Novel soluble HLA‐A2/MELAN‐A complexes selectively stain a differentiation defective subpopulation of CD8+ T cells in patients with melanoma

Multimeric MHC I‐peptide complexes containing phycoerythrin‐streptavidin are widely used to detect and investigate antigen‐specific CD8+ (and CD4+) T cells. Because such reagents are heterogeneous, we compared their binding characteristics with those of monodisperse dimeric, tetrameric and octameric complexes containing linkers of variable length and flexibility on Melan‐A‐specific CD8+ T cell clones and peripheral blood mononuclear cells (PBMC) from HLA‐A*0201⁺ melanoma patients. Striking binding differences were observed for different defined A2/Melan‐A_26‐35 complexes on T cells depending on their differentiation stage. In particular, short dimeric but not octameric A2/Melan‐A_26‐35 complexes selectively and avidly stained incompletely differentiated effector‐memory T cells clones and populations expressing CD27 and CD28 and low levels of cytolytic mediators (granzymes and perforin). This subpopulation was found in PBMC from all six melanoma patients analyzed and proliferated on peptide stimulation with only modest phenotypic changes. By contrast influenza matrix_58‐66 ‐specific CD8+ PBMC from nine HLA‐A*0201⁺ healthy donors were efficiently stained by A2/Flu matrix_58‐61 multimers, but not dimer and upon peptide stimulation proliferated and differentiated from memory into effector T cells. Thus PBMC from melanoma patients contain a differentiation defective sub‐population of Melan‐A‐specific CD8+ T cells that can be selectively and efficiently stained by short dimeric A2/Melan‐ A_26‐35 complexes, which makes them directly accessible for longitudinal monitoring and further investigation.     

结直肠癌患者外周血中CD4+CD45RA+ T和CD4+CD45RO+ T细胞的变化及其意义

目的 探讨外周血中CD4 CD45RA T细胞和CD4 CD45RO T细胞在结直肠癌中的变化及其临床意义。方法 采用流式细胞术检测60例结直肠癌患者手术前、术后1个月和3个月时,外周血的CD4 T细胞、CD4 CD45RA T细胞和CD4 CD45RO T细胞的比例。选取健康查体人群10例作为对照。结果 结直肠癌患者的CD4 T细胞与健康人群相比无差异。CD4 CD45RO T细胞比例明显增高,术后有显著下降,DukesA、B期患者尤其明显;而CD4 CD45RA T细胞比例正好相反。结论 CD4 CD45RA T细胞和CD4 CD45RO T细胞在肿瘤免疫中起重要作用,其表达同结直肠癌的分期和预后有密切关系。     

GM-CSF 基因修饰肿瘤细胞疫苗治疗前后黑素瘤患者CD8 +T细胞的变化及其对预后的影响

目的:探讨粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因修饰的肿瘤细胞疫苗(GM-CSF modified tumor cell vaccine,GVAX)治疗前后黑素瘤患者外周血免疫指标的变化及其对预后的影响。方法:收集2007年10月至2012年12月期间于天津医科大学肿瘤医院接受GVAX治疗的56例黑素瘤患者,采用流式细胞技术检测患者治疗前后外周血CD3+T细胞、CD4+T细胞、CD8+T细胞、调节性T细胞(Treg)、自然杀伤细胞(NK)及树突状细胞(DC1和DC2)的比例,分析治疗前后外周血各免疫细胞比例的变化,并探讨其与患者预后的关系。结果:GVAX治疗后患者外周血CD8+T细胞比例较前升高[(37.56±12.76)%vs(34.71±12.30)%,P=0.006],CD4+T细胞比例降低[(53.44±13.36)%vs(56.27±13.15)%,P=0.017],CD4+/CD8+比值降低[1.61(1.37)vs 1.75(0.71),P=0.009]。治疗后CD3+T、NK、Treg、DC1、DC2与治疗前的差异无统计学意义(P>0.05)。晚期患者GVAX治疗前外周血CD8+T细胞比例大于均值组与小于均值组相比,中位生存时间显著延长(29.16 vs 13.34个月,P=0.012)。结论:GVAX治疗黑素瘤可增强CD8+T细胞为主的抗肿瘤免疫应答,晚期患者外周血CD8+T细胞比例可作为预测GVAX疗效的指标,为判断预后起到一定的提示作用。     

Tim-3在恶性黑色素瘤患者外周血CD8+T细胞上的表达及临床意义

目的:本研究应用流式细胞术检测恶性黑色素瘤患者外周血CD8+T细胞中T细胞免疫球蛋白及黏蛋白-3(Tim-3)的表达情况,探讨Tim-3在恶性黑色素瘤进展中的作用.方法:应用流式细胞仪检测42例恶性黑色素瘤患者及38例健康体检者外周血CD8+T细胞上Tim-3的表达,同时分析其与恶性黑色素瘤患者临床病理特征的关系.结果:Tim-3在恶性黑色素瘤患者及健康体检者外周血CD8+T细胞中均有不同程度的表达,恶性黑色素瘤组Tim-3表达量为(15.89±6.49)％,健康对照组的表达量为(3.27±2.16)％,差异具有统计学意义(P＜0.01).Tim-3在CD8+T细胞中的表达水平与恶性黑色素瘤患者有无溃疡、淋巴结转移、临床分期、远处转移有关(P＜0.01),而与性别、年龄、Breslow分度无关(P＞0.05).结论:Tim-3在恶性黑色素瘤患者外周血CD8+T细胞上高水平表达,其表达水平与临床分期呈正相关,并有望成为恶性黑色素瘤早期预测及治疗的重要靶标.     

Lipopeptide-based melanoma cancer vaccine induced a strong MART-27-35-cytotoxic T lymphocyte response in a preclinal study

Identification of tumor antigens and their optimal antigenic peptides raised hopes for the development of peptide-based immunotherapeutic vaccine strategies for human melanoma, however. Synthetic peptides alone are not immunogenic enough, and adequate formulation is critical for elaboration of peptide vaccines. To improve formulation, we evaluated 2 lipopeptide constructs, both including HLA-A2-restricted MART 27-35-CD8+ T lymphocyte (CTL) epitope covalently linked to universal tetanus toxoid (TT) 830-843 helper T lymphocyte (HTL) epitope, in HLA-A2 transgenic mouse models that mimic human CTL responses in vivo. These 2 constructs only differed in the formulation of their lipid tail. We showed that lipopeptide constructs were strongly recognized, in vitro, by human MART 27-35 cytotoxic T cells derived from tumor-infiltrating lymphocytes. The transgenic Mice immunized with these 2 MART lipopeptide formulations containing covalently linked HTL-CTL epitopes induced strong MART 27-35 cytotoxic T cells. This CTL induction was critically dependent on the presence of the helper T lymphocyte epitope. These results also showed that a single palmitoyl-lysine chain is enough to assure immunogenicity of a given peptide and that the presence of a lipid tail bypass the need for adjuvant. These results support the selection of MART-lipopeptide melanoma vaccine for evaluation in a clinical trial.     

Blood monocytes sample MelanA/MART1 antigen for long‐lasting cross‐presentation to CD8+ T cells after differentiation into dendritic cells

Human blood monocytes are very potent to take up antigens. Like macrophages in tissue, they efficiently degrade exogenous protein and are less efficient than dendritic cells (DCs) at cross‐presenting antigens to CD8⁺ T cells. Although it is generally accepted that DCs take up tissue antigens and then migrate to lymph nodes to prime T cells, the mechanisms of presentation of antigens taken up by monocytes are poorly documented so far. In the present work, we show that monocytes loaded in vitro with MelanA long peptides retain the capacity to stimulate antigen‐specific CD8⁺ T cell clones after 5 days of differentiation into monocytes‐derived dendritic cells (MoDCs). Tagged‐long peptides can be visualized in electron‐dense endocytic compartments distinct from lysosomes, suggesting that antigens can be protected from degradation for extended periods of time. To address the pathophysiological relevance of these findings, we screened blood monocytes from 18 metastatic melanoma patients and found that CD14⁺ monocytes from two patients effectively activate a MelanA‐specific CD8 T cell clone after in vitro differentiation into MoDCs. This in vivo sampling of tumor antigen by circulating monocytes might alter the tumor‐specific immune response and should be taken into account for cancer immunotherapy.     

食管癌患者外周血MAGE-C2抗原特异性CD8+T细胞的功能及临床意义

目的:评价食管癌患者外周血恶性黑色素瘤相关抗原(melanoma-associated antigen,MAGE)-C2抗原特异性CD8+T细胞的功能,并分析其与临床病理参数的相关性.方法:密度梯度离心法获取130例食管癌初治患者(收集自2014年11月至2015年11月郑州大学第一附属医院胸外科)的PBMC,流式细胞术、RT-PCR方法筛选HLA-A2* 0201和MAGE-C2共阳性的患者,其PBMC培养7、14 d后加MAGE-C2(336-344)抗原肽1μg/m1、IL-2 100 μg/ml和CD3/CD28 Dynabeads 2μl处理,诱导扩增抗原特异性CD8+T细胞,流式细胞术检测CD8+T细胞表面CD107a和胞内IFN-γ的水平,并分析其与临床参数的相关性.结果:筛选出52例HLA-A2* 0201和MAGE-C2共阳性的患者,MAGE-C2(336-344)处理其PBMC后,MAGE-C2(336-344)处理组CD8+T细胞中CD107a+ IFN-γ+,CD107a+ IFN-γ-和CD107a IFN-γ+细胞的比例明显升高(P＜0.05);CDI07a的表达在早期、未发生淋巴结转移和高分化的食管癌患者明显高于晚期、发生淋巴结转移和低分化的癌症患者(P＜0.05);IFN-γ的表达在高分化的食管癌患者明显高于低分化的食管癌患者(P＜0.05).结论:成功诱导扩增MAGE-C2抗原特异性CD8+T细胞,并发现其功能与食管癌的分期和淋巴结转移相关,似可作为一种理想的免疫治疗靶点.     

Co-immunization with L-Myc enhances CD8+ or CD103+ DCs mediated tumor-specific multi-functional CD8+ T cell responses

Renal carcinoma shows a high risk of invasion and metastasis without effective treatment. Herein, we developed a chitosan (CS) nanoparticle-mediated DNA vaccine containing an activated factor L-Myc and a tumor-specific antigen CAIX for renal carcinoma treatment. The subcutaneous tumor models were intramuscularly immunized with CS-pL-Myc/pCAIX or control vaccine, respectively. Compared with single immunization group, the tumor growth was significantly suppressed in CS-pL-Myc/pCAIX co-immunization group. The increased proportion and mature of CD11c⁺ DCs, CD8⁺CD11c⁺ DCs and CD103⁺CD11c⁺ DCs were observed in the splenocytes from CS-pL-Myc/pCAIX co-immunized mice. Furthermore, the enhanced antigen-specific CD8⁺ T lymphocyte proliferation, cytotoxic T lymphocyte (CTL) responses, and multi-functional CD8⁺ T cell induction were detected in CS-pL-Myc/pCAIX co-immunization group compared with CS-pCAIX immunization group. Of note, the depletion of CD8 T cells resulted in the reduction of CD8⁺ T cells or CD8⁺CD11c⁺ DCs and the loss of anti-tumor efficacy induced by CS-pL-Myc/pCAIX vaccine, suggesting the therapeutic efficacy of the vaccine was required for CD8⁺ DCs and CD103⁺ DCs mediated CD8⁺ T cells responses. Likewise, CS-pL-Myc/pCAIX co-immunization also significantly inhibited the lung metastasis of renal carcinoma models accompanied with the increased induction of multi-functional CD8⁺ T cell responses. Therefore, these results indicated that CS-pL-Myc/pCAIX vaccine could effectively induce CD8⁺ DCs and CD103⁺ DCs mediated tumor-specific multi-functional CD8⁺ T cell responses and exert the anti-tumor efficacy. This vaccine strategy offers a potential and promising approach for solid or metastatic tumor treatment.     

Human papillomavirus (HPV) type 16-specific CD8+ T cell responses in women with high grade vulvar intraepithelial neoplasia

Human papillomavirus (HPV)-associated vulvar intraepithelial neoplasia (VIN) has serious sequelae for the sufferer. Current treatments are associated with poor response and high relapse rates. The development of HPV-specific T cell immunotherapies offers a new approach to treatment. This will require a detailed understanding of the spectrum of T cell responses induced by HPV antigens, and how effectively viral antigens can be accessed by the immune system. We have investigated the frequency and spectrum of HPV16-specific CD8+ T cell responses to three HPV16 antigens in 9 women with high grade VIN (VIN3). CD4-depleted populations of responder cells were screened against overlapping 30-35mer peptides covering the sequences of HPV16 E6, E7 and E4 using ELISPOT assays of IFN-gamma release. We demonstrated CD8+ T cell reactivity to one or more of the proteins in 6 of 9 patient samples. All 6 of these responders recognised peptides covering the E7 protein, 3 of 9 women responded to E6 peptides, but no reactivity was seen to E4. Our results suggest that HPV16-specific cytotoxic T cells (CTLs) are relatively common in women with persistent VIN3. The HPV-specific CTL response, however, seems to be ineffective. There is some evidence that there are problems associated with the processing and presentation of HPV antigens by the infected vulvar epithelium. It will be crucial to address this in the design of any T cell based therapy for HPV-associated VIN and vulval cancer.     

PD-L1+ dendritic cells in the tumor microenvironment correlate with good prognosis and CD8+ T cell infiltration in colon cancer

BackgroundThe prognostic value of tumor‐associated dendritic cells (DC) in colon cancer remains poorly understood. This may be in part due to the interchangeable expression of immunostimulatory and immunoinhibitory molecules on DC. Here we investigated the prognostic impact of CD11c⁺ DC co–expressing the immunoinhibitory molecule PD‐L1 and their spatial relationship with CD8⁺ T‐cells in patients treated for stage III colon cancer.MethodsTissue microarrays containing representative cores of central tumor, leading edge, and adjacent normal tissue from 221 patients with stage III colon cancer were immunostained for CD8, CD11c, PD‐L1, and cytokeratin using immunofluorescent probes. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kaplan‐Meier estimates and Cox regression were used to assess survival.ResultsIntratumoral CD8⁺ cell density (HR = .52, 95% confidence interval [CI] .33‐.83, P = .007), stromal CD11c⁺ cell density (HR = .52, 95% CI .33‐.83, P = .006), intratumoral CD11c⁺PD‐L1⁺ cell density (HR = .57, 95% CI .35‐.92, P = .021), and stromal CD11c⁺PD‐L1⁺ cell density (HR = .48, 95% CI .30‐.77, P = .003) on leading‐edge cores were all significantly associated with good survival. CD8⁺ cell density was positively correlated with both CD11c⁺ cell density and CD11c⁺PD‐L1⁺ cell density in tumor epithelium and stromal compartments.ConclusionHere we showed that PD‐L1‐expressing DC in the tumor microenvironment are associated with improved survival in stage III colon cancer and likely reflect an immunologically “hot” tumor microenvironment. Further investigation into the expression of immunomodulatory molecules by tumor‐associated DC may help to further elucidate their prognostic value.     

肿瘤外泌体对CD8+T细胞功能的影响及机制研究进展

外泌体（exosomes）是介导细胞间通讯的细胞外囊泡。它携带来源细胞的多种生物活性分子,并可将其输送给受体细胞,进而影响细胞功能。肿瘤来源外泌体可通过多种机制介导肿瘤的免疫逃逸。本文就肿瘤外泌体对肿瘤杀伤主力军CD8+T细胞的调控作用进行总结,分析其相关作用机制,以期为肿瘤免疫治疗的研发提供新的思路。     

Generation of CD8+ cytotoxic T lymphocytes against breast cancer cells by stimulation with mammaglobin-A-pulsed dendritic cells

Mammaglobin-A is exclusively expressed by breast cancer cells. Thus, mammaglobin-A-specific T cell immune responses may be useful for the design of new breast cancer-specific immunotherapies. We show herein that CD8+ T cells generated against recombinant mammaglobin-A-pulsed dendritic cells display a marked cytotoxic activity against mammaglobin-A-positive breast cancer cell lines. This study indicates the immunotherapeutic potential of this novel antigen for the treatment of breast cancer.     

Expression of Fas/FasL in CD8+ T and CD3+ Foxp3+ Treg Cells - Relationship with Apoptosis of Circulating CD8+ T Cells in Hepatocellular Carcinoma Patients

Aims: Dysfunction of the host immune system in cancer patients can be due to a number of factors, includinglymphocyte apoptosis. Several studies showed that Foxp3+T cells take part in inducing this process by expressingFasL in tumor patients. However, the relationship between apoptosis, CD8+T cells and Foxp3+T cells in HCCpatients is still unclear. The present study was designed to investigate the correlation between apoptosis levelsand Fas/FasL expression in CD8+T lymphocytes and Foxp3+T cells in patients with HCC. Methods: CD8+T cellsand CD3+Foxp3+T cells were tested from peripheral blood of HCC patients and normal controls and subjectedto multicolor flow cytometry. The expression of an apoptosis marker (annexin V) and the death receptor Fas inCD8+T cells and FasL in CD3+Foxp3+T cells were evaluated. Serum TGF-β1 levels in patients with HCC weremeasured by enzyme-linked immunosorbent assay. The relationship between apoptosis and Fas expression, aswell as FasL expression in CD3+Foxp3+T cells was then evaluated. Results: The frequency of CD8+T cells bindingannexin V and Fas expression in CD8+T cells, were all higher in HCC patients than normal controls and theproportion of apoptotic CD8+T cells correlated with their Fas expression. Serum TGF-β1 levels correlated inverselywith CD3+Foxp3+T cells. Conclusions: Fas/FasL interactions might lead to excessive turnover of CD8+T cellsand reduce anti-tumor immune responses in patients with HCC. Further investigations of apoptosis inductionin Fas+CD8+T cells in vitro are required.     

干细胞样CD8+ T细胞：肿瘤免疫疗法的新生力量

CD8+ T 细胞是抗肿瘤免疫应答的主要执行者。通过重塑CD8+ T 细胞杀伤肿瘤细胞的能力,免疫疗法已在抗肿瘤领域取得重大突破,但临床获益仅局限于部分患者和癌症类型。如何克服CD8+ T细胞功能障碍是肿瘤免疫疗法亟待解决的关键问题。近年来,多项研究揭示了CD8+ T细胞的干性调控机制,发现了干细胞样CD8+ T细胞具有自我更新和增殖能力,阐明了该细胞亚群在维持持续性肿瘤免疫治疗应答中的重要性。本文论述了干细胞样CD8+ T 细胞的分子与功能特征、CD8+ T 细胞干性的细胞内外影响因素,归纳总结了目前靶向CD8+ T细胞的干性重编程策略,进一步展望了靶向CD8+ T细胞干性程序来提高肿瘤免疫疗法疗效的思路和方法。     

Levels of circulating regulatory CD4+CD25+ T cells are decreased in breast cancer patients after vaccination with a HER2/neu peptide (E75) and GM-CSF vaccine

SummaryPurpose We are conducting clinical trials in breast cancer (BrCa) patients to test the HER2/neu peptide vaccine (E75). We have investigated the impact of this vaccine on circulating levels of regulatory T cells (T_reg) and the resulting effects on antitumor responses.Experimental design Twenty-two blood samples from healthy individuals and from 22 BrCa patients including pre- and post-vaccination samples from seven vaccinated HLA-A2⁺ patients were stained for CD4, CD25, and CD69 as well as CD8 and E75:HLA-A2 Ig dimer and quantified by flow cytometry. Cytotoxic activity against HER2/neu ⁺ tumors was measured by ⁵¹Cr-release. Serum from BrCa patients and normal subjects were analyzed for TGF-β levels.Results BrCa patients have a greater percentage of circulating T_reg (CD4⁺CD25⁺, 4.45% versus 2.96%; p = 0.007) than normal subjects. HLA-A2⁺ BrCa patients had more T_reg compared to the HLA-A2^– BrCa patients (CD4⁺CD25⁺, 5.63% versus 3.28%; p = 0.001). E75 vaccination increased circulating activated CD4⁺ T cells post-vaccination (CD4⁺CD69⁺, 1.23 versus 3.81%; p = 0.03). However, T_reg were significantly reduced after vaccination (CD4⁺CD25⁺, 5.31–1.81%; p < 0.0001). Furthermore, activated T_reg also decreased (CD4⁺CD25⁺CD69⁺, 0.23% versus 0.08%; p = 0.06). Importantly, post-vaccination decreases in T_reg were temporally associated with increased E75 vaccine-specific CD8⁺ T cells and corresponding HER2/neu ⁺ tumor cytotoxicity. Serum TGF-β levels were significantly elevated in BrCa patients compared to normals (3548 pg/ml versus 1007 pg/ml; p = 0.007). Four of seven vaccinated patients showed decreased serum TGF-β levels post-vaccination.Conclusions T_reg, are increased in BrCa patients along with serum levels of TGF-β. E75 vaccination resulted in CD4⁺ recruitment but was associated with a significant decrease in circulating T_reg and TGF-β levels in the majority of the vaccinated patients. Successful cancer vaccination strategies may require the alteration of complex immune interactions.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense     

CD4+/CD8+ T淋巴细胞在肝细胞癌组织中的浸润及与预后的相关性研究

目的 检测CD4⁺/CD8⁺ T淋巴细胞在肝细胞癌（hepatocellular carcinoma,HCC）组织中的浸润程度,并分析其与预后的相关性。方法 收集行肝切除术的HCC患者215例,采用免疫组化技术检测CD4⁺/CD8⁺ T淋巴细胞在HCC癌组织中的浸润程度,根据浸润情况比较患者肝切除术后无瘤生存率和总生存率。结果 CD4⁺ T淋巴细胞高浸润和低浸润比例分别为60.9%和39.1%。CD4⁺ T淋巴细胞高浸润组患者总生存率和无瘤生存率均显著高于低浸润组（P=0.015,P=0.038）。CD8⁺ T淋巴细胞高浸润和低浸润比例分别为34.9%和65.1%。CD8⁺ T淋巴细胞高浸润组患者的总生存率和无瘤生存率亦显著高于低浸润组患者（P=0.033,P=0.047）。结论 CD4⁺或CD8⁺ T淋巴细胞低浸润可能与HCC患者术后不良预后相关。     

CENPF as an independent prognostic and metastasis biomarker corresponding to CD4+ memory T cells in cutaneous melanoma

Owing to recent advances in immunotherapies, the overall survival of patients with skin cutaneous melanoma (SKCM) has increased; however, the 5‐year survival rate of metastatic patients remains poor. Skin cutaneous melanoma—upregulated genes were screened via analysis of differentially expressed genes ({"type":"entrez-geo","attrs":{"text":"GSE3189","term_id":"3189"}}GSE3189 and {"type":"entrez-geo","attrs":{"text":"GSE46517","term_id":"46517"}}GSE46517), and metastasis‐related oncogenes were identified via weighted gene coexpression network analysis of the {"type":"entrez-geo","attrs":{"text":"GSE46517","term_id":"46517"}}GSE46517 dataset. As confirmed by the Tumor Immune Estimation Resource, we found highly expressed centromere protein F (CENPF) in SKCM and its metastases. Immunostaining of human melanoma tissues demonstrated high CENPF expression. According to the Kaplan‐Meier survival curve log‐rank test, receiver‐operating characteristic curve, and univariate and multivariate analyses, the Cancer Genome Atlas (TCGA) database suggested CENPF be a typical independent predictor of SKCM. The CIBERSORT algorithm classified the types of the immune cells from {"type":"entrez-geo","attrs":{"text":"GSE46517","term_id":"46517"}}GSE46517 and showed higher proportion of CD4+ memory‐activated T cells in metastatic melanoma. Single‐sample gene set enrichment analysis of TCGA data confirmed the correlation between CENPF and activated CD4+ T cells. Centromere protein F was positively correlated with tumor mutational burden and CD4+ memory T cell markers (interleukin [IL]‐23A, CD28, and CD62L), negatively associated with memory T cell maintenance factors (IL‐7 and IL‐15) by correlation analysis. Moreover, immunofluorescence showed high coexpression of CENPF and IL23A, CD4 in melanoma. Upregulated CENPF might lead to premature depletion of CD4+ memory T cells and immunosuppression. Nomogram indicated CENPF clinical predictive value for 1‐, 3‐, 5‐, and 7‐year melanoma overall survival. Therefore, CENPF plays a vital role in the progression and metastasis of melanoma and can be an effective therapeutic target.     

Induction of CD8 T‐cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20‐mer NY‐ESO‐1f (NY‐ESO‐1 91‐110) peptide

Immunogenicity of a long 20‐mer NY‐ESO‐1f peptide vaccine was evaluated in a lung cancer patient TK‐f01, immunized with the peptide with Picibanil OK‐432 and Montanide ISA‐51. We showed that internalization of the peptide was necessary to present CD8 T‐cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T‐cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01‐restricted CD8 T‐cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02‐restricted CD8 T‐cell clones were defined and peptide/HLA tetramers were produced. NY‐ESO‐1 91‐101 on A*24:02, NY‐ESO‐1 92‐102 on B*35:01, NY‐ESO‐1 96‐104 on B*52:01 and NY‐ESO‐1 96‐104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T‐cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20‐mer NY‐ESO‐1f peptide harboring multiple CD8 T‐cell epitopes as an NY‐ESO‐1 vaccine. Characterization of CD8 T‐cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T‐cell responses comprised the dominant response.     

Glutamine deprivation impairs function of infiltrating CD8+ T cells in hepatocellular carcinoma by inducing mitochondrial damage and apoptosis

BACKGROUNDThe functions of infiltrating CD8⁺ T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment. Thus, the mechanisms of CD8⁺ T cell dysfunction have become a hot research topic, and there is increased interest on how changes in metabolomics correlate with CD8⁺ T cell dysfunction.AIMTo investigate whether and how glutamine metabolism affects the function of infiltrating CD8⁺ T cells in hepatocellular carcinoma.METHODSImmunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients. Differentially expressed genes in infiltrating CD8⁺ T cells in hepatocellular carcinoma were detected using RNA sequencing. Activated CD8⁺ T cells were co-cultured with Huh-7 cells for 3 d. The function and mitochondrial status of CD8⁺ T cells were analyzed by flow cytometry, quantitative real-time polymerase chain reaction, and transmission electron microscopy. Next, CD8⁺ T cells were treated with the mitochondrial protective and damaging agents. Functional alterations in CD8⁺ T cells were detected by flow cytometry. Then, complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected.RESULTSThere were a large number of infiltrating PD-1⁺CD8⁺ T cells in liver cancer tissues. Next, we co-cultured CD8⁺ T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8⁺ T cells. Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin (PRF1) and granzyme B (GZMB) by CD8⁺ T cells in the co-culture group. Meanwhile, JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8⁺ T cells of the co-culture group; additionally, the mitochondria of these cells were swollen. When CD8⁺ T cells were treated with the mitochondrial protective and damaging agents, their function was restored and inhibited, respectively, through the mitochondrial damage and apoptotic pathways. Subsequently, complete medium without glutamine was used to culture cells. As expected, CD8⁺ T cells showed functional downregulation, mitochondrial damage, and apoptosis.CONCLUSIONGlutamine deprivation impairs the function of infiltrating CD8⁺ T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.     

Predominant infiltration of macrophages and CD8(+) T Cells in cancer nests is a significant predictor of survival in stage IV nonsmall cell lung cancer

BACKGROUND.: The purpose of this study was to investigate whether tumor-infiltrating immune cells in biopsy specimens can be used to predict the clinical outcome of stage IV nonsmall cell lung cancer (NSCLC) patients. METHOD.: The authors performed an immunohistochemical study to identify and count the number of CD68(+) macrophages, c-kit(+) mast cells, and CD8(+) T cells in both cancer nests and cancer stroma in pretreatment biopsy specimens obtained from 199 patients with stage IV NSCLC treated by chemotherapy, and then analyzed for correlations between the number of immune cells and clinical outcome, including chemotherapy response and prognosis. RESULTS.: There was no correlation between the number of immune cells in either cancer nests or stroma and chemotherapy response. Patients with more tumor-infiltrating macrophages in cancer nests than in cancer stroma (macrophages, nests > stroma) had significantly better survival than nests < stroma cases median survival time (MST 440 days vs 199 days; P < .0001). Patients with more tumor-infiltrating CD8(+) T cells in cancer nests than in cancer stroma (CD8(+) T cells: nests > stroma) showed significantly better survival than in nests < stroma cases (MST 388 days vs 256 days; P = .0070). The proportion of tumor-infiltrating macrophages or CD8(+) T cells between cancer nests and stroma became independent prognostic factors in the multivariate analysis. Neither the number of mast cells in nests nor in stroma correlated with the clinical outcome. CONCLUSIONS.: Evaluation of the numbers of macrophages and CD8(+) T cells in cancer nests and stroma are useful biomarkers for predicting the prognosis of stage IV NSCLC patients treated with chemotherapy, but could fail to predict chemotherapy response. Cancer 2008. (c) 2008 American Cancer Society.