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Reduced MCMV Δm157 viral clearance in the absence of TSAd.

Moussa P, Abrahamsen G, Fodil N, Gopalakrishnan RP, Mancini M, Dissen E, Sæther PC, Wiltshire SA, Boivin GA, Caignard G, Spurkland A, Vidal SM - Sci Rep (2015)

Bottom Line: The T cell specific adapter protein (TSAd) is expressed in activated T cells and NK cells.While TSAd is beginning to emerge as a critical regulator of Lck and Itk activity in T cells, its role in NK cells has not yet been explored.We found that mice infected with MCMV Δm157 displayed reduced viral clearance in the spleen as well as reduced proliferation in spleen NK cells and CD8+ T cells in the absence of TSAd.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics and Department of Microbiology and Immunology, McGill University, Life Sciences Complex Montreal, QC, Canada.

ABSTRACT
The T cell specific adapter protein (TSAd) is expressed in activated T cells and NK cells. While TSAd is beginning to emerge as a critical regulator of Lck and Itk activity in T cells, its role in NK cells has not yet been explored. Here we have examined susceptibility to virus infections in a murine model using various viral infection models. We report that TSAd-deficient mice display reduced clearance of murine cytomegalovirus (MCMV) that lack the viral MHC class I homologue m157, which is critical for Ly49H-mediated NK cell recognition of infected cells. In this infection model, NK cells contribute in the early stages of the disease, whereas CD8+ T cells are critical for viral clearance. We found that mice infected with MCMV Δm157 displayed reduced viral clearance in the spleen as well as reduced proliferation in spleen NK cells and CD8+ T cells in the absence of TSAd. Though no other immunophenotype was detected in the infection models tested, these data suggests that in the absence of the Ly49H ligand activation, NK cell and CD8+ T cell responses may be compromised in TSAd-deficient mice.

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In vivo and in vitro Immunophenotying of NK cells in Sh2d2a+/+ and Sh2d2a−/− mice.Total leukocytes were isolated from mice uninfected or infected with 2 * 106 PFU of MCMV Δm157 for 5 days. Ex vivo spleen NK cell subsets were gated according to surface expression of maturation markers CD11b and CD27 (A–C) or expression of KLRG1 (D). Explanted lymphocytes from uninfected Sh2d2a+/+ and Sh2d2a−/− mice served to determine in vitro NK cell expression levels of IFNã upon stimulation with plate bound antibodies (x-axis), PMA/Ionomycin and IL12/IL18 (E). Total number of CD3-DX5+ splenocytes before and after infection with Δm157 for 5 days (F). NK proliferation was monitored by BrdU incorporation and staining with anti-BrdU in CD3-DX5+ splenocytes ex vivo. Total number (G) and frequency (H) of BrdU positive cells was determined by flow cytometry. Total number of CD3-CD8+ splenocytes before and after infection with Δm157 for 5 days (I). CD8+ T cell proliferation was monitored by BrdU incorporation and staining with anti-BrdU in CD3-DX5+ splenocytes ex vivo. Total number (J) and frequency (K) of BrdU positive cells was determined by flow cytometry. Explanted lymphocytes from uninfected Sh2d2a+/+ and Sh2d2a−/− mice served to determine in vitro levels of surface activating and inhibitory NK cell receptors (L). Results are expressed as mean +/− SEM. *<0.05, **<0.05.
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f4: In vivo and in vitro Immunophenotying of NK cells in Sh2d2a+/+ and Sh2d2a−/− mice.Total leukocytes were isolated from mice uninfected or infected with 2 * 106 PFU of MCMV Δm157 for 5 days. Ex vivo spleen NK cell subsets were gated according to surface expression of maturation markers CD11b and CD27 (A–C) or expression of KLRG1 (D). Explanted lymphocytes from uninfected Sh2d2a+/+ and Sh2d2a−/− mice served to determine in vitro NK cell expression levels of IFNã upon stimulation with plate bound antibodies (x-axis), PMA/Ionomycin and IL12/IL18 (E). Total number of CD3-DX5+ splenocytes before and after infection with Δm157 for 5 days (F). NK proliferation was monitored by BrdU incorporation and staining with anti-BrdU in CD3-DX5+ splenocytes ex vivo. Total number (G) and frequency (H) of BrdU positive cells was determined by flow cytometry. Total number of CD3-CD8+ splenocytes before and after infection with Δm157 for 5 days (I). CD8+ T cell proliferation was monitored by BrdU incorporation and staining with anti-BrdU in CD3-DX5+ splenocytes ex vivo. Total number (J) and frequency (K) of BrdU positive cells was determined by flow cytometry. Explanted lymphocytes from uninfected Sh2d2a+/+ and Sh2d2a−/− mice served to determine in vitro levels of surface activating and inhibitory NK cell receptors (L). Results are expressed as mean +/− SEM. *<0.05, **<0.05.

Mentions: In order to assess whether there was a difference in NK maturation between Sh2d2a−/− mice and wild type controls, levels of CD11b and CD27 on the surface of NK cells were analyzed 5 days post infection with Δm157 MCMV. No differences in any of the stages were observed between Sh2d2a−/− mice and their wild type littermates (Figure 4A–C). The level of the activation marker KLRG1on NK cells also showed no difference prior to and after 5 days of Δm157 MCMV infection (Figure 4D). Thus, the maturity and activation profile of NK cells are similar in the two groups of mice.


Reduced MCMV Δm157 viral clearance in the absence of TSAd.

Moussa P, Abrahamsen G, Fodil N, Gopalakrishnan RP, Mancini M, Dissen E, Sæther PC, Wiltshire SA, Boivin GA, Caignard G, Spurkland A, Vidal SM - Sci Rep (2015)

In vivo and in vitro Immunophenotying of NK cells in Sh2d2a+/+ and Sh2d2a−/− mice.Total leukocytes were isolated from mice uninfected or infected with 2 * 106 PFU of MCMV Δm157 for 5 days. Ex vivo spleen NK cell subsets were gated according to surface expression of maturation markers CD11b and CD27 (A–C) or expression of KLRG1 (D). Explanted lymphocytes from uninfected Sh2d2a+/+ and Sh2d2a−/− mice served to determine in vitro NK cell expression levels of IFNã upon stimulation with plate bound antibodies (x-axis), PMA/Ionomycin and IL12/IL18 (E). Total number of CD3-DX5+ splenocytes before and after infection with Δm157 for 5 days (F). NK proliferation was monitored by BrdU incorporation and staining with anti-BrdU in CD3-DX5+ splenocytes ex vivo. Total number (G) and frequency (H) of BrdU positive cells was determined by flow cytometry. Total number of CD3-CD8+ splenocytes before and after infection with Δm157 for 5 days (I). CD8+ T cell proliferation was monitored by BrdU incorporation and staining with anti-BrdU in CD3-DX5+ splenocytes ex vivo. Total number (J) and frequency (K) of BrdU positive cells was determined by flow cytometry. Explanted lymphocytes from uninfected Sh2d2a+/+ and Sh2d2a−/− mice served to determine in vitro levels of surface activating and inhibitory NK cell receptors (L). Results are expressed as mean +/− SEM. *<0.05, **<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363830&req=5

f4: In vivo and in vitro Immunophenotying of NK cells in Sh2d2a+/+ and Sh2d2a−/− mice.Total leukocytes were isolated from mice uninfected or infected with 2 * 106 PFU of MCMV Δm157 for 5 days. Ex vivo spleen NK cell subsets were gated according to surface expression of maturation markers CD11b and CD27 (A–C) or expression of KLRG1 (D). Explanted lymphocytes from uninfected Sh2d2a+/+ and Sh2d2a−/− mice served to determine in vitro NK cell expression levels of IFNã upon stimulation with plate bound antibodies (x-axis), PMA/Ionomycin and IL12/IL18 (E). Total number of CD3-DX5+ splenocytes before and after infection with Δm157 for 5 days (F). NK proliferation was monitored by BrdU incorporation and staining with anti-BrdU in CD3-DX5+ splenocytes ex vivo. Total number (G) and frequency (H) of BrdU positive cells was determined by flow cytometry. Total number of CD3-CD8+ splenocytes before and after infection with Δm157 for 5 days (I). CD8+ T cell proliferation was monitored by BrdU incorporation and staining with anti-BrdU in CD3-DX5+ splenocytes ex vivo. Total number (J) and frequency (K) of BrdU positive cells was determined by flow cytometry. Explanted lymphocytes from uninfected Sh2d2a+/+ and Sh2d2a−/− mice served to determine in vitro levels of surface activating and inhibitory NK cell receptors (L). Results are expressed as mean +/− SEM. *<0.05, **<0.05.
Mentions: In order to assess whether there was a difference in NK maturation between Sh2d2a−/− mice and wild type controls, levels of CD11b and CD27 on the surface of NK cells were analyzed 5 days post infection with Δm157 MCMV. No differences in any of the stages were observed between Sh2d2a−/− mice and their wild type littermates (Figure 4A–C). The level of the activation marker KLRG1on NK cells also showed no difference prior to and after 5 days of Δm157 MCMV infection (Figure 4D). Thus, the maturity and activation profile of NK cells are similar in the two groups of mice.

Bottom Line: The T cell specific adapter protein (TSAd) is expressed in activated T cells and NK cells.While TSAd is beginning to emerge as a critical regulator of Lck and Itk activity in T cells, its role in NK cells has not yet been explored.We found that mice infected with MCMV Δm157 displayed reduced viral clearance in the spleen as well as reduced proliferation in spleen NK cells and CD8+ T cells in the absence of TSAd.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics and Department of Microbiology and Immunology, McGill University, Life Sciences Complex Montreal, QC, Canada.

ABSTRACT
The T cell specific adapter protein (TSAd) is expressed in activated T cells and NK cells. While TSAd is beginning to emerge as a critical regulator of Lck and Itk activity in T cells, its role in NK cells has not yet been explored. Here we have examined susceptibility to virus infections in a murine model using various viral infection models. We report that TSAd-deficient mice display reduced clearance of murine cytomegalovirus (MCMV) that lack the viral MHC class I homologue m157, which is critical for Ly49H-mediated NK cell recognition of infected cells. In this infection model, NK cells contribute in the early stages of the disease, whereas CD8+ T cells are critical for viral clearance. We found that mice infected with MCMV Δm157 displayed reduced viral clearance in the spleen as well as reduced proliferation in spleen NK cells and CD8+ T cells in the absence of TSAd. Though no other immunophenotype was detected in the infection models tested, these data suggests that in the absence of the Ly49H ligand activation, NK cell and CD8+ T cell responses may be compromised in TSAd-deficient mice.

Show MeSH
Related in: MedlinePlus