Cytotoxic Function Of CD8 Research Articles

Aldehyde dehydrogenase 2-mediated aldehyde metabolism promotes tumor immune evasion by regulating the NOD/VISTA axis

BackgroundAldehyde dehydrogenase 2 (ALDH2) is a crucial enzyme involved in endogenous aldehyde detoxification and has been implicated in tumor progression. However, its role in tumor immune evasion remains unclear.MethodsHere, we...

High glucose-upregulated PD-L1 expression through RAS signaling-driven downregulation of PTRH1 leads to suppression of T cell cytotoxic function in tumor environment

BackgroundNearly 80% of patients with pancreatic cancer suffer from glucose intolerance or diabetes. Pancreatic cancer complicated by diabetes has a more immunosuppressive tumor microenvironment (TME) and is associated with a worse prognosis. The relationship between glucose metabolism and programmed cell death-Ligand 1 (PD-L1) is close and complex. It is important to explore the regulation of high glucose on PD-L1 expression in pancreatic cancer and its effect on infiltrating immune effectors in the tumor microenvironment.MethodsDiabetic murine models (C57BL/6) were used to reveal different immune landscape in euglycemic and hyperglycemic pancreatic tumor microenvironment. Bioinformatics, WB, iRIP [Improved RNA Binding Protein (RBP) Immunoprecipitation]-seq were used to confirm the potential regulating role of peptidyl-tRNA hydrolase 1 homolog (PTRH1) on the stability of the PD-L1 mRNA. Postoperative specimens were used to identify the expression of PD-L1 and PTRH1 in pancreatic cancer. Co-culturing T cells with pancreatic cancer cells to explore the immunosuppressive effect of pancreatic tumor cells.ResultsOur results revealed that a high dose of glucose enhanced the stability of the PD-L1 mRNA in pancreatic tumor cells by downregulating PTRH1 through RAS signaling pathway activation following epidermal growth factor receptor (EGFR) stimulation. PTRH1 overexpression significantly suppressed PD-L1 expression in pancreatic cells and improved the proportion and cytotoxic function of CD8+ T cells in the pancreatic TME of diabetic mice.ConclusionsPTRH1, an RBP, plays a key role in the regulation of PD-L1 by high glucose and is closely related to anti-tumor immunity in the pancreatic TME.

Synergy of heterodimeric IL-15 immunotherapy and a Fatty Acid Metabolism Modulator enhances intratumoral lymphocyte metabolism and eradicates EO771 murine breast tumors

Abstract Tumor-infiltrating cytotoxic CD8 +T cells frequently acquire an altered state of differentiation referred to as “exhaustion”, which prevents control of tumor growth. We study hetIL-15, a cytokine that stimulates the generation, proliferation and cytotoxic function of CD8 +T and NK cells. The objectives of this study was to assess the effects of hetIL-15 immunotherapy in TNBC tumors, to evaluate the metabolic profile of the tumor-infiltrating T cells and to study any potential synergy with a Fatty Acid Metabolism Modulator (FAMM). Using the EO771 orthotopic breast cancer model we monitored the efficacy of locoregional administration of hetIL-15 in combination with a FAMM. We monitored the effect of treatment on tumor growth, survival of mice and the status of tumor-infiltrating immune cells using Seahorse and flow cytometry. hetIL-15 locoregional administration resulted in complete tumor regression in 40% of the treated animals and increased survival. The infiltrating cytotoxic CD8 +T and NK cells were increased in hetIL-15 treated tumors and showed enhanced activation and proliferation. Their metabolic flux analysis confirmed a robust rise in Oxygen Consumption Rate (OCR) with substantial increase of spare respiratory capacity. Combined treatment of hetIL-15 immunotherapy and FAMM resulted in increased mitochondrial function, FA uptake and OCR. In addition, combination therapy resulted in statistically significant tumor growth delay and complete eradication of the tumors in 85% of mice. Our results indicate that metabolic reprogramming of tumor-specific CD8 +T cells might represent a strategy to promote survival in the metabolically hostile Tumor Microenvironment (TME), enhancing the clinical efficacy of immunotherapy.

Akkermansia supplementation reverses the tumor-promoting effect of the fecal microbiota transplantation in ovarian cancer

Ovarian cancer (OC) remains a clinical challenge for its difficulty in early diagnosis and insensitivity to treatments. Gut microbiota modulate multiple carcinoma progression through immunoregulation. The relationship between OC and gut microbiota has not been fully characterized. We find that the feces of patients with OC demonstrate different characteristics from benign controls. After fecal microbiota transplantation (FMT) from patients with OC into OC-bearing mice, the tumor development accelerates. Further, an Akkermansia supplementation with FMT significantly suppresses OC progression in mice. RNA sequencing of tumors shows that Tcell activation pathways are upregulated after Akkermansia supplementation with FMT. Moreover, acetate accumulation accompanies Akkermansia abundance elevation, which is associated with enhanced interferon γ (IFNγ) secretion of CD8+ Tcells and also its tumor-killing property. This work highlights the importance of protective gut microbiome in immune surveillance of OC, which connects accumulation of acetate and the cytotoxic function of CD8+ Tcells by increasing IFNγ secretion.

Obesity accelerates immune evasion of non-small cell lung carcinoma via TFEB-dependent upregulation of Siglec-15 and glycolytic reprogramming

Although obesity contributes to tumor incidence and progression in various cancers, whether obesity impacts the tumor microenvironment (TME) of non-small cell lung cancer (NSCLC) remains largely under-explored. We generated NSCLC xenograft model in diet-induced obese mice and identified that TFEB is critical to accelerate obesity-related NSCLC progression with mimic intrinsic functions on tumor biology. Mechanically, TFEB binds directly to Siglec-15 promoter to upregulate Siglec-15 expression and binds to Hk2 and Ldha promoters to enhance glycolytic flux in NSCLC cells, which restrain the expansion and cytotoxic function of CD8+ T cells while maintain suppressive Treg cells in TME, jointly promoting immune evasion of NSCLC cells in obesity. Blocking tumor TFEB improves the therapeutic efficiency of anti-PD-1 in obese mice. Altogether, our data identify essential roles of TFEB in remodeling immunosuppressive TME and promoting NSCLC development in obesity, providing scientific rational for TFEB as a potential biomarker to predict immune checkpoint blockade efficiency in obese NSCLC patients.

Splicing factor SRSF1 is essential for CD8 T cell function and host antigen-specific viral immunity.

Cytotoxic CD8 T cells are crucial for the host antigen-specific immune response to viral pathogens. Here we report the identification of an essential role for the serine/arginine-rich splicing factor (SRSF) 1 in CD8 T cell homeostasis and function. Specifically, SRSF1 is necessary for the maintenance of normal CD8 T lymphocyte numbers in the lymphoid compartment, and for the proliferative capacity and cytotoxic function of CD8 T cells. Furthermore, SRSF1 is required for antigen-specific IFN-γ cytokine responses in a viral infection challenge in mice. Transcriptomics analyses of Srsf1-deficient T cells reveal that SRSF1 controls proliferation, MAP kinase signaling and IFN signaling pathways. Mechanistically, SRSF1 controls the expression and activity of the Mnk2/p38-MAPK axis at the molecular level. Our findings reveal previously unrecognized roles for SRSF1 in the physiology and function of cytotoxic CD8 T lymphocytes and a potential molecular mechanism in viral immunopathogenesis.

Abstract 1361: Crosstalk between peripherally expanded T cells and Tfh-like cells in shaping effector functions of CD8 T cells in ovarian cancer

Abstract Background: Immune checkpoint blockade (ICB) of PD-1 and/or CTLA-4 can induce long-lasting clinical responses in several neoplasias. However, despite the successful response in some cancer, it is still ineffective for other malignant lesions such as ovarian cancer. Studies on T cell dynamics demonstrated that antitumor immune response from ICB is mediated by clonal replacement of CD8 T cells. Moreover, the presence of clones expanded in both tumor and peripheral tissue, also called dual-expanded, can predict response to ICB. To understand T cell dynamics in ovarian cancer, we characterized intratumoral and peripheral immune cells by single-cell transcriptome and TCR-sequencing. Methods and results: Using the 5’ Chromium assay (10X Genomics) on CD45+ cells isolated from 5 naïve-treatment patients undergoing cytoreductive surgery, we identified T cell states and classified clones by location (blood or tumor) and expansion pattern. In addition, the data revealed that clones expanded within the tumor were terminally exhausted (upregulation of PDCD1, HAVCR2, CTLA4, ENTPD1, and CXCL13 genes), while dual-expanded clones (expanded in blood and tumor) upregulated genes associated with the cytotoxic function of CD8 T cells, such as granzymes (GZMA and GZMB) and perforin (PRF1). Considering the importance of dual-expanded clones in antitumor immune response, we interrogated the cellular interactions they encountered in the tumor microenvironment. Using the tool NicheNet we identified IL21 as one of the top ligands mediating the interaction between dual-expanded clones and T follicular helper-like CD4+ cells (Tfh-like cells). Interestingly, the gene signature of Tfh-like cells showed that besides dual-expanded clones, these cells might attract other immune cells through CXCL13 and oxysterol metabolites. We identified B cells and plasmacytoid dendritic cells (pDCs) as the major cell types recruited by the CXCL13-CXCR5 axis and oxysterol-GPR183, respectively. Additionally, memory-like CD8 T cells were found to express GPR183, suggesting that a gradient of oxysterol might recruit them to a region defined by Tfh-like cells. Conclusions: Taken together, our study provides insights into additional functions of Tfh-like cells in the recruitment of immune cells through oxysterol gradient. A better understanding of the T cell dynamics and antitumor immune response can provide potential development of novel immunotherapy for the treatment of ovarian cancer. Citation Format: Mayra Carneiro, Cheng Zhao, Paméla Thébault, Yacine Bareche, Kurosh Rahimi, Jean-François Cailhier, Anne-Marie Mes-Masson, John Stagg, Sophie Petropoulos, Réjean Lapointe. Crosstalk between peripherally expanded T cells and Tfh-like cells in shaping effector functions of CD8 T cells in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1361.

Combined Application of Bevacizumab and Pd-1 Blockade Displays Durable Treatment Effects by Increasing the Infiltration And Cytotoxic Function of Cd8 + T Cells in Lung Cancer

Aim: VEGF/VEGFR inhibitors may help immune checkpoint inhibitors expand the population that will benefit from treatment. The authors investigated the efficacy of combined bevacizumab and PD-1 antibody. Materials & methods: C57BL/6J mice were injected subcutaneously with 1×106 Lewis lung carcinoma cells. The mice were intraperitoneally injected with 0.25mg anti-PD-1 inhibitors and/or 15mg/kg bevacizumab. Tumor tissues were harvested. The authors reported that a non-small cell lung cancer patient received 200 mg PD-1 antibodycombined with 7.5mg/kg bevacizumab as fourth-line treatment. Results: Bevacizumab combined with PD-1 antibody induced a strong and durable antitumor effect. Bevacizumab combined with PD-1 antibody improved abnormal tumor vessels and enhanced the cytotoxic function and infiltration of T lymphocytes. The patient's survival time was significantly prolonged. Conclusion: Bevacizumab combined with anti-PD-1 antibody induces a durable antitumor effect by increasing the infiltration and cytotoxic function of CD8+ T cells in lung cancer.

Elongation factor-2 kinase is a critical determinant of the fate and antitumor immunity of CD8+ T cells.

eEF-2K has important roles in stress responses and cellular metabolism. We report here a previously unappreciated but critical role of eEF-2K in regulating the fate and cytocidal activity of CD8+ T cells. CD8+ T cells from eEF-2K KO mice were more proliferative but had lower survival than their wild-type counterparts after their activation, followed by occurrence of premature senescence and exhaustion. eEF-2K KO CD8+ T cells were more metabolically active and showed hyperactivation of the Akt-mTOR-S6K pathway. Loss of eEF-2K substantially impaired the activity of CD8+ T cells. Furthermore, the antitumor efficacy and tumor infiltration of the CAR-CD8+ T cells lacking eEF-2K were notably reduced as compared to the control CAR-CD8+ T cells. Thus, eEF-2K is critically required for sustaining the viability and function of cytotoxic CD8+ T cells, and therapeutic augmentation of this kinase may be exploited as a novel approach to reinforcing CAR-T therapy against cancer.

Tanshinone IIA alleviates vitiligo by suppressing AKT mediated CD8+ T cells activation in a mouse model.

Tanshinone IIA has been reported to exhibit anti-inflammatory effects, while it is not clear whether Tanshinone IIA has protective role in vitiligo. Premelanosome (PMEL) CD8+ T cells were adoptive transferred into Krt14- Kitl* mice with Kit ligand (KITL) over-expressed, to construct the vitiligo model. Pdk1fl/fl and Stat3fl/fl mice were crossed with Cd8cre mice to establish Pdk1TKO and Stat3TKO mice. Tanshinone IIA (200 μg) was intravenous injected to treat vitiligo in mice every 3 days. The accumulation of macrophages and CD8+ T cells in the ear skin was assayed by flow cytometry. Bone marrow-derived macrophages (BMDMs) were induced and stimulated with lipopolysaccharides (LPS) and IL-4. It was found that Tanshinone IIA alleviated the development of vitiligo, impaired PMEL CD8+ T cells accumulation in the ear skin, and inhibited LPS-induced TNF-α, IL-6, and IL-1β expression and secretion in BMDMs, which could also inhibit IL-4-induced Arg-1 and Mrc-1 expression in BMDMs. In addition, Tanshinone IIA could inhibit the proliferation and cytotoxic function of CD8+ T cells indicated by the expression of Perforin, Granzymeb, and IFN-γ. Furthermore, Tanshinone IIA treated Pdk1TKO mice, not Stat3TKO mice, showed impaired PMEL CD8+ T cells accumulation in the ear skin. In summary, Tanshinone IIA alleviates vitiligo development with impaired CD8+ T cells accumulation and activation of Pdk1-Akt pathway.

Alphataxin, a Small-molecule Drug that Elevates Tumor-infiltrating CD4+ T Cells, in Combination with Anti-PD-1 Therapy, Suppresses Murine Renal Cancer and Metastasis

Abstract By promoting the cytotoxic function of CD8+ T cells, immune checkpoint inhibitor therapy, e.g. programmed cell death protein-1 (PD-1), effectively inhibits tumor growth in renal cell carcinoma. Yet, as many as 87% of cancer patients do not respond to immune checkpoint therapy. The function of cytotoxic CD8+ T cells integrally relies on CD4+ T helper cells. Remarkably, despite advances in immunotherapy, there are no pharmaceutical treatments that increase circulating CD4+ T cell counts. Nor has there been much attention given to tumor-infiltrating CD4+ T cells. The orally available small-molecule drug Alphataxin targets a pathway that regulates the number of circulating CD4+ T cells. We aimed to examine how Alphataxin affected tumor growth in a murine model of renal cell carcinoma. Alphataxin, in combination with anti-PD-1 antibody, significantly elevated the ratio of circulating and tumor-infiltrating CD4+/CD8+ T cells. In one study, following orthotopic implantation of syngeneic renal cell carcinoma, combination treatment resulted in 100% regression of tumor growth. Moreover, in mice implanted orthotopically with one log more tumor cells, doubling Alphataxin dose in combination treatment led to 100% regression in one-third of mice and 81% suppression of tumor growth in the remaining two-thirds of mice. Lung metastasis was evident in all mice except combination-treated mice. Orally available Alphataxin, the first and only drug developed to increase CD4+ T cells, in combination with anti-PD-1, is a powerful therapeutic method that provides long-term remission in renal cell carcinoma and potentially other T cell-responsive cancers by increasing the number of CD4+ tumor-infiltrating T cells.

Heterodimeric IL-15 in Cancer Immunotherapy.

Simple SummaryThe rapidly expanding field of cancer immunotherapy uses diverse technologies, including cytokines, T cells, and antibody administration, with the aim to induce effective immune responses leading to tumor control. Interleukin-15 (IL-15), a cytokine discovered in 1994, supports the homeostasis of cytotoxic immune cells and shows promise as an anti-tumor agent. Many studies have elucidated IL-15 synthesis, regulation and biological function and explored its therapeutic efficacy in preclinical cancer models. Escherichia coli-derived single-chain IL-15 was tested in the first in-human trial in cancer patients. Its effects were limited by the biology of IL-15, which in vivo comprises a complex of the IL-15 chain with the IL-15 receptor alpha (IL-15Rα) chain, together forming the IL-15 heterodimer (hetIL-15). Currently, single-chain IL-15 and several heterodimeric IL-15:IL-15Rα variants (hetIL-15, N-803 and RLI) are being tested in clinical trials. This review presents a summary of contemporary preclinical and clinical research on IL-15.Immunotherapy has emerged as a valuable strategy for the treatment of many cancer types. Interleukin-15 (IL-15) promotes the growth and function of cytotoxic CD8+ T and natural killer (NK) cells. It also enhances leukocyte trafficking and stimulates tumor-infiltrating lymphocytes expansion and activity. Bioactive IL-15 is produced in the body as a heterodimeric cytokine, comprising the IL-15 and the so-called IL-15 receptor alpha chain that are together termed “heterodimeric IL-15” (hetIL-15). hetIL-15, closely resembling the natural form of the cytokine produced in vivo, and IL-15:IL-15Rα complex variants, such as hetIL-15Fc, N-803 and RLI, are the currently available IL-15 agents. These molecules have showed favorable pharmacokinetics and biological function in vivo in comparison to single-chain recombinant IL-15. Preclinical animal studies have supported their anti-tumor activity, suggesting IL-15 as a general method to convert “cold” tumors into “hot”, by promoting tumor lymphocyte infiltration. In clinical trials, IL-15-based therapies are overall well-tolerated and result in the expansion and activation of NK and memory CD8+ T cells. Combinations with other immunotherapies are being investigated to improve the anti-tumor efficacy of IL-15 agents in the clinic.

NKG7 - regulating endosomal pathways?

Ng et al. have identified NKG7 as a regulator of inflammation in response to diverse immunological challenges. While NKG7 was required for the degranulation of cytotoxic cells, additional defects including reduced expansion and trafficking of CD8 T cells, and altered antigen presentation, were noted in NKG7-deficient mice. The precise mechanism by which NKG7 mediates its effects has not been resolved but may involve regulation of endosomal vesicle trafficking.

Abstract 5694: Complete regression of murine breast tumors by hetIL-15FC monotherapy correlates with infiltration by T, NK, cDC1 cells and a novel population of dendritic cells

Abstract Introduction: IL-15 cytokine stimulates the proliferation and cytotoxic function of CD8+ T and NK cells. Several preclinical models have supported the anti-tumor activity of IL-15. We have produced the native heterodimeric IL-15 (hetIL-15) of human, macaque or mouse and also fusions to the Fc antibody fragments (hetIL-15FC). Experimental procedures: We studied the therapeutic efficacy of hetIL-15 or hetIL-15FC immunotherapy in the murine EO771 orthotopic breast cancer model in syngeneic C57BL/6 mice. The effects of hetIL-15FC on immune cells were compared in tumors, lymph nodes and spleen by flow cytometry, immunohistochemistry (IHC) and gene expression profiling (Nanostring). We also assessed the metabolic profile of tumor infiltrating T cells. Results: hetIL-15 therapy delayed the primary tumor growth and increased overall survival. hetIL-15 or hetIL-15FC peritumoral administration resulted in tumor regression in 40% of the treated animals and resistance to tumor re-challenge. Therefore, monotherapy induced long term immunological memory able to block tumor growth. Both CD8+ T and NK cells were increased in hetIL-15FC treated tumors; phenotypic analysis showed they were activated and in an active proliferation state. Transcriptomic analysis confirmed the activated state of the T and NK cells, whereas metabolic flux analysis of the infiltrated CD8+T cells from treated mice confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity (as a measure of mitochondrial reserve), which supports an activated/non exhausted phenotype of these hetIL-15 treated effector cells. In addition, peritumoral hetIL-15FC administration resulted in an increase of infiltrating, conventional type 1 dendritic cells (cDC1s), suggesting mechanisms of DC-lymphocyte interactions in the tumor. We also observed a new population of intra-tumoral DCs induced by hetIL-15, CD24+ F4/80high cDC2s (F4/80high DCs). The new DC population was negatively correlated with the tumor size of EO771 tumor bearing mice. Phenotypic profiling of F4/80high DCs identified expression of several cDC1 specific markers, including CD103 and IRF8 and t-SNE analysis clustered F4/80high DCs close to cDC1s and macrophages. Conclusion: Our results show complete eradication of EO771 tumors by hetIL-15FC treatment, correlating with different tumor infiltrating immune cell types. We propose that the anti-cancer activity of hetIL-15 in primary tumors is orchestrated by the interplay of CD8+T cells, cDC1s and a novel subset of antigen presenting cells with a distinct phenotypic profile. hetIL-15 supported a favorable metabolic profile of intra-tumoral effector lymphocytes, important for their function. Effective hetIL-15 treatment leads to development of long-term anti-tumor memory. Citation Format: Sevasti Karaliota, Dimitris Stellas, Vasiliki Stravokefalou, Bethany Nagy, Cristina Bergamaschi, Barbara K. Felber, George N. Pavlakis. Complete regression of murine breast tumors by hetIL-15FC monotherapy correlates with infiltration by T, NK, cDC1 cells and a novel population of dendritic cells [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 5694.

Abstract 6651: Combination of hetIL-15 with chemotherapy in triple negative breast and pancreatic cancer mouse models increases tumor necrosis and alleviates metastatic disease

Abstract Introduction: We evaluated the potential therapeutic benefit of conventional chemotherapy combined with hetIL-15 immunotherapy in two mouse models of pancreatic and breast cancer. IL-15 is an important cytokine that stimulates the proliferation and cytotoxic functions of CD8+ T cells and NK cells. We have produced the native heterodimeric form of IL-15 (hetIL-15), which has advanced in clinical trials due to its anticancer activities. Study design and methods: We assessed the efficacy of hetIL-15 immunotherapy in combination with the chemotherapeutic agents gemcitabine or doxorubicin on the growth of primary pancreatic and breast tumors, and on the metastatic disease. We used the genetically engineered mouse model (GEMM) KPC of pancreatic cancer (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre) and the orthotopic 4T1 triple negative breast cancer (TNBC) model. We analyzed the tumor infiltrating lymphocytes (TILs) by flow cytometry and immunohistochemistry (IHC) and evaluated the extent of necrosis on the primary tumors by H&E and cytokeratin staining. We also evaluated the lung metastatic burden by histology. Results: The combination of hetIL-15 and doxorubicin significantly delayed 4T1 tumor growth. In addition, histological analysis showed an increase in the percentage of the necrotic areas of the primary tumors in the combination group. hetIL-15 treated groups also showed increased necrotic areas of the primary pancreatic tumors, although tumor growth showed no significant change compared to the control group. Flow analysis of TILs showed increased CD8+/CD4+ T cell ratios in tumors of hetIL-15 treated groups in both cancer models, which was confirmed by IHC. Importantly, the evaluation of the metastatic disease showed that hetIL-15 treated animals in both cancer models had reduced metastatic foci and in some cases were completely disease free. Chemotherapy did not significantly reduce the numbers of the total metastatic lesions but reduced their size. Conclusions: Chemotherapy can be combined with hetIL-15 treatment in both breast and pancreatic mouse models. Despite small beneficial results in the size of primary tumor, histological evaluation revealed increased tumor necrosis and potential synergy of chemotherapy and immunotherapy. The observed decreased metastatic burden in the lungs in both cancer models indicates that hetIL-15 strongly reduces metastases in several cancer types and suggests that hetIL-15 is a promising therapeutic agent against metastatic disease. Citation Format: Dimitris Stellas, Vasiliki Stravokefalou, Sevasti Karaliota, Bethany Nagy, Serguei Koslov, Konstantinos Dimas, Barbara K. Felber, George N. Pavlakis. Combination of hetIL-15 with chemotherapy in triple negative breast and pancreatic cancer mouse models increases tumor necrosis and alleviates metastatic disease [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 6651.

Sophoridine suppresses macrophage-mediated immunosuppression through TLR4/IRF3 pathway and subsequently upregulates CD8+ T cytotoxic function against gastric cancer

Gastric cancer is one of the most common and deadly neoplasms with limited effective treatments. The emergence of the immunotherapy has brought great expectations for cancer patients. Sophoridine is extracted from the seeds of sophora alopecuroides and has various pharmacological actions including anti-tumor, anti-inflammatory, anti- arrhythmia and anti-virus. However, the effect of Sophoridine on gastric cancer microenvironment immunity and its underling mechanism remains poorly known. This study was aimed to investigate the effect of Sophoridine on the polarization status of gastric tumor-associated macrophages (TAMs) and its underlying mechanism. We isolated primary bone marrow-derived macrophages (BMDMs) and primary CD8+ T cells to perform coculture assay. Sophoridine educated TAMs polarize to M1-TAMs and suppressed M2-TAMs polarization through TLR4/IRF3 axis. Sophoridine-treated TAMs exhibited stronger pro-inflammatory function through upregulation the expression of INOS, IFN-β and IL-12α, and downregulation the expression of Arg-1, CD206 and IL-10. Sophoridine -primed TAMs increased the proliferation and cytotoxic function of CD8+ T by upregulating the expression of Granzyme-B, TNF-α and Perforin, and downregulated the expression of CD8+ T cells function exhaustion markers PD-1, Tim-3 and Lag-3. Furthermore, Sophoridine inhibited the migration ability of macrophage by decrease the CCR2 expression. Thus, Sophoridine acted on macrophages and CD8+ T cells to reshape gastric cancer immune microenvironment. Our studies provided preclinical basis for clinical application of Sophoridine.

Metabolite of Carbamazepine Activates NLRP3 Inflammasome in Keratinocytes and Regulates CD8+ T-Cell Skin Trafficking in SJS/TEN

Background: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute cutaneous adverse drug reactions involving keratinocyte death and CD8+ T lymphocyte-mediated immune response. Drugs, especially anti-epileptic drugs such as carbamazepine (CBZ) are the most important etiologic agents in patients with SJS/TEN. Carbamazepine-10,11-epoxide (CBZE), a pharmacologically active metabolite of CBZ, oxidated by cytochrome P450 enzyme 3A4 (CYP3A4) in the liver, and is also distributed in the epidermis of skin. Why do patients of SJS/TEN develop a violent CD8+ T cells immune response to the epidermis of skin is unknown. Methods: Sera and blister fluid of six patients with SJS and four patients with TEN were obtained from Xijing Hospital. The levels of interleukin-1β (IL-1β), IL-18, Granzyme B, and granulysin were assessed by ELISA and quantitative real-time PCR. The mechanism results were confirmed with transient transfection of small interfering RNAs into cultured keratinocytes, western blot, migration assay, flow cytometry, and immunohistochemistry. Findings: Increased expression of NLRP3 inflammasome in lesions of patients with SJS/TEN. Increased levels of interleukin-1β (IL-1β) and IL-18 significantly correlated with body surface area (BSA), SCORe of Toxic Epidermal Necrosis (SCORTEN), and levels of Granzyme B in sera, blister fluid and lesions of patients with SJS/TEN. CBZE induces NLRP3 inflammasome activation in SJS/TEN keratinocytes. Activation of NLRP3 inflammasome in SJS/TEN keratinocytes upregulates the expression of CXCL9/CXCL10, activates CD8+ T lymphocytes, and increases the expression of cytotoxic markers via signaling of IL-1β/IL-1R or IL-18/IL-18R. This promotes chemotactic ability in CD8+ T lymphocytes of patients with SJS/TEN. Interpretation: To our knowledge, this study shows for the first time that CBZE induces activation of NLRP3 inflammasome and increases activation, migration and cytotoxic functions of CD8+ T lymphocytes in skin, thereby contributing to the dysregulated response in patients with SJS/TEN. Funding: This work was supported by the National Natural Science Foundation of China (81371738, 31400782, 81573038, 81472885). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The investigational protocol was approved by the Ethics Committee of the Fourth Military Medical University, according to the guidelines of Declaration of Helsinki.

The PD-1/PD-L1 Pathway Affects the Expansion and Function of Cytotoxic CD8+ T Cells During an Acute Retroviral Infection.

Cytotoxic CD8+ T lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. The checkpoint receptor PD-1 suppresses the functionality of virus-specific CD8+ T cells during chronic infection. However, the role of the PD-L1/PD-1 pathway during the acute phase of infections has not been well characterized. In the current study the effects of PD-1 or PD-L1 deficiency on the CD8+ T cell response against Friend retroviral (FV) infection of knockout mice was analyzed during acute infection. We observed an enhanced proliferation, functional maturation, and reduced apoptosis of effector CD8+ T cells in the absence of PD-1 or PD-L1. The knockout of PD-L1 had a stronger effect on the functionality of CD8+ T cells than that of PD-1. Augmented CTL responses were associated with an improved control of FV replication. The strong phenotype of FV-infected PD-L1 knockout mice was independent of the interaction with CD80 as an additional receptor for PD-L1. Furthermore, we performed a detailed analysis of the production of different granzymes in virus-specific CD8+ T cells and observed that especially the simultaneous production of multiple granzymes in individual T cells (multifunctionality) was under the control of the PD-1/PD-L1 pathway. The findings from this study allow for a better understanding of the development of antiviral cytotoxic immunity during acute viral infections.

Cytotoxic effector functions of T cells are not required for protective immunity against fatal Rickettsia typhi infection in a murine model of infection: Role of TH1 and TH17 cytokines in protection and pathology.

Endemic typhus caused by Rickettsia (R.) typhi is an emerging febrile disease that can be fatal due to multiple organ pathology. Here we analyzed the requirements for protection against R. typhi by T cells in the CB17 SCID model of infection. BALB/c wild-type mice generate CD4+ TH1 and cytotoxic CD8+ T cells both of which are sporadically reactivated in persistent infection. Either adoptively transferred CD8+ or CD4+ T cells protected R. typhi-infected CB17 SCID mice from death and provided long-term control. CD8+ T cells lacking either IFNγ or Perforin were still protective, demonstrating that the cytotoxic function of CD8+ T cells is not essential for protection. Immune wild-type CD4+ T cells produced high amounts of IFNγ, induced the release of nitric oxide in R. typhi-infected macrophages and inhibited bacterial growth in vitro via IFNγ and TNFα. However, adoptive transfer of CD4+IFNγ-/- T cells still protected 30–90% of R. typhi-infected CB17 SCID mice. These cells acquired a TH17 phenotype, producing high amounts of IL-17A and IL-22 in addition to TNFα, and inhibited bacterial growth in vitro. Surprisingly, the neutralization of either TNFα or IL-17A in CD4+IFNγ-/- T cell recipient mice did not alter bacterial elimination by these cells in vivo, led to faster recovery and enhanced survival compared to isotype-treated animals. Thus, collectively these data show that although CD4+ TH1 cells are clearly efficient in protection against R. typhi, CD4+ TH17 cells are similarly protective if the harmful effects of combined production of TNFα and IL-17A can be inhibited.

Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes.

Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy and have the advantage over mesenchymal stem cells (MSCs) of large-scale manufacturing and banking potential and thus prompt availability, it is important to understand how MAPCs interact with immune cells to validate their widespread therapeutic applicability. Cytotoxic immune effector cells play a crucial role in immune homeostasis and in the pathogenesis of some autoimmune diseases. This study assessed for the first time the in vitro influence of a clinical-grade human MAPC product (MultiStem) on the cytotoxic function of CD8+ T cells (CTLs) by evaluating the immunogenicity of MAPCs and the susceptibility of MAPCs toward CTL-mediated lysis and by analyzing the mechanism of MAPC-mediated modulation of CTL functionality. These results may represent a highly relevant contribution to the current knowledge and, in combination with the results of future phase II/III trials using MultiStem, could lead to an intriguing continuation of stem cell-based research for immunotherapy.