The CD4/CD8 ratio as a marker T-cell activation, senescence and activation/exhaustion in treated HIV-infected children and young adults

Abstract

We explored the associations of the CD4/CD8 ratio with markers of immunoactivation, immunosenescence and T-cell subsets, in 37 vertically HIV-infected children and adolescents. CD4/CD8 ratio inversion was associated with higher frequencies of activated, senescent and activated/exhausted CD4⁺and CD8⁺ T-cells, and a skewed T-cell phenotype from naive toward effector memory which persisted after the multivariate analysis. Thus, the CD4/CD8 ratio may identify patients with higher immunoactivation despite ART.

Immunoactivation has demonstrated to be a strong predictor of disease progression in HIV-infection, and one of the underlying causes leading to immunosenescence, premature aging and adverse outcomes among patients with access to modern ART regimens ^[1]. The effects of chronic immunoactivation and immunosenescence are likely to be more pernicious in vertically HIV-infected individuals, since their immune system coevolves since birth with the virus.

Inversion of the CD4/CD8 ratio (<1), a hallmark of untreated HIV infection, is a surrogate marker of immunosenescence and independently predicts all-cause mortality in the general population ^[2–5]. Although a failure to normalize the CD4/CD8 ratio is commonly observed HIV-infected patients after starting ART, its biological significance and clinical relevance remain unknown.

We hypothesized that a low CD4/CD8 ratio despite ART could be a predictor of increased immunoactivation and immunosenescence in vertically-HIV infected children and adolescents.

HIV-infected children and adolescents on stable ART were enrolled in a cross-sectional multicenter study. Exclusion criteria included acute or opportunistic infections and chronic inflammatory diseases. The study was approved by the Ethics Committee and all parents or legal guardians and children over 12 years gave written informed consent. Plasma viral load was measured using the Cobas TaqMan HIV-1 assay (Roche Diagnostics Systems, Inc., Branchburg, New Jersey, USA) with a detection limit of 50 copies/μl. CD4⁺ and CD8⁺ T-cell counts were determined using a Gallios flow cytometer and data analyzed using Kaluza software (Beckman Coulter, Fullerton, California, USA). T-cell activation was characterized by HLADR⁺CD38⁺ expression, senescence by CD57⁺ CD28⁻, and activation/exhaustion by HLADR⁺PD-1⁺, in CD4⁺ and CD8⁺ T-cells. T-cell subsets were defined as follows: naive (CD45RA⁺CD27⁺), central memory (CD45RO⁺CD27⁺) and effector memory (CD45RO⁺CD27⁻).

Patients were classified according to the presence of a normal (≥1) or an inverted (<1) CD4/CD8 ratio. Mann–Whitney tests were used for independent two-group comparisons and the Spearman correlation coefficient to analyze the correlation between continuous variables. Independent associations between CD4⁺ and CD8⁺ T-cells expressing markers of activation, senescence and activation/exhaustion (as dependent variables), with a limited subset of independent variables (including the CD4/CD8 ratio) were explored by a series of multivariate linear regression models. The crude correlation coefficients between the percentage of CD4⁺ and CD8⁺ T-cells with phenotypes of activation, senescence and activation/exhaustion and the CD4/CD8 ratio were calculated, followed by consecutive multivariate analyses adjusting by age, time on viral suppression, HIV-1 RNA less than 50 copies/μl, CD4⁺ T-cell nadir and accumulated ART exposure for all dependent variables. Statistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, Illinois, USA).

Thirty-seven vertically HIV-infected children and adolescents were included, with a mean age of 16.4 ± 3.8 years. Twenty-eight patients (75.7%) presented viral load less than 50 copies/μl. Median accumulated exposure to ART was 13.8 years (8.6–15.5), and mean time on viral suppression 4.8 years (1.8–7.3). Median CD4 nadir was 310 cells/μl, median CD4 cell count 783 cells/μl and median CD4/CD8 ratio 1.24. Sixteen (43.2%) patients showed CD4/CD8 ratio inversion (<1).

Main correlations between the CD4/CD8 ratio and clinical and immunological variables are shown in Fig. 1. The CD4/CD8 ratio positively correlated with CD4⁺ T-cell count, accumulated ART exposure and time on viral suppression. In contrast, the CD4/CD8 ratio inversely correlated with age and CD8⁺ T-cell count. No significant correlation was observed with the CD4⁺ T-cell nadir. The CD4/CD8 ratio negatively correlated with the percentages of activated and senescent CD4⁺ and CD8⁺ T-cells, and a strong negative correlation with T-cells expressing the activated/exhausted phenotype was observed.

Fig. 1: CD4/CD8 ratio relations with age, therapy, immunovirological variables, CD8⁺ T-cell activation, senescence and activation/exhaustion.The CD4/CD8 ratio was positively correlated with CD4⁺ T-cell count (r = 0.614, P < 0.001) (a), and inversely correlated with CD8⁺ T-cell count (r = −0.494, P = 0.002) (b) and age (r = −0.342, P = 0.038) (c). There was a positive correlation between CD4/CD8 ratio and cumulative antiretroviral therapy (ART) exposure (r = 0.339, P = 0.040) (d) and time with undetectable viral load (r = 0.457, P = 0.004) (e). No significant correlation was observed with the CD4⁺ T-cell nadir (r = 0.164, P = 0.332) (f). CD4/CD8 ratio was negatively correlated with the percentages of activated CD8⁺ T-cells (r = −0.414, P = 0.012) (g) and senescent CD8⁺ T-cells (r = −0.389, P = 0.017) (h). CD4/CD8 ratio strongly correlated with CD8⁺ T-cells expressing the activated/exhausted phenotype (r = −0.752, P < 0.001).

Compared with those with normal CD4/CD8 ratio, patients with an inverted CD4/CD8 ratio showed increased CD4⁺ and CD8⁺ T-cell activation (2.8 vs. 1.8% and 10.3 vs. 3.4%, respectively), senescence (0.2 vs. 0.04% and 19.6 vs. 11.0%, respectively) and activation/exhaustion (5.0 vs. 2.2% and 25.0 vs. 14%, respectively) (all comparisons P < 0.05, except for CD4⁺ T-cell senescence, P = 0.095). Regarding T-cell subpopulations, CD4/CD8 ratio inversion was associated with lower frequencies of naive CD4⁺ and CD8⁺ T-cells (54.0 vs. 61% and 31.5 vs. 48.6%, respectively) and increased effector memory CD8⁺ T-cells (10.2 vs. 5.2%) (all comparisons P < 0.05).

After consecutive linear regression models, adjusting by age, time on viral suppression, HIV-1 RNA less than 50 copies/μl, CD4⁺ T-cell nadir and accumulated ART exposure, the CD4/CD8 ratio remained independently associated with CD4⁺ T-cell activation (B = −1.1, P = 0.003), CD8⁺ T-cell activation (B = −6.1, P = 0.024), CD8⁺ T-cell senescence (B = −5.5, P = 0.016) and CD8⁺ T-cell activation/exhaustion (B = −15.5, P = 0.001), but not with CD4⁺ T-cell senescence (B = −0.786, P = 0.074) or CD4⁺ T-cell activation/exhaustion (B = −2.4, P = 0.343).

In our study in vertically HIV-infected children and adolescents, we found an independent inverse association between the CD4/CD8 ratio and the frequencies of T-cells expressing markers of activation, senescence and activation/exhaustion. Inversion of the CD4/CD8 ratio was also associated with lower levels of naïve CD4⁺ and CD8⁺ T-cells, supporting the hypothesis that an inverted CD4/CD8 ratio despite peripheral CD4⁺ T-cell restoration may reflect an underlying immunoactivation and immunosenescence.

In the general population, inversion of the CD4/CD8 ratio is considered a surrogate marker of the so-called immune risk profile ^[5], characterized by an increase in the number of CD8⁺CD28^- T-cells and associated with cytomegalovirus (CMV)-specific T-cells ^[2]. Moreover, the CD4/CD8 ratio has directly been associated with all-cause mortality in two European cohorts ^[4,6,7]. Many of these immunological alterations observed in the elderly are similar to those that take place in HIV-infected patients ^[8], such as expansion of CMV-specific T-cells ^[9], decreased levels of naïve CD8⁺ T-cells and increased memory CD8⁺ T-cells ^[8,10–12]. Thus, the elderly show lower CD4/CD8 ratio than younger patients ^[13], and on the contrary, children display higher CD4/CD8 ratio than adults ^[14]. This consideration must be kept in mind when interpreting our results given the wide range of age of our study population (6–23 years), and accordingly age was considered as a potential confounder in multivariate analyses.

Few studies have addressed the significance of the CD4/CD8 ratio in HIV infection. Before the introduction of the highly active ART in 1996, the CD4/CD8 ratio was identified as a predictor of disease progression ^[15]. More recently, a low CD4/CD8 ratio at initiation of ART has been associated with the prevalence and volume of coronary plaques ^[16]. We have previously reported an association between the CD4/CD8 ratio and immunoactivation in HIV-infected adults with long-term viral suppression ^[17]. Immunoactivation and immunosenescence are also associated with other surrogate markers of coronary events in HIV-infected patients, such as carotid intima-media thickness ^[18,19]. Of interest, we found increased subclinical atherosclerosis and immunoactivation in a study in vertically HIV-infected children and adolescents ^[20], and we have recently described in HIV-infected adults with long-term viral suppression that the CD4/CD8 ratio correlates with markers of age-associated disease ^[21]. Hence, if the CD4/CD8 ratio is further validated as a marker of the accelerated aging syndrome that characterizes treated HIV infection, HIV-infected children and adolescents failing to normalize the CD4/CD8 ratio despite ART should be carefully monitored for the prompt detection and early treatment of noninfectious disorders related to premature aging, as the effects of immunoactivation/immunosenescence on clinical outcomes will be probably stronger on vertically HIV-infected patients, whose immune system has developed in the presence of the virus since birth or even before. Also, the CD4/CD8 ratio might be of great interest for identifying the best candidates to be enrolled in clinical trials aiming to reduce immunoactivation. Importantly, as the CD4/CD8 ratio is available in clinical practice, its use to identify patients with higher levels of immunoactivation and consequently a higher risk of non-AIDS events might be easily implemented in clinical settings.

Our results demand a cautious interpretation given the small sample size and warrant confirmation in larger studies. Unfortunately, we were unable to explore the associations between the CD4/CD8 ratio, levels of bacterial translocation and the frequency of CMV-specific T-cells or CMV serostatus, which is a study limitation, as they could have helped explain the relationship between the CD4/CD8 ratio and immunosenescence in treated HIV infection.

In conclusion, our data suggest that a low CD4/CD8 ratio in vertically HIV-infected children and adolescents on ART is associated with increased levels of T-cells with markers of activation, senescence and activation/exhaustion, and a skewed T-cell phenotype from naïve toward effector memory. Thus, an inverted CD4/CD8 ratio may identify patients with ongoing immunoactivation despite ART, a finding that may have implications both in diagnostic and therapeutic settings.

Acknowledgements

The authors would like to particularly acknowledge all the children and adolescents as well as their families for their participation in this study. The authors also acknowledge the Spanish Paediatric HIV infection Group CORISPE for its collaboration.

Conflicts of interest

There are no conflicts of interest.

Author's contributions to the work: T.S., study design, data analysis, and writing. S.S.-V., study design, data analysis, and writing. L.D., laboratory tests. M.G.-T., recruitment, data collection and analysis Hospital Doce de Octubre. MD: Gurbindo: recruitment, data collection and analysis Hospital Gregrorio Marañón. M.I.deJ.: recruitment, data collection and analysis Hospital La Paz. M.M., recruitment, data collection and analysis Hospital Carlos III. J.R., recruitment, data collection and analysis Hospital de Getafe. J.Z.: study design, statistical analysis, S.M.: study design, data analysis and writing. M.A. M.-F. study design, data analysis and writing.

Funding: This work was partially supported by a Small Grant Award from the European Society of Pediatric Infectious Diseases (ESPID). T.S. and S.S-V. are funded by grants from the Spanish Ministry of Science and Innovation (Ayudas para Contratos de Formación en Investigación Río Hortega). L.D. is co-funded by the Spanish Ministry of Science and Innovation. The Paediatric HIV BioBank, integrated in the Spanish AIDS Research Network, is supported by the Instituto de Salud Carlos III, Spanish Health Ministry (Grant no. RD06/0006/0035) and the Fundación para la Investigación y Prevención del SIDA en España (FIPSE) (Grant no. 361910-10).

Summary: In a group of 37 vertically HIV-infected children, a low CD4/CD8 ratio was associated with increased levels of activated, senescent and activated/exhausted T-cells, and may be used as a marker of immunoactivation, both in diagnostic and therapeutic settings.