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Residual immune activation in combined antiretroviral therapy-treated patients with maximally suppressed viremia

Guihot, Amélie; Dentone, Chiara; Assoumou, Lambert; Parizot, Christophe; Calin, Ruxandra; Seang, Sophie; Soulié, Cathia; Marcelin, Anne-Geneviève; Calvez, Vincent; Autran, Brigitte; Katlama, Christine; Costagliola, Dominique; Carcelain, Guislaine

doi: 10.1097/QAD.0000000000000815
Research Letter

Residual immune activation was studied in 51 HIV-infected individuals, 16 with viral load between 1 and 20 copies/ml and 35 with viral load less than 1 copy/ml, and compared with results in 20 healthy blood donors. Higher T-cell activation and IP-10/CXCL10, MIG/CXCL9 and sCD14 plasma levels persisted in both HIV+ groups. The proportion of activated HLA-DR+ CD4 T cells was inversely correlated with the CD4 nadir and the current CD4 cell counts.

aSorbonne Universités, UPMC Univ Paris 06


cAPHP, Hôpital Pitié Salpêtrière, Département d’Immunologie, Paris, France

d Infectious Diseases Department, Sanremo Hospital, Italy and Center of Excellence for Biomedical Research, University of Genoa, Italy

eSorbonne Universités, UPMC Univ Paris 06, UMR_S 1136, Institut Pierre Louis d’Epidémiologie et de Santé Publique

fINSERM, UMR_S 1136, Institut Pierre Louis d’Epidémiologie et de Santé Publique

gAP-HP, Hôpital Pitié-Salpêtrière, Maladies Infectieuses et Tropicales

hAPHP, Hôpital Pitié Salpêtrière, Laboratoire de Virologie, Paris, France.

*Drs Guihot and Dentone contributed equally to this article.

Correspondence to Dr Guislaine Carcelain, Département d’immunologie, Hôpital Pitié-Salpêtrière, 83 bd de l’Hôpital, 75013 Paris, France. Tel: +33 1 42 17 79 30; fax: +33 1 42 17 74 90; e-mail:

Received 21 April, 2015

Revised 9 July, 2015

Accepted 9 July, 2015

Chronic immune activation and inflammation are a hallmark of HIV infection [1,2] and represent a key component of HIV pathogenesis [3]. Such persistent immune activation has been shown to increase cardiovascular risk, favors loss of bone mineral density, neurocognitive disorders or increase incidence of malignant diseases [4–15]. Although combined antiretroviral therapy (cART) is efficient in reducing both cellular and soluble activation markers, some of them remain elevated in virologically controlled patients [16–21]. Whether a step further down in viral suppression of plasma HIV-RNA can be associated to a lesser degree of immune activation and inflammation is an important question. In this cross-sectional study, we aimed to characterize the residual immune activation and inflammation using cellular and soluble markers in HIV-infected individuals with maximally suppressed viremia (viral load ≤20 copy/ml) compared with results in healthy blood donors. We also evaluated whether residual immune activation and inflammation were different depending on having an HIV-1 plasma viral load below or above 1 copy/ml.

HIV-infected individuals under cART for at least 6 months who achieved a controlled viral load were enrolled consecutively during a 1.5-month period (from 2 February, 2010 to 19 March, 2010) in the Infectious Diseases Department of the Pitié Salpêtrière Hospital, in Paris, France, and compared with 20 HIV-negative healthy blood donors whose samples were provided by the Etablissement Français du Sang of the Pitié Salpêtrière Hospital. T cell classical activation markers were studied on fresh peripheral blood by multiparameter flow cytometry (CD4-PC7, CD8-FITC, HLA-DR-APC-Cy7, CD38-PE, CD3-APC on a FACS Canto I; Becton Dickinson, Franklin Lakes, New Jersey, USA). Monocytes were subdivided on the basis of CD14-PerCP (BD Biosciences, San Diego, California, USA) and CD16-PC7 (Beckman Coulter, Fullerton, California, USA) expression into classical (CD14+ CD16−), intermediate (CD14+ CD16+/low) and nonclassical (CD14low CD16hi) monocytes. HLA-DR-APC-Cy7, CD38-PE, and CD11b-APC (BD Biosciences) activation markers were studied on subpopulations. A large panel of soluble immune activation and inflammation markers was studied in plasma including MCP-1/CCL2, IP-10/CXCL10 and MIG/CXCL9 (BD CBA, FACS Canto I, Becton Dickinson, Franklin Lakes), and interleukin-6, interleukin-18 and sCD14 (ELISA arrays R&D Quantikine HS, R&D Systems, Minneapolis, Minnesota, USA). Plasma HIV RNA was measured using an ultrasensitive technique (threshold ≤1 copy/ml), as previously described [22]. HIV-infected patients were classified in 2 groups according to the level of plasma viral load; viral load less than or equal to 1 copy/ml (n = 35) and 1 <viral load <20 copies/ml (n = 16). Activation and inflammation markers were compared between each of these two HIV-infected groups and the group of HIV-negative healthy blood donors and between the two HIV-infected groups using Mann–Whitney U test. Association between activation and inflammation markers and immunovirological parameters, in HIV-infected patients, was assessed using a nonparametric Spearman test. All reported P values were two-tailed, with a significance level of 0.05. Analyses were performed with SPSS version 19.0 for Windows (SPSS Inc, Chicago, Illinois, USA).

A total of 51 HIV-infected patients were enrolled, 35 males and 16 females with the following baseline characteristics: median age 47 years [interquartile range (IQR), 42–56], Nadir CD4 cell count 212 cells/mm3 (IQR, 99–313), antiretroviral therapy duration 8.9 years (IQR, 4.0–13.7), time of undetectable viral load 2.0 years (IQR, 0.9–4.4), current CD4+ cell count 593 cells/mm3 (IQR, 407–750), current CD8+ cell count 697 cells/mm3 (IQR, 537–897), current CD4+/CD8+ ratio 0.8 (IQR, 0.6–1.2). Four were HCV and two were HBV coinfected (one with HCV and HBV coinfection).

Both persistent abnormal CD4+ and CD8+ T cell activation with regard to HLA-DR expression was observed in the viral load less than or equal to 1 copy/ml group. Abnormal CD4+ but not CD8+ T cell activation was observed in the 1< viral load ≤20 cp/mL group, when compared with HIV-negative healthy blood donors (Fig. 1a and b). Overall, the CD38 expression was normal in both HIV+ groups (data not shown). The results were similar when excluding the five patients with HCV and/or HBV co-infection (data not shown). Monocyte subpopulations (classical, intermediate, and nonclassical) did not differ between HIV-infected patients and HIV-negative healthy blood donors, as well as the expression of activation markers CD38, HLA-DR and CD11b (data not shown). However, IP-10, MIG and sCD14 plasma levels were significantly higher in both groups of HIV-infected patients when compared with HIV-negative subjects (Fig. 1c, d, and e), whereas other plasma inflammation markers such as interleukin-6, interleukin-18 and MCP-1 (monocyte chemoattractant protein-1) were not different between groups (data not shown). When considering the whole HIV+ group, the CD4 nadir (ρ = −0.379, P = 0.006 Fig. 1F) and the current CD4 cell counts (ρ = −0.464; P = 0.0006 Fig. 1G) were found to be significantly associated with the proportion of HLA-DR+CD4 cells.

Fig. 1

Fig. 1

Taken together, these results show for the first time that an immune CD4 and CD8 T-cell activation characterized by an increased HLA-DR expression but not by an elevated CD38 expression persists despite an optimal virological control supported by the ultrasensitive viral load less than or equal to 1 copy/ml in HIV-infected patients on cART for nearly 10 years. This discrepancy without CD38 expression on CD8 T cells may be linked to undetectable viral loads below 1 copy/ml [23,24], whereas persisting low grade antigenic presentation by defective viruses could lead to HLA-DR expression on T cells [25,26]. Here we demonstrate that the current CD4 activation measured by HLA-DR expression is driven by both the CD4 T cell depletion at time of nadir and the current CD4 T cell depletion. In multivariable linear regression analysis, CD4+HLA-DR+ cells were independently associated with these two parameter (β = –0.14, P = 0.05 for nadir CD4 T cell count and β = –0.77, P = 0.001 for current CD4 T cell count). Noteworthy, the current CD4 T cell count was most strongly associated with the CD4 T cell activation. Both immune parameters are hallmarks of immune reconstitution, suggesting the residual CD4 activation observed here might reflect ongoing immune restoration. This T cell activation goes along with innate activation markers, which remain elevated independently of the CD4 nadir or counts, that is the interferon-γ-inducible chemokines, IP-10 (interferon-γ-inducible protein 10), MIG (monokine induced by γ interferon), and the sCD14 level, despite the absence of monocytic activation.

These results also show some dissociation between activation and inflammatory markers in context of full viral suppression even below 1 copy/ml, and raise the hypothesis that undetectable virus production, current lymphoid homeostatic proliferation and some residual gut mucosal lymphoid alterations differentially influence this residual immune activation. Finally, these results emphasize more than ever the need to treat HIV infection as soon as possible to avoid the establishment of the permanent scar of the initial CD4+ T cell depletion.

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Conflicts of interest

This work has been partially presented to the CROI 2011 International Conference in Boston, Massachusetts, USA (Abstract #D-198).

There are no conflict of interest.

Authors Contributions: A.G., C.D., C.K., B.A., D.C. and G.C. designed the study and wrote the article; A.G., C.D., S.S., R.C., C.S., A.G.M. and V.C. selected patients; C.P., C.D. and A.G. performed immunological assays; C.S., A.G.M. and V.C. did the virological assays. L.A., C.D. and D.C. performed the statistical analysis.

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