Melanoma-specific CD8 Research Articles

Toll-like receptor 4 gene polymorphism influences dendritic cell in vitro function and clinical outcomes in vaccinated melanoma patients.

Toll-like receptor 4 (TLR4) is expressed on dendritic cells (DCs), sensing environmental danger molecules that induce their activation and maturation. Recently, we reported a method for the production of therapeutic DCs against melanoma, called tumor antigen-presenting cells (TAPCells), using a heat-shocked allogeneic melanoma cell lysate (TRIMEL) as an activation factor and antigen provider. Since TRIMEL contains endogenous TLR4 ligands, we evaluated the role of TLR4 in TAPCells differentiation by antibody neutralization and the association of a Tlr4 polymorphism (896A/G) (Asp299Gly), determined by PCR-RFLP, with the in vitro activation capacity and the clinical outcome of TAPCells-vaccinated patients. Antibody blocking of monocyte TLR4 inhibited surface expression, determined by flow cytometry, of the major histocompatibility complex class I, CCR7, CD80, CD83 and CD86 on TAPCells, reduced interleukin (IL)-6 and tumor necrosis factor -α gene expression evaluated by qRT-PCR, and also inhibited the TAPCells-mediated interferon-γ (IFN-γ) secretion of melanoma-specific CD8(+) T cells determined by ELISpot (p < 0.01). Moreover, CD8(+) T-cell activation capacity was significantly reduced in TAPCells bearing the TLR4 Asp299Gly receptor (p < 0.05). Finally, TAPCells-vaccinated stage-IV melanoma patients bearing the Tlr4 896G allele showed a shortened post-therapy median survival rate compared with those carrying the Tlr4 896A allele (p < 0.05; log-rank test). Our results indicate that TLR4 is a key receptor for the tumor lysate-mediated in vitro generation of clinically efficient antigen-presenting cells. Further analysis of patients included in different vaccine protocols is necessary for definitively establishing a role for TLR4 polymorphism in clinical responses.

Transferred melanoma-specific CD8 + T cells persist, mediate tumor regression, and acquire central memory phenotype

Adoptively transferred tumor-specific T cells offer the potential for non-cross-resistant therapy and long-term immunoprotection. Strategies to enhance in vivo persistence of transferred T cells can lead to improved antitumor efficacy. However, the extrinsic (patient conditioning) and intrinsic (effector cell) factors contributing to long-term in vivo persistence are not well-defined. As a means to enhance persistence of infused T cells in vivo and limit toxicity, 11 patients with refractory, progressive metastatic melanoma received cyclophosphamide alone as conditioning before the infusion of peripheral blood mononuclear cell-derived, antigen-specific, CD8(+) cytotoxic T-lymphocyte (CTL) clones followed by low-dose or high-dose IL-2. No life-threatening toxicities occurred with low-dose IL-2. Five of 10 evaluable patients had stable disease at 8 wk, and 1 of 11 had a complete remission that continued for longer than 3 y. On-target autoimmune events with the early appearance of skin rashes were observed in patients with stable disease or complete remission at 4 wk or longer. In vivo tracking revealed that the conditioning regimen provided a favorable milieu that enabled CTL proliferation early after transfer and localization to nonvascular compartments, such as skin and lymph nodes. CTL clones, on infusion, were characterized by an effector memory phenotype, and CTL that persisted long term acquired phenotypic and/or functional qualities of central memory type CTLs in vivo. The use of a T-cell product composed of a clonal population of antigen-specific CTLs afforded the opportunity to demonstrate phenotypic and/or functional conversion to a central memory type with the potential for sustained clinical benefit.

Trogocytosis Is a Gateway to Characterize Functional Diversity in Melanoma-Specific CD8+ T Cell Clones

Trogocytosis, the transfer of membrane patches from target to immune effector cells, is a signature of tumor-T cell interaction. In this study, we used the trogocytosis phenomenon to study functional diversity within tumor-specific T cell clones with identical TCR specificity. MART-1(26-35)-specific CD8 T cell clones, which differed in their trogocytosis capacity (low [2D11], intermediate [2G1], high [2E2]), were generated from melanoma patients. Functional evaluation of the clones showed that the percentage of trogocytosis-capable T cells closely paralleled each clone's IFN-γ and TNF-α production, lysosome degranulation, and lysis of peptide-pulsed targets and unmodified melanoma. The highly cytotoxic 2E2 clone displayed the highest TCR peptide binding affinity, whereas the low-activity 2D11 clone showed TCR binding to peptide-MHC in a CD8-dependent manner. TCR analysis revealed Vβ16 for clones 2E2 and 2G1 and Vβ14 for 2D11. When peptide-affinity differences were bypassed by nonspecific TCR stimulation, clones 2E2 and 2D11 still manifested distinctive signaling patterns. The high-activity 2E2 clone displayed prolonged phosphorylation of ribosomal protein S6, an integrator of MAPK and AKT activation, whereas the low-activity 2D11 clone generated shorter and weaker phosphorylation. Screening the two clones with identical TCR Vβ by immunoreceptor array showed higher phosphorylation of NK, T, and B cell Ag (NTB-A), a SLAM family homophilic receptor, in clone 2E2 compared with 2G1. Specific blocking of NTB-A on APCs markedly reduced cytokine production by CD8 lymphocytes, pointing to a possible contribution of NTB-A costimulation to T cell functional diversity. This finding identifies NTB-A as a potential target for improving anti-cancer immunotherapy.

Phase II trial of the regulatory T cell-depleting agent, denileukin diftitox, in patients with unresectable stage IV melanoma

BackgroundWe previously found that administration of an interleukin 2/diphtheria toxin conjugate (DAB/IL2; Denileukin Diftitox; ONTAK) to stage IV melanoma patients depleted CD4+CD25HIFoxp3+ regulatory T cells and expanded melanoma-specific CD8+ T cells. The goal of this study was to assess the clinical efficacy of DAB/IL2 in an expanded cohort of stage IV melanoma patients.MethodsIn a single-center, phase II trial, DAB/IL2 (12 μg/kg; 4 daily doses; 21 day cycles) was administered to 60 unresectable stage IV melanoma patients and response rates were assessed using a combination of 2-[18 F]-fluoro-2-deoxy-glucose (FDG)-positron emission tomography (PET) and computed tomography (CT) imaging.ResultsAfter DAB/IL2 administration, 16.7% of the 60 patients had partial responses, 5% stable disease and 15% mixed responses. Importantly, 45.5% of the chemo/immuno-naïve sub-population (11/60 patients) experienced partial responses. One year survival was markedly higher in partial responders (80 ± 11.9%) relative to patients with progressive disease (23.7 ± 6.5%; p value < 0.001) and 40 ± 6.2% of the total DAB/IL2-treated population were alive at 1 year.ConclusionsThese data support the development of multi-center, randomized trials of DAB/IL2 as a monotherapy and in combination with other immunotherapeutic agents for the treatment of stage IV melanoma.Trial registrationNCT00299689

New perspectives on the role of vitiligo in immune responses to melanoma.

Melanoma-associated vitiligo is the best-studied example of the linkage between tumor immunity and autoimmunity. Although vitiligo is an independent positive prognostic factor for melanoma patients, the autoimmune destruction of melanocytes was long thought to be merely a side effect of robust anti-tumor immunity. However, new data reveal a key role for vitiligo in supporting T cell responses to melanoma. This research perspective reviews the history of melanoma-associated vitiligo in patients, the experimental studies that form the basis for understanding this relationship, and the unique characteristics of melanoma-specific CD8 T cells found in hosts with vitiligo. We also discuss the implications of our recent findings for the interpretation of patient responses, and the design of next-generation cancer immunotherapies.

Detection of Intra-Tumor Self Antigen Recognition during Melanoma Tumor Progression in Mice Using Advanced Multimode Confocal/Two Photon Microscope

Determining how tumor immunity is regulated requires understanding the extent to which the anti-tumor immune response “functions” in vivo without therapeutic intervention. To better understand this question, we developed advanced multimodal reflectance confocal/two photon fluorescence intra-vital imaging techniques to use in combination with traditional ex vivo analysis of tumor specific T cells. By transferring small numbers of melanoma-specific CD8+ T cells (Pmel-1), in an attempt to mimic physiologic conditions, we found that B16 tumor growth alone was sufficient to induce naive Pmel-1 T cell proliferation and acquisition of effector phenotype. Tumor -primed Pmel-1 T cells, are capable of killing target cells in the periphery and secrete IFNγ, but are unable to mediate tumor regression. Within the tumor, Pmel-1 T cells have highly confined mobility, displaying long term interactions with tumor cells. In contrast, adoptively transferred non tumor-specific OT-I T cells show neither confined mobility, nor long term interaction with B16 tumor cells, suggesting that intra-tumor recognition of cognate self antigen by Pmel-1 T cells occurs during tumor growth. Together, these data indicate that lack of anti-tumor efficacy is not solely due to ignorance of self antigen in the tumor microenvironment but rather to active immunosuppressive influences preventing a protective immune response.

Heat-Shock Induction of Tumor-Derived Danger Signals Mediates Rapid Monocyte Differentiation into Clinically Effective Dendritic Cells

This study characterizes, biologically and clinically, a novel type of dendritic cells (DC) produced in the short term and called tumor antigen-presenting cells (TAPCells). In particular, we identified factors present in a lysate derived from heat-shocked allogeneic melanoma cells (TRIMEL) that are associated with TAPCells' enhanced capability to induce CD8(+) T-cell responses in vitro and in vaccinated melanoma patients. First, extensive phenotypic and functional characterization of TAPCells was performed, followed by vaccination of 45 melanoma patients with four doses of TAPCells over a period of 2 months. Specific delayed-type hypersensitivity (DTH) reaction was analyzed posttreatment and correlated with overall survival rates. Furthermore, heat-shock (HS)-induced factors present in TRIMEL and their effects on DC activation were identified and studied. TRIMEL induced a committed, mature, DC-like phenotype in TAPCells and effectively activated melanoma-specific CD4(+) and CD8(+) T cells. Clinically, 64% of vaccinated patients showed positive DTH reaction against TRIMEL, and this was associated with improved overall survival. HS treatment of tumor cells increased calreticulin (CRT) plasma membrane translocation and induced the release of high-mobility group box 1 proteins (HMGB1). Both CRT and HMGB1 mobilization were associated with enhanced TAPCells' maturation and antigen (Ag) cross-presentation, respectively. DTH infiltration analysis revealed the presence of CD8(+)/CD45RO(+) T cells, thus confirming TAPCells' ability to cross-present Ags in vivo. Our results indicate that lysates derived from heat-shocked tumor cells are an optimal source of tumor-associated Ags, which are crucial for the generation of DCs with improved Ag cross-presentation capacity and clinically effective immunogenicity.

Selective Transduction of Dendritic Cells in Human Lymph Nodes and Superior Induction of High-avidity Melanoma-reactive Cytotoxic T Cells by a CD40-targeted Adenovirus

Targeted delivery of tumor antigen genes to dendritic cells (DCs) using adenoviral (Ad) vectors holds great potential for cancer immunotherapy. We previously showed that CD40 targeting of Ad vectors enhanced specific transduction of DC in human skin, while simultaneously ensuring their stable maturation and superior allogeneic T-cell stimulatory capacity. In this study, we evaluated whether CD40-targeted Ad encoding the full-length melanoma antigen recognized by T cells-1 (CD40-Ad-MART-1) could be used to efficiently and selectively transduce conventional and plasmacytoid DC to prime melanoma-specific CD8(+) T-effector cells in human melanoma-draining sentinel lymph nodes (SLNs). CD40 targeting of Ad was achieved using a bispecific fusion protein, binding and neutralizing the Ad fiber knob through soluble coxsackie and adenovirus receptor while retargeting the virus to hCD40 through the tumor necrosis factor-like domain of mCD40L. Selective transduction of conventional and plasmacytoid DC subsets by CD40-Ad was observed in suspensions of human melanoma-draining SLN. Moreover, CD40-Ad-MART-1 enhanced the expansion of functional MART-1-specific CD8(+) T cells from SLN with concomitant decreases in CD4:CD8 T-cell ratios and CD4(+)CD25(hi)FoxP3(+) regulatory T-cell rates. Additional studies revealed that transduction and activation of monocyte-derived DCs with CD40-Ad-MART-1 significantly enhanced their priming efficiency of functional CD8(+) effector T cells with high avidity. These findings provide preclinical evidence of possible efficacy of this approach for cancer immunotherapy.

Melanoma‐targeted chemo‐thermo‐immuno (CTI)‐therapy using N‐propionyl‐4‐S‐cysteaminylphenol‐magnetite nanoparticles elicits CTL response via heat shock protein‐peptide complex release

Melanogenesis substrate, N-propionyl-4-S-cysteaminylphenol (NPrCAP) is specifically taken up by melanoma cells and inhibits their growth by producing cytotxic free radicals. By taking advantage of this unique chemical agent, we have established melanoma-targeting intracellular hyperthermia by conjugating NPrCAP with magnetite nanoparticles (NPrCAP/M) upon exposure to an alternating magnetic field (AMF). This treatment causes cytotoxic reaction as well as heat shock responses, leading to elicitation of antitumor immune response, which was proved by tumor rechallenge test and CTL induction. We found the level of heat shock protein 72 (Hsp72) to be increased in the cell lysate and culture supernatant after intracellular hyperthermia. Melanoma-specific CD8(+) T-cell response to dendritic cells loaded with hyperthermia-treated tumor lysate was enhanced when compared with non-treated tumor lysate. When heat shock protein, particularly Hsp72, was immuno-depleted from hyperthermia-treated tumor cell lysate, specific CD8(+) T-cell response was abolished. Thus, it is suggested that antitumor immune response induced by hyperthermia using NPrCAP/M is derived from the release of HSP-peptide complex from degraded tumor cells. Therefore, this chemo-thermo-immuno (CTI)-therapy might be effective not only for primary melanoma but also for distant metastasis because of induction of systemic antimelanoma immune responses.

Cytokine Working Group Study of Lymphodepleting Chemotherapy, Interleukin-2, and Granulocyte-Macrophage Colony-Stimulating Factor in Patients With Metastatic Melanoma: Clinical Outcomes and Peripheral-Blood Cell Recovery

Recovery of lymphocyte populations after lymphocyte depletion is implicated in therapeutic immune pathways in animal models and in patients with cancer. We sought to evaluate the effects of chemotherapy-induced lymphodepletion followed by granulocyte-macrophage colony-stimulating factor (GM-CSF) and high-dose interleukin-2 (IL-2) therapy on clinical response and the recovery of lymphocyte subcompartments in patients with metastatic melanoma. This was a two-stage phase II trial design. Patients with measurable metastatic melanoma were treated with intravenous cyclophosphamide (60 mg/kg, days 1 and 2) and fludarabine (25 mg/m(2), day 3 through 7) followed by two 5-day courses of intravenous high-dose bolus IL-2 (600,000 U/kg; days 8 through 12 and 21 through 25). GM-CSF (250 microg/m(2)/d beginning day 8) was given until granulocyte recovery. Lymphocyte recovery profiles were determined by flow cytometric phenotyping at regular intervals, and clinical outcome was assessed by Response Evaluation Criteria in Solid Tumors (RECIST). The trial was stopped at the end of stage 1 with four of 18 objective responses noted. Twelve patients had detailed lymphocyte subcompartments evaluated. After lymphodepletion, we observed an induction of regulatory cells (CD4+ T regulatory cells; CD8+ T suppressor cells) and of T memory cells (CD8+ T central memory cells; T effector memory RA+ cells). Expansion of circulating melanoma-specific CD8(+) cells was observed in one of four HLA-A2-positive patients. Chemotherapy-induced lymphodepletion modulates the homeostatic repopulation of the lymphocyte compartment and influences recovering lymphocyte subpopulations. Clinical activity seems similar to standard high-dose aldesleukin alone.

Local Administration of PF-3512676 CpG-B Instigates Tumor-Specific CD8+ T-Cell Reactivity in Melanoma Patients

Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. Local administration of PF-3512676 (formerly known as CpG 7909) has shown immunostimulatory effects of both dendritic cell and T-cell subsets in the melanoma SLN. Here, we set out to ascertain whether these PF-3512676-induced immunostimulatory effects translate into higher frequencies of melanoma-specific CD8(+) T cells. Twenty-four stage I to III melanoma patients were randomized to preoperative local administration of either PF-3512676 or saline. CD8(+) T cells from SLN and peripheral blood were tested for reactivity by IFN-gamma ELISPOT assay against several HLA-A1/A2/A3-restricted epitopes derived from various melanoma-associated antigens (MAA) in 21 of 24 enrolled patients. Frequencies of natural killer (NK) cells and frequencies and maturation state of dendritic cell subsets in the SLN were determined by flow cytometry. Melanoma-specific CD8(+) T-cell response rates against >1 MAA epitope in the SLN were 0 of 11 for the saline group versus 5 of 10 for the PF-3512676-administered group (P = 0.012). Of these 5 responding patients, 4 also had a measurable response to >1 MAA epitope in the blood. Increased frequencies in the SLN of both MAA-specific CD8(+) T cells and NK cells correlated to CpG-induced plasmacytoid dendritic cell maturation. These data show an increase in melanoma-specific CD8(+) T-cell frequencies as well as an increased effector NK cell rate after a single dose of PF-3512676 and thus support the utility of local PF-3512676 administration as adjuvant treatment in early-stage melanoma to try and halt metastatic spread.

Induction of broad repertoire of melanoma associated antigen-specific CD4+ T cells by dendritic cell vaccine loaded with killed allogeneic melanoma cells in patients with metastatic melanoma

3029 Background: Tumor-specific CD4+ T cells play an important role in antitumor immunity. CD4+ T cells recognizing melanoma associated antigens can be spontaneously generated in patients with metastatic melanoma as demonstrated by the infiltration of such cells at tumor sites; however, they are often dysfunctional. We analyzed tumor-specific CD4+ T cell repertoires in the blood of metastatic melanoma patients, and examined whether dendritic cell (DC) vaccine loaded with killed allogeneic melanoma cells administered to metastatic melanoma patients can induce and/or improve melanoma-specific CD4+ T cell immunity. Methods: PBMCs were obtained from ten metastatic melanoma patients who received at least four monthly DC vaccine injections (GM-IL-4 DC vaccine loaded with killed allogeneic melanoma cells, matured with CD40L and TNFa). Melanoma-specific T cell repertoires at baseline and after vaccinations were assessed with EPIMAX approach using overlapping peptide libraries spanning MART-1, gp100, and TRP-1 antigens. The frequencies of CD4+ T cells responding to the identified peptides were assessed by intracellular cytokine detection assay with flow cytometry. Results: At baseline, four out of ten patients displayed gp100-specific CD4+ T cells which secreted IFNg and/or IL-2, while none displayed MART-1-specific CD4+ T cells. Nine gp100 CD4+ T cell epitopes were identified in total. The T cells lacked proliferative capacity (seven out of nine gp100-specific T cells) in spite of cytokine secretion in response to peptide stimulation. We found PD-1 expression on specific CD4+ T cells which lacked the proliferative capacity. Analysis of post-vaccine showed the presence of proliferative gp100-specific CD4+ T cells in four patients. One of these patients experienced a complete response and another one a long-term survival. The frequencies of specific CD4+ T cells secreting IL-2 and/or IFNg were increased at post-vaccine. Functional MART-1 and TRP-1-specific CD4+ T cells were also induced in both patients. Conclusions: DC vaccine loaded with killed tumor cells can prime broad repertoire of tumor-specific CD4+ T cells as well as revert dysfunction of pre-existing specific T cells. No significant financial relationships to disclose.

Distinct Role for CD8 T Cells toward Cutaneous Tumors and Visceral Metastases

The growth of immunogenic tumors in immunocompetent individuals is one of the oldest conundrums in tumor immunology. Although the ability of mouse CD8+ T cells to control transplanted tumors is well documented, little is known about their impact on autochthonous tumors. To gain insight into the role of CD8+ T cells during the course of cancer development, we produced a novel model of spontaneous melanoma. The metallothionein (MT)-ret/AAD mouse is transgenic for the RET oncogene and the chimeric MHC molecule AAD (alpha1-alpha2 domains of HLA-A2 linked to alpha3 domain of H2-Dd). This model recapitulates the natural history of human melanoma, and expression of the AAD molecule makes it suitable for analyzing CD8+ T cell responses directed against peptide Ags that have been previously identified in HLA-A2+ melanoma patients. We found that, as tumors grow, mice develop a broad melanoma-specific CD8+ T cell response. Occurrence of cutaneous nodules is not affected by CD8+ T cell depletion, showing that although CD8+ T cells are functional, they have no effect on established cutaneous tumors. However, depleted mice die from visceral disease much earlier than controls, showing that CD8+ T cells control metastasis spreading and disease progression. Antigenic modulation is observed in visceral metastases, suggesting that visceral nodules may be subject to immunoediting. Our data demonstrate that growth of melanoma in the MT-ret/AAD model involves several tolerance mechanisms sequentially. They also reveal a different role for CD8+ T cells toward early stage of cutaneous tumors and late visceral metastatic stage of the disease.

A novel strategy for rapid and efficient isolation of human tumor-specific CD4 + and CD8 + T-cell clones

Adoptive therapy with antigen-specific T cells is a promising approach for the treatment of infectious diseases and cancer. However, cloning of antigen-specific T cells by the traditional approach of limiting dilution is a time-consuming, laborious, and inefficient process. Here, we describe a novel flow cytometric strategy for rapid isolation of human tumor antigen-specific T-cell clones by using T-cell receptor (TCR) Vβ antibodies in combination with carboxyfluorescein succinimidyl ester (CFSE)-based proliferation assay. The CFSE dilution following antigen stimulation identified proliferating antigen-specific T cells, and the TCRVβ antibodies allowed distinguishing T cells at the clonal level from a heterogeneous T-cell population. This method of TCRVβ/CFSE dilution was used for the isolation of four different human lymphoma and melanoma-specific CD4 + and CD8 + T-cell clones reactive against defined and undefined tumor antigens. Isolated tumor-specific T-cell clones could be expanded to large numbers ex vivo while maintaining phenotype, function, and tumor antigen specificity. The method was simple, efficient, and reproducible, and may have potential application for the development of adoptive immunotherapeutic strategies.

Both Langerhans cells and interstitial DC cross‐present melanoma antigens and efficiently activate antigen‐specific CTL

Dendritic cells (DC) have a unique capacity to present external antigens to CD8(+) T cells, i.e. cross-presentation. However, it is not fully established whether the ability to cross-presentation is restricted to a unique subset of DC in humans. Here, we show that two major myeloid DC subsets, i.e. Langerhans cells (LC) and interstitial DC (Int-DC), have the ability to cross-present antigens to CD8(+) T cells in vitro. LC and Int-DC were obtained from DC generated by culturing human CD34(+)-hematopoietic progenitor cells with GM-CSF, FLT3-L, and TNF-alpha (CD34-DC). Both DC subsets were able to capture necrotic/apoptotic allogeneic melanoma cells and present antigens to CD8(+) T cells, resulting in efficient priming of naive CD8(+) T cells into CTL capable of killing melanoma cells. Strikingly, a single stimulation with either subset (LC or Int-DC) or total CD34-DC loaded with necrotic/apoptotic melanoma cells was sufficient to activate melanoma-specific memory CD8(+) T cells obtained from patients with metastatic melanoma to become effective CTL. Thus, this study provides the rationale to use CD34-DC loaded with necrotic/apoptotic allogeneic melanoma cells in a clinical trial.

Induction of Antigen-Specific CD8+ T cells in Melanoma Patients using a PrimeBoost Therapuetic Vaccine (48.18)

Abstract Induction of tumour-specific CD8+ T cell responses in melanoma patients is the major aim of antigen-specific immunotherapy. This study evaluated a prime-boost immunotherapeutic vaccine (Hi-8™ MEL) in stage III/IV melanoma patients using vectors encoding a polyepitope string of seven CD8+ T cell epitopes from five melanoma antigens. T cell responses were evaluated in cryopreserved PBMC samples taken before and during treatment. CD8+ T cells specific for the immunodominant Melan-A epitope were detected by ex vivo tetramer assay in 24/36 (67%) of all patients and in 10/11 (91%) of patients that received the highest treatment dose. Eleven of 36 (31%) patients mounted an ex vivo IFN-γ ELISPOT response: 9/36 (25%) to Melan-A and 3/36 (8%) to other epitopes in the string (NY-ESO-1 and MAGE-3). Notably, the Melan-A-tetramer+ CD8+ T cells in one patient reached 7.25% and acquired a CD45RA−CCR7− effector/memory phenotype. This patient also exhibited an ELISPOT response and a partial clinical response (&amp;gt;12 months). Across the study, tetramer responders showed a median survival of 86 weeks versus 37 weeks for non-responders (p&amp;lt;0.001), indicating that Hi-8™ MEL immunotherapy can elicit melanoma-specific CD8+ T cell responses that are associated with tumour control and increased survival. Additional immune assays are underway to further elucidate the nature of these efficacious T cell responses.

A Novel Strategy for Generation of Human Tumor-Specific T Cell Clones for Adoptive Transfer.

Adoptive T-cell therapy using donor lymphocyte infusions is a promising approach for treating hematological malignancies. But, efficacy is limited by the induction of graft-versus-host disease. Transfer of tumor-specific T-cell clones could enhance the graft-versus-tumor effect and eliminate graft-versus-host disease. However, isolating antigen-specific T-cell clones by the traditional limiting dilution approach is a time-consuming and laborious process. Here, we describe a novel strategy for rapidly cloning tumor-specific T cells. Lymphoma-specific T-cell lines were generated from two follicular lymphoma patients by repeated in vitro stimulation of lymphocytes isolated from tumor or blood with autologous soluble CD40 ligand-activated tumor cells. After four in vitro stimulations at 10-day intervals in the presence of IL-2 and IL-15, T-cell lines were found to be predominantly CD4+ T cells and produced significant amounts of TNF-a, GM-CSF, and IFN-γ in response to autologous tumor cells. The tumor reactivity was MHC class II restricted suggesting that it was mediated by CD4+ T cells. Staining with a TCR Vb antibody panel, a set of monoclonal antibodies against 24 human TCR Vb families, revealed that certain Vb families were overrepresented in each CD4+ T-cell line. In patient 1, 51% of CD4+ T cells were Vb1 positive, and in patient 2, 27% of CD4+ T cells were Vb8 positive. To clone lymphoma-specific T cells, CD4+ T-cell lines were labeled with CFSE and stimulated with autologous tumor cells. After 9 days of in vitro expansion in the presence of IL-2 and IL-15, CD4+ T-cell lines were stained with an anti-human CD4-APC monoclonal antibody and an anti-human TCR Vb-PE monoclonal antibody for each CD4+ T-cell line. Proliferating Vb1 cells from patient 1 and Vb8 cells from patient 2 were identified by their reduction in CFSE staining, and CD4+TCRV b +CFSEdim cells were sorted by flow cytometer. Monoclonality of the sorted cells was confirmed by PCR using a panel of optimized primers specific for 24 TCR Vb families, by TCR Vb spectratype analysis, and finally, by sequencing the TCR Vb gene used by each T-cell clone. Sorted tumor-specific T-cell clones could be expanded to large numbers using a 14-day rapid expansion protocol with allofeeder PBMCs, and confirmed to retain specificity against autologous tumor cells in a cytokine induction assay. This approach was also successfully used to isolate melanoma-specific CD8+ T-cell clones from two patients. We conclude that this approach is highly reproducible, rapid, and efficient for generating antigen-specific T-cell clones for adoptive T-cell therapy against human malignancies in the autologous or allogeneic setting.

New Generation Vaccine Induces Effective Melanoma-Specific CD8+ T Cells in the Circulation but Not in the Tumor Site

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.

Tumor-Specific CD8+ T Cell Reactivity in the Sentinel Lymph Node of GM-CSF–Treated Stage I Melanoma Patients is Associated with High Myeloid Dendritic Cell Content

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

Immunization of Stage IV Melanoma Patients with Melan-A/MART-1 and gp100 Peptides plus IFN-α Results in the Activation of Specific CD8+ T Cells and Monocyte/Dendritic Cell Precursors

The use of IFN-alpha in clinical oncology has generally been based on the rationale of exploiting its antiproliferative and antiangiogenic activities. However, IFN-alpha also exhibits enhancing effects on T-cell and dendritic cell functions, which may suggest a novel use as a vaccine adjuvant. We have carried out a pilot phase I-II trial to determine the effects of IFN-alpha, administered as an adjuvant of Melan-A/MART-1:26-35(27L) and gp100:209-217(210M) peptides, on immune responses in stage IV melanoma patients. In five of the seven evaluable patients, a consistent enhancement of CD8(+) T cells recognizing modified and native MART-1 and gp100 peptides and MART-1(+)gp100(+) melanoma cells was observed. Moreover, vaccination induced an increase in CD8(+) T-cell binding to HLA tetramers containing the relevant peptides and an increased frequency of CD45RA(+)CCR7(-) (terminally differentiated effectors) and CD45RA(-)CCR7(-) (effector memory) cells. In all patients, treatment augmented significantly the percentage of CD14(+) monocytes and particularly of the CD14(+)CD16(+) cell fraction. An increased expression of CD40 and CD86 costimulatory molecules in monocytes was also observed. Notably, postvaccination monocytes from two of the three patients showing stable disease or long disease-free survival showed an enhanced antigen-presenting cell function and capability to secrete IP10/CXCL10 when tested in mixed leukocyte reaction assays, associated to a boost of antigen and melanoma-specific CD8(+) T cells. Although further clinical studies are needed to show the adjuvant activity of IFN-alpha, the present data represent an important starting point for considering a new clinical use of IFN-alpha and new immunologic end points, potentially predictive of clinical response.