Intact IL-12 signaling is necessary for the generation of human natural killer cells with enhanced effector function after restimulation
Authors: Venkateswara R. Simhadri, John L. Mariano, Olatz Zenarruzabeitia, Christine M. Seroogy, Steven M. Holland, Hye Sun Kuehn, Sergio D. Rosenzweig, Francisco Borrego
Journal: Journal of Allergy and Clinical Immunology (2014)

Natural killer (NK) cells are lymphocytes that are important for host defense against viral and bacterial infections, as well as malignant transformation. They have the capacity to kill target cells and produce cytokines on activation without previous sensitization. Although NK cells have been traditionally categorized as members of the innate immune system, recent studies have shown that NK cells have the ability to modify their effector function based on previous cytokine stimulation, activating receptor–mediated stimulation, or both.1Cooper M.A. Elliott J.M. Keyel P.A. Yang L. Carrero J.A. Yokoyama W.M. Cytokine-induced memory-like natural killer cells.Proc Natl Acad Sci U S A. 2009; 106: 1915-1919Crossref PubMed Scopus (543) Google Scholar, 2Paust S. von Andrian U.H. Natural killer cell memory.Nat Immunol. 2011; 12: 500-508Crossref PubMed Scopus (185) Google Scholar, 3Sun J.C. Beilke J.N. Lanier L.L. Adaptive immune features of natural killer cells.Nature. 2009; 457: 557-561Crossref PubMed Scopus (1137) Google Scholar The first evidence that NK cells can remember came from studies showing that they mediated recall responses to haptens in mice lacking T and B cells using a contact hypersensitivity model.4O'Leary J.G. Goodarzi M. Drayton D.L. von Andrian U.H. T cell- and B cell-independent adaptive immunity mediated by natural killer cells.Nat Immunol. 2006; 7: 507-516Crossref PubMed Scopus (668) Google Scholar NK cell memory has also been demonstrated in viral infections by using murine models.3Sun J.C. Beilke J.N. Lanier L.L. Adaptive immune features of natural killer cells.Nature. 2009; 457: 557-561Crossref PubMed Scopus (1137) Google Scholar In these models NK cells acquired long-lived memory to influenza A, vesicular stomatitis virus, and HIV antigens.5Paust S. Gill H.S. Wang B.Z. Flynn M.P. Moseman E.A. Senman B. et al.Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.Nat Immunol. 2010; 11: 1127-1135Crossref PubMed Scopus (524) Google Scholar Additionally, development of long-lived NK cell memory to mouse cytomegalovirus infection was shown to be dependent on IL-12–mediated signals.3Sun J.C. Beilke J.N. Lanier L.L. Adaptive immune features of natural killer cells.Nature. 2009; 457: 557-561Crossref PubMed Scopus (1137) Google Scholar, 6Sun J.C. Madera S. Bezman N.A. Beilke J.N. Kaplan M.H. Lanier L.L. Proinflammatory cytokine signaling required for the generation of natural killer cell memory.J Exp Med. 2012; 209: 947-954Crossref PubMed Scopus (216) Google Scholar Even without exposure to specific antigens, ex vivo short exposure of mouse and human NK cells to activating cytokines, such as IL-12, IL-15, and IL-18, elicits "memory-like" properties that are defined as enhanced effector functions after restimulation.1Cooper M.A. Elliott J.M. Keyel P.A. Yang L. Carrero J.A. Yokoyama W.M. Cytokine-induced memory-like natural killer cells.Proc Natl Acad Sci U S A. 2009; 106: 1915-1919Crossref PubMed Scopus (543) Google Scholar, 7Ni J. Miller M. Stojanovic A. Garbi N. Cerwenka A. Sustained effector function of IL-12/15/18-preactivated NK cells against established tumors.J Exp Med. 2012; 209: 2351-2365Crossref PubMed Scopus (272) Google Scholar, 8Romee R. Schneider S.E. Leong J.W. Chase J.M. Keppel C.R. Sullivan R.P. et al.Cytokine activation induces human memory-like NK cells.Blood. 2012; 120: 4751-4760Crossref PubMed Scopus (389) Google Scholar In this study we tested the role of IL-12–mediated signals in the in vitro generation of human NK cells with enhanced effector function after restimulation. In our opinion the best method to address this issue would involve the use of PBMCs from patients with a deficiency in IL-12 or IL-12 receptor (IL-12R) because we could then exclude that the NK cells used in the experiments had been activated at any time in vivo by IL-12. We have previously described a 19-month-old patient with an IL-12Rβ1 deficiency caused by a complex mutation at exon 14 in the IL12RB1 gene (c.1623_1624delinsTT; p.Q541X).9Gruenberg D.A. Anover-Sombke S. Gern J.E. Holland S.M. Rosenzweig S.D. Torgerson T.R. et al.Atypical presentation of IL-12 receptor beta1 deficiency with pneumococcal sepsis and disseminated nontuberculous mycobacterial infection in a 19-month-old girl born to nonconsanguineous US residents.J Allergy Clin Immunol. 2010; 125: 264-265Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar We showed that IFN-γ production was markedly decreased after stimulation of PBMCs with PHA or PHA plus IL-12 and that there was diminished signal transducer and activator of transcription 4 phosphorylation after IL-12 stimulation.9Gruenberg D.A. Anover-Sombke S. Gern J.E. Holland S.M. Rosenzweig S.D. Torgerson T.R. et al.Atypical presentation of IL-12 receptor beta1 deficiency with pneumococcal sepsis and disseminated nontuberculous mycobacterial infection in a 19-month-old girl born to nonconsanguineous US residents.J Allergy Clin Immunol. 2010; 125: 264-265Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Here we specifically tested whether the NK cells from this patient responded to IL-12 stimulation. Flow cytometric analyses showed this patient's NK cells did not produce IFN-γ when they were stimulated with IL-12 plus IL-15 or IL-12 plus IL-18 (Fig 1). On the other hand, approximately 20% of the NK cells from the age-matched healthy control subject produced IFN-γ when they were stimulated with IL-12 plus IL-15, and 85% of the NK cells produced IFN-γ when they were stimulated with IL-12 plus IL-18 (Fig 1). When NK cells were stimulated with a combination of the 3 cytokines, almost all NK cells from the healthy control subject produced IFN-γ, whereas only 12% of the NK cells from the patient expressed IFN-γ. Furthermore, on a per-cell basis, the NK cells from the patient produced less IFN-γ than the NK cells from the control subject, as shown by a median fluorescence intensity of 921 for the IFN-γ producing NK cells from the patient versus a median fluorescence intensity of 35,205 for the IFN-γ–producing NK cells from the healthy control subject. We excluded that the patient had a defect in the IFNG gene or its regulation because her NK cells and those from the control subject produced similar amounts of IFN-γ in response to phorbol 12-myristate 13-acetate plus ionomycin (Fig 1). As expected, these results corroborated that NK cells from an IL-12Rβ1–deficient patient do not respond to IL-12. Next, we evaluated the role of IL-12–mediated signals in the generation of NK cells with enhanced effector functions after restimulation or "memory-like" NK cells, as reported,1Cooper M.A. Elliott J.M. Keyel P.A. Yang L. Carrero J.A. Yokoyama W.M. Cytokine-induced memory-like natural killer cells.Proc Natl Acad Sci U S A. 2009; 106: 1915-1919Crossref PubMed Scopus (543) Google Scholar, 8Romee R. Schneider S.E. Leong J.W. Chase J.M. Keppel C.R. Sullivan R.P. et al.Cytokine activation induces human memory-like NK cells.Blood. 2012; 120: 4751-4760Crossref PubMed Scopus (389) Google Scholar after preactivation with cytokines (Fig 2). We stimulated PBMCs with IL-12 plus IL-15 plus IL-18 for 16 hours, followed by washes and a 7-day rest period in vitro, with survival supported by low concentrations of IL-15. After the resting period, cells were harvested and coincubated with 721.221 target cells (EBV-transformed B cells). The expression of CD107 (CD107a and CD107b) as a surrogate for degranulation triggered by 721.221 tumor cells was measured by using flow cytometry. Preactivated NK cells from the healthy control subject degranulated more than nonpreactivated NK cells after coincubation with 721.221 cells (15.4% vs 9.4%; Fig 2, A, left panel). However, preactivated and nonpreactivated NK cells from the patient showed similar degranulation after coincubation with 721.221 cells (4.3% vs 4%; Fig 2, A, left panel). These results suggest there is no significant increase in the degranulation capabilities after preactivation with cytokines in NK cells lacking IL-12–mediated signaling. We also measured TNF-α production by NK cells after coincubation with 721.221 cells (Fig 2, A, right panel). Similar to the degranulation results, preactivated NK cells from the healthy control subject produced more TNF-α than nonpreactivated NK cells (0.51% vs 0.1%; Fig 2, A, right panel), whereas there were no differences in TNF-α production when preactivated and nonpreactivated NK cells from the patient were compared (0.02% vs 0.04%; Fig 2, A, right panel). A patient with a different homozygous null IL12RB1 mutation (c.1495C>T; p. Q499X) and impaired signal transducer and activator of transcription 4 phosphorylation on IL-12 stimulation (see Fig E1 in this article's Online Repository at www.jacionline.org) was also tested to confirm that the observed results were related to the impaired IL-12 signaling rather than with a particular mutation. Similar to the results obtained with cells from the first patient, preactivated NK cells from the healthy control subject produced more IFN-γ than nonpreactivated NK cells after restimulation with 721.221 cells (20.4% vs 14.7%) and IL-15 plus IL-18 (54.1% vs 29.7%; Fig 2, B). However, preactivated and nonpreactivated NK cells from the second patient showed similar IFN-γ production after restimulation with 721.221 cells (1.29% vs 3.06%) and IL-15 plus IL-18 (3.60% vs 1.94%; Fig 2, B). These results indicate that IL-12Rβ1–deficient NK cells do not have the ability to acquire enhanced effector functions after restimulation. In addition to the defect in restimulation, the IL-12Rβ1–deficient NK cells also have a defect in primary activation. The deficiency in IFN-γ production after target and IL-15 plus IL-18 stimulation and in the absence of preactivation suggests that human NK cells require in vivo priming with IL-12 to acquire full functional reactivity and is consistent with a study by Guia et al.10Guia S. Cognet C. de Beaucoudrey L. Tessmer M.S. Jouanguy E. Berger C. et al.A role for interleukin-12/23 in the maturation of human natural killer and CD56+ T cells in vivo.Blood. 2008; 111: 5008-5016Crossref PubMed Scopus (50) Google Scholar Our observations are partly in contradiction to those published by Romee et al,8Romee R. Schneider S.E. Leong J.W. Chase J.M. Keppel C.R. Sullivan R.P. et al.Cytokine activation induces human memory-like NK cells.Blood. 2012; 120: 4751-4760Crossref PubMed Scopus (389) Google Scholar who showed that after preactivation of human NK cells with IL-12 plus IL-15, restimulation with K562 target cells led to an increase in IFN-γ production without an increase in the cell-surface expression of CD107a on NK cells. Although we do not fully understand the reasons for this discrepancy, the use of different target cells and staining with both anti-CD107a and anti-CD107b mAbs in our experiments might explain the differences. In fact, our results are in accordance with those obtained with the mouse cytomegalovirus infection model, which demonstrated that degranulation by mouse memory NK cells was enhanced after stimulation with anti-NK1.1 mAb3Sun J.C. Beilke J.N. Lanier L.L. Adaptive immune features of natural killer cells.Nature. 2009; 457: 557-561Crossref PubMed Scopus (1137) Google Scholar and that the generation of memory NK cells was dependent on IL-12–mediated signals.6Sun J.C. Madera S. Bezman N.A. Beilke J.N. Kaplan M.H. Lanier L.L. Proinflammatory cytokine signaling required for the generation of natural killer cell memory.J Exp Med. 2012; 209: 947-954Crossref PubMed Scopus (216) Google Scholar Here, using human IL-12Rβ1–deficient NK cells, we have demonstrated the essential role of IL-12–mediated signals in the generation of human NK cells with enhanced effector function after restimulation. These findings provide the rationale for including brief stimulations or preactivation with IL-12 in combination with other cytokines, such as IL-15 and IL-18, to generate NK cells with enhanced effector functions (ie, "memory-like") that might be of great relevance in the field of NK cell–based immunotherapy and might also unveil a new aspect of pathophysiology to be considered in IL-12Rβ1–deficient patients.