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Basic Science

Analyses of Mitochondrial DNA and Immune Phenotyping Suggest Accelerated T-Cell Turnover in Treated HIV

Massanella, Marta PhD*,†; Karris, Maile Y. MD*; Pérez-Santiago, Josué PhD*,‡; Yek, Christina MD*,§; Vitomirov, Andrej BA*; Mehta, Sanjay R. MD*,║,¶

Author Information
JAIDS Journal of Acquired Immune Deficiency Syndromes: November 1, 2018 - Volume 79 - Issue 3 - p 399-406
doi: 10.1097/QAI.0000000000001824



Case–control studies have demonstrated that HIV-infected individuals experience age-related comorbidities (eg, diabetes, cardiovascular disease, and renal disease) 10 years earlier than HIV-uninfected age-matched controls.1 This premature aging has been attributed to HIV-associated chronic immune activation, persistent low-grade inflammation, epigenetic changes, and direct biologic effects on aging-related pathways.2,3 However, recent work suggests that aging of HIV-infected individuals may not be as accelerated as previously believed,4,5 and the differential between infected and uninfected persons may decrease with time. These studies and most others studies characterizing premature aging in HIV-infected individuals examined cardiovascular and metabolic complications. However, other physiologic systems may also be affected by HIV infection through inflammation or direct viral effects. The immune system is one such entity, and senescence in this system manifests as reduced breadth and potency of the immune response.6 This results in reduced responses to vaccination,7,8 increased rates of infection,9 and reduced immune surveillance.10 Immunosenescence is also associated with immune dysregulation that results in persistent immune activation and the senescence-associated secretory phenotype, resulting in the chronic low-level inflammation seen in with aging and in HIV-infected individuals.11–13

The pathogenesis of aging and cellular senescence is complex, but includes mitochondrial alterations within cells.13 Damage to mitochondria is believed to play a role in many age-related pathologies, and natural again is associated with increases in mitochondrial DNA (mtDNA) mutations and declines in mitochondrial function (reviewed in Ref. 14). We and others have previously demonstrated that mitochondrial DNA deletions accumulate with age in the brain15,16 and in muscle.17–19 Specifically, the proportion of mtDNA carrying the “common deletion,” a 4977bp deletion that affects several transfer RNA and respiratory chain genes, has also been associated with aging-related phenomena such as corneal thickening, hearing loss, loss of skin turgor, and sarcopenia.17,20–25 If this deletion is present in a high enough proportion of the mtDNA within a cell, the cell will be incapable of meeting its metabolic demands.26 This deletion is more common in older populations and in longer-lived cells often involved in degenerative diseases.17,27–29 We hypothesized that because HIV infection results in cumulative mitochondrial toxicity from direct viral effects and antiretroviral therapies (ARTs),30–32 immune cells from infected individuals may have greater mitochondrial DNA pathology than immune cells from age-matched uninfected individuals. We further hypothesized that the degree of mitochondrial DNA pathology would correlate with greater immunosenescence and immune activation. To address this question, we measured immunosenescence, immune activation, and mitochondrial DNA changes in circulating T-cells from persons living with HIV older than 40 years and compared them with aged-matched HIV-uninfected controls.


Study Cohort

The study sample consisted of 13 HIV-infected individuals over the age of 40 who had been on HIV therapy for more than 2 years and 10 healthy uninfected controls well-matched for age enrolled between July and December 2014. Individuals with cancer, hepatitis C, diabetes, or on immunosuppressive medications were excluded from the study. The study was approved by the Human Research Protections Program at the University of California San Diego (UCSD). All HIV-infected individuals were recruited from the UCSD Owen clinic and the UCSD Antiviral Research Center. Control subjects were recruited from UCSD general internal medicine clinics. After providing informed consent, sociodemographic and clinical data were obtained from each subject, and 100 and 8 cc of fresh whole blood (in acid citrate dextrose and EDTA tubes, respectively) were collected for further analysis.

Determination of Absolute T-Cell Counts

Absolute counts of T-cells in whole blood were determined by flow cytometry (Accuri; BD biosciences, Franklin Lakes, NJ). First, the absolute lymphocyte count was determined using a CD45-PerCP-Cy.5.5 antibody in combination with perfect count microspheres (Life technologies, Carlsbad, CA). Then, the percentage of the T-cells was determined using the following antibody combination: CD3-APC, CD4-FITC, and CD8-PE (all antibodies were from BD Biosciences). Absolute count of each cellular population was calculated as follows: (X × Y)/100, where X is the percentage of each subset and Y is the absolute count of lymphocytes.


The levels of proliferation (Ki67 FITC, tube 1), immune activation (CCR5 FITC, HLA-DR BV421, and CD38 BV605, tube 2), and immunosenescence (CD57 FITC and KLRG1 BV421, tube 3) in CD4+ and CD8+ T-cell subsets were evaluated (common backbone for all tubes: CD3 APC-H7, CD4 PerCP-Cy5.5, CD8 V500, CD45RA AF700, CD27 PE-Cy7, CCR7 PE-CF594, CD58 PE, and CD95 APC). Briefly, 150 µL of whole blood/tube was incubated for 20 minutes at room temperature with the antibody combinations. Cells were then lysed for 10 minutes at room temperature in FACS Lysing solution (BD Biosciences), washed in phosphate-buffered saline and fixed in phosphate-buffered saline containing 1% formaldehyde (Sigma, Burlington, MA), before acquisition in an FACS Canto (BD Biosciences). For tube 1, cells were washed after lysis and then fixed and permeabilized using FoxP3 staining buffer set (eBiosciences, San Diego, CA) for 30 minutes at 4°C and then incubated with anti-Ki67 for 30 minutes at 4°C. CD4 (CD3+CD4+CD8) and CD8 (CD3+CD8+CD4) T-cell subsets were defined as: TN: CD45RA+CD27+CCR7+CD95CD58, TSCM: CD45RA+CD27+CCR7+CD95brCD58br, TCM: CD45RACD27+CCR7+, Ttm: CD45RACD27+CCR7, TEMRA−: CD45RACD27CCR7, and TEMRA+: CD45RA+CD27CCR7. For each subset, the expression of proliferation, activation, and senescence makers was evaluated.

Quantification of mtDNA Within T-Cell Subsets

Blood from acid citrate dextrose tubes was immediately processed for isolation of peripheral blood mononuclear cells using Ficoll–Hypaque density gradients. Peripheral blood mononuclear cells were divided in 2 aliquots to negatively select CD4+ or CD8+ T-cells (Easysep CD4 and CD8 enrichment kit; Stem cell, Vancouver, CA). Enriched CD4 and CD8 T-cells were then labeled (CCR7 FITC, CD58 PE, CD45RA-PE-CF594, CD3 PerCP-Cy5.5, CD27 PE-Cy7, CD95 APC, and CD4 or CD8 APC-H7) for live-cell sorting (MoFlo; Beckman Coulter, Brea, CA) to obtain populations of TN, TSCM, TCM, TTM, and TEM CD4+ and CD8+ T-cells (Figure 1, Supplemental Digital Content, After sorting, sorted subsets were centrifuged 300g for 5 minutes, and the supernatants were carefully discarded. Cell pellets were resuspended in a lysis buffer [0.05% Nonidet P-40 and Tween-20 (Sigma), 0.1 mg/mL proteinase K (Invitrogen, Carlsbad, CA)] and digested for 30 minutes at 65°C. Proteinase K was inactivated by heating digested samples at 95°C for 15 minutes. Cell lysates were stored at −20°C until use. We measured the mitochondrial DNA (mtDNA) copy number per cell for each subset using droplet digital polymerase chain reaction (BioRad, Hercules, CA) directly from cell lysates. Quantification was performed as previously described16 using a primer–probe set designed to target a gene on the mitochondrial genome, MT-ND2.33 A second primer–probe set targeting human RPP30 was used as a cell-copy control because each cell contains 2 copies of this gene. In addition to quantifying the total mtDNA copy number, the proportion of mtDNA carrying the “common deletion” was also measured. As previously described,16 we measured the proportion of mtDNA carrying this deletion using a primer–probe combination targeting the bridge sequence formed by the ends of the mitochondrial genome left by the common deletion.33 This proportional measure of the common deletion was used as a surrogate measure of mitochondrial somatic damage.

Statistical Analyses

All statistical analyses were performed using the R statistical package. Normality of the levels of mtDNA and relative presence of the “common deletion” were assessed using a Shapiro test with a significance cutoff of P < 0.05. Given that log transformation did not normalize data, analysis was performed with nontransformed data. Differences in mtDNA levels and relative presence of the “common deletion” between HIV-infected and HIV-uninfected study groups were assessed by the Student t test and by using analysis of variance when comparing the individuals infected <15 and >15 years with the uninfected controls. If data failed normality, a Kruskal–Wallis test was used to assess differences between study groups. No corrections for multiple comparisons were made given the exploratory nature of this study.


Study Participant Characteristics

Thirteen virally suppressed HIV-infected individuals with a median age of 51 (range: 42–68 years) and 13 HIV-uninfected controls with a median age of 50.5 (range: 45–72) were enrolled in the study. Data from 3 uninfected individuals were subpar and not included in the analysis. HIV-infected individuals were on therapy for a median of 15 years (range: 2–29 years). Five individuals had previous exposures to d4T, ddI, ddC, or AZT (medications known to cause mitochondrial toxicity), but no participants were currently on those medications. The median CD4+ T-cell nadir for the HIV-infected group was 175 cells per mm3 (range: 0–628 cells/mm3), and there were no differences in CD4+ or CD8+ T-cells percentage in each subset between the infected and uninfected groups (Figure 2, Supplemental Digital Content, Additional characteristics are presented in Table 1.

Study Cohort

T-Cell Activation in Persons Living With HIV Compared to Age-Matched Controls

Immune activation as measured by dual expression (HLADR+CD38+) was higher in HIV-infected individuals than HIV-uninfected individuals in the mature CD4+ T-cell subsets (TTMP = 0.025, TEMRA−P = 0.0025, Fig. 1A). This remained true whether the individual had been on treatment for <15 years or >15 years (TTMP = 0.04/P = 0.09, TEMP = 0.07/0.003, Figure 3A, Supplemental Digital Content, No differences in immune activation were seen in CD8+ T-cell subsets as measured by differences in HLADR+CD38+ expression (Fig. 1B). Strangely, infected person with <15 years of treatment trended toward lower immune activation in TN, TSCM, and TTM CD8+ T-cell subsets than uninfected controls (TNP = 0.06, TSCMP = 0.096, and TTMP = 0.096) (Figure 3B, Supplemental Digital Content,

Immune activation. Immune activation measured by CD38+HLADR+ expression in CD4+ (panel A) and CD8+ (panel B) T-cell subsets. The TEM CD4+ T-cell subset from HIV-infected individuals was more likely to demonstrate immune activation (by HLADR+CD38+ expression). P values between age-matched infected and uninfected individuals are indicated.

T-Cell Senescence in Persons Living With HIV Compared to Age-Matched Controls

To evaluate differences in T-cell senescence between HIV-infected and HIV-uninfected individuals, we evaluated expression of KRLG1 and CD57 on subsets of CD4+ and CD8+ T-cells. Long-lived TN and TSCM CD4+ T-cell subsets from HIV-uninfected individuals were less senescent (TNP = 0.02, TSCMP = 0.07). However, the TTM subset was more senescent in uninfected individuals (TTMP = 0.02) (Fig. 2A). When senescence was defined by dual expression of KRGL1+CD57+, the CD4+TTM T-cell subset continued to seem more senescent in the HIV-uninfected population (TTMP = 0.001), while again the HIV-infected individuals had higher levels of CD57+KRLG1+ in CD4+TN cells (TNP = 0.03), although the numbers of cells were very low (Figure 4A, Supplemental Digital Content, KRGL1+CD57+ expression differences in CD4+ T-cells were more pronounced when uninfected persons were compared with HIV-infected persons treated for >15 years (uninfected vs. treated for >15 years, P = 0.001) (Figure 5A, Supplemental Digital Content,

Senescence. Senescence measured by KRLG1+ expression in CD4+ (panel A) and CD8+ (panel B) T-cell subsets. CD4+ (TN, TSCM, and TTM) and CD8+ (TEMRA+) T-cells from uninfected individuals were more likely to be senescent. P values between age-matched infected and uninfected individuals are indicated.

Regarding the CD8+ T-cell compartment, HIV-uninfected individuals had higher proportions of senescent TEMRA+ CD8+ T-cells as measured by KRLG1+ (P = 0.003, Fig. 2B) and CD57+KRLG1+ (P = 0.007, Figure 4B, Supplemental Digital Content, compared with the HIV-infected individuals. The lower observed senescence of TEMRA+ CD8+ T-cells in HIV-infected persons did not differ when individuals were stratified by treatment duration when compared with uninfected controls (Figure 5B, Supplemental Digital Content,

T-Cell Proliferation in Persons Living With HIV Compared to Age-Matched Controls

To evaluate differences in T-cell proliferation between HIV-infected and HIV-uninfected individuals, we evaluated expression of Ki67 on subsets of CD4+ and CD8+ T-cells. We found that CD4+ T-cells from HIV-infected individuals were more likely to express Ki67 molecule than HIV-uninfected individuals (P = 0.06), and this was most pronounced in the TEM population (P = 0.011) (Figure 6, Supplemental Digital Content,

Relative Mitochondrial DNA Abundance in Peripheral Blood T-Cells

To assess whether mitochondrial toxicity contributed to differences in immune activation and senescence, we evaluated mitochondrial DNA (mtDNA) copy number, measured by copies of MT-ND2, and the relative abundance of the mitochondrial common deletion in CD4+ and CD8+ T-cell subsets (Fig. 3). No differences in total mtDNA in CD4+ T-cell subsets were observed between infected and uninfected individuals. However, CD4+ T-cell subsets demonstrated lower mtDNA copy numbers in TTM than in TSCM (P = 0.030) and TEM (P = 0.09) (Fig. 3A). A similar trend was observed between TTM and TSCM from uninfected controls (P = 0.08).

Mitochondrial DNA measurements. Mitochondrial DNA copy number (ND2) and the relative proportion of mitochondrial DNA carrying the common deletion (RACD) in CD4+ (panels A and C) and CD8+ (panels B and D) T-cells. CD8+ (TSCM) from infected individuals had higher mtDNA levels and lower proportions of mtDNA with the common deletion. Across other T-cell subtypes, this was not statistically significant but visually trended in that direction. Overall, this suggests increased cell turnover in the T-cells of infected individuals.

More differences were observed in mtDNA from CD8+ T-cells. In HIV-infected subjects, TSCM had higher levels of mtDNA than in any other subset (TNP = 0.04, TCMP = 0.09, TTMP = 0.08, and TEMP = 0.0002) (Fig. 3B). The TSCM subset also trended toward higher mtDNA levels in the HIV-infected group compared with uninfected controls (P = 0.057). By contrast, uninfected individuals showed higher levels of mtDNA in TN compared with TTM (P = 0.05) and TEM (P = 0.006), and TCM showed higher levels of mtDNA than TEM (P = 0.002). The 3 subjects on treatment for the shortest period (2, 8, and 9 years) had the highest levels of MT-ND2 detected in the TCM and TTM subsets of both CD4+ and CD8+ populations, but we did not observe any significant differences between individuals on treatment for less than or more than 15 years.

Relative Abundance of Mitochondrial DNA Carrying the Common Deletion in T-Cells

The relative abundance of mtDNA carrying the common deletion (RACD) did not differ between HIV-infected and HIV-uninfected individuals in any T-cell subsets except for CD4+ and CD8+ TSCM cells, where a higher RACD was seen in uninfected individuals (P = 0.06 and P = 0.003, respectively) (Figs. 3C, D). This difference in CD8+ TSCM cells was most prominent when uninfected persons were compared to those with HIV infection >15 years (P = 0.002) (Figure 7, Supplemental Digital Content, RACD negatively correlated with MT-ND2 only in the CD4+ and CD8+ TEM subsets (CD4+: r2 = −0.31, P value = 0.005, CD8+: r2 = −0.31, P value = 0.005).

Correlation of Mitochondrial DNA With Senescence, Activation, and Proliferation Markers

We did not identify any associations between average mtDNA or RACD and senescence, activation, or proliferation markers in CD4+ or CD8+ T-cell subset populations. However, we did note that the direction of correlation between proliferation and RACD in T-cell subsets was nearly always negative, but that high RACD in mature CD4+ T-cell subsets was associated with proliferation of CD4+TSCM (Tables 1 and 2, Supplemental Digital Content,

Correlation of Mitochondrial DNA With Age and Years Living With HIV

We next evaluated the correlation between age and mtDNA content per cell. In CD4+ and CD8+ TN subsets (when combining both HIV-infected and HIV-uninfected individuals), we observed a correlation between age and mtDNA copies per cell (CD4+TN: r2 = 0.25, P = 0.014; CD8+TN: r2 = 0.245, P = 0.015). Consistent with this, we also found that age correlated with the RACD in the mature subsets of both CD4+ and CD8+ populations (CD4+TTM: r2 = 0.23, P = 0.02; CD8+TTM: r2 = 0.22, P = 0.025; and CD8+TEM: r2 = 0.28, P = 0.009) (Figure 8, Supplemental Digital Content,

No correlations were observed between age and mtDNA in the infected or uninfected population alone. However, there was a trend toward a correlation between the number of years infected with HIV with mtDNA levels in CD4+TN (r2 = 0.29, P = 0.09), and with the RACD in the CD4+TTM subset (r2 = 0.41, P = 0.03). The time on ART also correlated with the RACD of the CD4+TTM subset (r2 = 0.51, P = 0.014) (Figure 8, Supplemental Digital Content,


This cross-sectional study is one of the first to examine the relationship of mtDNA with immunosenescence and immune activation in the setting of aging with HIV. We observed that HIV-infected individuals on suppressive ART demonstrated higher levels of CD4+ T-cell immune activation than HIV-negative persons particularly in the TTM and TEM subsets, consistent with the function of these cells and previously reported literature.34,35 Unexpectedly, age-matched uninfected controls in our cohort had higher proportions of KLRG1+ senescent T-cells in more mature CD4+ and CD8+ subsets. We observed this to be true for CD57+KLRG1+ CD4+TTM cells as well. The seeming discordance between immune activation and immunosenescence could suggest that (1) higher levels of ongoing cellular inflammation in HIV-infected individuals do not result in terminal differentiation of lymphocytes36–38 or (2) HIV infection and chronic immune activation may drive increased CD4+ and CD8+ T-cell death39,40 and increased cellular turnover (in response to T-cell depletion), preventing the accumulation of senescent cells. This second hypothesis was supported by our observation that CD4+ T-cells (particularly, the TEM subset) demonstrated higher rates of proliferation than controls. The TEM subset also demonstrated an inverse relationship between mtDNA and RACD, suggesting increased cell turnover. We observed higher mtDNA and a smaller RACD in the long-lived stem cell subset (TSCM) of HIV-infected individuals compared with HIV-uninfected individuals, which is consistent with increased mitochondrial turnover in the setting of cellular proliferation, as suggested by Ross et al.41 Although not always statistically significant, the direction of correlation between T-cell subset RACD and Ki67 expression (proliferation), and RACD and mtDNA content was always negative.

By contrast, we observed an increase in mtDNA per cell with increasing age in the long-lived TN subset in both HIV-infected and HIV-uninfected individuals. This was most pronounced in the HIV-infected individuals with infection >15 years. The TN population from HIV-infected individuals also had a higher proportion of CD57+KLRG1+ CD4+ TN than HIV-uninfected individuals, which may suggest a shift to reduced cellular replicative capacity. We did not identify a relationship between RACD and mtDNA content in this subset. Unfortunately, further exploration of this concept was beyond the scope of this study, but others have demonstrated an association between increased senescence and reduced mitophagy,42 which may lead to accumulation of damaged mtDNA.

This pilot cross-sectional study is limited by its small sample size, and the clinical heterogeneity of the subjects (ie, significant variations in duration of infection and time on ART). Furthermore, we did not evaluate or measure other potential factors that could contribute to or impact inflammation and immune aging, including smoking history, cytomegalovirus status, antiretroviral regimen, measures of nuclear DNA replicative capacity (eg, telomerase activity), cellular division (eg, T-cell receptor excision circles), and other intracellular physiologic processes.43,44 Specifically, the antiretroviral exposure of individuals on treatment >15 years was more likely to include medications with a higher mitochondrial toxicity. As in other studies, measurements of mitochondrial DNA and RACD were across populations of cells, and the distribution of these measures was unlikely to be uniform. We also confined our evaluation of T-cell immune activation and immunosenescence to the peripheral blood and therefore cannot confirm that our findings were not due to differences in T-cell trafficking to effector sites. Finally, as an exploratory study, we did not statistically correct for multiple comparisons, limiting the interpretation of our findings.

In summary, we did not observe clear and consistent evidence of immunosenescence or mitochondrial signals of aging, despite higher levels of cellular immune activation in the peripheral blood of HIV-infected persons.34,35 Our data suggest this may be due to higher cell turnover in individuals with more recent HIV infection. This is different than what we observed in our study of mtDNA in the brain, where neurons and other glial cells live for extended periods. Previous studies attempting to quantify the accelerated aging of HIV-infected individuals have estimated a 2- to 10-year increase in biologic age greater than HIV-uninfected individuals of the same chronologic age.45,46 Our study suggests that in the first few years of treated HIV infection, immune activation continues to drive turnover of T-cells, a process that seems to decrease with time on therapy. Thus, using peripheral T-cells to assess aging may underestimate biologic age, particularly in more recently infected individuals, and limits our ability to use these cells to evaluate the effects of HIV on aging. The results of this study are limited by the heterogeneity of the participant population and small sample study, and therefore, further work into this area is warranted.


The authors thank the participants in our study, as well as the Translational Virology and Flow Cores of the University of California of San Diego Center for AIDS Research, which supported this project.


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immunosenescence; mitochondrial DNA; HIV; aging; common deletion

Supplemental Digital Content

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