RESUMO
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
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
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.