Increased susceptibility of CD4+ T cells from elderly individuals to HIV-1 infection and apoptosis is associated with reduced CD4 and enhanced CXCR4 and FAS surface expression levels
© Heigele et al. 2015
Received: 5 August 2015
Accepted: 2 October 2015
Published: 9 October 2015
Elderly HIV-1 infected individuals progress to AIDS more frequently and rapidly than people becoming infected at a young age. To identify possible reasons for these differences in clinical progression, we performed comprehensive phenotypic analyses of CD4+ T cells from uninfected young and elderly individuals, and examined their susceptibility to HIV-1 infection and programmed death.
Peripheral blood mononuclear cells (PBMCs) from older people contain an increased percentage of central memory and Th17 CD4+ T cells that are main target cells of HIV-1 and strongly reduced proportions of naïve T cells that are poorly susceptible to HIV-1. Unstimulated T cells from elderly individuals expressed higher levels of activation markers, death receptors, and the viral CXCR4 co-receptor than those from young individuals but responded poorly to stimulation. CD4+ T cells from older individuals were highly susceptible to CXCR4- and CCR5-tropic HIV-1 infection but produced significantly lower quantities of infectious virus than cells from young individuals because they were highly prone to apoptosis and thus presumably had a very short life span. The increased susceptibility of T cells from the elderly to HIV-1 infection correlated directly with CXCR4 and inversely with CD4 expression. The levels of apoptosis correlated with the cell surface expression of FAS but not with the expression of programmed death receptor 1 (PD1) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).
Increased levels of activated and highly susceptible HIV-1 target cells, reduced CD4 and enhanced CXCR4 cell surface expression, together with the high susceptibility to FAS-induced programmed cell death may contribute to the rapid CD4+ T cell depletion and accelerated clinical course of infection in elderly HIV-1-infected individuals.
Aging and HIV-1 infection affect one another and share many characteristics: both are associated with diminished T cell functionality, low production of naïve T cells, loss of regenerative capacity, an accumulation of aging T cells, reduced memory T cell populations, and lower numbers of properly functioning CD8+ cytotoxic T cells (CTLs) [1–4]. Furthermore, many non-AIDS but age-related illnesses, such as hepatic, pulmonary, cardiovascular and renal disease, as well as diabetes mellitus, dementia and arthritis, are substantially more frequent in HIV-1-infected individuals than in age-matched uninfected people [5–7]. Notably, this accelerated aging in virally infected individuals continues to be a problem under highly effective antiretroviral therapy (ART), most likely because even in the absence of detectable viral loads the levels of inflammation remain higher than in healthy uninfected individuals [7, 8].
Aging and HIV-1 infection also have synergistic effects because progression to AIDS occurs substantially faster in elderly infected individuals. Adjusted for general aging effects on mortality, the median survival for those who became HIV-1 infected at ages 25–34 was 11 years, as compared to 4.4 years in those who were infected at 65 years or older . Although elderly HIV-infected individuals progress to AIDS more rapidly than younger people, they usually achieve lower viral loads, possibly due to better adherence to treatment . Possible reasons for the frequent and accelerated disease progression in elderly HIV-infected patients are an age-associated decrease in thymic function , replicative senescence of the immune system associated with accelerated telomere shortening , and lower CD4 cell counts at baseline [10, 12]. Other factors proposed to contribute to the greater risk of disease progression in elderly HIV-infected patients are increased expression of the CCR5 HIV-1 co-receptor , reduced production of IL-2 and its receptor that may promote immunosenescence , and reduced CD8+ cytotoxic T cell function . However, the strongest predictors of progression to AIDS in HIV-infected humans of all ages are increased levels of immune activation and apoptosis [5, 15, 16]. Chronic inflammation increases the T cell turnover rates and thus the exhaustion of the regenerative capacity of the immune system associated with AIDS [17, 18].
It is conceivable that age is an important factor in AIDS progression because the regenerative capacity of people becoming HIV-1 infected at an older age is a priori reduced so that they already start with a handicap. However, the immune systems of elderly and younger individuals may also react differently to HIV-1 infection. In fact, recent data suggest that the levels of T cell activation, inflammation and apoptosis are particularly high in older HIV-infected patients [7, 19]. The reasons for these age-dependent differences are currently poorly understood and further studies seem highly warranted since they may provide new insights into the mechanisms underlying the sustained inflammation that drives progression to AIDS and thus ultimately help to optimize antiretroviral therapy in HIV-1-infected individuals. To identify possible reasons for the increased rates and accelerated kinetics of AIDS progression in elderly individuals, we analyzed which properties may render CD4+ T cells from the elderly particularly susceptible to HIV-1 infection and depletion.
Young and elderly differ in CD4+ T cell subsets that are targets for HIV-1 infection
CD4+ T cells from elderly individuals are highly susceptible to HIV-1 infection
To further examine the impact of donor age on the susceptibility of CD4+ T cells to HIV-1 infection and replication, we used X4 and R5 HIV-1 NL4-3 reporter viruses co-expressing the enhanced version of the green fluorescent protein (eGFP) and Nef via an internal ribosome entry site (IRES) [25, 26]. These HIV-1 reporter constructs express all viral proteins and replicate in infected PBMC cultures. Importantly, they readily allow determining both the efficiency of virus production, as well as the frequency and phenotype of HIV-1-infected (GFP+) and uninfected (GFP−) cells in FACS-based assays.
CD4+ T cells from elderly individuals show high levels of apoptosis
The levels of apoptosis in HIV-infected CD4+ T cells correlate with FAS expression
Inverse correlation between CD4 cell surface expression and virus production
Finally, we examined whether differences in the levels of activation of HIV-1-infected T cells from young and elderly individuals might also affect the efficiency of viral replication. We found that HIV-1-infected (GFP+) T cells expressed substantially higher levels of the early T cell activation marker CD69 than uninfected GFP- cells irrespective of the donor’s age or viral co-receptor tropism (Additional file 1: Figure S2A). In contrast, the levels of CD25 expression did not differ significantly between uninfected and infected T cells (Additional file 1: Figure S2B). However, CD25 expression in the HIV-1-infected population of the elderly was slightly increased compared to the young age group (Additional file 1: Figure S2B). To better assess whether this modest difference in the late state of T cell activation might affect viral LTR activity, we determined the MFIs of eGFP expression in HIV-infected primary T cells. eGFP is expressed together with Nef by the regular HIV-1 LTR promoter and thus represents an indicator of the viral transcriptional activity. However, the MFIs of eGFP expression did not differ significantly between the young and elderly age groups (Additional file 1: Figure S2C). Thus, age-dependent differences in the efficiency of HIV-1 infection are not due to differences in the efficiency of viral gene expression.
In the present study, we identified several differences in the phenotypes and properties of CD4+ T cells from young and elderly people that may play a role in the high susceptibility of older individuals to HIV-induced immunodeficiency [6, 29]. PBMC cultures from the elderly contain high percentages of TCM cells that are primary targets for HIV-1 replication but low percentages of naïve CD4+ T cells, which are relatively resistant to HIV-1 infection [30, 31]. Furthermore, the proportion of CCR6+ Th17 cells, reported to be highly permissive for HIV-1 infection  was significantly enhanced in elderly individuals. Finally, we observed a slightly higher frequency of follicular helper CD4+ T cells (Tfh) that regulate antigen-specific B cell immunity and may represent a major compartment for HIV-1 replication  in the elderly (Fig. 1b). Thus, an increased age was associated with an increased frequency of CD4+ T cells subsets known to be highly susceptible to HIV infection.
In addition to the age-dependent differences in T cell subsets, T cells from the elderly expressed significantly lower levels of the primary CD4 receptor and higher quantities of the CXCR4 co-receptor of HIV-1 entry compared to T cells from young individuals. These findings are in agreement with published data, reporting an increased frequency of CD4low T cells , and significant increases in CXCR4 surface expression  in uninfected elderly individuals. At first view, the inverse correlation between CD4 expression and the efficiency of HIV-1 replication may seem striking. Previous cell culture studies have shown, however, that reduced levels of CD4 cell surface expression may facilitate efficient virus release and enhance the infectivity of progeny HIV-1 virions [35–37]. The advantage of low levels of CD4 cell surface expression is perhaps most evident from the fact that HIV-1 uses three of its ten gene products (Env, Vpu and Nef) to down-modulate its primary receptor from the cell surface . Increased levels of CXCR4 expression in T cells from the elderly may also seem surprising since this receptor is predominantly expressed on naive CD4+ T cells  that are significantly reduced in the elderly (Fig. 1a). However, increased surface CXCR4 expression in the elderly has been observed previously and may be due to altered ubiquitination of this co-receptor . Enhanced CXCR4 expression might facilitate viral entry and helps to explain why the percentages of X4 HIV-1-infected GFP+ cells were significantly higher in the old age group. Our results thus suggest that low CD4 and high CXCR4 expression both contribute to the high susceptibility of T cells from the elderly to HIV-1 infection.
Our results support the presumption that the overall efficiency of HIV-1 replication depends on a delicate balance between the susceptibility of the CD4+ target T cells to HIV-1 infection and the levels of programmed death that might limit the life span of the infected cells and thus the time frame of virus production. These findings help to explain why HIV-1 may cause more damage in the elderly even at lower viral loads . On the one hand, increased levels of T cell activation and CXCR4 cell surface expression together with reduced CD4 expression levels render CD4+ T cells from the elderly highly susceptible to HIV-1 infection and replication. On the other hand, HIV-1-infected PBMC cultures from the elderly showed very high levels of apoptosis and the short span of infected CD4+ T cells was frequently associated with reduced virus production. Follow-up studies using HIV-1 reporter constructs co-expressing Nef and eGFP confirmed that PBMC cultures from the elderly show particularly higher levels of HIV-1-infected cells but reduced virus production efficiencies on a per cell basis after stimulation with CD3/CD28 beads due to accelerated cell death (Fig. 4). Increased numbers of activated CD4+ T cells that are highly susceptible to HIV-1 infection together with an increased sensitivity of virally infected cells to programmed death might help to explain why older HIV-1-infected patients show a more rapid loss of CD4+ T cells and progression to AIDS than younger individuals .
We found a highly significant correlation between the levels of FAS expression on CD4+ T cells prior to stimulation and the levels of apoptosis in HIV-1-infected T cells following stimulation with CD3/CD28 beads. It is known that the frequency of FAS expressing CD4+ T cells  and the susceptibility to FAS-induced apoptosis  is increased in elderly individuals. Our observation that the levels of FAS but not of TRAIL and FAS-L expression correlate significantly with the levels of apoptosis in HIV-1-infected T cells and are strongly increased in the elderly, suggests that this death receptor plays a relevant role in the high susceptibility of old individuals to HIV-induced immunodeficiency. In agreement to previous studies , we detected higher CTLA-4 expression in unstimulated T cells from the elderly. It has been reported that CTLA-4 signaling may result in high CCR5 expression and enhanced susceptibility to viral infection . In the present study, however, the levels of CCR5 cell surface expression did not differ significantly between the groups of the young and the elderly. Upon stimulation with CD3/CD28 beads the CTLA-4 expression levels were even significantly reduced in the elderly (Fig. 2f) and the role of this inhibitory receptor for the pathogenesis of HIV-1 infection in vivo remains to be elucidated. Another factor that was expressed at significantly higher levels in unstimulated CD4+ T cells from the elderly was PD1, which is associated with T cell exhaustion and disease progression . Thus, CD4+ T cells of the elderly show higher basal expression levels of death receptors and activation markers but react poorly to stimulation. In agreement with previous studies [45–47], the levels of FAS were strongly increased in T cells from the elderly. Most importantly, they correlated significantly with the levels of apoptosis in HIV-1-infected CD4+ T cells suggesting that FAS-mediated cell death may play an important role in the high sensitivity of elderly people to HIV-1 induced immune damage.
To approximate the in vivo situation, we used primary CD4+ T cells from young and elderly individuals, exposed them to HIV-1 particles containing regular Env glycoproteins, and performed most stimulations with CD3/CD28 beads. Nonetheless, the relevance of our findings for the difference in the rates of disease progression between young and elderly people remains to be determined. For example, it will be interesting to further examine whether the increased levels of CXCR4 expression in the elderly is associated with an increased frequency of the CCR5 to CXCR4 co-receptor switch that is associated with rapid progression to AIDS in untreated HIV-1-infected individuals [48, 49]. Finally, our observation that high sensitivity of CD4+ T cells to FAS-mediated apoptosis may contribute to rapid disease progression in elderly HIV-infected individual suggests that this death receptor may be a useful target for the prevention of inflammation and CD4+ T cells depletion.
People becoming HIV-infected at an older age do not only start with a handicap because their immune system has reduced regenerative capacity but also provide a particular susceptible environment for HIV-1 infection and immune damage.
Samples from young and elderly individuals
This study was approved by the Ethics Committee of Ulm University Medical Center. The age range of the young blood donors was 18–25 years (22.1 ± 2.2; n = 14) and of the elderly donors 50–86 years (62.6 ± 9.1; n = 16). Blood was collected from donors, which provided written consent or obtained from the blood donation center, Ulm. To the best of our knowledge, all blood donors were clinically healthy at the time of sampling.
Isolation and culture of PBMCs
Peripheral blood mononuclear cells (PBMCs) from healthy human donors were isolated using lymphocyte separation medium (Biocoll separating solution, Biochrom) and stimulated for 3 days with human T-activator CD3/CD28 Dynabeads at a bead to cell ratio of 1:1 (Life technologies, 11132D) and 10 ng/ml IL-2 in RPMI1640 medium with 10 % FCS prior to infection. For FACS analysis in unstimulated PBMCs cells were incubated without stimulus in supplemented RPMI overnight.
T cell subsets and receptor expression
One day post isolation FACS analysis was used to determine the percentages of various CD4+ T cell subsets, like naïve (TN), effector memory (TEM), central memory (TCM), T helper 17 cells (Th17), follicular helper T cells (Tfh) and regulatory T cells (Treg). For this the following antibodies were used: CD4-APC H7 (BD Biosciences, 560158), CD45RA-PE Cy7 (BD Biosciences, 560675), CD45RO-PerCP Cy5.5 (BD Biosciences, 560607), CCR7-APC (BioLegend, 353214), CCR6-PE (BD Biosciences, 559562), CXCR5-PerCP Cy5.5 (BioLegend, 335001), CD127-PE Cy7 (BD Biosciences, 560822), CD25-Horizon V450 (BD Biosciences, 560355). Further, the expression of different receptors on unstimulated as well as stimulated CD4+ T cells was determined using the following antibodies: CD4-APC H7, TCR-CD3-APC H7 (BD Biosciences, 560275), CD28-PerCP Cy5.5 (BD Biosciences, 560685), MHC-I-Horizon V450 (BD Biosciences, 561346), FAS-PE (BD Biosciences, 556641), FAS-L-PE (BioLegend, 306407), PD1-APC (BD Biosciences, 558694), PD1-L-PE Cy7 (BD Biosciences, 558017), TRAIL-PE (BD Biosciences, 550516), CTLA-4-PE (BD Biosciences, 555853), CD69-Horizon V450 (BD Biosciences, 560740), CD25-PE Cy7 (BD Biosciences, 557741), CXCR4-PerCP Cy5.5 (BD Biosciences, 560670) and CCR5-PE Cy7 (BD Biosciences, 557752). Cells were analyzed using the BD FACS Canto II with FACSDiva software.
For FACS analysis X4- (env wild type) and R5- (env V3 loop 92th014.12)  tropic reporter NL4-3 proviral constructs encoding an internal ribosome entry site (IRES) and the eGFP gene upstream of the nef gene were used [25, 26]. For wild type replication kinetics proviral constructs of X4-tropic HIV-1 NL4-3 and SG3 as well as R5-tropic HIV-1 NL4-3 and JR-CSF were used.
To generate viral stocks, 293T cells were transfected with the proviral constructs as described . The medium was changed after overnight incubation, and virus was harvested 24 h later. For replication kinetics, virus stocks were quantified using a p24 antigen capture assay provided by the NIH AIDS Research and Reference Reagent Program.
HIV-1 replication in PBMCs
PBMCs were isolated from young and old blood donors and stimulated with CD3/CD28 beads or PHA in the presence of IL-2. Three days post stimulation PBMCs were infected with p24 normalized wild type NL4-3 X4 or R5, SG3 X4, JRCSF R5 viruses for wild type replication kinetics and with NL4-3 X4 or R5 GFP reporter viruses [25, 26] for GFP kinetics. One day post infection the inoculum was removed, cells were washed and fresh medium containing IL-2 was added. Cells were cultured for 10 days and supernatant samples were taken every 2–3 days. The extent of viral replication was determined by reverse transcriptase (RT) assay as described previously . As standard a serial dilution of a HIV-1 NL4-3 virus stock was used and 30 ng/ml p24 was defined as 100 %. In addition FACS analysis were done to determine the percentages of infected cells (GFP+) in the GFP kinetics.
Apoptosis and activation in infected PBMC cultures
PBMCs were isolated from young and old blood donors and stimulated with CD3/CD28 beads and IL-2. Three days post stimulation PBMCs were infected with NL4-3 X4 or R5 reporter viruses. Two days post infection a second stimulus (beads + IL-2) was added. FACS analysis was done 1 day post second stimulus to determine CD69 expression levels and 2 days post second stimulus to determine CD25 expression levels, apoptosis, apoptotic cell populations, and death rates. For this the following antibodies and dyes were used: CD69-PE Cy7 (BD Biosciences, 557745), CD25-PerCP Cy5.5 (BD Biosciences, 560503), AnnexinV-PE (BD Biosciences, 556421) and fixable viability stain 450 (FVS) (BD Biosciences, 562247). Antibodies used to distinguish TN, TEM and TCM cells in the apoptotic population are mentioned above.
Groups were compared using a two-tailed Student’s t test. The PRISM package version 4.0 (Abacus Concepts, Berkeley, CA, USA) was used for all calculations. In most experiments data from 14 young and 16 elderly blood donors were available for comparison.
AH, SJ and KR performed the experiments. AH and FK designed the study, evaluated the data, prepared the figures and wrote the manuscript. All authors read and approved the final manuscript.
The authors thank Daniel Sauter, Dre van der Merwe and Jan Münch for helpful comments and suggestions and Susanne Engelhart, Martha Mayer and Daniela Krnavek for excellent technical assistance. This work was supported by the Hector foundation. SJ is a member of the research training program cellular and molecular mechanisms in aging (CEMMA) that is funded by the Deutsche Forschungsgemeinschaft (DFG) and part of the International Graduate School in Molecular Medicine Ulm (IGradU).
The authors declare that they have no competing interests.
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