Identification of novel Lck‐derived T helper epitope long peptides applicable for HLA‐A2+ cancer patients as cancer vaccine

The present study attempted to identify T helper epitope long peptides capable of inducing cytotoxic T lymphocytes (CTL) from Lck antigen (p56^Lck), the src family tyrosine kinase, which is known to be aberrantly expressed in metastatic cancers cells, in order to develop a long peptide‐based cancer vaccine for HLA‐A2⁺ cancer patients. Based on the biding motif to the HLA‐DR and HLA‐A2 alleles, 94 peptides were prepared from the Lck antigen. These peptides were screened for their reactivity to immunoglobulin G (IgG) from plasma of cancer patients, followed by testing of their ability to induce both CD4⁺ and CD8⁺ T lymphocytes showing not only peptide‐specific IFN‐γ production but cytotoxicity against HLA‐A2⁺ cancer cells from peripheral blood mononuclear cells (PBMC) of HLA‐A2⁺ cancer patients. Among 94 peptides tested, the three T helper epitope long peptides and their inner CTL epitope short peptides with HLA‐A2 binding motifs were frequently recognized by IgG of cancer patients, and efficiently induced both CD4⁺ IFN‐γ⁺ and CD8⁺ IFN‐γ⁺ T lymphocytes. Patients' PBMC stimulated with these long peptides showed cytotoxicity against HLA‐A2⁺ Lck⁺ cancer cells in HLA‐class I and HLA‐class II dependent manners. These three peptides might be useful for long peptide‐based vaccines for HLA‐A2⁺cancer patients with Lck⁺ tumor cells.     

Discovery of chemotherapy‐associated ovarian cancer antigens by interrogating memory T cells

According to the immunogenic cell death hypothesis, clinical chemotherapy treatments may result in CD8⁺ and CD4⁺ T‐cell responses against tumor cells. To discover chemotherapy‐associated antigens (CAAs), T cells derived from ovarian cancer (OC) patients (who had been treated with appropriate chemotherapy protocols) were interrogated with proteins isolated from primary OC cells. We screened for immunogenicity using two‐dimensional electrophoresis gel‐eluted OC proteins. Only the selected immunogenic antigens were molecularly characterized by mass‐spectrometry‐based analysis. Memory T cells that recognized antigens associated with apoptotic (but not live) OC cells were correlated with prolonged survival in response to chemotherapy, supporting the model of chemotherapy‐induced apoptosis as an adjuvant of anti‐tumor immunity. The strength of both memory CD4⁺ and CD8⁺ T cells producing either IFN‐γ or IL‐17 in response to apoptotic OC antigens was also significantly greater in Responders to chemotherapy than in nonresponders. Immunogenicity of some of these antigens was confirmed using recombinant proteins in an independent set of patients. The T‐cell interrogation system represents a strategy of reverse tumor immunology that proposes to identify CAAs, which may then be validated as possible prognostic tumor biomarkers or cancer vaccines.     

Mutational activation of the epidermal growth factor receptor down‐regulates major histocompatibility complex class I expression via the extracellular signal‐regulated kinase in non–small cell lung cancer

The efficacy of programmed cell death–1 (PD‐1) blockade in patients with non–small cell lung cancer (NSCLC) positive for epidermal growth factor receptor (EGFR) gene mutations has been found to be limited, but the underlying mechanisms for this poor response have remained obscure. Given that the recognition by T cells of tumor antigens presented by major histocompatibility complex class I (MHC‐I) molecules is essential for an antitumor immune response, we examined the effects of EGFR tyrosine kinase inhibitors (TKIs) on MHC‐I expression in NSCLC cell lines. Appropriate EGFR‐TKIs increased MHC‐I expression at the mRNA and cell surface protein levels in NSCLC cells positive for EGFR mutations including those with the T790M secondary mutation. Trametinib, an inhibitor of the extracellular signal–regulated kinase (ERK) kinase MEK, also increased MHC‐I expression, whereas the phosphatidylinositol 3‐kinase (PI3K) inhibitor buparlisib did not, suggesting that the MEK‐ERK pathway mediates the down‐regulation of MHC‐I expression in response to EGFR activation. Immunohistochemical analysis of EGFR‐mutated NSCLC specimens obtained before and after EGFR‐TKI treatment also revealed down‐regulation of phosphorylated forms of EGFR and ERK in association with up‐regulation of MHC‐I, an increased number of infiltrating CD8⁺ T cells, and increased PD‐1 ligand 1 expression after such treatment. Our results thus suggest that mutational activation of EGFR inhibits MHC‐I expression through the MEK‐ERK pathway in NSCLC and thereby contributes to the poor response of such tumors to immunotherapy. Further studies are warranted to evaluate the relation between EGFR‐MEK‐ERK signaling in and the immune response to EGFR‐mutated NSCLC.     

Identification of NY‐BR‐1‐specific CD4+ T cell epitopes using HLA‐transgenic mice

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer‐related deaths among women. The differentiation antigen NY‐BR‐1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen‐specific CD4⁺ effector T cells, NY‐BR‐1 was screened for the presence of HLA‐restricted CD4⁺ T cell epitopes that could be included in immunological treatment approaches. Upon NY‐BR‐1‐specific DNA immunization of HLA‐transgenic mice and functional ex vivo analysis, a panel of NY‐BR‐1‐derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY‐BR‐1‐specific, HLA‐DRB1*0301– or HLA‐DRB1*0401‐restricted CD4⁺ T cell lines from splenocytes of peptide immunized HLA‐transgenic mice. Notably, all four CD4⁺ T cell lines recognized human HLA‐DR‐matched dendritic cells (DC) pulsed with lysates of NY‐BR‐1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4⁺ T cells specific for all four CD4⁺ T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4⁺ T cell responses against the new epitopes are not deleted nor inactivated by self‐tolerance mechanisms. Our results present the first NY‐BR‐1‐specific HLA‐DRB1*0301– and HLA‐DRB1*0401‐restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer.     

CD80 expression in an HLA-A2-positive human non-small cell lung cancer cell line enhances tumor-specific cytotoxicity of HLA-A2-positive T cells derived from a normal donor and a patient with non-small cell lung cancer

To generate non-small cell lung cancer (NSCLC)–reactive lymphocytes, we transfected an HLA-A2-expressing human NSCLC line (1355) with the cDNA encoding the lymphocyte co-stimulatory molecule CD80. Following selection in G418, 1355.7 demonstrated stable cell-surface expression of CD80. Allogeneic mixed lymphocyte tumor cell cultures (MLTCs) were established in 600 IU/ml IL-2 using HLA-A2⁺ normal donor peripheral blood mononuclear cells (PBMCs) stimulated with 1355-P (parental), 1355.7 or IL-2 alone. In 7 of 9 MLTCs, those stimulated with 1355.7 demonstrated enhanced growth after 30 to 45 days of culture. The predominant lymphocyte to grow in all MLTCs was a CD3⁺αβ⁺CD4⁺ T cell. In one case, lymphocytes stimulated with 1355.7 (MLTC 2389.7) exhibited preferential lysis of 1355. MLTC 2389.7 was cloned by limiting dilution, and 2 resultant cloids were shown to be NSCLC-reactive and dependent on both MHC class I and CD3 in their recognition of tumor cells. Additionally, allogeneic MLTCs were established using three HLA-A2⁺ NSCLC patients' PBMC. The predominant lymphocyte to grow in these MLTCs was a CD3⁺ αβ⁺CD8⁺ T cell. In cytotoxicity studies, MLTC-UKY25.7 demonstrated preferential lysis of 1355-P, 1355.7 and an HLA-A2⁺ NSCLC cell line, 1650. Lymphocytes from this MLTC did not lyse K562, Daudi or an HLA-A2^– NSCLC cell line, 647. Our data suggest that CD80-expressing NSCLC tumor cells may enhance the generation of specific CTLs in vitro. These CTLs could be important reagents for use in cellular immunotherapy and/or in isolating tumor antigens for potential tumor vaccine development. Int. J. Cancer 78:685–694, 1998. © 1998 Wiley-Liss, Inc.     

The influence of myeloid‐derived suppressor cells on angiogenesis and tumor growth after cancer surgery

While myeloid‐derived suppressor cells (MDSCs) have been reported to participate in the promotion of angiogenesis and tumor growth, little is known about their presence and function during perioperative period. Here, we demonstrated that human MDSCs expressing CD11b⁺, CD33⁺ and HLA‐DR^– significantly increased in lung cancer patients after thoracotomy. CD11b⁺ CD33⁺ HLA‐DR^– MDSCs isolated 24 hr after surgery from lung cancer patients were more efficient in promoting angiogenesis and tumor growth than MDSCs isolated before surgical operation in allograft tumor model. In addition, CD11b⁺ CD33⁺ HLA‐DR^– MDSCs produced high levels of MMP‐9. Using an experimental lung metastasis mouse model, we demonstrated that the numbers of metastases on lung surface and Gr‐1⁺ CD11b⁺ MDSCs at postoperative period were enhanced in proportion to the degree of surgical manipulation. We also examined that syngeneic bone marrow mesenchymal stem cells (BMSCs) significantly inhibited the induction and proliferation of Gr‐1⁺ CD11b⁺ MDSCs and further prevented lung metastasis formation in the mice undergoing laparotomy. Taken together, our results suggest that postoperatively induced MDSCs were qualified with potent proangiogenic and tumor‐promotive ability and this cell population should be considered as a target for preventing postoperative tumor metastasis.     

Long‐term prognostic significance of interleukin‐17‐producing T cells in patients with non‐small cell lung cancer

The presence of interleukin (IL)‐17‐producing T cells has recently been reported in non‐small cell lung cancer (NSCLC) patients. However, the long‐term prognostic significance of these populations in NSCLC patients remains unknown. In the present study, we collected peripheral blood from 82 NSCLC patients and 22 normal healthy donors (NC). Percentages of IL‐17‐producing CD4⁺T (Th17), CD8⁺T (Tc17) and γδT cells (γδT17) were measured to determine their association with clinical outcomes and overall survival (OS) in NSCLC. All NSCLC patients were followed up until July 2018. Median follow‐up time was 13.5 months (range 1‐87 months). The 3‐ and 5‐year survival rate was 27% and 19.6%, respectively. We found that Th17 cells and γδT17 cells were significantly increased, whereas Tc17 cells were markedly decreased in patients with NSCLC compared with those in NC. In addition, Th17 cells were significantly positively associated with T helper type 1 cells (Th1), whereas γδT17 cells were significantly negatively associated with γδT ⁺ interferon (IFN)‐γ⁺ cells. High percentages of peripheral Tc17 cells were significantly associated with favorable 5‐year OS (P = .025), especially in patients with early TNM stage (P = .016). Furthermore, high percentages of peripheral Th17 cells were positively associated with favorable 5‐year OS in patients with late TNM stage (P = .002). However, no significant association was observed between γδT17 cells and OS, regardless of the TNM stage. In conclusion, our findings suggest that enhanced Th17 and reduced Tc17 cells in the peripheral blood could be a significant predictor of a favorable prognosis for NSCLC patients.     

Cancer immunotherapy using novel tumor‐associated antigenic peptides identified by genome‐wide cDNA microarray analyses

Recent genome‐wide cDNA microarray analysis of gene expression profiles in comprehensive tumor types coupled with isolation of cancer tissues by laser‐microbeam microdissection have revealed ideal tumor‐associated antigens (TAAs) that are frequently overexpressed in various cancers including head and neck squamous cell cancer (HNSCC) and lung cancer, but not in most normal tissues except for testis, placenta, and fetal organs. Preclinical studies using HLA‐transgenic mice and human T cells in vitro showed that TAA‐derived CTL‐epitope short peptides (SPs) are highly immunogenic and induce HLA‐A2 or ‐A24‐restricted CTLs. Based on the accumulated evidence, we carried out a phase II clinical trial of the TAA‐SP vaccine in advanced 37 HNSCC patients. This study showed a significant induction of TAA‐specific CTLs in the majority of patients without serious adverse effects. Importantly, clinical responses including a complete response were observed in this study. Another phase II clinical trial of therapeutic TAA‐SP vaccine, designed to evaluate the ability of prevention of recurrence, is ongoing in HNSCC patients who have received curative operations. Further studies in human preclinical studies and in vivo studies using HLA class I transgenic mice showed TAA‐derived long peptides (TAA‐LPs) have the capacity to induce not only promiscuous HLA class II‐restricted CD4⁺ T helper type 1 cells but also tumor‐specific CTLs through a cross‐presentation mechanism. Moreover, we observed an augmentation of TAA‐LP‐specific T helper type 1 cell responses and tumor antigen‐spreading in HNSCC patients vaccinated with TAA‐SPs. This accumulated evidence suggests that therapeutic TAA‐SPs and LPs vaccines may provide a promising cancer immunotherapy.     

CD40‐independent natural killer‐cell help promotes dendritic cell vaccine‐induced T‐cell immunity against endogenous B‐cell lymphoma

It is well established that an interplay between natural killer (NK) cells and dendritic cells (DCs) gives rise to their reciprocal activation and provides a Th1‐biased cytokine milieu that fosters antitumor T‐cell responses. Ex vivo‐differentiated DCs transferred into mice strongly stimulate endogenous NK cells to produce interferon (IFN)‐γ and initiate a cascade that eventually leads to cytotoxic T‐lymphocyte responses. We show that the ability of exogenous DCs to trigger this pathway obviates CD40 signaling and CD4⁺ T‐cell help and depends on a preceding maturation step. Importantly, this mechanism was also effective in endogenously arising tumors where IFN‐γ production is compromised in contrast to transplantable tumors. In c‐myc‐transgenic mice developing spontaneous lymphomas, injection of unpulsed DCs caused NK‐cell activation and induced CD8⁺ T cells capable of recognizing the lymphoma cells. Animals treated with unpulsed DCs showed a survival benefit compared to untreated myc mice. Hence, tumor immunity induced by DC‐based vaccines not only depends on specific antigens loaded on the DCs. Rather, DC vaccines generate broader immune responses, because endogenous DCs presenting tumor antigens may also become stimulated by NK cells that were activated by exogenous DCs. Thus, the DC/NK‐cell/cytotoxic T lymphocyte axis may commonly have relevance for DC‐based vaccination protocols in clinical settings.     

IgG1 anti‐epidermal growth factor receptor antibodies induce CD8‐dependent antitumor activity

Anti‐EGFR monoclonal antibodies (mAb) like Cetuximab are commonly used for treatment of EGFR⁺ solid tumors mainly by exerting their therapeutic effect through inhibition of signal transduction. Additionally, IgG1 is a potent mediator of antibody‐dependent cytotoxicity (ADCC). In case of the IgG1, Cetuximab induction of ADCC in vivo is controversially discussed. In our study, we investigated the efficiency of Cetuximab‐mediated ADCC in a humanized mouse tumor model in vivo and analyzed the contribution of immunologic processes toward antitumor activity. Therefore, we used immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA‐A2 and adoptively transferred human HLA‐A2⁺ PBMC after engraftment of human epidermoid cell carcinoma A431. Here, we show that high doses of anti‐EGFR mAb induced strong tumor regression independent of the immune system. However, tumor regression by low doses of anti‐EGFR mAb treatment was ADCC dependent and mediated by tumor infiltrating CD8⁺ T effector cells. This novel mechanism of ADCC conducted by CD8⁺ T effector cells was restricted to IgG1 anti‐EGFR mAb, dependent of binding to CD16 on T cells and could be inhibited after EGFR blockade on tumor cells. Furthermore, CD8⁺ T effector cell‐mediated ADCC was enhanced in the presence of IL‐15 and strongly improved after glycosylation of anti‐EGFR mAb indicating the potential of glycoengineered therapeutic mAb as efficient biologicals in cancer therapy.     

Tumor cells transduced with the MHC class II Transactivator and CD80 activate tumor-specific CD4+ T cells whether or not they are silenced for invariant chain

The specificity and potency of the immune system make immunotherapy a potential strategy for the treatment of cancer. To exploit this potential, we have developed cell-based cancer vaccines consisting of tumor cells expressing syngeneic MHC class II and costimulatory molecules. The vaccines mediate tumor regression in mice and activate human CD4+ T cells in vitro. Previous vaccines were generated by transducing MHC II negative tumor cells with a single HLA-DR allele. Because expression of multiple MHC II alleles would facilitate presentation of a broader repertoire of tumor antigens, we have now transduced tumor cells with the MHC class II transactivator (CIITA), a regulatory gene that coordinately increases expression of all MHC II alleles. Previous studies in mice indicated that coexpression of the MHC II accessory molecule invariant chain (Ii) inhibited presentation of endogenously synthesized tumor antigens and reduced vaccine efficacy. To determine if Ii expression affects presentation of MHC class II-restricted endogenously synthesized tumor antigens in human tumor cells, HLA-DR-MCF10 breast cancer cells were transduced with the CIITA, CD80 costimulatory molecule gene, and with or without small interfering RNAs (siRNA) specific for Ii. Ii expression is silenced >95% in CIITA/CD80/siRNA transductants; down-regulation of Ii does not affect HLA-DR expression or stability; and Ii(+) and Ii(-) transductants activate human CD4+ T cells to DRB1*0701-restricted HER-2/neu epitopes. Therefore, tumor cells transduced with the CIITA, CD80, and with or without Ii siRNA present endogenously synthesized tumor antigens and are potential vaccines for activating tumor-specific CD4+ T cells.     

Infiltration of tumor‐associated macrophages is involved in tumor programmed death‐ligand 1 expression in early lung adenocarcinoma

Programmed death‐ligand 1 (PD‐L1) is an immune modulator that promotes immunosuppression by binding to programmed death‐1 of T‐lymphocytes. Although tumor cell PD‐L1 expression has been shown to be associated with the clinical response to anti–PD‐L1 antibodies, its concise regulatory mechanisms remain elusive. In this study, we evaluated the associations of tumor PD‐L1 expression and immune cell infiltrating patterns in 146 cases of early lung adenocarcinoma (AC) to investigate the possible extrinsic regulation of tumor PD‐L1 by immune cells. Using immunohistochemistry, cell surface PD‐L1 expression in tumor cells was observed in 18.5% of stage 0‐IA lung AC patients. Tumor PD‐L1 positivity was significantly associated with stromal invasion, which was accompanied by increased tumor‐associated macrophages (TAM), CD8⁺ cytotoxic T cells and FoxP3⁺ regulatory T cells. Among these immune cells, TAM and CD8⁺ T cells significantly accumulated in PD‐L1‐positive carcinoma cell areas, which showed a tumor cell nest‐infiltrating pattern. Although CD8⁺ T cells are known to induce tumor PD‐L1 expression via interferon‐? production, the increased TAM within tumors were also associated with tumor cell PD‐L1 positivity, independently of CD8⁺ T cell infiltration. Our in vitro experiments revealed that PD‐L1 expression in lung cancer cell lines was significantly upregulated by co–culture with M2‐differentiated macrophages; expression of PD‐L1 was reduced to baseline levels following treatment with a transforming growth factor‐β inhibitor. These results demonstrated that tumor‐infiltrating TAM are extrinsic regulators of tumor PD‐L1 expression, indicating that combination therapy targeting both tumor PD‐L1 and stromal TAM might be a possible strategy for effective treatment of lung cancer.     

Interleukin‐6‐mediated resistance to immunotherapy is linked to impaired myeloid cell function

High serum levels of interleukin‐6 (IL‐6) correlate with poor prognosis and chemotherapy resistance in several cancers. The underlying mechanisms and its effects on immunotherapy are largely unknown. To address this, we developed a human papillomavirus type 16 (HPV16)‐associated tumor model expressing IL‐6 to investigate the impact of tumor‐expressed IL‐6 during cisplatin chemotherapy and HPV16 synthetic long peptide vaccination as immunotherapy. The effects of tumor‐produced IL‐6 on tumor growth, survival and the tumor microenvironment were analyzed. Our data demonstrated that tumor‐produced IL‐6 conferred resistance to cisplatin and therapeutic vaccination. This was not caused by a changed in vitro or in vivo growth rate of tumor cells, or a changed sensitivity of tumor cells to chemotherapy or T‐cell‐mediated killing. Furthermore, no overt differences in the frequencies of tumor‐infiltrating subsets of T cells or CD11b⁺ myeloid cells were observed. IL‐6, however, affected the systemic and local function of myeloid cells, reflected by a strong reduction of major histocompatibility complex (MHC) class II expression on all major myeloid cell subtypes. Resistance to both therapies was associated with a changed intratumoral influx of MHC class II⁺ myeloid cells toward myeloid cells with no or lower MHC class II expression. Importantly, while these IL‐6‐mediated effects provided resistance to the immunotherapy and chemotherapy as single therapies, their combination still successfully mediated tumor control. In conclusion, IL‐6‐mediated therapy resistance is caused by an extrinsic mechanism involving an impaired function of intratumoral myeloid cells. The fact that resistance can be overcome by combination therapies provides direction to more effective therapies for cancer.     

Melanoma‐associated chondroitin sulphate proteoglycan as a new target antigen for CD4+ T cells in melanoma patients

Melanoma‐associated chondroitin sulfate proteoglycan (MCSP) (also known as high molecular weight‐melanoma‐associated antigen) represents an interesting target antigen for cancer immunotherapy which is expressed on human melanomas and other tumors such as breast carcinomas, gliomas, neuroblastomas and acute leukemias. MCSP seems to play an important functional role in melanoma as it is involved in tumor cell migration, invasion and angiogenesis. In this study, we isolated CD4⁺ T helper cells from the blood of a healthy donor, recognizing a peptide from the MCSP core protein presented by HLA‐DBR1*1101 molecules. T cell reactivity against the identified peptide could be detected in the blood of healthy donors and melanoma patients. MCSP specific T cells from the blood of a patient could be readily expanded by repeated peptide stimulation and recognized MCSP and HLA‐DR expressing tumor cells. Our findings suggest that vaccination against MCSP helper T cell epitopes might be a promising approach to fight melanoma. © 2009 Wiley‐Liss, Inc.     

The variation of CD4+CD25+ regulatory T cells in the periphery blood and tumor microenvironment of non-small cell lung cancer patients and the downregulation effects induced by CpG ODN

The aim of the study was to observe the variation of CD4⁺CD25⁺ regulatory T cells in periphery blood and tumor microenvironment of non-small cell lung cancer (NSCLC) patients and the effects of CpG ODN. The proportion of CD4⁺CD25⁺ regulatory T cells, Foxp3 gene expression, levels of tumor growth factor-β (TGF-β) and immunoreactive fibronectin-γ (IFN-γ) in the periphery blood of 30 NSCLC patients and 30 healthy volunteers were compared. These indicators were compared before and after CpG ODN treatment. Foxp3 gene expression in the tumor microenvironment of NSCLC patients was also observed. The results showed CD4⁺CD25⁺ regulatory T cell proportion, Foxp3 expression and TGF-β levels in the periphery blood of NSCLC patients were higher than those of healthy volunteers (p < 0.05), and these indicators of patients were significantly decreased after CpG ODN 2006 treatment (p < 0.05). Foxp3 expression in the metastatic lymph nodes was higher than that in the non-metastatic ones of NSCLC patients (p = 0.000). In conclusion, a rise in the proportion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells was demonstrated in the periphery blood and tumor microenvironments of NSCLC patients. CpG ODN 2006 downregulated the CD4⁺CD25⁺Foxp3⁺ regulatory T cells proportion and TGF-β levels in the periphery blood of these patients.     

Enhancement of DNA vaccine potency by antigen linkage to IFN‐γ‐inducible protein‐10

DNA vaccines have emerged as an attractive approach to generate antigen‐specific T‐cell immune response. Nevertheless, the potency of DNA vaccines still needs to be improved for cancer immunotherapy. In this study, we explored whether functional linkage of a Th1‐polarizing chemokine, IP‐10, to a model tumor antigen, human papillomavirus type 16 (HPV‐16) E7, enhanced DNA vaccine potency. IP‐10 linkage changed the location of E7 from the nucleus to the endoplasmic reticulum and led to the secretion of functionally chemoattractive chimeric IP‐10/E7 protein. In addition, this linkage drastically enhanced the endogenous processing of E7 antigen through MHC class I. More importantly, we found that C57BL/6 mice intradermally vaccinated with IP‐10/E7 DNA exhibited a dramatic increase in the number of E7‐specific CD4⁺ Th1 T‐cells and CD8⁺ T‐cells and, consequently, were strongly resistant over the long term to E7‐expressing tumors compared to mice vaccinated with wild‐type E7 DNA. Thus, because of the increase in tumor antigen‐specific T‐cell immune responses obtained through both enhanced antigen presentation and chemoattraction, vaccination with DNA encoding IP‐10 linked to a tumor antigen holds great promise for treating tumors.     

Tumor‐reactive CD8+ T‐cell responses after vaccination with NY‐ESO‐1 peptide,CpG 7909 and Montanide® ISA‐51: association with survival

Peptide‐based vaccines have led to the induction of antigen‐specific CD8⁺ T‐cell responses in patients with NY‐ESO‐1 positive cancers. However, vaccine‐induced T‐cell responses did not generally correlate with improved survival. Therefore, we tested whether a synthetic CpG 7909 ODN (deoxycytidyl‐deoxyguanosin oligodeoxy‐nucleotides) mixed with NY‐ESO‐1 peptide p157‐165 and incomplete Freund's adjuvants (Montanide® ISA‐51) led to enhanced NY‐ESO‐1 antigen‐specific CD8⁺ immune responses in patients with NY‐ESO‐1 or LAGE‐1 expressing tumors. Of 14 HLA‐A2+ patients enrolled in the study, 5 patients withdrew prematurely because of progressive disease and 9 patients completed 1 cycle of immunization. Nine of 14 patients developed measurable and sustained antigen‐specific CD8⁺ T‐cell responses: Four had detectable CD8+ T‐cells against NY‐ESO‐1 after only 2 vaccinations, whereas 5 patients showed a late‐onset but durable induction of NY‐ESO‐1 p157‐165 specific T‐cell response during continued vaccination after 4 months. In 6 patients, vaccine‐induced antigen‐specific T‐cells became detectable ex vivo and reached frequencies of up to 0.16 % of all circulating CD8⁺ T‐cells. Postvaccine T‐cell clones were shown to recognize and lyse NY‐ESO‐1 expressing tumor cell lines in vitro. In 6 of 9 patients developing NY‐ESO‐1‐specific immune responses, a favorable clinical outcome with overall survival times of 43+, 42+, 42+, 39+, 36+ and 27+ months, respectively, was observed.     

Targeting high‐grade B cell lymphoma with CD19‐specific T cells

Adoptive T cell therapy is an important additional treatment option for malignant diseases resistant to chemotherapy. Using a murine high‐grade B cell lymphoma model, we have addressed the question whether the B cell differentiation antigen CD19 can act as rejection antigen. CD19^?/? mice inoculated with CD19⁺ B cell lymphoma cells showed higher survival rates than WT mice and were protected against additional tumor challenge. T cell depletion prior to tumor transfer completely abolished the protective response. By heterotypic vaccination of CD19^?/? mice against murine CD19, survival after tumor challenge was significantly increased. To define protective epitopes within the CD19 molecule, T cells collected from mice that had survived the tumor transfer were analyzed for IFNγ secretion in response to CD19‐derived peptides. The majority of mice exhibited a CD4⁺ T cell response to CD19 peptide 27, which was the most dominant epitope after CD19 vaccination. A peptide 27‐specific CD4⁺ T cell line protected CD19^?/? mice against challenge with CD19⁺ lymphoma and also cured a significant proportion of WT mice from recurrent disease in a model of minimal residual disease after chemotherapy. In conclusion, our data highlight CD19‐specific CD4⁺ T cells for adoptive T cell therapy of B cell lymphomas.     

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.