B-cell differentiation, survival and functions are impaired at several different levels in HIV-1-infected individuals [1,2]. Immature, less functional transitional B cells are increased in the circulation, in association with lymphopenia . Tissue-like memory B cells, a subset that includes most HIV-1-specific B cells, are present at higher number in HIV-1-infected patients and are characterized by an exhausted phenotype with the expression of several inhibitory receptors [4,5]. The ratio of resting memory B cells is decreased in patients [6,7], and in viraemic patients, a subset of apoptosis-sensitive activated memory B cells emerges with high expression of the Fas death receptor . In line with the data that link Fas expression with active viral replication, antiretroviral therapy (ART) initiated during primary infection decreases Fas expression and improves B-cell survival . In addition, early initiation of therapy preserved memory B-cell responses [10,11]. However, ART did not completely normalize Fas expression and B-cell survival , and viral suppression in chronically infected patients did not lead to restoration of memory B cells , suggesting the role of viraemia-independent mechanisms in decreasing B-cell survival. In addition, higher Fas expression was observed on both naive and memory B cells, indicating that increased Fas-mediated apoptosis might not be exclusively affecting the activated memory subset .
To better understand Fas regulation on B cells during HIV-1 infection, we performed a detailed analysis on the potential effects of viraemia, lymphopenia and B-cell modulatory cytokines on Fas expression of various B-cell subsets in HIV-1-infected patients. In addition, we analysed whether the altered expression of other B-cell regulatory molecules, PD-1 and LAIR1, can have cumulative effects with Fas upregulation on modulating the homeostasis of different B-cell subpopulations.
Participants and methods
Blood samples were obtained from 15 HIV-1-uninfected blood donors and from 54 HIV-1 patients (52 men and two women), divided into three different groups. First, viraemic individuals (n = 19) left untreated as their CD4+ T-cell counts remained higher than 350 cells/μl. Mean CD4+ T-cell count was 499 ± 44 cells/μl in the treatment-naive patients and viral load ranged between 200 and 132 000 copies/ml. The second group was composed of ART-treated, nonlymphopenic patients (n = 26) with undetectable viraemia (<25 copies/ml) and CD4+ T-cell counts higher than 200 cells/μl (mean = 486 ± 225 cells/μl). The third group was composed of ART-treated CD4+ T-cell lymphopenic patients (n = 11), with CD4+ T-cell counts below 200 cells/μl for a period of 3–20 years (average = 9.4 years). In this latter group, viral load ranged between undetectable and low (<100 copies/ml) levels, and mean CD4+ T-cell count was 149 ± 48 cells/μl. Among these patients, CD4+ T-cell count did not improve in six individuals despite successful control of HIV-1 replication for at least 2 years, suggesting a major impairment in T-cell homeostasis.
The age (mean age ± SD) of the individuals included in the different cohorts was 43 ± 9 for controls, 50 ± 9 for ART-treated nonlymphopenic patients, 40 ± 12 for treatment-naive patients and 46 ± 10 for ART-treated, CD4 T-cell lymphopenic patients. The ethical committee at Karolinska Institutet approved the study, and informed consent was obtained from all individuals included.
It is noteworthy that CD4+ T-cell counts were similar in the viraemic and in the ART-treated, nonlymphopenic cohorts, making it possible to evaluate the effects of viraemia on Fas expression independently of CD4+ T-cell depletion. In addition, viraemia was very low/undetectable in the cohort of lymphopenic patients, allowing us to study the effects of CD4+ T-cell depletion independently of viral replication.
Peripheral blood mononuclear cells were isolated by Ficoll gradient centrifugation (Lymphoprep; Axis-Shield PoC As, Rodelokka, Norway) and then stored frozen until required for analysis.
The following antibodies were used for immunostainings: V450 BD Horizon-labelled anti-CD27, fluorescein isothiocyanate-labelled anti-CD19, phycoerythrin (PE) or allophycocyanin-labelled anti-Fas, PE-Cy5-labelled anti-CD21, PE-Cy7-labelled anti-CD10, PE-labelled anti-PD-1 and PE-labelled anti-LAIR1, all from BD Pharmingen (San Diego, California, USA). Viable cells were detected using Near-IR-Live/Dead kit (Invitrogen, Carlsbad, California, USA). Fluorescence intensities were measured with FACS LSR II (Becton Dickinson, San Jose, California, USA) and data analysed with FlowJo v. 8.4.4 software (Tree Star Inc., Ashland, Oregon, USA). The peripheral B-cell populations CD21+CD27− naive, CD21+CD27+ resting memory, CD21lowCD27− tissue-like memory, CD21lowCD27+ activated memory, CD10+CD27− immature transitional and CD10+CD27+ germinal centre-like B cells were identified as we previously reported  and as shown on supplementary Fig. 1, http://links.lww.com/QAD/A273.
The levels of interleukin (IL)-2, IL-7 and interferon-gamma (IFN-γ) in serum samples obtained from HIV-1-infected and noninfected individuals were measured with the Milliplex Map kit, High Sensitivity Human Cytokine Immunoassay (Millipore Corporation, Billerica, Massachusetts, USA).
Statistical analyses were performed using Prism (version 5.0a for Mac OS X, GraphPad Software Inc., La Jolla, California, USA), One-way analysis of variance (ANOVA) with Newman–Keuls posttest and, to measure correlation between cytokine levels, Spearman test were used.
Potential effects of HIV-1 viraemia on Fas expression of B cells
In line with previously published data , the frequency of Fas-positive B cells was higher within the group of patients that presented with detectable viraemia than in nonviraemic patients (P < 0.0001). We could, however, not detect a significant correlation between viral load and Fas expression on B cells (P = 0.9). In patients naive to therapy, we found a significantly elevated Fas expression on the naive, transitional, tissue-like memory, activated and resting memory and germinal centre founder B-cell subsets, as compared with ART-treated patients with similar CD4+ T-cell counts (Fig. 1a and b). As expected, the CD21low activated memory B cells showed the highest Fas expression; we detected, however, only a modest increase in the frequency of this subset in viraemic individuals (mean frequency ± SD was 2.4 ± 1.4, 2.0 ± 1.2 and 4.1 ± 1.9% in noninfected controls, in ART-treated, nonlymphopenic and in untreated patients, respectively) that did not reach statistical significance (Fig. 1a). When compared with noninfected controls, Fas expression was significantly elevated on all studied B-cell subsets of viraemic patients. Although Fas expression of resting memory B cells was lower in ART-treated, nonlymphopenic patients than in the viraemic, nonlymphopenic cohort, the frequency of Fas-expressing resting memory B cells remained significantly elevated in the ART-treated group as compared with noninfected individuals (Fig. 1a and b).
Increased Fas expression in patients with CD4 T-cell lymphopenia
We detected a significantly increased Fas expression on all studied B-cell subsets in ART-treated lymphopenic patients as compared with the ART-treated nonlymphopenic cohort or noninfected individuals (Fig. 1). Importantly, the frequency of the CD21highCD27+ resting memory B cells was lower and the Fas expression of these cells was significantly elevated in all groups of HIV-1-infected patients when compared with noninfected individuals, indicating that the impairment of this subset is not reversed by the therapy-induced CD4+ T-cell restoration and viral suppression.
Cytokines associated with T-cell activation might increase Fas expression of naive and transitional B cells
We studied the serum IL-2, IL-7 and IFN-γ levels in HIV-1-infected and noninfected individuals (Fig. 2). IL-2 and IFN-γ levels might reflect T-cell activation and IL-7 has often been found elevated in lymphopenic HIV-1-infected patients . In addition, we have recently shown that these cytokines can prime resting B cells to Fas-mediated apoptosis . We found slightly higher IL-7 and IL-2 serum concentrations in ART-treated, nonlymphopenic patients than in controls or in ART-treated CD4+ T-cell lymphopenic patients, although none of the differences reached statistical significance. The surprisingly low levels of IL-7 in CD4+ T-cell depleted individuals (Fig. 2a) possibly reflects the deleterious effect of prolonged lymphopenia on lymphoid niches specialized in IL-7 production and T-cell maintenance . We found a strong positive correlation between IL-7 and IFN-γ concentrations (P < 0.0001, r = 0.72), as well as between IL-2 and IFN-γ concentrations (P < 0.0001, r = 0.69) in the serum of HIV-1-infected patients; suggesting a concerted regulation of cytokine production associated with T-cell activation and also the possibility of an inter-regulatory effect of γ-chain using cytokines and IFN-γ on each others expression  (Fig. 2b).
Importantly, the levels of IL-7, IL-2 and IFN-γ in blood correlated with Fas expression on B cells in the group of ART-treated, nonlymphopenic patients, supporting the hypothesis that γ-chain using cytokines and IFN-γ might participate in priming B cells for Fas-mediated apoptosis (Fig. 2c). We did not detect a significant correlation between the levels of IL-7, IL-2 or IFN-γ and Fas expression on B cells of patients naive to therapy or in the lymphopenic cohort. When analysing Fas expression on different B-cell populations in the ART-treated, nonlymphopenic patients, we found that serum IL-7 levels correlated with Fas expression of naive (r = 0.46, P < 0.05) and immature transitional B cells (r = 0.41, P < 0.05) and, similarly, IFN-γ levels correlated with Fas expression of naive (r = 0.58, P < 0.01) and immature transitional B cells (r = 0.42, P < 0.05). IL-2 levels correlated with Fas expression of the total B-cell population only.
Unique regulation of Fas, LAIR1 and PD-1 expression on B cells during HIV-1 infection
Although Fas might play a pivotal role in increased B-cell turnover during HIV-1 infection, other molecules have also been implicated in B-cell immunopathology, including LAIR1 and PD-1 [9,15–17]. We aimed at assessing whether HIV-1 infection could induce a coordinated modulation of various B-cell regulatory molecules, possibly by skewing B-cell differentiation towards particular phenotypes, as suggested by the initial observation of viraemia-induced accumulation of Fashigh activated memory B cells . We compared the expression pattern of Fas, LAIR1 and PD-1 in the cohorts of ART-treated lymphopenic (n = 11), ART-treated nonlymphopenic (n = 20), viraemic (n = 11) and noninfected individuals (n = 15). LAIR1 was downregulated on all memory B-cell subsets in HIV-1-infected individuals, independently of the levels of viral replication or CD4+ T-cell depletion, as suggested by the similarly decreased LAIR1 expression in all cohorts of HIV-1-infected individuals (Fig. 3a). PD-1, on the contrary, was significantly upregulated only on the resting memory B cells, when viraemic individuals were compared with noninfected controls (Fig. 3b).
Decreased memory B-cell maintenance during HIV-1 infection might contribute to loss of serological memory against various pathogens and decreased vaccine efficiency . Several molecular pathways have been implicated in the dysregulation of memory B-cell homeostasis, including the death receptor Fas that is highly expressed on the CD21lowCD27+ activated memory B cells accumulating in viraemic individuals , PD-1 that might contribute to the loss of memory B cells in rapid disease progressors  and LAIR1, a molecular break for lymphocyte activation, that is downregulated on memory B cells during HIV-1 infection [9,15]. In this study, we aimed at clarifying which B-cell subsets are affected by the altered Fas expression in HIV-1-infected individuals and we also analysed the potential contribution of viraemia, lymphopenia and T-cell activation in the modulation of Fas levels on B cells. In addition to Fas, we analysed LAIR1 and PD-1 expression in the same cohorts to understand whether the different B-cell regulatory pathways might have an additive action on B-cell homeostasis during HIV-1 infection.
Overall, we showed that viraemia and lymphopenia can both contribute to elevated Fas expression on all B-cell subpopulations. We detected a decreased ratio of the CD21highCD27+ resting memory B cells and increased Fas expression on these cells, even when nonviremic, nonlymphopenic patients were compared with noninfected individuals, indicating that the homeostasis of resting memory B cells shows the highest sensitivity for perturbations induced by HIV-1 infection. In addition, our data suggest that the cytokines IL-7 and IFN-γ might influence Fas expression on the less differentiated, normally Faslow/Fasneg naive and transitional B-cell populations and predominantly when viral replication is controlled by ART.
On the contrary to the previously described single mechanism leading to altered Fas expression on B cells during HIV-1 infection, via the increased ratio of Fas+ activated memory B cells in viraemic individuals , our data indicate that Fas expression on B cells is regulated by multiple factors and that Fas expression is altered on all peripheral B-cell subsets of HIV-1-infected individuals.
Limited information is available on the role of LAIR1 in B-cell regulation during HIV-1 infection. This molecule is widely expressed on various immune cells, probably providing a steady-state inhibitory signal for cellular activation upon binding to collagen . Accordingly, LAIR1 downregulation might lead to a lower threshold for B-cell activation, in terms of antigen affinity and concentration, contributing to a generalized B-cell activation instead of highly specific B-cell responses. Our data indicating a decreased LAIR1 expression in the memory subsets, irrespectively of viraemia or CD4+ T-cell depletion, confirmed earlier reports showing that LAIR1 downregulation occurs already during primary infection and it is not normalized upon ART . A parallel increase of Fas and downregulation of LAIR1 expression on memory B-cell subsets indicates the possibility of a cumulative effect of these pathways in the increase of memory B-cell turnover. Our data also indicate that the resting memory subset remains affected by Fas upregulation and the loss of LAIR1 even after successful HIV control and CD4+ T-cell preservation in ART-treated patients.
An important role of PD-1 in the regulation of memory B-cell maintenance has been indicated by showing the accelerated loss of PD-1high memory B cells in rapidly versus slowly progressing SIV-infected rhesus macaques . In vivo, PD-1 blockade promoted both SIV-specific and non SIV-specific humoral immune responses, probably as a result of the increased T-cell functionality and B-cell maintenance [16,17]. Here we showed a significantly elevated PD-1 expression within the resting memory subset of viremic individuals as compared to noninfected controls, indicating that PD-1 might contribute to the reduced maintenance of this subset in patients with uncontrolled viral replication.
Overall, we detected a unique expression pattern for the three B-cell regulatory molecules that we analysed: Fas expression was increased on all B-cell populations in patients characterized by viraemia or lymphopenia; LAIR1 expression decreased only on memory B cells and independently of viraemia or lymphopenia, whereas PD-1 expression was upregulated only on resting memory B cells of viraemic individuals. These results suggest that independent regulatory pathways affect Fas, LAIR1 and PD-1 expression levels on the various B-cell subsets, instead of a coordinated shift in their expression as result of an altered distribution of peripheral B-cell subpopulations in HIV-1-infected individuals. The three pathways might, on the other hand, act simultaneously in various disease stages characteristic of HIV-1 infection. In the presence of viraemia, B-cell maintenance could be affected via a generalized increase of Fas expression and higher PD-1 expression among resting memory B cells. Lymphopenia and cytokines associated with T-cell activation might contribute to further Fas upregulation. Successful control of HIV-1 replication and preservation of higher CD4+ T-cell counts decrease Fas expression on most B-cell subsets and PD-1 expression on the resting memory B cells. Fas expression remains, however, elevated on resting memory cells possibly contributing to their impaired maintenance. LAIR1, expressed at a lower level within the memory subsets of all groups of studied patients, might lead to further contraction of the resting memory subset by pushing B-cell differentiation towards the activated memory and the exhausted tissue-like memory subsets.
Understanding the mechanisms leading to impaired B-cell memory in HIV-1-infected individuals is essential to be able to induce and maintain immunity against infecting pathogens. Our study indicated that the control of viraemia and preservation of CD4+ T-cell counts can alleviate the impairment of B-cell maintenance induced by PD-1 and Fas upregulation. On the contrary, the decreased resting memory B-cell population and their increased Fas expression, together with the decreased LAIR1 expression on memory subsets, indicate that the B-cell homeostasis remains permanently altered in ART-treated patients. The beneficial effects of early ART initiation on the maintenance of serological memory have been repeatedly demonstrated [10,11], and it will be important to learn whether earlier ART initiation could eliminate all measurable defects of memory B-cell homeostasis.
B.R. contributed to study design, analysed data and wrote the article; S.S. and S.A. performed experiments, analysed data and wrote the article; S.P. performed experiments, B.H. contributed with patient samples, D.S. performed experiments, T.P.H. performed experiments, and F.C. contributed to study design and wrote the article.
The work of the authors is supported by grants received from the Swedish MRC, the Swedish International Development Agency (SIDA-SAREC), the EU Fp7 Collaborative project NGIN (Health-F3-2007-201433), the Fp6 Network of Excellence Europrise and the Karolinska Institutet. Financial support was also provided through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and the Karolinska Institutet.
Conflicts of interest
The authors have not declared any conflicts of interest.
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