CD4+ T cells do not normalize even after long-term virologically suppressive HAART in a subset of HIV-infected patients – ‘immunological non-responders’ (INRs). This phenomenon is more relevant in individuals delaying therapy until severe CD4+ T lymphopenia and may result in elevated AIDS and non-AIDS events [1,2].
Despite several associations with clinical–epidemiological factors and immune–virological pathways (reviewed in ), we still miss a comprehensive view of the immune–functional correlates of augmented clinical risk in patients starting HAART late in the disease and not adequately reconstituting CD4+.
Compared to other persistent infections, chronic untreated HIV disease features disturbed T lymphocytes differentiation with prevailing memory/activated T cells failing to acquire functional competence that is indicative of continuous antigen-driven stimulation [4,5]. In progressive HIV/AIDS, CD4+ interleukin (IL-2)-producing T cells are nearly absent, resulting in an impairment of CD8+ response with altered memory/effector protection upon antigen encounter [4,6,7].
Given that HAART only sporadically reconstitute T-cell maturation and function, we hypothesized that patients with inefficient CD4+ recovery upon virologically suppressive HAART will not improve T-lymphocyte maturation and helper/cytotoxic function.
To test this hypothesis, we cross-sectionally investigated parameters of T-lymphocyte maturation (CD4+ and CD8+ cell surface expression of CD45RA, CCR7, CD7) and function toward HIV and CMV antigens in a strictly characterized cohort of INRs as compared to patients with full viroimmunological recovery (full responders).
Patients and methods
This cross-sectional, observational study was conducted at the Clinics of Infectious Diseases, ‘Luigi Sacco’ and ‘San Paolo’ Hospitals, University of Milan. Inclusion criteria were CD4+ nadir less than 100 cells/μl, stable HAART for at least 24 months, HIV-RNA less than 50 copies/ml over the last 6 months and written consent to participate to the study.
On the basis of 24-month CD4+ responses, patients were divided into two groups: full responders, HIV-RNA less than 50 copies/ml, CD4+ 500 cells/μl or more; INRs, HIV-RNA less than 50 copiers/ml, CD4+ 200 cells/μl or less.
Peripheral blood immunephenotype was evaluated with the following mAbs: CD4-FITC, CD4-PC5, CD8-PC5, CD45RA-FITC (Beckman-Coulter, Milan, Italy), CCR7-PE (R&D, Milan, Italy), CD7-PE (Immunotech) (EPICS XL flow cytometer, Beckman-Coulter).
For interferon-γ (IFN-γ) ELISPOT, 96-well plates were incubated overnight at 4°C with a coating monoclonal human IFN-γ (Thermo Scientific) at 10 μg/ml. The plates were then washed with phosphate-buffered saline (PBS) and blocked with a solution of bovine serum albumin (BSA; Sigma-Aldrich, Milan, Italy) at 4% for 1 h at room temperature (RT). After discarding blocking solution, 2.5 × 105 cells per well peripheral blood mononuclear cells (PBMCs) were stimulated with medium, a pool of GAG peptides at 5 μmol/l or CMV grade 2 Antigen (Microbix Biosystem, Mississauga, Ontario, Canada) at 10 μg/ml in the presence of neutralizing anti-CD4+ monoclonal Ab (R&D) (0.1 μg/ml per well). Plates were incubated for 24 h at 37°C in 7% CO2; a biotinylated anti-IFNγ Ab (Thermo Scientific, Waltham, Massachusetts, USA) at 1 μg/ml was added for 2 h at RT, followed by incubation with horseradish peroxidase (HRP)-conjugated streptavidin for 30 min. Cytokine-producing cells were detected using the AEC chromogen substrate kit (Sigma-Aldrich) and reported as the number of spot-forming units (SFUs) per 106 cells after background cytokine secretion subtraction (EliScan Aelvis, Aelvis GmbH, Hannover, Germany).
For intracellular cytokine measurements, PBMCs were incubated for 18 h with medium, pool of GAG peptides and cytomegalovirus (CMV) grade 2 antigen (Microbix Biosystems) in the presence of anti-CD28 (R&D). Brefeldin A (10 μg/ml) (Sigma-Aldrich) was added during the last 6 h. PBMCs were stained with CD4-PC5 or CD8-PC5, IL-2-PE, IFN-γ-FITC (Beckman-Coulter).
Mann–Whitney nonparametric test was used for continuous variables and χ2-test and Fisher's exact test for categorical variables (SPSS Statistics 17.1; SPSS, Chicago, Illinois, USA).
Fifty-four HIV-positive patients were recruited: 34 INRs and 20 full responders. Patients were comparable in clinical–epidemiological and HIV-related parameters (Table 1). In particular, INRs and full responders presented comparable median [interquartile range (IQR)] age: [43 (40–49) and 41 (38–47) years, respectively; P = 0.327] (Table 1). As per inclusion criteria, INRs displayed lower median absolute CD4+ cell count [INRs = 166 (119–180) cells/μl; full responders = 947 (843–1018) cells/μl; P < 0.001). At the time of analysis, a higher proportion of protease inhibitor-based regimens was shown in INRs than in full responders (88 vs. 50%; P = 0.002) (Table 1).
We first assessed peripheral T-cell maturation. CD4+ naive CD45RA+CCR7+ frequencies were significantly reduced and CD4+ effector memory CD45RA−CCR7− frequencies were increased in INR compared with full responder patients. Thus, CD4+CD45RA+CCR7+ was 17.30% (8.35–28.05) in INRs and 34.55% (24.92–43.60) in full responders (P < 0.001); CD4+CD45RA−CCR7− was 46.85% (31.75–62.32) in INRs and 35.30% (17.32–43.42) in full responders (P < 0.001) (Fig. 1a).
A similar trend was shown in CD8+ T-cell frequencies: CD8+CD45RA+CCR7+ was 10.50% (5.94–23.15) in INRs and 27.85% (18.07–40.27) in full responders (P < 0.001); CD8+CD45RA−CCR7− was 54.35% (42.12–69.57) in INRs and 41.30% (28.10–57.60) in full responders (P = 0.06) (Fig. 1b).
No differences were detected in CD4+ central memory CD45RA−CCR7+ and CD4+ terminally differentiated CD45RA+CCR7− proportions. Thus, CD4+CD45RA−CCR7+ was 26.55% (13.30–40.02) in INRs and 24.65% (19.05–43.80) in full responders (P = 0.9); CD4+CD45RA+CCR7− was 2.25% (1.11–5.47) in INRs and 6.09% (1.85–8.83) in full responders (P = 0.1) (Fig. 1a).
Comparable data were shown in CD8+ frequencies: CD8+CD45RA−CCR7+ was 4% (1.64–12.35) in INRs and 3.14% (1.81–9.87) in full responders (P = 0.4); CD8+CD45RA+CCR7− was 24.2% (15.1–29.4) in INRs and 20.75% (15.85–25.65) in full responders (P = 0.5) (Fig. 1b).
Given the HIV-driven outgrowth of Th2-committed CD7−CD4+, we investigated CD7 expression on CD4+. Significantly reduced frequencies of CD7+CD4+ [INRs = 76.0% (69.1–84.4); full responders = 88.5% (86.5–90.0); P < 0.001) and higher CD7−CD4+ [INRs = 24.05% (15.7–30.9); full responders = 11.5% (9.8–14); P < 0.001) were seen in INRs than in full responders (Fig. 1c).
Given that T-cell maturation conditions function, we next investigated HIV-specific and CMV-specific CD4+/CD8+ function. Because more than 90% of HIV-infected patients with available anti-CMV serology were CMV antibody positive, CMV-specific ELISPOT/intracellular cytokines were analysed in patients with both known and unknown CMV serology.
Compared to full responders, significantly lower Gag-specific IFN-γ ELISPOT response were seen in INRs [0 SFCs/106 cells (0–4) vs. 8 SFC/106 cells (0–40), respectively; P = 0.04] (Fig. 1d). Accordingly, Gag-specific IFN-γ+CD8+ were also reduced in INRs, even if this trend did not reach statistical significance [INRs = 0% (0–0.14); full responders = 0.08% (0–0.20); P = 0.33]. No differences were shown in Gag-specific IFN-γ+CD4+ between groups [INRs = 0.10% (0–0.50); full responders = 0.10% (0–0.35); P = 0.82] (Fig. 1e).
A trend towards reduced median Gag-specific IL-2+CD4+ and IL-2+CD8+ was also seen in INRs compared with full responders, reaching significance for IL-2+CD8+ [CD4+: INRs = 0.08% (0–0.21), full responders = 0.12% (0–0.24), P = 0.71; CD8+: INRs = 0% (0–0.20), full responders = 0.17% (0–1.10), P = 0.08] (Fig. 1e).
No differences were detected in CMV-specific IFN-γ ELISPOT [INRs = 7.8 SFCs/106 cells (0–272); full responders = 50.3 SFCs/106 cells (0–114); P = 0.52] (Fig. 1f). Finally, intracellular cytokine staining confirmed no differences in the median frequency of CMV-specific IFNγ+ and IL-2+ CD4+/CD8+ [IFN-γ+CD4+: INRs = 0.15% (0–0.62), full responders = 0% (0–0.22), P = 0.27; IFN-γ+CD8+: INRs = 0.09% (0–0.29), full responders = 0.1% (0–0.40), P = 0.82; IL-2+CD4+: INRs = 0.10% (0–0.21), full responders = 0.10% (0–0.50), P = 0.78; IL-2+CD8+: INRs = 0.02% (0–0.20), full responders = 0.08% (0–0.50), P = 0.36] (Fig. 1g).
Given that intracellular cytokine profile reflects the presence of CMV, we performed a secondary analysis excluding 15 out of 54 (28%) patients (five full responders and 10 INRs) with no detectable response to CMV. No differences in CMV-specific IFN-γ ELISPOT [INRs = 100 SFCs/106 cells (4.6–1167); full responders = 64.2 SFCs/106 cells (44–783); P = 0.56] and in the median CMV-specific IFNγ+ and IL-2+ CD4+/CD8+ frequency between INRs and full responders were confirmed [IFN-γ+CD4+: INRs = 0.32% (0.10–0.97), full responders = 0.09% (0–1.47), P = 0.19; IFN-γ+CD8+: INRs = 0.1% (0.09–0.49), full responders = 0.13% (0–0.54), P = 0.84; IL-2+CD4+: INRs = 0.12% (0.08–0.39), full responders = 0.17% (0.04–0.60), P = 0.60; IL-2+CD8+: INRs = 0.14% (0–0.37), full responders = 0.29% (0.07–0.53), P = 0.42].
Data herein show the following immune alterations in HIV-positive patients failing CD4+ recovery on long-term virologically suppressive HAART: skewed peripheral CD4+/CD8+ T-lymphocyte maturation; expansion of type 2 committed CD7−CD4+; and reduced circulating HIV-specific CD8+ but comparable CMV-specific T cells.
Compared to full responders, INRs failed to expand unprimed naive T-subset while accumulating more differentiated effector-memory cells. Furthermore, in INRs, we observed a substantial expansion of CD4+ with downregulated CD7 expression. Such late-differentiated immunephenotype is reminiscent of a highly stressed, immunosenescent immunity proper of advanced untreated HIV/AIDS [4,5,8]. HIV infection is indeed characterized by skewed T-lymphocyte maturation, with an accumulation of preterminally differentiated CD45RA−CCR7− and a reduction in memory-quiescent CCR7+, memory-effector CCR7− and thymic-derived CD45RA+CCR7+ T subset [4,5]. This contrasts with other chronic infections where such profile is present in primary infection but is rapidly modified to protective CD45RA+CCR7− phenotypes ; differently, no further maturation is seen after primary infection in HIV disease, reflecting late-differentiated T-cell consumption resulting from T-cell activation/turnover and lack of CD4-helper activity . Given that HAART is only sporadically associated with the restoration of a physiological T-lymphocyte maturation [5,6], our data for the first time describe a senescent, highly differentiated T-cell maturation in INRs compared to patients with full immune-reconstitution. Furthermore, because CD4+CD7− T lymphocytes are characterized by Th0/Th2-comittment with poor IL-2/IFN-γ secretion, our findings suggest that T-helper dysfunction is present in INRs, possibly hampering CD8+ function . The CD7 molecule is in fact involved in T-cell activation and its downmodulation on CD4+ has been described during aging and in several clinical settings characterized by chronic T-cell turnover . During HIV/AIDS, a substantial expansion of the CD4+CD7− subset has been described and attributed a role in Th1/Th2 cytokine switch , with additional prognostic value in disease progression , and only partial recovery by HAART .
Thus, we set out to determine whether the highly differentiated maturation profile in INRs may affect T-lymphocyte function.
Interestingly, INRs displayed a tendency towards reduced HIV-specific IL-2/IFN-γ-producing CD8+ T cells, whereas CMV-specific T-cell responses are comparable in INRs and full responders. Our finding that few INRs seem to mount excellent responses to HIV suggests that lack of CD4+ recovery does not necessarily imply complete loss of virus-specific T cells.
Several pathways might be involved in the impairment of T-cell maturation/function seen in INRs: continuous antigenic stimulation [13–15]; antigen-independent dysregulation ; and systemic challenge by co-infecting viruses . Persistent low-level HIV-viremia has been shown in the background of a suppressed viral burden  and has been proposed to contribute to immune dysfunction in patients with low HAART-driven CD4+ gain [18,19]. Despite a presumed role for CMV in immunosenescence , our data fail to detect differences in CMV-specific responses between patients. Furthermore, should CMV be responsible for skewed T-cell maturation/function, we would expect a significant expansion of late-differentiated T cells with greatest cytotoxic potential [5,9,20]. That INRs show substantial accumulation of intermediately differentiated vis-à-vis reduced late-differentiated and thymic-derived T subsets in an IL-2-deprived environment implies continuous antigenic challenge. This might result in reduced cytotoxic/proliferative potential and exhausted reserves, failing HIV-specific T-cell replenishment. Furthermore, expanding CD45RA−CCR7− T cells are not able to recirculate through lymphnodes, thus jeopardizing memory control over HIV-mediated damage.
Although our findings on patients starting HAART late in disease should not be generalized to the whole HIV-infected population, data herein demonstrate that in long-term treated HIV infection, lack of CD4+ help recovery results in inadequate expansion of HIV-specific CD8+, providing mechanistic explanations of increased clinical risk in INRs. In particular, the finding of a highly differentiated/senescent T-cell maturation with impaired reconstitution of HIV-specific CD8+ function in patients failing CD4+ recovery provide rationale for possible therapeutic approaches aimed at controlling excessive immune activation such as immunosuppressive drugs, cell cycle inhibitors, CCR5 antagonists and drugs blocking type 1 interferons .
Fondo Interno Ricerca Scientifica e Tecnologica (FIRST) 2008 – Università degli Studi di Milano, and Istituto Superiore di Sanità, ‘National research program on AIDS’, Italy.
G.M. coordinated the project and experiments, analysed data and wrote the paper, L.G. helped in writing the paper, D.T. coordinated and ran laboratory experiments, F.B. reviewed patients' clinical records and performed statistical analysis, G.A. reviewed patients' clinical records, L.F. collected patients' clinical and laboratory data, L.M. performed flow cytometry data, M.G. and M.C. edited the manuscript, A.G. and A.d.M participated in the project design and edited the manuscript.
We are thankful to Tiziana Formenti for excellent typing assistance; we particularly thank all the patients participating in the study, and the staff of the Institute of Infectious Diseases and Tropical Medicine, ‘Luigi Sacco’ and ‘San Paolo’ Hospital who cared for the patients.
Presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Chicago, Illinois, USA, 17–20 September 2007, abstract no. H1016, and the 11th European AIDS Conference (EACS), Madrid, Spain, 24–27 October 2007, abstract no. P2.7/01.
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