Multiple receptors and ligands are involved in NK cell-mediated lysis of activated CD4⁺ T cells.

<p>Role of (A) activating and (B) inhibitory NK receptors in NK cell degranulation. Left column: representative histograms (of n≥3) for surface expression of ligands on activated (thick black line) and resting CD4⁺ T cells (thin black line). Isotype-matched control Ig are represented by dashed line (activated CD4⁺ T) and filled histogram (resting CD4⁺ T). Middle- and right column: NK and CD4⁺ T cells were activated for 4 days <i>in vitro</i> as described, and co-cultured for 4 hours with 10 ug/mL mAb (or relevant isotype-matched control Ig). Degranulation is shown for CD56^dim (middle column) and CD56^bright (right column) NK cells. Representative histograms of surface expression of receptors on activated (thick black line) and resting NK cells (thin black line). Isotype-matched control Ig are represented by dashed line (activated NK) and filled histogram (resting NK). * <i>P</i><0.05, ** <i>P</i><0.005, *** <i>P</i><0.001. (C) Sorted IL-2-activated CD56^dim and CD56^bright NK cells were co-cultured with ⁵¹Cr-labeled activated CD4⁺ T cells in a ⁵¹Cr-release assay with human IgG4 isotype control (•) or anti-NKG2A mAb (○). Data represents n = 3 experiments.</p

NK cells mediate TRAIL-dependent cytotoxicity towards activated CD4⁺ T cells.

<p>Autologous NK cells and CD4⁺ T cells were isolated and activated for 4 days as described. Resting NK and CD4⁺ T cells were unstimulated in media for 4 days. (A) Representative histograms for flow cytometric analysis of surface expression of TRAIL-R1 (DR4) and TRAIL-R2 (DR5) on activated CD4⁺ T cells (thick black line) and resting CD4⁺ T cells (thin black line). Isotype-matched control Ig are represented by dashed line (activated CD4⁺ T) and filled histogram (resting CD4⁺). (B) Histograms are representative of TRAIL surface expression on activated NK cells (thick black line) and resting NK cells (thin black line). Isotype-matched control Ig are represented by dashed line (activated NK) and filled histogram (resting NK). (C) Sorted IL-2-activated CD56^dim and CD56^bright NK cells were co-cultured with ⁵¹Cr-labeled activated CD4⁺ T cells in a ⁵¹Cr-release assay with isotype-matched control Ig (•) or anti-TRAIL mAb (○). Experiment shown is representative of n = 3.</p

Activated NK cells kill activated, but not resting, CD4⁺ T cells.

<p>(A) Representative gating strategy. NK cells were defined as viable, CD3⁻ singlets. NK cell subsets were defined based on expression of CD16 and CD56. (B) Activation of CD4⁺ T cells was confirmed by CD69 upregulation. CD4⁺ T cells were activated for 4 days with anti-CD3+anti-CD28 Dynabeads (propionate was added on day 3). Resting CD4⁺ T cells were unstimulated in media for 4 days. (C–D) NK cells were cultured for 4 days in IL-2, and CD4⁺ T cells were activated as described. Resting CD4⁺ T cells were unstimulated for 4 days in culture. Autologous NK cells and CD4⁺ T cells were co-cultured at an E∶T ratio of 1∶1 for 4 hours with FITC-conjugated anti-CD107a+anti-CD107b mAb. Flow cytometry was performed to determine CD107a/b expression on NK cell subsets. Data shown in (C) are for a representative donor, (D) are for n = 9. Data represent mean ± SEM. * <i>P</i><0.05; *** <i>P</i><0.001. (E) Sorted CD56^dim (□/▪) and CD56^bright (○/•) NK cells were cultured in media (□/○) or IL-2 (▪/•) for 4 days, and co-cultured with ⁵¹Cr-labeled activated CD4⁺ T cells in a ⁵¹Cr-release assay. Experiment shown is representative of n = 3.</p

NKG2D and LFA-1 act synergistically in mediating NK cell degranulation.

<p>NK and CD4⁺ T cells were activated for 4 days <i>in vitro</i> as described, and co-cultured for 4 hours with FITC conjugated anti-CD107a+anti-CD107b mAbs. 10 ug/mL blocking antibodies (or relevant isotype-matched control Ig) were added where indicated. Flow cytometry was performed to assess degranulation of (A) CD56^dim and (B) CD56^bright NK cells. (C) CD56^dim NK cells were gated based on surface expression of CD94/NKG2A, and degranulation of each subset was evaluated. * <i>P</i><0.05.</p

CD56^bright NK cells have a higher cytotoxic potential towards activated CD4⁺ T cells.

<p>Autologous NK cells and CD4⁺ T cells were isolated, and CD4⁺ T cells were activated for 4 days as described. NK cells were activated as indicated: 200 IU/mL IL-2, 25 ng/mL IL-4, 25 ng/mL IL-7, 25 ng/mL IL-9, 5 ug/mL IL-15, 100 ug/mL IL-21, 50 ug/mL IL-12, 0.25 mg/mL IL-18 or 100 U/mL IFN-αA. NK cells and CD4⁺ T cells were co-cultured for 4 hours with FITC-conjugated anti-CD107a+anti-CD107b antibodies. Flow cytometry was performed to determine degranulation of (A) CD56^dim NK cells and (B) CD56^bright NK cells. Data represent mean ± SEM of n≥4 experiments. Statistical significance is calculated in comparison to resting NK cells (media) co-cultured with activated CD4⁺ T cells. * <i>P</i><0.05, ** <i>P</i><0.005, *** <i>P</i><0.001.</p

Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation-4

*= 0.001, Wilcoxon rank sum test. WT, wildtype; KO, knockout.<p><b>Copyright information:</b></p><p>Taken from "Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation"</p><p>http://arthritis-research.com/content/10/2/R42</p><p>Arthritis Research & Therapy 2008;10(2):R42-R42.</p><p>Published online 15 Apr 2008</p><p>PMCID:PMC2453761.</p><p></p

Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation-7

Hanges. Phosphate-buffered saline-injected mice showed minimal signs of inflammation. Staining for macrophages was strongly positive. CD3-positive T cells were also present. Staining specificity was confirmed using, as primary antibody, nonimmune isotype-matched antibodies. Fibrin deposition was assessed by fibrin immunohistochemistry.<p><b>Copyright information:</b></p><p>Taken from "Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation"</p><p>http://arthritis-research.com/content/10/2/R42</p><p>Arthritis Research & Therapy 2008;10(2):R42-R42.</p><p>Published online 15 Apr 2008</p><p>PMCID:PMC2453761.</p><p></p

Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation-5

Reatment given 16 hours prior to soluble tissue factor injection. Platelet counts were performed 40 hours after injection of antibody. Footpad swelling was greatly reduced in mice treated with antiplatelet antibody. Antiplatelet antibody-treated mice, n = 10; control, rabbit normal serum-treated mice, n = 10. *< 0.05, **< 0.01, test.<p><b>Copyright information:</b></p><p>Taken from "Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation"</p><p>http://arthritis-research.com/content/10/2/R42</p><p>Arthritis Research & Therapy 2008;10(2):R42-R42.</p><p>Published online 15 Apr 2008</p><p>PMCID:PMC2453761.</p><p></p

Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation-3

Ur different protease activated receptors (PARs). PAR-1-deficient mice. PAR-2-deficient mice. PAR-3-deficient mice. PAR-4-deficient mice. In each experiment, footpad swelling was assessed in the PAR-deficient mice and their littermates (+/+ or +/-) as controls.<p><b>Copyright information:</b></p><p>Taken from "Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation"</p><p>http://arthritis-research.com/content/10/2/R42</p><p>Arthritis Research & Therapy 2008;10(2):R42-R42.</p><p>Published online 15 Apr 2008</p><p>PMCID:PMC2453761.</p><p></p

Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation-2

or untreated mice (n = 7), were injected with 1 μg soluble tissue factor. Results from all treated groups were significantly reduced (< 0.05 by test) compared with the control group. Wildtype mice were treated with ancrod (n = 7) or with phosphate-buffered saline (n = 7) and then injected with 1 μg soluble tissue factor. Results from ancrod-treated mice are significantly different from the control group, at all time points (< 0.05 by test).<p><b>Copyright information:</b></p><p>Taken from "Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation"</p><p>http://arthritis-research.com/content/10/2/R42</p><p>Arthritis Research & Therapy 2008;10(2):R42-R42.</p><p>Published online 15 Apr 2008</p><p>PMCID:PMC2453761.</p><p></p