Checkpoint blockade for cancer therapy: revitalizing a suppressed immune system.

Immune checkpoint receptors are crucial molecules for fine-tuning immune responses. Checkpoint signaling dampens T cell activation to avoid autoimmunity and the destructive effects of an excessive inflammatory response. It is well established that tumors use several mechanisms to avoid elimination by the immune system, and one involves hijacking these checkpoint pathways. Checkpoint blockade therapy utilizes monoclonal antibodies to release the brakes from suppressed T cells, allowing them to be activated and recover their antitumor activity. This therapeutic approach has revolutionized cancer immunotherapy, and extraordinary increases in overall survival were noted, first with anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) and subsequently with anti-PD-1 (programmed cell death receptor-1) in melanoma and other malignancies.

Mucin 1-specific active cancer immunotherapy with tecemotide (L-BLP25) in patients with multiple myeloma: an exploratory study.

Patients (n = 34) with previously untreated, slowly progressive asymptomatic stage I/II multiple myeloma or with stage II/III multiple myeloma in stable response/plateau phase following conventional anti-tumor therapy were immunized repeatedly with the antigen-specific cancer immunotherapeutic agent tecemotide (L-BLP25). Additionally, patients were randomly allocated to either single or multiple low doses of cyclophosphamide to inhibit regulatory T cells (Treg). Immunization with tecemotide resulted in the induction/augmentation of a mucin 1-specific immune response in 47% of patients. The immune responses appeared to involve a Th1-like cellular immune response involving CD4 and CD8 T cells. The rate of immune responses was similar with single versus multiple dosing of cyclophosphamide and in patients with vs. without pre-existing mucin 1 immunity. On-treatment reductions in the slope of M-protein concentration over time (but not fulfilling clinical criteria for responses with conventional anti-tumor agents) were observed in 45% of evaluable patients, predominantly in those without versus with pre-existing mucin 1 immunity and in patients with early stage disease. No differences were seen in patients receiving single or multiple cyclophosphamide dosing. Treatment with tecemotide was generally well tolerated. Repeated vs. single dosing of cyclophosphamide had no impact on Treg numbers and was stopped after a case of fatal encephalitis that was assessed as possibly study-related. Tecemotide immunotherapy induces mucin 1-specific cellular immune responses in a substantial proportion of patients, with preliminary evidence of changes in the M-protein concentration time curve in a subset of patients.

T cells from indolent CLL patients prevent apoptosis of leukemic B cells in vitro and have altered gene expression profile.

T cells may have a role in sustaining the leukemic clone in chronic lymphocytic leukemia (CLL). In this study, we have examined the ability of T cells from CLL patients to support the survival of the leukemic B cells in vitro. Additionally, we compared global gene expression of T cells from indolent CLL patients with healthy individuals and multiple myeloma (MM) patients. Apoptosis of purified leukemic B cells was inhibited in vitro when co-cultured with increasing numbers of autologous T cells (p < 0.01) but not autologous B and T cells of normal donors. The anti-apoptotic effect exceeded that of the anti-apoptotic cytokine IL-4 (p = 0.002) and was greater with CD8+ cells (p = 0.02) than with CD4+ cells (p = 0.05). The effect was depended mainly on cell-cell contact although a significant effect was also observed in transwell experiments (p = 0.05). About 356 genes involved in different cellular pathways were deregulated in T cells of CLL patients compared to healthy individuals and MM patients. The results of gene expression profiling were verified for 6 genes (CCL4, CCL5 (RANTES), XCL1, XCL2, KLF6, and TRAF1) using qRT-PCR and immunoblotting. Our results demonstrate that CLL-derived T cells can prevent apoptosis of leukemic B cells and have altered expression of genes that may facilitate the survival of the leukemic clone.

Expression of MHC Class I on breast cancer cells correlates inversely with HER2 expression.

HER2 is a promising target for immunotherapeutic interventions with T cell-based approaches since it is amplified and overexpressed in 20-30% of breast cancers. However, several previous studies including ours showed that HER2-overexpressing tumors may escape cytotoxic T lymphocyte-mediated lysis by downregulating MHC Class I and components of the antigen-processing machinery. The aims of the present study were to analyze the relationship between HER2 and MHC Class I expression and to elucidate the mechanisms underlying MHC Class I downregulation in breast cancer. We explored expression of HER2, MHC Class I, PTEN, Ki67, estrogen and progesterone expression in 70 breast cancer patients by immunohistochemistry (IHC) and analyzed their correlation. We also explored the components of the signal transduction pathway that are involved in the regulation of MHC Class I expression using small-interfering RNAs targeting HER2 as well as an inhibitor of HER2 signaling. HER2 expression in breast cancers correlated inversely with MHC Class I expression analyzed by IHC. HER2 depletion by small-interfering RNAs resulted in MHC Class I upregulation. Moreover, MHC Class I expression on breast cancer cell lines was upregulated by PD98059, an inhibitor of mitogen-associated protein kinases, in a dose-dependent manner. Thus, agents that target the MAPK signaling pathway may increase MHC Class I expression in breast cancer cells.

Vaccination with dendritic cells loaded with tumor apoptotic bodies (Apo-DC) in patients with chronic lymphocytic leukemia: effects of various adjuvants and definition of immune response criteria.

We previously demonstrated that autologous dendritic cells that have endocytosed apoptotic bodies of chronic lymphocytic leukemia (CLL) cells (Apo-DC) can stimulate antileukemic T cell responses in vitro. In this phase I study, we vaccinated 15 asymptomatic CLL patients at five time points with Apo-DC administered intradermally either alone (cohort I), or in combination with subcutaneous granulocyte-macrophage-colony-stimulating-factor (GM-CSF) (cohort II) or with GM-CSF and intravenous low-dose cyclophosphamide (cohort III). Aim of the study was to evaluate the safety and immunogenicity of Apo-DC alone or in combination with GM-CSF and low-dose cyclophosphamide in CLL patients. All patients completed the vaccination schedule without dose-limiting toxicity. No objective clinical responses were seen. Vaccine-induced leukemia-specific immune responses were evaluated by IFN-γ ELISpot and proliferation assays over a 52 weeks observation period and immune response criteria were defined. According to these criteria, 10/15 patients were defined as immune responders. The frequency of immune-responding patients was higher in cohorts II (3/5) and III (5/5) than in cohort I (2/5). In order to further characterize the induced immune response, estimation of secreted cytokines and CD107-degranulation assay were performed. Clustering of T and CLL cells was observed in CD107-degranulation assay and visualized by confocal microscopy. Additionally, assessment of regulatory T cells (T(regs)) revealed their significantly lower frequencies in immune responders versus non-responders (P < 0.0001). Cyclophosphamide did not reduce T(regs) frequency. In conclusion, vaccination with Apo-DC + GM-CSF and cyclophosphamide was safe and elicited anti-CLL immune responses that correlated inversely with T(regs) levels. Lack of clinical responses highlights the necessity to develop more potent vaccine strategies in B cell malignancies.

A synthetic peptide homologous to IL-10 functional domain induces monocyte differentiation to TGF-ß+ tolerogenic dendritic cells.

We have previously demonstrated that IT9302, a nonameric peptide homologous to the C-terminal domain of human IL-10, mimics several effects of the cytokine including down-regulation of the antigen presentation machinery and increased sensitivity of tumor cells to NK-mediated lysis. In the present report, we have explored a potential therapeutic utility for IT9302 related to the ex vivo production of tolerogenic dendritic cells (DCs). Our results indicate that IT9302 impedes human monocyte response to differentiation factors and reduces antigen presentation and co-stimulatory capacity by DCs. Additionally, peptide-treated DCs show impaired capacity to stimulate T-cell proliferation and IFN-γ production. IT9302 exerts its effect through mechanisms, in part, distinct from IL-10, involving STAT3 inactivation and NF-κB intracellular pathway. IT9302-treated DCs display increased expression of membrane-associated TGF-ß, linked to a more effective induction of foxp3+ regulatory T cells. These results illustrate for the first time that a short synthetic peptide can promote monocytes differentiation to tolerogenic DCs with therapeutic potential for the treatment of autoimmune and transplantation-related immunopathologic disease.

Lapatinib inhibits receptor phosphorylation and cell growth and enhances antibody-dependent cellular cytotoxicity of EGFR- and HER2-overexpressing esophageal cancer cell lines.

Lapatinib is a dual tyrosine kinase inhibitor of the EGFR and HER2 tyrosine kinase domains. EGFR is expressed in 33.3% and HER2 in 30.3% of esophageal squamous cell carcinomas (ESCCs). To explore the potential utility of Lapatinib for therapy of ESCC patients, we evaluated the effect of Lapatinib on a panel of ESCC cell lines. EGFR and HER2 expression by the cell lines was established, and the effects of Lapatinib on inhibition of the phosphorylation of HER2, antiproliferative effect, apoptosis-inducing activity and accumulation of HER2 and EGFR on cell surface were evaluated. Additionally, the combined effect of Lapatinib together with Herceptin or Cetuximab on cell-mediated cytotoxicity was evaluated. Lapatinib inhibited HER2 phosphorylation in HER2-overexpressing, HER2 gene amplification positive ESCC cell line. Lapatinib also inhibited cell proliferation, induced apoptosis and caused the surface accumulation of HER2 and EGFR in ESCC cell lines. Addition of Lapatinib increased Herceptin-mediated antibody-dependent cell-mediated cytotoxicity by 15-25% with three ESCC target cell lines. Similarly, Cetuximab-mediated antibody-dependent cell-mediated cytotoxicity also increased by 15-30% in two ESCC cell lines on addition of Lapatinib. Cumulatively, the data indicate that Lapatinib has activity in EGFR- and/or HER2-expressing ESCC cells, and the combination therapy of Lapatinib and Cetuximab/Herceptin is a promising strategy in ESCC.

T cell recognition of HLA-A2 restricted tumor antigens is impaired by the oncogene HER2.

The HER2 oncogene is frequently over-expressed in human cancers and a promising target for immune therapy. Previous studies have shown that over-expression of mouse or rat HER2 leads to markedly reduced levels of major histocompatibility complex (MHC) class I and molecules of the antigen processing and presentation machinery (APM), thus resulting in a phenotype promoting tumor escape from the immune system. Our study focuses on analyzing the effect of HER2 on MHC class I antigen presentation and sensitivity to tumor-antigen specific cytotoxic T lymphocytes (CTLs) in HLA-A2.1(+) melanoma cell lines. We demonstrate significant inverse correlations both between the expression of HER2 and total MHC class I surface expression as well as between HER2 and HLA-A2. A significant reduction of HLA-A2 levels was found when melanoma and carcinoma cell lines were transfected with a human HER2 gene. A signaling-competent HER2 molecule was crucial for the observed HLA-A2 down-regulation, as transfectants expressing high levels of HER2 mutated in the tyrosine signaling domain did not show altered HLA-A2 expression. Importantly, the human melanoma cell line EST049 demonstrated reduced HER2 and melanoma antigen-specific recognition by CTLs upon HER2 transfection. In addition, high expression of HER2 prevented both IFN-γ mediated HLA-A2 up-regulation and improved recognition by HLA-A2-restricted CTLs in treated cells. Moreover, key APM molecules were down-regulated by HER2. These findings implicate that HER2 over-expressing tumors may be more prone to escape from HLA-A2 restricted CTLs suggesting that immunotherapy approaches inducing an integrated humoral, cellular and innate immune response would be most effective.

Silencing of ROR1 and FMOD with siRNA results in apoptosis of CLL cells.

We have previously demonstrated that ROR1 and FMOD (fibromodulin) are two genes upregulated in chronic lymphocytic leukaemia (CLL) cells compared to normal blood B cells. In this study, siRNAs were used to specifically silence ROR1 and FMOD expression in CLL cells, healthy B cells and human fibroblast cell lines. siRNA treatment induced a specific reduction (75-95%) in FMOD and ROR1 mRNA. Western blot analysis with specific antibodies for FMOD and ROR1 demonstrated that the proteins were significantly downregulated 48 h after siRNA treatment. Silencing of FMOD and ROR1 resulted in statistically significant (P ≤ 0·05-0·001) apoptosis of CLL cells but not of B cells from normal donors. Human fibroblast cell lines treated with FMOD and ROR1 siRNA did not undergo apoptosis. This is the first report demonstrating that ROR1 and FMOD may be involved in the survival of CLL cells. ROR1 in particular is further explored as potential target for therapy in CLL.

Regulatory T cells in cancer.

At the present time, regulatory T cells (Tregs) are an integral part of immunology but the route from discovery of "suppressive" lymphocytes in the 1980s to the current established concept of Tregs almost 20 years later has been a rollercoaster ride. Tregs are essential for maintaining self-tolerance as defects in their compartment lead to severe autoimmune diseases. This vitally important function exists alongside the detrimental effects on tumor immunosurveillance and antitumor immunity. Beginning with the identification of CD4(+)CD25(+) Tregs in 1995, the list of Treg subsets, suppressive mechanisms, and knowledge about their various origins is steadily growing. Increase in Tregs within tumors and circulation of cancer patients, observed in early studies, implied their involvement in pathogenesis and disease progression. Several mechanisms, ranging from proliferation to specific trafficking networks, have been identified to account for their systemic and/or local accumulation. Since various immunotherapeutic approaches are being utilized for cancer therapy, various strategies to overcome the antagonistic effects exerted by Tregs are being currently explored. An overview on the biology of Tregs present in cancer patients, their clinical impact, and methods for modulating them is given in this review. Despite the extensive studies on Tregs in cancer many questions still remain unanswered. Even the paradigm that Tregs generally are disadvantageous for the control of malignancies is now under scrutiny. Insight into the specific role of Tregs in different types of neoplasias is the key for targeting them in a way that is beneficial for the clinical outcome.

Systemic immune effects of adjuvant chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide and/or radiotherapy in breast cancer: a longitudinal study.

Immunotherapy is being increasingly utilized for adjuvant treatment for breast cancer (BC). We have previously described immune functions during primary therapy for BC. The present study describes immune recovery patterns during long-term, unmaintained follow-up after completion of adjuvant therapy.A group of patients with primary BC had been treated with adjuvant radio-chemotherapy (RT + CT) 5-fluorouracil, epirubicin and cyclophosphamide (FEC) (n = 21) and another group with radiotherapy (RT) (n = 20) alone. Immunological testing of NK and T-cell functions was performed initially at the end of adjuvant treatment and repeated after 2, 6 and 12 months. NK cell cytotoxicity was significantly higher (P < 0.05) at all time-points in patients than in age-matched controls and did not differ between the two treatments groups during one year observation. In contrast, lower numbers of CD4 T-cells and lower expression of CD28 on T-cells was observed particularly in RT + CT patients and did not normalize during the observation period. The numbers of T(reg) cells (CD4(+)CD25(high)) were low in the RT + CT group during follow-up, as well as expression of TCRxi, Zap70, p56(lck), P59(fyn) and PI3 k in CD4(+) cells. In contrast, expression of intracellular cytokines (IFN-gamma, IL-2, IL-4) in CD4 and CD8 T cells were significantly higher in RT + CT patients than in the RT group and the difference increased during follow-up. In conclusion, NK-cell cytotoxicity increased during unmaintained long-term follow-up whereas CD4 and regulatory T cells as well as signal transduction molecules remained low following adjuvant radio-chemotherapy.

The future of cancer vaccines for non-small-cell lung cancer: ongoing trials.

Lung cancer represents one of the malignancies in which the 3 elements of conventional therapy (ie, surgery, radiotherapy, and chemotherapy) have limited effectiveness in curbing progressive disease. In this context, there is burgeoning interest in the use of vaccine therapy as a nontoxic adjunct to increase the treatment success rates over those obtained with traditional regimens alone. Several clinical trials using a variety of vaccination strategies have been reported or are ongoing. In this review, we have provided an overview of these trials, with a special focus on the clinical efficacy of the vaccines. The future prospects and challenges of vaccine therapy in lung cancer have also been discussed.

Immune response, depression and fatigue in relation to support intervention in mammary cancer patients.

GOAL OF WORK: To study the effect of support intervention on immune function in breast cancer patients. MATERIALS AND METHODS: Breast cancer patients from an ongoing prospective randomised quality-of -life study were chosen for assaying immune functions in relation to a support-group intervention program running on a residential basis. Twenty-one women received adjuvant-combined radio-chemotherapy (CT-RT) and 20 women radiotherapy (RT). Eleven CT-RT and ten RT patients were randomised to support-group intervention, the rest served as controls. Immune tests for NK cells and NK-cell cytotoxicity, as well as lymphocyte subpopulations and response to antigen were performed before intervention, 2, 6, and 12 months later, in parallel to controls and healthy volunteers (n = 11). Depression, anxiety and fatigue were evaluated by the Hospital Anxiety and Depression (HAD) and the Norwegian Fatigue questionnaire. The density of NK cell receptors and in vitro quantitation of functional NK cytotoxicity against K562 cell line were evaluated. Four-colour flow cytometry was used to detect signal transduction molecules and cytokine expression. T-cell proliferate response to purified protein derivate (PPD) antigen was evaluated. RESULTS: No significant immune effect of support intervention could be found. The immune variables were severely disarranged compared to healthy volunteers but showed a statistically significant improvement over time. The majority of patients suffered from fatigue but had low scores for depression and anxiety. CONCLUSION: No effect on immune parameters could be detected from support intervention. The long-lasting immune suppression might override a putative effect of the intervention. Low depression scores may contribute to the absence of a detectable effect.

Long-term idiotype vaccination combined with interleukin-12 (IL-12), or IL-12 and granulocyte macrophage colony-stimulating factor, in early-stage multiple myeloma patients.

PURPOSE AND EXPERIMENTAL DESIGN: Twenty-eight patients with immunoglobulin G myeloma stages I to II were immunized i.d. over 110 weeks with autologous M protein combined with interleukin-12 (IL-12; n = 15) or with IL-12 and granulocyte macrophage colony-stimulating factor (GM-CSF; n = 13). Idiotype-specific T-cell responses were assessed by [(3)H]thymidine incorporation, enzyme-linked immunospot assay, and delayed-type hypersensitivity reaction. RESULTS: Based on these three assays, idiotype-specific immune responses were noted in 5 of 15 (33%) patients in the IL-12 group and in 11 of 13 (85%) patients in the GM-CSF/IL-12 group (P < 0.01). Immune response was seen only in patients with M-component concentration of <50 g/L. Three of 16 (19%) responders showed a gradually increasing idiotype-specific T-cell response, whereas 11 of 16 (69%) patients showed initial response, which then disappeared rapidly; the latter pattern was frequently associated with subsequent progressive disease. Immune nonresponse was associated with an increase in the numbers of CD4(+)/CD25(+) cells (regulatory T cells), which was absent in responding patients. Median time to progression for immune responders (n = 16) was 108 weeks compared with 26 weeks for nonresponders (n = 12; P = 0.03). CONCLUSIONS: These results indicate that idiotype immunization of myeloma patients with GM-CSF and IL-12 may induce specific T-cell response more frequently than with IL-12 alone and that immune response may correlate with time to progression and nonresponse with increased numbers of regulatory T cells.

Telomerase (hTERT 611-626) serves as a tumor antigen in B-cell chronic lymphocytic leukemia and generates spontaneously antileukemic, cytotoxic T cells.

OBJECTIVE: Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase. In B-cell chronic lymphocytic leukemia (B-CLL), telomerase activity is increased in about 75% of patients. The aim of this study was to analyze whether B-CLL patients with telomerase-positive leukemic cells had naturally occurring, telomerase-specific T cells that might be utilized for immune-mediated lysis of autologous tumor cells. METHODS: Spontaneous T-cell immunity and cytotoxicity against hTERT was explored in B-CLL. Nineteen of 25 B-CLL patients (76%) expressed hTERT (reverse transcriptase polymerase chain reaction) and 10 were selected for specific T-cell analysis against hTERT. RESULTS: The stimulation index (SI) of T cells from seven telomerase-positive patients stimulated with a 16aa hTERT peptide (611-626) loaded onto dendritic cells (DC) was 33.9 +/- 15.4 (mean SI +/- standard error of mean) and 13.2 +/- 5.6 against a Ras control peptide (p = 0.05), whereas the corresponding SI values for three telomerase-negative patients were 5.3 +/- 5.3 against the hTERT 611-626 peptide and 10.3 +/- 6.5 against the Ras peptide, respectively; and for three healthy controls, 5.4 +/- 0.9 against the hTERT 611-626 peptide and 4.5 +/- 1.0 against the Ras peptide (both not significant). Blocking experiments revealed that the specific responses were major histocompatibility complex (MHC) class I and MHC class II restricted. DC pulsed with the hTERT-peptide generated MHC class I-restricted, hTERT-specific cytotoxic T lymphocytes in six of seven telomerase-positive patients; mean cytotoxicity of hTERT-stimulated T cells was 49.8% +/- 9.3% vs 13.1 +/- 2.9% for Ras-stimulated T cells (p < 0.05). In three of three telomerase-negative patients, no hTERT-specific cytotoxic T lymphocytes could be expanded. CONCLUSION: Telomerase-positive B-CLL patients have spontaneously occurring cytotoxic hTERT-specific T cells. This antigen might be explored as a therapeutic vaccine in B-CLL.

Signaling molecules and cytokine production in T cells of patients with B-cell chronic lymphocytic leukemia: long-term effects of fludarabine and alemtuzumab treatment.

Fludarabine and alemtuzumab are routinely used for treatment of B-cell chronic lymphocytic leukemia (B-CLL). The present study aimed to compare the expression of signaling molecules and cytokine production by T cells of B-CLL patients in long-term unmaintained remission/plateau phase following fludarabine or alemtuzumab treatment with that of indolent/untreated B-CLL patients and healthy donors. The frequency and intensity of TCR-CD3zeta chain, p56lck, p59fyn, ZAP-70, PI3-kinase and interferon (IFN)-gamma/interleukin (IL)-4 production in CD4 and CD8 T cells was examined by flow cytometry. T-cell function was assessed by stimulation with purified protein derivative (PPD) and phytohemagglutinin (PHA). Despite a reduction in number, the expression of IFN-gamma/IL-4 in T-cells in patients was significantly higher than in healthy donors. The intensity of most signaling molecules in treated patients was relatively unaffected vs. healthy donors but lower than untreated-indolent patients. However, the total number of T cells which expressed each of the signaling molecules was decreased in patients, with no difference between fludarabine- and alemtuzumab-treated patients. The T-cell response to PHA but not PPD was reduced in treated patients. The results suggest that, despite some alterations in signaling molecules and a reduction in T-cell number, overall T-cell functions may be relatively well preserved long-term after treatment with fludarabine and alemtuzumab.

Clinical results of vaccine therapy for cancer: learning from history for improving the future.

Active, specific immunotherapy for cancer holds the potential of providing an approach for treating cancers, which have not been controlled by conventional therapy, with very little or no associated toxicity. Despite advances in the understanding of the immunological basis of cancer vaccine therapy as well as technological progress, clinical effectiveness of this therapy has often been frustratingly unpredictable. Hundreds of preclinical and clinical studies have been performed addressing issues related to the generation of a therapeutic immune response against tumors and exploring a diverse array of antigens, immunological adjuvants, and delivery systems for vaccinating patients against cancer. In this chapter, we have summarized a number of clinical trials performed in various cancers with focus on the clinical outcome of vaccination therapy. We have also attempted to draw objective inferences from the published data that may influence the clinical effectiveness of vaccination approaches against cancer. Collectively the data indicate that vaccine therapy is safe, and no significant autoimmune reactions are observed even on long term follow-up. The design of clinical trials have not yet been optimized, but meaningful clinical effects have been seen in B-cell malignancies, lung, prostate, colorectal cancer, and melanoma. It is also obvious that patients with limited disease or in the adjuvant settings have benefited most from this targeted therapy approach. It is imperative that future studies focus on exploring the relationship between immune and clinical responses to establish whether immune monitoring could be a reliable surrogate marker for evaluating the clinical efficacy of cancer 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.