RESUMO
Limited representation of intratumoral immune cells is a major barrier to tumor control. However, simply enhancing immune responses in tumor-draining lymph nodes or through adoptive transfer may not overcome the limited ability of tumor vasculature to support effector infiltration. An alternative is to promote a sustained immune response intratumorally. This idea has gained traction with the observation that many tumors are associated with tertiary lymphoid structures (TLS), which organizationally resemble lymph nodes. These peri- and intratumoral structures are usually, but not always, associated with positive prognoses in patients. Preclinical and clinical data support a role for TLS in modulating immunity in the tumor microenvironment. However, there appear to be varied functions of TLS, potentially based on their structure or location in relation to the tumor or the origin or location of the tumor itself. Understanding more about TLS development, composition, and function may offer new therapeutic opportunities to modulate antitumor immunity.
Assuntos
Imunidade , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , Estruturas Linfoides Terciárias/imunologia , Microambiente Tumoral/imunologia , Animais , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/metabolismo , Neoplasias/mortalidade , Neoplasias/patologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Prognóstico , Estruturas Linfoides Terciárias/metabolismoRESUMO
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.
Assuntos
Antígenos de Neoplasias/imunologia , Imunofenotipagem , Melanoma Experimental/imunologia , Neoplasias Cutâneas/imunologia , Animais , Linhagem Celular Tumoral/transplante , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas B-raf/genética , Pele/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Análise Serial de Tecidos , beta Catenina/genéticaRESUMO
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.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Proteínas de Neoplasias/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/uso terapêutico , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/uso terapêutico , Humanos , Injeções Intramusculares , Pessoa de Meia-Idade , Proteínas de Neoplasias/uso terapêutico , Projetos Piloto , Resultado do TratamentoRESUMO
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.
Assuntos
Vacinas Anticâncer/imunologia , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/metabolismo , Fatores Imunológicos/uso terapêutico , Imunoterapia/métodos , Interferon gama/uso terapêutico , Melanoma/terapia , Linfócitos T/imunologia , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/imunologia , Movimento Celular , Células Cultivadas , ELISPOT , Feminino , Seguimentos , Humanos , Linfócitos do Interstício Tumoral/patologia , Masculino , Melanoma/mortalidade , Melanoma/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fragmentos de Peptídeos/imunologia , Análise de Sobrevida , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
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.
Assuntos
Aminoquinolinas/uso terapêutico , Antígenos de Neoplasias/imunologia , Antineoplásicos/uso terapêutico , Vacinas Anticâncer/imunologia , Melanoma/terapia , Fragmentos de Peptídeos/imunologia , Neoplasias Cutâneas/terapia , Linfócitos T/efeitos dos fármacos , Administração Tópica , Idoso , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Terapia Combinada , Citocinas/genética , Citocinas/metabolismo , ELISPOT , Feminino , Humanos , Imiquimode , Linfócitos do Interstício Tumoral/patologia , Masculino , Melanoma/secundário , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Cutâneas/secundário , Linfócitos T/imunologia , Receptor 7 Toll-Like/agonistas , Transcriptoma/imunologia , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Quimiocina CXCL10/metabolismo , Interferon gama/metabolismo , Melanoma/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 6 Toll-Like/agonistas , Linhagem Celular Tumoral , Humanos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Th17 cells represent a subset of CD4+ T helper cells that secrete the proinflammatory cytokine IL-17. Th17 cells have been ascribed both a beneficial role in promoting clearance of pathogenic fungi and bacteria, and a pathogenic role in autoimmune diseases. Here we identify the tyrosine phosphatase SHP-1 as a critical regulator of Th17 development, using 3 complementary approaches. Impaired SHP-1 activity through genetic deletion of SHP-1, transgenic expression of an inducible dominant negative SHP-1, or pharmacologic inhibition of SHP-1 strongly promotes the development of Th17. Ex vivo Th17 skewing assays demonstrate that genetic or pharmacologic disruption of SHP-1 activity in T cells results in a hyper-response to stimulation via IL-6 and IL-21, 2 cytokines that promote Th17 development. Mechanistically, we find that SHP-1 decreases the overall cytokine-induced phosphorylation of STAT3 in primary CD4+ T cells. These data identify SHP-1 as a key modifier of IL-6-and IL-21-driven Th17 development via regulation of STAT3 signaling and suggest SHP-1 as a potential new therapeutic target for manipulating Th17 differentiation in vivo.
Assuntos
Diferenciação Celular/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Células Th17/fisiologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Colite/genética , Colite/imunologia , Colite/metabolismo , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Interleucina-6/metabolismo , Interleucinas/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Th17/metabolismoRESUMO
Tertiary lymphoid structures (TLS) are ectopic lymphoid structures that can arise in human cancers and are associated with improved overall survival (OS) and response to immune checkpoint blockade (ICB) in several cancers, including non-desmoplastic metastatic melanoma (NDMM). Desmoplastic melanoma (DM) has one of the highest response rates to ICB, and we previously identified that primary DM (PDM) contains TLS. Despite the association of TLS with survival and ICB response, it is unknown whether TLS or associated markers of immune activity can differ between PDM and NDMM. We hypothesized that PDM would contain higher frequencies of TLS than NDMM, that T and B-cell densities and proliferation would be greater in TLS of PDM than TLS of NDMM, and that proliferation rates of T and B-cells in PDM TLS would be concordant with those of intratumoral lymphocytes. We found that four features of TLS in PDM distinguish them from TLS in NDMM. TLS were peritumoral in NDMM but intratumoral in PDM. CD8+ T-cell and CD20+ B-cell densities and proliferative fractions were higher in PDM TLS than NDMM TLS. Additionally, the proliferative fractions of T- and B-cells were concordant between the TLS and tumor site in PDM and discordant in NDMM. Collectively, these data suggest that TLS and associated immune markers can differ across melanoma subsets and suggest that PDM TLS may be more immunologically active and have enhanced immune cell trafficking between tumor and TLS compared to NDMM.
Assuntos
Linfócitos T CD8-Positivos , Melanoma , Estruturas Linfoides Terciárias , Humanos , Biomarcadores , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Melanoma/imunologia , Melanoma/patologia , Estruturas Linfoides Terciárias/imunologia , Estruturas Linfoides Terciárias/patologiaRESUMO
The importance of regulatory T cells (Tregs) for immune tolerance is well recognized, yet the signaling molecules influencing their suppressive activity are relatively poorly understood. In this article, through in vivo studies and complementary ex vivo studies, we make several important observations. First, we identify the cytoplasmic tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) as an endogenous brake and modifier of the suppressive ability of Tregs; consistent with this notion, loss of SHP-1 expression strongly augments the ability of Tregs to suppress inflammation in a mouse model. Second, specific pharmacological inhibition of SHP-1 enzymatic activity via the cancer drug sodium stibogluconate potently augmented Treg suppressor activity both in vivo and ex vivo. Finally, through a quantitative imaging approach, we directly demonstrate that Tregs prevent the activation of conventional T cells and that SHP-1-deficient Tregs are more efficient suppressors. Collectively, our data reveal SHP-1 as a critical modifier of Treg function and a potential therapeutic target for augmenting Treg-mediated suppression in certain disease states.
Assuntos
Tolerância Imunológica/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Citometria de Fluxo , Immunoblotting , Imunoprecipitação , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
This study was designed to screen for preliminary evidence of predictive markers of melanoma response to PD-1 blockade. We hypothesized that the following immune markers would be positive predictors of response: increased densities of CD103 + CD8 + T cells or Th1 lineage T-bet + T cells, high expression of CXCL9-11 and presence of tertiary lymphoid structures. Conversely, we hypothesized that the high expression of barrier molecules would be a negative predictor of response. Patients with advanced melanoma treated with pembrolizumab were identified, and clinical response as well as overall survival data were collected. Tumor samples were evaluated by multiplex immunofluorescence histology. All statistical analyses were performed in R Studio and Microsoft Excel using the Mann-Whitney U test, chi-square test, Spearman's rank correlation and Kaplan-Meier survival curves. Sixty-five advanced melanoma patients were identified, of whom 46 met inclusion criteria and were included in this study. Increased densities ( P = 0.04) and proportions ( P = 0.02) of CD8 + T cells expressing CD103 + were associated with complete response (CR) to pembrolizumab. Improved survival was associated with increased proportions of CD8 + cells expressing CD103 ( P = 0.0085) as well as decreased density of periplakin + cells ( P = 0.012) and periplakin + SOX10 + cells ( P = 0.0012). The density and proportion of CD8 + T cells expressing CD103 + positively correlated with PD-L1 expression, though PD-L1 expression was not significantly correlated with outcomes. This screening study found that increased density and proportion of CD8 + T cells expressing CD103 and decreased density of periplakin were associated with positive outcomes in patients with melanoma metastases treated with pembrolizumab and may warrant further study.
Assuntos
Melanoma , Segunda Neoplasia Primária , Neoplasias Cutâneas , Humanos , Antígeno B7-H1 , Biomarcadores/metabolismo , Linfócitos T CD8-Positivos , Linfócitos do Interstício Tumoral , Melanoma/patologia , Receptor de Morte Celular Programada 1RESUMO
BACKGROUND: A vaccine containing 6 melanoma-associated peptides to stimulate helper T cells (6MHP) is safe, immunogenic, and clinically active. A phase I/II trial was designed to evaluate safety and immunogenicity of 6MHP vaccines plus programmed death 1 (PD-1) blockade. PARTICIPANTS AND METHODS: Participants with advanced melanoma received 6MHP vaccines in an incomplete Freund's adjuvant (6 vaccines over 12 weeks). Pembrolizumab was administered intravenously every 3 weeks. Tumor biopsies at baseline and day 22 were analyzed by multiplex immunohistochemistry. Primary end points were safety (Common Terminology Criteria for Adverse Events V.4.03) and immunogenicity (ex vivo interferon-γ ELISpot assay). Additional end points included changes in the tumor microenvironment (TME) and clinical outcomes. RESULTS: Twenty-two eligible participants were treated: 6 naïve to PD-1 antibody (Ab) and 16 PD-1 Ab-experienced. Median follow-up was 24.4 months. Most common treatment-related adverse events (any grade) included injection site reactions, fatigue, anemia, lymphopenia, fever, elevated aspartate aminotransferase, pruritus, and rash. Treatment-related dose-limiting toxicities were observed in 3 (14%) participants, which did not cross the study safety bound. A high durable T cell response (Rsp) to 6MHP was detected in only one participant, but twofold T cell Rsps to 6MHP were detected in 7/22 (32%; 90% CI (16% to 52%)) by week 13. Objective clinical responses were observed in 23% (1 complete response, 4 partial responses), including 4/6 PD-1 Ab-naïve (67%) and 1/16 PD-1 Ab-experienced (6%). Overall survival (OS) was longer for PD-1 Ab-naïve than Ab-experienced participants (HR 6.3 (90% CI (2.1 to 28.7)). In landmark analyses at 13 weeks, OS was also longer for those with T cell Rsps (HR 6.5 (90% CI (2.1 to 29.2)) and for those with objective clinical responses. TME evaluation revealed increased densities of CD8+ T cells, CD20+ B cells, and Tbet+ cells by day 22. CONCLUSIONS: Treatment with the 6MHP vaccine plus pembrolizumab was safe, increased intratumoral lymphocytes, and induced T cell Rsps associated with prolonged OS. The low T cell Rsp rate in PD-1 Ab-experienced participants corroborates prior murine studies that caution against delaying cancer vaccines until after PD-1 blockade. The promising objective response rate and OS in PD-1 Ab-naïve participants support consideration of a larger study in that setting.
Assuntos
Vacinas Anticâncer , Melanoma , Linfócitos T CD8-Positivos , Humanos , Melanoma/tratamento farmacológico , Receptor de Morte Celular Programada 1 , Microambiente Tumoral , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
The presence of tertiary lymphoid structures (TLS) is correlated with prolonged patient survival in a variety of solid cancers, including melanoma. However, few methods have been described that could enable a more comprehensive understanding of the organization and functionality of TLS in solid cancers. In this chapter, we describe multiplex immunohistochemistry and microscopy approaches for identifying, characterizing, and quantifying TLS and intra-tumoral immune infiltrates in melanoma. The described methods are not limited to melanoma alone and could be used to evaluate tertiary lymphoid structures in a wide variety of human cancers.
Assuntos
Biomarcadores Tumorais/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Melanoma/metabolismo , Estruturas Linfoides Terciárias/metabolismo , Microambiente Tumoral , Humanos , Imuno-Histoquímica , Linfócitos do Interstício Tumoral/patologia , Melanoma/patologia , Estruturas Linfoides Terciárias/patologiaRESUMO
The density of tumour-infiltrating lymphocytes (TILs) in melanoma is correlated with improved clinical prognosis; however, standardized TIL immunotyping and quantification protocols are lacking. Herein, we provide a review of the technologies being utilized for the immunotyping and quantification of melanoma TILs.
Assuntos
Imunofenotipagem , Linfócitos do Interstício Tumoral , Melanoma , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Melanoma/imunologia , Melanoma/patologiaRESUMO
BACKGROUND: Tertiary lymphoid structures (TLSs) are immune aggregates in peripheral tissues that may support adaptive immune responses. Their presence has been associated with clinical response to checkpoint blockade therapy (CBT), but it is unknown whether TLS have prognostic significance independent of CBT in melanoma. We hypothesized that TLS in melanoma metastases would be associated with increased intratumoral lymphocyte infiltration, but that the intra-TLS immunological milieu would be distinct from the intratumoral immunological milieu. We also hypothesized that the presence of TLS would be associated with improved survival, and that TLS maturation or intra-TLS lymphocyte activity would also correlate with survival. METHODS: Cutaneous melanoma metastases (CMM) from 64 patients were evaluated by multiplex immunofluorescence for the presence and maturation status of TLS. Intra-TLS lymphocyte density, proliferation and B-cell Ig somatic hypermutation (AID+) were analyzed, as were markers of T-cell exhaustion and Th1/Tc1 differentiation. Associations between TLS maturation and intra-TLS immunologic activity were assessed, as well as associations with intratumoral immune cell infiltration. Independent associations with overall survival (OS) were assessed using log-rank tests and Cox proportional hazards models. RESULTS: TLS were identified in 30 (47%) of 64 CMM (TLS+) and were associated with increased intratumoral lymphocyte infiltration. However, proliferation of intra-TLS lymphocytes did not correlate with intratumoral lymphocyte proliferation. Most were early TLS; however, subsets of primary or secondary follicle-like TLS were also present. TLS+ lesions were associated with lower risk of tumor recurrence after metastasectomy and with improved OS in multivariate analyses (HR 0.51, p=0.04). OS was longer for TLS with low fractions of CD21+ B-cells (HR 0.29, p=0.02) and shorter for those with low AID+ fraction of B-cells (HR 2.74, p=0.03). CONCLUSIONS: The presence of TLS in CMMs is associated with improved OS in patients treated with surgery before CBT, but TLS vary widely in maturation state, in proportions of proliferating T and B cells, and in markers of B cell function, including AID and CD21. Importantly, these features have additional prognostic significance, which suggest that some TLS may have regulatory function, while others functioning to support antigen-driven immune responses, depending on the cellular composition and activation status.
Assuntos
Linfócitos B/metabolismo , Melanoma/genética , Neoplasias Cutâneas/genética , Hipermutação Somática de Imunoglobulina , Estruturas Linfoides Terciárias/genética , Linfócitos B/patologia , Feminino , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Masculino , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Análise de Sobrevida , Estruturas Linfoides Terciárias/metabolismo , Estruturas Linfoides Terciárias/patologia , Microambiente Tumoral , Melanoma Maligno CutâneoRESUMO
BACKGROUND: tumor-infiltrating lymphocytes are prognostic in many human cancers. However, the prognostic value of lymphocytes infiltrating glioblastoma (GBM), and roles in tumor control or progression are unclear. We hypothesized that B and T cell density, and markers of their activity, proliferation, differentiation, or function, would have favorable prognostic significance for patients with GBM. METHODS: initial resection specimens from 77 patients with IDH1/2 wild type GBM who received standard-of-care treatment were evaluated with multiplex immunofluorescence histology (mIFH), for the distribution, density, differentiation, and proliferation of T cells and B cells, as well as for the presence of tertiary lymphoid structures (TLS), and IFNγ expression. Immune infiltrates were evaluated for associations with overall survival (OS) by univariate and multivariate Cox proportional hazards modeling. RESULTS: in univariate analyses, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.36, p = 0.001) and CD20+ cells (HR 0.51, p = 0.008), as well as CD8+Tbet+ cells (HR 0.46, p = 0.004), and RORγt+ cells (HR 0.56, p = 0.04). Conversely, IFNγ intensity was associated with diminished OS (HR 0.59, p = 0.036). In multivariable analyses, adjusting for clinical variables, including age, resection extent, Karnofsky Performance Status (KPS), and MGMT methylation status, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.15, p < 0.001), and higher ratios of CD8+ cells to CD4+ cells (HR 0.31, p = 0.005). Diminished OS was associated with increases in patient age (HR 1.21, p = 0.005) and higher mean intensities of IFNγ (HR 2.13, p = 0.027). CONCLUSIONS: intratumoral densities of proliferating CD8 T cells and higher CD8/CD4 ratios are independent predictors of OS in patients with GBM. Paradoxically, higher mean intensities of IFNγ in the tumors were associated with shorter OS. These findings suggest that survival may be enhanced by increasing proliferation of tumor-reactive CD8+ T cells and that approaches may be needed to promote CD8+ T cell dominance in GBM, and to interfere with the immunoregulatory effects of IFNγ in the tumor microenvironment.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Glioblastoma/imunologia , Glioblastoma/patologia , Linfócitos B/imunologia , Biomarcadores Tumorais/metabolismo , Agregação Celular , Contagem de Células , Diferenciação Celular , Proliferação de Células , Feminino , Humanos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Análise de SobrevidaRESUMO
Tumor-associated tertiary lymphoid structures (TA-TLS) are associated with enhanced patient survival and responsiveness to cancer therapies, but the mechanisms underlying their development are unknown. We show here that TA-TLS development in murine melanoma is orchestrated by cancer-associated fibroblasts (CAF) with characteristics of lymphoid tissue organizer cells that are induced by tumor necrosis factor receptor signaling. CAF organization into reticular networks is mediated by CD8 T cells, while CAF accumulation and TA-TLS expansion depend on CXCL13-mediated recruitment of B cells expressing lymphotoxin-α1ß2. Some of these elements are also overrepresented in human TA-TLS. Additionally, we demonstrate that immunotherapy induces more and larger TA-TLS that are more often organized with discrete T and B cell zones, and that TA-TLS presence, number, and size are correlated with reduced tumor size and overall response to checkpoint immunotherapy. This work provides a platform for manipulating TA-TLS development as a cancer immunotherapy strategy.
Assuntos
Fibroblastos Associados a Câncer/patologia , Neoplasias/imunologia , Neoplasias/patologia , Estruturas Linfoides Terciárias/imunologia , Animais , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Proliferação de Células , Humanos , Imunoterapia , Ativação Linfocitária/imunologia , Receptor beta de Linfotoxina/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias/terapia , Peritônio/patologia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de SinaisRESUMO
Ipilimumab (IPI) can enhance immunity to the cancer-testis antigen NY-ESO-1. A clinical trial was designed to assess safety, immunogenicity, and clinical responses with IPI + NY-ESO-1 vaccines and effects on the tumor microenvironment (TME). Patients with measurable NY-ESO-1+ tumors were enrolled among three arms: A) IPI + NY-ESO-1 protein + poly-ICLC (pICLC) + incomplete Freund's adjuvant (IFA); B) IPI + NY-ESO-1 overlapping long peptides (OLP) + pICLC + IFA; and C) IPI + NY-ESO-1 OLP + pICLC. Clinical responses were assessed by irRC. T cell and Ab responses were assessed by ex vivo IFN-gamma ELIspot and ELISA. Tumor biopsies pre- and post-treatment were evaluated for immune infiltrates. Eight patients were enrolled: 5, 2, and 1 in Arms A-C, respectively. There were no DLTs. Best clinical responses were SD (4) and PD (4). T-cell and antibody (Ab) responses to NY-ESO-1 were detected in 6 (75%) and 7 (88%) patients, respectively, and were associated with SD. The breadth of Ab responses was greater for patients with SD than PD (p = .036). For five patients evaluable in the TME, treatment was associated with increases in proliferating (Ki67+) CD8+ T cells and decreases in RORγt+ CD4+ T cells. T cell densities increased for those with SD. Detection of T cell responses to NY-ESO-1 ex vivo in most patients suggests that IPI may have enhanced those responses. Proliferating intratumoral CD8+ T cells increased after vaccination plus IPI suggesting favorable impact of IPI plus NY-ESO-1 vaccines on the TME. List of Abbreviations: Ab = antibody; CTCAE = NCI Common Terminology Criteria for Adverse Events; DHFR/DHRP = dihydrofolate reductase; DLT = Dose-limiting toxicity; ELISA = enzyme-linked immunosorbent assay; IFA = incomplete Freund's adjuvant (Montanide ISA-51); IFNγ = Interferon gamma; IPI = Ipilimumab; irRC = immune-related response criteria; mIFH = multispectral immunofluorescence histology; OLP = NY-ESO-1 overlapping long peptides; PBMC = peripheral blood mononuclear cells; PD = Progressive disease; pICLC = poly-ICLC (Hiltonol), a TLR3/MDA-5 agonist; RLT = Regimen-limiting Toxicity; ROI = regions of interest; RT = room temperature; SAE = serious adverse event; SD = stable disease; TEAE = treatment-emergent adverse events; TLR = toll-like receptor; TME = tumor microenvironment; TRAE = treatment-related adverse events.
Assuntos
Vacinas Anticâncer , Melanoma , Antígenos de Neoplasias , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Vacinas Anticâncer/efeitos adversos , Humanos , Ipilimumab/uso terapêutico , Leucócitos Mononucleares , Masculino , Melanoma/tratamento farmacológico , Microambiente TumoralRESUMO
In a variety of solid tumors, the presence of higher densities of tumor-infiltrating lymphocytes or tertiary lymphoid structures (TLS) are correlated with prolonged patient survival. Murine studies are usually required to define mechanisms that govern immunologic infiltrate in tumors. However, few methods have been described that could enable a more comprehensive understanding of the functionality of intratumoral immune infiltrate and TLS in solid murine cancers. In this chapter, we describe multiplex immunohistochemistry and microscopy approaches for identifying, characterizing, and quantifying intratumoral immune infiltrate and TLS in murine tumor models.
Assuntos
Neoplasias , Estruturas Linfoides Terciárias , Animais , Humanos , Imuno-Histoquímica , Linfócitos do Interstício Tumoral , Camundongos , Microambiente TumoralRESUMO
BACKGROUND: Immunogenicity of cancer vaccines is impacted by adjuvants and schedule, but systematic assessments of their effects have not been performed. Montanide ISA-51, an incomplete Freund's adjuvant (IFA), is used in many vaccine trials, but concerns have been raised about negative effects in murine studies. We found in humans that IFA enhances systemic immune responses and that repeat vaccination at one site (same site vaccination (SSV)) creates tertiary lymphoid structures (TLS) in the vaccine site microenvironment (VSME). We hypothesized that vaccination with peptides+IFA+pICLC or SSV×3 with peptides in IFA would create an immunogenic milieu locally at the VSME, with activated dendritic cells (DC), TLS-associated chemokines and a Th1-dominant VSME. METHODS: Biopsies of the VSME were obtained from participants on two clinical trials who were immunized with multiple melanoma peptides (MELITAC 12.1) in adjuvants comprising IFA and/or the TLR3-agonist pICLC. Biopsies were obtained either a week after one vaccine or a week after SSV×3. Controls included normal skin and skin injected with IFA without peptides. Gene expression analysis was performed by RNAseq. RESULTS: VSME samples were evaluated from 27 patients. One vaccine with peptides in pICLC+IFA enhanced expression of CD80, CD83, CD86 (p<0.01), CD40 and CD40L (p<0.0001) over normal skin; these effects were significantly enhanced for SSV with peptides+IFA. Vaccines containing pICLC increased expression of TBX21 (T-bet) but did not decrease GATA3 over normal skin, whereas SSV with peptides in IFA dramatically enhanced TBX21 and decreased GATA3, with high expression of IFNγ and STAT1. SSV with peptides in IFA also reduced arginase-1 (ARG1) expression and enhanced expression of TLR adapter molecules TICAM-1 (TRIF) and MYD88. Furthermore, SSV with IFA and peptides also enhanced expression of chemokines associated with TLS formation. CONCLUSIONS: These findings suggest that SSV with peptides in IFA enhances CD40L expression by CD4 T cells, supports a Th1 microenvironment, with accumulation of activated and mature DC. Increased expression of TLR adaptor proteins after SSV with peptides in IFA might implicate effects of the skin microbiome. Reduced ARG1 may reflect diminished suppressive myeloid activity in the VSME. TRIAL REGISTRATION NUMBER: (NCT00705640, NCT01585350).
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Adjuvante de Freund/administração & dosagem , Lipídeos/administração & dosagem , Melanoma/terapia , Neoplasias Cutâneas/terapia , Vacinação/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/imunologia , Arginase/metabolismo , Biópsia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Vacinas Anticâncer/imunologia , Ensaios Clínicos Fase I como Assunto , Feminino , Adjuvante de Freund/imunologia , Humanos , Imunização Secundária/métodos , Imunogenicidade da Vacina , Injeções Intralesionais , Lipídeos/imunologia , Masculino , Manitol/administração & dosagem , Manitol/análogos & derivados , Manitol/imunologia , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Ácidos Oleicos/administração & dosagem , Ácidos Oleicos/imunologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Pele/imunologia , Pele/patologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Receptores Toll-Like/metabolismo , Microambiente Tumoral/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Adulto JovemRESUMO
Desmoplastic melanomas (DM) have unique and challenging clinical presentations and histomorphology. A characteristic feature is the presence of scattered lymphoid aggregates. However, the nature of these aggregates is not defined. We hypothesized that they may be tertiary lymphoid structures (TLS), and may be associated with programmed death ligand 1 (PD-L1) expression. We searched our tissue database for 'pure' DMs and for scars as control tissues, collected clinical information, and reviewed H&E histology. We performed multispectral imaging after staining for CD8, CD20, PNAd, FoxP3, CD83, and Ki67, and assessed PD-L1 expression by immunohistochemistry. Pure DM samples were evaluable in 11 patients. All had desmoplastic stroma and lymphoid aggregates on H&E. The lymphoid aggregates of eight of the 11 (72%) DM samples and only three of the 11 scars contained features of TLS, defined as distinct clusters of B cells and CD8 T cells, CD83 dendritic cells in T-cell zones, and PNAd vasculature resembling high endothelial venules. PD-L1 was expressed by at least 1% of melanoma cells in six and by at least 5% of immune cells in 10 of the 11 DM samples. We found that most lymphoid aggregates in DM are organized, classical TLS. PD-L1 expression was detected in most cases and was highest in two cases of DM with TLS. However, low PD-L1 expression in some cases suggests that some DM cells may be unresponsive to interferon-γ. TLS support antigen presentation and T-cell responses in chronic inflammation and cancer. Their presence in DM likely reflects an adaptive immune response, which may be enhanced with immune therapies.