Human natural killer T (NKT) cells are the cells of innate immune system that can respond rapidly to the antigen stimulus1 and known to act as a bridge between innate and adaptive immune response.2,3 The most widely studied human NKT cells are the type I or invariant NKT (iNKT) cells, which express an invariant T-cell receptor comprising Vα24-Jα18 rearrangement paired with Vβ11,4,5 and recognize the glycolipid antigen presented by CD1d.4,5 The NKT cells can be subdivided into CD4+ and CD4−subsets that have distinct functional profiles.6,7
On activation, the NKT cells secrete cytokines, such as interferon γ (IFN-γ), interleukin 4 (IL-4), and IL-17,8 that can subsequently activate other cells of immune system such as dendritic cells (DC), NK cells, and CD4 and CD8 T cells.9,10 NKT cells play a role in various pathological conditions such as cancer,11–13 autoimmune diseases,14,15 and various viral, bacterial, fungal, and parasitic infections.16,17 HIV infection is known to influence both quality and quantity of the NKT cells.18–26 The NKT cells are found to be depleted in early HIV infection and further decreased after seroconversion.20 However, limited information is available on the role of NKT cell quantity and functionality in the HIV disease progression. One study has shown that the HIV-infected individuals with lower frequency of NKT cells have increased risk of cancers,11 whereas another study has shown that the NKT cell number within 1–5 years of seroconversion is not predictive of disease progression.20 We hypothesize that the NKT cells might be preserved in HIV-infected individuals who did not show disease progression. To test this hypothesis, we used the long-term nonprogressors (LTNPs) as a model of absence of disease progression. The NKT cells and their major subpopulations CD4+ and CD4− NKT cells were characterized for their frequency and activation, exhaustion, and senescence status along with their proliferation ability in LTNPs and the findings were compared with those obtained from the HIV-infected individuals with progressive disease.
MATERIALS AND METHODS
Thirty-two LTNPs (10 M/22 F) (HIV-infected individuals who are asymptomatic without combination antiretroviral therapy (cART) and maintained a stable CD4 count of ≥500 cells/mm3 for a period of 7 or more years) were enrolled in the study from the ongoing LTNPs cohort along with 40 progressors (20 M/20 F) (HIV-infected patients with CD4 count ≤500 cells/mm3 and not fulfilling the criteria for LTNPs) from the outpatient clinics of the National AIDS Research Institute, Pune. The clinical and laboratory data analysis showed that all study participants were negative for hepatitis coinfection at the time of enrolment. cART was initiated in 18 of the 40 progressors (10 M/8 F). To determine the effects of cART on NKT cells, these patients were followed up for 12 months post-ART. Additionally, 35 (21 M/14 F) HIV seronegative healthy individuals were also enrolled for comparison. The demographic and clinical details of the study participants are given in (Supplemental Digital Content, Table 1, https://links.lww.com/QAI/A977). There was no significant difference in age of the participants from all study groups. At the enrolment visit, the median CD4 count was 684 cells/mm3 (interquartile range [IQR] = 546–865 cells/mm3), 319 cells/mm3 (IQR = 180–381 cells/mm3), and 944 cells/mm3 (IQR = 756–1092 cells/mm3) in the LTNPs, progressors, and healthy controls (HCs), respectively. The median CD4 counts was significantly higher in the LTNPs than in the progressors (P < 0.0001) and lower than seen in the HCs (P = 0.001). The plasma viral load (pVL) was significantly lower (median =3.41 log10 copies/mL, IQR = 2.04–4.17) in LTNPs than in progressors (median = 4.52 log10 copies/mL, IQR = 3.03–6.16), (P < 0.0001). All 18 progressors in whom cART was initiated had undetectable pVL at 12 months, whereas the median CD4 count at cART initiation visit was 192 cells/mm3 (IQR = 129–293 cells/mm3) and at 12 months post-cART it was increased to 372 cells/mm3 (IQR = 286–417 cells/mm3).
Twenty milliliters whole blood sample was collected from the study participants. From the 18 participants who were put on cART, the sample was collected before and at 12 months post-ART. The plasma and peripheral blood mononuclear cells (PBMCs) were separated as previously described,27 and stored at −80°C and −196°C, respectively, until tested. The study was approved by institutional ethics committee, and all subjects gave written informed consent for participation.
CD4 Count and Viral Load Estimation
CD4 T-cell counts were quantified by flow cytometry (FACSCalibur; Becton-Dickinson, San Jose, CA) using TruCount kit (Becton-Dickinson) as described previously,27 and pVL was measured as RNA copies per milliliter by Abbott m2000rt HIV-1 real-time polymerase chain reaction according to the manufacturer's instructions.
NKT Cell Characterization
The NKT cells were identified as CD3+CD4+ or CD3+CD4− cells that are double positive for their invariant T-cell receptor; Vα24 and Vβ11,28 and further assessed for the expression of activation (CD38, CD69, and HLA-DR), exhaustion (PD-1) and senescence (CD57) markers using multicolor flow cytometry. The cryopreserved PBMCs were revived and rested overnight in RPMI with 10% fetal bovine serum, at 37°C with 5% CO2. The next day, the PBMCs were resuspended in 1 mL phosphate-buffered saline and then stained with violet amine reactive dye (Invitrogen, Carlsbad, CA) for 30 minutes at room temperature for differentiation of dead and live cells. The samples having more than 90% viability were considered for further analysis. The cells were washed again and incubated with a cocktail of antibodies [anti Vα24 PE, anti Vβ11 FITC (Beckman Coulter, Marseilles, France), anti-CD57 APC (Biolegend, San Diego, CA), anti-CD4 PETR (Invitrogen), anti-CD3 APC, anti-CD8 PECy7, anti-CD38 PerCpCy5.5, anti-CD69 PECy7, anti-PD1 PerCpCy5.5, anti-HLA-DR PECy7 (all from BD Biosciences, San Jose, CA)] for 30 minutes at room temperature. After washing, the cells were fixed in 3% paraformaldehyde, acquired on FACSAria-I (BD Biosciences) and analyzed using FACSDiva software version 5.03 (BD Biosciences).
To determine the percentage of Vα24+Vβ11+-positive NKT cells, lymphocytes were first gated on the basis of forward and side scatter and second gate was set on live lymphocytes using side scatter and violet amine reactive viability dye. Minimum 100,000 events of live CD3+ T lymphocytes were analyzed. A third gate was set on Vα24+Vβ11+ live lymphocyte and percentage of NKT cells was calculated on CD3+ live lymphocytes (Fig. 1A). Single-stained controls were used to set compensation parameters and fluorescence-minus one controls were used to set gates.
Assessment of Proliferation Ability of NKT Cells
The proliferation ability of α-GalCer stimulated NKT cells was assessed as described previously.24 Briefly, after revival and resting overnight, 1 × 106 PBMCs were incubated in triplicate in RPMI 1640 with 10% fetal bovine serum, 100 ng/mL α-GalCer (Funakoshi, Tokyo, Japan), and 50 IU/mL recombinant human IL-2 (Roche Diagnostics GmbH, mannheim, Germany). The medium was replenished at day 3 and day 7, and the culture was analyzed for NKT cells frequency at day 0 and 13 on flow cytometry as described earlier.
GraphPad Prism version 5.01 software was used for statistical analyses. Differences in variables between the study groups were analyzed with Mann–Whitney U test and Spearman test was used for the correlation analysis. The mean of triplicate experiments for proliferation assessment was considered for the analysis. Changes in the parameter over the time were analyzed with paired t test. P value of <0.05 was considered as significant. To calculate the depletion in the NKT cells caused by HIV infection, we used the median value of NKT cell percentages obtained from the healthy controls as we did not have the preinfection value from the HIV-infected study participants. The percent depletion was calculated as the ratio of the frequency of NKT cells (either total, or CD4+ and CD4−) from each HIV-infected subjects to the median frequency in uninfected healthy controls, and subtracted from 100 as described previously.29 The percent of NKT cells restoration was calculated as the ratio of the frequency after cART to the frequency before cART, and subtracted from 100.
LTNPs Showed Significantly Higher Frequencies of NKT Cells
The NKT cell frequencies in LTNPs were significantly higher with median frequency (0.12%) of all T cells as compared with those in the progressors (median; 0.04%) (P < 0.0001) and lower than seen in the healthy controls (median = 0.24%) (Fig. 1B, left panel and Supplemental Digital Content, Table 1, https://links.lww.com/QAI/A977). NKT cell frequencies in HIV-infected participants (LTNPs and progressors) were significantly associated with higher CD4 counts (r = 0.38; P = 0.001) (Fig. 1C, left panel), and moderately with lower pVL (r = −0.23; P = 0.04) (Fig. 1D, left panel). The percentages of both CD4+ and CD4− NKT cells (obtained as a proportion of NKT cells) were significantly lower in the progressors compared with LTNPs and healthy controls (Fig. 1B, middle and right panel), and also associated with higher CD4 counts (r = 0.58; P < 0.0001, r = 0.52; P < 0.0001 for CD4+ and CD4− NKT cells, respectively) (Fig. 1C, middle and right panel) and lower pVL (r = −0.26; P = 0.02, r = −0.24; P = 0.03 for CD4+ and CD4− NKT cells, respectively) when the LTNPs and progressors were analyzed together (Fig. 1D, middle and right panel).
NKT Cells From LTNPs Showed Less Activation, Exhaustion, and Aging
Activation of the immune cells is necessary for their function but it also induces early apoptosis. Hence, we analyzed the percentage of activated NKT cells in the study groups. We found significantly lower frequency of activated (expressing either CD38 or CD69 or HLA-DR) NKT cells in LTNPs compared with those observed in the progressors (P ≤ 0.001 for all) (Fig. 2B) and in the patients with suppressive 12-month cART (P ≤ 0.001 for all) (Fig. 2B), but significantly higher compared with the healthy controls (P ≤ 0.03 for all) (Fig. 2B). The percentages of CD38+, CD69+, and HLA-DR+ NKT cells were inversely associated with CD4 count (P < 0.0001 for all) (Fig. 2C) and positively with pVL values in case of CD38+ NKT cells (P = 0.01), and HLA-DR+ NKT cells (P = 0.01) (Fig. 2D), whereas no association was observed with CD69 expressing NKT cells.
The activated CD4+ and CD4− NKT cells were inversely correlated with CD4 count (P ≤ 0.05 for all) (Supplemental Digital Content, Figure 1A and 1B, https://links.lww.com/QAI/A977), and positively with pVL values in case of CD4+CD38+ NKT cells (r = 0.32; P = 0.04), CD4+CD69+ NKT cells (r = 0.44; P = 0.005) and CD4+HLA-DR+ NKT cells (r = 0.38; P = 0.02) (Supplemental Digital Content, Figure 1C, https://links.lww.com/QAI/A977). Although the association between pVL and CD4−CD38+ (r = 0.1; P = 0.57) or CD4−CD69+ (r = 0.29; P = 0.07) NKT cells showed a trend of positive association, it did not reach the significance.
Furthermore, we wanted to explore whether the NKT cells in HIV infection show premature aging (senescence) and also whether these cells are in exhaustion stage. Hence, we determined the expression of CD57; as a senescence marker and the expression of Program Death 1 (PD1), as an immune exhaustion marker on NKT cells. Although the percentages of CD57 and PD1 expressing NKT cells were significantly higher in HIV-infected groups compared with the healthy controls (P < 0.05; Fig. 3B), within HIV-infected groups, the percentages were lower in LTNPs in comparison with that in the progressors and in the patients of cART at 12 months post-ART (P < 0.05; Fig. 3B). The frequencies of CD57+ and PD1+ NKT cells were correlated with higher CD4 count (P < 0.0001 for both Fig. 3C), and lower pVL [CD57+ NKT cells (P = 0.0001), and PD1+ NKT cells (P = 0.001)] (Fig. 3D). Similarly, the frequencies of CD4+ and CD4− NKT cells expressing CD57 and PD1 were inversely correlated with CD4 count (CD4+CD57+ NKT cells: r = −0.43, P = 0.0005; CD4+PD1+ NKT cells: r = −0.31, P = 0.01; CD4−CD57+ NKT cells: r = −0.35, P = 0.005; CD4−PD1+ NKT cells: r = −0.26, P = 0.04) (Supplemental Digital Content, Figure 2A and 2B, https://links.lww.com/QAI/A977). The association with higher pVL was found to be moderate and only with CD4− NKT cells (CD57+CD4-NKT cells: r = 0.33, P = 0.04; PD1+CD4− NKT cells: r = 0.37, P = 0.02) (Supplemental Digital Content, Figure 2C, https://links.lww.com/QAI/A977), whereas the CD57+CD4+ (r = 0.23; P = 0.15) and PD1+CD4+ (r = 0.21; P = 0.19) NKT cell frequencies did not show any association with pVL.
Depletion of NKT Cells Was Lower in LTNPs and Was Similar in Case of the CD4+ and CD4− NKT Cells
In vitro, HIV preferentially depletes CD4+ NKT cells over CD4+ conventional T cells,18,19 although limited information is available about the in vivo situation. We observed that compared with proxy preinfection values (median of total as well as CD4+ and CD4− NKT cell frequencies of HCs), the estimated depletion in total NKT cells was significantly lower in LTNPs compared with the progressors (Fig. 4A). However, in LTNPs, the estimated depletion was similar for CD4+ and CD4− NKT cells (Fig. 4B). Whereas in the progressors, the estimated depletion was significantly higher in case of CD4+ NKT cells (Fig. 4C).
NKT Cells From LTNPs Had Higher Proliferation Ability
We further wanted to analyze the proliferation ability of the NKT cells in HIV infection in response to the stimulus because the proliferation could be considered as one of the marker for functionality.24,30 The ability of NKT cells to proliferate was assessed as the percent fold increase in the NKT cell percentage after α-GalCer stimulation. The percentage fold increase in the α-GalCer stimulated NKT cells was significantly higher in healthy controls as compared with HIV-infected population (P = 0.001). Whereas within the HIV-infected groups, LTNPs showed significantly higher percent fold increase in comparison with the progressors (P = 0.002) (Fig. 4D). NKT cell proliferation ability was significantly associated with higher CD4 counts (r = 0.69; P < 0.0001) (Fig. 4E), and with lower pVL (r = −0.48; P = 0.02) (Fig. 4F) and also with lower frequencies of CD57 and PD1 expressing NKT cells (r = −0.46, P = 0.01; r = −0.49, P = 0.006; respectively) (Fig. 4G).
NKT Cells Frequencies and Functional Ability Was Partially Restored After Suppressive cART
To determine the influence of suppressive cART on NKT cells, we studied 18 patients before and at 12 months after cART. We observed that the NKT cells percentages were significantly increased after 12 months of cART as compared with the percentages before initiation (P = 0.001 by Wilcoxon signed rank test, Fig. 5A) but were still significantly lower than those observed in LTNPs (P = 0.04; Fig. 1B, left panel). Similar restoration was observed in the frequencies of subsets of NKT cells also (P = 0.03 for CD4+ and P = 0.006 for CD4− NKT cells) (Fig. 1B, middle and right panel, respectively). To compare the restoration between the NKT cell subsets, we calculated the degree of restoration. The degree of restoration was similar in both subsets of NKT cells (P > 0.05) (Fig. 5F). We further assessed the impact of ART on activation and exhaustion status of NKT cells. We observed that the frequencies of CD38+ and CD57+ NKT cells were significantly reduced at 12 months of cART (P = 0.002 and 0.01, respectively) (Figs. 5B, D). Although there was a downward trend in HLA-DR, and PD1 expressing NKT cells at 12 months post-cART, it was not statistically significant (Figs. 5C, E). We observed that cART-treated patients restored the proliferation ability as compared with the ART-naive progressors (P = 0.02) (Fig. 4D), although it was slightly lower as compared with the proliferation ability of NKT cells from LTNPs.
The importance of the innate immune response in control of HIV disease progression is recognized recently. We wanted to assess the role of NKT cells in HIV disease progression in HIV-1 C–infected population. Hence, we assessed the frequencies of NKT cells and its subsets in Indian population where HIV-1 C is the predominant subtype. We found that although HIV-1 C infection compromised the quantity and quality of NKT cells, supporting previous observation in HIV-1 B infection and simian immunodeficiency virus infection,19,20,23 the degree of impairment was lower in LTNPs as compared with the progressors. Hence, we determined whether the NKT cell frequencies can predict the HIV disease outcome. We observed that the frequencies of NKT cells in our Indian cohort were associated with higher CD4 count and lower pVL. The previous literature has shown that such association could be seen in early HIV infection but not in chronic phase of HIV infection.29 A study by Van der Vliet et al20 has found no association between the NKT cell frequencies and pVL and CD4 count, but the HIV disease status of the participants was not clear and few of them were on ART. It might be possible that in our LTNP cohort, the NKT cells have not experienced significant depletion in the early course of infection or the NKT cells would have reappeared after the initial depletion as suggested earlier.18 It has also been reported that compromised function of NKT cells in acute hepatitis C infections influenced the outcomes the infection.31 We do not have the NKT cell frequencies from early course of HIV infection in our LTNP cohort; however, the observation in hepatitis C infection indirectly supports our speculation about absence of early depletion of NKT cells in LTNPs.
It is known that the HIV-1 can infect CD4+ NKT cell in the same manner as conventional CD4+ T cells.16 It is thus expected that the CD4− NKT cells are not infected by HIV and hence would not be depleted.29 However, we observed lower frequency of both CD4+ and CD4− NKT cells in LTNPs and progressors as compared with HIV-uninfected controls and in case of LTNPs, the estimated depletion was similar in both the subsets. Also, both CD4+ and CD4− NKT population have increased expression of activation and exhaustion markers. Hence, it might be possible that the chronic immune activation caused by HIV is responsible for non–HIV-mediated apoptosis of the CD4− NKT cells also.
Chronic immune activation in HIV infection is responsible for pan-activation across the immune cells and known to be associated with faster disease progression.32,33 The lesser frequencies of activated NKT cells in LTNPs thus might reflect overall less immune activation, contributing to the sustained CD4 count and absence of disease progression. Along with the activation of immune system, HIV induces aging (senescence) and exhaustion in both innate and adaptive immune cells.8,34–36 The NKT cell population from LTNPs showed lower expression of senescence (CD57) and exhaustion (PD-1) markers. The aged and exhausted cells are known to be impaired in cytokine secretion.24,37 The NKT cells are known to secrete IFN-γ to further stimulate natural killer cells.9 The lesser secretion of these cytokines might result in compromised NK cell function. Our Indian LTNP cohort has shown higher NK cell quantity and functionality as compared to the individuals with progressive infection and it was associated with lower pVL.27 Hence, we can argue that the NKT cells with reduced senescence and exhaustion marker might be competent in activating NK cells, which has an important effect on the lysing of infected cells and reducing viral load. Higher frequencies of CD57 and PD-1–positive NKT cells in the progressive HIV infection in our study support this argument.
The proliferated NKT cells have ability to mount further activation of other immune cells, and thus an important functionality. We observed that the proliferating ability of NKT cells is compromised in HIV infection. It is also known that the NKT cells proliferate after stimulation by antigen presenting cells such as DC. Because HIV infection is known to compromise DC function, it is possible that the compromised DC function has contributed to impaired proliferating ability of NKT cells in HIV infection. Within HIV-infected population however, we observed that the NKT cells from LTNPs have greater ability to proliferate on stimulation as compared with the progressors. It is possible that this could lead to optimum cytokine secretion helping the NK cells and CD8+ T cells to mount an efficient killing of the virally infected cells. It has been shown that NKT cells facilitate antitumor activity by secondary activation of NK cells and other lymphocyte via cytokine-mediated activation.6–8 Hence, preserved proliferating ability might result in optimum cytokine secretion in LTNPs and further in stimulation of other effector cells such as NK cells and CD8+ T cells.
cART in HIV-infected individuals restore the CD4 count and suppress the viral load below undetectable levels. The reports on NKT cell restoration after cART showed variable results.20,24,38,39 A study by Van der Vliet et al20 showed rapid recovery of the circulating NKT cells within the first year of HAART, whereas other studies have shown partial or no restoration on NKT cells after ART.19,24,40 These variable results might be due to the differences in the study population, baseline immune activation, CD4 counts, pVL, etc.41 It has also been shown that the kinetics of NKT reconstitution appear to be slower than that of conventional CD4+ T cells.40,42 Our study showed that cART was responsible for partial restoration of NKT cells frequencies. It has also reduced the percentage of activated, aged, and apoptotic NKT cells and also improved the proliferating ability. Because the role of NKT cells have been implicated in protection against neoplasia and in fighting against number of pathogens, the restoration on NKT cells in HIV infection after cART would add to the benefit of cART in improving the quality of life and thus supports cART initiation in early course HIV infection.
In summary, although the mechanism of the anti-HIV effect of NKT cells may be nonspecific, the preservation of NKT cells with reduced activation, exhaustion, and aging in LTNPs might be one of the important factors involved in controlling the HIV-1 infection and halting the disease progression. The restoration of NKT cells after cART would be important in preventing other infections. The functional ability of these NKT cells in terms of cytokine secretion and longitudinal analysis of these cells in LTNP cohort will further add to our understanding in the role of NKT cells and the impact of HIV-1 on these innate immune cells.
The authors thank the study participants and the staff of the clinic, Immunology (Mr. Amol Kokare and Mrs Shubhangi Bichare) and Virology laboratory (Mr. Rajkumar londhe and Mrs Vaishali Chimanpure) for their help. The investigators thank the Indian Council of Medical Research (ICMR), Government of India for supporting Mr. Dharmendra Singh with Senior Research Fellowship.
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