Detection and Significance of Cytotoxic Cell Subsets in Biopsies of HCV-Infected Human Livers

Chronic viral hepatitis C still remains the clinical challenge. Attempts of the immune system to cope with this infection are unsatisfactory. There is a conviction that the main site of interaction between virus (Hepatitis C virus, HCV) and immune system is in situ, i.e., in liver. Natural killer (NK) cells appeared relevant in the acute hepatitis. Less is known about the immune response in the chronic HCV infection. The aim of this study was to evaluate the prevalence of various cytotoxic cell subsets in chronic HCV⁺ liver tissue and to seek links between them and laboratory data of patients. Sections from paraffin blocks of liver biopsy tissues of HCV⁺ untreated patients were subjected to the reaction with antibodies vs. cytotoxic cell subsets and immunohistochemistry. Positive cells were searched in cellular infiltrates in portal areas and in liver parenchyma. They were classified on the “Yes” or “No” basis. Majority of liver biopsies exhibited cellular infiltrates in portal spaces and as single cells in liver parenchyma. Infiltrates consisted of CD8⁺ T cells, CD56⁺ NK ones, including CD158i⁺ and CD158b⁺. The latter were rarely seen. There were also granzyme B⁺ cells. The most abundant were NKG2D⁺ cells, much more common than NK CD56⁺ ones. It implied that NKG2D was also expressed on T cells. Prevalence of NKG2D⁺ cells correlated with high activity of liver enzymes such as alanine aminotransferase, aspartate aminotransferase and a greater histological severity of liver injury. NKG2D⁺ cells form the bulk of cells infiltrating HCV-infected human liver. Correlation of NKG2D⁺ cells with some laboratory parameters of patients suggests their role in hepatitis C pathogenesis.     

Enhancement of cytokine‐driven NK cell IFN‐γ production after vaccination of HCMV infected Africans

Human cytomegalovirus (HCMV) infection drives the phenotypic and functional differentiation of NK cells, thereby influencing the responses of these cells after vaccination. NK cell functional differentiation is particularly advanced in African populations with universal exposure to HCMV. To investigate the impact of advanced differentiation on vaccine‐induced responses, we studied NK‐cell function before and after vaccination with Trivalent Influenza Vaccine (TIV) or diphtheria, tetanus, pertussis, inactivated poliovirus vaccine (DTPiP) in Africans with universal, lifelong HCMV exposure. In contrast to populations with lower prevalence of HCMV infection, no significant enhancement of NK‐cell responses (IFN‐γ, CD107a, CD25) occurred after in vitro re‐stimulation of post‐vaccination NK cells with TIV or DTPiP antigens compared to pre‐vaccination baseline cells. However, both vaccinations resulted in higher frequencies of NK cells producing IFN‐γ in response to exogenous IL‐12 with IL‐18, which persisted for up to 6 months. Enhanced cytokine responsiveness was restricted to less differentiated NK cells, with increased frequencies of IFN‐γ⁺ cells observed within CD56^brightCD57^?, CD56^dimCD57^?NKG2C^? and CD56^dimCD57^?NKG2C⁺ NK‐cell subsets. These data suggest a common mechanism whereby different vaccines enhance NK cell IFN‐γ function in HCMV infected donors and raise the potential for further exploitation of NK cell “pre‐activation” to improve vaccine effectiveness.     

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.     

Reduced LAK cytotoxicity of peripheral blood mononuclear cells in patients with bladder cancer: Decreased LAK cytotoxicity caused by a low incidence of CD56+ and CD57+ mononuclear blood cells

The cytotoxicity of unstimulated peripheral blood mononuclear cells (US-PBMC), phytohemagglutinin (PHA)-stimulated PBMC (PS-PBMC) and interleukin-2 (IL-2)-activated PBMC (LAK cells) was assessed in patients with noninvasive and invasive transitional-cell bladder cancer and compared with those determined in healthy controls. The differences in the cytotoxicities were correlated with specific changes in the subsets of peripheral blood mononuclear cells (PBMC). PBMC from 37 patients and 13 healthy controls were tested against the bladder cancer cell line T24 in⁵¹Cr-release assays. The PBMC subsets were analyzed using monoclonal antibodies against T cells, natural killer (NK) -cells, monocytes, and activation markers. The cytotoxicities of US-PBMC, PS-PBMC, and LAK cells were all significantly lower in the cancer patients than in the controls (P<0.05). The percentages of PBMC positive for the NK-cell markers CD56 and CD57 were lowest in the patients and were correlated to the decrease in cytotoxicity. Depletion of CD56⁺ or CD57⁺ cells from PBMC prior to or after 2 days stimulation with IL-2 demonstrated that these cells are the major source of LAK-cell cytotoxicity and showed that the reduced ability of bladder cancer patient PBMC to develop LAK-cell cytotoxicity is a result of a low incidence of CD56⁺ and CD57⁺ cells in the blood. These findings indicate that IL-2 therapy alone might not be a sufficient therapy of bladder cancer patients.     

Increased proportion of the CD56bright NK cell subset in patients chronically infected with hepatitis C virus (HCV) receiving interferon‐αand ribavirin therapy

Natural killer (NK) cells are implicated in the regulation of a protective immune response in patients chronically infected with hepatitis C virus (HCV), but effects of interferon‐α/ribavirin therapy on NK cell subsets and the consequences of viral clearance during therapy remain unclear. Samples were collected from chronically infected patients (n = 34) at baseline and from a subset after 3–10 months on pegylated interferon‐α and ribavirin therapy (n = 19). NK cells present in cryopreserved PBMC were characterized by flow cytometry. Before therapy, the frequency of CD3?CD56+ NK cells was lower in patients than uninfected controls. Therapy increased proportions of CD56^bright NK cells. Frequencies of CD56^dim NK cells declined slightly while perforin and CD16 expression on CD56^dim NK cells decreased compared to baseline samples. Evaluation of NK cell subsets at baseline did not identify patients able to achieve sustained virological response following therapy. However, therapy may promote the expansion of NK cells able to produce interferon‐γ, while minimizing cytotoxicity to limit liver damage. J. Med. Virol. 82:568–574, 2010. © 2010 Wiley‐Liss, Inc.     

The effects of direct-acting antiviral agents on the frequency of myeloid-derived suppressor cells and natural killer cells in patients with chronic hepatitis C

Currently, hepatitis C antiviral therapy is entering a new era with the use of direct-acting antiviral (DAA) agents. However, the precise immunological influences of DAA therapy in patients with chronic hepatitis C (CHC) are insufficiently understood. This study aimed to investigate the effects of DAA therapy on the frequency of myeloid-derived suppressor cells (MDSCs), T lymphocytes, and natural killer (NK) cells in patients with CHC. Thirty-two treatment-naive CHC patients were treated with DAA therapy, and the frequency of immune cells was analyzed by flow cytometry at various time points during and after therapy. Sixteen healthy donors were recruited for comparison. DAA therapy decreased the frequency of MDSCs and monocytic MDSCs in patients with CHC to a normal level. DAA therapy also increased the CD8⁺ T and NK cell levels in patients with CHC. In addition, activation (NKp30 and NKp46) and inhibitory (NKG2A) receptors on NK cells were downregulated to yield an NK cell phenotype resembling that observed in the healthy controls. This study provides insight into the normalization of immune cell levels under DAA therapy and indicates that restoration of the immune system in patients with CHC strongly supports long-term curative hepatitis C virus eradication.     

Non‐neutralizing epitopes induce robust hepatitis C virus (HCV)‐specific antibody‐dependent CD56+ natural killer cell responses in chronic HCV‐infected patients

Natural killer (NK) cell‐mediated antibody‐dependent cellular cytotoxicity (NK‐ADCC) is of considerable interest in viral infection. However, little is known about NK‐ADCC responses in chronic hepatitis C virus (HCV) infection. In this study, impaired non‐specific antibody‐dependent CD56⁺ NK cell responses were observed in chronic HCV infection, as shown by decreased degranulation (extracellular CD107a expression) and interferon (IFN)‐γ production in response to antibody‐bound P815 cells. A peptide pool composed of epitopes recognized by anti‐HCV‐E1/E2 antibodies could induce pronounced HCV‐specific antibody‐dependent NK cell responses in sera from approximately half the chronic HCV carriers. Additionally, HCV‐specific epitopes with the capacity to induce robust NK‐ADCC activity were identified. Five linear NK‐ADCC epitopes (aa211‐aa217, aa384‐aa391, aa464‐aa475, aa544‐aa551 and aa648‐aa659 of the HCV envelope) were identified and do not overlap with putative linear neutralizing epitopes. This study revealed the dysfunctional characteristics of antibody‐dependent CD56⁺ NK cell responses in chronic HCV carriers. The key non‐neutralizing NK‐ADCC epitopes identified in this study may act as new targets for immunological intervention.     

MAIT be different–persisting dysfunction after DAA‐mediated clearance of chronic hepatitis C virus infection

MAIT cells are an abundant innate‐like T‐cell subset that is defined by the invariant T‐cell receptor (iTCR) V‐alpha chain Vα7.2‐Jα33. Little is currently known about their frequency and function in chronic hepatitis C virus (HCV) infection and their fate after therapy‐mediated HCV elimination by direct acting antivirals (DAA). In this issue of the European Journal of Immunology, Hengst et al. [Eur. J. Immunol. 2016. 46: 2204‐2210] give important novel insights into the biological role of MAIT cells in a relevant human chronic viral infection by showing that first, MAIT cells are only present at low frequencies in chronic HCV infection; second, circulating MAIT cells in HCV patients also display an altered phenotype; third, they are impaired in their MR‐1‐dependent effector functions and finally, and maybe most importantly, MAIT‐cell frequency and function was not restored after HCV elimination by DAA therapy. These results suggest that MAIT cells are severely affected by a chronic human viral infection in their frequency and function and that this impairment is not reversed after HCV elimination. This is in contrast to rapid DAA‐mediated restorations of NK‐cell and CD8⁺ T‐cell functions, and indicates a differential impact of chronic infection and clearance on different immune cell subsets.     

High-dimensional immune phenotyping of blood cells by mass cytometry in patients infected with hepatitis C virus

ObjectivesChronic hepatitis C virus (HCV) infection affects the immune system. Whether elimination of HCV with direct-acting antivirals (DAA) restores immunity is unclear. We used mass cytometry to get a broad and in-depth assessment of blood cell populations of patients with chronic HCV before and after DAA therapy.MethodsBefore and 12 weeks after sustained virological response (SVR12) to DAA therapy, 22 cell populations were analysed by mass cytometry in blood collected from ten healthy control individuals and 20 HCV-infected patients with (ten patients) or without (ten patients) human immunodeficiency virus (HIV) infection.ResultsHCV infection altered the frequency of 14/22 (64%) blood cell populations. At baseline, the frequencies (median, interquartile range (IQR); control, HCV, HCV/HIV) of intermediate monocytes (1.2, IQR 0.47–1.46; 1.76, IQR 0.83–2.66; 0.78, IQR 0.28–1.77), non-classical monocytes (1.11, IQR 0.49–1.26; 0.9, IQR 0.18–0.99; 0.54, IQR 0.28–1.77), conventional dendritic cells type 2 (0.55, IQR 0.35–0.59; 0.31, IQR 0.16–0.38; 0.19, IQR 0.11–0.36) and CD56^dim natural killer cells (8.08, IQR 5.34–9.79; 4.72, IQR 2.59–6.05) 3.61, IQR 2.98–5.07) were reduced by 35% to 65%, particularly in HCV/HIV co-infected patients. In contrast, activated double-negative T cells (0.07, IQR 0.06–0.10; 0.10, IQR 0.09–0.19; 0.19, IQR 0.12–0.25), activated CD4 T cells (0.28, IQR 0.21–0.36; 0.56, IQR 0.33–0.77; 0.40, IQR 0.22–0.53) and activated CD8 T cells (0.23, IQR 0.14–0.42; 0.74, IQR 0.30–1.65; 0.80, IQR 0.58–1.16) were increased 1.4 to 3.5 times. Upon stimulation with Toll-like receptor ligands, the expression of cytokines was up-regulated in 7/9 (78%) and 17/19 (89%) of the conditions in HCV- and HCV/HIV-infected patients, respectively. Most alterations persisted at SVR12.ConclusionsChronic HCV and HCV/HIV infections induce profound and durable perturbations of innate and adaptive immune homeostasis.     

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.     

The immune potential of decidua-resident CD16+CD56+ NK cells in human pregnancy

Human CD56⁺CD3^? NK cells can be subdivided into two different subsets according to the expression pattern of CD56 and CD16. CD56^+/brightCD16^? (CD16^?) NK cells are prominently cytokine producers with little cytotoxicity whereas CD56^+/dimCD16⁺ (CD16⁺) NK cells are efficient killers with poorer cytokine production potential. In human pregnancy, CD56⁺ decidual (d)NK cells accumulate in the maternal fetal interface to regulate placental immunity and development. Unlike peripheral blood (pb)NK cells, the majority of dNK cells are CD56 positive with limited CD16 reactivity. Our results demonstrated that in normal and pathological pregnancies, CD16⁺ dNK cells are a unique population in comparison to CD16^? dNK subset. The expression of NK activation receptors CD335, CD336, CD244 and CD314 on CD16⁺ dNK subpopulation was lower than that on CD16^? dNK cells. Upon cytokine stimulation with rhIL-12/15/18 or TGFβ blockade, the CD16⁺ dNK subset exhibited more robust response on the expression of IFNG, IL-8 and CD107a, compared to that of the CD16^? dNK subpopulation. Functions of the CD16⁺ dNK subset were shown to be independent of cellular interaction with trophoblast cells. Studies of preeclamptic patients revealed lower proportions of CD16⁺ dNK cells, suggesting potential protective roles of these cells during normal gestations.. Therefore, we suggest that the CD16⁺ dNK subset, through compensating CD16^? dNK cell function, is an indispensable component to regulate decidual immune response and to support placentation.     

SHORT COMMUNICATION: CD3− CD56+ CD16+ Natural Killer Cells and Improvement of Pregnancy Outcome in IVF/ICSI Failure After Additional IVIG‐Treatment

Citation Heilmann L, Schorsch M, Hahn T. CD3^? CD56⁺ CD16⁺ Natural killer cells and improvement of pregnancy outcome in IVF/ICSI failure after additional IVIG‐treatment. Am J Reprod Immunol 2010; 63: 263–265 Problem The purpose of this retrospective, observational study was to investigate whether additional treatment with intravenous immunglobulin (IVIG) increased the rate of successful pregnancies after repeated implantation failure (RIF). The retrospective data were compared with data of patients without IVIG‐therapy from the meta‐analysis of Clark et al. Method of study A total of 188 women with 226 treatment cycles between 2007 and 2009 were evaluated for IVIG therapy. The percentage of NK cells was measured two times before a new embryo transfer (only women with NK cell percentages >12% were included) and after embryo transfer at a positive pregnancy test. Results In comparison with the meta‐analysis of Clark et al., we observed a pregnancy rate of 50.5%, an implantation rate of 21% and a miscarriage rate of 16.8%. In 42%/IVIG‐ patient or 34.9%/embryo transfer, we observed a live born baby. The live born rate per embryo was 16.6%. In accordance with the study of Kwak et al., we indicate a decrease in the NK cells in patients with improved pregnancy outcome. Conclusion In a subgroup of RIF‐patients with high level of CD56⁺ CD16⁺ NK‐cells the additional application of IVIG leads to a favourable pregnancy outcome.     

Increased number of CD16CD56 NK cells in peripheral blood mononuclear cells after allogeneic cord blood transplantation

In the present study, we investigated subpopulations of natural killer (NK) cells and the expression of stimulatory and inhibitory NK receptors after adult blood and bone marrow transplantation (BBMT) and cord blood transplantation (CBT). There were significant increases in CD16⁺CD56^dim cell proportion and in absolute number in peripheral blood mononuclear cells (PBMC) during a period of 4–9 months after CBT compared with these in normal PBMC, cord blood (CB), and in PBMC after BBMT. Also, increased numbers of CD16⁺CD56^dim NK cells were sustained in some patients until 4 years after CBT. This CD16⁺CD56^dim cell subset after CBT exhibited decreased expression of NKG2A compared with that in CB and increased expression of NKG2C. Purified CD16⁺CD56^dim cells from patients 8–9 months after CBT exhibited significantly higher levels of cytolytic activity against K562 than did purified CD16⁺CD56^bright cells and also whole PBMC. The CD16⁺CD56^dim cell subset with a high level of cytolytic activity significantly increased after CBT, and these cells may be responsible for NK cell–mediated immunity after CBT.     

Immediate major dynamic changes in the T- and NK-cell subset composition after cardiac transplantation

Early kinetics of lymphocyte subsets involved in tolerance and rejection following heart transplantation (HTx) are barely defined. Here, we aimed to delineate the early alloimmune response immediately after HTx. Therefore, blood samples from 23 heart-transplanted patients were collected before (pre-), immediately (T0), 24 hours (T24), and 3 weeks (3 wks) after HTx. Immunophenotyping was performed using flow cytometry. A significant increase was detected for terminally differentiated (TEMRA) CD4⁺ or CD8⁺ T cells and CD56^dimCD16⁺ NK cells immediately after HTx linked to a decrease in naïve CD8⁺ and CM CD4⁺ T as well as CD56^brightCD16⁻ NK cells, returning to baseline levels at T24. More detailed analyses revealed increased CD69⁺CD25⁻ and diminished CD69⁻CD25⁻CD4⁺ or CD8⁺ T-cell proportions at T0 associated with decreasing S1PR1 expression. Passenger T and NK cells were found at low frequencies only in several patients at T0 and did not correlate with lymphocyte alterations. Collectively, these results suggest an immediate, transient shift toward memory T and NK cells following HTx. Opposite migratory properties of naïve versus memory T and NK cells occurring in the early phase after HTx could underlie these observations and may impinge on the development of allo-specific immune responses.     

Increased expressions of NKp44, NKp46 on NK/NKT-like cells are associated with impaired cytolytic function in self-limiting hepatitis E infection

We have characterized the NK/NKT-like cells in patients with self-limiting hepatitis E infection. The distribution of peripheral NK/NKT-like cells, expressions of activation receptors, cytotoxic potential and effector function of NK/NKT-like cells from fresh peripheral blood mononuclear cells of 86 acute patients, 101 recovered and 54 control individuals were assessed. Activated NKT-like (CD16⁺ CD56⁺ CD3⁺) cells were high in the patient groups. On CD56⁺ CD3^? cells, NKp44 and NKp46 expressions were high in the acute patients, whereas NKp30, NKp44, NKp46 and NKG2D were high in the recovered individuals. On CD56⁺ CD3⁺ cells, NKp44, NKp46 and NKG2D expressions were high in the recovered but NKp30 was low in both the patient groups. Collectively, the current study elucidates the role of NK/NKT-like cells demonstrating phenotypic alterations of activated NKT-like cells and activation receptors, lack of CD107a expression and functional impairment of peripheral NK/NKT-like cells in self-limiting hepatitis E infection.     

Substance P primes lipoteichoic acid- and Pam3CysSerLys4-mediated activation of human mast cells by up-regulating Toll-like receptor 2

We investigated the distribution of natural killer (NK) cell subsets, their activating and inhibitory receptors, and their cytolytic potential, in primary human immunodeficiency virus (HIV)-infected (PHI) individuals at baseline and during 1 year of follow-up with or without antiretroviral therapy, and compared the results with those obtained in treatment-naïve, chronically HIV-infected (CHI) individuals, and HIV-seronegative (HN) healthy individuals. The proportion of the CD56^dim and CD56^bright subsets decreased with disease progression, whereas that of the CD56⁻ CD16⁺ subset increased. In the CD56^dim subset, the proportion of cells with natural cytotoxicity receptors (NCRs) decreased with disease progression, and their cytolytic potential was reduced. Conversely, the CD56^bright subset was characterized by a high proportion of NCR-positive, killer cell immunoglobulin-like receptor (KIR)-positive NKG2A⁺ cells in both CHI and PHI individuals, which was associated with an increase in their cytolytic potential. During the 1 year of follow-up, the PHI individuals with high viraemia levels and low CD4⁺ T-cell counts who received highly active antiretroviral therapy (HAART) had a similar proportion of NK subsets to CHI individuals, while patients with low viraemia levels and high CD4⁺ T-cell counts who remained untreated had values similar to those of the HN individuals. Our results indicate a marked perturbation of the NK cell compartment during HIV-1 infection that is multifaceted, starts early and is progressive, primarily involves the CD56^bright subset, and is partially corrected by effective HAART.     

Use of an anti-CD16 antibody for in vivo depletion of natural killer cells in rhesus macaques

Non-human primates serve as key animal models for a variety of viral infections. To evaluate the contribution of natural killer (NK) cells to the immune-mediated control of these viruses in macaque monkeys, we have described a method for depleting NK cells in vivo by administration of anti-human CD16 mouse monoclonal antibody. Using a fluorometric NK-cell cytotoxicity assay, we show that most NK-cell cytotoxicity in rhesus monkey peripheral blood mononuclear cells resides in the CD16⁺ and/or CD159A⁺ subset of lymphocytes. The anti-human CD16 antibody, 3G8, binds to subsets of rhesus monkey lymphocytes and monocytes but not to neutrophils. Intravenous administration of 10–50 mg/kg of 3G8 to normal rhesus monkeys resulted in anti-CD16 antibody persistence in the plasma for 1–3 weeks. This treatment also depleted 80–90% of CD3^– CD159A⁺ lymphocytes, putative NK cells, from blood for at least 1 week and was associated with the loss of NK-cell cytotoxicity when evaluated by in vitro assays. Using this method, transient depletion of NK cells from two rhesus monkeys chronically infected with simian immunodeficiency virus failed to cause changes in virus replication. These studies describe a non-human primate model for in vivo NK-cell depletion and suggest a limited role for cytotoxic CD16⁺ NK cells in controlling AIDS virus replication during chronic infection.     

Impaired calcium mobilization in natural killer cells from chronic fatigue syndrome/myalgic encephalomyelitis patients is associated with transient receptor potential melastatin 3 ion channels

Transient receptor potential melastatin subfamily 3 (TRPM3) ion channels play a role in calcium (Ca²⁺) cell signalling. Reduced TRPM3 protein expression has been identified in chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) patients. However, the significance of TRPM3 and association with intracellular Ca²⁺ mobilization has yet to be determined. Fifteen CFS/ME patients (mean age 48·82 ± 9·83 years) and 25 healthy controls (mean age 39·2 ± 12·12 years) were examined. Isolated natural killer (NK) cells were labelled with fluorescent antibodies to determine TRPM3, CD107a and CD69 receptors on CD56^dimCD16⁺NK cells and CD56^brightCD16^dim/– NK cells. Ca²⁺ flux and NK cytotoxicity activity was measured under various stimulants, including pregnenolone sulphate (PregS), thapsigargin (TG), 2‐aminoethoxydiphenyl borate (2APB) and ionomycin. Unstimulated CD56^brightCD16^dim/– NK cells showed significantly reduced TRPM3 receptors in CFS/ME compared with healthy controls (HC). Ca²⁺ flux showed no significant difference between groups. Moreover, PregS‐stimulated CD56^brightCD16^dim/–NK cells showed a significant increase in Ca²⁺ flux in CFS/ME patients compared with HC. By comparison, unstimulated CD56^dimCD16⁺ NK cells showed no significant difference in both Ca²⁺ flux and TRPM3 expression. PregS‐stimulated CD56^dimCD16⁺ NK cells increased TRPM3 expression significantly in CFS/ME, but this was not associated with a significant increase in Ca²⁺ flux. Furthermore, TG‐stimulated CD56^dimCD16⁺ NK cells increased K562 cell lysis prior to PregS stimulation in CFS/ME patients compared with HC. Differential expression of TRPM3 and Ca²⁺ flux between NK cell subtypes may provide evidence for their role in the pathomechanism involving NK cell cytotoxicity activity in CFS/ME.     

Spontaneous and natural cytotoxicity receptor‐mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus‐infected chimpanzees

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV‐infected chimpanzees. In HCV‐infected chimpanzees increased spontaneous cytotoxicity was observed in CD94^high/dimCD16^pos and CD94^lowCD16^pos subsets. By contrast, increased natural cytotoxicity receptor (NCR)‐ mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94^dimCD16^neg subset. Our findings suggest that spontaneous and NCR‐mediated cytotoxicity are effector functions of distinct NK subsets in HCV‐infected chimpanzees.     

CD56(+dim) and CD56(+bright) cell activation and apoptosis in hepatitis C virus infection

CD3- CD56(+dim) natural killer (NK) cells, which are cytotoxic against virally infected cells, may be important in hepatitis C virus (HCV)-infected patients who are successfully treated with pegylated interferon (PEG-IFN)-alpha. We used flow cytometry to enumerate activated (CD69+) and apoptotic (annexin-V+) dim (CD3- CD56(+dim)) and bright (CD3- CD56(+bright)) NK cells obtained from HCV-infected patients before treatment (n=16) and healthy controls (n=15) in the absence and presence of pegylated interferon (PEG-IFN)-alpha-2b. A subset of HCV-infected patients, subsequently treated with PEG-IFN-alpha-2b in vivo, was determined to have a sustained virological response (SVR, n=6) or to not respond (NR) to treatment (n=5). In the absence of IFN, activated dim (CD3- CD56(+dim) CD69+) NK cells were significantly decreased (P=0.04) while activated apoptotic dim (CD3- CD56(+dim)CD69+ annexin-V+) NK cells tended to be increased (P=0.07) in SVR patients compared with NR patients. Activated bright (CD3-CD56(+bright)CD69+) and activated apoptotic bright (CD3- CD56(+bright)CD69+ annexin-V+) NK cells were significantly correlated (P=0.02 and P=0.01, respectively) with increasing hepatic inflammation. These findings suggest that in the absence of PEG-IFN, activated dim (CD3- CD56(+dim)CD69+) NK cell turnover may be enhanced in SVR compared with NR patients and that activated bright (CD3- CD56(+bright)CD69+) NK cells may play a role in liver inflammation.