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).
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.
We thank all the adolescents and young adults who agreed to participate in the study.
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vertical HIV infection; immune activation; immune exhaustion; cytokine; PD-1; adolescents
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