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Dual Requirement of Cytokine and Activation Receptor Triggering for Cytotoxic Control of Murine Cytomegalovirus by NK Cells.

Parikh BA, Piersma SJ, Pak-Wittel MA, Yang L, Schreiber RD, Yokoyama WM - PLoS Pathog. (2015)

Bottom Line: The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice.While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood.These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

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m157-specific cytotoxicity in vivo is greatly enhanced by poly(I:C) and dependent on IFN-I and degranulation.(A-D) Mice were i.p. injected with 100 μg poly(I:C), PBS, 200 μg anti-NK1.1, 200 μg anti-Ly49H and/or isotype control. After 16 hours 2x106 differentially CFSE-labeled splenocytes were injected intravenously. Three hours after adoptive transfer splenocytes were analyzed by flow cytometry. (A) Representative histograms showing splenic CD19+CFSE+ cells. Numbers indicate percentage of m157-Tg CFSElow and WT CFSEhigh cells. (B-D) Specific lysis of m157-Tg targets. (E) Mice were injected IP with 100 ug poly(I:C) or PBS. After 16 hours the spleens were harvested and NK cells were analyzed for GzmB. Data are representative of two to three independent experiments with individual points representing a single mouse.
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ppat.1005323.g006: m157-specific cytotoxicity in vivo is greatly enhanced by poly(I:C) and dependent on IFN-I and degranulation.(A-D) Mice were i.p. injected with 100 μg poly(I:C), PBS, 200 μg anti-NK1.1, 200 μg anti-Ly49H and/or isotype control. After 16 hours 2x106 differentially CFSE-labeled splenocytes were injected intravenously. Three hours after adoptive transfer splenocytes were analyzed by flow cytometry. (A) Representative histograms showing splenic CD19+CFSE+ cells. Numbers indicate percentage of m157-Tg CFSElow and WT CFSEhigh cells. (B-D) Specific lysis of m157-Tg targets. (E) Mice were injected IP with 100 ug poly(I:C) or PBS. After 16 hours the spleens were harvested and NK cells were analyzed for GzmB. Data are representative of two to three independent experiments with individual points representing a single mouse.

Mentions: To directly determine the requirement for both Ly49H and IFN-I activation for cytotoxicity in vivo, we determined the in vivo cytotoxic elimination of m157-expressing cells (Fig 6A). Following injection of equal numbers of congenic m157-Tg and WT splenocytes differentially labeled with CFSE into control-treated mice, we found there were low levels (15–35%) of m157-specific elimination (Fig 6A and 6B), consistent with previous findings on m157-specific rejection under steady-state conditions [37, 38]. Anti-NK1.1 depletion abrogated m157-specific elimination of target cells, confirming NK cell dependence. However, mice pretreated with poly(I:C) exhibited 3-fold higher levels of m157-specific rejection as compared to control-treated mice (Fig 6A and 6B). Blockade of Ly49H prevented m157-expressing target elimination in poly(I:C)-treated mice, confirming the role of Ly49H (Fig 6C). Similar results were obtained in B6.BxD8 mice specifically lacking the Ly49H receptor. Specific elimination was also abrogated in Jinx mice (Fig 6D), indicating that m157-expressing target elimination in vivo in poly(I:C)-treated mice is due to cytotoxicity. The m157-specific killing was 3-fold lower in IFNAR1-/- mice treated with poly(I:C) as compared to WT control mice, showing IFN-I signaling affects m157-specific rejection (Fig 6D). This defective cytotoxic capacity in IFNAR1-/- mice correlated with a lack of increased GzmB production in response to poly(I:C) (Fig 6E). Conversely, Jinx mice failed to eliminate m157-expressing targets (Fig 6D) even though GzmB levels were similar to WT mice (Fig 6E), highlighting the requirement of both degranulation and cytokine activation for optimal Ly49H-dependent cytotoxicity in vivo.


Dual Requirement of Cytokine and Activation Receptor Triggering for Cytotoxic Control of Murine Cytomegalovirus by NK Cells.

Parikh BA, Piersma SJ, Pak-Wittel MA, Yang L, Schreiber RD, Yokoyama WM - PLoS Pathog. (2015)

m157-specific cytotoxicity in vivo is greatly enhanced by poly(I:C) and dependent on IFN-I and degranulation.(A-D) Mice were i.p. injected with 100 μg poly(I:C), PBS, 200 μg anti-NK1.1, 200 μg anti-Ly49H and/or isotype control. After 16 hours 2x106 differentially CFSE-labeled splenocytes were injected intravenously. Three hours after adoptive transfer splenocytes were analyzed by flow cytometry. (A) Representative histograms showing splenic CD19+CFSE+ cells. Numbers indicate percentage of m157-Tg CFSElow and WT CFSEhigh cells. (B-D) Specific lysis of m157-Tg targets. (E) Mice were injected IP with 100 ug poly(I:C) or PBS. After 16 hours the spleens were harvested and NK cells were analyzed for GzmB. Data are representative of two to three independent experiments with individual points representing a single mouse.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4697817&req=5

ppat.1005323.g006: m157-specific cytotoxicity in vivo is greatly enhanced by poly(I:C) and dependent on IFN-I and degranulation.(A-D) Mice were i.p. injected with 100 μg poly(I:C), PBS, 200 μg anti-NK1.1, 200 μg anti-Ly49H and/or isotype control. After 16 hours 2x106 differentially CFSE-labeled splenocytes were injected intravenously. Three hours after adoptive transfer splenocytes were analyzed by flow cytometry. (A) Representative histograms showing splenic CD19+CFSE+ cells. Numbers indicate percentage of m157-Tg CFSElow and WT CFSEhigh cells. (B-D) Specific lysis of m157-Tg targets. (E) Mice were injected IP with 100 ug poly(I:C) or PBS. After 16 hours the spleens were harvested and NK cells were analyzed for GzmB. Data are representative of two to three independent experiments with individual points representing a single mouse.
Mentions: To directly determine the requirement for both Ly49H and IFN-I activation for cytotoxicity in vivo, we determined the in vivo cytotoxic elimination of m157-expressing cells (Fig 6A). Following injection of equal numbers of congenic m157-Tg and WT splenocytes differentially labeled with CFSE into control-treated mice, we found there were low levels (15–35%) of m157-specific elimination (Fig 6A and 6B), consistent with previous findings on m157-specific rejection under steady-state conditions [37, 38]. Anti-NK1.1 depletion abrogated m157-specific elimination of target cells, confirming NK cell dependence. However, mice pretreated with poly(I:C) exhibited 3-fold higher levels of m157-specific rejection as compared to control-treated mice (Fig 6A and 6B). Blockade of Ly49H prevented m157-expressing target elimination in poly(I:C)-treated mice, confirming the role of Ly49H (Fig 6C). Similar results were obtained in B6.BxD8 mice specifically lacking the Ly49H receptor. Specific elimination was also abrogated in Jinx mice (Fig 6D), indicating that m157-expressing target elimination in vivo in poly(I:C)-treated mice is due to cytotoxicity. The m157-specific killing was 3-fold lower in IFNAR1-/- mice treated with poly(I:C) as compared to WT control mice, showing IFN-I signaling affects m157-specific rejection (Fig 6D). This defective cytotoxic capacity in IFNAR1-/- mice correlated with a lack of increased GzmB production in response to poly(I:C) (Fig 6E). Conversely, Jinx mice failed to eliminate m157-expressing targets (Fig 6D) even though GzmB levels were similar to WT mice (Fig 6E), highlighting the requirement of both degranulation and cytokine activation for optimal Ly49H-dependent cytotoxicity in vivo.

Bottom Line: The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice.While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood.These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

Show MeSH
Related in: MedlinePlus