CD3^BrightCD56⁺T Cell Associated With Peg-IFNα Treatment Nonresponse In HBV Patients And MicroRNA Transcriptomes Of Distinct Human NK Cell

Part 1. Clinical research on HBV therapyHepatitis B virus is one of the most prevalent problems around the world. Although the introduction of the hepatitis B virus vaccine measures, the worldwide prevalence of HBV infection has fallen, there are still about 400 million chronic hepatitis B carriers,25% of whom are Chinese. Chronic hepatitis B virus infection is a urgent health concern that patients with hepatitis B are at high risk of developing hepatic decompensation, cirrhosis, and even carcinoma. Thus, effective treatment schemes are of great importance.Hepatitis B virus replication is a critical point of liver injury and disease progression, and therefore, the ultimate goal of HBV therapy is to suppress the virus DNA levels and achieve HBeAg or HBsAg seroconversion. Currently, pegylated interferon-alpha (peg-IFNα) and nucleoside/nucleotide analogues (NA) are the most commonly prescribed treatments. Pegylated interferon have better treatment effect due to the immunomodulating activity. However, the response rate after 48 weeks of peg-IFNα is only 30% in HBeAg-positive patients. The poor sustained response rates in patients reflect the dysfunctional immune activity to the virus, especially in the liver.CD56+ T cells generally account for about 50% of T cells in the liver and have the antiviral activity against hepatic viruses via IFNy secretion. Indeed, CD56+ T cells are a more potent source of IFNy than NK cell. CD56+ T cells play an important part in immune responses to several viruses. The poor sustained response rates in peg-IFN therapy suggest that CD56+ T cells may be in a compromised situation in CHB patients, that of a subdued antiviral state.As a result, the aims of our research is to investigate the reason of poor sustained response rates in CHB patients. We focus on the role of CD56+ T cells in peg-IFNα treatment of CHB patients. We wish to present a new auxiliary clinical indicator for evaluating the efficacy of peg-IFNα therapy in CHB patients.In this study, we evaluated 85 new cases of untreated HBeAg positive CHB patients who were systematically treated with peg-IFNα for 48 weeks, with a 24-week post-treatment observation period. Time points to detect readouts including clinical indexes and immunostaining were determined at 0,12,24,36,48,60 and 72 weeks after the first peg-IFNa injection. The majority of our results are shown as follows:1. A majority of CHB patients preferentially harbor a CD3brightCD56+ T cell populationBased on published studies, CD56+ T cells may represent a unique cell population with a significant immunoregulatory role in CHB patients. To further characterize these CD56+ T cells, we analyzed this cell population in live PBMC lymphocytes from CHB patients and healthy control individuals by flow cytometry. We found that the mean fluorescence intensity (MFI) of CD3 expression was significantly higher on CD56+ T cells from CHB patients than from healthy controls. Based on the CD3 MFI expression on CD56+ T cells, we classified each individual CHB patient and healthy control into one of 2 groups:one group that primarily harbored CD3brightCD56+ T cells and another group that primarily harbored CD3dimCD56+ T cells. Of the 85 total CHB patients enrolled in the study, a majority (55/85,64.7%) harbored the CD3brightCD56+ T cell population; in contrast, only 10/33 healthy controls (30.3%) harbored the CD3brightCD56+ T cell population. Indeed, the proportion of individuals harboring CD3brightCD56+ T cells was much higher in CHB individuals than in healthy controls. Taken together, these results suggest that CHB patients preferentially harbor CD3brightCD56+ T cells.2. CHB patients harboring CD3brightCD56+ T cells exhibit a poor therapeutic response to peg-IFNaWe next determined whether there was a correlation between the presence of CD3brightCD56+ T cells in CHB patients and their response to therapy. Among the 69 from the total 85 CHB patients who had complete clinical data, we found that the clinical indexes of CHB patients harboring the CD3brightCD56+ T cell population were significantly higher at different time points after treatment. What’s more, a larger proportion of CHB patients harboring CD3brightCD56+ T cells (42/47,89.4%) were nonresponse to the treatment than the patients harboring CD3dimCD56+ T cells (13/22, 59.1%). Futhermore, the CD3 MFI on CD56+ T cells had a competitive advantage in nonresponse patients compared to the response patients. Collectively, these results suggested that CHB patients harboring CD3brightCD56+ T cells exhibited a relatively poor therapeutic response to peg-IFNα treatment.3. CD3brightCD56+ T cells express low CD8 levelsBased on the poor treatment effect we observed in CHB patients harboring CD3brightCD56+ T cells, we hypothesized that these CD3brightCD56+ T cells were dysfunctional compared to other CD56+ T cells. When comparing between the CHB patient groups harboring CD3brightCD56+ T cells or CD3dimCD56+ T cells, CD3brightCD56+ T cells remarkably exhibited lower CD8 expression than CD3dimCD56+ T cells, even after peg-IFNa treatment. With respect to the treatment effect, nonresponse patients had the lowest frequency of CD8+CD56+ T cells. In summary, CD3brightCD56+ T cells expressed low CD8 levels throughout peg-IFNa treatment, and nonresponse CHB patients harbored less CD8+CD56+ T cells than response patients.4. CD3brightCD56+ T cells express high inhibitory NKG2A and CD94 receptorsTo next explore whether CD3brightCD56+ T cells were undergoing any inhibition, we assessed their phenotypic expression of inhibitory molecules. Remarkably, within the corresponding CHB patient groups, CD3brightCD56+ T cells displayed higher expression of the inhibitory molecule NKG2A compared to CD3dimCD56+ T cells. During peg-IFNa therapy, NKG2A maintained a higher expression on CD3brightCD56+ T cells compared to CD3dimCD56+ T cells. With respect to the treatment effect, nonresponse patients had the highest frequency of NKG2A+CD56+ T cells compared to response patients. Taken together, our results showed that CD3brightCD56+ T cells expressed high levels of the inhibitory heterodimers NKG2A and CD94 throughout peg-IFNa treatment, and nonresponse CHB patients harbored more NKG2A+CD56+ and CD94+CD56+ T cells than response patients.5. CD3brightCD56+ T cells exhibit dysfunctional antiviral responsesIFNγ reportedly plays a key role in the host antiviral response, and peg-IFNa therapy is thought to potentiate IFNy secretion by antiviral effector cells. Based on the inhibitory phenotype we observed on CD3brightCD56+ T cells, we hypothesized that these T cells had reduced antiviral function and tested this idea by evaluating their ability to secrete IFNy in vitro. Intracellular staining of IFNy on CD56+ T cells revealed that CD3brightCD56+ T cells produced lower IFNy levels compared to CD3dimCD56+ T cells. During peg-IFNa therapy, IFNy and CD107a expression in CD3brightCD56+ T cells were consistently lower throughout the treatment period. Thus, our results suggested that CD3brightCD56+ T cells had dysfunctional antiviral activity.6. Tim-3 upregulation correlates with CD3brightCD56+ T cell dysfunction during peg-IFNα treatmentTim-3 reportedly suppresses several effector cells in CHB patients. However, Tim-3 expression was similar between CD3brightCD56+ and CD3brightCD56+ T cells before peg-IFNα treatment. Tim-3 expression rapidly increased on CD3brightCD56+ but not CD3dimCD56+, T cells during peg-IFNα treatment. Moreover, Tim-3 expression on CD3brightCD56+ T cells negatively correlated with IFNγ secretion. Taken together, the negative correlation between Tim-3 upregulation and IFNγ secretion on CD56+ T cells might provide a plausible explanation for the observed CD3brightCD56+ T cell dysfunction during peg-IFNα treatment in CHB patients harboring this CD56+ T cell populationTaken together, we identified a previously unrecognized population of CD56+ T cells based the CD3 mean fluorescence intensity—the CD3brightCD56+ T cell population—and further characterized that this population had subdued function in CHB patients. We revealed that a majority of CHB patients preferentially harbored this CD3brightCD56+ T cell population and that a larger proportion of CHB patients harboring CD3brightCD56+ T cells (42/47,89.4%) were nonresponse to the peg-IFNa treatment. Futhermore, We found that inhibitory Tim-3 expression on CD3brightCD56+ T cells was rapidly upregulated during peg-IFNα treatment, which might have led to their observed dysfunction.Part 2. Basic research on NK cellsNK cell functions against malignancy and infection as the body’s first line of defense. In different tissues and organs, NK cells display mutiple developmental and functional status. For instance, CD56bright NK cells, which are immature and function mainly by cytokine production, are relatively dominant in decidua. In contrast, the majority of peripheral blood NK cells are CD56dim, a phenotype exhibit relatively mature developmental stage, with a cytotoxicity function.MicroRNAs which are short noncoding RNA molecules act as key regulators in various cell types. Recently, studies have shown that miRNA are required for the proper maturation and function of NK cells, however little is known about the differential miRNA expression profiles in different phenotypes and functions of human NK cell subsets.In this work, we focus on the detailed miRNA expression profiles of three human NK populations isolated from different tissue compartments (peripheral blood, cord blood, and decidua). Using this miRNA expression data, we identified miR-362-5p plays a regulatory role human NK cell function. The detailed result description are as follows:1. Preferentially expressed miRNAs in NK cell populationsWe determined the miRNA expression profiles of various lymphocyte subsets, and found several miRNAs which were preferentially expressed two-fold upregulated in a single NK cell type (pNK, cNK, dNK). Futhermore, seven miRNAs that were specifically up-regulated in all NK cell population compared to CD56+ T and T cells were selected, one of which is miR-362-5p.2. Differential miRNA expression analysis in various human NK populations.Next, we looked for differential miRNA expression in the different human NK cell populations. We performed pairwise comparisons between each of the two NK populations, and found that the difference was more apparent between the dNK and pNK subsets. Futher analysis revealed that miR-362-5p is highly expressed in human pNK cells, indicating an important regulatory role in NK cell function.3. CYLD is a target of miR-362-5p in human NK cells.Based on these results, we paid close attention on miR-362-5p. First, we should identify the mRNA targets regulated by miR-362-5p in human NK cells that might be involved in human NK cell function. We used TargetScan to make bio-informative predictions of the potential miR-362-5p target genes, and proved that miR-362-5p directly regulated CYLD mRNA via dual-luciferase reporter assay. PCR and western blot futher demonstrated that CYLD is a target of miR-362-5p in human NK cells.4. Overexpression or inhibition of miR-362-5p acts through CYLD to regulate human NK cell function.To investigate the role of miR-362-5p in NK cells, we transfected with miR-362-5p mimics into dNK cells via nucleofaction, and found miR-362-5p caused a significant increase in effector function in human dNK cells. In addition, we transfected with miR-362-5p inhibitor into pNK cells in which miR-362-5p are highly expressed, pNK cells showed a dramatic decrease in their cytotoxic activity and cytokine release. Futher analysis demonstrated that miR-362-5p regulates human NK cell effector function by targeting CYLD. This result indicated that miR-362-5p promotes human NK function.Taken together, the miRNA transcriptomes of human dNK, cNK, and pNK cells are presented in this study, thus opening avenues for future research on molecular regulation of the distinct NK populations into clearer focus. In addition, we revealed that a novel miRNA, miR-362-5p, could promote NK cell effector function. Our data offer a new views and provide resources for investigating the roles of miRNAs in human NK cell biology.