Intrahepatic and peripheral blood phenotypes of natural killer and T cells: differential surface expression of killer cell immunoglobulin‐like receptors

Deep characterization of the frequencies, phenotypes and functionalities of liver and peripheral blood natural killer (NK), natural killer T (NKT) and T cells from healthy individuals is an essential step to further interpret changes in liver diseases. These data indicate that CCR7, a chemokine essential for cell migration through lymphoid organs, is almost absent in liver NK and T cells. CD56^bright NK cells, which represent half of liver NK cells, showed lower expression of the inhibitory molecule NKG2A and an increased frequency of the activation marker NKp44. By contrast, a decrease of CD16 expression with a potential decreased capacity to perform antibody‐dependent cellular cytotoxicity was the main difference between liver and peripheral blood CD56^dim NK cells. Liver T cells with an effector memory or terminally differentiated phenotype showed an increased frequency of MAIT cells,T‐cell receptor‐γδ (TCR‐γδ) T cells and TCR‐αβ CD8⁺ cells, with few naive T cells. Most liver NK and T cells expressed the homing markers CD161 and CD244. Liver T cells revealed a unique expression pattern of killer cell immunoglobulin‐like receptors (KIR) receptors, with increased degranulation ability and higher secretion of interferon‐γ. Hence, the liver possesses a large amount of memory and terminally differentiated CD8⁺ cells with a unique expression pattern of KIR activating receptors that have a potent functional capacity as well as a reduced amount of CCR7, which are unable to migrate to regional lymph nodes. These results are consistent with previous studies showing that liver T (and also NK) cells likely remain and die in the liver.     

Tissue Distribution Dynamics of Human NK Cells Inferred from Peripheral Blood Depletion Kinetics after Sphingosine‐1‐Phosphate Receptor Blockade

Human natural killer (NK) cell subsets differentially distribute throughout the organism. While CD56^dim and CD56^bright NK cell subsets similarly reside in the bone marrow (BM), the CD56^dim population predominantly accumulates in non‐lymphoid tissues and the CD56^bright counterpart in lymphoid tissue (LT). The dynamics with which these NK cell subsets redistribute to tissues remains unexplored. Here, we studied individuals newly exposed to fingolimod, a drug that efficiently blocks sphingosine‐1‐phosphate (S1P)‐directed lymphocyte – including NK cell – egress from tissue to blood. During an observation period of 6h peripheral blood depletion of CD56^bright NK cells was observed 3 h after first dose of fingolimod, with 40–50% depletion after 6 h, while a decrease of the numbers of CD56^dim NK cells did not reach the level of statistical significance. In vitro, CD56^bright and CD56^dim NK cells responded comparably to the BM‐homing chemokine CXCL12, while CD56^bright NK cells migrated more efficiently in gradients of the LT‐homing chemokines CCL19 and CCL21. In conjuncture with these in vitro studies, the indirectly observed subset‐specific depletion kinetics from blood are compatible with preferential and more rapid redistribution of CD56^bright NK cells from blood to peripheral tissue such as LT and possibly also the inflamed central nervous system. These data shed light on an unexplored level at which access of NK cells to LT, and thus, for example antigen‐presenting cells, is regulated.     

Back to the drawing board: Understanding the complexity of hepatic innate lymphoid cells

Recent studies of immune populations in nonlymphoid organs have highlighted the great diversity of the innate lymphoid system. It has also become apparent that mouse and human innate lymphoid cells (ILCs) have distinct phenotypes and properties. In this issue of the European Journal of Immunology, Harmon et al. [Eur. J. Immunol. 2016. 46: 2111–2120] characterized human hepatic NK‐cell subsets. The authors report that hepatic CD56^bright NK cells resemble mouse liver ILC1s in that they express CXCR6 and have an immature phenotype. However, unlike mouse ILC1s, they express high levels of Eomes and low levels of T‐bet, and upon stimulation with tumor cells, secrete low amounts of cytokines. These unexpected findings further support the differences between human and mouse immune populations and prompt the study of the role of hepatic ILC subsets in immune responses.     

Correlation Between Natural Cytotoxicity Receptors and Intracellular Cytokine Expression of Peripheral Blood NK Cells in Women with Recurrent Pregnancy Losses and Implantation Failures

Problem Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. We investigated whether women with recurrent pregnancy losses (RPLs) and implantation failures have aberrant correlation between NCRs and intracellular cytokine expression of NK cells. Method of study Peripheral blood NK cells (CD56^dim and CD56^bright) were analyzed for NCRs (NKp46, NKp44 and NKp30) and cytokine expression (TNF‐α, IFN‐γ, IL‐4, IL‐10) using flow cytometry in RPL (n = 22), implantation failures (n = 23) or controls (n = 15). Results In type 1 cytokine studies, CD56^bright/NKp30⁺ cells in controls (r = 0.696, P < 0.05) were positively correlated with CD56^bright/IFN‐γ⁺/TNF‐α⁺ cells. CD56^bright/NKp46⁺ cells in implantation failures (r = ?0.76, P < 0.01) were negatively correlated with CD56^bright/IFN‐γ⁺/TNF‐α^? cells. RPL did not have any correlation. In type 2 cytokine studies, CD56⁺/NKp46⁺ cells (r = 0.758, P < 0.01) and CD56⁺/NKp30⁺ cells (r = 0.637, P < 0.05) were positively correlated with CD56^bright/IL‐4⁺/IL‐10⁺ cells in controls. CD56⁺/NKp30⁺ cells in implantation failures (r = ?0.778, P < 0.05) were negatively correlated with CD56^bright/IL‐10⁺/IL‐4⁺ cells. There were no correlations in RPL. Conclusion Recurrent pregnancy losses and implantation failures have lack of, or negative correlation between NCRs and intracellular cytokines expression. This observation suggests that excessive pro‐inflammatory cytokine expression in NK cells in RPL and implantation failures may be exerted through the NCRs or interruption of signal transduction processes.     

Murine CXCR3+CD27bright NK cells resemble the human CD56bright NK‐cell population

Human NK cells can be subdivided into CD56^dim and CD56^bright NK cells, which exhibit different phenotypical and functional characteristics. As murine NK cells lack CD56 or a distinct correlate, direct comparative studies of NK cells in mice and humans are limited. Although CD27 is currently proposed as a feasible subset marker in mice, we assume that the usage of this marker alone is insufficient. We rather investigated the expression of the chemokine receptor CXCR3 for its suitability for distinguishing murine NK‐cell subsets with simultaneous consideration of CD27. Compared with CXCR3^? NK cells, exerting stronger cytotoxic capability, CXCR3⁺ NK cells displayed an activated phenotype with a lower expression of Ly49 receptors, corresponding to human CD56^bright NK cells. Also in common with human CD56^bright NK cells, murine CXCR3⁺ NK cells exhibit prolific expansion as well as robust IFN‐γ, TNF‐α and MIP‐1α production. We additionally demonstrated changes in both CXCR3 and CD27 expression upon NK‐cell activation. In summary, CXCR3 serves as an additional applicable marker for improved discrimination of functionally distinct murine NK‐cell subsets that comply with those in humans.     

CD56bright NK cells after hematopoietic stem cell transplantation are activated mature NK cells that expand in patients with low numbers of T cells

We studied early NK‐cell recovery in 29 allografted patients undergoing different lymphoreductive regimens. Already at 2 wk after graft take, the number of NK cells had reached (supra)normal levels but NK‐cell subsets were skewed. The number of CD56^dimCD16^bright NK cells was low and correlated strongly with the level of hematopoiesis, whereas the number of the more abundant NK cells expressing high levels of CD56 did not. Post‐transplant CD56^bright NK cells (ptCD56^bright) differed from CD56^bright NK cells in normal controls (CD56^bright) in being HLA‐DR‐ and perforin‐positive, CCR7^?, CD27^?, CD127^? and mostly c‐kit^?. CD56^bright from normal controls stimulated by IL‐15 in vitro (NK^IL‐15) acquired all the characteristics distinguishing CD56^bright from ptCD56^bright. IL‐2 exerted similar effects. Moreover, when cultured without cytokines, ptCD56^bright, CD56^bright and NK^IL‐15 responded similarly by upregulating CD127 and c‐kit but not CCR7. IL‐12 stimulated IFN‐γ production in ptCD56^bright, whereas CD56^bright responded only to IL‐12 plus IL‐15. Hence, ptCD56^bright have all the features of cytokine‐stimulated CD56^bright. Because only patients with low numbers of T cells had high numbers of ptCD56^bright, we conclude that ptCD56^bright are activated CD56^bright that expand while competing with T cells for the elevated post‐transplant level of IL‐15.     

TGF‐β affects development and differentiation of human natural killer cell subsets

Human peripheral blood NK cells may be divided into two main subsets: CD56^brightCD16^? and CD56^dimCD16⁺. Since TGF‐β is known to influence the development of many leukocyte lineages, its effects on NK cell differentiation either from human CD34⁺Lin^? hematopoietic progenitor/stem cells in vitro or from peripheral blood NK cells were investigated. TGF‐β represses development of NK cells from CD34⁺ progenitors and inhibits differentiation of CD16⁺ NK cells. Moreover, TGF‐β also results in conversion of a minor fraction of CD56^brightCD16⁺ cells found in peripheral blood into CD56^brightCD16^? cells, highlighting a possible role of the former as a developmental intermediate and of TGF‐β in influencing the genesis of NK subsets found in blood.     

Activating NK cell receptor expression/function (NKp30, NKp46, DNAM-1) during chronic viraemic HCV infection is associated with the outcome of combined treatment

Specific NK cell killer inhibitory receptor (KIR):HLA haplotype combinations have been associated with successful clearance of acute and chronic HCV infection. Whether an imbalance of activating NK cell receptors also contributes to the outcome of treatment of chronic HCV infection, however, is not known. We studied peripheral NK cell phenotype and function in 28 chronically viraemic HCV genotype I treatment‐naïve patients who underwent treatment with pegylated IFN‐α and ribavirin. At baseline, chronically infected patients with sustained virological response (SVR) had reduced CD56^brightCD16^+/? cell populations, increased CD56^dullCD16⁺ NK cell proportions, and lower expression of NKp30, DNAM‐1, and CD85j. Similarly, reduced NK cell IFN‐γ production but increased degranulation was observed among nonresponding (NR) patients. After treatment, CD56^brightCD16^+/? NK cell numbers increased in both SVR and NR patients, with a parallel significant increase in activating NKp30 molecule densities in SVR patients only. In vitro experiments using purified NK cells in the presence of rIL‐2 and IFN‐α confirmed upregulation of NKp30 and also of NKp46 and DNAM‐1 in patients with subsequent SVR. Thus, differences in patient NK cell receptor expression and modulation during chronic HCV‐1 infection are associated with subsequent outcome of standard treatment. Individual activating receptor expression/function integrates with KIR:HLA genotype carriage to determine the clearance of HCV infection upon treatment.     

TLR activation of tumor‐associated macrophages from ovarian cancer patients triggers cytolytic activity of NK cells

We analyzed the functional outcome of the interaction between tumor‐associated macrophages (TAMs) and natural killer (NK) cells. TAMs from ascites of ovarian cancer patients displayed an alternatively activated functional phenotype (M2) characterized by a remarkably high frequency and surface density of membrane‐bound IL‐18. Upon TLR engagement, TAMs acquired a classically activated functional phenotype (M1), released immunostimulatory cytokines (IL‐12, soluble IL‐18), and efficiently triggered the cytolytic activity of NK cells. TAMs also induced the release of IFN‐γ from NK cells, which however was significantly lower compared with that induced by in vitro‐polarized M2 cells. Most tumor‐associated NK cells displayed a CD56^bright, CD16^neg or CD56^bright, CD16^dim phenotype, and very poor cytolytic activities, despite an increased expression of the activation marker CD69. They also showed downregulation of DNAM‐1, 2B4, and NTB‐A activating receptors, and an altered chemokine receptor repertoire. Importantly however, when appropriately stimulated, NK cells from the patients, including those cells isolated from ascites, efficiently killed autologous TAMs that expressed low, “nonprotective” levels of HLA class I molecules. Overall, our data show the existence of a complex tumor microenvironment in which poorly cytolytic/immature NK cells deal with immunosuppressive tumor‐educated macrophages.     

CD8+ NK cells are predominant in chimpanzees,characterized by high NCR expression and cytokine production,and preserved in chronic HIV‐1 infection

HIV‐1 infection in humans results in an early and progressive NK cell dysfunction and an accumulation of an “anergic” CD56^? CD16⁺ NK subset, which is characterised by low natural cytotoxicity receptor expression and low cytokine producing capacity. In contrast to humans, chimpanzee NK cells do not display a distinguishable CD56^bright and CD56^dim subset but, as shown here, could be subdivided into functionally different CD8⁺ and CD8^? subsets. The CD8⁺ NK cells expressed significantly higher levels of triggering receptors including NKp46 and, upon in vitro activation, produced more IFN‐γ, TNF‐α and CD107 than their CD8^? counterparts. In addition, chimpanzee CD8^? NK cells had relatively high levels of HLA‐DR expression, suggestive of an activated state. Killing inhibitory receptors were expressed only at low levels; however, upon in vitro stimulation, they were up‐regulated in CD8⁺ but not in CD8^? NK cells and were functionally capable of inhibiting NKp30‐triggered killing. In contrast to HIV‐1‐infected humans, infected chimpanzees maintained their dominant CD8⁺ NK cell population, with high expression of natural cytotoxicity receptors.     

Low-dose IL-2 induces CD56bright NK regulation of T cells via NKp44 and NKp46

Low-dose interleukin (IL)-2 has shown clinical benefits in patients with autoimmune and inflammatory diseases. Both regulatory T cells (T_regs) and natural killer (NK) cells are increased in response to low-dose IL-2 immunotherapy. The role of regulatory T cells in autoimmune diseases has been extensively studied; however, NK cells have not been as thoroughly explored. It has not been well reported whether the increase in NK cells is purely an epiphenomenon or carries actual benefits for patients with autoimmune diseases. We demonstrate that low-dose IL-2 expands the primary human CD56^bright NK cells resulting in a contact-dependent cell cycle arrest of effector T cells (T_effs) via retention of the cycle inhibitor p21. We further show that NK cells respond via IL-2R-β, which has been shown to be significant for immunity by regulating T cell expansion. Moreover, we demonstrate that blocking NK receptors NKp44 and NKp46 but not NKp30 could abrogate the regulation of proliferation associated with low-dose IL-2. The increase in NK cells was also accompanied by an increase in T_reg cells, which is dependent on the presence of CD56^bright NK cells. These results not only heighten the importance of NK cells in low-dose IL-2 therapy but also identify key human NK targets, which may provide further insights into the therapeutic mechanisms of low-dose IL-2 in autoimmunity.     

Adaptive reconfiguration of the human NK‐cell compartment in response to cytomegalovirus: A different perspective of the host‐pathogen interaction

As discussed in this review, human cytomegalovirus (HCMV) infection in healthy individuals is associated with a variable and persistent increase of NK cells expressing the CD94/NKG2C activating receptor. The expansion of NKG2C⁺ NK cells reported in other infectious diseases is systematically associated with HCMV co‐infection. The functionally mature NKG2C^bright NK‐cell subset expanding in HCMV⁺ individuals displays inhibitory Ig‐like receptors (KIR and LILRB1) specific for self HLA class I, and low levels of NKp46 and NKp30 activating receptors. Such reconfiguration of the NK‐cell compartment appears particularly marked in immunocompromised patients and in children with symptomatic congenital infection, thus suggesting that its magnitude may be inversely related with the efficiency of the T‐cell‐mediated response. This effect of HCMV infection is reminiscent of the pattern of response of murine Ly49H⁺ NK cells against murine CMV (MCMV), and it has been hypothesized that a cognate interaction of the CD94/NKG2C receptor with HCMV‐infected cells may drive the expansion of the corresponding NK‐cell subset. Yet, the precise role of NKG2C⁺ cells in the control of HCMV infection, the molecular mechanisms underlying the NK‐cell compartment redistribution, as well as its putative influence in the response to other pathogens and tumors remain open issues.     

Dysregulation of regulatory CD56bright NK cells/T cells interactions in multiple sclerosis

Recent evidence has shown that CD56^bright NK cells, a subset of NK cells abundant in lymph nodes, may have an immunoregulatory function. In multiple sclerosis (MS), expansion of CD56^bright NK cells has been associated to successful response to different treatments and to remission of disease during pregnancy; how whether they exert immunoregulation in physiologic conditions and whether this is impaired in MS is not known. We dissected the immunoregulatory role of CD56^bright NK cells function in healthy subjects (HS) and compared it with that of untreated MS subjects or patients with clinically isolated syndrome suggestive of MS (CIS). We found that CD56^bright NK cells from HS acquire, upon inflammatory cues, the capability of suppressing autologous CD4+T cell proliferation through direct cytotoxicity requiring engagement of natural cytotoxicity receptors (NCRs) and secretion of granzyme B. CD56^bright NK cells from patients with MS/CIS did not differ in frequency and share a similar phenotype but displayed a significantly lower ability to inhibit autologous T cell proliferation. This impairment was not related to deficient expression of NCRs or granzyme B by CD56^bright NK cells, but to increased HLA-E expression on T cells from MS/CIS subjects, which could enhance the inhibitory effect mediated by NKG2A that is homogeneously expressed on CD56^bright NK cells. The defect in controlling autologous T cells by CD56^bright NK cells in MS/CIS might contribute to the excess of autoimmune response that is associated to disease development.     

CD56bright natural killer cell subsets: Characterization of distinct functional responses to interleukin-2 and the c-kit ligand

Natural killer (NK) cells are bone marrow-derived large granular lymphocytes that express the CD56 surface antigen. The CD56^bright NK subset represents approximately 10 % of all NK cells and is thought to be the least differentiated NK cell component in blood. The most mature NK cell expresses CD56 at low density and CD16 (FcRγIII) at high density, whereas CD56^bright NK cells either lack CD 16 (CD56^brightCD16^?) or express it at low density (CD56^brightCD16^dim). c-kit is a tyrosine kinase receptor which is expressed on both CD34⁺ hemato-poietic precursor cells and CD56^bright NK cells. In the current study, we characterize interleukin (IL)-2 receptor (IL-2R) and c-kit expression in each of the CD56^bright subsets. Both the CD56^brightCD16^? and CD56^brightCD16^dim NK subsets express the high-affinity IL-2R and the c-kit receptor when isolated from fresh blood. However, each CD56^bright NK cell subset has distinct functional responses to IL-2, the c-kit ligand (KL), or both. Activation of the high-affinity IL-2R on CD56^brightCD16^? NK cells induces a proliferative response that is significantly weaker than that observed in the CD56^brightCD16^dim NK cell subset. Incubation of the CD56^brightCD16^? NK cell subset with KL significantly enhances IL-2-induced proliferation, while KL has no such effect on the CD56^brightCD16^dim NK subset. Activation of the high-affinity IL-2R in both CD56^bright subsets induces lymphokine-activated killer (LAK) activity, but the addition of KL has no effect on LAK activity. Co-stimulation of either CD56^bright subset with IL-12 and concentrations of IL-2 that only saturate the high-affinity IL-2R induces substantial interferon (IFN)-γ production. The addition of KL to this co-stimulatory signal enhances IFN-γ production in both CD56^bright NK subsets. The distinct functional responses to IL-2 and KL seen in the CD56^brightCD16^? and CD56^brightCD16^dim NK subsets provide insight into IL-2R signaling and suggest that each phenotype identifies a discrete stage of NK cell differentiation.