Figure 1
Figure 1. BM NK cell chemokine receptor expression and chemotactic response profile to CXCL12, CXCL10, and CCL3 during development. (A) Cell surface expression of chemokine receptors on developing NK cells. BM cells from individual C57BL/6 mice were stained with anti-CXCR4, anti-CXCR3, or anti-CCR5 mAb in combination with antibodies that identify pNK (anti-CD122, anti-CD3ϵ, anti-CD19, anti-NK1.1, and DX5), iNK, and mNK (anti-CD3ϵ, anti-NK1.1, and DX5) cells. CD3− lymphocytes were gated on the CD122+lin− population (top left dot plot) and on the NK1.1+DX5− or NK1.1+DX5+ population (top right dot plot). Shown are the histogram plot overlays of chemokine receptor antibody staining (empty histogram) against isotype control Ab (gray histogram). To analyze CCR1 expression, intracellular staining was performed on permeabilized cells using Alexafluor 647–conjugated goat polyclonal anti-CCR1 (empty histogram) or normal IgG (gray histogram). Numbers in the histograms indicate the percentage of positive cells of a representative experiment of at least 3 performed. Control staining was always less than 2%. (B) The chemotactic response of NK cells changes during development in the BM. In vitro chemotaxis assay of cells isolated from BM of individual animals was performed as described in “Methods.” One million cells were allowed to migrate in response to recombinant murine CXCL10 and CCL3 and human CXCL12 (used at 200 ng/mL) for 90 minutes. At the end of the experiment, duplicate wells were pooled, and cells were centrifuged and stained with the antibody cocktail used to identify pNK, iNK, and mNK cells (C indicates medium alone). Data are expressed as percentage of input cells and represent the means plus or minus SD of percentage migration of cells from a total of 8 to 12 animals analyzed in independent experiments. Student t test was performed by comparing migration of different BM NK cell subsets with each other; *P < .05. Migration of pNK versus iNK is not significantly different and thus omitted for sake of simplicity. (C) CXCL12 induces migration of pNK cells and promotes their chemotaxis to CCL3 and CXCL10. BM pNK cell migration was performed after cell pretreatment with 5 μg/mL CXCL12. Cells were washed and allowed to migrate in response to the indicated chemokines for 90 minutes. Migrated cells were then collected and stained with anti-CD122, anti-CD3ϵ, anti-CD19, anti-NK1.1, and DX5. Migrating pNK cells were identified as CD122+/lin− cells and quantified as positive events in 150 seconds acquisitions. C indicates control. Data are expressed as percentage of input cells and represent the means plus or minus SD of 6 independent experiments. Student t test was performed by comparing migration of untreated versus CXCL12-pretreated pNK cells. *P < .05.

BM NK cell chemokine receptor expression and chemotactic response profile to CXCL12, CXCL10, and CCL3 during development. (A) Cell surface expression of chemokine receptors on developing NK cells. BM cells from individual C57BL/6 mice were stained with anti-CXCR4, anti-CXCR3, or anti-CCR5 mAb in combination with antibodies that identify pNK (anti-CD122, anti-CD3ϵ, anti-CD19, anti-NK1.1, and DX5), iNK, and mNK (anti-CD3ϵ, anti-NK1.1, and DX5) cells. CD3− lymphocytes were gated on the CD122+lin− population (top left dot plot) and on the NK1.1+DX5− or NK1.1+DX5+ population (top right dot plot). Shown are the histogram plot overlays of chemokine receptor antibody staining (empty histogram) against isotype control Ab (gray histogram). To analyze CCR1 expression, intracellular staining was performed on permeabilized cells using Alexafluor 647–conjugated goat polyclonal anti-CCR1 (empty histogram) or normal IgG (gray histogram). Numbers in the histograms indicate the percentage of positive cells of a representative experiment of at least 3 performed. Control staining was always less than 2%. (B) The chemotactic response of NK cells changes during development in the BM. In vitro chemotaxis assay of cells isolated from BM of individual animals was performed as described in “Methods.” One million cells were allowed to migrate in response to recombinant murine CXCL10 and CCL3 and human CXCL12 (used at 200 ng/mL) for 90 minutes. At the end of the experiment, duplicate wells were pooled, and cells were centrifuged and stained with the antibody cocktail used to identify pNK, iNK, and mNK cells (C indicates medium alone). Data are expressed as percentage of input cells and represent the means plus or minus SD of percentage migration of cells from a total of 8 to 12 animals analyzed in independent experiments. Student t test was performed by comparing migration of different BM NK cell subsets with each other; *P < .05. Migration of pNK versus iNK is not significantly different and thus omitted for sake of simplicity. (C) CXCL12 induces migration of pNK cells and promotes their chemotaxis to CCL3 and CXCL10. BM pNK cell migration was performed after cell pretreatment with 5 μg/mL CXCL12. Cells were washed and allowed to migrate in response to the indicated chemokines for 90 minutes. Migrated cells were then collected and stained with anti-CD122, anti-CD3ϵ, anti-CD19, anti-NK1.1, and DX5. Migrating pNK cells were identified as CD122+/lin− cells and quantified as positive events in 150 seconds acquisitions. C indicates control. Data are expressed as percentage of input cells and represent the means plus or minus SD of 6 independent experiments. Student t test was performed by comparing migration of untreated versus CXCL12-pretreated pNK cells. *P < .05.

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