Naturally processed and HLA-B8-presented HPV16 E7 epitope recognized by T cells from patients with cervical cancer

Several major histocompatibility complex (MHC) alleles have been reported to present peptides derived from the HPV16 E7 oncoprotein to T cells. We describe an overrepresentation of the HLA-B8 allele (28.44%) in cervical cancer patients as compared to the MHC class I allele frequency in a local healthy control population (18.80%) and the identification of an HLA-B8-binding peptide TLHEYMLDL (HPV16 E7(7-15)), which is able to drive HPV16 E7-specific and MHC class I-restricted T-cell responses in peripheral blood lymphocytes from healthy individuals. TLHEYMLDL-specific T cells recognize the naturally processed and presented peptide on HPV16+ cervical cancer cells transfected with the HLA-B8 gene defined by IFN-gamma production. This peptide epitope is also recognized by freshly harvested tumor-infiltrating T cells or T cells from tumor-draining lymph nodes from patients with cervical cancer determined by flow cytometry as well as by tetramer in situ staining. HLA-B8-restricted HPV E7(7-15)-specific T cells reside predominantly in the CD8+ CD45RA+ CCR7+ precursor or in the differentiated CD8+ CD45RA+ CCR7- T-cell population.     

Reversed CD4/CD8 ratios of tumor-infiltrating lymphocytes are correlated with the progression of human cervical carcinoma.

BACKGROUND: To investigate the clinical significance of tumor-infiltrating lymphocytes (TILs) within the tumor milieu of human cervical carcinoma, the authors quantitatively measured and compared the subpopulations of lymphocytes infiltrating the neoplastic cervix. METHODS: A total of 30 patients with Stage Ia-IIa cervical carcinoma were enrolled. TILs were isolated from tissue specimens by means of a mechanical dispersal technique, and the immunocyte subsets were quantified with dual-color flow cytometry. Bulky tumor was defined as tumor size >4 cm in greatest dimension according to the 1995 staging of the International Federation of Gynecology and Obstetrics. RESULTS: The CD4/CD8 ratios of TILs were reversed in both cervical squamous cell carcinoma (n = 20) and cervical adenocarcinoma (n = 10). The proportion of CD4(+) T cells was significantly lower in tumors from patients with lymph node metastasis (n = 8) than in those from patients without lymph node metastasis (n = 22) (24.5 vs. 32.7, P = 0.001), as was the reversed CD4/CD8 ratio (0.50 vs. 0.81, P = 0.001). The proportion of CD4(+) T cells was much lower in bulky tumors (n = 5) than in nonbulky tumors (n = 25) (21.4 vs. 32.5, P < 0.001), reflecting in a more strongly reversed CD4/CD8 ratio (0.41 vs. 0.81, P = 0.001). CONCLUSIONS: Decreased proportions of tumor-infiltrating CD4+ T cells with reversed CD4/CD8 ratios are highly correlated with rapid tumor growth and lymph node metastasis in cervical carcinoma. The regional immune escape is of prognostic importance with regard to cancer progression.     

Effector function of human tumor-specific CD8 T cells in melanoma lesions: a state of local functional tolerance

Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.     

Systemic and local human papillomavirus 16-specific T-cell immunity in patients with head and neck cancer

Squamous cell carcinomas of the head and neck (HNSCC), in particular those of the oropharynx, can be caused by human papilloma virus Type 16 (HPV16). Whereas these HPV-induced oropharyngeal carcinomas may express the HPV16 E6 and E7 oncoproteins and are associated with better survival, the nonvirally induced HNSCC are associated with overexpression of p53. In this study we assessed the presence of systemic and local T cells reactive against these oncoproteins in HNSCC. An exploratory study on the presence, type and function of HPV16- and/or p53-specific T cells in the blood, tumor and/or metastatic lymph node as measured by several immune assays was performed in an unselected group of 50 patients with HNSCC. Tumor tissue was tested for HPV DNA and the overexpression of p53 protein. Almost all HPV16+ tumors were located in the oropharynx. Circulating HPV16- and p53-specific T cells were found in 17/47 and 7/45 tested patients. T cells were isolated from tumor cultures and/or lymph nodes of 20 patients. HPV16-specific T cells were detected in six of eight HPV+ tumors, but in none of the 12 HPV-tumors. Tumor-infiltrating p53-specific T cells were not detected. In depth analysis of the HPV16-specific T-cell response revealed that this response comprised a broad repertoire of CD4+ T-helper Type 1 and 2 cells, CD4+ regulatory T cells and CD8+ T cells reactive to HPV16. The local presence of HPV16-specific T-cell immunity in HPV16-induced HNSCC implicates a role in the antitumor response and support the development of immunotherapy for HNSCC.     

Immune activation status of CD8+ T cells infiltrating non-small cell lung cancer

In a variety of human cancers, the presence of tumor-infiltrating T lymphocytes (TILs) is associated with tumor regression and favorable prognosis. Local interferon (IFN)-gamma secretion from activated T cells is supposed to induce a specific immune response leading to tumor-specific cytotoxicity. Nonetheless, significance and properties of TILs still remains controversial in lung cancer patients. We determined CD8+ T cell counts in 31 patients with non-small cell lung cancer (NSCLC) by immunohistochemistry, and assessed T-cell immune activation status in a subset of patients by measuring IFN-gamma mRNA expression by quantitative PCR (TaqMan). Semi-quantitative immunohistochemical analysis revealed significantly higher CD8+ T cell counts within the tumor as when compared to the invasive margin. CD8+ T cells immune activation status, represented in the IFN-gamma/CD8 mRNA ratio, correlated with the median number of CD8+ T cells presented at the tumor-host interface. Neither tumor histology and grade, nor CD8+ T cell counts and IFN-gamma/CD8 ratio could demonstrate an influence on overall survival in these patients. Our results indicate that CD8+ T cells infiltrating the tumor cell nests may be inadequately activated and thus incapable of mounting an effective anti-tumor immune response.     

Nodal metastasis in cervical cancer occurs in clearly delineated fields of immune suppression in the pelvic lymph catchment area

In cervical cancer, high frequencies of regulatory T cells (Tregs) and immunosuppressive PD-L1⁺CD14⁺ antigen-presenting cells dominate the microenvironment of tumor-positive lymph nodes (LN+). It is unknown whether this is restricted to LN+ or precedes metastasis, emanating from the primary tumor and spreading through tumor-draining lymph nodes (TDLNs). To investigate immunosuppression in the lymphatic basin of cervical tumors, all dissected TDLNs of five cervical cancer patients (in total 9 LN+ and 74 tumor-negative lymph nodes (LN−)) were analyzed for FoxP3⁺ Tregs, CD8⁺ T cells, HLA-DR⁺- and PD-L1⁺ myeloid cells by immunohistochemistry.Tregs and PD-L1⁺ cells were found to form an immunosuppressive cordon around metastatic tumor cells. Importantly, whereas high HLA-DR⁺- and PD-L1⁺ cell rates were strongly associated with LN+, elevated Treg levels and decreased CD8⁺ T cell/Treg ratios were found similar in LN+ and adjacent LN−, as compared to LN− at more distant anatomical localizations. These data suggest that delineated fields of Treg-associated immune suppression in anatomically co-localized TDLNs enable metastasis by creating metastatic niches. This may be of importance for decision-making regarding (surgical) intervention in cervical cancer. Future efforts should include the implementation of immunotherapeutic regimens to overcome this immune suppression, establish loco-regional control and halt systemic tumor spread.     

Induction of tumor-specific CD4+ and CD8+ T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine.

PURPOSE: The study aims to evaluate the effect of a human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides vaccine on the antigen-specific T-cell response in cervical cancer patients. EXPERIMENTAL DESIGN: Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51. HPV16-specific T-cell immune responses were analyzed by evaluating the magnitude, breadth, type, and polarization by proliferation assays, IFN gamma-ELISPOT, and cytokine production and phenotyped by the T-cell markers CD4, CD8, CD25, and Foxp3. RESULTS: Vaccine-induced T-cell responses against HPV16 E6 and E7 were detected in six of six and five of six patients, respectively. These responses were broad, involved both CD4(+) and CD8(+) T cells, and could be detected up to 12 months after the last vaccination. The vaccine-induced responses were dominated by effector type CD4(+)CD25(+)Foxp3(-) type 1 cytokine IFN gamma-producing T cells but also included the expansion of T cells with a CD4(+)CD25(+)Foxp3(+) phenotype. CONCLUSIONS: The HPV16 E6 and E7 synthetic long peptides vaccine is highly immunogenic, in that it increases the number and activity of HPV16-specific CD4(+) and CD8(+) T cells to a broad array of epitopes in all patients. The expansion of CD4(+) and CD8(+) tumor-specific T cells, both considered to be important in the antitumor response, indicates the immunotherapeutic potential of this vaccine. Notably, part of the vaccine-induced T cells display a CD4(+)CD25(+)Foxp3(+) phenotype that is frequently associated with regulatory T-cell function, suggesting that strategies to disarm this subset of T cells should be considered as components of immunotherapeutic modalities against HPV-induced cancers.     

肝癌荷瘤小鼠调节性T细胞数量的改变及其与肿瘤生长的关系

目的研究肝癌荷瘤小鼠调节性T细胞数量的改变及其与肿瘤生长的关系。方法采用小鼠肝癌细胞系H22接种BALB／c小鼠,建立肝癌模型;采用流式细胞术方法检测CD4^＋ CD25^＋T／CD4^＋T细胞的比例;以RT-PCR和流式细胞术检测Foxp3基因的表达。以免疫磁珠分选法纯化CD4^＋CD25^＋T和CD4^＋CD25^-T细胞;在体外,用3H-TdR掺入法检测T细胞的增殖情况;在体内,观察荷瘤小鼠来源的CD4^＋CD25^＋T细胞对肿瘤生长的作用。结果（1）荷瘤小鼠在引流淋巴结中,CD4^＋CD25^＋T细胞占CD4＋T细胞（18．80％±0．06％）比例增高,与对照组（9．50％±0．03％）相比,差异有统计学意义（P〈0．01）;在非引流淋巴结（LN）和脾脏（SP）中,荷瘤小鼠CD4^＋CD25^＋T／CD4^＋T比例分别为16．28％±0．02％和17．28％±0．06％,与对照组9．50％±0．03％和11．08％±0．04％相比,差异有统计学意义（P〈0．01,P〈0．05）;同时,调节性T细胞特异性标志Foxp3 mRNA的表达也升高。在同一只荷瘤小鼠中,引流淋巴结中CD4^＋CD25^＋T细胞数量（18．8％±0．06％）较对侧非引流淋巴结（16．28％±0．02％）略有升高,但差异无统计学意义（P〉0．05）。（2）从荷瘤小鼠中纯化的CD4^＋CD25^＋T细胞,在体外对抗CD3单抗的刺激无反应,但能抑制CD4^＋CD25^-T细胞的增殖。（3）CD4^＋CD25^＋T／CD4＋T比例与肿瘤大小呈正相关,并且可以抑制CD4^＋CD25^-T细胞的抗肿瘤效应。结论肝癌细胞在小鼠体内的生长可以提高调节性T细胞的数量,其数量的高低与肿瘤的大小呈正相关,提示清除调节性T细胞将是肿瘤免疫治疗的策略之一。     

The detection of circulating human papillomavirus‐specific T cells is associated with improved survival of patients with deeply infiltrating tumors

A detailed analyses of HPV‐specific immunity was performed in a large group of patients with HPV‐induced cervical cancer (CxCa) in relation to HLA‐types and prognostic factors. Patients were HLA‐typed and HPV16/18‐specific T‐cell immunity was assessed by proliferation assay and cytometric bead array using freshly isolated PBMC and by phenotypic analysis of HPV‐specific T cells. The results were analyzed in relation to known disease‐related HLA‐types (DR7, DR13, DR15/DQ06), invasion‐depth and size of tumor, lymph node (LN) status and disease free survival. In total 119 HLA‐typed patients with CxCa were analyzed. Patients expressing the HLA‐DR13 haplotype were underrepresented as compared to the Dutch population (p = 0.014), whereas HLA‐DR7 was overrepresented in patients with HPV16+ CxCa (p = 0.006). In 29 of 94 patients (31%) from whom blood could be tested, a proliferative response to HPV16/18 was detected, which was associated with increased numbers of HPV‐specific CD4+CD25+ (activated) T cells (p = 0.03) and HPV‐specific CD4+CD25+FoxP3‐positive T cells (p = 0.04). The presence of both FoxP3‐positive and negative HPV‐specific CD4+CD25+ T cells was significantly correlated (p = 0.01). Interestingly, the detection of HPV‐specific proliferation was associated with invasion depth (p = 0.020) but not with HLA type, tumor size nor LN status. Moreover, the detection of HPV‐specific immunity was associated with an improved disease free survival (p = 0.04) in patients with deeply infiltrating tumors. In conclusion, HPV‐specific proliferative T‐cell response, comprising higher percentages of HPV‐specific CD25+ and CD25+FoxP3‐positive CD4+T cells, are more frequently detected in patients with deep infiltrating CxCa tumors and associated with an improved survival.     

The T-cell receptor repertoire of tumor-infiltrating regulatory T lymphocytes is skewed toward public sequences

The accumulation of CD4(+) T regulatory cells (Treg) in tumor tissue is a widely described phenomenon in mouse models and in human cancer patients. Understanding the mechanisms by which Treg migrate and accumulate in tumors is important because they strongly influence the potential efficacy of many immunotherapies. In this study, we used immunoscope technology to analyze the T-cell receptor (TCR) repertoire of tumor-infiltrating T cells in non-TCR transgenic mice. Both tumor-infiltrating Tregs and T effector cells (Teff) displayed sequence profiles in the CDR3 region that were characteristic of biased repertoires seen during clonal cell expansions, implying that strong T-cell responses have occurred within the tumor tissue. By comparing the TCR sequences of tumor-infiltrating Tregs, we obtained evidence of the presence of so-called public TCR sequences that are common to many individuals yet were tumor-specific in nature. Such comparisons also suggested that the Treg-Teff conversion process is not an active process at the tumor site or tumor-draining lymph nodes. Our findings strongly suggest that Treg infiltration of tumor tissue is followed by marked proliferation of a few dominant T-cell clones in the tumor.     

Human papilloma virus specific T cells infiltrating cervical cancer and draining lymph nodes show remarkably frequent use of HLA-DQ and -DP as a restriction element

Human papillomavirus (HPV)-induced malignancies are frequently infiltrated by lymphocytes. To comprehend the contribution of HPV-specific T cells in this anti-tumor response we developed a method that allowed the analysis of the presence and specificity of cervix-infiltrating and draining lymph node resident T cells in a group of 74 patients with cervical malignancies, 54 of which were induced by HPV16 or HPV18. We detected the presence of HPV16 or HPV18-specific T cells in at least 23 of the 54 HPV-16 or -18 positive patients, and not in the 20 controls. Detailed studies resulted in the identification of 17 novel CD4+ and CD8+ T cell epitopes and their HLA-restriction elements, and also revealed that the HPV-specific immune response was aimed at both E6 and E7 and showed no preferential recognition of immunodominant regions. Unexpectedly, the vast majority of the CD4+ T cell epitopes were presented in the context of the less abundantly expressed HLA-DQ and HLA-DP molecules. Since the identified T cell epitopes constitute physiological targets in the immune response to HPV16 and HPV18 positive tumors they will be valuable for detailed studies on the interactions between the tumor and the immune system. This is crucial for the optimization of cancer immunotherapy in patients with pre-existing tumor-immunity.     

CD4+ T cells are able to promote tumor growth through inhibition of tumor-specific CD8+ T-cell responses in tumor-bearing hosts

Modulation of the immune response by established tumors may contribute to the limited success of therapeutic vaccination for the treatment of cancer compared with vaccination in a preventive setting. We analyzed the contribution of the CD4+ T-cell population to the induction or suppression of tumor-specific CD8+ T cells in a tumor model in which eradication of tumors crucially depends on CD8+ T cell-mediated immunity. Vaccine-mediated induction of protective antitumor immunity in the preventive setting (i.e., before tumor challenge) was CD4+ T cell dependent because depletion of this T-cell subset prevented CD8+ T-cell induction. In contrast, depletion of CD4+ cells in mice bearing established E1A+ tumors empowered the mice to raise strong CD8+ T-cell immunity capable of tumor eradication without the need for tumor-specific vaccination. Spontaneous eradication of tumors, which had initially grown out, was similarly observed in MHC class II-deficient mice, supporting the notion that the tumor-bearing mice harbor a class II MHC-restricted CD4+ T-cell subset capable of suppressing a tumor-specific CD8+ T-cell immune response. The deleterious effects of the presence of CD4+ T cells in tumor-bearing hosts could be overcome by CD40-triggering or injection of CpG. Together these results show that CD4+ T cells with a suppressive activity are rapidly induced following tumor development and that their suppressive effect can be overcome by agents that activate professional antigen-presenting cells. These observations are important for the development of immune interventions aiming at treatment of cancer.     

非小细胞肺癌患者引流区淋巴结中CD4+CD25+调节性T细胞的检测及临床意义

目的分析非小细胞肺癌(NSCLC)患者引流区淋巴结中CD4 CD25 调节性T细胞在淋巴结局部免疫抑制状态的形成以及在肺癌发生发展中的作用。方法手术切除53例NSCLC患者引流区淋巴结,采用双标记的间接免疫荧光法检测CD4 CD25 调节性T细胞数量,实时荧光定量逆转录-聚合酶链反应(RT-PCR)法检测细胞因子TGF-β1、IL-10的表达水平,常规免疫组化方法检测CD8 T细胞的数量。结果NSCLC患者引流区转移淋巴结中,CD4 CD25 调节性T细胞(28.80%±8.06%)明显高于未转移淋巴结(15.48%±4.66%,P<0.01)。随肺癌的进展,引流区淋巴结中CD4 CD25 调节性T细胞数量增多。在转移的纵隔淋巴结(N2)和肺内淋巴结(N1)中,CD4 CD25 调节性T细胞数量分别为32.58%±7.52%和22.76%±4.67%(P<0.01)。在进展期(Ⅲ)和早期(Ⅰ Ⅱ)NSCLC患者引流区淋巴结中,CD4 CD25 调节性T细胞数量分别为30.42%±7.47%和16.22%±4.88%(P< 0.01)。NSCLC患者引流区淋巴结中的CD4 CD25 调节性T细胞数量与其自身的CD8 T细胞的数量呈负相关(r=-0.756,P<0.001)。在NSCLC患者引流区淋巴结中,CD4 CD25 调节性T细胞数量与抑制性细胞因子TGF-β1和IL-10的表达水平呈正相关(TGF-β1:r=0.645,P<0.001;IL-10:r=0.769,P<0.001)。结论NSGLC患者引流区淋巴结的CD4 CD25 调节性T细胞数量与肺癌的发展密切相关。一方面,检测肺癌患者引流区淋巴结的免疫状况为评价NSCLC患者疾病的进展程度和预后提供了一个新的免疫学指标;另一方面,在NSCLC的生物治疗中,控制CD4 CD25 调节性T细胞数量,阻断其发挥免疫抑制作用,具有广阔的临床应用前景。     

Differential MHC class II component expression in HPV-positive cervical cancer cells: implication for immune surveillance

Effective eradication of human papillomavirus (HPV)-positive tumors may require CD8+ and CD4+ T-cell-mediated immune responses. Ectopic expression of MHC class II surface molecules has been described in the context of cervical cancer, but coexpression with other components of the MHC class II antigen presentation pathway has not been addressed. We have evaluated the MHC class II antigen presentation pathway in malignant squamous epithelium of HPV+ cervical cancer lesions by in situ costaining HLA-DR with CLIP or DMA/DMB. Cervical cancer cells exhibit 3 MHC class II phenotypes: (i) DR+/CLIP+ or DM+; (ii) DR+/CLIP- or DM-; and (iii) DR-/CLIP+ or DM+. The identical profile has been identified in HPV+ ME180 cells, which serve as a target for HLA-DR4-restricted and HPV68, E7-specific CD4+ T cells. IFN-gamma pretreatment of ME180 cells, associated with differential trafficking of MHC class II molecules, is necessary for effective T-cell recognition. Although proinflammatory cytokines may facilitate MHC class II-restricted antigen recognition in tumor cells, different phenotypes of the MHC class II antigen presentation pathway may be associated with evasion from CD4+-mediated cellular immune responses.     

Tumor-specific CD8+ T cell reactivity in the sentinel lymph node of GM-CSF-treated stage I melanoma patients is associated with high myeloid dendritic cell content.

PURPOSE: Impaired immune functions in the sentinel lymph node (SLN) may facilitate early metastatic events during melanoma development. Local potentiation of tumor-specific T cell reactivity may be a valuable adjuvant treatment option. EXPERIMENTAL DESIGN: We examined the effect of locally administered granulocyte/macrophage-colony stimulating factor (GM-CSF) on the frequency of tumor-specific CD8+ T cells in the SLN and blood of patients with stage I melanoma. Twelve patients were randomly assigned to preoperative local administration of either recombinant human GM-CSF or NaCl 0.9%. CD8+ T cells from SLN and peripheral blood were tested for reactivity in an IFNgamma ELISPOT assay against the full-length MART-1 antigen and a number of HLA-A1, HLA-A2, and HLA-A3-restricted epitopes derived from a range of melanoma-associated antigens. RESULTS: Melanoma-specific CD8+ T cell response rates in the SLN were one of six for the control group and four of six for the GM-CSF-administered group. Only one patient had detectable tumor-specific CD8+ T cells in the blood, but at lower frequencies than in the SLN. All patients with detectable tumor-specific CD8+ T cells had a percentage of CD1a+ SLN-dendritic cells (DC) above the median (i.e., 0.33%). This association between above median CD1a+ SLN-DC frequencies and tumor antigen-specific CD8+ T cell reactivity was significant in a two-sided Fisher's exact test (P = 0.015). CONCLUSIONS: Locally primed antitumor T cell responses in the SLN are detectable as early as stage I of melanoma development and may be enhanced by GM-CSF-induced increases in SLN-DC frequencies.     

Circulating human papillomavirus type 16 specific T-cells are associated with HLA Class I expression on tumor cells, but not related to the amount of viral oncogene transcripts

Human papillomavirus (HPV) is a necessary factor in the pathogenesis of cervical cancer. Circulating HPV-specific T-cells responding to the E6 and E7 HPV proteins can be detected only in half of cervical cancer patients. Potential explanations for the absence of this response are lack of sufficient amounts of antigen to activate the immune response or local immune escape mechanisms. We studied the relationship between HPV 16 E6/E7 oncogene mRNA expression, human leukocyte antigen (HLA) expression on tumor cells and the presence of circulating E6- and E7-specific T-cell responses in cervical cancer patients. The amount of antigen was assessed by HPV E6/E7 mRNA expression levels measured by quantitative polymerase chain reaction. HLA Class I and Class II expression on tumor cells was analyzed by immunohistochemistry. A proliferative HPV-specific T-cell response was detected in 15/29 patients. The amount of HPV E6/E7 mRNA was not related to the presence of immune response. HLA Class I expression was downregulated in 19 patients and completely lost in 7 patients. HLA Class II expression was upregulated in 18 patients. HLA Class I expression on tumor cells showed a strong correlation with immunity (p = 0.001). Explicitly, all patients with complete HLA loss lacked HPV specific T-cell responses. The presence of circulating HPV-specific T-cells might reflect ongoing antitumor response that is sustained by CD8+ T-cells killing HLA Class I positive cancer cells. We hypothesize that HLA Class I expression status on tumor cells might as well influence the response to HPV E6/E7 directed immunotherapy.     

Human autologous tumor-specific T cells in malignant melanoma

T cell lines and clones with autologous tumor-specific activity have been developed in malignant melanoma by stimulating peripheral blood lymphocytes (PBL), lymph node lymphocytes or tumor-infiltrating lymphocytes (TIL) with autologous melanoma cells in the presence of recombinant interleukin 2 (rIL2). T-cell lines and clones have been developed with specific cytotoxicity and/or proliferative responses for autologous melanoma targets but not for allogeneic melanoma tumor cells, autologous normal cells or natural killer (NK)-sensitive targets. The concentration of rIL2 is critical for the generation of autologous tumor-specific T-cell lines, with low rIL2 concentrations (up to 800 IU/ml) facilitating the growth of T-cell lines with tumor-specific activity. The T-cell receptor (TCR) and the CD3 antigen are involved in specific cytotoxicity and/or proliferative responses of these T-cell lines and clones. An oligoclonal pattern of -chain TCR gene rearrangements was observed on T-cell lines and clones with autologous tumor-specific cytotoxicity, suggesting that they are comprised of T cells that have undergone a clonal expansion in response to particular antigen. Autologous tumor-specific cytotoxic T cells are HLA-restricted and recognize on the melanoma tumor cells HLA Class I or possibly Class II antigens plus a tumor-specific determinant. TIL from patients with metastatic melanoma have unique characteristics in comparison with PBL and lymph node lymphocytes and they appear to contain substantial proportions of T cells that have been locally sensitized to autologous tumor cells. Single stimulation of TIL with autologous tumor cells in the presence of rIL2 is sufficient for the generation of T cell lines with autologous tumor-specific activity, whereas, multiple stimulation of PBL and lymph node lymphocytes was required to achieve the same purpose. TIL-derived T cell lines have been expanded in rIL2 in vitro by at least 1,500-fold without losing their activity. Approximately, 40% of the patients exhibited complete or partial responses to adoptive immunotherapy with melanoma TIL and rIL2.     

Anti-4-1BB monoclonal antibody enhances rejection of large tumor burden by promoting survival but not clonal expansion of tumor-specific CD8+ T cells

Anti-4-1BB monoclonal antibody (mAb) has been shown to induce antitumor immunity by a CD4/CD8-dependent mechanism, but its direct effect on tumor-specific CD8+ T cells in tumor rejection is unclear. Here we used transgenic CD8+ T cells against the unmutated tumor rejection antigen P1A to analyze whether this mAb can promote CD8+ T-cell function against large tumors in the absence of CD4+ T-helper cells. RAG-2(-/-) mice were challenged with P1A-expressing plasmacytoma J558. Once tumor size reached a diameter of 0.85-1.75 cm, mice were treated with P1A-specific CD8+ CTL (P1CTL) in conjunction with anti-4-1BB mAb or control IgG. All of the mice showed a partial regression of tumor, but mice treated with anti-4-1BB mAb exhibited markedly enhanced tumor rejection, delayed tumor progression, and prolonged survival. Correspondingly, we observed a substantial increase in the number of P1CTL in anti-4-1BB mAb-treated mice. Surprisingly, anti-4-1BB mAb did not accelerate division of the tumor-specific CD8+ T cells, and the increase in tumor-specific T-cell number was due to reduced activation-induced cell death. These results indicate that anti-4-1BB mAb can promote CD8+ T cell-mediated protection against large tumors in the absence of CD4+ T-cell help by promoting P1CTL survival without increasing initial clonal expansion.     

Glucocorticoid-induced tumor necrosis factor receptor stimulation enhances the multifunctionality of adoptively transferred tumor antigen-specific CD8+ T cells with tumor regression

We have reported for the first time the significance of effector T-cell multifunctionality in antitumor immunity, suggesting that the appearance of multifunctional/polyfunctional tumor-specific CD8⁺ T cells in vivo is a critical determinant of the success of antitumor immunotherapy, and a strategy to induce multifunctionality in effector cells is required for the successful immunotherapy of hosts with progressing tumor. Glucocorticoid-induced tumor necrosis factor receptor (GITR) stimulation has been shown to enhance antitumor immune response. However, its functional impact on adoptively transferred T cells remains unclear. Here, we analyzed the impact of GITR stimulation in vivo on the functional profiles of adoptively transferred CD8⁺ T cells specific for murine fibrosarcoma CMS5. GITR stimulation was found to enhance multifunctionality (interferon (IFN)-γ and tumor necrosis factor (TNF)-α production and CD107a mobilization as a degranulation marker) in transferred cells at the single-cell level. These cells exhibited upregulated expression of CD25 in draining lymph nodes and increased infiltration in tumor. Mice that received T-cell therapy with GITR stimulation showed reduced Foxp3⁺CD4⁺ T cells among tumor infiltrating lymphocytes and increased in vivo cytotoxic T lymphocytes (CTL) activity even with progressing tumor, resulting in enhanced tumor regression. These data strengthen the idea that effector T-cell multifunctionality is a sensitive immune correlate for successful immunotherapy against malignancy and provide an immunological rationale for effective T-cell therapy combined with GITR stimulation. ( Cancer Sci 2009; 100: 1317–1325)