Reply to: “Flow cytometry makes all the difference”
Authors: Barbara Oliviero, Dalila Mele, Stefania Varchetta, Mario U. Mondelli
Journal: Journal of Hepatology (2013)

Flow cytometry makes all the differenceJournal of HepatologyVol. 59Issue 4PreviewWe read with interest the reported association between natural killer (NK) cell phenotype and treatment outcome in chronic HCV infection [1]. There is growing evidence that NK cell function and phenotype, in particular NKp46 expression, are associated with liver inflammation, treatment outcome, and may even impinge upon the ability to mount a robust CD4+ T-cell response during the successful IFN-α based treatment of HCV [2–6]. However, this remains a complex field with often apparently conflicting reports within the literature. Full-Text PDF Open AccessNatural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infectionJournal of HepatologyVol. 59Issue 1PreviewA substantial proportion of patients with chronic hepatitis C virus infection treated with pegylated interferon α/ribavirin fail to achieve sustained virological response (SVR). Since growing evidence suggests that innate immunity may influence treatment responses, we examined natural killer (NK) cell phenotypic and functional changes during standard antiviral therapy. Full-Text PDF Does it? No, it doesn’tWe thank Pembroke and colleagues for the comments to our paper which, we are pleased to note, is generating interest in the small community of liver immunologists. Contrary to our data, they found no difference in the percentage of CD56dim/CD16neg NK cells between patients developing a sustained virological response (SVR) and those who failed treatment, and hypothesize that the higher proportion of CD56dim/CD16neg NK cells at baseline and on treatment, which we describe to be associated with SVR in patients with chronic HCV infection, could be due to increased proportions of dead cells in this particular NK subset. Curiously, retrospective flow cytometric analysis of their mononuclear cell samples stored in liquid nitrogen revealed that the vast majority of CD56dim/CD16neg NK cells (mean 95%) were actually composed of dead cells. In view of this unexpected finding, Pembroke concludes that our data are not compatible with those of others (which?), being flawed by a purportedly high number of dead cells in this specific NK-cell subset. Beside any technical consideration, and even allowing for this being indeed the cause of our findings, the first comment that comes to one’s mind is why would dead NK cells concentrate in SVR patients only and not in non-responders (NR) or relapsers and why would they be confined to the CD56dim/CD16neg subset? Unfortunately, we have no plausible explanation for either question, nor do we have evidence of such a high percentage of dead cells in our preparations, particularly in the CD56dim/CD16neg subset.To check for possible flaws in our analysis, we also went back to our frozen samples, and examined the proportions of dead cells in the various NK-cell subsets as indicated by Pembroke. Moreover, we repeated the analysis of the prevalence of CD56dim/CD16neg NK cells in an independent set of 10 patients, 5 SVR and 5 NR, to check whether there was any peculiarity in our original cohort of patients [[1]Oliviero B. Mele D. Degasperi E. Aghemo A. Cremonesi E. Rumi M.G. et al.Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection.J Hepatol. 2013; 59: 38-44Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. Fig. 1A–F shows the gating strategy to identify CD56dimCD16neg NK cells in a representative SVR patient. Lymphocytes were gated by morphological features (A) and NK cells identified as CD56pos/CD3neg (B) which were analyzed on the basis of CD56 and CD16 expression without excluding dead cells (C). Alternatively, dead cells were excluded by gating ethidium monoazide (EMA) positive events which represented only 4.3% of the total number of events (D). Interestingly, the proportion of CD56dim/CD16neg NK (F) obtained from NK cells after exclusion of dead cells (E) was virtually identical to that obtained without excluding EMApos cells (C), indicating that our data were not influenced by an allegedly high concentration of dead cells in the CD56dim/CD16neg NK subset, as suggested by Pembroke et al. Moreover, Fig. 1G clearly shows that there were no differences between the proportions of dead cells (mean ± SEM) in the CD56dim/CD16neg and CD56bright or CD16pos NK subpopulations in SVR and NR patients. Finally, we were gratified to confirm that, as already reported in our manuscript, the percentages of CD56dim/CD16neg among SVR and NR were significantly different (mean ± SEM): 21.9% ± 4.3 vs. 6% ± 1.1, respectively, p = 0.007, by including all cells, and 21.5% ± 4.2 vs. 5.8% ± 1.05, respectively, p = 0.007, after exclusion of dead cells, also in this independent set of patients.We believe that the above strongly corroborates our recently published findings [[1]Oliviero B. Mele D. Degasperi E. Aghemo A. Cremonesi E. Rumi M.G. et al.Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection.J Hepatol. 2013; 59: 38-44Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar] and we are particularly grateful to Pembroke for allowing us to dispel any possible doubt regarding the quality of our data.Conflict of interestThe authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Does it? No, it doesn’tWe thank Pembroke and colleagues for the comments to our paper which, we are pleased to note, is generating interest in the small community of liver immunologists. Contrary to our data, they found no difference in the percentage of CD56dim/CD16neg NK cells between patients developing a sustained virological response (SVR) and those who failed treatment, and hypothesize that the higher proportion of CD56dim/CD16neg NK cells at baseline and on treatment, which we describe to be associated with SVR in patients with chronic HCV infection, could be due to increased proportions of dead cells in this particular NK subset. Curiously, retrospective flow cytometric analysis of their mononuclear cell samples stored in liquid nitrogen revealed that the vast majority of CD56dim/CD16neg NK cells (mean 95%) were actually composed of dead cells. In view of this unexpected finding, Pembroke concludes that our data are not compatible with those of others (which?), being flawed by a purportedly high number of dead cells in this specific NK-cell subset. Beside any technical consideration, and even allowing for this being indeed the cause of our findings, the first comment that comes to one’s mind is why would dead NK cells concentrate in SVR patients only and not in non-responders (NR) or relapsers and why would they be confined to the CD56dim/CD16neg subset? Unfortunately, we have no plausible explanation for either question, nor do we have evidence of such a high percentage of dead cells in our preparations, particularly in the CD56dim/CD16neg subset.To check for possible flaws in our analysis, we also went back to our frozen samples, and examined the proportions of dead cells in the various NK-cell subsets as indicated by Pembroke. Moreover, we repeated the analysis of the prevalence of CD56dim/CD16neg NK cells in an independent set of 10 patients, 5 SVR and 5 NR, to check whether there was any peculiarity in our original cohort of patients [[1]Oliviero B. Mele D. Degasperi E. Aghemo A. Cremonesi E. Rumi M.G. et al.Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection.J Hepatol. 2013; 59: 38-44Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. Fig. 1A–F shows the gating strategy to identify CD56dimCD16neg NK cells in a representative SVR patient. Lymphocytes were gated by morphological features (A) and NK cells identified as CD56pos/CD3neg (B) which were analyzed on the basis of CD56 and CD16 expression without excluding dead cells (C). Alternatively, dead cells were excluded by gating ethidium monoazide (EMA) positive events which represented only 4.3% of the total number of events (D). Interestingly, the proportion of CD56dim/CD16neg NK (F) obtained from NK cells after exclusion of dead cells (E) was virtually identical to that obtained without excluding EMApos cells (C), indicating that our data were not influenced by an allegedly high concentration of dead cells in the CD56dim/CD16neg NK subset, as suggested by Pembroke et al. Moreover, Fig. 1G clearly shows that there were no differences between the proportions of dead cells (mean ± SEM) in the CD56dim/CD16neg and CD56bright or CD16pos NK subpopulations in SVR and NR patients. Finally, we were gratified to confirm that, as already reported in our manuscript, the percentages of CD56dim/CD16neg among SVR and NR were significantly different (mean ± SEM): 21.9% ± 4.3 vs. 6% ± 1.1, respectively, p = 0.007, by including all cells, and 21.5% ± 4.2 vs. 5.8% ± 1.05, respectively, p = 0.007, after exclusion of dead cells, also in this independent set of patients.We believe that the above strongly corroborates our recently published findings [[1]Oliviero B. Mele D. Degasperi E. Aghemo A. Cremonesi E. Rumi M.G. et al.Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection.J Hepatol. 2013; 59: 38-44Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar] and we are particularly grateful to Pembroke for allowing us to dispel any possible doubt regarding the quality of our data. We thank Pembroke and colleagues for the comments to our paper which, we are pleased to note, is generating interest in the small community of liver immunologists. Contrary to our data, they found no difference in the percentage of CD56dim/CD16neg NK cells between patients developing a sustained virological response (SVR) and those who failed treatment, and hypothesize that the higher proportion of CD56dim/CD16neg NK cells at baseline and on treatment, which we describe to be associated with SVR in patients with chronic HCV infection, could be due to increased proportions of dead cells in this particular NK subset. Curiously, retrospective flow cytometric analysis of their mononuclear cell samples stored in liquid nitrogen revealed that the vast majority of CD56dim/CD16neg NK cells (mean 95%) were actually composed of dead cells. In view of this unexpected finding, Pembroke concludes that our data are not compatible with those of others (which?), being flawed by a purportedly high number of dead cells in this specific NK-cell subset. Beside any technical consideration, and even allowing for this being indeed the cause of our findings, the first comment that comes to one’s mind is why would dead NK cells concentrate in SVR patients only and not in non-responders (NR) or relapsers and why would they be confined to the CD56dim/CD16neg subset? Unfortunately, we have no plausible explanation for either question, nor do we have evidence of such a high percentage of dead cells in our preparations, particularly in the CD56dim/CD16neg subset. To check for possible flaws in our analysis, we also went back to our frozen samples, and examined the proportions of dead cells in the various NK-cell subsets as indicated by Pembroke. Moreover, we repeated the analysis of the prevalence of CD56dim/CD16neg NK cells in an independent set of 10 patients, 5 SVR and 5 NR, to check whether there was any peculiarity in our original cohort of patients [[1]Oliviero B. Mele D. Degasperi E. Aghemo A. Cremonesi E. Rumi M.G. et al.Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection.J Hepatol. 2013; 59: 38-44Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. Fig. 1A–F shows the gating strategy to identify CD56dimCD16neg NK cells in a representative SVR patient. Lymphocytes were gated by morphological features (A) and NK cells identified as CD56pos/CD3neg (B) which were analyzed on the basis of CD56 and CD16 expression without excluding dead cells (C). Alternatively, dead cells were excluded by gating ethidium monoazide (EMA) positive events which represented only 4.3% of the total number of events (D). Interestingly, the proportion of CD56dim/CD16neg NK (F) obtained from NK cells after exclusion of dead cells (E) was virtually identical to that obtained without excluding EMApos cells (C), indicating that our data were not influenced by an allegedly high concentration of dead cells in the CD56dim/CD16neg NK subset, as suggested by Pembroke et al. Moreover, Fig. 1G clearly shows that there were no differences between the proportions of dead cells (mean ± SEM) in the CD56dim/CD16neg and CD56bright or CD16pos NK subpopulations in SVR and NR patients. Finally, we were gratified to confirm that, as already reported in our manuscript, the percentages of CD56dim/CD16neg among SVR and NR were significantly different (mean ± SEM): 21.9% ± 4.3 vs. 6% ± 1.1, respectively, p = 0.007, by including all cells, and 21.5% ± 4.2 vs. 5.8% ± 1.05, respectively, p = 0.007, after exclusion of dead cells, also in this independent set of patients. We believe that the above strongly corroborates our recently published findings [[1]Oliviero B. Mele D. Degasperi E. Aghemo A. Cremonesi E. Rumi M.G. et al.Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection.J Hepatol. 2013; 59: 38-44Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar] and we are particularly grateful to Pembroke for allowing us to dispel any possible doubt regarding the quality of our data. Conflict of interestThe authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.