Cancer cell-derived exosomal circUSP7 induces CD8+ T cell dysfunction and anti-PD1 resistance by regulating the miR-934/SHP2 axis in NSCLC

Abstract Background CD8+ T cells play a critical role in the innate antitumour immune response. Recently, CD8+ T cell dysfunction has been verified in various malignant cancers, including non-small cell lung cancer (NSCLC). However, the molecular biological mechanisms of CD8+ T cell dysfunction in human NSCLC are still unclear. Methods The expression of circular ubiquitin-specific protease-7 (circUSP7) in NSCLC tissues, exosomes, and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Exosomes were isolated from the culture medium of NSCLC cells and the plasma of NSCLC patients using an ultracentrifugation method and the ExoQuick Exosome Precipitation Solution kit. The exosomes were then characterized by transmission electronic microscopy (TEM), NanoSight and western blotting. The role of circUSP7 in CD8+ T cell dysfunction was assessed by enzyme-linked immunosorbent assay (ELISA). In vivo circular RNA (circRNA) precipitation (circRIP), RNA immunoprecipitation (RIP), and luciferase reporter assays were performed to explore the molecular mechanisms of circUSP7 in CD8+ T cells. In a retrospective study, the clinical characteristics and prognostic significance of circUSP7 in NSCLC tissues were determined. Results The expression levels of circUSP7 were higher in human NSCLC tissues than in matched adjacent nontumour tissues. Increased levels of circUSP7 indicate poor clinical prognosis and CD8+ T cell dysfunction in patients with NSCLC. The circUSP7 found in NSCLC patient plasma is predominantly secreted by NSCLC cells in an exosomal manner, and circUSP7 inhibits IFN-γ, TNF-α, Granzyme-B and Perforin secretion by CD8+ T cells. Furthermore, circUSP7 inhibits CD8+ T cell function by upregulating the expression of Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) via sponging miR-934. Finally, we show that circUSP7 may promote resistance to anti-PD1 immunotherapy in NSCLC patients. Conclusions Exosomal circUSP7 is predominantly secreted by NSCLC cells and contributes to immunosuppression by promoting CD8+ T cell dysfunction in NSCLC. CircUSP7 induces resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for NSCLC patients.

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Nuclear Factor of Activated T Cells Regulates Neutrophil Recruitment, Systemic Inflammation, and T-Cell Dysfunction in Abdominal Sepsis

Infection and Immunity ◽

10.1128/iai.01569-14 ◽

2014 ◽

Vol 82 (8) ◽

pp. 3275-3288 ◽

Cited By ~ 14

Author(s):

Su Zhang ◽

Lingtao Luo ◽

Yongzhi Wang ◽

Maria F. Gomez ◽

Henrik Thorlacius

Keyword(s):

T Cells ◽

T Cell ◽

Systemic Inflammation ◽

Abdominal Sepsis ◽

Neutrophil Recruitment ◽

Luciferase Reporter ◽

Nuclear Factor ◽

Activated T Cells ◽

Cell Dysfunction ◽

T Cell Dysfunction

ABSTRACTThe signaling mechanisms regulating neutrophil recruitment, systemic inflammation, and T-cell dysfunction in polymicrobial sepsis are not clear. This study explored the potential involvement of the calcium/calcineurin-dependent transcription factor, nuclear factor of activated T cells (NFAT), in abdominal sepsis. Cecal ligation and puncture (CLP) triggered NFAT-dependent transcriptional activity in the lung, spleen, liver, and aorta in NFAT-luciferase reporter mice. Treatment with the NFAT inhibitor A-285222 prior to CLP completely prevented sepsis-induced NFAT activation in all these organs. Inhibition of NFAT activity reduced sepsis-induced formation of CXCL1, CXCL2, and CXCL5 chemokines and edema as well as neutrophil infiltration in the lung. Notably, NFAT inhibition efficiently reduced the CLP-evoked increases in HMBG1, interleukin 6 (IL-6), and CXCL5 levels in plasma. Moreover, administration of A-285222 restored sepsis-induced T-cell dysfunction, as evidenced by markedly decreased apoptosis and restored proliferative capacity of CD4 T cells. Along these lines, treatment with A-285222 restored gamma interferon (IFN-γ) and IL-4 levels in the spleen, which were markedly reduced in septic mice. CLP-induced formation of regulatory T cells (CD4+CD25+Foxp3+) in the spleen was also abolished in A-285222-treated animals. All together, these novel findings suggest that NFAT is a powerful regulator of pathological inflammation and T-cell immune dysfunction in abdominal sepsis. Thus, our data suggest that NFAT signaling might be a useful target to protect against respiratory failure and immunosuppression in patients with sepsis.

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Blimp-1–mediated CD4 T cell exhaustion causes CD8 T cell dysfunction during chronic toxoplasmosis

Journal of Experimental Medicine ◽

10.1084/jem.20151995 ◽

2016 ◽

Vol 213 (9) ◽

pp. 1799-1818 ◽

Cited By ~ 35

Author(s):

SuJin Hwang ◽

Dustin A. Cobb ◽

Rajarshi Bhadra ◽

Ben Youngblood ◽

Imtiaz A. Khan

Keyword(s):

T Cells ◽

T Cell ◽

Critical Role ◽

Cd8 T Cell ◽

Cd4 T Cell ◽

T Cell Exhaustion ◽

Cell Exhaustion ◽

Cell Dysfunction ◽

T Cell Dysfunction

CD8, but not CD4, T cells are considered critical for control of chronic toxoplasmosis. Although CD8 exhaustion has been previously reported in Toxoplasma encephalitis (TE)–susceptible model, our current work demonstrates that CD4 not only become exhausted during chronic toxoplasmosis but this dysfunction is more pronounced than CD8 T cells. Exhausted CD4 population expressed elevated levels of multiple inhibitory receptors concomitant with the reduced functionality and up-regulation of Blimp-1, a transcription factor. Our data demonstrates for the first time that Blimp-1 is a critical regulator for CD4 T cell exhaustion especially in the CD4 central memory cell subset. Using a tamoxifen-dependent conditional Blimp-1 knockout mixed bone marrow chimera as well as an adoptive transfer approach, we show that CD4 T cell–intrinsic deletion of Blimp-1 reversed CD8 T cell dysfunction and resulted in improved pathogen control. To the best of our knowledge, this is a novel finding, which demonstrates the role of Blimp-1 as a critical regulator of CD4 dysfunction and links it to the CD8 T cell dysfunctionality observed in infected mice. The critical role of CD4-intrinsic Blimp-1 expression in mediating CD4 and CD8 T cell exhaustion may provide a rational basis for designing novel therapeutic approaches.

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Regulating T Cell Population Alleviates SLE by Inhibiting mTORC1/C2 in MRL/lpr Mice

Frontiers in Pharmacology ◽

10.3389/fphar.2020.579298 ◽

2021 ◽

Vol 11 ◽

Author(s):

Dongya Zhang ◽

Meiling Wang ◽

Guoping Shi ◽

Peng Pan ◽

Jianjian Ji ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Cells ◽

Lupus Erythematosus ◽

Critical Role ◽

Mammalian Target Of Rapamycin ◽

T Cell Proliferation ◽

Systemic Lupus ◽

Cell Dysfunction ◽

T Cell Dysfunction

It’s well known that the mammalian target of rapamycin (mTOR) exerts a critical role in the regulator of immune cells and is associated with T cells dysfunction in patients with systemic lupus erythematosus (SLE). Antigen-induced T-cell proliferation via mTORC1 suppressed by Rapamycin has been used to improve SLE primarily. Previously it has showed that INK128, a highly potent, specific orally inhibitor of mTORC1 and mTORC2, significantly attenuates SLE in pristine-induced lupus mice. Herein we compared the cure effects of INK128 and rapamycin on lupus mice. We treated MRL/lpr mice with INK128 or rapamycin at 12 weeks-age. The effect of the two inhibitors on the lupus mice was determined by immunohistochemistry. The effect of the two inhibitors on T cell populations was investigated by flow cytometry. The mTOR signaling was measured by Western Blot. INK128 remarkably alleviated SLE by reducing splenomegaly, renal inflammation and damage, and resuming T-cell dysfunction. The more effective of INK128 on SLE than rapamycin. INK128 effectively suppressed mTORC1 and mTORC2 activity in T cells, but rapamycin just suppressed mTORC1 activity. Thus, our results show that INK128 is can effectively alleviate SLE and be used as one of the potential clinical therapeutic candidates for SLE.

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Tim-3 adaptor protein Bat3 is a molecular checkpoint of T cell terminal differentiation and exhaustion

Science Advances ◽

10.1126/sciadv.abd2710 ◽

2021 ◽

Vol 7 (18) ◽

pp. eabd2710

Author(s):

Chen Zhu ◽

Karen O. Dixon ◽

Kathleen Newcomer ◽

Guangxiang Gu ◽

Sheng Xiao ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Adaptor Protein ◽

Transcriptional Analysis ◽

T Cell Exhaustion ◽

Cell Exhaustion ◽

Cell Dysfunction ◽

Autoreactive T Cell ◽

Autoimmune Conditions ◽

T Cell Dysfunction

T cell exhaustion has been associated with poor prognosis in persistent viral infection and cancer. Conversely, in the context of autoimmunity, T cell exhaustion has been favorably correlated with long-term clinical outcome. Understanding the development of exhaustion in autoimmune settings may provide underlying principles that can be exploited to quell autoreactive T cells. Here, we demonstrate that the adaptor molecule Bat3 acts as a molecular checkpoint of T cell exhaustion, with deficiency of Bat3 promoting a profound exhaustion phenotype, suppressing autoreactive T cell–mediated neuroinflammation. Mechanistically, Bat3 acts as a critical mTORC2 inhibitor to suppress Akt function. As a result, Bat3 deficiency leads to increased Akt activity and FoxO1 phosphorylation, indirectly promoting Prdm1 expression. Transcriptional analysis of Bat3−/− T cells revealed up-regulation of dysfunction-associated genes, concomitant with down-regulation of genes associated with T cell effector function, suggesting that absence of Bat3 can trigger T cell dysfunction even under highly proinflammatory autoimmune conditions.

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TIGIT Induces (CD3+) T Cell Dysfunction by Inhibiting Glucose Metabolism and its Reversal by TIGIT and/or PD-1 Blockade in Colorectal Cancer

10.21203/rs.3.rs-641407/v1 ◽

2021 ◽

Author(s):

qi shao ◽

Lei Wang ◽

maoling yuan ◽

Xiaohong Jin ◽

changping wu

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

T Cell ◽

Glucose Metabolism ◽

Tumor Growth ◽

Tumor Cells ◽

Peripheral Blood ◽

Cancer Tissue ◽

Cell Dysfunction ◽

T Cell Dysfunction

Abstract Background: T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an immunosuppressive receptor expressed on the surface of immune cells, suppressing immune responses by activating the intracellular negative regulatory signals. TIGIT plays an important role in the pathogenesis of various tumors, but its immune escape in colorectal cancer remains unclear.Methods: In this study, TIGIT expression in the peripheral blood and tissue microarrays was detected flow cytometry and immunofluorescence and its relationship with prognosis was evaluated. The proliferation and cytokines of TIGIT+ T cells were measured. Glucose metabolism and key enzymes were detected by qPCR or western blot. After establishing the co-cultured system and xenotransplant models, TIGIT antibody alone or combined with PD-1 antibody was blocked to observe the tumor growth.Results: We found that the proportion of CD3+TIGIT+ T cells was increased in peripheral blood and cancer tissue in colorectal cancer patients when compared with the healthy donors. These cells exhibited functional defects, low proliferative activity, impaired cytokine production and reduced glucose metabolism. A strong association was also observed between the elevated TIGIT expression and poor prognosis. In the in vitro co-culture assays of T cells and tumor cells, the suppressed glucose metabolic activity of T cells was reversed by TIGIT blockade. In addition, this blockade induced the apoptosis and reduced G2/M transit in tumor cells. The antitumor efficacy of TIGIT Ab therapy was further demonstrated in a human colorectal xenograft mice model while co-blockers of TIGIT and PD-1 exhibited synergistic suppressing effects on tumor growth.Conclusions: It is suggest that while TIGIT induces CD3+ T cell dysfunction in colorectal cancer, co-targeting TIGIT and PD-1 can lead to an effective antitumor response and may serve as a novel therapeutic strategy for colorectal patients.

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Combination cancer immunotherapy targeting PD-1 and GITR can rescue CD8+T cell dysfunction and maintain memory phenotype

Science Immunology ◽

10.1126/sciimmunol.aat7061 ◽

2018 ◽

Vol 3 (29) ◽

pp. eaat7061 ◽

Cited By ~ 44

Author(s):

Bei Wang ◽

Wen Zhang ◽

Vladimir Jankovic ◽

Jacquelynn Golubov ◽

Patrick Poon ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Programmed Cell Death ◽

Cancer Immunotherapy ◽

Effector Memory ◽

Cell Phenotype ◽

Checkpoint Inhibition ◽

Cell Dysfunction ◽

T Cell Dysfunction

Most patients with cancer do not develop durable antitumor responses after programmed cell death protein 1 (PD-1) or programmed cell death ligand 1(PD-L1) checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing of more than 2000 tumor-infiltrating CD8+T cells in mice receiving both PD-1 and GITR (glucocorticoid-induced tumor necrosis factor receptor–related protein) antibodies and found that this combination synergistically enhanced the effector function of expanded CD8+T cells by restoring the balance of key homeostatic regulators CD226 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), leading to a robust survival benefit. Combination therapy decreased CD8+T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1–Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy.

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663 Media based on the metabolic composition of tumor interstitial fluid reveals persistent T cell dysfunction induced through arginine deprivation and exposure to the oncometabolite phosphoethanolamine

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.663 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A691-A691

Author(s):

Yupeng Wang ◽

Chufan Cai ◽

Dayana Rivadeneira ◽

Alexander Muir ◽

Greg Delgoffe

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Interstitial Fluid ◽

Cytokine Production ◽

Cell Dysfunction ◽

Kennedy Pathway ◽

T Cell Dysfunction ◽

Tumor Interstitial Fluid

BackgroundWhile CD8 T cells are crucial for anti-tumor immunity, tumor infiltrating CD8 T cells encounter stressors which deviate their differentiation to a dysfunctional, exhausted phenotype. T cell functions are closely regulated by T cell metabolism, and the dysfunctional vasculature in tumor tissues and the deregulated metabolism of tumor cells lead to depletion of nutrients and accumulation of metabolic wastes in the tumor microenvironment (TME). Thus, the unbalanced levels of the nutrients and the metabolic wastes might skew the metabolism of T cells thus contributing to T cell dysfunction.MethodsOvalbumin-specific OT-I cells were activated with SIINFEKL/IL2 and cultured with IL2. The tumor interstitial fluid media (TIFM) was formulated based on the concentrations of the metabolites measured in the tumor interstitial fluid of pancreatic ductal adenocarcinoma.1 Purified arginine and phosphoethanolamine (PEtn) were used to change their levels in TIFM/RPMI1640 culture. Expression level of cytokines and PD-1 was measured by flow cytometry.ResultsWe sought to determine how T cells would differentiate, in vitro, if they were exposed only to the metabolites present in the TME. Using media formulated to model the metabolic composition of tumor interstitial fluid (TIFM),1 we show that CD8 T cells develop features of exhausted T cells in the TIFM culture: reduced proliferation, increased expression of PD-1 and decreased cytokine production. Using 'dropout' and 'add-back' approaches, we found arginine levels as a major contributor to the proliferation defect observed in TIFM-cultured T cells. Arginine was sufficient to restore proliferative capacity to T cells cultured in TIFM, but had no effect on the inhibited cytokine production. We then asked which metabolites were enriched in the TIFM, finding that PEtn, an intermediate in the ethanolamine branch of the Kennedy pathway and an oncometabolite enriched in the interstitial of many solid tumors, up-regulates PD-1 expression and compromises the cytokine production of the cells in culture. Depletion of Pcyt2, the metabolizing enzyme of PEtn and the rate limiting enzyme in the Kennedy pathway, makes CD8 T cells resistant to the effects of PEtn.ConclusionsOur data shows that the metabolic environment in the TME can be recapitulated in vitro and is sufficient to drive T cell dysfunction. Arginine depletion acts as a major inhibitor of T cell proliferation in the TME, but the oncometabolite PEtn drives a hypofunctional effector fate of T cells. Targeting PEtn metabolism via Pcyt2 depletion or inhibition is a potential target to reinvigorate T cells and enhance anti-tumor immunity.ReferenceSullivan MR, Danai LV, Lewis CA, Chan SH, Gui DY, Kunchok T, Dennstedt EA, Vander Heiden MG, Muir A. Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability. Elife 2019;;8:e44235. doi: 10.7554/eLife.44235. PMID: 30990168; PMCID: PMC6510537.

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GB Virus C E2 Inhibits PD-1-Mediated T Cell Signaling Dysfunction during Chronic Viral Infection

Proceedings ◽

10.3390/proceedings2020050062 ◽

2020 ◽

Vol 50 (1) ◽

pp. 62

Author(s):

Nirjal Bhattarai ◽

Jennifer L. Welch ◽

Jinhua Xiang ◽

Muthu Saravanan Manoharan ◽

Jeffrey A. Martinson ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Viral Infections ◽

Activated T Cells ◽

E2 Protein ◽

Gb Virus C ◽

Cell Dysfunction ◽

Chronic Viral Infections ◽

T Cell Dysfunction ◽

Virus C

Background: Program death receptor 1 (PD-1) is a co-inhibitory receptor that is upregulated and contributes to T cell dysfunction (exhaustion) during chronic viral infections, including HIV and HCV. GB virus C (GBV-C) is a persistent human virus, and co-infection is associated with reduced immune activation and improved clinical outcomes in HIV- and Ebola-infected individuals. Methods: PD-1 levels were measured by flow cytometry on CD38+ T cells from 45 HIV-infected individuals, 20 of whom were co-infected with GBV-C. Jurkat cell lines that stably express GBV-C E2 protein and vector control were used to purify total cellular RNA before, and 24 h following, activation using anti-CD3/CD28 treatment. Gene expression was analyzed by RNA-seq and qRT-PCR. Results: HIV-infected individuals with GBV-C viremia had reduced PD-1 expression on activated CD4+ and CD8+ T cells compared to HIV-infected GBV-C negative individuals. GBV-C particles and GBV-C E2 protein each inhibited PD-1 expression on T cells in vitro. Consistent with this, GBV-C E2 reduced gene expression of PD-1, and its ligand PD-L1, in both resting and activated T cells. GBV-C E2 regulated transcription of the PD-1 signaling pathway and T cell activation associated genes, without downregulation of the interferon-stimulated and innate immunity-related genes needed to resolve viral infections. Conclusions: Our current understanding of chronic RNA virus infections is that upregulation of PD-1 with T cell exhaustion is critical for viral persistence. However, these data demonstrate that GBV-C infection reduced PD-1 expression on activated T cells during HIV infection, and that the GBV-C E2 protein inhibits PD-1 signaling in T cells. This may preserve T cell function and contribute to the lack of immune deficiency in people with chronic GBV-C infection. Understanding the mechanisms by which GBV-C E2 alters PD-1 signaling may aid in the development of novel immunomodulatory therapeutics to prevent T cell dysfunction (exhaustion) during chronic viral infections.

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Resilience of T cell-intrinsic dysfunction in transplantation tolerance

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1910298116 ◽

2019 ◽

Vol 116 (47) ◽

pp. 23682-23690 ◽

Cited By ~ 2

Author(s):

Michelle L. Miller ◽

Christine M. McIntosh ◽

Ying Wang ◽

Luqiu Chen ◽

Peter Wang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cardiac Transplantation ◽

T Cell Memory ◽

Cell Dysfunction ◽

Antigen Persistence ◽

T Cell Dysfunction ◽

Alloreactive T Cell ◽

Donor Specific Tolerance ◽

Specific Tolerance

Following antigen stimulation, naïve T cells differentiate into memory cells that mediate antigen clearance more efficiently upon repeat encounter. Donor-specific tolerance can be achieved in a subset of transplant recipients, but some of these grafts are rejected after years of stability, often following infections. Whether T cell memory can develop from a tolerant state and whether these formerly tolerant patients develop antidonor memory is not known. Using a mouse model of cardiac transplantation in which donor-specific tolerance is induced with costimulation blockade (CoB) plus donor-specific transfusion (DST), we have previously shown that systemic infection with Listeria monocytogenes (Lm) months after transplantation can erode or transiently abrogate established tolerance. In this study, we tracked donor-reactive T cells to investigate whether memory can be induced when alloreactive T cells are activated in the setting of tolerance. We show alloreactive T cells persist after induction of cardiac transplantation tolerance, but fail to acquire a memory phenotype despite becoming antigen experienced. Instead, donor-reactive T cells develop T cell-intrinsic dysfunction evidenced when removed from the tolerant environment. Notably, Lm infection after tolerance did not rescue alloreactive T cell memory differentiation or functionality. CoB and antigen persistence were sufficient together but not separately to achieve alloreactive T cell dysfunction, and conventional immunosuppression could substitute for CoB. Antigen persistence was required, as early but not late surgical allograft removal precluded the acquisition of T cell dysfunction. Our results demonstrate transplant tolerance-associated T cell-intrinsic dysfunction that is resistant to memory development even after Lm-mediated disruption of tolerance.

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