Selection-associated High Mobility Group Box Research Articles

HO-1 attenuates testicular ischaemia/reperfusion injury by activating the phosphorylated C-jun-miR-221/222-TOX pathway

AimsHeme oxygenase (HO-1) affords protection against ischaemia/reperfusion (I/R) injury; however, its effects on testicular I/R injury remain poorly explored. Herein, we aimed to examine the effects of HO-1 on testicular I/R injury and elucidate the underlying mechanism. MethodsUsing the TALEN technique, we knocked out the HO-1 gene from rats. In vivo: Thirty hmox+/+ and 30 hmox−/− rats were randomly assigned to six groups: sham-operated (sham), I/R (the left testicle torsion/detorsion) 0 d,I/R 1d, I/R 3d, I/R 7d and I/R 28d. In vitro: GC-1 were suffered from: control,H/R (oxygen-deprivation/reoxygenation),H/R + HO-1 siRNA,H/R + c-Jun siRNA or H/R + HO-1 siRNA + c-jun.We performed immunofluorescence and immunohistochemistry experiments to detect HO-1 nuclear translocation. Flow cytometry was used to detect cell apoptosis and analyse the cell cycle. High-resolution miRNA, mRNA sequencing, reverse transcription-quantitative PCR, and western blotting were performed to identify testicular I/R injury-related genes strongly conserved in HO-1 knockout rats. A double luciferase reporter assay was performed to verify the relationship between C-jun and miR-221/222. Main findingsIn vivo, HO-1 improved the pathological damage induced by testicular I/R. In GC-1 cells, we confirmed the nuclear translocation of HO-1 and its protective effect against hypoxia/reoxygenation (H/R) damage. Accordingly, HO-1 protein itself, rather than heme metabolites, might play a key role in testicular I/R. Gene sequencing was performed to screen for miR221/222 and its downstream gene, thymocyte selection-associated high mobility group box (TOX). HO-1 increased c-Jun phosphorylation in the H/R group, knocked down c-Jun in GC-1 cells, and decreased miR-221/222 expression. Inhibition of HO-1 expression decreased the expression of c-Jun and miR-221/222, which was rescued by adding c-Jun. Dual-luciferase reporter assay confirmed the interaction between c-Jun and miR-221/222. ConclusionsHO-1 could exert a protective effect against testicular I/R via the phosphorylated c-Jun-miR-221/222-TOX pathway.

E3 ligase TRIM65 alleviates intestinal ischemia/reperfusion injury through inhibition of TOX4-mediated apoptosis

Intestinal ischemia-reperfusion (II/R) injury is an urgent clinical disease with high incidence and mortality, and impaired intestinal barrier function caused by excessive apoptosis of intestinal cells is an important cause of its serious consequences. Tripartite motif-containing protein 65 (TRIM65) is an E3 ubiquitin ligase that is recently reported to suppress the inflammatory response and apoptosis. However, the biological function and regulation of TRIM65 in II/R injury are totally unknown. We found that TRIM65 was significantly decreased in hypoxia-reoxygenation (H/R) induced intestinal epithelial cells and II/R-induced intestine tissue. TRIM65 knockout mice markedly aggravated intestinal apoptosis and II/R injury. To explore the molecular mechanism of TRIM65 in exacerbating II/R-induced intestinal apoptosis and damage, thymocyte selection-associated high mobility group box factor 4 (TOX4) was screened out as a novel substrate of TRIM65 using the yeast two-hybrid system. TRIM65 binds directly to the N-terminal of TOX4 through its coiled-coil and SPRY structural domains. Immunofluorescence confocal microscopy showed that they can co-localize both in the cytoplasm and nucleus. Furthermore, TRIM65 mediated the K48 ubiquitination and degradation of TOX4 depending on its E3 ubiquitin ligase activity. In addition, TRIM65 inhibits H/R-induced intestinal epithelial apoptosis via TOX4. In summary, our results indicated that TRIM65 promotes ubiquitination and degradation of TOX4 to inhibit apoptosis in II/R. These findings provide a promising target for the clinical treatment of II/R injury.

KLRG1-expressing CD8+ T cells are exhausted and polyfunctional in patients with chronic hepatitis B.

Killer cell lectin-like receptor G1 (KLRG1) has traditionally been regarded as an inhibitory receptor of T cell exhaustion in chronic infection and inflammation. However, its exact role in hepatitis B virus (HBV) infection remains elusive. CD8+ T cells from 190 patients with chronic hepatitis B were analyzed ex vivo for checkpoint and apoptosis markers, transcription factors, cytokines and subtypes in 190 patients with chronic hepatitis B. KLRG1+ and KLRG1- CD8+ T cells were sorted for transcriptome analysis. The impact of the KLRG1-E-cadherin pathway on the suppression of HBV replication mediated by virus-specific T cells was validated in vitro. As expected, HBV-specific CD8+ T cells expressed higher levels of KLRG1 and showed an exhausted molecular phenotype and function. However, despite being enriched for the inhibitory molecules, thymocyte selection-associated high mobility group box protein (TOX), eomesodermin (EOMES), and Helios, CD8+ T cells expressing KLRG1 produced significant levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, perforin, and granzyme B, demonstrating not exhausted but active function. Consistent with the in vitro phenotypic assay results, RNA sequencing (RNA-seq) data showed that signature effector T cell and exhausted T cell genes were enriched in KLRG1+ CD8+ T cells. Furthermore, in vitro testing confirmed that KLRG1-E-cadherin binding inhibits the antiviral efficacy of HBV-specific CD8+ T cells. Based on these findings, we concluded that KLRG1+ CD8+ T cells are not only a terminally exhausted subgroup but also exhibit functional diversity, despite inhibitory signs in HBV infection.

Evaluation of mRNA Expressions of TOX and NR4As in CD8+ T cells in Acute Leukemia.

Thymocyte selection-associated high mobility group box protein (TOX) and members of the nuclear receptor 4A (NR4A) are known as transcription factors involved in T cell exhaustion. To evaluate the mRNA expression of TOX and NR4A1-3 in CD8+ T cells in acute leukemia. Blood samples were obtained from 21 ALL and 6 AML patients as well as 20 control subjects. CD8+ T cells were isolated using MACS. Relative gene expression of TOX and NR4A1-3 was then evaluated using qRT-PCR. Comparison of mRNA expression of TOX in CD8+ T cells showed no significant difference among the study groups (p>0.05), while the expression of NR4A1 was significantly lower in AML patients than in the control group (p=0.0006). Also, the expression of NR4A2 and NR4A3 was significantly lower in both ALL (p=0.0049 and p=0.0005, respectively) and AML (p=0.0019 and p=0.0055, respectively) patients. NR4As expressions were found to be lower in CD8+ T cells from patients with AML and ALL compared to controls, whereas the mRNA expression of TOX showed no significant difference. Although TOX and NR4As are associated with CD8+ T cell exhaustion in solid tumors, they might play different roles in acute leukemia, which requires further investigation.

Acertannin prevents azoxymethane/dextran sulfate sodium-induced colon cancer growth by inhibiting the colonic expression of interleukin-1β, monocyte chemoattractant protein-1, cyclooxygenase-2, and thymocyte selection-associated high mobility group box proteins (TOX)/TOX2 in C57BL/6J mice

Colon cancer was the second leading cause of cancer-related death in 2019. We herein investigated the effects of acertannin containing Acer species on azoxymethane (AOM)/dextran sulfate sodium (DDS)-induced colon cancer growth and changes in the colonic levels of interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1, IL-10, and programmed cell death-1 (PD-1). Colorectal carcinogenesis was induced by an intraperitoneal injection of AOM (10 mg/kg) on days 0 and 27. Mice were given 1% (w/v) DSS drinking water ad libitum on days 7–14, 32–33, and 35–38. Acertannin (30 and 100 mg/kg) was orally administered on days 1–16, discontinued for 11 days (days 16–26), and then administered again on days 27–41. The colonic levels of cytokines, a chemokine, and PD-1 were measured using the respective ELISA kits.The number and area of tumors in mice treated with acertannin (100 mg/kg) decreased by 53.9 and 63.1%, respectively. Furthermore, the colonic levels of IL-1β, MCP-1, IL-10, and PD-1 showed reductions of 57.3, 62.9, 62.8, and 100%, respectively, while the numbers of cyclooxygenase-2 (COX-2)-, thymocyte selection-associated high mobility group box proteins (TOX)/TOX2-, PD-1-, and signal transducer and activator of transcription 3 (STAT3) phosphorylation-positive numbers decreased by 79.6, 77.9, 93.8, and 100%, respectively. In conclusion, the inhibitory effects of acertannin on AOM/DSS-induced colon tumor growth appear to be associated with reductions in the colonic levels of IL-1β, MCP-1, IL-10, and PD-1 through the down-regulated expression of COX-2 and TOX/TOX2 in the tumor microenvironment.

TOX regulates T lymphocytes differentiation and its function in tumor.

Thymocyte selection-associated high mobility group box protein (TOX) is expressed differently at all T lymphocytes development stages. Owing to more advanced scientific and technological means, including single-cell sequencing technology, heterogeneity of T lymphocytes and TOX has gradually been revealed. Further exploration of such heterogeneity will help us comprehend the developmental stage and functional characteristics of T lymphocytes in greater detail. Emerging evidence supports its regulation not only in exhausting, but also in activating T lymphocytes, thereby verifying TOX heterogeneity. TOX can be used not only as a latent intervention target for tumor diseases and chronic infections, and a therapeutic strategy for autoimmune diseases, but also as a critical factor predicting the drug response and overall survival of patients with malignant tumors.

Higher thymocyte selection-associated high mobility group box (TOX) expression predicts poor prognosis in patients with ovarian cancer

BackgroundOvarian cancer is one of the most lethal gynecologic malignancies with a dismal prognosis that poses a serious threat to human health, highlighting the need for more knowledge about what is required for identifying some biomarkers for early diagnosis, prediction of prognosis and disease monitoring. TOX, a critical transcription factor related to the development of malignancies that contributing to lymphocytes not just T cells, had been proved prognostic value in some spectrum of cancers. Here, we aimed to study the prognostic role of TOX in ovarian cancer.ResultsWe found that TOX was not only expressed in CD8 T cells but also tumor cells. TOX expression score was higher in ovarian cancer tissues and correlated with survival status. Survival analysis revealed that ovarian cancer patients with high TOX expression score generally shorter overall survival and disease-free survival times. Univariate and Multivariate Cox demonstrated that TOX expression score could be used as an independent prognostic factor for patients with ovarian cancer.ConclusionTOX expression in ovarian cancer could be a promising tool for predict overall survival of ovarian cancer patients.

Manipulating Offense and Defense Signaling to Fight Cold Tumors with Carrier-Free Nanoassembly of Fluorinated Prodrug and siRNA.

Chemoimmunotherapy has shown great potential to activate an immune response, but the immunosuppressive microenvironment associated with T cell exhaustion remains a challenge in cancer therapy. The proper immune-modulatory strategy to provoke a robust immune response is to simultaneously regulate T-cell exhaustion and infiltration. Here, a new kind of carrier-free nanoparticle is developed to simultaneously deliver chemotherapeutic drug (doxorubicin, DOX), cytolytic peptide (melittin, MPI), and anti-TOX small interfering RNA (thymocyte selection-associated high mobility group box protein, TOX) using a fluorinated prodrug strategy. In this way, the enhanced immunogenic cell death (ICD) induced by the combination of DOX and MPI can act as "offense" signaling to increase CD8+ T-cell infiltration, while the decreased TOX expression interfered with siTOX can serve as "defense" signaling to mitigate CD8+ T-cell exhaustion. As a result, the integration of DOX, MPI, and siTOX in such a bifunctional system produced a potent antitumor immune response in liver cancer and metastasis, making it a promising delivery platform and effective strategy for converting "cold" tumors into "hot" ones.

TOX Expression in Mycosis Fungoides and Sezary Syndrome

Mycosis fungoides (MF) and Sezary syndrome (SS) are the two most common type of cutaneous T-cell lymphoma (CTCL). Currently, no markers can be clearly related to prognosis or to differential diagnosis between early stages and inflammatory benign diseases (IBD). The thymocyte selection-associated high mobility group box factor (TOX), has been proposed as a possible marker in differential diagnosis between early CTCL stages and IBD. Recently TOX has been related to prognosis. We aimed to investigate whether TOX may be a diagnostic or prognostic marker. MF and SS biopsies between 2010 and 2020 were retrieved. New tissues slides were stained with an anti-TOX antibody, (Clone NAN448B). On each slide, 5 fields were examined at high magnification (400×), to evaluate the percentage of marker-positivity in a quantitative way. Thirty-six patients (12 females and 24 males) and 48 biopsies were collected. Nine patients had multiple biopsies. TOX expression in MF/SS cases showed an increase from early to advanced phases. TOX was not regarded as a prognostic marker due to the absence of significant changes by comparing early MF cases with reactive conditions. TOX statistical significance increased in patients alive with disease and in those dead of disease (p = 0.013 and = 0.0005, respectively) as compared with patients in complete remission. Our results show that TOX should be regarded more as a prognostic than a diagnostic marker.

TOX promotes follicular helper T cell differentiation in patients with primary Sjögren's syndrome.

Whether naive CD4+ T cells are dysregulated and associated with the overactivation of CD4+ T cells in primary SS (pSS) remains unclear. We aimed to explore the role and underlying mechanism of naive CD4+ T cells in pSS. We examined the activation, proliferation and differentiation of naive CD4+ T cells from pSS patients and healthy controls. Differentially expressed genes were identified using RNA sequencing, and were overexpressed or silenced to determine the gene regulating follicular helper T (Tfh) cells. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) with chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) was performed to explore the epigenetic mechanism. Naive CD4+ T cells were treated with pSS-related cytokines to explore the upstream signalling pathway. pSS naive CD4+ T cells had higher potentials of activation, proliferation and differentiation towards Tfh cells. Thymocyte selection-associated high mobility group box protein (TOX) was upregulated in pSS naive CD4+ T cells and promoted T cell activation and Tfh cell polarization. TOX silencing in pSS naive CD4+ T cells downregulated B cell lymphoma 6 (BCL6) expression and altered levels of multiple Tfh-associated genes. ChIP-seq analysis implied that TOX bound to the BCL6 locus, where there were accessible regions found by ATAC-seq. IFN-α induced TOX overexpression, which was attenuated by Janus kinase (JAK) and signal transducer and activator of transcription 1 (STAT1) inhibitors. Our data suggest that TOX in pSS naive CD4+ T cells is upregulated, which facilitates Tfh cell differentiation. Mechanistically, IFN-α induces TOX overexpression in naive CD4+ T cells through JAK-STAT1 signalling and TOX regulates BCL6 expression. Therefore, IFN-α-JAK-STAT1 signalling and TOX might be potential therapeutic targets in pSS.

Transcriptional Pattern Analysis of Virus-Specific CD8+ T Cells in Hepatitis C Infection: Increased Expression of TOX and Eomesodermin During and After Persistent Antigen Recognition.

Thymocyte selection-associated high mobility group box (TOX) has been described to be a key regulator in the formation of CD8+ T cell exhaustion. Hepatitis C virus (HCV) infection with different lengths of antigen exposure in acute, chronic, and after resolution of HCV infection is the ideal immunological model to study the expression of TOX in HCV-specific CD8+ T cells with different exposure to antigen. HCV-specific CD8+ T cells from 35 HLA-A*01:01, HLA-A*02:01, and HLA-A*24:02 positive patients were analyzed with a 16-color FACS-panel evaluating the surface expression of lineage markers (CD3, CD8), ectoenzymes (CD39, CD73), markers of differentiation (CD45RO, CCR7, CD127), and markers of exhaustion and activation (TIGIT, PD-1, KLRG1, CD226) and transcription factors (TOX, Eomesodermin, T-bet). Here, we defined on-target T cells as T cells against epitopes without escape mutations and off-target T cells as those against a “historical” antigen mutated in the autologous sequence. TOX+HCV-specific CD8+ T cells from patients with chronic HCV and on-target T cells displayed co-expression of Eomesodermin and were associated with the formation of terminally exhausted CD127-PD1hi, CD39hi, CD73low CD8+ T cells. In contrast, TOX+HCV-specific CD8+ T cells in patients with off-target T cells represented a progenitor memory Tex phenotype characterized by CD127hi expression and a CD39low and CD73hi phenotype. TOX+HCV-specified CD8+ T cells in patients with a sustained virologic response were characterized by a memory phenotype (CD127+, CD73hi) and co-expression of immune checkpoints and Eomesodermin, indicating a key structure in priming of HCV-specific CD8+ T cells in the chronic stage, which persisted as a residual after therapy. Overall, the occurrence of TOX+HCV-specific CD8+ T cells was revealed at each disease stage, which impacted the development of progenitor Tex, intermediate Tex, and terminally exhausted T cell through an individual molecular footprint. In sum, TOX is induced early during acute infection but is modulated by changes in viral sequence and antigen recognition. In the case of antigen persistence, the interaction with Eomesodermin leads to the formation of terminally exhausted virus-specific CD8+ T cells, and there was a direct correlation of the co-expression of TOX and Eomes and terminally exhausted phenotype of virus-specific CD8+ T cells.

Two hydroxyflavanones isolated from Scutellaria baicalensis roots prevent colitis-associated colon cancer in C57BL/6 J mice by inhibiting programmed cell death-1, interleukin 10, and thymocyte selection-associated high mobility group box proteins TOX/TOX2

BackgroundColorectal cancer was the second leading cause of mortality in 2019 and the number of new colorectal cancer cases was the highest in 2018 and 2019 in Japan. PurposeThe present study investigated the inhibitory effects of 2(S)-2′,5,6′,7-tetrahydroxyflavanone and 2 (R), 3(R)-2′,3,5,6′-7-pentahydroxyflavanone on the incidence and growth of tumors in azoxymethane (AOM) plus dextran sulfate sodium (DSS)-treated mice. MethodsThe intraperitoneal administration of AOM (10 mg/kg) on day 0 induced colorectal carcinogenesis. Mice were given free and unlimited access to drinking water containing 1.5% (w/v) DSS on days 5 - 8, 30 - 33, and 56 - 57. They were orally administered tetra- and penta-hydroxyflavanones (10 and 30 mg/kg) for 10, 11, and 14 days followed by discontinuation intervals of 20 and 15 days. Cytokine, chemokine, programmed cell death-1 (PD-1), cyclooxygenase (COX)-2, and thymocyte selection-associated high mobility group box protein (TOX)/TOX2 expression levels were measured using their respective ELISA kits and an immunohistochemical analysis. ResultsThe number and area of tumors decreased by 60.6 and 72.9% in mice administered 10 mg/kg tetra- and pentahydroxyflavanones, respectively, with reductions of 95.0 and 87.0% in Ki-67-positive cells, 91.7 and 92.7% in COX-2-postive cells, and 83.1 and 93.8% in TOX/TOX2-positive cells, respectively, in the colon. On the other hand, two tera- and pentahydroxyflavanone had no effect on p53 (a tumor suppressor by cell cycle arrest and apoptosis)-positive cells. The administration of 10 mg/kg tetra- and pentahydroxyflavanones to AOM/DSS-treated mice also resulted in decreases of 59.5 and 42.5% in IL-10 levels and 58.1 and 93.9% in PD-1 levels, respectively, in the colon. ConclusionThe inhibitory effects of tetra- and pentahydroxyflavanones on the growth of colon tumors in AOM/DSS-treated mice appear to be associated with decreases in the colon levels of IL-10 and PD-1 through the down-regulated expression of COX-2 and CD8+ T-cell exhaustion by TOX/TOX2 in the tumor microenvironment.

Comorbidity between lung cancer and COVID-19 pneumonia: role of immunoregulatory gene transcripts in high ACE2-expressing normal lung.

SARS-CoV-2 (COVID-19) elicits a T-cell antigen-mediated immune response of variable efficacy. To understand this variability, we explored transcriptomic expression of angiotensin-converting enzyme 2 (ACE2, the SARS-CoV-2 receptor) and of immunoregulatory genes in normal lung tissues from patients with non-small cell lung cancer (NSCLC). This study used the transcriptomic and the clinical data for NSCLC patients generated during the CHEMORES study [n = 123 primary resected (early-stage) NSCLC] and the WINTHER clinical trial (n = 32 metastatic NSCLC). We identified patient subgroups with high and low ACE2 expression (p = 1.55 × 10-19) in normal lung tissue, presumed to be at higher and lower risk, respectively, of developing severe COVID-19 should they become infected. ACE2 transcript expression in normal lung tissues (but not in tumor tissue) of patients with NSCLC was higher in individuals with more advanced disease. High-ACE2 expressors had significantly higher levels of CD8+ cytotoxic T lymphocytes and natural killer cells but with presumably impaired function by high Thymocyte Selection-Associated High Mobility Group Box Protein TOX (TOX) expression. In addition, immune checkpoint-related molecules - PD-L1, CTLA-4, PD-1, and TIGIT - are more highly expressed in normal (but not tumor) lung tissues; these molecules might dampen immune response to either viruses or cancer. Importantly, however, high inducible T-cell co-stimulator (ICOS), which can amplify immune and cytokine reactivity, significantly correlated with high ACE2 expression in univariable analysis of normal lung (but not lung tumor tissue). We report a normal lung immune-tolerant state that may explain a potential comorbidity risk between two diseases - NSCLC and susceptibility to COVID-19 pneumonia. Further, a NSCLC patient subgroup has normal lung tissue expressing high ACE2 and high ICOS transcripts, the latter potentially promoting a hyperimmune response, and possibly leading to severe COVID-19 pulmonary compromise.

Hist-O-06 - Image-guided lymph node core-needle biopsy predicts survival in mycosis fungoides and Sézary syndrome

<h3>Background:</h3> The prognosis of Sézary syndrome (SS) and mycosis fungoides (MF) depends on lymph node (LN) involvement. The usefulness of LN image-guided core-needle biopsies (CNBs), instead of surgical sampling, has been poorly evaluated. <h3>Objectives:</h3> To determine the prognostic value of LN CNB in MF/SS. <h3>Methods:</h3> A retrospective search was conducted to identify all LN biopsy specimens of MF/SS between 2008 and 2019. Biopsies were staged according to the International Society for Cutaneous Lymphomas/European Organisation for Research and Treatment of Cancer (ISCL/EORTC) criteria. We performed immunolabelling and determined the tumour clone frequency (TCF) by high-throughput sequencing of the T-cell receptor beta locus. <h3>Results:</h3> We included 119 consecutive biopsies from 100 patients, 45 with MF and 55 with SS. N1, N2 and N3 stages were diagnosed in 34 (29%), 26 (22%) and 59 (49%) cases, respectively. The TCF, Ki67 index, and percentage of cells positive for thymocyte selection-associated high mobility group box protein (TOX), programmed cell death protein 1 (PD1), killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2) and cluster of differentiation (CD)30 were all positively correlated with the N stage. Median overall survival (OS) for N1/N2 vs. N3 patients was 42 months (range 26–not reached) vs. 14 months (range 5–30), respectively (p<0.001). In univariate analyses, an age >75 years, LN short-axis diameter >15 mm, N3 stage, presence of large-cell transformation, TOX >60%, PD1 >25%, Ki67 >30%, KIR3DL2 >15%, CD30 >10% and TCF >25% were identified as adverse prognostic factors. In multivariate analyses, only an age >75 years and Ki67 index >30% were associated with reduced OS. We developed a new prognostic index associating the N stage and the Ki67 index, which better discriminates N3 patients with poor prognosis. <h3>Conclusions:</h3> CNB allows an objective assessment of the LN involvement in MF/SS, relevant for staging and prognosis.

Image-guided lymph node core-needle biopsy predicts survival in mycosis fungoides and Sézary syndrome.

The prognosis of Sézary syndrome (SS) and mycosis fungoides (MF) depends on lymph node (LN) involvement. The usefulness of LN image-guided core-needle biopsies (CNBs), instead of surgical sampling, has been poorly evaluated. To determine the prognostic value of LN CNB in MF/SS. A retrospective search was conducted to identify all LN biopsy specimens of MF/SS between 2008 and 2019. Biopsies were staged according to the International Society for Cutaneous Lymphomas/European Organisation for Research and Treatment of Cancer (ISCL/EORTC) criteria. We performed immunolabelling and determined the tumour clone frequency (TCF) by high-throughput sequencing of the T-cell receptor beta locus. We included 119 consecutive biopsies from 100 patients, 45 with MF and 55 with SS. N1, N2 and N3 stages were diagnosed in 34 (29%), 26 (22%) and 59 (49%) cases, respectively. The TCF, Ki67 index, and percentage of cells positive for thymocyte selection-associated high mobility group box protein (TOX), programmed cell death protein 1 (PD1), killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2) and cluster of differentiation (CD)30 were all positively correlated with the N stage. Median overall survival (OS) for N1/N2 vs. N3 patients was 42months (range 26-not reached) vs. 14months (range 5-30), respectively (P<0·001). In univariate analyses, an age>75years, LN short-axis diameter>15mm, N3 stage, presence of large-cell transformation, TOX>60%, PD1>25%, Ki67>30%, KIR3DL2>15%, CD30>10% and TCF>25% were identified as adverse prognostic factors. In multivariate analyses, only an age>75years and Ki67 index>30% were associated with reduced OS. We developed a new prognostic index associating the N stage and the Ki67 index, which better discriminates N3 patients with poor prognosis. CNB allows an objective assessment of the LN involvement in MF/SS, relevant for staging and prognosis.

TOX-expressing terminally exhausted tumor-infiltrating CD8+ T cells are reinvigorated by co-blockade of PD-1 and TIGIT in bladder cancer.

Exhausted T cells in the tumor microenvironment are major targets of immunotherapies. However, the exhaustion status of CD8+ tumor-infiltrating lymphocytes (TILs) in bladder cancer has not been comprehensively evaluated. Herein, we examined distinct exhaustion status of CD8+ TILs based on the level of programmed cell death-1 (PD-1) and thymocyte selection-associated high mobility group box protein (TOX) expression in urothelial bladder cancer. We also evaluated the reinvigoration of exhausted CD8+ TILs upon ex vivo treatment with inhibitory checkpoint blockers. TOX-expressing PD-1highCD8+ TILs had the highest expression of immune checkpoint receptors (ICRs), the most terminally exhausted features, and the highest tumor antigen reactivity among PD-1+CD8+ TILs. Bladder cancer patients with a high percentage of PD-1highTOX+CD8+ TILs had more progressed T-cell exhaustion features and higher programmed death-ligand 1 expression in tumor tissues. TIGIT was the most frequent co-expressed ICR on PD-1+CD8+ TILs, and TIGIT blockade enhanced the PD-1 blockade-mediated cytokine production by CD8+ TILs from bladder cancer patients. Our findings provide an improved understanding of the heterogeneous exhaustion status of CD8+ TILs and additional immunotherapy strategies to improve outcomes of bladder cancer patients.

Increased Frequency of TOX+ CD39+ TIGIT+ CD73- CD8+ T Cells in Patients with Newly Diagnosed AML

Increased Frequency of TOX+ CD39+ TIGIT+ CD73- CD8+ T Cells in Patients with Newly Diagnosed AML

TOX defines the degree of CD8+ T cell dysfunction in distinct phases of chronic HBV infection

ObjectiveChronic hepatitis B virus (HBV) infection is characterised by HBV-specific CD8+ T cell dysfunction that has been linked to Tcell exhaustion, a distinct differentiation programme associated with persisting antigen recognition....

Mechanisms of T-Cell Exhaustion in Pancreatic Cancer.

T-cell exhaustion is a phenomenon that represents the dysfunctional state of T cells in chronic infections and cancer and is closely associated with poor prognosis in many cancers. The endogenous T-cell immunity and genetically edited cell therapies (CAR-T) failed to prevent tumor immune evasion. The effector T-cell activity is perturbed by an imbalance between inhibitory and stimulatory signals causing a reprogramming in metabolism and the high levels of multiple inhibitory receptors like programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and Lymphocyte-activation gene 3 (Lag-3). Despite the efforts to neutralize inhibitory receptors by a single agent or combinatorial immune checkpoint inhibitors to boost effector function, PDAC remains unresponsive to these therapies, suggesting that multiple molecular mechanisms play a role in stimulating the exhaustion state of tumor-infiltrating T cells. Recent studies utilizing transcriptomics, mass cytometry, and epigenomics revealed a critical role of Thymocyte selection-associated high mobility group box protein (TOX) genes and TOX-associated pathways, driving T-cell exhaustion in chronic infection and cancer. Here, we will review recently defined molecular, genetic, and cellular factors that drive T-cell exhaustion in PDAC. We will also discuss the effects of available immune checkpoint inhibitors and the latest clinical trials targeting various molecular factors mediating T-cell exhaustion in PDAC.

Expression pattern, regulation, and clinical significance of TOX in breast cancer.

Thymocyte selection-associated high mobility group box protein (TOX) is a transcription factor implicated in the regulation of T cell exhaustion during chronic infection and cancer. While TOX is being targeted for cancer immunotherapy, limited information is available about its significance in breast cancer and other solid tumors. We performed a comprehensive analysis of TOX gene expression, its epigenetic regulation, protein localization, relation to tumor infiltrating immune cell composition, and prognostic significance in breast cancer using publicly available datasets. Our results suggest an inverse correlation between TOX expression and DNA methylation in tumor cells. However, its expression is elevated in tumor infiltrating immune cells (TIICs), which may compensates for the total TOX levels in the tumor as a whole. Furthermore, higher TOX levels in tumors are associated with T cell exhaustion signatures along with presence of active inflammatory response, including elevated levels of T cell effector cytokines. Survival analysis also confirmed that higher expression of TOX is associated with better prognosis in breast cancer. Therefore, expression of TOX may serve as a novel prognostic marker for this malignancy.