Further Evidence That the Expression of CD38 and HLA-DR + in CD8 + Lymphocytes Does Not Correlate to Disease Progression in HIV-1 Vertically Infected Children

Abstract

Background: In adults, an increase in CD8⁺CD38⁺ T cell levels is a strong indicator of disease progression in HIV infection. However, in children, data are conflicting. Slow-progressing children (SPC) provide an exceptional resource for the investigation and clarification of the immunological and virological mechanisms of natural control of HIV infection and can be used to investigate prognostic indicators of disease progression. Objectives: To investigate the immune activation status and T regulatory (Treg) cell levels in SPC. Study design: A cross-sectional study was carried out on 28 children 8 years old and older who were vertically infected with HIV. The children were stratified into 3 groups according to their clinical outcome: SPC (anti-retroviral-therapy-naïve; ≥8 years-old; CD4 ≥20%; viral load <25,000 copies), IF/VF (anti-retroviral-therapy but with no therapeutic response), and IS/VS (anti-retroviral therapy with good therapeutic response). Uninfected children (NI) were assessed as healthy control group. Results: A higher percentage of activated CD8⁺ T cells were found in all HIV infected children, regardless of the evolution of disease. The activation of CD8⁺ T cells was not associated with either viral load or the percentage of CD4⁺ T cells. In addition, Treg cell levels did not show any correlation with the clinical outcome or the activation status of CD8⁺ T cells. Conclusions: HIV-1-infected children presented an increased percentage of activated CD8⁺ T cells and an unaltered percentage of Treg cells, regardless of their clinical evolution. Thus, these immunological parameters should not be used for prognostic evaluation.

Keywords

HIV slow-progressing children CD38 Treg HLA-DR

Background

Slow-progressing children (SPC) provide an exceptional resource for the investigation and clarification of the immunological and virological characteristics of HIV infection. In addition, they can aid in clarifying the underlying mechanisms of natural infection control and can be used to investigate prognostic indicators of disease progression.¹

CD8⁺ T lymphocytes, which have a role in the in vitro control of HIV replication, are known to influence the process that underlies slow progression.² HIV infection induces lymphocyte activation, which results in the increased expression of surface T lymphocyte activation markers such as CD38 and HLA-DR.^3,4 In adults, an increase in CD8⁺CD38⁺T cell levels is a strong indicator of disease progression.³ However, in children, increased CD8⁺CD38⁺T cell levels has been described as a survival indicator by some authors and has been associated with advanced stages of infection by others.^5,6 Likewise, in some studies, the increase in HLA-DR expression in CD8⁺CD38⁺T cells has been proposed as an HIV-1 infection progression marker in both adults and children.⁷

It is still not clear how the immune system responds to this hyperactivation state. As explored in a review by Terzieva, several recent studies have defined regulatory T cells (Treg cells) as a key population in the regulation of immune activation during HIV-1 infection.⁸ Initially the Treg cells were characterized as CD4CD25⁺, but lately it was demonstrated that their regulatory function depends on the expression of the transcription factor FoxP3. However, longitudinal studies of Treg cell frequencies during the course of HIV-1 infection have provided divergent results.^9

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Objectives

The aim of this study was to assess the relationship between immune activation, Treg cells, and the infection progression profile of children vertically infected with HIV-1.

Study Design

All works were approved by the Ethical Committee of Universidade Federal de Minas Gerais (COEP). A cross-sectional study was performed on 28 children vertically infected with HIV stratified into 3 groups according to their clinical outcome: SPC (n = 9, age >8 years old, antiviral-treatment naive, CD4 ≥ 20% and/or ≥ 500 cells/mm³; viral load <25 000 copies/mL), immunological failure/virological failure ([IF/VF]; n = 10, age >6 years old, children on highly active antiretroviral therapy (HAART) but with IF and VF) and immunological success/virological success ([IS/VS]; n = 9; age >6 years old, children on HAART but with IS and VS). Noninfected children ([NI] n = 9) were also studied as healthy controls. Viral load was measured by b-DNA assay, and T cell subsets were evaluated by 3-color flow cytometry. Cytofluorimetric data acquisition was performed with a FacsCalibur instrument (BD Biosciences, Mountain View, California). CELLQUEST TM software provided by the manufacturer was used for data acquisition and analysis. Statistical analysis was performed using Prism 5.01. As all data sets were nonparametric in nature, statistical analysis was performed using Kruskal-Wallis variance analysis followed by Dunn test for comparison between groups. Spearman’s tests were used for correlation. Significant differences at P < .05 are identified in the figure and in the table.

Results

The immunological and clinical characteristics of all patients are shown in Table 1. As shown in Figure 1A, the infected children presented significantly higher percentages of CD8⁺CD38⁺ cells in the peripheral blood when compared to the NI, regardless of the disease evolution profile. The infected children also presented significantly higher percentages of CD8⁺HLA-DR⁺ cells in the population of CD8⁺ lymphocytes, as shown in Figure 1B. Among the groups of infected patients, no relevant difference was observed in relation to either marker used above. In addition, we examined whether there was any correlation between CD8⁺CD38⁺, CD8⁺HLA-DR⁺ cells, and the percentage of CD4 count. As shown in Figure 1C and D, no significant correlation was observed between the percentages of CD4 count and activated CD8⁺ T lymphocytes in any of the assessed infected patient groups.

Figure 1.

A and B, Percentage of CD8+ T cells expressing CD38 (A) or HLA-DR+ (B) in noninfected children (NI), slow progressing children (SPC), children on highly active antiretroviral therapy (HAART) with immunological and virological success (IS and VS) and children on HAART with immunological and virological failure (IF and VF). Significant differences at P < .05 are identified by letters a, b, and c when compared NI with SPC, IS/VS, and IF/VF, respectively. C and D, Correlation between the percentage of CD4 lymphocyte and (C) CD8+CD38+ T cells (SPC [□] r = .285, P = .457; IF/VF [•] r = .289, P = .417; IS/VS [▴] r = .475, P = .197; NI [◇] r = .252, P = .455) and (D) CD8+HLA-DR+ T cells (SPC [□] r = .327, P = .145, IF/VF [•] r = .429, P = .216, IS/VS [▴] r = .375, P = .105, NI [◇] r = .186, P = .585). E and F, Correlation between the percentage of CD4CD25high lymphocytes and the percentage of (E) CD8+CD38+ T cells (SPC [□], P = .527; IF/VF [•] r = .381, P = .277; IS/VS [▴] r = .321, P = .399; NI (◇) r = .413, P = .236) and (F) CD8+HLA-DR+ T cells (SPC [□] r = .429, P = .07; IF/VF [•] r = .159, P = .660; IS/VS [▴] r = 0.312, P = .414; NI [◇] r = .056, P = .878).

Table 1.

Demographic, Clinical, Immunological, and Virological Characteristics of the Children Included in This Study

	Noninfected Group	HIV-Infected Children
	NI	SPC	IF/VF	IS/VS
n	11	9	10	9
Median age, years	9.6 ± 2.5	10.9 ± 2.7	12.4 ± 2.8	8.4 ± 2.3
Sex, male/female	5/6	5/4	8/2	4/5
CDC classification
N	N/A	0	0	0
A	N/A	7	0	1
B	N/A	2	2	1
C	N/A	0	8	7
Immunological profile
Median CD4, % (IQR 25%-75%)	37.33 (34.29-42.87)	30.00 (23.63-34.69)	15.69^a,^b (4.24-26.81)	33.24 (29.62-38.69)
Median CD4 count, cells/mm³ (IQR 25%-75%)	898 (834-1224)	910 (365-1102)	303^a-c (149-427)	777 (584-1131)
Median CD8, % (IQR 25%-75%)	24.90 (21.96-29.21)	39.79^a,^b (38.56-48,36)	47.79^a,^b (42.09-60,01)	33.57 (29.13-35.73)
Median CD8 count, cells/mm³ (IQR 25%-75%)	674 (555-760)	1177 (836-1446)	966 (568-1372)	681 (584-1131)
Virological profile
Viral load <50 copies/mL (%)	N/A	1 (11%)	0	4 (44%)
Median plasma viral load, log (IQR 25%-75%)	N/A	4.14 (3.86-4.37)	4.40 (4.03-4.56)	2.23^d (1.90-2.23)
Antiretroviral therapy
Antiretroviral therapy naive	N/A	9	0	0
On treated	N/A	0	10	9

Abbreviations: NI, noninfected children; SPC, slow-progressing children; HAART, highly active antiretroviral therapy; IF/VF, children on HAART with immunological and virological failure; IS/VS, children on HAART with immunological and virological success; IQR: interquartile range; N/A: not applicable.

^a Significant differences between NI (P < .05).

^b Significant differences between IS/VS (P < 05).

^c Significant differences between SPC (P < .05).

^d Significant differences between IF/VF (P < .05).

Next, we assessed whether the activation of CD8⁺ T cells was associated with virological evolution in HIV-infected children by examining the possible correlation of CD8⁺CD38⁺ and CD8⁺HLA-DR⁺ cells with viral load. In our patients, no significant correlation between the viral load and CD8⁺CD38⁺ and CD8⁺HLA-DR⁺ lymphocyte percentages was observed in any of the groups of infected patients assessed (data not shown).

Our results also showed that the percentage of CD4CD25^high cells, a potential T regulatory cell subset, was similar in all groups of children (data not shown). In order to verify whether Treg cells were associated with immunological activation in HIV-infected children, we assessed the correlation between CD4CD25^high cells and CD8⁺CD38⁺ and CD8⁺HLA-DR⁺ lymphocytes. As shown in Figure 1E and F, no significant correlation between the percentage values of CD4CD25^high cells and the percentage values of CD8⁺CD38⁺ and CD8⁺HLA-DR⁺ lymphocytes was observed in any infected patient group.

Discussion

HIV infection is known to cause significant immunological impairment, including the increased expression of activation markers, such as CD38 and HLA-DR on CD8⁺ T lymphocytes, and this increased expression has been associated with particular clinical outcomes.^4,14,15 The T cell activation has been related to the immune system’s attempt to control viral replication in HIV-1-infected children.¹⁶ But it has also been proposed as one of the main mechanisms responsible for the depletion of CD4 count and the subsequent development of the disease.^16,17

In our study, high activation of CD8⁺ T cells was found in all groups of HIV-1-infected children, regardless of their profile of clinical evolution. In fact, we did not observe any association between CD8⁺ T cell activation and CD4 count percentage or viral load. This high expression of activation marker can be explained by immune repopulation that is known to occur in children despite persistent viremia.¹⁸ It has been reported that CD38 is expressed early in hematopoietic cells, with downregulation during the cell maturation process and reexpression upon cell activation. This cyclic nature of CD38 expression during lymphopoietic ontogeny may explain the presence of high expression of CD38 in HIV-infected children.

Another explanation for these divergent results can be attributed to the differences among the characteristics of studied patients and differences in the stage of HIV infection at which these cells were examined. In many other studies, the classification of children with slow progression includes children under a treatment either with or without protease inhibitors.^17,19 In the present study, we only assessed in SPC group, children that were therapy naive, and thus free of treatment pressure.

Considering the important regulatory role of Treg cells in the immune system, it is possible that this cell population also influences the immunopathology of HIV-1 chronic infection.^8,9 Longitudinal studies on the frequencies of Treg cells in the adult HIV-1-infected population have provided divergent results. Eggena et al observed that Treg depletion was strongly associated with the activation and depletion of CD4 count. They suggested that Treg depletion contributes to the chronic immune activation observed in HIV infection during disease progression, corroborating data from the other studies.^9,10 Thus, the loss of immunoregulatory mechanisms in the more serious clinical forms of disease would contribute to immune response unbalance, triggering powerful inflammatory activity and the subsequent greater reduction in CD4 count. Nevertheless, Cao et al verified that Treg proportions are high during the course of HIV-1 infection in adults and that the magnitude of this rise is associated with the progression of the disease.²⁰ Recently, Freguja and colleagues reported an elegant study that evaluated the relationship between viral load, immune activation, and Treg cells in HIV-1-infected children. The study showed a strong relationship between viral load, immune activation, and expansion of Tregs, suggesting that viral load induces a rise in immune activation that leads to an expansion of Tregs.²¹ Contrary to these findings, in our study, we did not observe any correlation between Treg cells and the status of the immunological activation of CD8⁺ T cells in infected children in any analyzed groups. It is possible that these divergent results may be partially attributed to the differences in the definitions of Treg cells, since we did not evaluate FoxP3 expression.

In conclusion, based on our data, the percentage of activated T cells and the percentage of CD4CD25^high cells may not be a good prognostic biomarker in HIV-infected children, since in our population, children with distinguished clinical outcome present similar profiles.

Footnotes

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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