Generation, transcriptomic states, and clinical relevance of CX3CR1+ CD8 T cells in melanoma.

Recent progress in single-cell profiling technologies has revealed significant phenotypic and transcriptional heterogeneity in tumor-infiltrating CD8+ T cells. However, the transition between the different states of intratumoral antigen-specific CD8+ T cells remains elusive. Here, we sought to examine the generation, transcriptomic states, and the clinical relevance of melanoma-infiltrating CD8+ T cells expressing a chemokine receptor and T-cell differentiation marker, CX3C chemokine receptor 1 (CX3CR1). Analysis of single-cell datasets revealed two distinct human melanoma-infiltrating CD8+ T-cell clusters expressing CX3CR1 and PDCD1, whereas both clusters expressed genes associated with effector T cell function. No obvious impact of CX3CR1 expression in melanoma on the response to immune checkpoint inhibitor therapy was observed while increased pre- and on-treatment frequency of a CD8+ T-cell cluster expressing high levels of exhaustion markers was associated with poor response to the treatment. Adoptively transferred antigen-specific CX3CR1- CD8+ T cells differentiated into the CX3CR1+ subset in mice treated with FTY720, which inhibits lymphocyte egress from secondary lymphoid tissues, suggesting the intratumoral generation of CX3CR1+ CD8+ T cells rather than their trafficking from secondary lymphoid organs. Furthermore, analysis of adoptively transferred antigen-specific CD8+ T cells, in which the Cx3cr1 gene was replaced with a marker gene confirmed that CX3CR1+ CD8+ T cells could directly differentiate from the intratumoral CX3CR1- subset. These findings highlight that tumor antigen-specific CX3CR1- CD8+ T cells can fully differentiate outside the secondary lymphoid organs, and generate CX3CR1+ CD8+ T cells in the tumor microenvironment, which are distinct from CD8+ T cells that express markers of exhaustion.

CD40 and CD80/86 signaling in cDC1s mediate effective neoantigen vaccination and generation of antigen-specific CX3CR1+ CD8+ T cells.

The use of tumor mutation-derived neoantigen represents a promising approach for cancer vaccines. Preclinical and early phase human clinical studies have shown the successful induction of tumor neoepitope-directed responses; however, overall clinical efficacy of neoantigen vaccines has been limited. One major obstacle of this strategy is the prevailing lack of sufficient understanding of the mechanism underlying the generation of neoantigen-specific CD8+ T cells. Here, we report a correlation between antitumor efficacy of neoantigen/toll-like receptor 3 (TLR3)/CD40 agonists vaccination and an increased frequency of circulating antigen-specific CD8+ T cells expressing CX3C chemokine receptor 1 (CX3CR1) in a preclinical model. Mechanistic studies using mixed bone marrow chimeras identified that CD40 and CD80/86, but not CD70 signaling in Batf3-dependent conventional type 1 dendritic cells (cDC1s) is required for the antitumor efficacy of neoantigen vaccine and generation of neoantigen-specific CX3CR1+ CD8+ T cells. Although CX3CR1+ CD8+ T cells exhibited robust in vitro effector function, in vivo depletion of this subset did not alter the antitumor efficacy of neoantigen/TLR3/CD40 agonists vaccination. These findings indicate that the vaccine-primed CX3CR1+ subset is dispensable for antitumor CD8+ T cell responses, but can be used as a blood-based T-cell biomarker for effective priming of CD8+ T cells as post-differentiated T cells. Taken together, our results reveal a critical role of CD40 and CD80/86 signaling in cDC1s in antitumor efficacy of neoantigen-based therapeutic vaccines, and implicate the potential utility of CX3CR1 as a circulating predictive T-cell biomarker in vaccine therapy.

T-cell CX3CR1 expression as a dynamic blood-based biomarker of response to immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICI) have revolutionized treatment for various cancers; however, durable response is limited to only a subset of patients. Discovery of blood-based biomarkers that reflect dynamic change of the tumor microenvironment, and predict response to ICI, will markedly improve current treatment regimens. Here, we investigate CX3C chemokine receptor 1 (CX3CR1), a marker of T-cell differentiation, as a predictive correlate of response to ICI therapy. Successful treatment of tumor-bearing mice with ICI increases the frequency and T-cell receptor clonality of the peripheral CX3CR1+CD8+ T-cell subset that includes an enriched repertoire of tumor-specific and tumor-infiltrating CD8+ T cells. Furthermore, an increase in the frequency of the CX3CR1+ subset in circulating CD8+ T cells early after initiation of anti-PD-1 therapy correlates with response and survival in patients with non-small cell lung cancer. Collectively, these data support T-cell CX3CR1 expression as a blood-based dynamic early on-treatment predictor of response to ICI therapy.

A Critical Role of CD40 and CD70 Signaling in Conventional Type 1 Dendritic Cells in Expansion and Antitumor Efficacy of Adoptively Transferred Tumor-Specific T Cells.

In vivo expansion of adoptively transferred CD8+ T cells is a critical determinant of successful adoptive T cell therapy. Emerging evidence indicates Batf3-dependent conventional type 1 dendritic cells (cDC1s) rarely found within the tumor myeloid compartment are crucial for effector T cell recruitment to the tumor microenvironment. However, the role of cDC1s in expansion of tumor-specific CD8+ T cells remains unclear. In this article, we addressed the role of cDC1s and their costimulatory molecules, CD40, CD70, and CD80/CD86, in expansion and antitumor efficacy of adoptively transferred in vitro-primed CD8+ T cells recognizing nonmutated tumor-associated self-antigens. We found that TLR/CD40-mediated expansion and antitumor efficacy of adoptively transferred tumor-specific CD8+ T cells were abrogated in Batf3-/- mice. Further mechanistic studies using mixed bone marrow chimeric mice identified that CD40 and CD70 but not CD80/CD86 signaling in cDC1s played a critical role in expansion and antitumor efficacy of adoptively transferred CD8+ T cells. Moreover, induction and activation of cDC1s by administration of FMS-like tyrosine kinase 3 ligand (Flt3L) and TLR/CD40 agonists augmented expansion of adoptively transferred CD8+ T cells, delayed tumor growth, and improved survival. These findings reveal a key role for CD40 and CD70 signaling in cDC1s and have major implications for the design of new vaccination strategies with adoptive T cell therapy.

CX3CR1-CD8+ T cells are critical in antitumor efficacy but functionally suppressed in the tumor microenvironment.

Although blockade of the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) immune checkpoint has revolutionized cancer treatment, how it works on tumor-infiltrating CD8+ T cells recognizing the same antigen at various differentiation stages remains elusive. Here, we found that the chemokine receptor CX3CR1 identified 3 distinct differentiation states of intratumor CD8+ T cell subsets. Adoptively transferred antigen-specific CX3CR1-CD8+ T cells generated phenotypically and functionally distinct CX3CR1int and CX3CR1hi subsets in the periphery. Notably, expression of coinhibitory receptors and T cell factor 1 (Tcf1) inversely correlated with the degree of T cell differentiation defined by CX3CR1. Despite lower expression of coinhibitory receptors and potent cytolytic activity, in vivo depletion of the CX3CR1hi subset did not alter the antitumor efficacy of adoptively transferred CD8+ T cells. Furthermore, differentiated CX3CR1int and CX3CR1hi subsets were impaired in their ability to undergo proliferation upon restimulation and had no impact on established tumors upon second adoptive transfer compared with the CX3CR1- subset that remained effective. Accordingly, anti-PD-L1 therapy preferentially rescued proliferation and cytokine production of the CX3CR1- subset and enhanced antitumor efficacy of adoptively transferred CD8+ T cells. These findings provide a better understanding of the phenotypic and functional heterogeneity of tumor-infiltrating CD8+ T cells and can be exploited to develop more effective immunotherapy.

Low DMT1 Expression Associates With Increased Oxidative Phosphorylation and Early Recurrence in Hepatocellular Carcinoma.

BACKGROUND: Despite a high rate of recurrences, long-term survival can be achieved after the resection of hepatocellular carcinoma (HCC) with effective local treatment. Discovery of adverse prognostic variables to identify patients with high risk of recurrence could improve the management of HCC. Accumulating evidence showing a link between carcinogenesis and increased expression of iron import proteins and intracellular iron prompted us to investigate a role of divalent metal-ion transporter-1 (DMT1) that binds and regulates a variety of divalent metals in HCC. MATERIALS AND METHODS: Clinical and gene expression data from RNA seq in 369 HCC patients were obtained from The Cancer Genome Atlas. Disease-free survival was compared between DMT1 high- and low-expressing tumors, and gene set enrichment analysis was conducted. RESULTS: Patients with lower expression of DMT1 exhibited significantly worse disease-free survival compared with the DMT1 high group (P = 0.044), notably in advanced-stage patients (P = 0.008). DMT1 expression did not differ in etiologies, stages, and differentiation status of HCC. Interestingly, DMT1 expression levels inversely associated with cellular respiratory function in HCC. Furthermore, gene set enrichment analysis revealed that metabolism-related gene sets such as glycolysis, oxidative phosphorylation, and reactive oxygen species pathway were significantly enriched in the DMT1 low-expressing HCC. CONCLUSIONS: Low DMT1 expression associates with increased oxidative phosphorylation as well as glycolysis and identifies early recurrence in HCC patients after surgical treatment.

Prevention of hepatocellular carcinoma: Focusing on antioxidant therapy.

Oxidative stress has been investigated in the context of alcoholic liver injury for many years and shown to be a causal factor of chronic hepatitis C (CHC), nonalcoholic steatohepatitis (NASH), drug-induced liver injury, Wilson's disease, and hemochromatosis. In CHC, it has been demonstrated that oxidative stress plays an important role in hepatocarcinogenesis. In cases with persistent hepatitis due to failure of hepatitis C virus eradication, or chronic liver disease, such as NASH, the treatment of which remains unestablished, it is important to reduce serum alanine aminotransferase levels and prevent liver fibrosis and development of hepatocellular carcinoma. This also suggests the importance of antioxidant therapy. Among treatment options where it would be expected that anti-inflammatory activity plays a role in their confirmed efficacy for chronic hepatitis, iron depletion therapy, glycyrrhizin, ursodeoxycholic acid, Sho-Saiko-To, and vitamin E can all be considered antioxidant therapies. To date, however, the ability of these treatments to prevent cancer has been confirmed only in CHC. Nevertheless, anti-inflammatory and anti-fibrotic effects have been demonstrated in other liver diseases and these therapies may potentially be effective for cancer prevention.

Increased duodenal iron absorption through up-regulation of divalent metal transporter 1 from enhancement of iron regulatory protein 1 activity in patients with nonalcoholic steatohepatitis.

UNLABELLED: Increased hepatic iron accumulation is thought to be involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). Hepatic iron accumulation, as well as oxidative DNA damage, is significantly increased in NASH livers. However, the precise mechanism of iron accumulation in the NASH liver remains unclear. In this study, 40 cases with a diagnosis of NASH (n = 25) or simple steatosis (SS; n = 15) by liver biopsy were enrolled. An oral iron absorption test (OIAT) was used, in which 100 mg of sodium ferrous citrate was administered to each individual. The OIAT showed that absorption of iron from the gastrointestinal (GI) tract was increased significantly in NASH patients, compared to SS and control subjects. Iron reduction therapy was effective in patients with NASH, who exhibited iron deposition in the liver and no alanine aminotransferase improvement after other therapies (n = 9). Serum hepcidin concentration and messenger RNA (mRNA) levels of divalent metal transporter 1 (DMT1) also were significantly elevated in patients with NASH. OIAT results were correlated with grade of liver iron accumulation and DMT1 mRNA levels. Then, we demonstrated that DMT1 mRNA levels increased significantly in Caco-2/TC7 cell monolayers cultured in transwells with serum from NASH patients. An electrophoresis mobility shift assay showed activation of iron regulatory protein (IRP) in those cells, and IRP1 small interfering RNA clearly inhibited the increase of DMT1 mRNA levels. CONCLUSION: In spite of elevation of serum hepcidin, iron absorption from the GI tract increased through up-regulation of DMT1 by IRP1 activation by humoral factor(s) in sera of patients with NASH.

[Bendamustine-rituximab therapy is effective for transformed follicular lymphoma with significant expression of p53].

We describe a patient with transformed follicular lymphoma(FL), expressing p53 but remaining in complete remission(CR) due to bendamustine-rituximab(BR)therapy. She was a 64-year-old female diagnosed with stage IV FL(grade 3A)in July 2007 when she was admitted with right lower abdominal pain and body weight loss. Colonoscopy revealed Bauhin' valve lymphoma of the terminal ileum, and computed tomography(CT)scan showed lymphadenopathy, involving the cervical, mediastinal para-aortic lymph nodes and right tonsil. She received chemotherapy with eight courses of CHOP therapy with rituximab and achieved CR. Two and a half years later, mediastinal lymph node swelling relapsed, and ibritumomab tiuxetan therapy induced the second CR. After ten months, however, a third relapse occurred as a submucosal tumor(SMT)of the stomach. Gastric SMT biopsy showed diffuse large B cell lymphoma(DLBCL)transformation with immunohistochemical expression of p53. Although gastric SMT disappeared after radiotherapy, which achieved the third CR, lymph node swelling was detected again in the para-aortic and-iliac artery lymph nodes in September 2011. Subsequently, she was treated with five courses of BR therapy, because bendamustine had been reported to be effective for p53 gene-deficient B cell neoplasms. The therapy was successful and achieved the fourth CR, demonstrating that BR therapy was effective for p53-expressing DLBCL.

Increased hepatic oxidative DNA damage in patients with nonalcoholic steatohepatitis who develop hepatocellular carcinoma.

BACKGROUND: The rate of onset of hepatocellular carcinoma (HCC) in patients with nonalcoholic steatohepatitis (NASH) has been reported recently to be comparable to that of patients with chronic hepatitis C. However, the precise mechanism contributing to carcinogenesis in the former remains unclear. Although increased oxidative stress is presumed to play a role in carcinogenesis in patients with NASH, this relationship remains to be directly proven. In this study, we investigated the involvement of oxidative DNA damage in hepatocarcinogenesis in patients with NASH. METHODS: Patients with nonalcoholic fatty liver disease who were treated at our university hospital were eligible for enrolment in the study(n = 49). The study cohort included 30 patients with NASH without HCC (NASH without HCC), six HCC patients with NASH (NASH-HCC), and 13 patients with simple steatosis. Quantitative immunohistochemistry with a KS-400 image analyzing system was used for 8-hydroxy-2'-deoxyguanosine (8-OHdG) detection. RESULTS: The 8-OHdG content in the liver tissue of NASH-HCC patients was significantly different from that in the other patients. The median immunostaining intensity was 8.605 in the NASH-HCC cases, which was significantly higher than that in the cases of NASH without HCC (4.845; P = 0.003). Multivariate analysis using hepatic 8-OHdG content as a factor in addition to age and fasting blood sugar revealed a significant difference in clinicopathological factors between NASH-HCC and NASH without HCC cases. Old age (P = 0.015) and high relative immunostaining intensity for intrahepatic 8-OHdG (P = 0.037) were identified as independent factors. CONCLUSIONS: 8-OHdG content in liver tissue may serve a marker of oxidative stress and could be a particularly useful predictor of hepatocarcinogenesis.

[Complete response by rituximab monotherapy in a patient with leukemic mantle cell lymphoma diagnosed by fluorescence in situ hybridization analysis].

An 85-year-old male was admitted to a hospital with abdominal discomfort in October, 2010. Severe splenomegaly and mild para-aortic lymphoadenopathy were detected. In addition, an increase in atypical lymphocytes was noticed by bone marrow analyses with weak positive staining of cyclin D1. Subsequently, the fluorescence in situ hybridization(FISH)method confirmed cyclin D1 reconstruction, and the fusion signals of the BCL1 and IgH genes were detected, thus providing a definitive diagnosis of leukemic mantle cell lymphoma. After treatment with rituximab monotherapy, the Ki-67 index was stabilized to within 10%, and complete response was obtained.

[Chronic hepatitis C presenting with hepatic involvement by chronic lymphocytic leukemia responding to polyethylene glycol interferon-α-2b].

Hepatic involvement by chronic lymphocytic leukemia(CLL)is common, but rarely presents with liver injury. We report a case of chronic hepatitis C(CH-C)in a patient who had suffered from liver injury as a result of hepatic involvement by CLL. A 74-year-old man was hospitalized because of an examination confirming him positive for leukocytosis. Computer tomography scan showed mild hepatosplenomegaly, lymph node swelling of the neck and axilla, and abdominal lymphadenopathy. He was diagnosed as CLL by bone marrow examination. His laboratory data revealed hepatis C virus(HCV)antibody-positive, and elevated levels of both aminotransferase and HCV-RNA. Liver biopsy demonstrated significant CLL involvement of portal areas and a mild T lymphocyte invasion of centrilobular and portal areas. After treatment with polyethylene glycol interferon (PEG-IFN)-α-2b for CH-C, the CLL count was decreased in both peripheral blood and the liver. The hematological response was significantly correlated with the disappearance of HCV-RNA. The patient has maintained a sustained virological response(SVR)status for the past 9 months after PEG-IFN-α-2b administration. Thus, PEG-IFN-α-2b therapy could be effective not only for CH-C but also for hepatic involvement of CLL as seen in our patient.