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Intact IL-12 signaling is necessary for the generation of human natural killer cells with enhanced effector function after restimulation  Venkateswara.

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Presentation on theme: "Intact IL-12 signaling is necessary for the generation of human natural killer cells with enhanced effector function after restimulation  Venkateswara."— Presentation transcript:

1 Intact IL-12 signaling is necessary for the generation of human natural killer cells with enhanced effector function after restimulation  Venkateswara R. Simhadri, PhD, John L. Mariano, BS, Olatz Zenarruzabeitia, PhD, Christine M. Seroogy, MD, Steven M. Holland, MD, Hye Sun Kuehn, PhD, Sergio D. Rosenzweig, MD, PhD, Francisco Borrego, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 134, Issue 5, Pages e1 (November 2014) DOI: /j.jaci Copyright © 2014 The Authors Terms and Conditions

2 Fig 1 Decreased IFN-γ production by IL-12Rβ1–deficient NK cells in response to IL-12 in combination with IL-15, IL-18, or both. PBMCs were stimulated with different combinations of IL-12 (10 ng/mL), IL-15 (10 ng/mL), and IL-18 (50 ng/mL) for 16 hours or with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (1 μmol/L) for 6 hours. For the last 6 to 8 hours, monensin was added to the culture. Then cell-surface receptor expression and intracellular IFN-γ production were determined by using flow cytometry. Lymphocytes were electronically gated based on forward- and side-scatter parameters, and NK cells were identified by the CD3−CD56+ phenotype. Journal of Allergy and Clinical Immunology  , e1DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

3 Fig 2 Absence of enhanced effector functions after restimulation by IL-12Rβ1−deficient NK cells preactivated with IL-12 plus IL-15 plus IL-18. A, PBMCs were cultured for 16 hours with either low concentrations of IL-15 (1 ng/mL; ie, nonpreactivated) or with IL-12 (10 ng/mL), IL-15 (10 ng/mL), and IL-18 (50 ng/mL; ie, preactivated). Next, cells were washed with PBS and cultured in Iscove modified medium supplemented with 10% human AB serum plus 1 ng/mL IL-15. After 7 days, cells were harvested and incubated with cells for 6 hours in the presence of anti-CD107a and anti-CD107b mAbs. The secretion inhibitor monensin was added for the last 5 hours. Then CD107a/b expression (left panel) and TNF-α production (right panel) by NK cells were determined by using flow cytometry. Lymphocytes were electronically gated based on forward- and side-scatter parameters, and NK cells were identified by CD3−CD56+ phenotype. B, PBMCs from the healthy control subject and the second patient were cultured as in Fig 2, A. After 7 days, cells were harvested and incubated with cells or with IL-15 plus IL-18 for 16 hours. Monensin was added for the last 5 hours. Then the IFN-γ production by NK cells in response to restimulation with cells (left panel) and IL-15 plus IL-18 (right panel) was measured by using flow cytometry. Journal of Allergy and Clinical Immunology  , e1DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

4 Fig E1 Genetic and phenotypic characterization of IL-12Rβ1 deficiency in the second patient. A, Coding exons and adjacent splicing sites within the IL12RB1 gene from the second patient were PCR amplified and sequenced with the Sanger technique. Results showed a mutation at exon 13, adding to a premature stop codon. The test result was IL12RB1 NM_ c.1495C>T, p.Q499X homozygous. B, IL-12Rβ1 cell-surface expression on PBMCs from 2 healthy donors and the second patient. Cells were stimulated with anti-CD3 and anti-CD28 (1 μg/mL) antibodies plus IL-2 (10 ng/mL) for 5 days. After that, cells were treated with IL-12 (100 ng/mL) for 30 minutes. Lymphocytes were electronically gated based on forward- and side-scatter parameters. Histograms in gray show the binding of isotype control antibody, and histograms in black show the binding of anti–IL-12Rβ1 antibody. PE, Phycoerythrin. C, PBMCs were cultured as in Fig E1, B. After that, cells were treated with IL-12 (100 ng/mL) for 30 minutes, and signal transducer and activator of transcription 4 phosphorylation (p-STAT4) was detected by means of flow cytometric analysis. Lymphocytes were electronically gated based on forward- and side-scatter parameters. Binding of anti–p-STAT4 antibody to unstimulated (black) and stimulated (red) cells is shown. Journal of Allergy and Clinical Immunology  , e1DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

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