RESUMO
Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting.
Assuntos
Anticorpos , Bioensaio , Europa (Continente) , Estados UnidosRESUMO
Interleukin (IL) 17-secreting CD4(+) helper T cells (Th17 cells) are essential for host defense at mucosal surfaces, and Th17 cell dysregulation can result in autoimmunity. Exposure to microbial products, such as bacterial LPS, can affect the ability of dendritic cells (DCs) to polarize Th17 cells. Acyloxyacyl hydrolase (AOAH) is a mammalian enzyme expressed by antigen (Ag)-presenting cells that deacylates and thereby inactivates LPS in host tissues. We hypothesized that inactivation of intestinal microbiota-derived LPS by AOAH influences the ability of DCs to polarize and generate Th17 effector cells. We found that LPS-containing Gram-negative microbiota augmented the differentiation of Ag-specific Th17 cells, and identified a colonic DC subset (CD103(+)CD11b(+)ALDH(-)) displaying a unique capacity to both express AOAH and polarize Th17 cells. Compared with WT, these Aoah(-/-) colonic DCs produce less IL-6, resulting in diminished Ag-specific Th17 polarization and increased regulatory T-cell induction in vitro. Oral administration of LPS led to reduced IL-6 production from CD103(+)CD11b(+)ALDH(-) colonic DCs in Aoah(-/-) mice compared with Aoah(+/+) mice, resulting in an abrogated Ag-specific Th17 response in the colon after mucosal immunization that could be rescued by systemic delivery of recombinant IL-6. These data identify the ability of AOAH to modulate microbiota signals that drive Th17 polarization and influence mucosal T-cell immunity, and suggest that host pathways to handle microbiota-derived products may be targeted to modulate Th17 responses in the context of inflammatory disorders or infection at mucosal surfaces.
Assuntos
Hidrolases de Éster Carboxílico/deficiência , Colo/metabolismo , Células Dendríticas/citologia , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/metabolismo , Células Th17/citologia , Animais , Células Apresentadoras de Antígenos/citologia , Antígenos CD/metabolismo , Células da Medula Óssea/citologia , Antígeno CD11b/metabolismo , Linfócitos T CD4-Positivos/citologia , Hidrolases de Éster Carboxílico/fisiologia , Endotoxinas/metabolismo , Feminino , Citometria de Fluxo , Inflamação , Cadeias alfa de Integrinas/metabolismo , Interleucina-6/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteínas Recombinantes/metabolismo , Linfócitos T/citologia , Receptores Toll-Like/metabolismoRESUMO
BACKGROUND & AIMS: Treatment of inflammatory bowel disease would benefit from specific targeting of therapeutics to the intestine. We developed a strategy for localized delivery of the immunosuppressive cytokine interleukin (IL)-27, which is synthesized actively in situ by the food-grade bacterium Lactococcus lactis (LL-IL-27), and tested its ability to reduce colitis in mice. METHODS: The 2 genes encoding mouse IL-27 were synthesized with optimal codon use for L lactis and joined with a linker; a signal sequence was added to allow for product secretion. The construct was introduced into L lactis. Colitis was induced via transfer of CD4(+)CD45RB(hi) T cells into Rag(-/-) mice to induce colitis; 7.5 weeks later, LL-IL-27 was administered to mice via gavage. Intestinal tissues were collected and analyzed. RESULTS: LL-IL-27 administration protected mice from T-cell transfer-induced enterocolitis and death. LL-IL-27 reduced disease activity scores, pathology features of large and small bowel, and levels of inflammatory cytokines in colonic tissue. LL-IL-27 also reduced the numbers of CD4(+) and IL-17(+) T cells in gut-associated lymphoid tissue. The effects of LL-IL-27 required production of IL-10 by the transferred T cells. LL-IL-27 was more effective than either LL-IL-10 or systemic administration of recombinant IL-27 in reducing colitis in mice. LL-IL-27 also reduced colitis in mice after administration of dextran sodium sulfate. CONCLUSIONS: LL-IL-27 reduces colitis in mice by increasing the production of IL-10. Mucosal delivery of LL-IL-27 could be a more effective and safer therapy for inflammatory bowel disease.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Enterocolite/imunologia , Fatores Imunológicos/administração & dosagem , Doenças Inflamatórias Intestinais , Interleucina-10/imunologia , Interleucinas/administração & dosagem , Mucosa Intestinal/imunologia , Lactococcus lactis , Administração Oral , Animais , Modelos Animais de Doenças , Fatores Imunológicos/farmacologia , Interleucinas/imunologia , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Linfócitos T , Transformação BacterianaRESUMO
Mechanisms underlying modern increases in prevalence of human inflammatory diseases remain unclear. The hygiene hypothesis postulates that decreased microbial exposure has, in part, driven this immune dysregulation. However, dietary fatty acids also influence immunity, partially through modulation of responses to microbes. Prior reports have described the direct effects of high-fat diets on the gut microbiome and inflammation, and some have additionally shown metabolic consequences for offspring. Our study sought to expand on these previous observations to identify the effects of parental diet on offspring immunity using mouse models to provide insights into challenging aspects of human health. To test the hypothesis that parental dietary fat consumption during gestation and lactation influences offspring immunity, we compared pups of mice fed either a Western diet (WD) fatty acid profile or a standard low-fat diet. All pups were weaned onto the control diet to specifically test the effects of early developmental fat exposure on immune development. Pups from WD breeders were not obese or diabetic, but still had worse outcomes in models of infection, autoimmunity, and allergic sensitization. They had heightened colonic inflammatory responses, with increased circulating bacterial LPS and muted systemic LPS responsiveness. These deleterious impacts of the WD were associated with alterations of the offspring gut microbiome. These results indicate that parental fat consumption can leave a "lard legacy" impacting offspring immunity and suggest inheritable microbiota may contribute to the modern patterns of human health and disease.
Assuntos
Gorduras na Dieta/efeitos adversos , Imunidade Inata/imunologia , Fenômenos Fisiológicos da Nutrição Materna/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Animais , Bactérias , Imunoprecipitação da Cromatina , Colo/imunologia , Colo/microbiologia , Dieta , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , GravidezRESUMO
Substance-P and hemokinin-1 are proinflammatory neuropeptides with potential to promote type 1 immunity through agonistic binding to neurokinin-1 receptor (NK1R). Dendritic cells (DCs) are professional antigen-presenting cells that initiate and regulate the outcome of innate and adaptive immune responses. Immunostimulatory DCs are highly desired for the development of positive immunization techniques. DCs express functional NK1R; however, regardless of their potential DC-stimulatory function, the ability of NK1R agonists to promote immunostimulatory DCs remains unexplored. Here, we demonstrate that NK1R signaling activates therapeutic DCs capable of biasing type 1 immunity by inhibition of interleukin-10 (IL-10) synthesis and secretion, without affecting their low levels of IL-12 production. The potent type 1 effector immune response observed following cutaneous administration of NK1R-signaled DCs required their homing in skin-draining lymph nodes (sDLNs) where they induced inflammation and licensed endogenous-conventional sDLN-resident and -recruited inflammatory DCs to secrete IL-12. Our data demonstrate that NK1R signaling promotes immunostimulatory DCs, and provide relevant insight into the mechanisms used by neuromediators to regulate innate and adaptive immune responses.
Assuntos
Células Dendríticas/imunologia , Imunidade Celular/imunologia , Interleucina-12/imunologia , Receptores da Neurocinina-1/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Citometria de Fluxo , Imunização/métodos , Imunofenotipagem , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Receptores da Neurocinina-1/agonistas , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
T helper 17 (Th17) cells play an important role in mucosal host defense through production of the signature cytokines IL-17 and IL-22. Prostaglandin E2 (PGE2) has been shown to enhance IL-17 production by mature Th17 cells. However, when present during Th17 cell differentiation, we found that PGE2 inhibited the transcription factor IRF4 and suppressed production of IL-17 but not IL-22. We show that IRF4 was required for IL-17 expression but inhibited IL-22 expression, highlighting the potential for discordant regulation of these two cytokines in Th17 cells. The pathogenic fungus Cryptococcus neoformans produces PGE2, and we found that it uses PGE2- and IRF4-dependent mechanisms to specifically inhibit induction of IL-17 during Th17 cell differentiation. Blockade of host PGE2 during infection led to increased IL-17 production from CD4(+) T cells and increased survival of mice. These findings suggest that host- or pathogen-derived PGE2 can act directly on Th17 cells during differentiation to inhibit IL-17-dependent antimicrobial responses.
Assuntos
Cryptococcus neoformans/metabolismo , Dinoprostona/metabolismo , Fatores Reguladores de Interferon/antagonistas & inibidores , Interleucina-17/biossíntese , Células Th17/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Criptococose/imunologia , Cryptococcus neoformans/patogenicidade , Fatores Reguladores de Interferon/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucinas/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Células Th17/metabolismo , Interleucina 22RESUMO
Human skin-migratory dendritic cells (DCs) have the ability to prime and bias Th1 and Th2 CD4+ T lymphocytes. However, whether human cutaneous DCs are capable of initiating proinflammatory Th17 responses remains undetermined. We report that skin-migratory DCs stimulate allogeneic naive CD4+ T cells that differentiate simultaneously into two distinct effector Th17 and Th1 populations capable of homing to the skin, where they induce severe cutaneous damage. Skin-migratory Langerhans cells (smiLCs) were the main cutaneous DC subset capable of inducing Th17 responses dependent on the combined effects of IL-15 and stabilized IL-6, which resulted in IL-6 trans-signaling of naive CD4+ T cells. Different from smiLCs, purified skin-migratory dermal DCs did not synthesize IL-15 and were unable to bias Th17 responses. Nevertheless, these dermal DCs were capable of differentiating Th17 cells in mixed leukocyte cultures supplemented with IL-15 and stabilized IL-6. Overall, our data demonstrate that human epidermal smiLCs induce Th17 responses by mechanisms different from those previously described and highlight the need to target clinical treatments based on these variations.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Mediadores da Inflamação/fisiologia , Interleucina-17/fisiologia , Pele/imunologia , Pele/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Técnicas de Cocultura , Células Dendríticas/patologia , Humanos , Interleucina-15/biossíntese , Interleucina-15/fisiologia , Interleucina-6/metabolismo , Interleucina-6/fisiologia , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Teste de Cultura Mista de Linfócitos , Técnicas de Cultura de Órgãos , Transdução de Sinais/imunologia , Pele/patologia , Linfócitos T Auxiliares-Indutores/patologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/patologiaRESUMO
Dendritic cells (DCs) are the preferred targets for immunotherapy protocols focused on stimulation of cellular immune responses. However, regardless of initial promising results, ex vivo generated DCs do not always promote immune-stimulatory responses. The outcome of DC-dependent immunity is regulated by proinflammatory cytokines and neuropeptides. Proinflammatory neuropeptides of the tachykinin family, including substance P (SP) and hemokinin-1 (HK-1), bind the neurokinin 1 receptor (NK1R) and promote stimulatory immune responses. Nevertheless, the ability of pro-inflammatory tachykinins to affect the immune functions of DCs remains elusive. In the present work, we demonstrate that mouse bone marrow-derived DCs (BMDCs) generated in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), express functional NK1R. Signaling via NK1R with SP, HK-1, or the synthetic agonist [Sar(9)Met(O(2))(11)]-SP rescues DCs from apoptosis induced by deprivation of GM-CSF and IL-4. Mechanistic analysis demonstrates that NK1R agonistic binding promotes DC survival via PI3K-Akt signaling cascade. In adoptive transfer experiments, NK1R-signaled BMDCs loaded with Ag exhibit increased longevity in draining lymph nodes, resulting in enhanced and prolonged effector cellular immunity. Our results contribute to the understanding of the interactions between the immune and nervous systems that control DC function and present a novel approach for ex vivo-generation of potent immune-stimulatory DCs.
Assuntos
Células Dendríticas/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Mediadores da Inflamação/farmacologia , Receptores da Neurocinina-1/fisiologia , Taquicininas/farmacologia , Transferência Adotiva , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Células da Medula Óssea/metabolismo , Antígenos CD40/metabolismo , Antígenos CD40/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Células Dendríticas/transplante , Ativação Enzimática/efeitos dos fármacos , Imunidade Celular/genética , Imunidade Celular/fisiologia , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores da Neurocinina-1/agonistas , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Taquicininas/metabolismoRESUMO
The proinflammatory capacities of the skin and the presence of high numbers of resident dendritic cells (DCs) constitute an ideal microenvironment for successful immunizations. Regardless of the ability of DCs to respond to local inflammatory signals in an immunostimulatory fashion, the immune functions of skin-resident DCs remain controversial, and epidermal Langerhans cells (LCs) have been referred to recently as anti-inflammatory/protolerogenic APCs. Substance P (SP), released by skin nerve fibers, is a potent proinflammatory neuropeptide that favors development of skin-associated cellular immunity. SP exerts its proinflammatory functions by binding with high affinity to the neurokinin 1 receptor (NK1R). In this study, we tested whether signaling skin cells via the NK1R promotes humoral and cellular immunity during skin genetic immunizations. We used the gene gun to deliver transgenic (tg) Ag to the skin of C57BL/6 mice and the selective NK1R agonist [Sar(9)Met (O(2)) (11)]-SP as a potential proinflammatory Th1-biasing adjuvant. Our strategy expressed tg Ag exclusively in the epidermis and induced a preferential migration of activated LCs to skin-draining lymph nodes. Local administration of the NK1R agonist during skin genetic immunizations increased significantly the expression of tg Ag by a mechanism involving the translocation of NF-kappaB into the nuclei of cutaneous DCs homing to skin-draining lymph nodes. Importantly, our immunization approach resulted in Th1 and T cytotoxic (CTL)-1 bias of effector T cells that supported cellular and Ab-mediated immune responses. We demonstrate that signaling skin cells via the NK1R provides the adjuvant effect which favors the immunostimulatory functions of LCs.