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Prolonged Antiretroviral Therapy in Adolescents With Vertical HIV Infection Leads to Different Cytokine Profiles Depending on Viremia Persistence

Munhoz, Luiz Gustavo Cano BSc, MSc*,†; Garcia Spina, Fernanda BSc, MSc*; Machado, Daisy Maria MD, PhD*; Gouvea, Aída MD, PhD*; Succi, Regina Célia De Menezes MD, PhD*; Diaz, Ricardo Sobhie MD, PhD; De Moraes-Pinto, Maria Isabel MD, PhD*

The Pediatric Infectious Disease Journal: November 2019 - Volume 38 - Issue 11 - p 1115–1120
doi: 10.1097/INF.0000000000002446
HIV Reports
Free
SDC

Background: We investigated immune activation, exhaustion markers and cytokine expression upon stimulation in adolescents with vertical HIV infection.

Methods: Thirty adolescents receiving antiretroviral therapy (ART) for vertical HIV infection, including 12 with detectable viral load (HIV/DET), 18 with undetectable viral load (HIV/UND) and 30 control adolescents without HIV infection (CONTROL), were evaluated for immune activation and programmed cell death protein-1 expression by flow cytometry, and 21 cytokines by Luminex Multiple Analyte Profiling technology after in vitro peripheral blood phytohemagglutinin stimulation.

Results: Lower CD4+ T cells and higher T cell activation and exhaustion markers were noted on CD4+ T and on CD8+ T cells and memory subsets from HIV/DET group, who also produced lower in vitro IFN-gamma, IL-10, IL-13, IL-17A, IL-5 and IL-6 than HIV/UND group. HIV/UND were comparable with CONTROL group in respect to CD4+ T cell counts and T cell activation and exhaustion markers, but with higher in vitro production of ITAC (a chemokine with leukocyte recruitment function), IL-4 and IL-23. An inverse correlation between cytokine production and programmed cell death protein-1 expression on CD4+ T and CD8+ T subsets was detected.

Conclusions: Persistent viremia despite ART leads to T cell activation and immune exhaustion with low cytokine production, whereas viral suppression by ART leads to parameters similar to CONTROL, although a different cytokine profile is observed, indicating residual HIV impact despite absence of detectable viremia.

From the *Division of Pediatric Infectious Diseases

Infectious Diseases Division, Department of Medicine, Federal University of São Paulo, São Paulo, Brazil.

Accepted for publication July 15, 2019.

Supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (2013/21853-1) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (148888/2016-0).

The authors have no conflicts of interest to disclose.

L.G.C.M., F.G.S., and M.I.M-P. conceived and designed the study. F.G.S. managed data collection. L.G.C.M. analyzed the data. L.G.C.M. and M.I.M-P. wrote the article. D.M.M., R.C.M.S., and A.G. collected data and discussed results. R.S.D. discussed results and provided support during article writing. All authors have read and approved the final article.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.pidj.com).

Address for correspondence: Maria Isabel de Moraes-Pinto, MD, PhD, Rua Pedro de Toledo, 781, 9° andar, São Paulo, SP-04039-032, Brazil. E-mail: m.isabelmp@uol.com.br.

Despite the increase in life expectancy provided by antiretroviral therapy (ART),1 ART immediately after birth was not available to individuals who were vertically infected nearly 2 decades. In the absence of ART, vertically HIV-infected children tend to maintain high viral loads and progress more rapidly towards AIDS than adults.2

Exposure to HIV without early treatment leads to a chronic infection with persistent immune activation and loss of function of effector cells, increase of inhibitory co-receptors expression and modification in the cytokine expression pattern.3 Programmed cell death protein-1 (PD-1) regulates inflammation and homeostasis, and its sustained expression is observed mainly in persistent chronic viral infections such as HIV.4 PD-1 is expressed on CD4+ T and CD8+ T cells when they are activated,5 interacting with different cell populations.6

Evaluation of children with HIV infection with respect to cytokine profile is scarce.7 Most studies usually do not separately consider groups on and not on ART, or those with undetectable or detectable viral load.7–9

We assessed adolescents and young adults with vertical HIV infection who, like many children born in the 1990s, had ART introduced late in the course of the disease. Immune activation and PD-1 expression were evaluated on CD4+ T and CD8+ T cell subsets and 21 cytokines were assessed in cell supernatant after in vitro stimulation.

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MATERIALS AND METHODS

Study Population

This is a cross-sectional study approved by Universidade Federal de São Paulo research ethics committee (#2.362.224). All patients or parents/guardians signed an informed consent prior to enrollment. Sixty adolescents and young adults were enrolled between June 2015 and June 2016 to the following groups: youth with vertical HIV infection receiving ART with detectable viral load (HIV/DET, n = 12) or with undetectable viral load (HIV/UND, n = 18) and youth without HIV infection (CONTROL, n = 30). Youth with HIV infection had to be in regular follow-up at the Pediatric AIDS Clinic, receiving ART and with CD4+ T cells above 200/mm3. Exclusion criteria for all participants were pregnancy and any type of infection present 15 days before study entry. CONTROL individuals were not using any immunosuppressive drug and were free of any chronic, autoimmune, neoplastic or allergic disease.

An 8-mL peripheral blood sample was collected from each participant. HIV RNA was quantified on plasma with HIV-1 06L18 kit at m2000 RealTime System (Abbott, Des Plaines, IL).

HIV/UND individuals were those who maintained viral load below 40 copies/mL for prolonged periods (median duration = 8.6 years) and had a maximum of 4 nonconsecutive viral blips. A viral blip was defined as a detectable viral load episode in low levels, with an undetectable viral load immediately before and after the blip episode.

CONTROL group individuals underwent standard HIV serologic testing before study entry using Determine HIV-1/2 kit rapid test (Abbott, Tokyo, Japan).

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Immune Phenotyping, Immune Activation and Exhaustion

Whole blood was stained with fluorescent-conjugated antibodies against CD3, CD4, CD8, CD38, HLA-DR, PD-1, CCR7 and CD45RA (BD). Immune activation was defined by expression of both HLA-DR and CD38 markers and exhaustion was determined by PD-1 expression on CD4+ T and CD8+ T cells. PD-1 expression was also analyzed on naïve (CCR7+CD45RA+), central memory (CCR7+CD45RA), effector memory (CCR7-CD45RA) and effector (CCR7CD45RA+) cells (Table, Supplemental Digital Content 1, http://links.lww.com/INF/D580, and Figures, Supplemental digital content 2–4, http://links.lww.com/INF/D581, http://links.lww.com/INF/D582, http://links.lww.com/INF/D583).

Samples were run on LSR Fortessa Flow Cytometer (BD, San Jose, CA) using FACSDiva software (BD, San Jose, CA) and analyzed on FlowJo software (TreeStar, Ashland, OR).

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Blood Culture and Cell Stimulation

Heparin whole blood was diluted 1 in 10 in supplemented RPMI 1640 medium (Gibco, New York, NY). To one 1-mL aliquot, 5 μg/mL of phytohemagglutinin (Gibco) were added and to the other aliquot no stimulus was added. The plate was placed at 5% CO2 and 37°C for 7 days. Cell supernatant was stored at −80°C.

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Cytokine Quantification in Cell Culture Supernatant

Cytokine quantification was performed by Luminex Multiple Analyte Profiling technology technique in supernatant of whole blood samples stimulated in vitro with phytohemagglutinin10 using MILLIPLEX MAP HSTCMAG28SPX21 (Merck Millipore, Darmstadt, Germany) kit and performed in Luminex Multiplex MagPix plate reader (Luminex, Austin, TX).

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Statistical Analysis

Quantitative variables were evaluated by Mann-Whitney and Kruskal-Wallis tests; categorical variables were tested with χ2 test. Spearman’s rank correlation was employed to investigate association between variables. Statistical significance was set at P < 0.05. All analyses were performed using GraphPad Prism software (La Jolla, CA).

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RESULTS

Demographic characteristics were similar in the 3 groups and there were no significant differences in clinical characteristics between the HIV/DET and HIV/UND groups (Table 1).

TABLE 1

TABLE 1

CD4+ T cell counts were lowest in the HIV/DET group, which also had a trend to higher immune activation. The HIV/UND group was comparable with CONTROL group in respect to CD4+ T cell counts as well as expression of activation and exhaustion markers on all cell maturation subsets (Table 2).

TABLE 2

TABLE 2

The HIV/DET group had lower naïve, central memory and higher effector memory CD8+ T cell percentages when compared with CONTROL. The HIV/UND group had higher effector memory CD4+ T percentages and lower naïve CD8+ T percentages when compared with CONTROL (Table, Supplemental Digital Content 5, http://links.lww.com/INF/D584).

A higher mean fluorescence intensity of PD-1 on CD4+ T cells and a higher percentage of cells expressing this marker were observed in the HIV/DET group, who also had a higher percentage of central memory and effector memory cells expressing PD-1 when compared with HIV/UND and CONTROL groups.

CD8+ T cell counts were comparable among the 3 groups. However, a higher expression of activation markers and expression of PD-1, both in cell percentage and mean fluorescence intensity, was observed in HIV/DET when compared with HIV/UND and CONTROL groups. When maturation subsets were analyzed with respect to PD-1 expression, a higher expression was observed in effector memory subset of HIV/DET than the other groups (Table 2).

The HIV/DET group produced less IFN-gamma, IL-10, IL-13, IL-17A, IL-5 and IL-6 than the HIV/UND group and less IL-10, IL-17A and IL-6 than the CONTROL group. In contrast, the HIV/UND group had higher levels of ITAC, IL-4 and IL-23 than the CONTROL group (Table 3 and Figure 1).

TABLE 3

TABLE 3

Figure 1

Figure 1

In the HIV/DET group, there was a positive correlation for activation markers and PD-1 expression on CD8+ T cells (Table, Supplemental Digital Content 6, http://links.lww.com/INF/D585) and an inverse correlation between expression of PD-1 on naïve CD8+ T cells and production of ITAC, IFN-gamma, IL-10, IL-12, IL-13, IL-2, IL-21, IL-4, IL-23, IL-5, MIP-1B and TNF-alpha (Table, Supplemental Digital Content 7, http://links.lww.com/INF/D586). The expression of PD-1 on central memory CD4+ T cells was also inversely correlated with IL-10, IL-1B, IL-2, IL-21, IL-23, IL-6 and IL-8. For CD8+ T cells, the inverse correlation was significant for ITAC, Fractalkine, IFN-gamma, MIP-3a IL-12, IL-2, IL-21, IL-23, IL-5, IL-7, IL-8, MIP-1B and TNF-alpha (Table, Supplemental Digital Content 8, http://links.lww.com/INF/D587).

In HIV/UND group, an inverse correlation was observed between GM-CSF, IFN-gamma, IL-10, IL-12, IL-17A, IL-1B, IL-23, IL-6 and IL-8 levels and PD-1 expression on central memory CD8+ T cells (Table, Supplemental Digital Content 8, http://links.lww.com/INF/D587).

No significant correlations were observed between cytokine concentrations and PD-1 expression on effector memory CD4+ or CD8+ T cells (Table, Supplemental Digital Content 9, http://links.lww.com/INF/D588) or on effector CD4+ or CD8+ T cells (Table, Supplemental Digital Content 10, http://links.lww.com/INF/D589). Immune activation showed an inverse correlation with some cytokines for HIV/DET group, without a specific pattern (Table, Supplemental Digital Content 11, http://links.lww.com/INF/D590). The duration of undetectable viral load was not associated with CD4+ or CD8+ T cell counts, activation, exhaustion markers or in vitro cytokine production (Table, Supplemental Digital Content 12, http://links.lww.com/INF/D591).

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DISCUSSION

Adolescents and young adults with vertical HIV infection who are receiving ART but have detectable plasma viremia have lower CD4+ counts, higher activation and expression of PD-1 on CD4+ T and CD8+ T cells, and a different pattern of cytokine expression when compared with individuals with undetectable viral load and the control group.

Both HIV/DET and HIV/UND groups presented altered maturation subset percentages. The HIV/DET group had lower percentages of naïve and central memory, but higher effector memory subsets than CONTROL. HIV/UND maintained lower naïve and higher CD8 T effector memory subset percentages than CONTROL but lower than HIV/DET. Those findings suggest that adverse consequences of HIV infection persist despite absence of detectable viremia.11

HIV/DET group showed a lower production of cytokines when compared with HIV/UND and CONTROL groups. Despite the greater similarity of HIV/UND with CONTROL group, a differentiated pattern of cytokine expression persisted in the HIV/UND group.

The persistence of detectable HIV viral load was associated with higher expression of activation markers on CD4+ T cells, but especially on CD8+ T cells, as previously described.12,13 Some studies failed to associate higher immune activation of CD4+ T cells in individuals with detectable viral load compared with aviremic ones.14 It is therefore conceivable that duration of undetectability might account for part of those differences.

Unlike from other pediatric studies in which vertical and horizontal transmission were analyzed together,13,14 our study evaluated exclusively adolescents and young adults infected through vertical transmission who had a reliable record of duration of ART and of undetectable viral load. Adolescents infected horizontally were not included because they are usually older when infection takes place,15 with a more mature immune system and the duration of viral exposure is smaller when compared with an adolescent with vertical HIV infection.

The magnitude of viral replication in infants infected by HIV has already been demonstrated16 as well as its correlation with immune activation.11 Moreover, the decay of expression of activation markers in CD8+ T cells in children who show a good viral response to ART has already been described.17 Despite full access to ART, some of our patients had detectable viral load, which can be explained by adherence problems in the majority of cases. Adherence difficulties are well documented in adolescence due to several issues, such as acceptance by partners and drug side effects.18

A previous study of children with HIV infection showed that immune activation of CD4+ T and CD8+ T cells decreased and reached control group’s levels after ART introduction.19 In our study, the median duration of ART for all 30 youth with HIV infection was 15.7 years. The group with undetectable viral loads had T cell activation markers similar to those in the control group. However, those with detectable viral loads maintained high CD4+ T and CD8+ T cells activation.

Previous studies have established that immune activation in chronic HIV infection can lead to immune exhaustion.5 Apart from exhaustion markers being expressed more often among HIV-infected individuals when compared with healthy ones, the pattern of exhaustion marker expression on CD4+ T cell subsets among infected individuals is also unique.20

We observed higher expression of PD-1 on CD4+ T and CD8+ T cells in HIV/DET than CONTROL group, mainly on memory subsets. A previous study suggested that PD-1 expression on central memory and effector memory CD4+ T cells is associated with HIV infection progression.7 However, it is not clear whether PD-1 expression is evidence of immune exhaustion or only an indication recently activated cells.7

We also observed a widespread decrease of cytokine production by peripheral blood cells in youth with HIV infection who had detectable viral load despite ART. A shift from a TH1 to a TH2 cytokine expression in HIV infection was identified.21 The low IL-10 expression has already been associated with depletion of regulatory T cells mainly in the gut-associated lymphoid tissues. HIV infection also leads to destruction of TH17 cells in the gut-associated lymphoid tissues, with low IL-17A expression, increased immune T cell activation, injury to the gut barrier and microbial translocation22

The depletion of memory CD4+ T cells due to HIV infection may lead to a reduced expression of different cytokines. The loss of central memory subset can affect IL-2 production, as well as the loss of effector memory subset can affect IFN-gamma production.22 However, the low levels of Th1, Th2, Th17 and even of regulatory cytokine expression suggest that this reduction might be due to exhaustion of T cells.23 That is in line with the negative association between PD-1 expression on naive and central memory CD8+ T cell subsets and cytokine expression.

In contrast, individuals who had undetectable viral loads showed higher levels of ITAC, IL-4 e IL-23 cytokines than controls, suggesting that even very low viral load levels can alter the dynamics of the immune system.

The HIV/UND group produced IL-17A levels similar to control group and much higher than levels observed in HIV/DET. This could be explained by the high IL-23 levels. Fernandes et al24 demonstrated that increased IL-23 levels could represent an attempt of the immune system to restore homeostasis, since it has a primordial role in the induction of IL-17A expression.

ITAC is a chemokine that attracts cells of immune system and induces certain leukocytes to express cytokines. ITAC production was demonstrated25 in individuals infected by herpes simplex, and the ITAC-CXCR3 interaction might be associated with an increase in both IFN-gamma and IL-10 production by CD4+ T and CD8+ T lymphocytes. Interestingly, the HIV/UND group had IFN-gamma and IL-10 levels similar to those in CONTROL and higher than those in HIV/DET group.

A change in pattern of cytokine production in detectable viral load individuals who start ART was previously shown9; however, undetectable viral load must be achieved and sustained to maximize and maintain this effect. Despite this, immunologic differences still persist when HIV/UND individuals are compared with CONTROL group.

Previous studies demonstrated a correlation between the period of ART use and CD4+ T cell count increase16 and decrease of immune activation.19 Perhaps the absence of correlation between the undetectable period and the parameters analyzed was noted in our study because these patients have maintained undetectable viral loads for a median period of time of 8.6 years, a much longer period than those from other studies.14

Nonetheless, clear differences still remain between adolescents with vertical HIV infection and healthy controls. A hereditary factor of CD8+ T cells exhaustion might explain exhaustion of T cells despite viral control. This exhaustion would be transmitted to new generations of T cells, which would cause damage that could not be reversed by ART.26

Despite the small sample size, our cohort of adolescents with vertical HIV infection has been monitored for almost 2 decades, with constant and properly documented clinical and laboratorial follow-up, which makes the obtained results very robust.

A characteristic cytokine expression pattern was observed in HIV/UND group, reinforcing that HIV chronic infection, even with proper ART, can interfere with the immune system. This effect may occur because viral proteins can still be produced by latent infected cells with defective viruses.27

Studies are necessary to evaluate the influence of these markers on the immune response of these individuals and how an intervention at determinate checkpoints could be beneficial, such as PD-1 blockade and gene therapy to slow down T cell exhaustion in individuals who present such alterations.

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ACKNOWLEDGMENTS

We thank all the adolescents and young adults who agreed to participate in the study.

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Keywords:

vertical HIV infection; immune activation; immune exhaustion; cytokine; PD-1; adolescents

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