- Email: [email protected]
Targeting to sialoadhesin receptor improves antigen presentation to T cells
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
327
contrast, virus replication was not rescued by an identical 2-AP treatment of PAMs previously primed with IFN. These findings demonstrate not only differential sensitivity of several PRRSV strains to IFN, but also highlight differences in the host cell systems employed. The results suggest that PKR is involved in the antiviral state induced by IFN, specifically in MARC-145 cells, though it is apparent that there are other important factors contributing to host innate immunity to PRRSV infection, as exemplified by the differing results obtained when using PAMs.
to purified 146S FMDV antigen; under the same conditions, the prevalence of IFN-␥ positive cells (ELISPOT and flow cytometry assays) was decreased beneath the control value in 3 animals out of 4; this was reversed in the presence of 5 U/ml of IFN-␣. In conclusion, intracellular IFN-␣ is constitutively expressed in some populations of swine PBMC and AM and it seemingly differs from its mature, extracellular forms. Finally, minute amounts of IFN-␣ could significantly affect some Th cell functions.
doi:10.1016/j.vetimm.2008.10.248
Targeting to sialoadhesin receptor improves antigen presentation to T cells
Constitutive expression of interferon alpha by swine leukocytes
doi:10.1016/j.vetimm.2008.10.249
Massimo Amadori
Teresa Poderoso, Concepciön Revilla, Belen Alvarez, Sonia Chamorro, Paloma Martínez de La Riva, Fernando Alonso, Angel Ezquerra, Javier Domínguez ∗
Department of Animal Welfare and Immunoprophylaxis, Istituto Zooprofilattico Sperimentale, Brescia, Italy Keywords: Pig; Innate immunity; Interferon alpha
Dpto de Biotecnología, INIA, Madrid, Spain Keywords: Sialoadhesin; Antigen-presenting cell; Monoclonal antibody; Vaccine and swine
Species: Swine Interferon alpha (IFN-␣) plays an important role in the homeostatic regulation of the inflammatory/stress response, which does not conflict with its main functions in the innate immune system. Constitutive expression in healthy subjects is in line with this tenet and it was therefore investigated in swine leukocytes. The same low prevalence of IFN-␣ positive cells was shown by flow cytometry in control and virus-stimulated peripheral blood mononuclear cells (PBMC); the latter only were also positive for cytokine secretion. Results were confirmed with the same mAb by western blotting on NP-40 lysates of PBMC, spleen, lymph node cells and alveolar macrophages (AM) of control and H1 N1 influenza virus-infected pigs. PBMC and AM of control and virusinfected pigs expressed in fact a strong 32 kDa + minor bands, as opposed to spleen and lymph nodes. Interestingly, the signal in PBMC disappeared in western blotting but not in flow cytometry after overnight culture in vitro. Results were confirmed by immunoprecipitation of 35 Smethionine/cysteine, metabolically labelled PBMC of 4 healthy, SPF piglets. Intriguingly, 3 out of 4 lysates were also IFN-␥ positive (40 kDa band) with a seemingly inverse correlation to IFN-␣ expression. The possible paracrine role of IFN-␣ was investigated in vitro. Minute amounts of IFN-␣ (≥0.5 U/ml) significantly increased the in vitro survival of CD4+ T cells after both mitogen and foot-and-mouth disease virus (FMDV) recall antigen stimulation. Instead, the prevalence of double-positive CD4+/CD25high +, allegedly Treg cells was not increased. In the same model of recall Th2 response to FMDV O Manisa, as little as 5 U/ml IFN-␣ could significantly boost the in vitro serum-neutralizing (SN) antibody response; no antibody was secreted in control cultures without purified 146S FMDV antigen; the height of this response was inversely related to the number of antibody producing cells at day 12 (exhaustion phase). 3 H-thymidine incorporation was inhibited after exposure of these PBMC
E-mail address: [email protected] (J. Domínguez). Species: Other The main goal of vaccine biotechnology is to generate a protective and long-lasting immune response against pathogens. One of the strategies that are being investigated to enhance responses to weak vaccine antigens is targeting them to APC receptors by conjugation to specific antibodies. Sialoadhesin is a member of the family of sialic acid binding immunoglobulin-like lectins (Siglecs), whose expression is restricted to subsets of tissue macrophages, inflammatory monocytes and some dendritic cells. Here, we have analysed the expression of sialoadhesin in porcine tissues, its regulation by cytokines and its potential as an antigen targeting receptor. Porcine sialoadhesin was expressed in macrophages of the marginal zone of spleen and in dendritic cells derived from monocytes cultured with GM-CSF plus IL-4 (MoDC). Blood monocytes (Mo) were negative for this molecule but its expression could be induced after treatment with IFN-␣. To analyse if targeting antigen to sialoadhesin leads to an enhancement of T cell responses we compared the proliferative response elicited by a mAb to sialoadhesin (1F1) with that of an isotype matched control mAb (1D9). T cells from pigs immunized with a pool of mouse Igs were used as responder cells and MoDC or Mo cultured with IFN-␣ as APCs. MAb to sialoadhesin induced T cell proliferation at concentrations 100-fold lower than control mAb, suggesting an efficient role of sialoadhesin in antigen uptake and/or processing in APCs. Consistent with these data the Alexa 488-labeled 1F1 mAb but not the Alexa 488-labeled control 1D9 mAb was significantly internalized in macrophages and MoDC cultured at 37 ◦ C for 30 min. Experiments to evaluate in vivo the potential of sialoadhesin as a vaccine targeting receptor are in progress. doi:10.1016/j.vetimm.2008.10.250
327
contrast, virus replication was not rescued by an identical 2-AP treatment of PAMs previously primed with IFN. These findings demonstrate not only differential sensitivity of several PRRSV strains to IFN, but also highlight differences in the host cell systems employed. The results suggest that PKR is involved in the antiviral state induced by IFN, specifically in MARC-145 cells, though it is apparent that there are other important factors contributing to host innate immunity to PRRSV infection, as exemplified by the differing results obtained when using PAMs.
to purified 146S FMDV antigen; under the same conditions, the prevalence of IFN-␥ positive cells (ELISPOT and flow cytometry assays) was decreased beneath the control value in 3 animals out of 4; this was reversed in the presence of 5 U/ml of IFN-␣. In conclusion, intracellular IFN-␣ is constitutively expressed in some populations of swine PBMC and AM and it seemingly differs from its mature, extracellular forms. Finally, minute amounts of IFN-␣ could significantly affect some Th cell functions.
doi:10.1016/j.vetimm.2008.10.248
Targeting to sialoadhesin receptor improves antigen presentation to T cells
Constitutive expression of interferon alpha by swine leukocytes
doi:10.1016/j.vetimm.2008.10.249
Massimo Amadori
Teresa Poderoso, Concepciön Revilla, Belen Alvarez, Sonia Chamorro, Paloma Martínez de La Riva, Fernando Alonso, Angel Ezquerra, Javier Domínguez ∗
Department of Animal Welfare and Immunoprophylaxis, Istituto Zooprofilattico Sperimentale, Brescia, Italy Keywords: Pig; Innate immunity; Interferon alpha
Dpto de Biotecnología, INIA, Madrid, Spain Keywords: Sialoadhesin; Antigen-presenting cell; Monoclonal antibody; Vaccine and swine
Species: Swine Interferon alpha (IFN-␣) plays an important role in the homeostatic regulation of the inflammatory/stress response, which does not conflict with its main functions in the innate immune system. Constitutive expression in healthy subjects is in line with this tenet and it was therefore investigated in swine leukocytes. The same low prevalence of IFN-␣ positive cells was shown by flow cytometry in control and virus-stimulated peripheral blood mononuclear cells (PBMC); the latter only were also positive for cytokine secretion. Results were confirmed with the same mAb by western blotting on NP-40 lysates of PBMC, spleen, lymph node cells and alveolar macrophages (AM) of control and H1 N1 influenza virus-infected pigs. PBMC and AM of control and virusinfected pigs expressed in fact a strong 32 kDa + minor bands, as opposed to spleen and lymph nodes. Interestingly, the signal in PBMC disappeared in western blotting but not in flow cytometry after overnight culture in vitro. Results were confirmed by immunoprecipitation of 35 Smethionine/cysteine, metabolically labelled PBMC of 4 healthy, SPF piglets. Intriguingly, 3 out of 4 lysates were also IFN-␥ positive (40 kDa band) with a seemingly inverse correlation to IFN-␣ expression. The possible paracrine role of IFN-␣ was investigated in vitro. Minute amounts of IFN-␣ (≥0.5 U/ml) significantly increased the in vitro survival of CD4+ T cells after both mitogen and foot-and-mouth disease virus (FMDV) recall antigen stimulation. Instead, the prevalence of double-positive CD4+/CD25high +, allegedly Treg cells was not increased. In the same model of recall Th2 response to FMDV O Manisa, as little as 5 U/ml IFN-␣ could significantly boost the in vitro serum-neutralizing (SN) antibody response; no antibody was secreted in control cultures without purified 146S FMDV antigen; the height of this response was inversely related to the number of antibody producing cells at day 12 (exhaustion phase). 3 H-thymidine incorporation was inhibited after exposure of these PBMC
E-mail address: [email protected] (J. Domínguez). Species: Other The main goal of vaccine biotechnology is to generate a protective and long-lasting immune response against pathogens. One of the strategies that are being investigated to enhance responses to weak vaccine antigens is targeting them to APC receptors by conjugation to specific antibodies. Sialoadhesin is a member of the family of sialic acid binding immunoglobulin-like lectins (Siglecs), whose expression is restricted to subsets of tissue macrophages, inflammatory monocytes and some dendritic cells. Here, we have analysed the expression of sialoadhesin in porcine tissues, its regulation by cytokines and its potential as an antigen targeting receptor. Porcine sialoadhesin was expressed in macrophages of the marginal zone of spleen and in dendritic cells derived from monocytes cultured with GM-CSF plus IL-4 (MoDC). Blood monocytes (Mo) were negative for this molecule but its expression could be induced after treatment with IFN-␣. To analyse if targeting antigen to sialoadhesin leads to an enhancement of T cell responses we compared the proliferative response elicited by a mAb to sialoadhesin (1F1) with that of an isotype matched control mAb (1D9). T cells from pigs immunized with a pool of mouse Igs were used as responder cells and MoDC or Mo cultured with IFN-␣ as APCs. MAb to sialoadhesin induced T cell proliferation at concentrations 100-fold lower than control mAb, suggesting an efficient role of sialoadhesin in antigen uptake and/or processing in APCs. Consistent with these data the Alexa 488-labeled 1F1 mAb but not the Alexa 488-labeled control 1D9 mAb was significantly internalized in macrophages and MoDC cultured at 37 ◦ C for 30 min. Experiments to evaluate in vivo the potential of sialoadhesin as a vaccine targeting receptor are in progress. doi:10.1016/j.vetimm.2008.10.250