T-cell homeostasis alteration in HIV-1 infected subjects with low CD4 T-cell count despite undetectable virus load during HAART
Marziali, Marcoa; De Santis, Wladimirob; Carello, Rossellab; Leti, Wilmab; Esposito, Antonellac; Isgrò, Antonellaa; Fimiani, Caterinac; Sirianni, Maria Ca; Mezzaroma, Ivanoa; Aiuti, Fernandoa
From the aDepartment of Clinical Medicine, University of Rome “La Sapienza”, Italy
bDoctorate School of Research in Science of Immunotherapy, University of Rome “La Sapienza”, Italy
cSchool of Specialisation in Allergy and Clinical Immunology, University of Rome “La Sapienza”, Italy.
Received 18 May, 2006
Accepted 10 August, 2006
Correspondence to F. Aiuti, Department of Clinical Medicine, University of Rome “La Sapienza”, Viale dell'Università 37, 00185 Rome, Italy. Tel: +39 0649972017; fax:+39 064466209; e-mail: firstname.lastname@example.org
Objective: To investigate the pathogenesis of low CD4 T-cell count in subjects who are immunological non responders (InR) to HAART.
Design: Thirty-five HIV-positive subjects on HAART for at least 1 year, all with undetectable HIV-1 RNA, were studied. Patients were defined as InR according to a CD4 cell increase < 20% from CD4 cell baseline or CD4 cell count < 200/μl; subjects with a CD4 T-cell increase > 20% from baseline and a CD4 cell count > 200/μl were defined as immunological responders (IR). We performed a comprehensive study to characterize the immune response of InR.
Methods: The immunological phenotype of peripheral blood mononuclear cells, thymic naive T cells, T-cell receptor Vβ repertoire, serum concentration of interleukin (IL)-7, the expression of IL-7Rα on naive and memory CD4 and CD8 T cells, and regulatory T cells (Treg) were studied.
Results: In InR a significant reduction (P < 0.0001) of naive and thymic naive CD4 T cells was associated with a reduced expression of IL-7Rα in both cell subsets, with an increased serum concentration of IL-7 was observed. Furthermore, an increased immune activation with a reduced Treg frequency and increased number of expansions of Vβ families was observed.
Conclusions: The reduced expression of IL-7Rα associated with the persistent immune activation and the alteration of Treg frequencies in part explains the low level of CD4 T cells observed in InR.
After the introduction of HAART most patients who adhere to treatment show a good response, defined by a decrease of plasma viral load (pVL) to undetectable levels and an immunological reconstitution with a significant increase of CD4 T cell levels from baseline values .
However, two ‘paradoxical responses’ may occur during the course of antiviral treatment. The first is characterized by a CD4 T cell count increase despite a persistently detectable pVL. Such patients are defined as virological non-responders/immunological responders. This may be caused by the selection of mutant viruses with low fitness induced by HAART, or to a modest benefit from antiviral drugs, which causes only a partial reduction of pVL [2–4]. Other investigators have suggested that protease inhibitors may act on the immune system independently from their antiretroviral effects [5–7].
The second type of ‘paradoxical response’ occurs when the CD4 T-cell count does not increase despite a full suppression of viral replication: this condition includes virological responder/immunological non-responder individuals, briefly defined as immunological non-responders (InR) [8–10]. The reasons for this variability remain unclear. Immunological, virological and host-related factors may play an important role in the reconstitution of the immune system . Moreover, there is no agreement on the definition of sufficient immune response. In general an increase of up to 20% of the CD4 T-cell count from baseline during the first 6 months of HAART defines a good immunological response. The value of 200 CD4 T cells/μl is considered a critical threshold under which the immune reconstitution fails, and this occurs in 5–27% of patients receiving HAART [9,12]. In fact, CD4 T-cell count persistently < 250 cells/μl or a percentage of CD4 T cells < 17, has been considered a sign of poor immune reconstitution [13,14].
In InR subjects an impairment of the naive T-cell compartment has been described [13,15]. Inbalance of several cytokines such as interleukin (IL)-7 and abnormalities of the IL-7 receptor have also been implicated in CD4 T-cell depletion [16–24]. In addition, many studies have shown that chronic immune activation plays a central role in determining the CD4 T cell decrease [25–27] and during recent years the role of regulatory T cells (Treg) in modulating the immune response has gained importance . Immune responses are strictly regulated by Treg , and their deficiencies might play a role in the pathogenesis of chronic immune activation in HIV disease.
The aim of this study was to investigate the pathogenesis of low CD4 T-cell count in InR subjects. We evaluated the immunological phenotype of peripheral blood mononuclear cells (PBMC), thymic naive T cells, T-cell receptor (TCR) Vβ repertoire, serum concentration of IL-7, the expression of IL-7R (CD127) on naive and memory CD4 and CD8 T cells as well as Treg.
In the literature, some of these parameters have been the subject of specific studies in InR patients [13,15,30] but, to our knowledge, this is the first study to evaluate all of them together in the same group of patients.
Materials and methods
Two groups of chronically HIV-infected patients treated with HAART for at least 1 year were studied. All had achieved an undetectable plasma HIV RNA (< 50 copies/ml). The first group included all subjects who fulfilled the definition of InR, with CD4 T-cell increases < 20% from baseline and/or CD4 T-cell counts < 200/μl, and who began HAART between January 2002 and April 2005. They were 15 subjects from a pool of 186 patients who started antiretroviral therapy during that period. The second group consisted of 20 immunological responder subjects (IR) who showed a CD4 T-cell increase > 20% from baseline values and a CD4 T-cell count > 200/μl. They were selected from the same pool of subjects and were matched for baseline CD4 values. Fifteen normal HIV-1 negative blood donors were studied as controls. All patients and controls gave a written informed consent to the blood sampling for the study.
Flow cytometric analysis of CD4 and CD8 T-cell subsets and TCRBV repertoire
Two millilitres of whole blood were lysed using 40 ml Ortho lysing reagent (Ortho-Clinical Diagnostics, Raritan, New Jersey, USA) and were washed, labelled with a cocktail of four monoclonal antibodies per tube for 30 min at 4°C within 1 h of blood collection. Naive and memory T cells were investigated by evaluating the differential expression of CD45RA and CD62L on both CD4 and CD8 T cells. Thymic naive CD4 T cells were evaluated by the coexpression of CD45RA and CD31 . Treg were identified by CD4+CD25high, by CD4+CD25+CD62L+ and by CD4+CD25+CD27+ expression [28,32]. Allophycocyanin-conjugated anti-CD4, peridinin chlorophyll protein-conjugated anti-CD8, fluorescein isothiocyanate (FITC)-conjugated anti-CD45RA, phycoerythrin (PE)-conjugated anti-CD62L, PE-conjugated anti-CD31, PE-conjugated anti-CD127(IL-7Rα), FITC-conjugated anti-CD25, PE-conjugated anti-CD27, FITC-conjugated anti-HLA-DR, FITC-conjugated anti-CD95, PE-conjugated anti-CCR5 and PE-conjugated anti-CXCR4 were from Becton Dickinson (B-D Immunocytometry Systems, San Jose, California, USA). Anti-TCRBV antibodies were from IOTest Beta Mark (Immunotech, Marseille, France). All assays were performed according to manufacturers' instructions. After staining, cells were washed once in phosphate-buffered saline containing 2% foetal bovine serum, and were analysed on a FACSCalibur (B-D Immunocytometry Systems) using Cell Quest software. To determine marker expression on CD4 and CD8 cells, total lymphocytes were first identified and gated by forward and side scatter. The cells were then additionally gated for CD4 or CD8 expression. TCRBV gene segment denomination is in accordance with that of Wei et al. . The normal distribution of TCRBV families was obtained from 40 healthy subjects aged between 25–65 years.
Determination of viral load
Plasma HIV RNA levels were determined by reverse transcription-PCR (Amplicor Kit, Roche Molecular Systems, Branchburg, New Jersey, USA) and the threshold was 1.69 log10 HIV RNA copies/ml.
CD4 and CD8 T cells were separated by using CD4 and CD8 MicroBeads and MACS columns according to manufacturers' protocols (Miltenyi Biotec, Bergisch Gladbach, Germany). Total mRNA was extracted directly from 1 × 106 bead-coated cells using Trizol-LS Reagent (Gibco BRL, Grand Island, New York, USA) and Micro-carrier (Molecular Research Center, Cincinnati, Ohio, USA) and precipitated with isopropyl alcohol. The pelleted RNA was resuspended in diethyl-pyrocarbonate-treated water and the poly-(A)+ portion of total RNA was converted into cDNA using 2.5 μM oligo(dT) as primer for reverse transcription, 50 mM KCl, 10 mM Tris/HCl, 5 mM MgCl2, 1 mM each dNTP, 1 U/μl RNase Inhibitor, and 2.5 U/μl MULV reverse transcriptase (Applied Biosystems, Foster City, California, USA).
To analyse the TCRBV transcript size patterns cDNA samples were amplified by using a TCR[beta] C1/C2-specific primer. The third complementarity-determining region (CDR3) profile was then analysed with the Genescan software (Applied Biosystems). Analysis of the level of perturbation of TCRBV repertoire of HIV-infected patients was performed as previously described [34–37].
IL-7 serum concentration
IL-7 serum concentration was evaluated by standard ELISA assay purchased from R&D Systems (Minneapolis, Minnesota, USA). The assay was carried out according to the manufacturer's instructions.
Statistical analysis was performed by using StatView software version 5.0 (SAS Institute, Inc., Cary, North Carolina, USA). All data are expressed as mean values ± SD.
For the comparison between groups we used the Mann–Whitney U test; moreover all data were tested for regression analysis (ANOVA). P values < 0.05 were considered statistically significant.
Baseline characteristics of patients
As summarized in Table 1, no significant differences were observed at baseline between the two patient groups. In particular, demographic parameters (age and sex), risk factors, duration of disease, and immunological and virological parameters before HAART were not different.
Flow cytometric analysis of T-cell subsets
Mean CD4 T-cell levels after HAART were significantly lower in InR than in IR subjects (11.6 ± 5.2%, 163 ± 79 cells/μl versus 30.1 ± 9%, 574 ± 230 cells/μl; P < 0.0001). Among CD4 cells, naive and thymic naive cells were significantly reduced (17 ± 12.1%, 32 ± 32 cells/μl versus 40.3 ± 15%, 237 ± 178 cells/μl, P < 0.0001; 10.8 ± 7%, 18 ± 14 cells/μl versus 29.8 ± 13%, 173 ± 123 cells/μl, P < 0.0001, respectively; Fig. 1a and b). In addition, a positive correlation was observed between the percentage of naive and thymic naive CD4 T cells and the percentage of CD4 T cells (r = 0.672, P < 0.0001, and r = 0.653, P < 0.0001, respectively).
In InR subjects we also observed a significantly increased expression of activation markers (HLA-DR, CD95 and CCR5) (Fig. 2a). Moreover, a significant inverse correlation between CD4 cells expressing CD95 or HLA-DR and the percentage of CD4 (Fig. 3a and b) was observed.
In addition, InR patients showed a significant reduction of the CD4+CD25high Treg subset with respect to IR subjects and healthy controls (0.58 ± 0.35%, 1.26 ± 0.47% and 1.32 ± 0.5%, respectively); even the CD4 subset co-expressing CD25high and CD62L was significantly reduced among InR subjects (Fig. 2b). In 12 patients (six from the InR group and six from the IR group), the CD4 cells co-expressing CD25 and CD27 were lower among InR subjects (0.5 ± 0.4% among InR and 1.1 ± 0.2% among IR; P = 0.05). A positive correlation was observed between the percentage of CD4+CD25high cells and CD4 cells both as percentage and absolute count (Fig. 3c and d). Moreover, the same correlation was found between the absolute count of CD4+CD25high and naive (Fig. 3e) and thymic naive CD4 cells (data not shown). While analysing Treg as a proportion of CD4 T-cells (CD4/CD25highCD62Lhigh), we observed a variability among the studied subjects. Some patients showed a low CD4 count and a higher value of Treg, but this finding was not statistically significant (Fig. 3f). The percentage of CD4+CD25high was inversely correlated with the percentage of CD4 cells expressing the activation markers HLA-DR and CD95 (Fig. 3g and h).
Regarding the expression of IL-7R (CD127) on CD4 cells, we observed reduced CD127 both on CD4 T cells and naive CD4 T cells (CD4+CD45RA+CD62L); the percentage of CD4 T cells expressing CD127 was 69 ± 9 in the InR group compared with 88.1 ± 5.1 among IR subjects (P = 0.014) and 88.5 ± 2.9 among healthy controls (Fig. 4a). Among naive CD4 T cells, the percentage of cells expressing CD127 was 87 ± 6, 96 ± 3 and 99 ± 1, respectively (P = 0.006 for the comparison between InR and IR). CD4+CD127+ cells were 105 ± 73/μl among InR subjects, compared with 463 ± 121/μl among IR (P = 0.0004), while CD4+CD45RA+CD62L+CD127 cells were 30 ± 32/μl and 151 ± 53/μl, respectively (P = 0.0005) (Fig. 4c). A positive correlation was observed between the percentage of CD4 T cells and the percentage of CD4 cells expressing CD127 (r = 0.726, P = 0.0006). Moreover, the number of naive CD4 cells expressing CD127 was positively correlated with the absolute CD4 cell count (r = 0.754, P = 0.0003). In addition, a positive correlation was observed between the number of CD4+CD25high Treg and both CD4 and naive CD4 T cells expressing CD127 (r = 0.809, P < 0.0001 and r = 0.629, P = 0.0052, respectively).
Regarding CD8 T cells, no significant differences were observed in the evaluated subsets. Naive CD8 T cells were 23.5 ± 14% in InR, compared with 34.6 ± 20.3% (P = 0.105), HLA-DR+ subset was 26 ± 14% in InR compared with 22.9 ± 14.5% (P = 0.33), and CD8/CD127+ cells were 49.8 ± 21.9% among InR compared with 53 ± 9.8% (P = 0.72).
TCRBV repertoire analysis
Flow cytometric analysis of TCRBV repertoire showed increased number of expansions on CD4 cells from 12 InR subjects compared with 11 IR subjects (37 versus 16 expansions, respectively). The mean number of expansion per patient was 3.1 ± 1.8 and 1.4 ± 1.7, respectively (P = 0.06). With regard to TCRBV expression we define as expansion a value greater than the mean of the controls plus 3 SD. On CD8 T cells, the number of expansions was 21 among InR and 17 among IR, but this finding is not statistically significant.
Analysis of CDR3 spectratyping showed an increase of the CD4 perturbation level in InR patients versus IR subjects. Analysis of the deviation of patients' histograms from the normal distribution revealed significantly altered patterns in most BV genes examined in CD4 cells of InR subjects, while the level of CDR3 perturbation on CD8 cells was similar between the two groups (data not shown).
Serum IL-7 concentration
IL-7 serum concentration was higher in InR subjects with respect to IR patients and healthy controls (13.5 ± 4.4, 11.5 ± 2.9 and 10.6 ± 3.2 pg/ml, respectively, P = 0.05 for the comparison between InR and IR) (Fig. 4b). IL-7 concentration was inversely correlated with the percentage and the absolute count of CD4 T cells expressing CD127 (r = −0.738, P = 0.0026 and r = −0.564, P = 0.0358, respectively), with the percentage of CD4+CD127+ T lymphocytes (r = −0.691, P = 0.0062) and with the absolute count of naive CD4 cells expressing CD127 (r = −0.688, P = 0.0065).
We evaluated several immunological parameters in two groups of patients undergoing HAART, to better investigate the immunological response of InR patients and the pathogenesis of their low CD4 T-cell count, despite a persistent plasma HIV RNA < 50 copies/ml. In particular, we studied the naive T-cell compartment, the serum concentration of IL-7 and the expression of IL-7Rα on naive and memory CD4 and CD8 T cells. Activation markers and the role of Treg were also evaluated, as well as the TCR Vβ repertoire.
Our data confirm that in InR patients the naive CD4 T-cell subset is compromised. In fact, we observed a reduction of CD4 T cells and naive and thymic naive T cells with respect to IR subjects. A positive correlation was observed between the percentage of naive and thymic naive CD4 T cells and the percentage of CD4 T lymphocytes.
Mechanisms underlying CD4 T-cell homeostasis are not yet well understood. To this end, the study of the IL-7/IL-7Rα pathway is important. In fact, IL-7 is a key cytokine in the regulation of T-cell homeostasis, based on its effect on thymopoiesis, and it is a survival factor for naive and memory CD4 and CD8 T lymphocytes [16–20]. In addition, IL-7 acts as a co-stimulatory molecule upon T-cell activation [21,22] and promotes homeostatic proliferation of peripheral T cells in lymphopenic conditions [19,20,23]. Recently, a high IL-7 concentration, together with loss of IL-7Rα, has been associated with CD4 T-cell depletion in HIV patients. Rethi and colleagues demonstrated that T cells, isolated from HIV-infected subjects and cultured in the presence of IL-7, have a survival disadvantage, as compared to T cells from healthy individuals, suggesting that decreased responsiveness to IL-7 may play a role in disease progression . Moreover, IL-7 has been shown to be an important component for the establishment and maintenance of memory T cells [16–19]. In InR subjects we observed reduced expression of IL-7Rα both on CD4 T cells and on naive CD4 T cells, in comparison to IR patients. Furthermore, we found a positive correlation between the percentage of CD4 T cells and those expressing IL-7Rα. Serum concentration of IL-7 was increased in InR subjects, as compared with IR patients and healthy controls. The IL-7 level was inversely correlated with the expression of CD127 both on CD4 and naive CD4 T cells. In addition, the number of naive CD4 T cells was positively correlated with the absolute count of CD4 T cells. These data, taken together, seem to confirm the hypothesis that a defect in IL-7Rα expression might affect the rise of CD4 T cells in patients with a persistently suppressed viral load, thus playing a role in the pathogenesis of partial immune responses to HAART. Moreover, our results on thymic naive T cells support the hypothesis that deficiency of central CD4 regeneration might also play a role in the pathogenesis of low CD4 recovery .
It has been demonstrated that chronic immune activation plays a central role in determining CD4 T-cell decrease [25–27,38] and during the last years, the role of Treg in modulating the immune response has gained importance. In peripheral blood, Treg constitutively expressing a high level of CD25, defined as CD4+CD25high T cells, have regulatory functions [39,40]. Moreover, defects in the function of the CD4+CD25highCD62L+ cells, defined as suppressive Treg for their ability to down-regulate self-reactive T-cell responses, have been implicated in the pathogenesis of this immune activation. It has been demonstrated that Treg are depleted during chronic HIV infection and that their loss, together with the presence of plasma viral load, might contribute to chronic immune activation . In addition, patients with detectable HIV RNA have higher frequencies, compared to healthy donors and patients with an undetectable viral load, of Treg, which however, have a reduced function . These findings are consistent with the hypothesis that CD4 T cells and Treg decline independently, and thus they should be evaluated independently from each other, to better measure the decrease of Treg .
In our study, InR patients showed a significant reduction of CD4+CD25high Treg with respect to IR subjects and healthy controls. Moreover, even the CD4+CD25highCD62Lhigh subset is significantly reduced among InR patients. Eggena and colleagues  showed that this subset strongly suppresses anti-CD3-induced proliferation. In addition, it has been recently reported that the coexpression of CD25 and CD27 identifies FoxP3+ T cells with suppressor activity . Our data on the CD4+CD25+CD27+ subset, in a number of our subjects, confirm that Treg are diminished among InR patients.
Our results on the augmented activated T cells are consistent with findings of previous studies  suggesting that even patients with HAART-mediated viral load suppression show a significant higher percentage of activated T cells. Moreover, CD8 T-cell subsets, including activated cells, that did not differ between the two groups, do not seem to play a role in the pathogenesis of chronic immune activation in InR subjects. It might be hypothesised that in the case of InR patients immune activation may play a role independently from the viral load and that chronic immune activation in HIV infection may be at least in part due to the dysregulation of Treg. Most likely, the period preceding the beginning of HAART, with an active viral replication, is characterized by immune activation and dysregulation of Treg, and in InR subjects these factors may be persistent even when viral replication is effectively suppressed. Moreover, it is possible that even when the viral load is < 50 copies/ml, there might be a persistent low-grade replication, or the presence of viral blips, that, even if not clinically significant , might drive the persistent immune activation. In addition, there might be a discordance between plasma viral load and viral replication in reservoirs, such as bone marrow or gut-associated lymphoid tissue (GALT). We recently observed two patients with HIV-1 infection (followed for several months without HAART) who had persistent undetectable HIV-1 RNA using ultrasensitive methods and negative HIV DNA in PBMC, but positive proviral DNA in cells from GALT or bone marrow (C Fimiani et al. unpublished data). Finally, chronic immune activation might be driven by persistent immunological memory cells, such as occurs for several months after acute Epstein–Barr virus or cytomegolovirus infections .
Skewing of the CD4 TCR repertoire has been observed in advanced HIV infection and only partially normalized in patients undergoing fully suppressive HAART . The higher level of perturbation observed in InR subjects, both in flow cytometry analysis and in CDR3 spectratyping, confirms that these patients have an impaired immune reconstitution as evidenced by the lower CD4 T-cell count, and by the naive and thymic naive T-cell subset impairment.
In the course of HIV infection a dysregulation of the cytokine network has been observed, with Th2 polarization associated with disease progression [44,45]. We assessed the serum concentration of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13 and interferon (IFN)γ, and we did not find any significant difference, except for IFN-γ levels that were significantly lower in InR subjects (data not shown). According to recent data , these findings suggest that in patients with poor immunological response to HAART the dysregulation of the cytokine network does not play a central role.
The immunological defect of InR seems mainly restricted to CD4 T cells and may be the result of bone marrow impairment as in patients with idiopathic CD4 deficiency . In fact in InR patients, we found an altered clonogenic potential, in parallel with an increased expression of Fas/Fas ligand on the stem cells, as the result of increased apoptosis (A. Isgrò, unpublished data). In vivo and in vitro, hematopoiesis occurs in association with the complex network of cell types found in the stroma. A central function of stromal cells is IL-7 production . IL-7 primarily acts as a growth and anti-apoptotic factor for B and T cell precursors and its production is a critical step for the beginning of B and T lymphopoiesis, starting from stem cells. In HIV infection we have shown that IL-7 increases in parallel with CD4 T-cell depletion. Its levels normalize when subjects are treated with HAART and their CD4 T-cell numbers increase . Thus, the alteration of the IL7/IL7Rα pathway observed in InR patients may contribute to explain the CD4 T-cell defect.
In conclusion, the reduced expression of IL7Rα, the reduction of naive T cells and a persistent immune activation, accompanied by a reduced frequency of suppressor Treg, are the principal causes involved in the pathogenesis of immune CD4 recovery defect in InR subjects.
Sponsorship: This paper was supported by Istituto Superiore di Sanità grants nr. 40F.3, 40F.39 and 40F.53 (2004).
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Treg; interleukin-7; IL-7Rα; CD4 T cells; HAART
© 2006 Lippincott Williams & Wilkins, Inc.
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