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).
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).
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 .
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.
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