Loss of CCR7 Expression on CD56bright NK Cells Is Associated with a CD56dimCD16+ NK Cell-Like Phenotype and Correlates with HIV Viral Load

NK cells are pivotal sentinels of the innate immune system and distinct subpopulations in peripheral blood have been described. A number of studies addressed HIV-induced alterations of NK cell phenotype and functionality mainly focusing on CD56^dimCD16⁺ and CD56⁻CD16⁺ NK cells. However, the impact of HIV-infection on CD56^bright NK cells is less well understood. Here we report a rise of CD56^bright NK cells in HIV-infected individuals, which lack CCR7-expression and strongly correlate with HIV viral load. CCR7⁻CD56^bright NK cells were characterized by increased cytolytic potential, higher activation states and a more differentiated phenotype. These cells thus acquired a number of features of CD56^dimCD16⁺ NK cells. Furthermore, CD56^bright NK cells from HIV patients exhibited higher degranulation levels compared to uninfected individuals. Thus, chronic HIV-infection is associated with a phenotypic and functional shift of CD56^bright NK cells, which provides a novel aspect of HIV-associated pathogenesis within the NK cell compartment.     

Altered Expression of Natural Cytotoxicity Receptors and NKG2D on Peripheral Blood NK Cell Subsets in Breast Cancer Patients

Human natural killer (NK) cells are considered professional cytotoxic cells that are integrated into the effector branch of innate immunity during antiviral and antitumoral responses. The purpose of this study was to examine the peripheral distribution and expression of NK cell activation receptors from the fresh peripheral blood mononuclear cells of 30 breast cancer patients prior to any form of treatment (including surgery, chemotherapy, and radiotherapy), 10 benign breast pathology patients, and 24 control individuals. CD3⁻CD56^dimCD16^bright NK cells (CD56^dim NK) and CD3⁻CD56^brightCD16^dim/− NK cells (CD56^bright NK) were identified using flow cytometry. The circulating counts of CD56^dim and CD56^bright NK cells were not significantly different between the groups evaluated, nor were the counts of other leukocyte subsets between the breast cancer patients and benign breast pathology patients. However, in CD56^dim NK cells, NKp44 expression was higher in breast cancer patients (P = .0302), whereas NKp30 (P = .0005), NKp46 (P = .0298), and NKG2D (P = .0005) expression was lower with respect to healthy donors. In CD56^bright NK cells, NKp30 (P = .0007), NKp46 (P = .0012), and NKG2D (P = .0069) expression was lower in breast cancer patients compared with control group. Only NKG2D in CD56^bright NK cells (P = .0208) and CD56^dim NK cells (P = .0439) showed difference between benign breast pathology and breast cancer patients. Collectively, the current study showed phenotypic alterations in activation receptors on CD56^dim and CD56^bright NK cells, suggesting that breast cancer patients have decreased NK cell cytotoxicity.     

HIV Infection Is Associated with a Preferential Decline in Less-Differentiated CD56dim CD16+ NK Cells

HIV-1 infection is characterized by loss of CD56^dim CD16⁺ NK cells and increased terminal differentiation on various lymphocyte subsets. We identified a decrease of CD57⁻ and CD57^dim cells but not of CD57^bright cells on CD56^dim CD16⁺ NK cells in chronic HIV infection. Increasing CD57 expression was strongly associated with increasing frequencies of killer immunoglobulin-like receptors (KIRs) and granzyme B-expressing cells but decreasing percentages of cells expressing CD27⁺, HLA-DR⁺, Ki-67⁺, and CD107a. Our data indicate that HIV leads to a decline of less-differentiated cells and suggest that CD57 is a useful marker for terminal differentiation on NK cells.NK cells are effector cells of innate immunity which are pivotal as first-line defense against viral infections, such as HIV infection (14). Large genotypic studies demonstrated a delayed onset of AIDS in HIV-seropositive individuals carrying the activating receptor KIR3DS1 and/or alleles of the inhibiting receptor KIR3DL1 in conjunction with HLA-Bw4-80I (18, 19). Development of NK cells mainly takes place in the bone marrow, from which mature NK cells move out to reside and circulate in peripheral sites (13). Mature NK cells are characterized by granules which harbor granzymes and perforin. These NK cells are fully armed, “ready-to-go” effector cells (17).A number of NK cell abnormalities have been reported in HIV infection (9), including high activation status (2, 10), increased turnover (16), differential expression of activating and inhibitory receptors (20), impaired interaction with dendritic cells (12), and loss of CD56^dim CD16⁺ NK cells (23). CD56^dim CD16⁺ NK cells represent the largest NK cell subset in peripheral blood in healthy individuals. The expression of killer immunoglobulin-like receptors (KIRs) and CD57 are predominant features of this subpopulation (8, 15). CD57 expression on NK cells has been previously associated with replicative senescence on T and NK cells (4), raising the question of how HIV-1 infection alters CD57 expression on CD56^dim CD16⁺ NK cells.To the best of our knowledge, no one has addressed the phenotypic and functional properties of CD56^dim CD16⁺ NK cells that are preferentially lost during HIV infection. Here, we provide evidence that increasing CD57 expression indicates terminal differentiation in healthy individuals, as well in as HIV-infected subjects. We furthermore show that HIV infection is associated with preferential loss of less-differentiated cells, which are characterized by high activation status and turnover.In this study, blood samples from 37 HIV-seropositive individuals and 15 healthy subjects were analyzed; all HIV-infected patients were either antiretroviral therapy naïve or untreated for more than one year. The HIV-positive study cohort comprised 10 patients with a viral load of less than 2,000 copies/ml, 14 patients with a viral load ranging from 2,000/ml to 20,000 copies/ml, and 13 patients with a viral load above 20,000 copies/ml. CD4 T cell counts ranged from 180/μl to 1,355/μl, the average being 457.3/μl.The study was approved by the local ethics commission (Ethikkommission der Medizinischen Hochschule Hannover, Votum No. 3150), and all study participants gave informed written consent for their participation.Flow cytometric analysis was performed on cryopreserved peripheral blood mononuclear cells (PBMCs) as previously described (21, 22). A list of monoclonal antibodies employed in this study is available upon request. For intracellular analysis of granzyme B, perforin, and Ki-67, we used a fixation and permeabilization kit (Invitrogen). At least 1 million events were acquired for each sample, using either a FACSAria or LSR II flow cytometer (BD Biosciences). Data were analyzed with FlowJo (TreeStar). Lymphocytes were defined by forward and side scatter. CD3⁺, CD14⁺, CD19⁺, dead cells, and cell aggregates were removed from analysis based on peridinin chlorophyll protein and Viaprobe staining and gating on a plot of forward-scatter area versus forward-scatter height (Fig. ​(Fig.1A).1A). NK cells and their distinctive subpopulations were defined based on their CD56 and/or CD16 expression. Fluorescence-minus-one (FMO) staining was used to determine threshold values for the expression of specific markers.Open in a separate windowFIG. 1.HIV infection is associated with loss of CD57⁻ and CD57^dim but not CD57^bright CD56^dim CD16⁺ NK cells. (A) Representative gating scheme for identification of NK cells. NK cells were defined as CD3⁻ CD14⁻ CD19⁻ lymphocytes expressing either CD56 or CD16 or both. We divided CD56^dim CD16⁺ NK cells into three subsets based on their level of CD57 expression: CD57⁻, CD57^dim, and CD57^bright cells. Numbers on FACS plots indicate frequency of gated population. SSC-A, side scatter area; FSC-A, forward scatter area; FSC-W, forward scatter width. (B) Comparison of percentages of the CD57⁻, CD57^dim, and CD57^bright subpopulations in control subjects (n = 14) and HIV-seropositive individuals (n = 34) on CD56^dim CD16⁺ NK cells. ns, not significant (P > 0.05); **, P < 0.01; ***, P < 0.001. (C) Frequencies of CD57⁻, CD57^dim, and CD57^bright expressing CD56^dim CD16⁺ NK cells in relation to total NK cells in control subjects (n = 14) and HIV-seropositive individuals (n = 34). (D) Mean frequency of CD56^dim CD16⁺ NK cells in 14 control individuals and in 34 HIV-infected people and the distribution of CD57⁻, CD57^dim, and CD57^bright cells within CD56^dim CD16⁺ NK cells is shown. (E) Relationship between percentage of CD57^dim CD56^dim CD16⁺NK cells and percentage of CD56^neg CD16⁺ NK cells on total NK cells. Horizontal bars in dot plots show the means.NK cells as defined above were sorted from cryopreserved PBMCs on a FACSAria (purities ranged from 91% to 99%). An amount of 10⁵ NK cells was plated per well and stimulated with 10 ng/ml interleukin-15 (IL-15), 100 ng/ml IL-12, and 5 × 10⁴ K562 cells. A CD107a degranulation assay was performed as described previously (1, 12). GraphPad Prism (version 5.0) software was used for statistical evaluation of data. Correlation analysis was performed using the Pearson test. The unpaired t test was performed when two groups were compared, and all t tests were two tailed. Comparison of more than two groups was performed using one-way analysis of variance followed by Tukey''s post-hoc test. P values of less than 0.05 were considered significant.We found that CD57 on NK cells was predominantly expressed on the CD56^dim CD16⁺ population (Fig. ​(Fig.1A).1A). The expression patterns of CD57 allowed us to differentiate between three subfractions within CD56^dim CD16⁺ NK cells, namely, CD57⁻, CD57^dim, and CD57^bright cells. The frequency of the CD57^bright subpopulation on CD56^dim CD16⁺ NK cells was increased compared to the frequency of the CD57^dim subpopulation on CD56^dim CD16⁺ NK cells in HIV-seropositive patients but not in HIV-seronegative control subjects (Fig. ​(Fig.1B).1B). This relative increase was associated with substantial reductions of the CD57⁻ CD56^dim and the CD57^dim CD56^dim NK cell subpopulations of total NK cells in our HIV-seropositive cohort compared to these subpopulations in healthy control subjects (means, 36.6% versus 24.8% [P = 0.0002] and 22.4% versus 15.4% [P = 0.0001]), but the frequencies of CD57^bright CD56^dim NK cells within total NK cells were similar between HIV-infected patients and HIV-seronegative individuals (Fig. ​(Fig.1C).1C). In accordance with previously published data (3, 23), we could confirm that there is a relative loss of CD56^dim CD16⁺ NK cells in HIV infection (mean, 84.3% versus 67.0%, P = 0.0004) (Fig. ​(Fig.1D).1D). Our data indicate that this loss is predominantly due to decreased numbers of CD57⁻ CD56^dim and CD57^dim CD56^dim NK cells, leading to a relative overrepresentation of CD57^bright cells within CD56^dim CD16⁺ NK cells in HIV infection (Fig. ​(Fig.1C).1C). There was no significant correlation between the relative loss of CD57⁻ and CD57^dim NK cells and absolute numbers of CD56^dim CD16⁺ NK cells, but there was a significant inverse correlation between loss of CD57^dim NK cells and increasing percentages of CD56⁻ CD16⁺ cells (Pearson r = −0.54, P = 0.001) (Fig. ​(Fig.1E1E).To determine whether the relative decrease of CD57⁻ and CD57^dim NK cells was associated with parameters of HIV disease progression, we performed correlation analysis of the percentages of CD57⁻ or CD57^dim cells with viral load and CD4 T cell counts. We found no such correlations (Pearson r < 0.2 and P > 0.05 for all) (data not shown). A recent cross-sectional and longitudinal study demonstrated that changes in the NK cell compartment, as shown by down-modulation of Siglec-7 on CD56^dim NK cells, are associated with HIV viremia (5). The longitudinal data in the study indicated that the full restoration of NK cell pathologies required 24 months of antiviral treatment. This suggests that alterations in the NK cell compartment can be driven by HIV viral load but that these changes seem to require a significant amount of time.We next investigated the phenotypic and functional properties of the CD57⁻, CD57^dim, and CD57^bright subpopulations on CD56^dim CD16⁺ NK cells. For KIR2DL2/DL3/DS2, we detected increasing prevalences of KIR-expressing NK cells with increasing expression of CD57 in both healthy control subjects and HIV-infected blood donors (Fig. ​(Fig.2A).2A). As for KIR3DS1/DL1, we found an increase of KIR⁺-expressing NK cells between CD57⁻ and CD57^bright cells in control individuals and significant differences in percentages of KIR3DS1/DL1-expressing NK cells between CD57⁻ and CD57^dim, as well as between CD57⁻ and CD57^bright, NK cells in our HIV-positive cohort (Fig. ​(Fig.2A).2A). These results suggest that increasing CD57 expression is associated with higher numbers of KIR-expressing NK cells in control subjects and HIV-infected subjects.Open in a separate windowFIG. 2.Phenotypic characterization of the CD57⁻, CD57^dim, and CD57^bright subpopulations of CD56^dim CD16⁺ NK cells. Representative flow cytometry plots for one control and one HIV-infected subject and summary data for all individuals whose PBMCs were analyzed are shown. CD57⁻, CD57^dim, and CD57^bright NK cells are concatenated to visualize them in a single dot plot. Numbers in contour plots indicate percentages of gated events of the respective subset. (A) Percentages of KIR2DL2/DL3/DS2 and KIR3DS1/DL1-expressing CD57⁻, CD57^dim, and CD57^bright cells were analyzed in control individuals (n = 15) and HIV-infected subjects (n = 37). (B) Numbers of HLA-DR-expressing and CD27-expressing CD57⁻, CD57^dim, and CD57^bright cells in control individuals'' (n = 15) and HIV-infected subjects'' (n = 37) PBMCs were analyzed. Horizontal bars in dot plots show the means. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.We next addressed the question of whether increasing CD57 expression is linked to differential phenotypic properties of NK cells and analyzed the HLA-DR and CD27 expression of the CD57⁻, CD57^dim, and CD57^bright subpopulations on CD56^dim CD16⁺ NK cells. A significantly higher fraction of NK cells expressed HLA-DR in the CD57⁻ than in the CD57^bright subset in both healthy control individuals and HIV-infected subjects (Fig. ​(Fig.2B).2B). A considerably higher portion of NK cells was positive for HLA-DR in HIV-infected individuals than in control subjects (means, 3.2% versus 13.2% [P < 0.0001], 1.8% versus 10.4% [P = 0.001], and 0.9% versus 6.5% [P = 0.005] for CD57⁻, CD57^dim, and CD57^bright subpopulations, respectively). We furthermore detected marked differences in frequencies of cells expressing CD27, a member of the tumor necrosis factor (TNF) receptor family (24). CD57⁻ NK cells displayed the highest percentages of CD27⁺ cells, whereas CD57^bright cells were almost all negative for CD27, in both control individuals and HIV-seropositive subjects (Fig. ​(Fig.2B).2B). We thus show that increasing expression of CD57 is associated with differential activation status and differential phenotype.Next, we sought to determine whether CD57 is linked to differential functional phenotypes by assessing the intracellular expression of granzyme B, perforin, and Ki-67. The frequencies of perforin-expressing NK cells did not vary within the different CD57 subsets of CD56^dim CD16⁺ NK cells (Fig. ​(Fig.3A).3A). However, we found that CD57^bright cells displayed the highest frequencies of granzyme B⁺ in both control and HIV-seropositive subjects, whereas CD57⁻ cells exhibited the lowest percentages for granzyme B⁺ cells (Fig. ​(Fig.3A).3A). Conversely, when we studied the expression of Ki-67, we identified the opposite trend: less than 5% of CD57^bright cells in control individuals and less than 10% of CD57^bright cells in HIV-infected study subjects expressed Ki-67 (Fig. ​(Fig.3B).3B). The highest numbers of Ki-67⁺ cells were found in the CD57⁻ population.Open in a separate windowFIG. 3.Functional characterization of CD57⁻, CD57^dim, and CD57^bright cells within the CD56^dim CD16⁺ NK cell population. (A) Representative staining results for granzyme B and perforin and summary data for control (n = 14) and HIV-seropositive subjects (n = 36). Numbers in the concatenated contour plots indicate percentages of gated events of the respective subset. B cells were defined as the negative control for granzyme and perforin staining. (B) Percentages of Ki-67⁺ and CD107a⁺ cells on CD57⁻, CD57^dim, and CD57^bright cells within the CD56^dim NK cell population in control (n = 14 and n = 9, respectively) and HIV-seropositive (n = 36 and n = 21, respectively) subjects'' PBMCs were analyzed. Horizontal bars in dot plots show the means. NC, negative control; ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.We also assessed the presence of the degranulation marker CD107a on CD57⁻, CD57^dim, and CD57^bright subpopulations of CD56^dim CD16⁺ NK cells after stimulation with IL-12 and IL-15 and exposure to K562 cells. Similarly to what we had observed for Ki-67 expression, CD57⁻ cells were the most efficient at degranulation when compared with CD57^dim and CD57^bright cells in HIV-infected individuals. Comparison to healthy controls revealed that there was a higher expression of CD107a in HIV-seropositive subjects for each CD57 subset. However, the most effective degranulation occurred in the CD57⁻ and CD57^dim subsets, which are preferentially depleted in HIV infection.We focused our analysis on CD56^dim CD16⁺ NK cells because they constitute the largest NK cell subset in peripheral blood, they are the major NK cell subset expressing CD57 and KIRs, and they are the most prominent subpopulation for cytolytic activity. CD56^dim CD16⁺ cells but not CD56^bright CD16⁻ NK cells were reported to be decreased in HIV-infected subjects (23), which we could confirm in our experiments (data not shown). We did not find CD57 on CD56^bright CD16⁻ NK cells either in healthy or in HIV-infected individuals. CD57 has been described as a marker for replicative senescence, and its expression has been associated with shorter telomeres and diminished proliferative capacities on T and NK cells (4). The presence of this marker on CD56^dim CD16⁺ but not on CD56^bright CD16⁺ NK cells might explain why the latter subset was shown to proliferate more efficiently upon cytokine stimulation (6). We demonstrated that increasing CD57 expression on NK cells was associated with lower numbers of CD27-expressing cells, a marker which is mainly expressed by CD56^bright CD16⁻ NK cells (24). CD56^bright CD16⁻ cells were suggested to be early NK cells, which differentiate from CD34^dim CD45RA⁺ hematopoietic precursor cells with high expression of integrin α₄β₇ (11). These cells can furthermore give rise to CD56^dim CD16⁺ NK cells (7). Our data support this hypothesis, as we show that CD57 can be found on CD56^dim CD16⁺ NK cells but not on CD56^bright NK cells, whereas the opposite is observed for CD27.We demonstrate that differential CD57 expression is associated with distinct functional characteristics. We show for the first time that increasing expression of CD57 on CD56^dim CD16⁺ NK cells is associated with increasing prevalence of KIR⁺ and granzyme B⁺ cells. These cells appear to be more mature and differentiated in terms of KIR and granzyme B expression but less functionally active, as shown by decreased expression of Ki-67 and CD107a. We therefore propose that CD57 is not only a marker for replicative senescence but, in addition, a marker for terminal differentiation on NK cells, which is characterized by increased expression of KIR and higher granzyme B content and “counterbalanced” by decreased degranulation (CD107a) and decreased proliferation (Ki-67).Notably, we observed consistently higher frequencies of granzyme B⁺ cells in all three subsets within CD56^dim CD16⁺ NK cells from HIV-seropositive individuals than in healthy control subjects (means, 52.9% versus 78.7% [P < 0.0001], 65.3% versus 89.6% [P < 0.0001], and 76.5% versus 95.0% [P < 0.0001]for CD57⁻, CD57^dim, and CD57^bright subpopulations, respectively) (Fig. ​(Fig.1C).1C). Furthermore, HIV infection was associated with higher numbers of Ki-67-expressing NK cells (means, 8.4% versus 16.1% [P = 0.0005], 5.3% versus 11.6% [P = 0.0016], and 4.1% versus 6.2% [P = 0.04]) (Fig. ​(Fig.1C).1C). These changes, including the strong increase in HLA-DR-expressing NK cells, probably reflect the systemic immune activation in HIV-infected individuals.In summary, these findings support a view of a differential regulation of NK function and are in concordance with maturation of NK cells with high expression of CD57 on NK cells with a more terminally differentiated phenotype. Our data indicate that high turnover; activation status; and active degranulation as characterized by the expression of Ki-67, HLA-DR, and CD107a are mainly features of CD57⁻ and much less of CD57^dim NK cells. HIV infection is associated with increased activation, proliferation, and cytotoxicity during “early” stages of CD56^dim CD16⁺ NK cell differentiation compared to their occurrence in healthy controls, but those are the very cells that are significantly decreased in chronic HIV infection. A loss of these functionally more active NK cells may be a yet-unappreciated factor in overall NK cell pathology and a further possible explanation for the impairment of NK cells in their contribution to viral control in HIV infection.     

NK Cell Terminal Differentiation: Correlated Stepwise Decrease of NKG2A and Acquisition of KIRs

Background

Terminal differentiation of NK cells is crucial in maintaining broad responsiveness to pathogens and discriminating normal cells from cells in distress. Although it is well established that KIRs, in conjunction with NKG2A, play a major role in the NK cell education that determines whether cells will end up competent or hyporesponsive, the events underlying the differentiation are still debated.

Methodology/Principal Findings

A combination of complementary approaches to assess the kinetics of the appearance of each subset during development allowed us to obtain new insights into these terminal stages of differentiation, characterising their gene expression profiles at a pan-genomic level, their distinct surface receptor patterns and their prototypic effector functions. The present study supports the hypothesis that CD56^dim cells derive from the CD56^bright subset and suggests that NK cell responsiveness is determined by persistent inhibitory signals received during their education. We report here the inverse correlation of NKG2A expression with KIR expression and explore whether this correlation bestows functional competence on NK cells. We show that CD56^dimNKG2A⁻KIR⁺ cells display the most differentiated phenotype associated to their unique ability to respond against HLA-E+ target cells. Importantly, after IL-12 + IL-18 stimulation, reacquisition of NKG2A strongly correlates with IFN-γ production in CD56^dimNKG2A⁻ NK cells.

Conclusions/Significance

Together, these findings call for the reclassification of mature human NK cells into distinct subsets and support a new model, in which the NK cell differentiation and functional fate are based on a stepwise decrease of NKG2A and acquisition of KIRs.     

CD16 cross-linking induces increased expression of CD56 and production of IL-12 in peripheral NK cells

Human NK cells are classified into two populations according to the intensity of CD56 surface expression, as well as possession of CD16, FcRIII. CD56^dimCD16^bright make up 90% circulating NK cells, whereas CD56^brightCD16^-/dim comprises the remaining 10%. Here we report that peripheral NK cells upon CD16 cross-linking up-regulates the expression of activating markers and receptors such as CD25, CD69, NKp44, NKp30, CD40L and the intensity of CD56 expression. Additionally, co-culturing immature DCs with CD16 activated NK cells was found to significantly increase the expression of maturation markers on DCs. These results suggest that CD16 cross-linking on resting peripheral blood NK cells triggers the activation of these cells and induces the appearance of CD56^bright NK cells. The latter were found capable of producing pro-inflammatory cytokines, IFN-γ and TNF-α and notably IL-12.     

Reduction of the CD16?CD56bright NK Cell Subset Precedes NK Cell Dysfunction in Prostate Cancer

Background

Natural cytotoxicity, mediated by natural killer (NK) cells plays an important role in the inhibition and elimination of malignant tumor cells. To investigate the immunoregulatory role of NK cells and their potential as diagnostic markers, NK cell activity (NKA) was analyzed in prostate cancer (PCa) patients with particular focus on NK cell subset distribution.

Methods

Prospective data of NKA and NK cell subset distribution patterns were measured from 51 patients initially diagnosed with PCa and 54 healthy controls. NKA was represented by IFN-γ levels after stimulation of the peripheral blood with Promoca®. To determine the distribution of NK cell subsets, PBMCs were stained with fluorochrome-conjugated monoclonal antibodies. Then, CD16⁺CD56^dim and CD16⁻CD56^bright cells gated on CD56⁺CD3⁻ cells were analyzed using a flow-cytometer.

Results

NKA and the proportion of CD56^bright cells were significantly lower in PCa patients compared to controls (430.9 pg/ml vs. 975.2 pg/ml and 2.3% vs. 3.8%, respectively; p<0.001). Both tended to gradually decrease according to cancer stage progression (p for trend = 0.001). A significantly higher CD56^dim-to-CD56^bright cell ratio was observed in PCa patients (41.8 vs. 30.3; p<0.001) along with a gradual increase according to cancer stage progression (p for trend = 0.001), implying a significant reduction of CD56^bright cells in relation to the alteration of CD56^dim cells. The sensitivity and the specificity of NKA regarding PCa detection were 72% and 74%, respectively (best cut-off value at 530.9 pg/ml, AUC = 0.786).

Conclusions

Reduction of CD56^bright cells may precede NK cell dysfunction, leading to impaired cytotoxicity against PCa cells. These observations may explain one of the mechanisms behind NK cell dysfunction observed in PCa microenvironment and lend support to the development of future cancer immunotherapeutic strategies.     

Ageing is associated with a decline in peripheral blood CD56bright NK cells

Background  

Natural killer (NK) cells are cytotoxic lymphocytes that lack CD3 and express variable levels of CD16, CD56 and CD57. In recent years NK cells have been categorised into two major groups based on the level of CD56 expression. This phenotypic classification correlates with functional activity as CD56^bright NK cells are the major cytokine producing subset whereas CD56^dim NK cells exhibit greater cytotoxic activity. Previous studies have revealed a reduction in total NK cell numbers in association with ageing and this study sought to determine the potential influence of ageing on the number of NK cell subsets within peripheral blood.     

Active but not inactive granulomatosis with polyangiitis is associated with decreased and phenotypically and functionally altered CD56dim natural killer cells

BackgroundThe role of natural killer (NK) cells in granulomatosis with polyangiitis (GPA) is poorly understood. We recently reported that peripheral blood NK cell percentages correlate with the suppression of GPA activity (cohort I). The purpose of the current study was to further characterize NK cell subsets, phenotype and function in a second GPA cohort (cohort II).MethodsPeripheral blood lymphocyte subsets were analyzed at a clinical diagnostic laboratory. Clinical data were extracted from medical records and patients were grouped according to their activity state (remission vs. active/non-remission). Separate analysis (cohort II, n = 22) and combined analysis (cohorts I and II, n = 34/57) of NK cell counts/percentages was performed. NK cell subsets and phenotypes were analyzed by multicolor flow cytometry. Cytotoxicity assays were performed using ⁵¹Cr-labeled K562 target cells.ResultsIn cohort II, NK cell counts were lower than the lower limit of normal in active GPA, despite normal percentages due to lymphopenia. NK cell counts, but not other lymphocyte counts, were significantly higher in remission. Combined analysis of cohorts I and II confirmed decreased NK cell counts in active GPA and increased percentages in long-term remission. Follow-up measurements of six patients revealed increasing NK cell percentages during successful induction therapy. Multicolor analysis from cohort II revealed that in active GPA, the CD56^dim subset was responsible for decreased NK cell counts, expressed more frequently CD69, downregulated the Fc-receptor CD16 and upregulated the adhesion molecule CD54, the chemokine receptor CCR5 and the activating receptor NKG2C. In remission, these markers were unaltered or marginally altered. All other receptors investigated (NKp30, NKp44, NKp46, NKG2D, DNAM1, 2B4, CRACC, 41BB) remained unchanged. Natural cytotoxicity was not detectable in most patients with active GPA, but was restored in remission.ConclusionsNK cell numbers correlate inversely with GPA activity. Reduced CD56^dim NK cells in active GPA have an activated phenotype, which intriguingly is associated with profound deficiency in cytotoxicity. These data suggest a function for NK cells in the pathogenesis and/or modulation of inflammation in GPA. NK cell numbers, phenotype (CD16, CD69, NKG2C) or overall natural cytotoxicity are promising candidates to serve as clinical biomarkers to determine GPA activity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-016-1098-7) contains supplementary material, which is available to authorized users.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript> production within tumor microenvironment inversely correlated with infiltration of CD56<Superscript>dim</Superscript> NK cells in gastric and esophageal cancer: possible mechanisms of NK cell dysfunction

Human NK cells can be divided into two subsets, CD56^dimCD16(+)NK and CD56^brightCD16(−)NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed the relationship between CD56^dim/CD56^bright NK cells and H₂O₂ in tumor-infiltrating NK cells in patients with gastric (n = 50) and esophageal (n = 35) cancer. The ratio of CD56^dim NK cells infiltrating tumors gradually decreased according to disease progression. H₂O₂ was abundantly produced within tumor microenvironments, and there was an inverse correlation between CD56^dim NK cell infiltration and H₂O₂ production. CD56^dim NK cells are more sensitive to apoptosis induced by physiological levels of H₂O₂ than CD56^bright NK cells. Furthermore, the exposure of NK cells to H₂O₂ resulted in the impairment of ADCC activity. In conclusion, H₂O₂ produced within tumor microenvironments inversely correlated with the infiltration of CD56^dim NK cells, possibly due to their preferentially induced cell death. These observations may explain one of the mechanisms behind NK cell dysfunction frequently observed in tumor microenvironments.     

Homing Receptor Expression Is Deviated on CD56+ Blood Lymphocytes during Pregnancy in Type 1 Diabetic Women

Type 1 Diabetes Mellitus (T1DM) is characterized by an augmented pro-inflammatory immune state. This contributes to the increased risk for gestational complications observed in T1DM mothers. In normal pregnancies, critical immunological changes occur, including the massive recruitment of lymphocytes, particularly CD56^bright NK cells, into early decidua basalis and a 2^nd trimester shift towards Type 2 immunity. Decidual CD56^bright NK cells arise at least partly from circulating progenitors expressing adhesion molecules SELL and ITGA4 and the chemokine receptors CXCR3 and CXCR4. In vitro studies show that T1DM reduces interactions between blood CD56⁺ NK cells and decidual endothelial cells by reducing SELL and ITGA4-based interactions. To address the mechanisms by which specific lymphocyte subsets may be recruited from the circulation during pregnancy and whether these mechanisms are altered in T1DM, flow cytometry was used to examine eight peripheral blood lymphocyte subsets (Type 1 (IL18R1⁺) and Type 2 (IL1RL1⁺) CD56^bright NK, CD56^dim NK, NKT and T cells) from control and T1DM women. Blood was collected serially over pregnancy and postpartum, and lymphocytes were compared for expression of homing receptors SELL, ITGA4, CXCR3, and CXCR4. The decline of Type 1/Type 2 immune cells in normal pregnancy was driven by an increase in Type 2 cells that did not occur in T1DM. CD56^bright NK cells from control women had the highest expression of all four receptors with greatest expression in 2^nd trimester. At this time, these receptors were expressed at very low levels by CD56^bright NK cells from TIDM patients. Type 1/Type 2 NKT cell ratios were not influenced by either pregnancy or TIDM. Our results suggest that T1DM alters immunological balances during pregnancy with its greatest impact on CD56^bright NK cells. This implicates CD56^bright NK cells in diabetic pregnancy complications.     

Defining early human NK cell developmental stages in primary and secondary lymphoid tissues

A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56^brightCD16^+/− and CD56^dimCD16⁺ subsets within the different compartments and suggests that microenvironment may play a role in differential in situ development of the NK cell receptor repertoire of committed NK cells. Overall, differential in situ NK cell development and trafficking towards multiple tissues may give rise to a broad spectrum of mature NK cell subsets found within the human body.     

Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3⁻ CD56⁺ NK subsets in HIV⁺ and HCV⁺ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56^dim cell fraction compared to CD56^bright cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56^dim NK subset were observed in HIV⁺ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV⁺ and HCV⁺ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections.     

Long-term proliferation of functional human NK cells,with conversion of CD56<Superscript>dim</Superscript> NK cells to a CD56<Superscript>bright</Superscript> phenotype,induced by carcinoma cells co-expressing 4-1BBL and IL-12

4-1BB ligation co-stimulates T cell activation, and agonistic antibodies have entered clinical trials. Natural killer (NK) cells also express 4-1BB following activation and are implicated in the anti-tumour efficacy of 4-1BB stimulation in mice; however, the response of human NK cells to 4-1BB stimulation is not clearly defined. Stimulation of non-adherent PBMC with OVCAR-3 cells expressing 4-1BB ligand (4-1BBL) or IL-12 resulted in preferential expansion of the NK cell population, while the combination 4-1BBL + IL-12 was superior for the activation and proliferation of functional NK cells from healthy donors and patients with renal cell or ovarian carcinoma, supporting long-term (21 day) NK cell proliferation. The expanded NK cells are predominantly CD56^bright, and we show that isolated CD56^dimCD16⁺ NK cells can switch to a CD56^brightCD16⁻ phenotype and proliferate in response to 4-1BBL + IL-12. Whereas 4-1BB upregulation on NK cells in response to 4-1BBL required ‘help’ from other PBMC, it could be induced on isolated NK cells by IL-12, but only in the presence of target (OVCAR-3) cells. Following primary stimulation with OVCAR-3 cells expressing 4-1BBL + IL-12 and subsequent resting until day 21, NK cells remained predominantly CD56^bright and retained both high cytotoxic capability against K562 targets and enhanced ability to produce IFNγ relative to NK cells in PBMC. These data support the concept that NK cells could contribute to anti-tumour activity of 4-1BB agonists in humans and suggest that combining 4-1BB-stimulation with IL-12 could be beneficial for ex vivo or in vivo expansion and activation of NK cells for cancer immunotherapy.     

Extensive activation,tissue trafficking,turnover and functional impairment of NK cells in COVID-19 patients at disease onset associates with subsequent disease severity

The SARS-CoV-2 infection causes severe respiratory involvement (COVID-19) in 5–20% of patients through initial immune derangement, followed by intense cytokine production and vascular leakage. Evidence of immune involvement point to the participation of T, B, and NK cells in the lack of control of virus replication leading to COVID-19. NK cells contribute to early phases of virus control and to the regulation of adaptive responses. The precise mechanism of NK cell dysregulation is poorly understood, with little information on tissue margination or turnover. We investigated these aspects by multiparameter flow cytometry in a cohort of 28 patients hospitalized with early COVID-19.Relevant decreases in CD56^brightCD16+/- NK subsets were detected, with a shift of circulating NK cells toward more mature CD56^dimCD16+KIR+NKG2A+ and “memory” KIR+CD57+CD85j+ cells with increased inhibitory NKG2A and KIR molecules. Impaired cytotoxicity and IFN-γ production were associated with conserved expression of natural cytotoxicity receptors and perforin. Moreover, intense NK cell activation with increased HLA-DR and CD69 expression was associated with the circulation of CD69+CD103+ CXCR6+ tissue-resident NK cells and of CD34+DNAM-1^brightCXCR4+ inflammatory precursors to mature functional NK cells. Severe disease trajectories were directly associated with the proportion of CD34+DNAM-1^brightCXCR4+ precursors and inversely associated with the proportion of NKG2D+ and of CD103+ NK cells.Intense NK cell activation and trafficking to and from tissues occurs early in COVID-19, and is associated with subsequent disease progression, providing an insight into the mechanism of clinical deterioration. Strategies to positively manipulate tissue-resident NK cell responses may provide advantages to future therapeutic and vaccine approaches.     

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94⁺ NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94⁺ NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL⁺ CD56^dim NK cells, in contrast to the efficient responses by CD56^bright NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94⁺ KIR2DL^- NK cells may be uniquely beneficial.     

Regulation of Development of CD56brightCD11c+ NK-like Cells with Helper Function by IL-18

Human γδ T cells augment host defense against tumors and infections, and might have a therapeutic potential in immunotherapy. However, mechanism of γδ T cell proliferation is unclear, and therefore it is difficult to prepare sufficient numbers of γδ T cells for clinical immunotherapy. Recently, natural killer (NK)-like CD56^brightCD11c⁺ cells were shown to promote the proliferation of γδ T cells in an IL-18-dependent manner. In this study, we demonstrated that the NK-like CD56^brightCD11c⁺ cells could directly interact with γδ T cells to promote their sustained expansion, while conventional dendritic cells (DCs), IFN-α-induced DCs, plasmacytoid DCs or monocytes did not. We also examined the cellular mechanism underlying the regulation of CD56^brightCD11c⁺ cells. CD14⁺ monocytes pre-incubated with IL-2/IL-18 formed intensive interactions with CD56^intCD11c⁺ cells to promote their differentiation to CD56^brightCD11c⁺ cells with helper function. The development of CD56^brightCD11c⁺ cells was suppressed in an IFN-α dependent manner. These results indicate that CD14⁺ monocytes pretreated with IL-2/IL-18, but neither DCs nor monocytes, play a determining role on the development and proliferation of CD56^brightCD11c⁺ cells, which in turn modulate the expansion of γδ T cells. CD56^brightCD11c⁺ NK-like cells may be a novel target for immunotherapy utilizing γδ T cells, by overcoming the limitation of γδ T cells proliferation.     

CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity

The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56^bright, NK cells. Most NK cells in healthy human donors are CD45RA⁺CD45RO^-. The CD45RA^-RO⁺ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RA^dimRO^- (CD45RA^dim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RA^dim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RA^dim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells.     

Natural Killer Cell Cytokine Response to M. bovis BCG Is Associated with Inhibited Proliferation,Increased Apoptosis and Ultimate Depletion of NKp44+CD56bright Cells

Mycobacterium bovis BCG, a live attenuated strain of M. bovis initially developed as a vaccine against tuberculosis, is also used as an adjuvant for immunotherapy of cancers and for treatment of parasitic infections. The underlying mechanisms are thought to rely on its immunomodulatory properties including the recruitment of natural killer (NK) cells. In that context, we aimed to study the impact of M. bovis BCG on NK cell functions. We looked at cytotoxicity, cytokine production, proliferation and cell survival of purified human NK cells following exposure to single live particles of mycobacteria. We found that M. bovis BCG mediates apoptosis of NK cells only in the context of IL-2 stimulation during which CD56^bright NK cells are releasing IFN-γ in response to mycobacteria. We found that the presence of mycobacteria prevented the IL-2 induced proliferation and surface expression of NKp44 receptor by the CD56^bright population. In summary, we observed that M. bovis BCG is modulating the functions of CD56^bright NK cells to drive this subset to produce IFN-γ before subsequent programmed cell death. Therefore, IFN-γ production by CD56^bright cells constitutes the main effector mechanism of NK cells that would contribute to the benefits observed for M. bovis BCG as an immunotherapeutic agent.     

Identification and characterization of a tumor infiltrating CD56+/CD16− NK cell subset with specificity for pancreatic and prostate cancer cell lines

In a recent clinical trial, a patient exhibited regression of several pancreatic cancer metastases following the administration of the immune modulator Ipilimumab (anti-CTLA-4 antibody). We sought to characterize the immune cells responsible for this regression. Tumor infiltrating lymphocytes (TIL-2742) and an autologous tumor line (TC-2742) were expanded from a regressing metastatic lesion excised from this patient. Natural killer (NK) cells predominated in the TIL (92% CD56⁺) with few T cells (12% CD3⁺). A majority (88%) of the NK cells were CD56^brightCD16⁻. TIL-2742 secreted IFN-γ and GM-CSF following co-culture with TC-2742 and major histocompatibility complex mismatched pancreatic tumor lines. After sorting TIL-2742, the purified CD56⁺CD16⁻CD3⁻ subset showed reactivity similar to TIL-2742 while the CD56⁻CD16⁻CD3⁺ cells exhibited no tumor recognition. In co-culture assays, TIL-2742 and the NK subset expressed high reactivity to several pancreatic and prostate cancer cell lines and could lyse the autologous tumor as well as pancreas and prostate cancer lines. Reactivity was partially abrogated by blockade of TRAIL. We thus identified a unique subset of NK cells (CD56^brightCD16^dim) isolated from a regressing metastatic pancreatic cancer in a patient responding to Ipilimumab. This represents the first report of CD56⁺CD16⁻ NK cells with apparent specificity for pancreatic and prostate cancer cell lines and associated with tumor regression following the treatment with an immune modulating agent.     

Human CD56+ Cytotoxic Lung Lymphocytes Kill Autologous Lung Cells in Chronic Obstructive Pulmonary Disease

CD56+ natural killer (NK) and CD56+ T cells, from sputum or bronchoalveolar lavage of subjects with chronic obstructive pulmonary disease (COPD) are more cytotoxic to highly susceptible NK targets than those from control subjects. Whether the same is true in lung parenchyma, and if NK activity actually contributes to emphysema progression are unknown. To address these questions, we performed two types of experiments on lung tissue from clinically-indicated resections (n = 60). First, we used flow cytometry on fresh single-cell suspension to measure expression of cell-surface molecules (CD56, CD16, CD8, NKG2D and NKp44) on lung lymphocytes and of the 6D4 epitope common to MICA and MICB on lung epithelial (CD326+) cells. Second, we sequentially isolated CD56+, CD8+ and CD4+ lung lymphocytes, co-cultured each with autologous lung target cells, then determined apoptosis of individual target cells using Annexin-V and 7-AAD staining. Lung NK cells (CD56+ CD3−) and CD56+ T cells (CD56+ CD3+) were present in a range of frequencies that did not differ significantly between smokers without COPD and subjects with COPD. Lung NK cells had a predominantly “cytotoxic” CD56+ CD16+ phenotype; their co-expression of CD8 was common, but the percentage expressing CD8 fell as FEV₁ % predicted decreased. Greater expression by autologous lung epithelial cells of the NKG2D ligands, MICA/MICB, but not expression by lung CD56+ cells of the activating receptor NKG2D, correlated inversely with FEV₁ % predicted. Lung CD56+ lymphocytes, but not CD4+ or CD8+ conventional lung T cells, rapidly killed autologous lung cells without additional stimulation. Such natural cytotoxicity was increased in subjects with severe COPD and was unexplained in multiple regression analysis by age or cancer as indication for surgery. These data show that as spirometry worsens in COPD, CD56+ lung lymphocytes exhibit spontaneous cytotoxicity of autologous structural lung cells, supporting their potential role in emphysema progression.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00281229","term_id":"NCT00281229"}}NCT00281229