Cytotoxic T-cell Function Research Articles

Emerging and multifaceted role of neutrophils in lung cancer.

It has long been recognized that cigarette smoking is a shared risk factor for lung cancer and the debilitating lung disease, chronic obstructive pulmonary disease (COPD). As the severity of COPD increases, so does the risk for developing lung cancer, independently of pack years smoked. Neutrophilic inflammation increases with COPD severity and anti-inflammatories such as non-steroidal anti-inflammatory drugs (NSAIDs) can modulate neutrophil function and cancer risk. This review discusses the biology of tumour associated neutrophils (TANs) in lung cancer, which increase in density with tumour progression, particularly in smokers with non-small cell lung cancer (NSCLC). It is now increasingly recognized that neutrophils are responsive to the tumour microenvironment (TME) and polarize into distinct phenotypes that operate in an anti- (N1) or pro-tumorigenic (N2) manner. Intriguingly, the emergence of the pro-tumorigenic N2 phenotype increases with tumour growth, to suggest that cancer cells and the surrounding stroma can re-educate neutrophils. The neutrophil itself is a potent source of reactive oxygen species (ROS), arginase, proteases and cytokines that paradoxically can exert a potent immunosuppressive effect on lymphocytes including cytotoxic T cells (CTLs). Indeed, the neutrophil to lymphocyte ratio (NLR) is a systemic biomarker that is elevated in lung cancer patients and prognostic for poor survival outcomes. Herein, we review the molecular mechanisms by which neutrophil derived mediators can suppress CTL function. Selective therapeutic strategies designed to suppress pathogenic neutrophils in NSCLC may cooperate with immune checkpoint inhibitors (ICI) to increase CTL killing of cancer cells in the TME.

Combination of atezolizumab and pirfenidone in second-line and beyond NSCLC: A phase I/II study.

TPS2678 Background: Checkpoint inhibitors (CPI) targeting the PD1/PD-L1 axis significantly improved patient outcomes in stage IV non-small cell lung cancer (NSCLC). However, these patients will eventually develop resistance and progression. There is a need to identify novel treatment options. Poor response to PD-L1 antibody was correlated with increase in cancer-associated fibroblasts (CAF), which is known to interact with cytotoxic T cells (CTLs) by suppressing their function in a manner similar to regulatory T cells (Tregs). Production of cytokines by CAFs leads to impaired antitumor immunity by impairing CTL function (TGF beta) and prevent recruitment/mobilization of CTLs into tumors. These effects suggesting that CAF can be a therapeutic target in lung cancer resistant to checkpoint inhibitors. Pirfenidone (P) is approved to treat pulmonary fibrosis with anti-fibrotic effect by blocking the differentiation of fibroblasts into CAFs and suppress the production of TGF beta and TGF beta-induced signaling pathways/collagens. Atezolizumab is a humanized immunoglobulin (Ig) G1 monoclonal antibody that targets PD-L1 and inhibits the interaction between PD-L1 and its receptors, PD-1 and B7-1 (CD80), both of which function as inhibitory receptors expressed on T cells. We proposed a phase I/II trial to test the combination of atezolizumab (A) with P in patients with recurrent non-small cell lung cancer (NSCLC) after progression with CPI. The primary objective of phase I is to determine the maximum tolerated (MTD) dose of P in combination with A and assess the safety and tolerability of this combination. The secondary objective is to determine the efficacy of AP in all NSCLC participants treated in this study. Exploratory objectives include the measurement of circulating levels of TGF beta and research in expression of CAF related proteins. Methods: The initial phase I will enroll 3 patients using P at 801 mg po TID. A will be at 1200mg iv every 3 weeks. If there is ≤ 1 DLT, the study will proceed to phase II If there are 2- 3 DLT, P will be reduced to 534 mg TID. If there is ≤ 1 DLT, then this dose will proceed to phase II. If there is 2-3 DLT, then the study will be terminated. The phase II will enroll 16 patients to assess efficacy. Main inclusion criteria are patients with recurrent NSCLC after progression with first-line therapy CPI with or without chemotherapy, measurable disease, ECOG 0-2, and adequate organ function. Clinical trial information: NCT04467723.

Blockade of transforming growth factor β2 by anti-sense oligonucleotide improves immunotherapeutic potential of IL-2 against melanoma in a humanized mouse model

Background aimsIL-2 is a potent cytokine that activates natural killer cells and CD8+ cytotoxic T lymphocytes (CTLs) and has been approved for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, the medical use of IL-2 is restricted because of its narrow therapeutic window and potential side effects, including the expansion of regulatory T cells (Tregs). MethodsIn this study, the authors investigated the complementary effects of transforming growth factor-β2 (TGF-β2) anti-sense oligodeoxynucleotide (TASO) on the immunotherapeutic potential of IL-2 in a melanoma-bearing humanized mouse model. ResultsThe authors observed that the combination of TASO and IL-2 facilitated infiltration of CTLs into the tumor, thereby potentiating the tumor killing function of CTLs associated with increased granzyme B expression. In addition, TASO attenuated the increase in Tregs by IL-2 in the peripheral blood and spleen and also inhibited infiltration of Tregs into the tumor, which was partly due to decreased CCL22. Alteration of T-cell constituents at the periphery by TGF-β2 inhibition combined with IL-2 might be associated with the synergistic augmentation of serum pro-inflammatory cytokines (such as interferon γ and tumor necrosis factor α) and decreased ratio of Tregs to CTLs in tumor tissues, which consequently results in significant inhibition of tumor growth ConclusionsThese results indicate that the application of TASO improves IL-2-mediated anti-tumor immunity, thus implying that blockade of TGF-β2 in combination with IL-2 may be a promising immunotherapeutic strategy for melanoma.

Teasing out function from morphology: Similarities between primary cilia and immune synapses.

Immune synapses are formed between immune cells to facilitate communication and coordinate the immune response. The reorganization of receptors involved in recognition and signaling creates a transient area of plasma membrane specialized in signaling and polarized secretion. Studies on the formation of the immune synapse between cytotoxic T lymphocytes (CTLs) and their targets uncovered a critical role for centrosome polarization in CTL function and suggested a striking parallel between the synapse and primary cilium. Since these initial observations, a plethora of further morphological, functional, and molecular similarities have been identified between these two fascinating structures. In this review, we describe how advances in imaging and molecular techniques have revealed additional parallels as well as functionally significant differences and discuss how comparative studies continue to shed light on the molecular mechanisms underlying the functions of both the immune synapse and primary cilium.

Stem Cell-Derived Viral Antigen-Specific T Cells Suppress HIV Replication and PD-1 Expression on CD4+ T Cells.

The viral antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress the human immunodeficiency virus (HIV) infection. After adoptive transfer, PSC-CTLs can infiltrate into the local tissues to suppress HIV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs elicit the antiviral response remain to be fully elucidated. In this study, we generated the functional HIV-1 Gag epitope SL9-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the suppression of SL9-specific iPSC-CTLs on viral replication and the protection of CD4+ T cells. A chimeric HIV-1, i.e., EcoHIV, was used to produce HIV replication in mice. We show that adoptive transfer of SL9-specific iPSC-CTLs greatly suppressed EcoHIV replication in the peritoneal macrophages and spleen in the animal model. Furthermore, we demonstrate that the adoptive transfer significantly reduced expression of PD-1 on CD4+ T cells in the spleen and generated persistent anti-HIV memory T cells. These results indicate that stem cell-derived viral Ag-specific CTLs can robustly accumulate in the local tissues to suppress HIV replication and prevent CD4+ T cell exhaustion through reduction of PD-1 expression.

PD-L1 silencing inhibits triple-negative breast cancer development and upregulates T-cell-induced pro-inflammatory cytokines

Triple-negative breast cancer (TNBC) is an invasive tumor with a high incidence of distant metastasis and poor prognosis. In TNBC cells, high PD-L1 expression can induce an immunosuppressive tumor microenvironment, repressing the anti-tumoral immune responses. Although FDA-approved agents targeting the PD-1/PD-L1 axis are potent to eliminate tumoral cells, their immune-related adverse events have become worrisome. As the regulator of gene expression, siRNAs can directly target PD-L1 in breast cancer cells. The gene modification of tumoral PD-L1 can reduce our reliance on the current method of targeting the PD-L1/PD-1 axis. We initiated the study with bioinformatics analysis; the results indicated that TNBC and the MDA-MB-231 cells significantly overexpressed PD-L1 compared to other breast cancer subtypes and cell lines. Our results demonstrated that PD-L1 silencing substantially reduced PD-L1 expression at mRNA and protein levels in MDA-MB-231 cells.Moreover, our results demonstrated that PD-L1 knockdown reduced cancer cell proliferation and induced apoptosis via intrinsic and extrinsic apoptosis pathways. We observed that PD-L1 silencing effectively inhibited the migration of TNBC cells. Further investigation also displayed that silencing of PD-L1 in breast cancer cells induced T-cell cytotoxic function by upregulating the gene expression of pro-inflammatory cytokines, i.e., IL-2, IFN-γ, and TNF-α, and downregulating the gene expression of anti-inflammatory cytokines, i.e., IL-10, and TGF-β, in a co-culture system.

Potentiating CD8+ T cell antitumor activity by inhibiting PCSK9 to promote LDLR-mediated TCR recycling and signaling

Metabolic regulation has been proven to play a critical role in T cell antitumor immunity. However, cholesterol metabolism as a key component of this regulation remains largely unexplored. Herein, we found that the low-density lipoprotein receptor (LDLR), which has been previously identified as a transporter for cholesterol, plays a pivotal role in regulating CD8+ T cell antitumor activity. Besides the involvement of cholesterol uptake which is mediated by LDLR in T cell priming and clonal expansion, we also found a non-canonical function of LDLR in CD8+ T cells: LDLR interacts with the T-cell receptor (TCR) complex and regulates TCR recycling and signaling, thus facilitating the effector function of cytotoxic T-lymphocytes (CTLs). Furthermore, we found that the tumor microenvironment (TME) downregulates CD8+ T cell LDLR level and TCR signaling via tumor cell-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) which binds to LDLR and prevents the recycling of LDLR and TCR to the plasma membrane thus inhibits the effector function of CTLs. Moreover, genetic deletion or pharmacological inhibition of PCSK9 in tumor cells can enhance the antitumor activity of CD8+ T cells by alleviating the suppressive effect on CD8+ T cells and consequently inhibit tumor progression. While previously established as a hypercholesterolemia target, this study highlights PCSK9/LDLR as a potential target for cancer immunotherapy as well.

Abstract PS6-49: Intratumoral cytotoxic t-lymphocyte numbers and chemokine predict long-term survival of triple-negative breast cancer independently of tumor mutational burden

Abstract Background: Cytotoxic T-lymphocyte (CTL) infiltration into tumor is a positive prognostic factor in breast cancer. While high tumor mutation burden (TMB) is also considered as a predictor of tumor immunogenicity and response to immunotherapy. Triple-negative breast cancer (TNBC) has a higher TMB and accumulates more CTLs compared to other breast cancer subtypes. CTLs are identified by CD8 surface marker, which is encoded by CD8A gene. Granzyme B (GZMB) is a serine protease that is secreted by activated CTLs to induce apoptosis of the target cells. Chemokines, such as CXCL10 and CCL5 are key to the selective attraction of activated CTLs into tumors, as shown in multiple cancers. However, it remains unknown whether tumor infiltrating functional CTLs levels correlate with improved patient survival and are independent of TMB. In order to investigate it, we developed Functional Hotness Score (FHS) combining gene expressions of markers and attractants of activated CTL. Methods: Utilizing publicly available breast cancer cohorts, we established Functional Hotness Score (FHS), based on gene expression levels of CTL and chemokine markers in bulk tumors. The associations of FHS and breast cancer patient prognosis as well as distinct immunity markers were analyzed. Results: CD8, GZMB and CXCL10 combination resulted in the best prediction of the breas cancer patient prognosis. Thus, we established FHS based on the expression levels of these three genes. Breast cancer patients with the high-FHS tumors showed significantly better survival. FHS was lower in the metastatic breast cancers. Among breast cancer subtypes, triple-negative breast cancer (TNBC) showed the highest FHS. FHS predicted patient survival in hormone receptor (HR)-negative, especially TNBC, but not in HR-positive breast cancer. The high-FHS TNBCs showed not only higher CD8+ T cell infiltration, but also enhanced broader type-1 anti-cancer immunity. The patients with the high-FHS tumors showed better prognosis not only in high-TMB tumors but also in low-TMB TNBCs. The combination of high-TMB with high-FHS identified a unique subset of patients who do not recur over time. Conclusions: TNBCs with high-FHS based on the expression levels of CD8A, GZMB and CXCL10 showed improved prognosis with higher anti-cancer immunity regardless of TMB. FHS constitutes an independent prognostic marker of survival, particularly robust when combined with TMB in TNBCs. Citation Format: Eriko Katsuta, Li Yan, Mateusz Opyrchal, Pawel Kalinski, Kazuaki Takabe. Intratumoral cytotoxic t-lymphocyte numbers and chemokine predict long-term survival of triple-negative breast cancer independently of tumor mutational burden [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-49.

Synthesis of 6-epi-tuberiferin and the biological activities of tuberiferin, dehydrobrabrachylaenolide, 6-epi-tuberiferin, and their synthetic intermediates

Tuberiferin, 6-epi-tuberifelin, dehydrobrachylaenolide and two series of eudesmanolides, eudesmane-12,6 α-lactones and eudesmane-12,6β-lactones, were synthesized for the studies of the structure–activity relationships to explore novel anti-inflammatory, anti-cancer and crop disease prevention agents. The anti-inflammatory activities were tested by the inhibitory on the induction of inter-cellular adhesion molecule (ICAM-1), the permeation of leucocyte into inflammatory air pouch of murine, the killing function of cytotoxic T-lymphocytes (CTL), production of IL-1; The anti-cancer activities were established on the cytotoxic activities to six kinds of cell lines (P388, CCRF-CEM, VA-13, HepG2, QG-56, and WI-38). Results showed that Dehydrobrachylaenolide (an exo-endo cross conjugated dienone and α-methylene γ-lactone) was the most effective compound inhibiting ICAM-1 (IC50 3.0 μM) and the cell line VA-13 (IC50 0.45 μM); Compound 20 with an α-bromo-ketone moiety embraced the most potent inhibitory activity towards the permeation of leucocyte into inflammatory air pouches of murine in vivo (inhibitory ratio 54% at 10 mg); Compound 25 with an α-bromo-ketone and α-methylene trans-γ-lactone) showed the most significant inhibitory activity on the killing function of CTL (IC50 18 μM), as well as the cell lines of CCRF-CEM (IC50 1.1 μM) and P388 (IC50 1.21 μM) ; Tuberiferin (an α,β-unsaturated ketone and α- methylene γ-lactone) was on the top effective inhibitory on the production of IL-1; Compounds 19 with an α-bromo-ketone and α-methylene cis-γ-lactone exhibited the most potent inhibitory of QG-56 (IC50 12.5 μM); Compound 29 with an α-bromo-α,β-unsaturated ketone and α-methylene γ-lactone) showed significant inhibitory for HepG2 (IC50 1.23 μM) , though potently inhibited WI 38 (IC50 0.31 μM) as well. A conclusion may be reached that the α-Methylene γ-lactone moiety, exo-endo cross conjugated dienone moiety, and α-bromo-ketone withα-methylene moiety might be essential for eudesmanolides in the expression of their anti-inflammatory, anti-tumour biological activities. Similarly, the above mentioned key moieties are also responsible for the preventive activity of crop disease controlling.

ARID1A Mutation May Define an Immunologically Active Subgroup in Patients with Microsatellite Stable Colorectal Cancer.

AT-rich interactive domain 1A (ARID1A) is commonly mutated in colorectal cancer, frequently resulting in truncation and loss of protein expression. ARID1A recruits MSH2 for mismatch repair during DNA replication. ARID1A deficiency promotes hypermutability and immune activation in preclinical models, but its role in patients with colorectal cancer is being explored. The DNA sequencing and gene expression profiling of patients with colorectal cancer were extracted from The Cancer Genome Atlas and MD Anderson Cancer Center databases, with validation utilizing external databases, and correlation between ARID1A and immunologic features. IHC for T-cell markers was performed on a separate cohort of patients. Twenty-eight of 417 patients with microsatellite stable (MSS) colorectal cancer (6.7%) had ARID1A mutation. Among 58 genes most commonly mutated in colorectal cancer, ARID1A mutation had the highest increase with frameshift mutation rates in MSS cases (8-fold, P < 0.001). In MSS, ARID1A mutation was enriched in immune subtype (CMS1) and had a strong correlation with IFNγ expression (Δz score +1.91, P < 0.001). Compared with ARID1A wild-type, statistically significant higher expression for key checkpoint genes (e.g., PD-L1, CTLA4, and PDCD1) and gene sets (e.g., antigen presentation, cytotoxic T-cell function, and immune checkpoints) was observed in mutant cases. This was validated by unsupervised differential expression of genes related to immune response and further confirmed by higher infiltration of T cells in IHC of tumors with ARID1A mutation (P = 0.01). The immunogenicity of ARID1A-mutant cases is likely due to an increased level of neoantigens resulting from increased tumor mutational burden and frameshift mutations. Tumors with ARID1A mutation may be more susceptible to immune therapy-based treatment strategies and should be recognized as a unique molecular subgroup in future immune therapy trials.

Neutrophil Extracellular Traps, Local IL-8 Expression, and Cytotoxic T-Lymphocyte Absence in the Lungs of Fatal COVID-19: A Retrospective Histopathology Cohort Study

Background: Excessive inflammation is pathogenic in pneumonitis associated to severe COVID-19. Neutrophils are among the most abundantly present leukocytes in the inflammatory infiltrates and may form neutrophil extracellular traps (NETs) under the local influence of cytokines. NETs constitute a defence mechanism against bacteria but have also been shown to mediate tissue damage in a number of diseases. Methods: In this retrospective cohort study, sixteen immediate post-mortem lung biopsies were methodologically analysed as exploratory and validation cohorts. NETs were quantitatively analysed by multiplexed immunofluorescence and correlated with local levels of IL-8 mRNA expression and the density of CD8+ T-cell infiltration. SARS-CoV-2 presence in tissue was quantified by RT-PCR and immunohistochemistry.Findings: NETs were found in the lung interstitium and surrounding the bronchiolar epithelium with interindividual and spatial heterogeneity. NET density did not correlate with SARS-CoV-2 tissue viral load. NETs were associated with local IL-8 mRNA levels. NETs were also detected in pulmonary thrombi and in only one out of eight liver tissues in spatial fashion. NET focal presence negatively correlated with CD8+ T-cell infiltration in the lungs. Interpretation: Abundant neutrophils undergoing NETosis are found in the lungs of patients with fatal COVID-19, showing no correlation with viral loads. The strong association between NETs and IL-8 focal expression points to this chemokine as the potential causative factor. The function of cytotoxic T-lymphocytes in the immune responses against SARS-CoV-2 may be interfered by the presence of NETs.Funding Information: This study was supported by Banco Bilbao Vizcaya (BBVA) Foundation, “Ayudas a Equipos de Investigación Científica SARS-CoV-2 y COVID-19”. Declaration of Interests: I.M. reports receiving commercial research grants from BMS, Bioncotech, Alligator, Pfizer, Leadartis and Roche; has received speakers bureau honoraria from MSD; and is a consultant or advisory board member for BMS, Roche, Genmab, F-Star, Bioncotech, Bayer, Numab, Pieris, Alligator, and Merck Serono. C.E.A reports research grants from AstraZeneca. All other authors declare no competing interests.Ethics Approval Statement: This study was approved by the ethics committee of the University of Navarra, Spain (Approval 2020.192). Tissue collections were obtained with consent from a first-degree relative, following a protocol approved by the ethics committee of the University of Navarra (Protocol 2020.192p).

Application of a newly-developed cynomolgus macaque BiTE-mediated cytotoxic T-lymphocyte activity assay to various immunomodulatory agents in vitro

The immunotoxic potential of drug candidates is assessed through the examination of results from a variety of in vitro and in vivo immunophenotyping and functional study endpoints in pre-clinical studies. CD8+ cytotoxic T-lymphocyte (CTL) activity impairment by immunosuppressive agents is recognized to be a potentiating factor for decreased antiviral defense and increased cancer risk. A bi-specific T-cell engager (BiTE®)-mediated CTL activity assay that applies to ex vivo experimentation in non-human primates in the context of toxicology studies was successfully developed and applied in cynomolgus monkey regulatory studies. While an ex vivo analysis conducted in the context of repeat-dose toxicology studies focuses on the long-term impact on CTL function, an in vitro assay with the same experimental design captures acute effects in the presence of the test article. Here, the in vitro assay was applied to a list of drugs with known clinical immunomodulatory impact to understand the applicability of the assay. The results showed this assay was sensitive to a wide range of immunosuppressants directly targeting cell-intrinsic signaling pathways in activated CTL. However, agents executing immuno-modulation through inhibiting cytokines/cytokine receptors, co-stimulatory molecules, and cell adhesion and migration pathways did not impair the CTL activity in this short-term in vitro culture. In addition, anti-PD-1/PD-L1 immune checkpoint blockers enhanced the CTL activity. Taken together, the results here demonstrate that in concordance with their mechanism of action, the in vitro BiTE®-mediated CTL assay is applicable and sensitive to immunomodulatory agents acting via a variety of mechanisms.

Cytotoxic T-lymphocyte infiltration and chemokine predict long-term patient survival independently of tumor mutational burden in triple-negative breast cancer.

Background:Cytotoxic T-lymphocyte (CTL) infiltration into tumor is a positive prognostic factor in breast cancer. High tumor mutational burden (TMB) is also considered as a predictor of tumor immunogenicity and response to immunotherapy. However, it is unclear whether the infiltration of functional CTL simply reflects the TMB or represents an independent prognostic value.Methods:Utilizing The Cancer Genome Atlas (TCGA) breast cancer cohort, we established the Functional Hotness Score (FHS). The associations of FHS and breast cancer patient prognosis as well as distinct immunity markers were analyzed in a total of 3011 breast cancer patients using TCGA, METABRIC and metastatic breast cancer (MBC) cohort GSE110590.Results:We established FHS, based on CD8A, GZMB and CXCL10 gene expression levels of bulk tumors, which delivered the best prognostic value among some gene combinations. Breast cancer patients with the high-FHS tumors showed significantly better survival. FHS was lower in the MBCs. Triple-negative breast cancer (TNBC) showed the highest FHS among subtypes. FHS predicted patient survival in hormone receptor (HR)-negative, especially in TNBC, but not in HR-positive breast cancer. FHS predicted patient prognosis independently in TNBC. The high-FHS TNBCs showed not only higher CD8+ T cell infiltration, but also enhanced broader type-1 anti-cancer immunity. The patients with the high-FHS tumors showed better prognosis not only in high-TMB tumors but also in low-TMB TNBCs. The combination of high-TMB with high-FHS identified a unique subset of patients who do not recur over time in TNBC.Conclusion:TNBCs with high FHS based on the expression levels of CD8A, GZMB and CXCL10 showed improved prognosis with enhanced anti-cancer immunity regardless of TMB. FHS constitutes an independent prognostic marker of survival, particularly robustly when combined with TMB in TNBC.

Relationship between immune status after ATG treatment and PNH clone evolution in patients with severe aplastic anemia.

ObjectivesTo investigate the relationship between immune status and paroxysmal nocturnal hemoglobinuria (PNH) clonal evolution of severe aplastic anemia (SAA) patients who received anti‐human thymocyte globulin (ATG) treatment.MethodsThe clinical data of 102 SAA patients who received ATG were collected and retrospectively analyzed. The remission rate, remission time, response rate, hematopoietic, and immune status were compared. Malignant clones were also observed.ResultsThe remission rate of the group with PNH clones appeared after treatment was significantly higher than the group without PNH clones. The response rate at 12 months of the groups with PNH clones was significantly higher than the group without PNH clones. The recovery of Hb and Ret % of patients with PNH clones was earlier than the patients without PNH clones. The reduction of percentage of CD8+HLA‐DR+/CD8+ and Th1/Th2 ratio of patients with PNH clones was both earlier than the patients without PNH clones. Six patients developed myelodysplastic syndromes (MDS).ConclusionIn SAA patients with PNH clones, the cytotoxic T‐cell function and Th1 cell number recovered more quickly and had better response to IST. A small number of SAA patients with or without PNH clones developed MDS malignant clones.

Identification of a Novel Role for PD-1 Signaling in Promotion Tumor Proliferation in B-Cell Lymphoma

Identification of a Novel Role for PD-1 Signaling in Promotion Tumor Proliferation in B-Cell Lymphoma

The CAR T-Cell Mechanoimmunology at a Glance.

Chimeric antigen receptor (CAR) T‐cell transfer is a novel paradigm of adoptive T‐cell immunotherapy. When coming into contact with a target cancer cell, CAR T‐cell forms a nonclassical immunological synapse with the cancer cell and dynamically orchestrates multiple critical forces to commit cytotoxic immune function. Such an immunologic process involves a force transmission in the CAR and a spatiotemporal remodeling of cell cytoskeleton to facilitate CAR activation and CAR T‐cell cytotoxic function. Yet, the detailed understanding of such mechanotransduction at the interface between the CAR T‐cell and the target cell, as well as its molecular structure and signaling, remains less defined and is just beginning to emerge. This article summarizes the basic mechanisms and principles of CAR T‐cell mechanoimmunology, and various lessons that can be comparatively learned from interrogation of mechanotransduction at the immunological synapse in normal cytotoxic T‐cell. The recent development and future application of novel bioengineering tools for studying CAR T‐cell mechanoimmunology is also discussed. It is believed that this progress report will shed light on the CAR T‐cell mechanoimmunology and encourage future researches in revealing the less explored yet important mechanosensing and mechanotransductive mechanisms involved in CAR T‐cell immuno‐oncology.

Helpless Priming Sends CD8+ T Cells on the Road to Exhaustion.

Persistent antigen exposure in chronic infection and cancer has been proposed to lead to cytotoxic T lymphocyte (CTL) “exhaustion”, i.e., loss of effector function and disease control. Recent work identifies a population of poorly differentiated TCF-1+PD-1+ CD8+ T cells as precursors of the terminally exhausted CTL pool. These “predysfunctional” CTLs are suggested to respond to PD-1 targeted therapy by giving rise to a pool of functional CTLs. Supported by gene expression analyses, we present a model in which lack of CD4+ T cell help during CD8+ T cell priming results in the formation of predysfunctional CTLs. Our model implies that predysfunctional CTLs are formed during priming and that the remedy for CTL dysfunction is to provide “help” signals for generation of optimal CTL effectors. We substantiate that this may be achieved by engaging CD4+ T cells in new CD8+ T cell priming, or by combined PD-1 blocking and CD27 agonism with available immunotherapeutic antibodies.

Transcriptomic and Immunophenotypic Characterization of Tumor Immune Microenvironment in Squamous Cell Carcinoma of the Oral Tongue.

The tumor immune microenvironment of oral tongue squamous cell carcinoma may be accountable for differences in clinical behavior, particularly between different age groups. We performed RNA expression profiling and evaluated tumor infiltrating lymphocytes (TILs) and their T-cell subsets in order to assess the functional status of oral tongue squamous cell carcinoma tumor microenvironment and detect potentially clinically useful associations. Archival surgical pathology material from sixteen oral tongue squamous cell carcinoma patients was microscopically evaluated for TIL densities. RNA was extracted from macrodissected whole tumor sections and normal controls and RNA expression profiling was performed by the NanoString PanCancer IO 360 Gene Expression Panel. Immunostains for CD4, CD8 and FOXP3 were evaluated manually and by digital image analysis. Oral tongue squamous cell carcinomas had increased TIL densities, numerically dominated by CD4 + T cells, followed by CD8 + and FOXP3 + T cells. RNA expression profiling of tumors versus normal controls showed tumor signature upregulation in inhibitory immune signaling (CTLA4, TIGIT and PD-L2), followed by inhibitory tumor mechanisms (IDO1, TGF-β, B7-H3 and PD-L1). Patients older than 44years showed a tumor microenvironment with increased Tregs and CTLA4 expression. Immunohistochemically assessed CD8% correlated well with molecular signatures related to CD8 + cytotoxic T-cell functions. FOXP3% correlated significantly with CTLA4 upregulation. CTLA4 molecular signature could be predicted by FOXP3% assessed by immunohistochemistry (R2 = 0.619, p = 0.026). Oral tongue squamous cell carcinoma hosts a complex inhibitory immune microenvironment, partially reflected in immunohistochemically quantified CD8 + and FOXP3 + T-cell subsets. Immunohistochemistry can be a useful screening tool for detecting tumors with upregulated expression of the targetable molecule CTLA4.

Abstract 1023: CD73 inhibition with a novel orally bioavailable small molecule blocks adenosine production and rescues T-cells activation

Abstract Adenosine has emerged as a key immunosuppressive metabolite within the tumor microenvironment (TME). A persistently elevated concentration of adenosine in TME can impair immune control of tumor growth by diminishing cytotoxic T-cell responses and function of natural killer cells while augmenting the suppressive activity of myeloid and regulatory T-cells. The ectonucleotidase, CD73 mediates the final dephosphorylation step in the conversion of extracellular ATP to adenosine. Overexpression of CD73 is observed in many solid tumors and correlates with unfavorable clinical outcome. We aimed to overcome adenosine-driven immunosuppression by developing an orally bioavailable, best-in-class, small molecule inhibitor of CD73. Here, we show superior potency of our selective AMP-competitive CD73 inhibitor in blocking adenosine generation by multiple cell types. CD73 inhibitor activity was assessed by directly measuring the generation of adenosine from AMP by LC-MS/MS. In CD73 expressing cells, potent and complete inhibition of CD73 ectonucleotidase activity was observed. To test the functional consequence of CD73 inhibition, we interrogated the effects of CD73 inhibitors on CD8+ T-cell function upon exposure to AMP. CD73 expressed on T-cells is sufficient to drive adenosine generation from AMP resulting in severely suppressed proliferation, diminished production of inflammatory cytokines and reduced expression of activation markers. Our inhibitors successfully counteracted the effects of AMP and completely rescued all aspects of CD8+ T-cell activation. Interestingly, we found that the concentration of AMP in tumors may vary from micromolar to millimolar levels, underscoring the necessity of AMP-competitive CD73 inhibitors to be efficacious in a high AMP environment. We demonstrated that nanomolar concentrations of our CD73 inhibitors can efficiently rescue T-cell function in the presence of millimolar AMP concentrations. We are currently conducting biophysical studies to better understand this unique property of our inhibitors. We next aimed to identify an adenosine responsive gene signature in cytotoxic T-cells. RNA sequencing analysis of primary anti-CD3/28/2 activated CD8+ T-cells from human peripheral blood mononuclear cells revealed a unique set of genes persistently and similarly altered in response to both adenosine and AMP. Finally, we demonstrated that CD73 inhibitors can completely block gene expression changes of this AMP/adenosine-response signature. Discovery of an AMP/adenosine-response gene signature may help identify patients whose tumors harbor elevated adenosine signaling and would benefit from an orally bioavailable small molecule inhibitor of CD73 to reverse immunosuppression. Citation Format: Tatiana Zavorotinskaya, Brian Blank, Brenda Chan, Chelsea Chen, Yuping Chen, Xiaohui Du, Frank Duong, Lori Friedman, Tom Huang, Melissa R. Junttila, Wayne Kong, Todd C. Metzger, Jared T. Moore, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Natalie Yuen. CD73 inhibition with a novel orally bioavailable small molecule blocks adenosine production and rescues T-cells activation [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 1023.

Abstract 6616: Functional hotness score generated by representative functional cytotoxic T-lymphocytes predicts long-term survival of triple-negative breast cancer independently to the tumor mutational burden

Abstract Background: Cytotoxic T-lymphocytes (CTLs) infiltration in a bulk tumor is a positive prognostic factor in breast cancer. CTL recognition is believed to react to mutation-induced neoantigens, thus, higher tumor mutation burden (TMB) is considered as an important predictor of tumor immunogenicity and response to immunotherapy. Although majority of breast cancer have low tumor mutation burden (TMB), triple-negative breast cancer (TNBC) has higher TMB compared to other breast cancer subtypes. However, it is unclear if the prognostic value high CTL infiltration depends on the TMB as the primary driver of enhanced anti-cancer immunity or represents an independent prognostic marker which can be used as a complement to TMB to predict patients' outcomes. CTLs are identified by CD8 marker, is encoded by CD8A. Granzyme B (GZMB) is a serine protease that is secreted by functionally-active CTLs to induce apoptosis of the target cells. CXCL10 is a chemokine which selectively attracts activated CTLs into subsets of other cancers; but its role in breast cancer remains unknown. We investigated if TNBC infiltrated with high levels of functional CTLs, so called “hot tumors”, have improved survival independently of their TMB. Methods: Utilizing The Cancer Genome Atlas (TCGA) breast cancer cohort, we established Functional Hotness Score (FHS), based on the CD8A, GZMB and CXCL10 gene expression levels of bulk tumors. Total of 4149 breast cancer patients from publicly available multiple cohorts were analyzed to assess FHS and breast cancer patient prognosis as well as distinct immunity markers. Results: FHS predicted the breast cancer patient survival better than each consisting gene. The breast cancer patients with high FHS tumors showed significantly better survival in the testing cohort (TCGA), which was further confirmed in the validation cohort (METABRIC). FHS was higher in the primary breast cancer tissues with metastasis compared to that without metastasis in TCGA. Further, FHS was higher in the metastatic tumors compared to the primary tumors in GSE110590. Among breast cancer subtypes, TNBC showed highest FHS compared to other subtypes in testing TCGA, which was validated in the METABRIC cohort. The patients with high FHS tumor demonstrated significantly better long-term survival than that with low FHS only in the hormone receptor (HR)-negative breast cancers, but not in the HR-positive tumors. The high FHS TNBCs showed higher not only CD8-positive T cell infiltration, but also a broader type-1 anti-cancer immunity. To investigate if FHS predicts patient survival independent of TMB, the patients were divided into high and low TMB groups. Interestingly, the higher FHS patients showed better prognosis not only in the high TMB group but also in the low TMB among TNBC patients. The combination of high TMB with high FHS identified the unique subset of the patients who did not recur over time. Conclusion: In conclusion, TNBC with higher FHS based on the expression levels of CD8A, GZMB and CXCL10 showed improved prognosis with higher anti-cancer immunity regardless of TMB, and constituting an independent prognostic marker of survival, particularly when combined with TMB. Citation Format: Eriko Katsuta, Li Yan, Mateusz Opyrchal, Pawel Kalinski, Kazuaki Takabe. Functional hotness score generated by representative functional cytotoxic T-lymphocytes predicts long-term survival of triple-negative breast cancer independently to the tumor mutational burden [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 6616.