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Sublingual Immunotherapy Induce Systemic T-cell Tolerance in Naive Mice
the establishment of asthma model. And increased IL-10 and IFN-g production in recipients.
181
Airway Exposures to House Dust Extracts Provide Th2 Adjuvant Activity with Intermittent Delivery and Long Term Tolerogenic Activity with Daily Delivery G. M. Batzer, N. Ng, D. P. Lam, A. A. Horner; UCSD, La Jolla, CA. RATIONALE: While mechanistic understanding is limited, there is general agreement that living environments have a significant impact on allergic risk. These investigations characterized the immunomodulatory influence of airway house dust extract (HDE) exposures on the development of aeroallergen hypersensitivities. METHODS: Sterile HDEs were prepared from bedroom dust samples. In initial studies mice received 3 weekly intranasal (i.n.) immunizations with ovalbumin (OVA) and an optimized adjuvant dose of HDE. In subsequent experiments mice received weekly OVA, while HDEs were i.n. delivered weekly with OVA, daily at 1/7th the weekly dose (beginning 1 week before the first and ending with the last OVA immunization), or both. Alternatively, mice immunized as just described received additional weekly i.n. OVA/HDE immunizations beginning 1 month after primary exposures were completed. In analogous experiments, mice were immunized with OVA and LPS. Both OVA specific immune and airway hypersensitivity responses were assessed. RESULTS: Mice intranasally (i.n.) immunized with OVA alone had weak adaptive responses while mice i.n. co-immunized with HDE developed robust Th2-biased immune and airway hypersensitivity responses. Paradoxically, if mice received OVA weekly but HDEs were delivered daily at 1/7th the weekly immunization dose, they demonstrated evidence of both short and long term OVA tolerance. In additional studies LPS was found to mimic both the Th2 adjuvant and tolerogenic activities of HDEs. CONCLUSIONS: Experimental results with HDEs and purified LPS demonstrate that depending on airway exposure levels and frequency, stimulants of innate immunity that are ubiquitous in living environments can either promote or prevent the development of aeroallergen hypersensitivities. Funding: NIH/NIAID
182
Neonatal Education of Dendritic and T Cells by LPS results in Tolerance in an Adoptive Transfer Model of Airways Allergy Y. Wang, C. T. McCusker; McGill University, Montreal, PQ, CANADA. RATIONALE: We have shown that stimulation of the neonatal respiratory immune system with LPS directs subsequent mucosal responses toward the T-regulatory phenotype and prevents allergic respiratory disease in the adult mouse. In the present study we used adoptive transfer of DCs or CD41 T cells derived from LPS or PBS exposed mice to elucidate the roles of these cells in LPS-induced tolerance. METHODS: Newborn (3 days after birth) non-anesthetized DO11.10 mice received repeated intranasal (IN) application (exposure) of 1.0 mg of endotoxin LPS in each nostril. At 6 weeks of age, the mice sacrificed and CD11c1 DCs or CD41 T cells were isolated from spleen. DCs or Cd41 T cells then were adoptively transferred into 6 week old naı¨ve BALB/c mice via the trachea (DCs) or tail vein (CD41 cells). These mice were subsequently sensitized and challenge with OVA. RESULTS: Results show significant decrease in airway hyperresponsiveness to methacholine in mice who received DCs or CD41 T cells from LPS exposed mice compared with controls. IL10 levels were significantly increased in culture supernatants from splenocytes obtained from the animals following transfer of LPS-DCs and OVA sensitization and challenge. CONCLUSIONS: The results support the hypothesis that neonatal LPS exposure leads to maturation of airway DCs which play critical roles in directing subsequent CD41 T cell activation toward production of regulatory T cells in naı¨ve animals. Adoptive transfer of LPS-exposed CD41 T cells also transfers the tolerant phenotype suggesting that both mature DCs and T cells significantly influence de novo immune response to respiratory allergens. Funding: CIHR
183
Peripherally-Induced Allergen-Specific Foxp31 Regulatory T Cells are Essential for the Control of Chronic Allergic Lung Inflammation M. A. Curotto de Lafaille, N. Kutchukhidze, Y. Ding, S. Shen, J. J. Lafaille; New York University School of Medicine, New York, NY. RATIONALE: Allergen-specific Foxp31 Treg develop after allergen exposure via mucosa. Using a mouse model lacking naturally-occurring regulatory T cells (Treg), we investigated the role of peripherally-generated allergen-specific Foxp31 Treg in a) the establishment of mucosal tolerance, b) the regulation of acute and chronic allergic lung inflammation. METHODS: Tolerant or allergic inflammatory immune responses were studied in mice that bear a naı¨ve allergen-specific monoclonal T and B lymphocyte repertoire, lack thymus derived Treg, and carry either normal or deficient (scurfy) Foxp3 genes. Immune tolerance was induced by administration of antigen in the drinking water (oral) or intra-nasally (respiratory). Allergic sensitization and lung inflammation were induced by intra-peritoneal immunization with antigen in alum, followed by intranasal antigen challenge. RESULTS: We found that both respiratory and oral tolerance were drastically impaired in Foxp3 deficient mice, in which allergen-specific Treg failed to be induced in the periphery. In allergen sensitized mice receiving inhaled allergen chronically, Foxp3 deficiency led to persistent lung eosinophilia and fibrosis, and to increasing IL-4 and IgE production. In contrast, immunized mice carrying normal Foxp3 genes showed stabilized IgE levels and decreased eosinophilia and fibrosis upon chronic allergen inhalation. CONCLUSIONS: Peripherally-induced allergen-specific Foxp31 Tregs are essential to establish mucosal tolerance. In sensitized individuals submitted to chronic allergic exposure, peripherally-induced Foxp31 Tregs are essential to limit type 2 cytokine and IgE production and to resolve lung inflammation. Funding: NIH
184
Sublingual Immunotherapy Induce Systemic T-cell Tolerance in Naive Mice J. Brimnes, J. Kildsgaard, H. Jacobi, K. Lund; ALK-Abello, Hoersholm, DENMARK. RATIONALE: The mechanisms behind the effect of sublingual immunotherapy (SLIT) remain elusive. In the present study we have investigated the immunological response induced by SLIT treatment of naı¨ve mice. METHODS: Naı¨ve mice were SLIT treated with an extract of Phleum pratense (Phl p) for two to six weeks and at different concentrations followed by challenge with up to three intraperitoneal injections of alum adsorbed Phl p extract. Subsequently, the mice were sacrificed and serum antibodies and T-cell reactivity were measured. RESULTS: SLIT treatment alone did not lead to activation of systemic Tcells, as the Phl p specific in vitro proliferation did not exceed the background. When the SLIT treated mice were subsequently challenged intraperitoneally with alum adsorbed Phl p extract it could be demonstrated that SLIT leads to the induction of systemic T-cell tolerance. Both T cell proliferation as well as secretion of IFN -g, IL-4, IL-5 and IL-10 were reduced by more than 50% in SLIT treated mice compared to buffer-treated mice. This tolerance induction was both time and dose dependent. Furthermore, tolerance induced by SLIT treatment was achieved at concentrations at which peroral administration was not effective. Preliminary studies indicated that spleen cells from SLIT treated mice were able to transfer the tolerance to naı¨ve recipients. CONCLUSION: The results of the present study demonstrate for the first time that sublingual treatment of naı¨ve mice leads to the induction of systemic tolerance, which can be induced at lower concentrations than perorally induced tolerance. Funding: ALK-Abello
SATURDAY
Abstracts S47
J ALLERGY CLIN IMMUNOL VOLUME 119, NUMBER 1
181
Airway Exposures to House Dust Extracts Provide Th2 Adjuvant Activity with Intermittent Delivery and Long Term Tolerogenic Activity with Daily Delivery G. M. Batzer, N. Ng, D. P. Lam, A. A. Horner; UCSD, La Jolla, CA. RATIONALE: While mechanistic understanding is limited, there is general agreement that living environments have a significant impact on allergic risk. These investigations characterized the immunomodulatory influence of airway house dust extract (HDE) exposures on the development of aeroallergen hypersensitivities. METHODS: Sterile HDEs were prepared from bedroom dust samples. In initial studies mice received 3 weekly intranasal (i.n.) immunizations with ovalbumin (OVA) and an optimized adjuvant dose of HDE. In subsequent experiments mice received weekly OVA, while HDEs were i.n. delivered weekly with OVA, daily at 1/7th the weekly dose (beginning 1 week before the first and ending with the last OVA immunization), or both. Alternatively, mice immunized as just described received additional weekly i.n. OVA/HDE immunizations beginning 1 month after primary exposures were completed. In analogous experiments, mice were immunized with OVA and LPS. Both OVA specific immune and airway hypersensitivity responses were assessed. RESULTS: Mice intranasally (i.n.) immunized with OVA alone had weak adaptive responses while mice i.n. co-immunized with HDE developed robust Th2-biased immune and airway hypersensitivity responses. Paradoxically, if mice received OVA weekly but HDEs were delivered daily at 1/7th the weekly immunization dose, they demonstrated evidence of both short and long term OVA tolerance. In additional studies LPS was found to mimic both the Th2 adjuvant and tolerogenic activities of HDEs. CONCLUSIONS: Experimental results with HDEs and purified LPS demonstrate that depending on airway exposure levels and frequency, stimulants of innate immunity that are ubiquitous in living environments can either promote or prevent the development of aeroallergen hypersensitivities. Funding: NIH/NIAID
182
Neonatal Education of Dendritic and T Cells by LPS results in Tolerance in an Adoptive Transfer Model of Airways Allergy Y. Wang, C. T. McCusker; McGill University, Montreal, PQ, CANADA. RATIONALE: We have shown that stimulation of the neonatal respiratory immune system with LPS directs subsequent mucosal responses toward the T-regulatory phenotype and prevents allergic respiratory disease in the adult mouse. In the present study we used adoptive transfer of DCs or CD41 T cells derived from LPS or PBS exposed mice to elucidate the roles of these cells in LPS-induced tolerance. METHODS: Newborn (3 days after birth) non-anesthetized DO11.10 mice received repeated intranasal (IN) application (exposure) of 1.0 mg of endotoxin LPS in each nostril. At 6 weeks of age, the mice sacrificed and CD11c1 DCs or CD41 T cells were isolated from spleen. DCs or Cd41 T cells then were adoptively transferred into 6 week old naı¨ve BALB/c mice via the trachea (DCs) or tail vein (CD41 cells). These mice were subsequently sensitized and challenge with OVA. RESULTS: Results show significant decrease in airway hyperresponsiveness to methacholine in mice who received DCs or CD41 T cells from LPS exposed mice compared with controls. IL10 levels were significantly increased in culture supernatants from splenocytes obtained from the animals following transfer of LPS-DCs and OVA sensitization and challenge. CONCLUSIONS: The results support the hypothesis that neonatal LPS exposure leads to maturation of airway DCs which play critical roles in directing subsequent CD41 T cell activation toward production of regulatory T cells in naı¨ve animals. Adoptive transfer of LPS-exposed CD41 T cells also transfers the tolerant phenotype suggesting that both mature DCs and T cells significantly influence de novo immune response to respiratory allergens. Funding: CIHR
183
Peripherally-Induced Allergen-Specific Foxp31 Regulatory T Cells are Essential for the Control of Chronic Allergic Lung Inflammation M. A. Curotto de Lafaille, N. Kutchukhidze, Y. Ding, S. Shen, J. J. Lafaille; New York University School of Medicine, New York, NY. RATIONALE: Allergen-specific Foxp31 Treg develop after allergen exposure via mucosa. Using a mouse model lacking naturally-occurring regulatory T cells (Treg), we investigated the role of peripherally-generated allergen-specific Foxp31 Treg in a) the establishment of mucosal tolerance, b) the regulation of acute and chronic allergic lung inflammation. METHODS: Tolerant or allergic inflammatory immune responses were studied in mice that bear a naı¨ve allergen-specific monoclonal T and B lymphocyte repertoire, lack thymus derived Treg, and carry either normal or deficient (scurfy) Foxp3 genes. Immune tolerance was induced by administration of antigen in the drinking water (oral) or intra-nasally (respiratory). Allergic sensitization and lung inflammation were induced by intra-peritoneal immunization with antigen in alum, followed by intranasal antigen challenge. RESULTS: We found that both respiratory and oral tolerance were drastically impaired in Foxp3 deficient mice, in which allergen-specific Treg failed to be induced in the periphery. In allergen sensitized mice receiving inhaled allergen chronically, Foxp3 deficiency led to persistent lung eosinophilia and fibrosis, and to increasing IL-4 and IgE production. In contrast, immunized mice carrying normal Foxp3 genes showed stabilized IgE levels and decreased eosinophilia and fibrosis upon chronic allergen inhalation. CONCLUSIONS: Peripherally-induced allergen-specific Foxp31 Tregs are essential to establish mucosal tolerance. In sensitized individuals submitted to chronic allergic exposure, peripherally-induced Foxp31 Tregs are essential to limit type 2 cytokine and IgE production and to resolve lung inflammation. Funding: NIH
184
Sublingual Immunotherapy Induce Systemic T-cell Tolerance in Naive Mice J. Brimnes, J. Kildsgaard, H. Jacobi, K. Lund; ALK-Abello, Hoersholm, DENMARK. RATIONALE: The mechanisms behind the effect of sublingual immunotherapy (SLIT) remain elusive. In the present study we have investigated the immunological response induced by SLIT treatment of naı¨ve mice. METHODS: Naı¨ve mice were SLIT treated with an extract of Phleum pratense (Phl p) for two to six weeks and at different concentrations followed by challenge with up to three intraperitoneal injections of alum adsorbed Phl p extract. Subsequently, the mice were sacrificed and serum antibodies and T-cell reactivity were measured. RESULTS: SLIT treatment alone did not lead to activation of systemic Tcells, as the Phl p specific in vitro proliferation did not exceed the background. When the SLIT treated mice were subsequently challenged intraperitoneally with alum adsorbed Phl p extract it could be demonstrated that SLIT leads to the induction of systemic T-cell tolerance. Both T cell proliferation as well as secretion of IFN -g, IL-4, IL-5 and IL-10 were reduced by more than 50% in SLIT treated mice compared to buffer-treated mice. This tolerance induction was both time and dose dependent. Furthermore, tolerance induced by SLIT treatment was achieved at concentrations at which peroral administration was not effective. Preliminary studies indicated that spleen cells from SLIT treated mice were able to transfer the tolerance to naı¨ve recipients. CONCLUSION: The results of the present study demonstrate for the first time that sublingual treatment of naı¨ve mice leads to the induction of systemic tolerance, which can be induced at lower concentrations than perorally induced tolerance. Funding: ALK-Abello
SATURDAY
Abstracts S47
J ALLERGY CLIN IMMUNOL VOLUME 119, NUMBER 1