Levels Of Cytotoxic T Lymphocytes Research Articles

Naphthaleneoxypropargyl-Containing Piperazine as a Regulator of Effector Immune Cell Populations upon an Aseptic Inflammation.

This study investigated the effects of aseptic inflammation and heavy metal exposure on immune responses, as well as the potential immunomodulatory properties of the newly synthesized 1-[1-(2,5-dimethoxyphenyl)-4-(naphthalene-1-yloxy)but-2-ynyl]-4-methylpiperazine complexed with β-cyclodextrin (β-CD). Aseptic inflammation was induced by a subcutaneous injection of turpentine in rats, while heavy metal exposure was achieved through a daily administration of cadmium chloride and lead acetate. The levels of immune cell populations, including cytotoxic T lymphocytes (CTL), monocytes, and granulocytes, were assessed in the spleen. The results showed that aseptic inflammation led to decreased levels of CTL, monocytes, and granulocytes on the 14th day, indicating an inflammatory response accompanied by a migration of effector cells to the inflamed tissues. The exposure to cadmium chloride and lead acetate resulted in systemic immunotoxic effects, with reduced levels of B cells, CD4+ Th cells, monocytes, and granulocytes in the spleen. Notably, piperazine complexed with β-CD (the complex) exhibited significant stimulatory effects on CD4+, CD8+, and myeloid cell populations during aseptic inflammation, even in the presence of heavy metal exposure. These findings suggest the potential immunomodulatory properties of the complex in the context of aseptic inflammation and heavy metal exposure.

High CD8+tumor-infiltrating lymphocytes indicate severe exhaustion and poor prognosis in angioimmunoblastic T-cell lymphoma.

Exhaustion of CD8+ tumor-infiltrating lymphocytes (TILs), characterized by the overexpression of immune checkpoints (IC), is a major impediment to anti-tumor immunity. However, the exhaustion status of CD8+TILs in angioimmunoblastic T cell lymphoma (AITL) remains unclear. Therefore, we aimed to elucidate the exhaustion status of CD8+TILs in AITL and its influence on prognosis. The correlation between CD8+TILs and IC expression in AITL was analyzed using single-cell RNA sequencing (n = 2), flow cytometry (n = 20), and RNA sequencing (n = 20). Biological changes related to CD8+TILs exhaustion at different cytotoxic T lymphocyte (CTL) levels (mean expression levels of CD8A, CD8B, GZMA, GZMB, and PRF1) in AITL were evaluated using RNA sequencing (n = 20) and further validated using the GEO dataset (n = 51). The impact of CD8 protein expression and CTL levels on patient prognosis was analyzed using flow cytometry and RNA sequencing, respectively. Our findings demonstrated that the higher the infiltration of CD8+TILs, the higher was the proportion of exhausted CD8+TILs characterized by the overexpression of multiple IC. This was accompanied by extensive exhaustion-related biological changes, which suggested severe exhaustion in CD8+TILs and may be one of the main reasons for the poor prognosis of patients with high CD8+TILs and CTL. Our study comprehensively reveals the exhaustion status of CD8+TILs and their potential negative impact on AITL prognosis, which facilitates further mechanistic studies and is valuable for guiding immunotherapy strategies.

Key Factors and Parameter Ranges for Immune Control of Equine Infectious Anemia Virus Infection.

Equine Infectious Anemia Virus (EIAV) is an important infection in equids, and its similarity to HIV creates hope for a potential vaccine. We analyze a within-host model of EIAV infection with antibody and cytotoxic T lymphocyte (CTL) responses. In this model, the stability of the biologically relevant endemic equilibrium, characterized by the coexistence of long-term antibody and CTL levels, relies upon a balance between CTL and antibody growth rates, which is needed to ensure persistent CTL levels. We determine the model parameter ranges at which CTL and antibody proliferation rates are simultaneously most influential in leading the system towards coexistence and can be used to derive a mathematical relationship between CTL and antibody production rates to explore the bifurcation curve that leads to coexistence. We employ Latin hypercube sampling and least squares to find the parameter ranges that equally divide the endemic and boundary equilibria. We then examine this relationship numerically via a local sensitivity analysis of the parameters. Our analysis is consistent with previous results showing that an intervention (such as a vaccine) intended to control a persistent viral infection with both immune responses should moderate the antibody response to allow for stimulation of the CTL response. Finally, we show that the CTL production rate can entirely determine the long-term outcome, regardless of the effect of other parameters, and we provide the conditions for this result in terms of the identified ranges for all model parameters.

The epiphany derived from T-cell-inflamed profiles: Pan-cancer characterization of CD8A as a biomarker spanning clinical relevance, cancer prognosis, immunosuppressive environment, and treatment responses.

CD8A encodes the CD8 alpha chain of αβT cells, which has been proposed as a quantifiable indicator for the assessment of CD8+ cytotoxic T lymphocytes (CTLs) recruitment or activity and a robust biomarker for anti-PD-1/PD-L1 therapy responses. Nonetheless, the lack of research into the role of CD8A in tumor microenvironment predisposes to limitations in its clinical utilization. In the presented study, multiple computational tools were used to investigate the roles of CD8A in the pan-cancer study, revealing its essential associations with tumor immune infiltration, immunosuppressive environment formation, cancer progression, and therapy responses. Based on the pan-cancer cohorts of the Cancer Genome Atlas (TCGA) database, our results demonstrated the distinctive CD8A expression patterns in cancer tissues and its close associations with the prognosis and disease stage of cancer. We then found that CD8A was correlated with six major immune cell types, and immunosuppressive cells in multiple cancer types. Besides, epigenetic modifications of CD8A were related to CTL levels and T cell dysfunctional states, thereby affecting survival outcomes of specific cancer types. After that, we explored the co-occurrence patterns of CD8A mutation, thus identifying RMND5A, RNF103-CHMP3, CHMP3, CD8B, MRPL35, MAT2A, RGPD1, RGPD2, REEP1, and ANAPC1P1 genes, which co-occurred mutations with CD8A, and are concomitantly implicated in the regulation of cancer-related pathways. Finally, we tested CD8A as a therapeutic biomarker for multiple antitumor agents’ or compounds’ responsiveness on various cancer cell lines and cancer cohorts. Our findings denoted the underlying mechanics of CD8A in reflecting the T-cell-inflamed profiles, which has potential as a biomarker in cancer diagnosis, prognosis, and therapeutic responses.

Phenotypes of lymphocytes in exudate in atopic dermatitis

Aim. To determine subpopulations of lymphocytes in exudate in atopic dermatitis.
 Material and methods. The study included 80 patients with atopic dermatitis according to predetermined inclusion and exclusion criteria, who were under dispensary supervision at the Regional Dermatovenerologic Dispensary (Chita) from 2018 to 2021. Two groups (adolescents and adults) and two subgroups (patients with limited and widespread lesions) were formed. Skin exudate sampling was carried out during the period of exacerbation of the disease. The control group consisted of 30 practically healthy volunteers undergoing a medical examination at the same dispensary, having primary health records and meeting the inclusion criteria for the study. Lymphocyte phenotypes were determined in skin exudate by flow cytofluorometry. Statistical analysis was performed using Microsoft Excel, IBM SPSS Statistics version 25.0, and the ShapiroWilk test, MannWhitney and Wilcoxon U-test, KruskalWallis nonparametric analysis of variance were used. The data were presented by median and interquartile intervals Me (25%; 75%). The critical indicator of the significance level of the differences was p 0.05.
 Results. In the exudate of practically healthy volunteers, obtained by the skin window method, no subpopulations of lymphocytes were detected. In the skin exudate of adolescents with limited lesions, the content of lymphocytes was 149.00 (129.75; 157.75) cells/l, T-lymphocytes (CD3+CD19) 109.5 (96.25; 113.75) cells/l, among them 48.95% active, T-helpers 42.5 (39.25; 57.50) cells/l, natural killers (CD3+CD16+CD56+) 38.50 (36.25; 41.00) cells/l, however, with the widespread process, the level of lymphocytes increased by 11% (p1=0.002), T-helpers by 45%, natural killers by 27%, T-lymphocytes up to 125.00 (110.5;135.75) cells/l (p1=0.00001). These indicators in adults and the level of cytotoxic T-lymphocytes (CD3+CD8+) in adolescents and adults had no significant differences. The number of T-NK-killers (CD3+CD16+CD56+) in adolescents was higher with a widespread process 23.00 (11.75; 29.75) cells/l (p1=0.0001), in adults with a limited process 18.00 (10.25; 20.75) cells/l (p2=0.0001). The number of NKCD8+ (CD3CD16+CD56+CD8+) in adolescents with limited dermatosis was 22.50 (18.25; 26.00) cells/l, with widespread dermatosis 1.6 times more (p1=0.0001), in adults with a limited process 29.50 (25.25; 33.75) cells/l, with widespread 27% less (p1=0.0001).
 Conclusion. In the skin exudate in atopic dermatitis, cytotoxic T-lymphocytes, T-NK-killers, natural killers, CD8-positive NK-killers are detected.

HGG-56. Spatial mapping of the tumor micro-environment in pediatric glioma

High-grade glioma are the main cause of cancer-related death in children. The highly heterogeneous composition of the tumor cells and their interactions with the tumor micro-environment (TME), contribute substantially to the poor response to treatment and the high levels of morbidity and mortality. Here, we used high-dimensional, multiplexed immunohistochemistry to map the single-cell tissue architecture of 26 pediatric glioma samples covering 8 histologic diagnoses, allowing us to determine the spatial distribution of the various tumoral subtypes and how these interact with their local immune-microenvironment. Overall, this analysis showed that tumor grade anti-correlated with the amount of infiltrating cytotoxic T-lymphocytes (CTLs), which were typically more exhausted in the higher grade tumors. In addition, tumor associated macrophages were primarily infiltrating from the blood and presented an M2-like anti-inflammatory phenotype which became more extended with tumor grade. Using the spatial information, possible cell-cell interactions could be determined. In lower grade glioma, we observed an increased activation level of CTLs that were closely located to neighboring T-helper cells. In pediatric glioblastoma, on the other hand, CTLs, even though they were located close to a T-helper cell, could only minimally be activated, and showed more extended exhaustion when residing further away. Additionally, the activation of the CTLs was associated to the distance to the closest PD-L1 positive macrophage in pilocytic astrocytoma and desmoplastic infantile ganglioglioma. In conclusion, with the use of multiplex immunohistochemistry, we are able to study the tumor and TME of pediatric glioma in depth on a single-cell and spatial level, which allows us to further study the heterogeneous landscape of these tumors.

The impact of tumor-infiltrating lymphocytes on tumor features and pathological characteristics in breast cancer patients: the role of cytotoxic T lymphocytes and regulatory T cells.

Though tumor-infiltrating lymphocytes (TILs) have a predictive impact in cancer patients, their association with presentation and prognosis in breast cancer is less consistent. This study aimed to assess the level of infiltrating cytotoxic T lymphocytes (CTLs) and regulatory T lymphocytes (Tregs) and their association with the clinicopathological features of breast cancer. Tissue samples from female patients (n=153) diagnosed with primary invasive breast cancer were stained with CD8 (a CTL marker) and Foxp3 (a Treg marker) using immunohistochemistry. CTLs were distributed between tumor bed and stroma whereas Treg cells were mainly located in the stroma. The level of intratumoral CTLs correlated positively with Tregs in both tumor and stroma (rho=0.312, p<0.001 and rho=0.176, p=0.031; respectively). Stromal CTLs correlated positively with stromal Tregs (rho=0.319, p=0.005). Tumor size correlated inversely with the number of Treg cells in the tumor bed (rho= - 0.179, p=0.028). Tregs were associated with lymphovascular invasion status in the tumor bed (p=0.042). The ratio of intratumoral CTLs to Tregs was associated with estrogen receptor positivity and luminal subtype (p=0.029 and p=0.045, respectively). The median number of CTLs was significantly lower in patients using aspirin or antihypertensive medications compared to nonusers (p=0.024 and p=0.03, respectively). TILs were distributed differently in tumor tissues of breast cancer patients. CTLs infiltrates were found in both tumor bed and stroma while Tregs were dominant in the stroma. TILs were also distinctly associated with tumor features. The impact of TILs on prognosis and treatment outcomes in Jordanian breast cancer patients needs further investigation.

Immune responses of patients without cancer recurrence after a cancer vaccine over a long term.

The present study aimed to clarify the humoral and cellular immune responses of patients with cancer who experienced no recurrence over a long term after receiving a cancer vaccine. The immune kinetics were investigated in response to a personalized peptide vaccination (PPV) among 44 Japanese patients without an active tumor at entry to the vaccination: Lung adenocarcinoma (n=11); colon (n=18); and breast cancer (n=15) (9, 10, 12, 8 and 5 patients with stage I, II, III and IV recurrences, respectively). The patients' immunoglobulin G (IgG) and cytotoxic T lymphocyte (CTL) activities were measured using a multiplexed Luminex assay and an interferon-γ release assay, respectively. There were no severe adverse events related to the PPV other than a grade III injection site reaction. A potent boost in IgG or CTL at the end of the 1st vaccination cycle was observed in 77% of the patients (n=84). The IgG levels were sustained throughout the follow-up period, whereas the CTL levels declined and were transient. A total of 37 of the 44 patients (84%) had no recurrence, with a median follow-up of 67.6 months (interquartile range, 45.6-82.8 months). Overall, the PPV induced long-term humoral immunity with transient cellular immunity in the majority of patients with cancer without an active tumor at their entry to the PPV.

Potential PD-L1 expressing cytotoxic T-cell immunopathology in Alzheimer disease.

Growing evidence suggested that adaptive immunity involved Alzheimer's disease (AD). Peripheral blood mononuclear cell (PBMC) subpopulations and expression of immune checkpoints such as programmed death-ligand 1(PD-L1) and programmed cell death protein-1 (PD-1) could play roles in Alzheimer disease. The interaction of PD-L1/PD-1 lead PD-1 expressing T cells to attenuate T cell-activating signals. Recent studies had demonstrated the blockade of PD-L1/PD-1 axis modified brain pathology and restored cognitive performance in tauopathy murine model. However similar human experiment suggested controversial results in AD patients. To observe the changes of PBMC subpopulations and PD-L1/PD-1 expression in AD and healthy controls, we utilized flow cytometry, and applied t-distributed stochastic neighbor embedding (t-SNE) algorithm as a new approach to analyze results. AD patients (n=16) with different disease severity (CDR= 1 - 2) and healthy controls (n=16) were enrolled. Expression levels of PD-L1/PD-1 in AD patients and healthy controls were derived by flow cytometric analysis parameters. Moreover, a dimensionality reduction algorithm for visualized t-SNE, was implemented to analyze the PD-L1/PD-1 expressing cell clusters between AD patients and healthy controls. We found no significant PD-1 expression intensity difference between cytotoxic T lymphocytes (CTL) of AD patients and healthy controls. In AD patients, subpopulation of CTL expressing PD-L1 was elevated by 85.53%. Further, PD-L1 expressing CTL clusters between AD patients and healthy controls were compared. We observed that densities of PD-L1high CTL clusters are significantly higher than the healthy controls. Upregulation of PD-L1 and PD-L1 expressing CTL were increased in peripheral blood of AD patients, linking to newly found strong correlation between the level of PD-L1 positive CTL and markers which indicated negative immune climate. Abnormal adaptive immune response of CTL might associate with concurrent viral infection, intracellular bacterial infection, and underlying cancer in AD patients. Our results suggested a novel strategy to prevent AD progression with regulation of PD-L1 expressing CTL, and notation of underlying intracellular infection and malignancy for which could relate to dementia progression.

Tumor-Infiltrating Cytotoxic T Cells and Tumor-Associated Macrophages Correlate With the Outcomes of Neoadjuvant Chemoradiotherapy for Locally Advanced Rectal Cancer.

BackgroundTumor-infiltrating immune cells (TIICs) play a key role in immunoregulatory networks and are related to tumor development. Emerging evidence shows that these cells are associated with sensitivity to chemotherapy and radiotherapy. However, the predictive role of TIICs in the outcomes of neoadjuvant chemoradiotherapy (nCRT) for locally advanced rectal cancer (LARC) is unclear.MethodsImaging mass cytometry (IMC) was performed to comprehensively assess the immune status before nCRT in 6 patients with LARC (3 achieved pathological complete response (pCR), 3 did not) with matched clinicopathological parameters. Immunohistochemistry (IHC) for CD8, CD163 and Foxp3 on biopsy samples from 70 patients prior to nCRT and logistic regression analysis were combined to further evaluate its predictive value for treatment responses in an independent validation group.ResultsA trend of increased CD8+ cytotoxic T lymphocytes (CTLs) and decreased CD163+ tumor-associated macrophages (TAMs) and Foxp3+ regulatory T cells (Tregs) in the pCR group was revealed by IMC. In the validation group, CTLs and TAMs were strong predictors of the clinical response to nCRT. High levels of CTLs were positively associated with the pCR ratio (OR=1.042; 95% CI: 1.015~1.070, p=0.002), whereas TAMs were correlated with a poor response (OR=0.969; 95% CI: 0.941~0.998, p=0.036). A high density of TAMs was also associated with an advanced cN stage.ConclusionCTLs in the tumor microenvironment (TME) may improve the response to nCRT, whereas TAMs have the opposite effect. These results suggest that these cells might be potential markers for the clinical outcomes of nCRT and aid in the clinical decision-making of LARC for improved clinical outcomes.

Efficacy finding cohort of a cancer peptide vaccine, TAS0313, in treating recurrent glioblastoma.

2038 Background: TAS0313 is a cancer vaccine cocktail comprising three long peptides with a total of 12 cytotoxic T lymphocyte (CTL) epitope peptides. These peptides were derived from eight cancer-associated antigens (EGFR, KUA, LCK, MRP3, PTHRP, SART2, SART3, and WHSC2) that are overexpressed in various cancer types including glioblastoma (GBM). We performed a single-arm, multicenter efficacy confirmatory cohort study as part of the TAS0313 Phase I/II study in patients with recurrent GBM. Methods: The enrolled patients with histologically or cytologically confirmed grade IV GBM (including gliosarcoma, giant cell glioblastoma, and epithelioid glioblastoma) had at least one of the following HLA types: HLA-A*02:01, -A*02:06, -A*02:07, -A*11:01, -A*24:02, -A*31:01, or -A*33:03. Eligible patients were those who received standard radiotherapy and temozolomide, had a confirmed first or second recurrence, or progression with measurable disease outcomes with good performance status (Karnofsky Performance Status score &gt; 70). TAS0313 (27mg) was subcutaneously administered on days 1, 8, and 15 of cycles 1 and 2 and day 1 of cycle 3 or later in 21-day cycles until disease progression or unacceptable toxicity occurred. Tumor response was evaluated using The Response Assessment in Neuro-Oncology criteria. The antigen-specific CTL and immunoglobulin G (IgG) were analyzed by ELISPOT assay and Luminex assay, respectively, before and after treatment. The primary objective was to evaluate the efficacy of TAS0313. Secondary and exploratory methods were used to evaluate the safety and immune response of TAS0313. Results: As of 10th September 2020, 10 patients have been treated with TAS0313 (eight with GBM and two with gliosarcoma). The median age of participants was 56.5 [range, 33–69 years], and methylation status of the MGMT promoter was methylated in four patients, unmethylated in four, and unknown in two. The best overall response was partial response 1/10 (10.0%) and stable disease 4/10 (40.0%). One case showed 69.1% tumor shrinkage. The treatment of two patients was ongoing for over 7 months. The 6-month progression-free survival (PFS) rate was 25.0%, and the median PFS was 2.3 months. The most common adverse drug reactions (ADRs) were grade 1–2 injection site reactions and pyrexia. There were no grade 4 or 5 ADRs. In some patients, TAS0313 treatment was confirmed to increase CTL and IgG levels compared with pre-treatment samples. Conclusions: TAS0313 showed promising efficacy with expected immune responses and favorable safety and tolerability in patients with recurrent GBM. Further investigation of TAS0313 is warranted to validate our findings. Clinical trial information: JapicCTI-183824.

A versatile photothermal vaccine based on acid-responsive glyco-nanoplatform for synergistic therapy of cancer

The race is on for therapeutic agents that stop cancer. An effective vaccine offers a safe and promising approach for cancer immunotherapy. However, substantial barriers to immunotherapy in cancer vaccines include the low immunogenicity of cancer antigens and the immunosuppression commonly present in solid tumors, resulting in significant challenges for developing a clinically effective cancer vaccine. Here, the state of the art of synergistic therapy, which includes the photothermal effect combined with immunotherapy, was investigated to target tumors. For the first time, indocyanine green (ICG, referred to as I), imiquimod (R837, referred to as R) and a foreign cytotoxic T lymphocyte antigen peptide (CTL-Ap, referred to as Ap) with the sequence of SIINFEKL from ovalbumin (OVA) were encapsulated by acetalated dextran (AcDEX) to form nanoparticles (NPs) averaging 92 nm in diameter as an immunogen. Administration of the resulting multifunctional vaccine I-R-Ap-AcDEX NPs enhanced antitumor cytotoxic T lymphocyte (CTL) immunotherapy. On the one hand, subcutaneous immunization of the NPs allows foreign Ap to enter the major histocompatibility complex class I (MHC-I) cross-presentation pathway of antigen-presenting cells, thereby presenting Ap and eliciting high levels of Ap-specific CTLs. On the other hand, intratumor/intravenous injections of the NPs allow foreign Ap to enter tumor cells and present Ap through the MHC-I cross-presentation pathway. Ap-specific CTLs can kill Ap-presented tumor cells. Furthermore, the NPs generated near-infrared laser triggered the photothermal killing of tumor cells. To our knowledge, this is the first report of AcDEX NPs in antitumor photothermal therapy. Strikingly, systemic administration of the I-R-Ap-AcDEX NPs combined with near-infrared laser irradiation allowed for complete protection to mice from the tumors when applied to two non-OVA tumor models. This quite impressive result displays the great promise of synergistic therapy by the vaccine I-R-Ap-AcDEX NPs, an approach that harnesses the photothermal effect to boost antitumor immunotherapy.

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.

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.

Abstract P3-09-14: Regulation of gene expression and DNA methylation with cytotoxic T lymphocytes evaluation in subtypes of breast cancers

Abstract Introduction: Low cytotoxic T lymphocyte (CTLs) infiltration and poor immunogenicity in the breast cancer (BC) microenvironment is a challenge to treatment with immune checkpoint (ICK) inhibitors. However, ICK inhibitors were recently approved by the FDA in triple-negative breast cancer (TNBC), which are characterized by high levels of tumor infiltrating CTL. DNA methylation is a component of epigenetic modification involved in gene expression programming that can promote the progression of cancers, including BC. However, the association between transcriptional and methylation changes that modulate CTL infiltration in the tumor microenvironment in specific subtypes of breast cancer is still unclear. In this study, we compare transcriptional and methylation profile data in patients with different BC subtypes to identify targetable genes to improve CTL infiltration and ICK inhibitor efficacy in BC patients. Methods: We included The Cancer Genome Atlas (TCGA) RNA sequencing (n=1,212) and DNA methylation (Illumina Human Methylation 450K; n=783) databases to detect the gene expression and methylation profiles based on molecular subtypes: LumA vs TNBC, LumB vs TNBC, Her2+ vs TNBC. To be consistent with clinical diagnosis, our TNBC patients included all ER-, PR-, Her2 -/1+/2+ patients (n=192). Microenvironment Cell Populations-counter (MCPcounter) were used to determine CTL infiltration in BC subtypes. Genes with significantly different expression (p&amp;lt;0.01, LogFC≥|1.5|) and difference of mean methylation β value ≥|0.25| were integrated for Pearson correlation coefficient analysis with CTL MCPcounter score (r&amp;gt;|0.7|). Results: Comparing LumA and TNBC patients, hypermethylation was associated with 113 upregulated genes and 309 downregulated genes. Hypomethylation was associated with 156 upregulated genes and 104 downregulated genes. Additionally, in LumA tumors, 7 genes were differentially methylated and associated with CTL scores: 3 genes were downregulated and hypomethylated (AIM2, SEL1L3, TOX), and 4 genes were downregulated and hypermethylated (CD38, HLA-DOB, LAMP3, RUNX3). Comparing LumB and TNBC tumors, hypermethylation was associated with 158 upregulated genes and 525 downregulated genes. In these tumors, hypomethylation was associated with 178 upregulated genes and 168 downregulated genes. In this group, three genes (AIM2, GPR55, UBD) were downregulated, hypomethylated, and highly correlated with CTL scores. Finally, comparison of Her2+ BC with TNBC tumors showed that hypermethylation was associated with 39 upregulated genes and 223 downregulated genes. In this group, hypomethylation was associated with 54 upregulated genes and 31 downregulated genes. Among these genes, CHST2 gene was the only gene found downregulated with hypermethylated and associated with CTL score in Her2+ tumors. Conclusion: We found eleven genes that were differentially expressed, differentially methylated, and highly correlated with CTL infiltration were identified in LumA, LumB and Her2+ subtypes compared with TNBC. Specifically, interferon-inducible protein AIM2 is a gene that was altered in both LumA and LumB, is associated with response to ICK in murine models and may be a potential target to improve response to immune based therapy in these subtypes of BC patients. Citation Format: Yuanyuan(Daisy) Shen, Chi-Ren Shyu, Jonathan B. Mitchem, Yifeng Shi, Ayesha N. Shajahan-Haq. Regulation of gene expression and DNA methylation with cytotoxic T lymphocytes evaluation in subtypes of breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-14.

Type I interferon suppresses tumor growth through activating the STAT3-granzyme B pathway in tumor-infiltrating cytotoxic T lymphocytes

BackgroundType I interferons (IFN-I) have recently emerged as key regulators of tumor response to chemotherapy and immunotherapy. However, IFN-I function in cytotoxic T lymphocytes (CTLs) in the tumor microenvironment is largely unknown.MethodsTumor tissues and CTLs of human colorectal cancer patients were analyzed for interferon (alpha and beta) receptor 1 (IFNAR1) expression. IFNAR1 knock out (IFNAR-KO), mixed wild type (WT) and IFNAR1-KO bone marrow chimera mice, and mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO) were used to determine IFN-I function in T cells in tumor suppression. IFN-I target genes in tumor-infiltrating and antigen-specific CTLs were identified and functionally analyzed.ResultsIFNAR1 expression level is significantly lower in human colorectal carcinoma tissue than in normal colon tissue. IFNAR1 protein is also significantly lower on CTLs from colorectal cancer patients than those from healthy donors. Although IFNAR1-KO mice exhibited increased susceptibility to methylcholanthrene-induced sarcoma, IFNAR1-sufficient tumors also grow significantly faster in IFNAR1-KO mice and in mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO), suggesting that IFN-I functions in T cells to enhance host cancer immunosurveillance. Strikingly, tumor-infiltrating CTL levels are similar between tumor-bearing WT and IFNAR1-KO mice. Competitive reconstitution of mixed WT and IFNAR1-KO bone marrow chimera mice further determined that IFNAR1-deficient naïve CTLs exhibit no deficiency in response to vaccination to generate antigen-specific CTLs as compared to WT CTLs. Gene expression profiling determined that Gzmb expression is down-regulated in tumor-infiltrating CTLs of IFNAR1-KO mice as compared to WT mice, and in antigen-specific IFNAR1-KO CTLs as compared to WT CTLs in vivo. Mechanistically, we determined that IFN-I activates STAT3 that binds to the Gzmb promoter to activate Gzmb transcription in CTLs.ConclusionIFN-I induces STAT3 activation to activate Gzmb expression to enhance CTL effector function to suppress tumor development. Human colorectal carcinoma may use down-regulation of IFNAR1 on CTLs to suppress CTL effector function to evade host cancer immunosurveillance.

Abstract B077: Signatures of T-cell dysfunction and exclusion predict cancer immunotherapy response

Abstract Cancer treatment by immune checkpoint blockade (ICB) can bring long-lasting clinical benefits, but only a fraction of patients responds to treatment. To predict ICB response we developed TIDE, a computational method to model two primary mechanisms of tumor immune evasion: inducing T-cell dysfunction in tumors with high infiltration of cytotoxic T lymphocytes (CTL) and preventing T-cell infiltration in tumors with low CTL level. We identified signatures of T-cell dysfunction from large tumor cohorts by testing how the expression of each gene in tumors interacts with the CTL infiltration level to influence patient survival. We also modeled factors that exclude T-cell infiltration into tumors using expression signatures from immunosuppressive cells. Using this framework and pre-treatment RNA-Seq or NanoString tumor expression profiles, TIDE predicted the outcome of melanoma patients treated with first-line anti-PD1 or anti-CTLA4 more accurately than other biomarkers such as PD-L1 level and mutation load. TIDE also revealed new candidate ICB resistance regulators, such as SERPINB9, demonstrating utility for immunotherapy research. TIDE source code and a web application are available at http://tide.dfci.harvard.edu. Citation Format: Peng Jiang, Shengqing Gu, Deng Pan, Jingxin Fu, Avinash Sahu, Xihao Hu, Ziyi Li, Nicole Traugh, Xia Bu, Bo Li, Jun Liu, Gordon J Freeman, Myles A Brown, Kai W. Wucherpfennig, Xiaole Shirley Liu. Signatures of T-cell dysfunction and exclusion predict cancer immunotherapy response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B077.

Reactive versus Constitutive: Reconcile the Controversial Results about the Prognostic Value of PD-L1 Expression in cancer.

The prognostic value of programmed death-ligand 1 (PD-L1) has been controversial in recent studies. PD-L1 is known to play a major role in suppressing the immune response, yet increasing studies have reported that PD-L1 expression has a favorable prognostic value for cancer patients. This raises the concern about how to understand PD-L1 expression: merely an immune inhibitory signal, or more likely a reactive process to T-cell response that indicates cytotoxic T lymphocyte (CTL) level in a tumor? To solve this dilemma, an integrative investigation is required. We compared the PD-L1 expression between tumor cells and immune cells, and characterized the inter- and intra-tumor correlation between CTL and PD-L1 expression. The prognostic values between PD-L1 and CTL is compared across 15 solid cancers and 11 independent cohorts of ovarian cancer. PD-L1 and PD-L1-adjusted CTL are analyzed in immunotherapy dataset receiving nivolumab. We observed unexpected high concordance between the prognostic value of PD-L1 and CTL across different cancers and cohorts. We found primarily reactive rather than constitutive PD-L1 expression in most tumors. We revealed that PD-L1-adjusted CTL level, rather than the expression of PD-L1, effectively predicts the responders to immune checkpoint inhibitors. This study highlights the importance of PD-L1 expression, as primarily a signature of reacting efficiency of pre-existing anti-tumor immunity, in balancing the tumor microenvironment. Importantly, it suggests that the reactive efficiency of PD-L1 is more useful to predict the response to immunotherapy.

Signatures of T cell dysfunction and exclusion predict cancer immunotherapy response.

Cancer treatment by immune checkpoint blockade (ICB) can bring long-lasting clinical benefits, but only a fraction of patients respond to treatment. To predict ICB response, we developed TIDE, a computational method to model two primary mechanisms of tumor immune evasion: the induction of T cell dysfunction in tumors with high infiltration of cytotoxic T lymphocytes (CTL) and the prevention of T cell infiltration in tumors with low CTL level. We identified signatures of T cell dysfunction from large tumor cohorts by testing how the expression of each gene in tumors interacts with the CTL infiltration level to influence patient survival. We also modeled factors that exclude T cell infiltration into tumors using expression signatures from immunosuppressive cells. Using this framework and pre-treatment RNA-Seq or NanoString tumor expression profiles, TIDE predicted the outcome of melanoma patients treated with first-line anti-PD1 or anti-CTLA4 more accurately than other biomarkers such as PD-L1 level and mutation load. TIDE also revealed new candidate ICB resistance regulators, such as SERPINB9, demonstrating utility for immunotherapy research.

Abstract 4966: The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion

Abstract Human colon cancer, except for the small subset of microsatellite instable (MSI) colon cancer, does not respond to anti-PD-L1/PD-1 immune checkpoint inhibitor (ICI) immunotherapy. A well-established notion is that MSI colon cancer harbors various mutations that serve as neoantigens to generate tumor-specific cytotoxic T lymphocytes (CTLs). Because CTLs suppress tumor through inducing tumor cell apoptosis, we hypothesized that colon cancer cell intrinsic apoptosis resistance is potentially another mechanism underlying microsatellite stable (MSS) colon cancer nonresponse to ICI immunotherapy. If tumor cells are not sensitive to apoptosis induction, then tumor cells cannot be killed by CTLs regardless of how potent the CTLs are. To test this hypothesis, we analyzed MSI and MSS human colon carcinoma specimens and developed a small molecule that targets H3K9me3-mediated cell death pathways as a sensitizer for colon cancer ICI immunotherapy. As expected, we observed high level of CTL infiltration in all eight MSI colon carcinoma specimens examined. However, five of the nine MSS colon carcinoma specimens also exhibited high level of CTL infiltration, suggesting that CTL level is not the sole mechanism underlying MSS colon cancer non-response to ICI immunotherapy. Analysis of the TCGA database revealed that SUV39H1, a histone methyltransferase that catalyzes H3K9me3, is significantly elevated in human colon carcinoma specimens compared to normal human colon tissues. A SUV39H1 structure-based virtual chemical library screening in combination with functional assays identified a small-molecule SUV39H1 inhibitor. Further analysis of the top forty-three hits identified a potent SUV39H1 inhibitor. Structure modifications were then performed and a chemical synthesis procedure was developed to synthesize a novel SUV39H1 inhibitor, termed F5446. F5446 has an EC50 of 0.496 µM against SUV39H1 enzymatic activity and suppresses human colon carcinoma cell growth in a dose-dependent manner in vitro. F5446 suppresses colon carcinoma growth through upregulating key cell cycle regulators to induce cell cycle arrest at the S phase. Furthermore, F5446 also decreased H3K9me3 at the FAS promoter to upregulate Fas expression. Consequently, a sublethal dose of F5446 effectively overcame human colon carcinoma cell resistance to apoptosis induced by FasL, a ligand on activated CTLs in vitro. Treatment of colon carcinoma-bearing mice with F5446 and anti-PD-1 both significantly suppress tumor growth in vivo. Our data determined that F5446 is a novel SUV39H1 inhibitor that has the potential to be further developed as a sensitizer to overcome colon carcinoma resistance to ICI immunotherapy through activating the Fas-FasL pathway. Citation Format: Chunwan Lu, Dafeng Yang, John D. Klement, Aaron H. Colby, Mark W. Grinstaff, Cedric Pearce, Nicholas H. Oberlies, Thomas Albers, Iryna Lebedyeva, Kebin Liu. The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion [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 4966.