in the development of T cell exhaustion.

Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing.

Upon tumor antigen recognition, cytotoxic T lymphocytes (CTLs) and target cells form specialized supramolecular structures, called cytotoxic immunological synapses, which are required for polarized delivery of cytotoxic granules. In previous reports, we described the accumulation of connexin 43 (Cx43)-formed gap junctions (GJs) at natural killer (NK) cell–tumor cell cytotoxic immunological synapse. In this report, we demonstrate the functional role of Cx43-GJs at the cytotoxic immunological synapse established between CTLs and melanoma cells during cytotoxicity. Using confocal microscopy, we evaluated Cx43 polarization to the contact site between CTLs isolated from pMEL-1 mice and B16F10 melanoma cells. We knocked down Cx43 expression in B16F10 cells and evaluated its role in the formation of functional GJs and the cytotoxic activity of CTLs, by calcein transfer and granzyme B activity assays, respectively. We found that Cx43 localizes at CTL/B16F10 intercellular contact sites via an antigen-dependent process. We also found that pMEL-1 CTLs but not wild-type naïve CD8+ T cells established functional GJs with B16F10 cells. Interestingly, we observed that Cx43-GJs were required for an efficient granzyme B activity in target B16F10 cells. Using an HLA-A2-restricted/MART-1-specific CD8+ T-cell clone, we confirmed these observations in human cells. Our results suggest that Cx43-channels are relevant components of cytotoxic immunological synapses and potentiate CTL-mediated tumor cell killing.

Abstract 1200: AXL targeting enhances lymphocyte-mediated cytotoxicity of lung cancer cells

Abstract As immunotherapies are now used to treat a large proportion of NSCLC patients, defining mechanisms of immune resistance is critical. Immune resistance may arise from both genetic instability and tumor heterogeneity driven by microenvironmental stresses such as hypoxia that promotes carcinoma cell plasticity as well as extrinsic or intrinsic mechanisms of immune resistance. AXL, a member of the TAM receptor tyrosine kinase family is widely expressed human cancers and increasingly recognized for its role in cell plasticity and drug resistance. In this study, we used a model of hypoxia-induced tumor plasticity to generate multiple lung cancer clones with mesenchymal and epithelial features to address mechanisms of immune resistance. We demonstrate that AXL expression is dramatically increased in mesenchymal lung cancer clones. Moreover, expression of AXL in the cells was correlated with an increased cancer cell intrinsic resistance to both NK and CTL-mediated killing, Notably, small molecule AXL targeting potently sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte-mediated killing. Mechanistically, we showed that attenuation of AXL-dependent immune resistance to immune cells involved a novel molecular network comprising NF-κB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Congruently, higher ICAM1 and ULBP1 tumor expression, correlated with improved patient survival in two NSCLC cohorts. These results reveal a novel AXL-mediated immune escape regulatory pathway, suggest AXL as a novel candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC. Citation Format: Sallem Chouaib, Stephane Terry, Stephanie Buart, Agnete Engelsen, Gro Gausdal, James Lorens, Jean Paul Thiery, Fathia Mami-Chouaib. AXL targeting enhances lymphocyte-mediated cytotoxicity of lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1200.

Abstract 5754: Hypoxia-induced tumor plasticity and immune resistance involves an alteration of target recognition by a mechanism involving TGF-beta signaling

Abstract Backgound: Hypoxia is known to shape the tumor microenvironment of developing tumors and contributes to both tumor progression and escape to immune surveillance. Previous work using non-small cell lung carcinoma cells (NSCLC) showed that hypoxia promotes epithelial-mesenchymal transition (EMT) in a fraction of carcinoma cells and that acquisition of a more mesenchymal (Mes) phenotype by certain cancer subclones correlates with an increased propensity to resist cytotoxic T lymphocytes (CTL) attacks as compared to more Epithelial (Epi) cancer subclones. Moreover, numerous studies identified alterations or loss of major histocompatibility complex (MHC) class I as a potential mechanism of tumor escape in cancer. Here, we asked whether hypoxia-induced EMT could regulate MHC class I expression and whether that could impact on resistance to CTL-mediated killing. Methods and Results : By comparing CMH class I in various NSCLC cancer subclones that have experienced hypoxic stress, we found that cancer clones with enhanced Mes features have a reduced expression of CMH class I molecules, as compared to those with a more Epi phenotype. Transient siRNA knockdown of hypoxia effectors HIF-1α or HIF-2α did not affect the expression of CMH class I molecules suggesting that EMT effectors, rather that HIFs, directly regulate CMH class I expression. Among EMT effectors, TGF-β expression was found to be highly upregulated in Mes cancer clones. Moreover, treatment of these cells with TGF-βR inhibitors markedly upregulated CMH class I expression. and as target cells, increases their susceptibility to CTL-mediated killing. However, blocking of TGF-β using Ab to TGF-β during the coculture time had no significant effects on CTL-mediated killing. This suggests that the observed changes in target cell susceptibility occurs through TGF-β-mediated repression of MHC class I rather than by cancer cell production of TGF-β affecting CTL effector functions. Conclusions: These results suggest that the increase in TGF-β signaling, especially found in EMTed Mes carcinoma cells in response to hypoxic stress, contributes to downregulate CMH class I expression in these cells while coinciding with a reduced susceptibilty to CTL attacks. The development of agents targeting hypoxia or EMT components should provide new therapeutic opportunities in combination with current approaches. Citation Format: Stephane Terry, Stephanie Buart, Jean-Paul Thiery, Fathia Mami-Chouaib, Salem Chouaib. Hypoxia-induced tumor plasticity and immune resistance involves an alteration of target recognition by a mechanism involving TGF-beta signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5754.

Killing without poking holes

Cancer Immunology Given the success of T cell–centric cancer immunotherapies, there is considerable interest in understanding exactly how tumors sometimes evade this form of treatment. Kearney et al. carried out a series of genome-wide CRISPR screens to identify mechanisms of tumor immune evasion from cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Interferon-γ signaling and antigen presentation were critical for CTL-mediated killing of cancer cells, and TNF (tumor necrosis factor) signaling was a key effector mechanism for both CTL and NK cell antitumor activity. The same immune evasion mechanisms arose upon screening with perforin-deficient CTLs, suggesting that tumors evade the immune system by dampening the effects of cytokines, rather than reducing direct killing via perforin. Sci. Immunol. 3 , eaar3451 (2018).