IL-21 augments natural killer effector functions in chronically HIV-infected individuals - PubMed (original) (raw)
IL-21 augments natural killer effector functions in chronically HIV-infected individuals
Natasa Strbo et al. AIDS. 2008.
Abstract
Objective: This study addresses the interleukin (IL)-21 effects on resting peripheral blood natural killer (NK) cells in chronically HIV-infected individuals.
Design: The effects of IL-21 on perforin expression, proliferation, degranulation, interferon (IFN)-gamma production, cytotoxicity and induction of STAT phosphorylation in NK cells were determined in vitro.
Methods: Peripheral blood mononuclear cells from HIV-infected and healthy individuals were incubated in vitro for 6 h, 24 h or 5 days with IL-21 or IL-15. Percentages of perforin, IFN-gamma, CD107a, NKG2D and STAT3-5 positive cells were determined within NK cell populations. K562 cells were used as target cells in NK cytotoxicity assay.
Results: Frequency of CD56 cells in chronically HIV-infected individuals was diminished. Perforin expression in CD56 and CD56 was comparable in healthy and HIV-infected individuals. IL-15 upregulated perforin expression primarily in CD56 NK cells, whereas IL-21 upregulated perforin in both NK subsets. IL-21 and IL-15 upregulated CD107a and IFN-gamma, as well as NK cytotoxicity. IL-15 predominantly activated STAT5, whereas IL-21 activated STAT5 and STAT3. IL-15, but not IL-21 increased NK cell proliferation in uninfected and HIV-infected individuals.
Conclusion: IL-21 augments NK effector functions in chronically HIV-infected individuals and due to its perforin enhancing properties it has potential for immunotherapy or as a vaccine adjuvant.
Figures
Figure 1. In chronically HIV-infected individuals frequency of CD56dim cells is diminished compared to healthy controls
(a) Freshly isolated PBMC from healthy controls (n=6) and HIV-infected individuals (n=9) were analyzed for the frequency of CD56+ NK cells within gated CD3negative cells. Mean values for each group are indicated by a horizontal bar; (b) Scatter diagram representing the relationship between the CD4 counts and percentage of CD3negCD56+ cells in HIV–infected individuals; (c) Freshly isolated PBMC from healthy controls (n=6) and HIV infected individuals (n=9) were analyzed for the frequency of CD56dim and CD56bright NK cells. Mean values for each group are indicated by a horizontal bar. Spearman’s correlation coefficient was utilized to determine relationships between CD4 counts and percentage of CD3negative CD56+ cells. Non-parametric Mann-Whitney U test was used to evaluate differences between 2 groups. Differences were considered significant when p<0.05.
Figure 2. Effects of IL-21 and IL-15 on NK cell subset frequency and perforin expression
Freshly isolated PBMC from healthy control (n=4) and HIV-infected individuals (n=5) were cultured in medium or with IL-15 (50ng/ml), IL-21 (50ng/ml) for 5 days. The frequency of CD56bright (a) and CD56dim (b) cells within CD3negCD56+ cells is shown. Significance was determined by 1-way ANOVA with Tukey's Multiple Comparison test. (c) Representative dot plots of CD56 expression and matched isotype control (mouse IgG2b) within gated CD3neg cells from one HIV-infected individual are shown. Gates were set for CD56dim population when the intensity of CD56 expression was 10–100 times that of the matched isotype control and for CD56bright populations when the intensity of CD56 expression was 100–1000 times that of the matched isotype control. The percentage of perforin positive cells within (d) CD56bright and (e) CD56dim gates, with mean values for each group are shown. (f) Perforin mRNA expression following 5 hour stimulation with medium, IL-21 or IL-15 in purified CD56+ NK cells from healthy and HIV-infected individual as determined by quantitative RT-PCR.
Figure 3. Differential effects of IL-15 and IL-21 on frequency and proliferation of NK cells. IL-15, but not IL-21 induced proliferation of NK cells in both healthy and HIV-infected individuals
Freshly isolated PBMC from healthy control and HIV infected individuals were stained with CFSE and cultured in medium with IL-15 (50ng/ml), IL-21 (50ng/ml), or without cytokine for 5 days. Average number of cell divisions that the responding cells underwent (Division index) was calculated after gating on CD3negCD56+ cells and presented as mean ± SEM from healthy (n=5) controls and HIV-infected (n=10) individuals. Significance was determined by 1-way ANOVA with Tukey's Multiple Comparison test.
Figure 4. Rapid induction of degranulation marker, CD107a, perforin and IFN-γ in IL-15 and IL-21 treated NK cells
Freshly isolated PBMC from healthy controls were cultured in medium or with IL-15 (50ng/ml) or IL-21 (50ng/ml) for 24h followed by 6h stimulation with MHC-devoid NK-sensitive K562 cells (a) Representative dot plot of perforin MFI in gated CD3negCD56+ cells after 24h culture with IL-21 or IL-15. (b–d) Expression of perforin, CD107a, and IFN-γ in gated CD3negCD56+ cells after 6h co-culture of medium- or cytokine (IL-21/IL-15) pre-cultured PBMC with K562 cells. Paired t-test was used to evaluate differences between 2 groups. Differences were considered significant when p<0.05. e) Freshly isolated human NK cells (95% purity with <1% each of CD3+, CD20+ and HLA-DR+ cells) from HIV-infected individuals were cultured in medium or with IL-21 (50ng/ml) or IL-15 (50ng/ml) for 24 h and examined for perforin MFI and (f) for cytotoxicity, following coculture of 24 hour cytokine treated NK cells with NK sensitive targets, K562 cells labeled with PKH-26 at the ratios 6:1, 12.5:1, 25:1 for 4 h. Target cells were gated by side scatter and fluorescence (FL2). 7-AAD uptake was determined within gated, target cells. The percent lysis was determined as [(percent 7-AAD staining sample – percent 7-AAD staining of negative control)/(100-percent 7-AAD staining of negative control)] × 100. Significance was determined by 1-way ANOVA with Tukey's Multiple Comparison test.
Figure 5. IL-2 and IL-15 predominantly activate STAT5 in both healthy and HIV-infected CD3negCD56+ NK cells, while IL-21 activates STAT5 and STAT3
PBMCs isolated from (a) healthy and (b) HIV-infected individuals were cultured in medium or stimulated with IL-2 (1000IU/ml), IL-15 (50ng/ml) or IL-21 (50ng/ml) for 15 min and expression of phosphorylated epitopes for STAT3, 4 and 5 was determined on fixed and permeabilized cells. STAT expression depicted as mean ± SEM from healthy (n=5) controls and HIV-infected (n=3) individuals. Representative histograms of STAT3, STAT4 and STAT5 expression following medium, IL-15 or IL-21 treatment in gated CD3negCD56+ cells from (c) one healthy and (d) one HIV-infected individual are shown.
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