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The strength and durability of systemic anti-tumor immune responses induced by cancer vaccines depends on adjuvants to support an immunogenic vaccine site microenvironment (VSME). Adjuvants include water-in-oil emulsions with incomplete Freund's adjuvant (IFA) and combinations of toll-like receptor (TLR) agonists, including a preparation containing TLR4 and TLR9 agonists with QS-21 (AS15). IFA-containing vaccines can promote immune cell accumulation at the VSME, whereas effects of AS15 are largely unexplored. Therefore, we assessed innate and adaptive immune cell accumulation and gene expression at the VSME after vaccination with AS15 and compared to effects with IFA. We hypothesized that AS15 would promote less accumulation of innate and adaptive immune cells at the VSME than IFA vaccines. In two clinical trials, patients with resected high-risk melanoma received either a multipeptide vaccine with IFA or a recombinant MAGE-A3 protein vaccine with AS15. Vaccine site biopsies were obtained after one or multiple vaccines. T cells accumulated early after vaccines with AS15, but this was not durable or of the same magnitude as vaccination in IFA. Vaccines with AS15 increased durable expression of DC- and T cell-related genes, as well as PD-L1 and IDO1, suggesting complex activation and regulation of innate and adaptive immune function with AS15. These changes were generally greater with vaccines containing IFA, but IFA induced reduction in myeloid suppressor cells markers. Evidence of tertiary lymphoid structure (TLS) formation was observed with both adjuvants. Our findings highlight adjuvant-dependent changes in immune features at the VSME that may impact systemic immune responses.
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Inmunidad Adaptativa/inmunología , Vacunas contra el Cáncer/inmunología , Inmunidad Innata/inmunología , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Adyuvantes Inmunológicos/farmacología , Antígenos de Neoplasias/inmunología , Femenino , Adyuvante de Freund/inmunología , Humanos , Lípidos/inmunología , Masculino , Persona de Mediana Edad , Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Vacunación/métodos , Vacunas de Subunidad/inmunologíaRESUMEN
BACKGROUND: Cancer vaccines require adjuvants to induce effective immune responses; however, there is no consensus on optimal adjuvants. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC or TLR4 agonist lipopolysaccharide (LPS), combined with CD4 T cell activation, would support strong and durable CD8+ T cell responses, whereas addition of an incomplete Freund's adjuvant (IFA) would reduce magnitude and persistence of immune responses. PATIENTS AND METHODS: Participants with resected stage IIB-IV melanoma received a vaccine comprised of 12 melanoma peptides restricted by Class I MHC (12MP), plus a tetanus helper peptide (Tet). Participants were randomly assigned 2:1 to cohort 1 (LPS dose-escalation) or cohort 2 (polyICLC). Each cohort included 3 subgroups (a-c), receiving 12MP + Tet + TLR agonist without IFA (0), or with IFA in vaccine one (V1), or all six vaccines (V6). Toxicities were recorded (CTCAE v4). T cell responses were measured with IFNγ ELIspot assay ex vivo or after one in vitro stimulation (IVS). RESULTS: Fifty-three eligible patients were enrolled, of which fifty-one were treated. Treatment-related dose-limiting toxicities (DLTs) were observed in 0/33 patients in cohort 1 and in 2/18 patients in cohort 2 (11%). CD8 T cell responses to 12MP were detected ex vivo in cohort 1 (42%) and in cohort 2 (56%) and in 18, 50, and 72% for subgroups V0, V1, and V6, respectively. T cell responses to melanoma peptides were more durable and of highest magnitude for IFA V6. CONCLUSIONS: LPS and polyICLC are safe and effective vaccine adjuvants when combined with IFA. Contrary to the central hypothesis, IFA enhanced T cell responses to peptide vaccines when added to TLR agonists. Future studies will aim to understand mechanisms underlying the favorable effects with IFA. TRIAL REGISTRATION: The clinical trial Mel58 was performed with IRB (#15781) and FDA approval and is registered with Clinicaltrials.gov on April 25, 2012 (NCT01585350). Patients provided written informed consent to participate. Enrollment started on June 24, 2012.
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Adyuvantes Inmunológicos/administración & dosificación , Vacunas contra el Cáncer/administración & dosificación , Carboximetilcelulosa de Sodio/análogos & derivados , Adyuvante de Freund/administración & dosificación , Lípidos/administración & dosificación , Lipopolisacáridos/administración & dosificación , Melanoma/tratamiento farmacológico , Poli I-C/administración & dosificación , Polilisina/análogos & derivados , Receptores Toll-Like/agonistas , Vacunas de Subunidad/administración & dosificación , Adyuvantes Inmunológicos/efectos adversos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/efectos adversos , Carboximetilcelulosa de Sodio/administración & dosificación , Carboximetilcelulosa de Sodio/efectos adversos , Femenino , Adyuvante de Freund/efectos adversos , Humanos , Lípidos/efectos adversos , Lipopolisacáridos/efectos adversos , Masculino , Melanoma/inmunología , Poli I-C/efectos adversos , Polilisina/administración & dosificación , Polilisina/efectos adversos , Vacunas de Subunidad/efectos adversosRESUMEN
Immune-cell infiltration is associated with improved survival in melanoma. Human melanoma metastases may be grouped into immunotypes representing patterns of immune-cell infiltration: A (sparse), B (perivascular cuffing), and C (diffuse). Immunotypes have not been defined for murine melanomas, but may provide opportunities to understand mechanism-driving immunotype differences. We performed immunohistochemistry with immune-cell enumeration, immunotyping, and vascular density scoring in genetically engineered (Braf/Pten and Braf/Pten/ß-catenin) and transplantable (B16-F1, B16-OVA, and B16-AAD) murine melanomas. The transplantable tumors were grown in subcutaneous (s.c.) or intraperitoneal (i.p.) locations. Braf/Pten and Braf/Pten/ß-catenin tumors had low immune-cell densities, defining them as Immunotype A, as did B16-F1 tumors. B16-OVA (s.c. and i.p.) and B16-AAD s.c. tumors were Immunotype B, while B16-AAD i.p. tumors were primarily Immunotype C. Interestingly, the i.p. location was characterized by higher immune-cell counts in B16-OVA tumors, with counts that trended higher for B16-F1 and B16-AAD. The i.p. location was also characterized by higher vascularity in B16-F1 and B16-AAD tumors. These findings demonstrate that spontaneously mutated neoantigens in B16 melanomas were insufficient to induce robust intratumoral immune-cell infiltrates, but instead were Immunotype A tumors. The addition of model neoantigens (OVA or AAD) to B16 enhanced infiltration, but this most often resulted in Immunotype B. We find that tumor location may be an important element in enabling Immunotype C tumors. In aggregate, these data suggest important roles both for the antigen type and for the tumor location in defining immunotypes.
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Antígenos de Neoplasias/inmunología , Inmunofenotipificación , Melanoma Experimental/inmunología , Neoplasias Cutáneas/inmunología , Animales , Línea Celular Tumoral/trasplante , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfohidrolasa PTEN/genética , Proteínas Proto-Oncogénicas B-raf/genética , Piel/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Análisis de Matrices Tisulares , beta Catenina/genéticaRESUMEN
INTRODUCTION: Infiltration of non-small-cell lung cancer (NSCLC) by CD8+ T lymphocytes predicts improved patient survival; however, heterogeneity of intratumoral localization complicates this assessment. Strategies for tumor sampling may not accurately represent the whole tumor. We hypothesized that sampling strategies may alter the identification of tumors with high CD8 density and affect the prognostic significance. PATIENTS AND METHODS: Twenty-three primary NSCLC tumors were immunohistochemically stained for CD8 and were assessed using automated software with eight different sampling strategies or the whole tumor. Results of all sampling strategies were compared to the whole tumor counts (paired t tests, Pearson's r). Associations between CD8 densities and overall survival were assessed (log-rank test). RESULTS: Counts from all eight sampling strategies significantly correlated with whole tumor counts (p ≤ 0.001). However, the magnitude of CD8+ cell counts and categorization into high vs low infiltrate groups were affected by the sampling strategy. The most concordant values were derived from random sampling of 20 % of the tumor, a simulated core biopsy, or from sampling the tumor center. TIL infiltration was associated with survival when sampling the center (p = 0.038), but not the invasive margin (p > 0.2) or other strategies. CONCLUSION: Different tumor sampling strategies may yield discordant TIL density results and different stratification for risk assessment. Small biopsies may be particularly unrepresentative. Random sampling of larger tumor areas is recommended. Enumerating CD8+ T cells in the tumor center may have prognostic value.
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Linfocitos T CD8-positivos/inmunología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Neoplasias Pulmonares/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos T CD8-positivos/patología , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Linfocitos Infiltrantes de Tumor/patología , Pronóstico , Análisis de SupervivenciaRESUMEN
Therapeutic blockade of PD-1/PD-L1 can have dramatic therapeutic benefit in some patients; however, the prognostic associations of PD-1 and its ligands, in the absence of therapeutic blockade have not been definitively addressed. In particular, associations of PD-L2 with immune infiltrates and with outcome have yet to be explored. We hypothesized that surface expression of both PD-L1 and PD-L2 by melanoma cells would be associated with immune cell infiltration and with overall patient survival, independent of checkpoint blockade therapy. We also characterized the heterogeneity of their distribution within a tumor and within tumors of the same patient. Tissue microarrays of metastatic melanoma samples from 147 patients were quantified for CD8+, CD45, CD4+, CD3, CD163, CD20, CD138, FoxP3, PD-1, PD-L1 and PD-L2 markers by immunohistochemistry. Relationships between the proportions of PD-L1 and PD-L2 expressing tumor cells with the immune cell count, distribution (immunotype) and patient survival were studied. Expressions of both PD-L1 and PD-L2 correlated significantly with increasing densities of immune cells in the tumor specimens and with immunotype. Positive PD-L2 expression was associated with improved overall survival and the simultaneous positive expression of both PD-1 ligands showed a higher association with survival. Significant heterogeneity of PD-L1 and PD-L2 expressions within tumors were observed, however, they were less pronounced with PD-L2. In conclusion, both are markers of immune infiltration and PD-L2, alone or in combination with PD-L1, is a marker for prognosis in metastatic melanoma patients. Larger tumor samples yield more reliable assessments of PD-L1/L2 expression.
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INTRODUCTION: Optimal approaches to induce T cell infiltration of tumors are not known. Chemokines CXCL9, CXCL10, and CXCL11 support effector T cell recruitment and may be induced by IFN. This study tests the hypothesis that intratumoral administration of IFNγ will induce CXCL9-11 and will induce T cell recruitment and anti-tumor immune signatures in melanoma metastases. PATIENTS AND METHODS: Nine eligible patients were immunized with a vaccine comprised of 12 class I MHC-restricted melanoma peptides and received IFNγ intratumorally. Effects on the tumor microenvironment were evaluated in sequential tumor biopsies. Adverse events (AEs) were recorded. T cell responses to vaccination were assessed in PBMC by IFNγ ELISPOT assay. Tumor biopsies were evaluated for immune cell infiltration, chemokine protein expression, and gene expression. RESULTS: Vaccination and intratumoral administration of IFNγ were well tolerated. Circulating T cell responses to vaccine were detected in six of nine patients. IFNγ increased production of chemokines CXCL10, CXCL11, and CCL5 in patient tumors. Neither vaccination alone, nor the addition of IFNγ promoted immune cell infiltration or induced anti-tumor immune gene signatures. CONCLUSION: The melanoma vaccine induced circulating T cell responses, but it failed to infiltrate metastases, thus highlighting the need for combination strategies to support T cell infiltration. A single intratumoral injection of IFNγ induced T cell-attracting chemokines; however, it also induced secondary immune regulation that may paradoxically limit immune infiltration and effector functions. Alternate dosing strategies or additional combinatorial treatments may be needed to promote trafficking and retention of tumor-reactive T cells in melanoma metastases.
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Vacunas contra el Cáncer/inmunología , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/metabolismo , Factores Inmunológicos/uso terapéutico , Inmunoterapia/métodos , Interferón gamma/uso terapéutico , Melanoma/terapia , Linfocitos T/inmunología , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/inmunología , Movimiento Celular , Células Cultivadas , Ensayo de Immunospot Ligado a Enzimas , Femenino , Estudios de Seguimiento , Humanos , Linfocitos Infiltrantes de Tumor/patología , Masculino , Melanoma/mortalidad , Melanoma/patología , Persona de Mediana Edad , Estadificación de Neoplasias , Fragmentos de Péptidos/inmunología , Análisis de Supervivencia , Vacunas de Subunidad/inmunologíaRESUMEN
INTRODUCTION: Infiltration of cancers by T cells is associated with improved patient survival and response to immune therapies; however, optimal approaches to induce T cell infiltration of tumors are not known. This study was designed to assess whether topical treatment of melanoma metastases with the TLR7 agonist imiquimod plus administration of a multipeptide cancer vaccine will improve immune cell infiltration of melanoma metastases. PATIENTS AND METHODS: Eligible patients were immunized with a vaccine comprised of 12 melanoma peptides and a tetanus toxoid-derived helper peptide, and imiquimod was applied topically to metastatic tumors daily. Adverse events were recorded, and effects on the tumor microenvironment were evaluated from sequential tumor biopsies. T cell responses were assessed by IFNγ ELIspot assay and T cell tetramer staining. Patient tumors were evaluated for immune cell infiltration, cytokine and chemokine production, and gene expression. RESULTS AND CONCLUSIONS: Four eligible patients were enrolled, and administration of imiquimod and vaccination were well tolerated. Circulating T cell responses to the vaccine was detected by ex vivo ELIspot assay in 3 of 4 patients. Treatment of metastases with imiquimod induced immune cell infiltration and favorable gene signatures in the patients with circulating T cell responses. This study supports further study of topical imiquimod combined with vaccines or other immune therapies for the treatment of melanoma.
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Aminoquinolinas/uso terapéutico , Antígenos de Neoplasias/inmunología , Antineoplásicos/uso terapéutico , Vacunas contra el Cáncer/inmunología , Melanoma/terapia , Fragmentos de Péptidos/inmunología , Neoplasias Cutáneas/terapia , Linfocitos T/efectos de los fármacos , Administración Tópica , Anciano , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Terapia Combinada , Citocinas/genética , Citocinas/metabolismo , Ensayo de Immunospot Ligado a Enzimas , Femenino , Humanos , Imiquimod , Linfocitos Infiltrantes de Tumor/patología , Masculino , Melanoma/secundario , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Cutáneas/secundario , Linfocitos T/inmunología , Receptor Toll-Like 7/agonistas , Transcriptoma/inmunología , Vacunas de Subunidad/inmunologíaRESUMEN
Melanoma is known to be under latent immunosurveillance. Here, we studied four biomarkers of immunogenic cell stress and death (microtubule-associated proteins 1A/1B light chain 3B (MAP-LC3B, best known as LC3B)-positive puncta in the cytoplasm as a sign of autophagy; presence of nuclear HMGB1; phosphorylation of eIF2α; increase in ploidy) in melanoma cells, in tissue microarrays (TMA) from metastases from 147 melanoma patients. These biomarkers of immunogenicity were correlated with the density of immune cells infiltrating the metastases and expressing CD3, CD4(+), CD8(+), CD20, CD45, CD56, CD138, CD163, DC-LAMP or FOXP3. LC3B puncta positively correlated with the infiltration of metastases by CD163(+) macrophages, while expression of HMGB1 correlated with infiltration by FOXP3(+) regulatory T cells and CD56(+) lymphocytes. eIF2α phosphorylation was associated with an augmentation of nuclear diameters, reflecting an increase in ploidy. Interestingly, therapeutic vaccination led to a reduction of eIF2α phosphorylation suggestive of immunoselection against cells bearing this sign of endoplasmic reticulum (ER) stress. None of the stress/death-related biomarkers had a significant prognostic impact, contrasting with the major prognostic effect of the ratio of cytotoxic T lymphocytes (CTL) over immunosuppressive FOXP3(+) and CD163(+) cells. Altogether, these results support the idea of a mutual dialog between, on one hand, melanoma cells with their cell-intrinsic stress pathways and, on the other hand, immune effectors. Future work is required to understand the detailed mechanisms of this interaction.
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INTRODUCTION: Methods to induce T cell responses to protein vaccines have not been optimized. The immunostimulant AS15 has been administered with the recombinant MAGE-A3 protein (recMAGE-A3) i.m. but not i.d. or s.c. This study tests hypotheses that the i.d./s.c. route is safe and will increase CD4(+) and CD8(+) T cell responses to MAGE-A3. PATIENTS AND METHODS: Twenty-five patients with resected stage IIB-IV MAGE-A3(+) melanoma were randomized to immunization with recMAGE-A3 combined with AS15 immunostimulant (MAGE-A3 immunotherapeutic) either i.m. (group A, n = 13) or i.d./s.c. (group B, n = 12). Adverse events were recorded. Ab responses to MAGE-A3 were measured by ELISA. T cell responses to overlapping MAGE-A3 peptides were assessed in PBMC and a sentinel immunized node (SIN) after 1 in vitro stimulation with recMAGE-A3, by IFN-γ ELISPOT assay and by flow cytometry for multifunctional (TNF-α/IFN-γ) responses. RESULTS: Both routes of immunization were well tolerated without treatment-related grade 3 adverse events. All patients had durable Ab responses. For all 25 patients, the T cell response rate by ELISPOT assay was 30 % in SIN (7/23) but only 4 % (1/25) in PBMC. By flow cytometry, multifunctional CD8(+) T cell responses were identified in one patient in each group; multifunctional CD4(+) T cell response rates for groups A and B, respectively, were 31 and 64 % in SIN and 31 and 50 % in PBMC. CONCLUSION: The MAGE-A3 immunotherapeutic was well tolerated after i.d./s.c. administration, with trends to higher CD4(+) T cell response rates than with i.m. administration. This study supports further study of AS15 by i.d./s.c. administration.
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Adyuvantes Inmunológicos/uso terapéutico , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Proteínas de Neoplasias/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/uso terapéutico , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/uso terapéutico , Humanos , Inyecciones Intramusculares , Persona de Mediana Edad , Proteínas de Neoplasias/uso terapéutico , Proyectos Piloto , Resultado del TratamientoRESUMEN
We have identified eight genes whose expression in human melanoma metastases and ovarian cancers is associated with a lack of Th1 immune signatures. They encode molecules with mechanical barrier function in the skin and other normal tissues and include filaggrin (FLG), tumor-associated calcium signal transducer 2 (TACSTD2), and six desmosomal proteins (DST, DSC3, DSP, PPL, PKP3, and JUP). This association has been validated in an independent series of 114 melanoma metastases. In these, DST expression alone is sufficient to identify melanomas without immune signatures, while FLG and the other six putative barrier molecules are overexpressed in a different subset of melanomas lacking immune signatures. Similar associations have been identified in a set of 186 ovarian cancers. RNA-seq data from 471 melanomas and 307 ovarian cancers in the TCGA database further support these findings and also reveal that overexpression of barrier molecules is strongly associated with early patient mortality for melanoma (p = 0.0002) and for ovarian cancer (p < 0.01). Interestingly, this association persists for FLG for melanoma (p = 0.012) and ovarian cancer (p = 0.006), whereas DST overexpression is negatively associated with CD8+ gene expression, but not with patient survival. Thus, overexpression of FLG or DST identifies two distinct patient populations with low immune cell infiltration in these cancers, but with different prognostic implications for each. These data raise the possibility that molecules with mechanical barrier function in skin and other tissues may be used by cancer cells to protect them from immune cell infiltration and immune-mediated destruction.
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Clinical approaches to treat advanced melanoma include immune therapies, whose benefits depend on tumor-reactive T-cell infiltration of metastases. However, most tumors lack significant immune infiltration prior to therapy. Selected chemokines promote T-cell migration into tumors; thus, agents that induce these chemokines in the tumor microenvironment (TME) may improve responses to systemic immune therapy. CXCL10 has been implicated as a critical chemokine supporting T-cell infiltration into the TME. Here, we show that toll-like receptor (TLR) agonists can induce chemokine production directly from melanoma cells when combined with IFNγ treatment. We find that TLR2 and TLR6 are widely expressed on human melanoma cells, and that TLR2/6 agonists (MALP-2 or FSL-1) synergize with interferon-gamma (IFNγ) to induce production of CXCL10 from melanoma cells. Furthermore, melanoma cells and immune cells from surgical specimens also respond to TLR2/6 agonists and IFNγ by upregulating CXCL10 production, compared to treatment with either agent alone. Collectively, these data identify a novel mechanism for inducing CXCL10 production directly from melanoma cells, with TLR2/6 agonists +IFNγ and raise the possibility that intratumoral administration of these agents may improve immune signatures in melanoma and have value in combination with other immune therapies, by supporting T-cell migration into melanoma metastases.
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Antineoplásicos/farmacología , Quimiocina CXCL10/metabolismo , Interferón gamma/metabolismo , Melanoma/metabolismo , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 6/agonistas , Línea Celular Tumoral , Humanos , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Cancer cells display novel phosphopeptides in association with MHC class I and II molecules. In this study, we evaluated two HLA-A2-restricted phosphopeptides derived from the insulin receptor substrate (IRS)-2 and the cell-cycle regulator CDC25b. These proteins are both broadly expressed in multiple malignancies and linked to cancer cell survival. Two phosphopeptides, termed pIRS-21097-1105 and pCDC25b38-46, served as targets of strong and specific CD8 T-cell memory responses in normal human donors. We cloned T-cell receptor (TCR) cDNAs from murine CD8 T-cell lines specific for either pIRS-21097-1105 or pCDC25b38-46. Expression of these TCRs in human CD8 T cells imparted high-avidity phosphopeptide-specific recognition and cytotoxic and cytokine-secreting effector activities. Using these cells, we found that endogenously processed pIRS-21097-1105 was presented on HLA-A2(+) melanomas and breast, ovarian, and colorectal carcinomas. Presentation was correlated with the level of the Ser(1100)-phosphorylated IRS-2 protein in metastatic melanoma tissues. The highest expression of this protein was evident on dividing malignant cells. Presentation of endogenously processed pCDC25b38-46 was narrower, but still evident on HLA-A2(+) melanoma, breast carcinoma, and lymphoblastoid cells. Notably, pIRS-21097-1105-specific and pCDC25b38-46-specific TCR-expressing human CD8 T cells markedly slowed tumor outgrowth in vivo. Our results define two new antigens that may be developed as immunotherapeutic agents for a broad range of HLA-A2(+) cancers.
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Antígeno HLA-A2/inmunología , Proteínas Sustrato del Receptor de Insulina/inmunología , Neoplasias/inmunología , Fosfopéptidos/inmunología , Fosfatasas cdc25/inmunología , Animales , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Cancers produce soluble and cell-associated molecules that can suppress or alter antitumor immunity. Preclinical studies suggest the disease burden may alter the cytokine profile of helper T cell responses to cancer antigens. We studied cytokine production by helper T cells responding to vaccination with 6 melanoma helper peptides (6MHP) in blood and lymph nodes. METHODS: Twenty-three patients with stage IIIB-IV melanoma received a 6MHP vaccine. Antigen-reactive T cells from blood and draining lymph nodes were cultured, exposed to antigen, and then supernatants (days 2 and 5) were assayed for Th1 and Th2 cytokines. Results from 4 time points were compared to pre-vaccine levels. RESULTS: Cytokine responses to vaccinating peptides were observed in 83% of patients. Th1 favoring responses were most common (17 of 19 responders). The most abundant cytokines produced were IFN-γ and IL-5 in the PBMC's. IL-2 responses predominated in cells obtained from draining lymph nodes in 2-day culture but not in 5-day cultures. Patients with clinically measurable disease produced similar levels of total cytokine and similar degree of Th1 polarization as patients with no evidence of disease (NED). CONCLUSIONS: The MHC class II-associated peptides used in this study induced helper T cells with a Th1-biased cytokine response in both PBMC and sentinel immunized nodes. Most patients can mount a Th1 dominant response to these peptides. Future studies are needed to test newer vaccine adjuvants in combination with these peptides. TRIAL REGISTRATION: CDR0000378171, Clinicaltrials: NCT00089219.
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PURPOSE: Survival after amputation for melanoma is short; however, rare long-term survivors are reported. The mechanism for durable systemic tumor control in patients with regional failure is not known. To explore whether systemic tumor immunity may be implicated, tumor and circulating immune responses were examined in a patient who survived disease-free 14 years after hip disarticulation. METHODS: A 71-year-old female with extensive regional metastases of melanoma in the left lower extremity underwent amputation for palliative reasons. Tumor was collected at surgery, and blood was collected during follow-up. Tumor sections were evaluated for lymphocytic infiltration and NY-ESO-1 expression by immunohistochemistry. Cellular immune responses to defined tumor antigens were evaluated by ELISPOT assay, and antibody responses to a panel of tumor antigens were assayed by ELISA. RESULTS: The patient's tumor had minimal lymphocytic infiltrate (immunotype A). NY-ESO-1 was strongly expressed by the melanoma cells. Circulating T-cell responses to NY-ESO-1 peptides were observed 6 and 12 years postoperatively, and antibodies to NY-ESO-1 were detected 2-6 years after surgery. CONCLUSION: The patient described in this report experienced relentless regional tumor progression, with intravascular metastases, and then 14-year systemic disease-free survival after palliative resection, without evidence of melanoma recurrence before death from other causes. Her immune response to NY-ESO-1 likely failed to control established regional metastases because T cells were unable to infiltrate them. It is possible, however, that among other factors, the host immune response may have contributed to systemic protection.
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Amputación Quirúrgica/métodos , Melanoma/cirugía , Recurrencia Local de Neoplasia/cirugía , Neoplasias Cutáneas/cirugía , Anciano , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Femenino , Humanos , Extremidad Inferior/cirugía , Linfocitos/inmunología , Linfocitos/metabolismo , Melanoma/inmunología , Melanoma/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/metabolismo , Cuidados Paliativos , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Sobrevivientes , Factores de TiempoRESUMEN
Immune cells infiltrating the microenvironment of melanoma metastases may either limit or promote tumor progression, but the characteristics that distinguish these effects are obscure. In this study, we systematically evaluated the composition and organization of immune cells that infiltrated melanoma metastases in human patients. Three histologic patterns of immune cell infiltration were identified, designated immunotypes A, B, and C. Immunotype A was characterized by no immune cell infiltrate. Immunotype B was characterized by infiltration of immune cells limited only to regions proximal to intratumoral blood vessels. Immunotype C was characterized by a diffuse immune cell infiltrate throughout a metastatic tumor. These immunotypes represented 29%, 63%, and 8% of metastases with estimated median survival periods of 15, 23, and 130 months, respectively. Notably, from immunotypes A to C, there were increasing proportions of B cells and decreasing proportions of macrophages. Overall, the predominant immune cells were T cells (53%), B cell lineage cells (33%), and macrophages (13%), with natural killer and mature dendritic cells only rarely present. Whereas higher densities of CD8(+) T cells correlated best with survival, a higher density of CD45(+) leukocytes, T cells, and B cells also correlated with increased survival. Together, our findings reveal striking differences in the immune infiltrate in melanoma metastases in patients, suggesting microenvironmental differences in immune homing receptors and ligands that affect immune cell recruitment. These findings are important, not only by revealing how the immune microenvironment can affect outcomes but also because they reveal characteristics that may help improve individualized therapy for patients with metastatic melanoma.
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Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B/inmunología , Células Dendríticas/inmunología , Femenino , Humanos , Inmunofenotipificación , Subgrupos Linfocitarios/inmunología , Macrófagos/inmunología , Masculino , Melanoma/irrigación sanguínea , Melanoma/mortalidad , Melanoma/patología , Melanoma/secundario , Persona de Mediana Edad , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Sobrevivientes , Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Adulto JovenRESUMEN
Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell (DC) activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in 2 clinical trials of melanoma patients. In 1 study (Mel38), patients received 1 injection with an adjuvant mixture alone, composed of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature DCs, and vessels resembling high endothelial venules (HEVs). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DCs, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4and CD8 T lymphocytes, as well as FoxP3CD4 lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Adyuvante de Freund/administración & dosificación , Lípidos/administración & dosificación , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Piel/patología , Antígenos de Neoplasias/inmunología , Linfocitos B/patología , Comunicación Celular , Movimiento Celular , Microambiente Celular , Citocinas/metabolismo , Células Dendríticas/patología , Factores de Transcripción Forkhead/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Humanos , Inmunización Secundaria , Inmunomodulación , Inyecciones Intradérmicas , Melanoma/patología , Melanoma/terapia , Neovascularización Fisiológica , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Piel/irrigación sanguínea , Piel/inmunología , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapia , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patologíaRESUMEN
BACKGROUND: Melanoma vaccines have not been optimized. Adjuvants are added to activate dendritic cells (DCs) and to induce a favourable immunologic milieu, however, little is known about their cellular and molecular effects in human skin. We hypothesized that a vaccine in incomplete Freund's adjuvant (IFA) would increase dermal Th1 and Tc1-lymphocytes and mature DCs, but that repeated vaccination may increase regulatory cells. METHODS: During and after 6 weekly immunizations with a multipeptide vaccine, immunization sites were biopsied at weeks 0, 1, 3, 7, or 12. In 36 participants, we enumerated DCs and lymphocyte subsets by immunohistochemistry and characterized their location within skin compartments. RESULTS: Mature DCs aggregated with lymphocytes around superficial vessels, however, immature DCs were randomly distributed. Over time, there was no change in mature DCs. Increases in T and B-cells were noted. Th2 cells outnumbered Th1 lymphocytes after 1 vaccine 6.6:1. Eosinophils and FoxP3+ cells accumulated, especially after 3 vaccinations, the former cell population most abundantly in deeper layers. CONCLUSIONS: A multipeptide/IFA vaccine may induce a Th2-dominant microenvironment, which is reversed with repeat vaccination. However, repeat vaccination may increase FoxP3+T-cells and eosinophils. These data suggest multiple opportunities to optimize vaccine regimens and potential endpoints for monitoring the effects of new adjuvants. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00705640.
Asunto(s)
Inmunofenotipificación/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Melanoma/patología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Vacunación , Adyuvantes Inmunológicos/farmacología , Vacunas contra el Cáncer/inmunología , Diferenciación Celular/efectos de los fármacos , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Masculino , Persona de Mediana Edad , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunología , Factores de TiempoRESUMEN
BACKGROUND: Human cancer vaccines incorporating autologous tumor cells carry a risk of implantation and subsequent metastasis of viable tumor cells into the patient who is being treated. Despite the fact that the melanoma cell preparations used in a recent vaccine trial (Mel37) were gamma-irradiated (200 Gy), approximately 25% of the preparations failed quality control release criteria which required that the irradiated cells incorporate 3H-thymidine at no more than 5% the level seen in the non-irradiated cells. We have, therefore, investigated ultraviolet (UV)-irradiation as a possible adjunct to, or replacement for gamma-irradiation. METHODS: Melanoma cells were gamma- and/or UV-irradiated. 3H-thymidine uptake was used to assess proliferation of the treated and untreated cells. Caspase-3 activity and DNA fragmentation were measured as indicators of apoptosis. Immunohistochemistry and Western blot analysis was used to assess antigen expression. RESULTS: UV-irradiation, either alone or in combination with gamma-irradiation, proved to be extremely effective in controlling the proliferation of melanoma cells. In contrast to gamma-irradiation, UV-irradiation was also capable of inducing significant levels of apoptosis. UV-irradiation, but not gamma-irradiation, was associated with the loss of tyrosinase expression. Neither form of radiation affected the expression of gp100, MART-1/MelanA, or S100. CONCLUSION: These results indicate that UV-irradiation may increase the safety of autologous melanoma vaccines, although it may do so at the expense of altering the antigenic profile of the irradiated tumor cells.
Asunto(s)
Vacunas contra el Cáncer , Rayos gamma , Melanoma/terapia , Células Tumorales Cultivadas/efectos de la radiación , Rayos Ultravioleta , Apoptosis/efectos de la radiación , Western Blotting , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , InmunohistoquímicaRESUMEN
PURPOSE: A phase I/II trial was performed to evaluate the safety and immunogenicity of a novel melanoma vaccine comprising six melanoma-associated peptides defined as antigenic targets for melanoma-reactive helper T cells. Source proteins for these peptides include MAGE proteins, MART-1/MelanA, gp100, and tyrosinase. PATIENTS AND METHODS: Thirty-nine patients with stage IIIB to IV melanoma were vaccinated with this six-peptide mixture weekly at three dose levels, with a preceding phase I dose escalation and subsequent random assignment among the dose levels. Helper T-lymphocyte responses were assessed by in vitro proliferation assay and delayed-type hypersensitivity skin testing. Patients with measurable disease were evaluated for objective clinical response by Response Evaluation Criteria in Solid Tumors. RESULTS: Vaccination with the helper peptide vaccine was well tolerated. Proliferation assays revealed induction of T-cell responses to the melanoma helper peptides in 81% of patients. Among 17 patients with measurable disease, objective clinical responses were observed in two patients (12%), with response durations of 1 and 3.9+ years. Durable stable disease was observed in two additional patients for periods of 1.8 and 4.6+ years. CONCLUSION: Results of this study support the safety and immunogenicity of a vaccine comprised of six melanoma helper peptides. There is also early evidence of clinical activity.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/toxicidad , Melanocitos/inmunología , Melanoma/inmunología , Melanoma/prevención & control , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/prevención & control , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Diferenciación/inmunología , Antígenos de Neoplasias/inmunología , Proliferación Celular , Femenino , Humanos , Antígeno MART-1 , Masculino , Melanoma/patología , Glicoproteínas de Membrana/inmunología , Persona de Mediana Edad , Monofenol Monooxigenasa/inmunología , Proteínas de Neoplasias/inmunología , Estadificación de Neoplasias , Neoplasias Cutáneas/patología , Pruebas Cutáneas , Linfocitos T Colaboradores-Inductores/inmunología , Vitíligo/inducido químicamente , Antígeno gp100 del MelanomaRESUMEN
The effectiveness of peptide-based cancer vaccines depends on the ability of peptides to bind to MHC molecules on the surface of antigen-presenting cells, where they reconstitute epitopes for cytotoxic T lymphocytes (CTLs). Multivalent vaccines have advantages over single-peptide vaccines; however, peptides may compete for binding to the same MHC molecules. In particular, it is possible that peptides with high affinity for MHC molecules prevent the binding of lower-affinity peptides. However, only small numbers of peptide/MHC complexes per cell are required for CTL recognition. Thus, the authors hypothesized that competition of peptides for MHC binding would not significantly reduce CTL recognition of individual peptides within a multiple-peptide mixture, and this hypothesis was tested by a series of experiments performed in vitro. In multiple experiments, two peptides with different affinities for HLA-A*0201 molecules were mixed at various concentrations and pulsed onto HLA-A2 cells, which were then evaluated for susceptibility to lysis by HLA-A*0201-restricted CTLs. CTL recognition of the melanoma peptides gp100(154-162) (KTWGQYWQV), gp100(280-288) (YLEPGPVTA), and tyrosinase(369-377D) (YMDGTMSQV) was maintained even when target cells were co-pulsed with equimolar concentrations of peptides with comparable or higher affinity for HLA-A2. In some cases, CTL recognition was maintained even when the higher-affinity peptide was present at concentrations several orders of magnitude higher than the target peptide. In addition, CTLs generated by in vitro stimulation with a peptide mixture developed reactivity to three different peptides, at a level comparable to that obtained by stimulation with each individual peptide separately. These data suggest that CTLs can respond to multiple peptides presented on the same antigen-presenting cells and justify further investigation, in clinical trials, of multiple-peptide cancer vaccines.