Vaccination with tumor lysate-pulsed dendritic cells elicits antigen-specific, cytotoxic T-cells in patients with malignant glioma

The primary goal of this Phase I study was to assess the safety and bioactivity of tumor lysate-pulsed dendritic cell (DC) vaccination to treat patients with glioblastoma multiforme and anaplastic astrocytoma. Adverse events, survival, and cytotoxicity against autologous tumor and tumor-associated antigens were measured. Fourteen patients were thrice vaccinated 2 weeks apart with autologous DCs pulsed with tumor lysate. Peripheral blood mononuclear cells were differentiated into phenotypically and functionally confirmed DCs. Vaccination with tumor lysate-pulsed DCs was safe, and no evidence of autoimmune disease was noted. Ten patients were tested for the development of cytotoxicity through a quantitative PCR-based assay. Six of 10 patients demonstrated robust systemic cytotoxicity as demonstrated by IFN-gamma expression by peripheral blood mononuclear cells in response to tumor lysate after vaccination. Using HLA-restricted tetramer staining, we identified a significant expansion in CD8+ antigen-specific T-cell clones against one or more of tumor-associated antigens MAGE-1, gp100, and HER-2 after DC vaccination in four of nine patients. A significant CD8+ T-cell infiltrate was noted intratumorally in three of six patients who underwent reoperation. The median survival for patients with recurrent glioblastoma multiforme in this study (n = 8) was 133 weeks. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous tumor lysate-pulsed DC vaccine for patients with malignant glioma. We demonstrate for the first time the ability of an active immunotherapy strategy to generate antigen-specific cytotoxicity in brain tumor patients.     

Tumor lysate and IL-18 loaded dendritic cells elicits Th1 response,tumor-specific CD8+ cytotoxic T cells in patients with malignant glioma

In this study, we demonstrate that tumor lysate-loaded dendritic cells can elicit a specific CD8+ cytotoxic T lymphocyte response against autologous tumor cells in patients with malignant glioma. CTL from three of five patients expressed strong cytolytic activity against autologous glioma cells, did not lyse autologous lymphoblasts and were variably cytotoxic against the LAK-sensitive cell line Daudi. Also, DCs pulsed normal brain lysate failed to induce cytolytic activity against autologous glioma cells, suggesting the lack of autoimmune response. Two of five patients CD8+ T cells expressed a modest cytotoxicity against autologous glioma cells. CD8+ T cells isolated during these ineffective primings secreted large amounts of IL-10, less amounts of IFN-γ as detected by ELISA, Type 2 bias in the CD8+ T cell response accounts for the lack of cytotoxic effector function from these patients. Cytotoxicity against autologous glioma cells could be significantly inhibited by anti-HLA class I antibody. These data demonstrate that tumor lysate-loaded DC can be an effective tool in inducing glioma-specific CD8+ CTL able to kill autologous glioma cells in vitro. However, high levels of tumor specific tolerance in some patients may account for a significant barrier to therapeutic vaccination. Moreover, cytotoxic responses were augmented by transfecting DC with the gene for IL-18. For all five patients, CD8+T cells treated with IL18 transfected DC produced Th1 response. These results may have important implications for the treatment of malignant glioma patients with immunotherapy. DCs loaded with total tumor lysate and IL-18 may represent a method for inducing Th1 immunoresponses against the entire repertoire of glioma antigens.     

Induction and clonal expansion of tumor-specific cytotoxic T lymphocytes from renal cell carcinoma patients after stimulation with autologous dendritic cells loaded with tumor cells

Melanoma and renal cell carcinoma (RCC) are considered to be the most immunogenic tumors in humans. To generate conditions to induce primary T-cell responses against RCC and to allow further expansion of tumor-specific cytotoxic T lymphocytes (CTL) for adoptive transfer, peripheral blood mononuclear cells from RCC patients were stimulated with primary autologous tumor cells or monocyte-derived dendritic cells (DC) loaded with either tumor lysate (TU-LY) or apoptotic tumor cells (TU-AP). Whereas repetitive stimulation (4x) with tumor cells alone induced a predominant population of CD3(-) natural killer cells, 4 weeks of stimulation with tumor-loaded DC favored induction and expansion of CD4+ T cells (>80%). However, 2 weekly stimulation cycles with tumor-loaded DC followed by restimulation with autologous irradiated tumor cells alone were optimal for induction of tumor-specific CTL responses in vitro. Using these culture conditions a marked increase of CD4+ T cells was observed during the first 2 weeks of stimulation with tumor-loaded DC. Subsequent restimulation with autologous tumor cells alone gave rise to 500-fold expansion of CD8+ T cells. These CD8+ T cells were shown to exhibit strong major histocompatibility complex class I-restricted cytotoxic activity against the autologous tumor. Comparison of TU-LY and TU-AP as a source of tumor antigen for loading DC did not show any difference in stimulating tumor-specific CTL. Length pattern analysis of the complementary determining region 3 (CDR3) of the T-cell receptor Vbeta chain revealed expansion of oligoclonal CTL populations with outgrowth of 1 or 2 clones after prolonged stimulation with autologous tumor cells. Our study demonstrated an efficient method for generating tumor-specific CTL in vitro that may be used to identify tumor cell antigens or that can be expanded for adoptive T-cell transfer in tumor immunotherapy.     

CD4+ T helper responses in squamous cell carcinoma of the head and neck

Anti-tumor immunity plays an important role in the development of and protection from malignancy. However, there is a lack of information regarding induction of CD4+ T helper responses in patients with squamous cell carcinoma (SCCHN). To explore anti-tumor immune responses against SCCHN, a permanent cell line, Gun-1 was established from a squamous cell carcinoma of the hypopharynx. In addition to its characterization, we performed mixed lymphocyte-tumor cell cultures (MLTC) using peripheral blood lymphocytes and autologous tumor cells. Furthermore, T cell responses to wild type (wt) p53-derived peptides were assessed. Gun-1 cells overexpressed p53 and were negative for HLA-A2 expression. No tumor-specific or wt p53-specific CD8+ CTL lines could be established from peripheral blood mononuclear cells (PBMCs) of this patient. Autologous tumor-specific HLA-DR-restricted CD4+ T helper clone was obtained by limiting dilutions using bulk populations from MLTC. This clone produced IFN-gamma but not IL-5 in response to autologous tumor cells. In addition, CD4+ T cells were generated from the patient's PBMCs which responded to two HLA-DP5-restricted wt p53-derived peptides. Our results suggest that the immune cells specific for autologous tumor as well as wt p53-derived epitopes are present in the peripheral circulation of this cancer patient. However, helper-type CD4+ T lymphocytes represent the predominant anti-tumor response.     

A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced cancer.

PURPOSE: The objectives of this study were to assess the toxicity and immunological response induced by the intradermal (i.d) administration of tumor lysate-pulsed dendritic cells (DCs). EXPERIMENTAL DESIGN: Patients with stage IV solid malignancies were treated in cohorts that received 10(6), 10(7), and 10(8) DCs i.d. every 2 weeks for three vaccines. Each vaccine was composed of a mixture of half DCs pulsed with autologous tumor lysate and the other half with keyhole limpet hemocyanin (KLH). Peripheral blood mononuclear cells (PBMCs) harvested 1 month after the last immunization was compared with pretreatment PBMCs for immunological response. Delayed-type hypersensitivity reactivity to tumor antigen and KLH was also assessed. RESULTS: Fourteen patients received all three vaccines and were evaluable for toxicity and/or immunological monitoring. There were no grade 3 or 4 toxicities associated with the vaccines or major evidence of autoimmunity. Local accumulation of CD4(+) and CD8(+) T cells were found at the vaccination sites. There was a significant proliferative response of PBMCs to KLH induced by the vaccine. In 5 of 6 patients, the vaccine resulted in increased IFN-gamma production by PBMCs to KLH in an ELISPOT assay. Using the same assay, 3 of 7 patients' PBMCs displayed increased IFN-gamma production in response to autologous tumor lysate. One patient with melanoma also was observed to have an increased frequency of MART-1- and gp100-reactive CD8(+) T cells after vaccination. By delayed-type hypersensitivity testing, 8 of 9 and 4 of 10 patients demonstrated reactivity to KLH and autologous tumor, respectively. Two patients with melanoma experienced a partial and a minor response, respectively. CONCLUSION: The administration of tumor lysate-pulsed DCs is nontoxic and capable of inducing immunological response to tumor antigen. Additional studies are necessary to improve tumor rejection responses.     

EphA2 as a glioma-associated antigen: a novel target for glioma vaccines

EphA2 is a receptor tyrosine kinase and is frequently overexpressed in a wide array of advanced cancers. We demonstrate in the current study that the EphA2 protein is restrictedly expressed in primary glioblastoma multiforme and anaplastic astrocytoma tissues in comparison to normal brain tissues. To evaluate the possibility of targeting EphA2 in glioma vaccine strategies, we stimulated human leukocyte antigen (HLA) A2+ peripheral blood mononuclear cells (PBMCs) obtained from healthy donors and glioma patients with autologous dendritic cells (DCs) loaded with synthetic EphA2883-891 peptide (TLADFDPRV), which has previously been reported to induce interferon-gamma in HLA-A2+ PBMCs. Stimulated PBMCs demonstrated antigen-specific cytotoxic T lymphocyte (CTL) responses as detected by specific lysis of T2 cells loaded with the EphA2883 peptide as well as HLA-A2+ glioma cells, SNB19 and U251, that express EphA2. Furthermore, in vivo immunization of HLA-A2 transgenic HHD mice with the EphA2883-891 peptide resulted in the development of an epitope-specific CTL response in splenocytes, despite the fact that EphA2883-891 is an autoantigen in these mice. Taken together, these data suggest that EphA2883-891 may be an attractive antigen epitope for molecularly targeted glioma vaccines.     

Identification of a novel HLA-A*0201-restricted, cytotoxic T lymphocyte epitope in a human glioma-associated antigen, interleukin 13 receptor alpha2 chain.

PURPOSE: Interleukin 13 receptor alpha2-chain (IL-13Ralpha2) has been reported to be abundantly and specifically overexpressed in glioblastoma multiforme. Here we report the identification of a CTL epitope derived from the IL-13Ralpha2. EXPERIMENTAL DESIGN: Mature dendritic cells (DCs) were pulsed with each of the synthetic peptides that were designed, based on a binding affinity-based prediction and a proteosomal cleavage site prediction system, and used to stimulate autologous CD8+ T cells from an HLA-A2+ healthy donor. After four to six cycles of restimulation, the immunoreactivity of the T cells was analyzed for specific IFN-gamma production and CTL reactivity. RESULTS: Of the five peptides tested, IL-13Ralpha(345-354) (WLPFGFILI) induced a CD8(+) T-cell line that specifically produced IFN-gamma in response to HLA-A2+ T2 cells pulsed with the relevant peptide and lysed these cells. Peptide titration assays demonstrated that half-maximal lysis of IL-13Ralpha(345-354) peptide-reactive CD8(+) T cells required peptide loading concentration of approximately 5 nM. Perhaps most importantly, this CD8(+) T-cell line also displayed lytic activity against the HLA-A2+ human glioma cell lines that express IL-13Ralpha2. CONCLUSIONS: This novel CTL epitope may therefore serve as an attractive component of peptide-based vaccines to treat glioma and as a surrogate marker of T-cell immune responses in patients before and after therapy.     

负载自体肿瘤细胞裂解物的DC疫苗联合CIK治疗晚期肾癌的临床观察--附10例报告

背景与目的:肾癌的主要治疗手段是手术,但晚期肾癌术后复发率高,加上肾癌对化疗和放疗不敏感,因此,晚期肾癌预后不佳,需要寻找新的更有效的治疗方法。本研究通过负载自体肿瘤细胞裂解物的树突细胞(dendriticcells,DC)疫苗联合细胞因子诱导的杀伤细胞(cytokine-inducedkillercells,CIK)治疗10例晚期肾癌,观察近期的临床疗效,免疫学反应及副作用。方法:分离患者外周血单核细胞,体外经GM-CSF和IL-4诱导产生DC细胞,并负载自体肿瘤细胞裂解物;T淋巴细胞经IFN-γ、IL-2、CD3单抗、IL-1α体外诱导产生CIK细胞。所有患者在切除原发病灶后,接受每周一次的皮内DC疫苗注射治疗,至少8次治疗;CIK细胞过继细胞免疫治疗,每2周一次,至少接受4次治疗。临床疗效和免疫学反应分别通过影象学检查,外周血T淋巴细胞亚群改变和迟发性超敏(delayed-typehypersensitivity,DTH)反应进行评估。结果:(1)4例有可评价病灶的患者中1例部分缓解(PR),2例疾病稳定(SD),1例进展(PD);6例没有可评价病灶的患者中1例PD,1例失访,另外4例未见疾病进展。随访时间6～20个月(中位时间11个月)。(2)与治疗前比较,治疗2个月后患者CD3 、CD4 、CD4 /CD8 、CD56 明显升高(P<0.05)。(3)包括PR患者在内的6例患者DTH反应呈现阳性。(4)除一过性的发热、畏寒外没有其它不良反应出现。结论:负载自体肿瘤细胞裂解物的DC疫苗联合CIK细胞治疗晚期肾癌有一定的近期临床疗效,能诱导出特异的抗肾癌免疫反应,并且有良好的耐受性。     

Vaccination of recurrent glioma patients with tumour lysate-pulsed dendritic cells elicits immune responses: results of a clinical phase I/II trial

In this Phase I/II trial, the patient's peripheral blood dendritic cells were pulsed with an autologous tumour lysate of the glioma. Seven patients with glioblastoma and three patients with anaplastic glioma, ranging in age from 20 to 69 years, participated in this study. The mean numbers of vaccinations of tumour lysate-pulsed dendritic cells were 3.7 times intradermally close to a cervical lymph node, and 3.2 times intratumorally via an Ommaya reservoir. The percentage of CD56-positive cells in the peripheral blood lymphocytes increased after immunisation. There were two minor responses and four no-change cases evaluated by radiological findings. Dendritic cell vaccination elicited T-cell-mediated antitumour activity, as evaluated by the ELISPOT assay after vaccination in two of five tested patients. Three patients showed delayed-type hypersensitivity reactivity to the autologous tumour lysate, two of these had a minor clinical response, and two had an increased ELISPOT result. Intratumoral CD4+ and CD8+ T-cell infiltration was detected in two patients who underwent reoperation after vaccination. This study demonstrated the safety and antitumour effects of autologous tumour lysate-pulsed dendritic cell therapy for patients with malignant glioma.     

Generation of in vitro B-CLL specific HLA class I restricted CTL responses using autologous dendritic cells pulsed with necrotic tumor lysate

New approaches in the treatment of chronic B lymphocytic leukemia (B-CLL) have led to improved clinical response rates. In this setting there is a need to evaluate novel therapeutic approaches that aim to eradicate minimal residual B-CLL cells following an initial favorable response. The use of tumor lysate-pulsed dendritic cells (DC) represents a potentially important development in the field of cancer vaccination. B-CLL is ideally suited for DC-based vaccination since tumor cells are readily available (peripheral blood) and both known (tumor idiotype) and unknown antigens can be exploited to stimulate immune responses. In the current study we have evaluated the ability to stimulate in vitro autologous immune reactivity against target B-CLL cells using autologous DCs pulsed with B-CLL tumor lysate. Enhanced specific T cell IFN-gamma expression was detected in 9 of 14 patients evaluated. These responses were specific with increased levels of IFN-gamma mRNA measurable in T-cells stimulated with NC-DCs and not unpulsed DCs or DCs pulsed with normal B cell lysate. CTLs demonstrating increased levels of IFN-gamma mRNA also lysed autologous B-CLL targets cells in an MHC class 1-restricted manner by (51)chromium release assay. Priming target leukemic cells with CD40 ligand and IL-4 enhanced CTL killing. The effector CTL displayed negligible toxicity against NK susceptible target cells K-562 and spared CD19(+)CD5(-) normal B cells in cytotoxicity assays. The specificity of the CTL response was confirmed by blocking HLA class I molecules and cold target inhibition assays.     

肺癌肿瘤全溶物脉冲自体树突状细胞体外诱生T细胞抗肿瘤反应的研究

目的　探讨非小细胞肺癌 (NSCLC)患者全瘤溶解物 (WTL)冲击的自体树突状细胞 (DCs)瘤苗体外诱生T细胞介导的抗肿瘤反应。方法　在含重组人粒细胞 巨噬细胞 集落刺激因子 (rhGM CSF)和重组人白细胞介素 4 (rhIL 4 )的培养条件下 ,以 10例患者的贴壁外周血单核细胞 (PBMCs)衍生不成熟的树突状细胞 (imDCs) ,分别添加肿瘤坏死因子 (TNFα)、自体WTL或WTL TNFα诱导成熟 ,即分别为DCs/TNF、DCs/WTL和 DCs/WTL ,用流式细胞仪和混合淋巴细胞反应 (MLR) ,检测细胞表面分子的表达变化及其刺激异基因或同基因的T细胞增殖能力。将 DCs/WTL和自体T细胞共培养1～ 2周 ,以诱导细胞毒性T淋巴细胞 (CTL) ,用计数γ 干扰素 (IFN γ)释放的酶联免疫斑点 (ELISPOT)试验和乳酸脱氢酶 (LDH)释放的细胞毒试验 ,分别检测该CTL中识别自体肿瘤细胞释放特异性IFN γ的T细胞数和溶瘤活性。结果　以 30 μg/ml蛋白的WTL体外冲击自体 10 6imDCs,可导致上调DC表型 ,包括CD1a、CD83和CD86以及HLA DR的分子表达 ,与常规TNFα诱导成熟的DCs表型相似。虽然两者均能显著刺激异基因T细胞的增殖 ,但其刺激自体T淋巴细胞的增殖能力 ,DCs/WTL却显著高于DCs/TNF(P <0 .0 5 )。将体外与 DCs/WTL共培养的自体T细胞作为反应或效应细胞 ,再次     

Dendritic cell vaccination in glioblastoma patients induces systemic and intracranial T-cell responses modulated by the local central nervous system tumor microenvironment.

PURPOSE: We previously reported that autologous dendritic cells pulsed with acid-eluted tumor peptides can stimulate T cell-mediated antitumor immune responses against brain tumors in animal models. As a next step in vaccine development, a phase I clinical trial was established to evaluate this strategy for its feasibility, safety, and induction of systemic and intracranial T-cell responses in patients with glioblastoma multiforme. EXPERIMENTAL DESIGN: Twelve patients were enrolled into a multicohort dose-escalation study and treated with 1, 5, or 10 million autologous dendritic cells pulsed with constant amounts (100 mug per injection) of acid-eluted autologous tumor peptides. All patients had histologically proven glioblastoma multiforme. Three biweekly intradermal vaccinations were given; and patients were monitored for adverse events, survival, and immune responses. The follow-up period for this trial was almost 5 years. RESULTS: Dendritic cell vaccinations were not associated with any evidence of dose-limiting toxicity or serious adverse effects. One patient had an objective clinical response documented by magnetic resonance imaging. Six patients developed measurable systemic antitumor CTL responses. However, the induction of systemic effector cells did not necessarily translate into objective clinical responses or increased survival, particularly for patients with actively progressing tumors and/or those with tumors expressing high levels of transforming growth factor beta(2) (TGF-beta(2)). Increased intratumoral infiltration by cytotoxic T cells was detected in four of eight patients who underwent reoperation after vaccination. The magnitude of the T-cell infiltration was inversely correlated with TGF-beta(2) expression within the tumors and positively correlated with clinical survival (P = 0.047). CONCLUSIONS: Together, our results suggest that the absence of bulky, actively progressing tumor, coupled with low TGF-beta(2) expression, may identify a subgroup of glioma patients to target as potential responders in future clinical investigations of dendritic cell-based vaccines.     

Dendritic cells, pulsed with lysate of allogeneic tumor cells, are capable of stimulating MHC-restricted antigen-specific antitumor T cells

A variety of approaches have been used to deliver tumor-associated antigens (TAA) in conjunction with dendritic cells (DC) as cellular adjuvants. DC derived from monocytic precursors have been pulsed with whole tumor antigen using a variety of strategies and have been demonstrated to induce CD4+ and CD8+ antitumor responses. In the present study, monocyte-derived DC have been pulsed with lysate from an allogeneic melanoma cell line, A-375, and used to repeatedly stimulate T cells. The resultant T cells were examined for cytotoxic activity against A-375 targets as well as the HLA A2-positive melanoma cell line DFW. Uptake of FITC-labeled melanoma lysate by DC established that lysate of melanoma cells was efficiently endocytosed. Stimulation with lysate-pulsed DC resulted in strong proliferative responses by T cells, which could be inhibited by antibodies against both MHC class I and class II. T cells stimulated in vitro with lysate-pulsed DC demonstrated potent cytotoxicity against the melanoma targets which were blocked by antibodies against MHC class I. Lysate-pulsed DC also elicited IFN-gamma secretion by T cells as measured in an ELISPOT assay. We have also examined the ability of lysate-pulsed DC to present melanoma-associated antigens to T cells. ELISPOT assays with synthetic peptides of melanoma-associated antigens, such as gp100, mage1, NY-ESO, and MART-1, revealed that lysate-pulsed DC could stimulate T cells in an antigen-specific manner. The results demonstrate that lysate from allogeneic tumor cells may be used as a source of antigens to stimulate tumor-specific T cells in melanoma.     

MAGE-6 encodes HLA-DRbeta1*0401-presented epitopes recognized by CD4+ T cells from patients with melanoma or renal cell carcinoma.

CD4+ T cells modulate the magnitude and durability of CTL responses in vivo and may serve as potent effector cells within the tumor microenvironment. The current study was undertaken to define novel epitopes from the broadly expressed tumor antigen MAGE-6 that are recognized by CD4+ T cells. We have combined the use of a HLA-DR4/peptide binding algorithm with the IFN-gamma enzyme-linked immunospot assay to identify four nonoverlapping sequences derived from the MAGE-6 protein that served as CD4+ T-cell epitopes in HLA-DR4+ donors. Strikingly, patients with active melanoma or renal cell carcinoma failed to secrete IFN-gamma in response to MAGE-6-derived epitopes, whereas both normal donors and cancer patients with no current evidence of disease were responsive, particularly after short-term in vitro stimulations with peptide-pulsed dendritic cells. Importantly, peptide-specific CD4+ T cells also recognized HLA-DRbeta1*0401+ tumor cells that constitutively expressed the MAGE-6 protein and autologous HLA-DRbeta1*0401+ dendritic cells transfected with MAGE-6 cDNA-elicited CD4+ T cells that reacted against individual peptide epitopes in vitro. These data suggest that MAGE-6-derived epitopes could serve as useful vaccine candidate components and may provide an immune-monitoring index of clinically important Th1-type immunity in patients with renal cell carcinoma or melanoma.     

Vaccination of malignant glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration

In this Phase I trial, patients' peripheral blood dendritic cells were pulsed with peptides eluted from the surface of autologous glioma cells. Three biweekly intradermal vaccinations of peptide-pulsed dendritic cells were administered to seven patients with glioblastoma multiforme and two patients with anaplastic astrocytoma. Dendritic cell vaccination elicited systemic cytotoxicity in four of seven tested patients. Robust intratumoral cytotoxic and memory T-cell infiltration was detected in two of four patients who underwent reoperation after vaccination. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous peptide-pulsed dendritic cell vaccine for patients with malignant glioma.     

Dendritic cells fused with allogeneic colorectal cancer cell line present multiple colorectal cancer-specific antigens and induce antitumor immunity against autologous tumor cells.

The aim of antitumor immunotherapy is to induce CTL responses against autologous tumors. Previous work has shown that fusion of human dendritic cells and autologous tumor cells induce CTL responses against autologous tumor cells in vitro. However, in the clinical setting of patients with colorectal carcinoma, a major difficulty is the preparation of sufficient amounts of autologous tumor cells. In the present study, autologous dendritic cells from patients with colorectal carcinoma were fused to allogeneic colorectal tumor cell line, COLM-6 (HLA-A2(-)/HLA-24(-)), carcinoembryonic antigen (CEA)(+), and MUC1(+) as an alternative strategy to deliver shared colorectal carcinoma antigens to dendritic cells. Stimulation of autologous T cells by the fusion cells generated with autologous dendritic cells (HLA-A2(+) and/or HLA-A24(+)) and allogeneic COLM-6 resulted in MHC class I- and MHC class II-restricted proliferation of CD4(+) and CD8(+) T cells, high levels of IFN-gamma production in both CD4(+) and CD8(+) T cells, and the simultaneous induction of CEA- and MUC1-specific CTL responses restricted by HLA-A2 and/or HLA-A24. Finally, CTL induced by dendritic cell/allogeneic COLM-6 fusion cells were able to kill autologous colorectal carcinoma by HLA-A2- and/or HLA-A24-restricted mechanisms. The demonstration of CTL activity against shared tumor-associated antigens using an allogeneic tumor cell line, COLM-6, provides that the presence of alloantigens does not prevent the development of CTL with activity against autologous colorectal carcinoma cells. The fusion of allogeneic colorectal carcinoma cell line and autologous dendritic cells could have potential applicability to the field of antitumor immunotherapy through the cross-priming against shared tumor antigens and provides a platform for adoptive immunotherapy.     

Integration of autologous dendritic cell-based immunotherapy in the primary treatment for patients with newly diagnosed glioblastoma multiforme: a pilot study

Despite resection, radiochemotherapy, and maintenance temozolomide chemotherapy (TMZm), the prognosis of patients with glioblastoma multiforme (GBM) remains poor. We integrated immunotherapy in the primary standard treatment for eight pilot adult patients (median age 50 years) with GBM, to assess clinical and immunological feasibility and toxicity in preparation of a phase I/II protocol HGG-2006. After maximum, safe resection, leukapheresis was performed before radiochemotherapy, and four weekly vaccinations with autologous GBM lysate-loaded monocyte-derived dendritic cells were given after radiochemotherapy. Boost vaccines with lysates were given during TMZm. During the course of vaccination, immunophenotyping showed a relative increase in CD8+CD25+ cells in six of the seven patients, complying with the prerequisites for implementation of immunotherapy in addition to postoperative radiochemotherapy. In five patients, a more than twofold increase in tumor antigen-reacting IFN-γ-producing T cells on Elispot was seen at the fourth vaccination compared with before vaccination. In three of these five patients this more than twofold increase persisted after three cycles of TMZm. Quality of life during vaccination remained excellent. Progression-free survival at six months was 75%. Median overall survival for all patients was 24 months (range: 13–44 months). The only serious adverse event was an ischemic stroke eight months postoperatively. We conclude that tumor vaccination, fully integrated within the standard primary postoperative treatment for patients with newly diagnosed GBM, is feasible and well tolerated. The survival data were used to power a currently running phase I/II trial.     

An increase in CD4+CD25+FOXP3+ regulatory T cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme

The subpopulation of CD4+CD25+ immunoregulatory T (Tr) cells constitutes 5%-10% of CD4+ cells in humans. These cells play a crucial role in the control of tumor immune response. In this study, we evaluated the distribution of Tr cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme and examined the difference between the brain and autologous blood with respect to Tr cells. Glioma samples from 10 patients were classified as WHO grade IV astrocytoma. Control samples were obtained from patients undergoing resection of a seizure focus. The samples were analyzed by flow cytometry to determine the frequency of Tr cells and by real-time PCR for forkhead box P3 (FOXP3) expression. We then examined the expression of CD62L, CD45RO, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and assessed the functionality of Tr cells in vitro. There was a significant difference in the number of FOXP3-expressing CD4+CD25+ T cells within glioma-infiltrating lymphocytes as compared to controls (P < 0.01). This difference was further observed in studies of autologous patient blood and control blood. The expression level of FOXP3 mRNA was high in Tr cells and weak in CD4+CD25-T cells. Moreover, the expression of CD62L and CTLA-4 was elevated in glioma Tr cells as compared to that in the controls. These cells were also CD45RO positive. Functional assays confirmed the suppressive activity of Tr cells in patients with glioma. The expression of CD4+CD25+FOXP3+ T cells was significantly higher in patients with glioblastoma multiforme than in controls. This increase in the frequency of Tr cells that display suppressive activity might play a role in modulation of the immune response against glioma. In light of these findings, Tr cells may represent a potential target for immunotherapy of malignant brain tumors.     

T cell adoptive immunotherapy of newly diagnosed gliomas.

Patients with newly diagnosed gliomas were treated with adoptive transfer of ex vivo activated T lymphocytes, derived from lymph nodes (LNs) draining autologous tumor vaccines, to determine the long-term toxicity of this treatment. Twelve consecutive patients were enrolled: 2 with grade II astrocytoma, 4 with anaplastic gliomas, and 6 with glioblastoma multiforme. Patients were injected intradermally with short-term cultured autologous irradiated tumor cells, admixed with granulocyte macrophage colony-stimulating factor, to stimulate draining LNs. The LN cells were activated with staphylococcal enterotoxin A for 48 h and then cultured in medium containing interleukin 2 for an additional 6-8 days and subsequently transferred i.v. to the patients. The number of cells obtained from the LNs ranged from 9 x 10(7) to 1.1 x 10(9), and the median cell proliferation was 41-fold. The dose of T cells infused ranged from 0.6 to 5.5 x 10(10) with a median of 1.1 x 10(10), the majority of which were CD 4+ (mean, 71%). The entire treatment was performed as outpatient therapy and was associated with a toxicity of grade 2 or less, consisting mainly of fever, nausea, and myalgias during the first 24 h. There were no indications of late adverse events from this treatment even among three patients with follow-up greater than 2 years post T cell transfer. Moreover, four patients demonstrated partial regression of residual tumor. This Phase I clinical trial of adoptive immunotherapy for patients with newly diagnosed malignant gliomas demonstrates feasibility, lack of long-term toxicity, and several objective clinical responses.     

TRANCE- and CD40 ligand-matured dendritic cells reveal MHC class I-restricted T cells specific for autologous tumor in late-stage ovarian cancer patients.

PURPOSE: The use of mature dendritic cells (DCs) presenting tumor-associated antigens (TAAs) to trigger tumor-specific T cells in vivo or in vitro represents a promising approach for cancer immunotherapy. We hypothesized that tumor antigens, mostly unidentified, are present on ovarian tumor cells and that mature DCs could be used to generate tumor-specific responses in unprimed patients. We also sought to measure preexisting antitumor immunity in patients with advanced ovarian cancer. EXPERIMENTAL DESIGN: Autologous DCs from 10 patients with ovarian cancer were pulsed with killed autologous primary tumors as a source of TAAs. DCs were then cultured in the presence of tumor necrosis factor alpha + TRANCE (tumor necrosis factor-related activation-induced cytokine) to induce maturation. Mature TAA-pulsed DCs were used in vitro to stimulate tumor-specific peripheral blood T cells. RESULTS: TRANCE and CD40 ligand were effective at maturing DCs. T-cell lines were generated in vitro that were capable of secreting IFN-gamma in response to autologous tumor. These tumor-specific T cells were MHC class I restricted. The frequency of tumor-specific T cells in uncultured cells from malignant ascites fluid and peripheral blood was measured in the same patients. CONCLUSIONS: IFN-gamma-secreting tumor-specific T cells were demonstrated at baseline in uncultured T cells from some unvaccinated ovarian cancer patients; however, the T cells could not kill autologous tumor. These data demonstrate that mature DCs presenting tumor antigens from engulfed autologous tumors can be used to augment antitumor immunity in vitro in patients with epithelial ovarian cancer. The results support the feasibility of therapeutic vaccination of ovarian cancer patients.