HIV-1 infection induces a multilayered dysregulation of B lymphocytes that in conjunction with the affectation of the T-cell compartment and the destruction of the lymphoid organs compromise the humoral response [1–3]. Chronic HIV-1 infection leads to a B-cell hyperactivation status characterized by high frequency of plasmablast/plasma cells and hypergammaglobulinemia [4,5]. HIV-1 can directly stimulate B cells by the interaction of envelope glycoprotein gp120 with C-type lectins, B-cell receptor (BCR), or α4β7 integrin expressed on the surface of B cells and by promoting the production of B-cell activation factor, interleukin-10, transforming growth factor-ß, or ferritin by B or innate cells [6–8]. Moreover, the homeostasis of B-cell subsets is drastically affected by HIV infection at all maturation stages, from immature/transitional  to plasma cells . However, memory B (MEB) cells seem to be the most affected subset. The CD27+ MEB compartment is reduced  and an aberrant tissue-like MEB repertoire is expanded in viremic individuals . Furthermore, the marginal zone-like B cell population is also reduced in HIV-1-infected individuals [12,13]. Antiretroviral treatment (ART) normalizes some aspects of B-cells homeostasis, such as the activation status and the number of transitional B cells . However, ART is ineffective in recovering the marginal zone-like and the CD27+ MEB, leading to impaired responses to polysaccharide vaccines and weakening the protection acquired after vaccination. Therefore, HIV-1-infected individuals show increased susceptibility to infection by encapsulated bacteria and need to be revaccinated against common pathogens [12,15]. Accordingly, it has been reported that HIV-1-infected individuals can develop a functional hyposplenism  that, ultimately, might be responsible for irreversible loss of the marginal zone-like B cells.
HIV-1 also disrupts the class switch recombination  that apart from somatic hypermutation is pivotal in the generation of an effective humoral response. HIV-1 also reduces the lifespan of B cells by increasing their susceptibility to apoptosis [18,19]. Interestingly, ART successfully recovers B-cell derangement in early-treated individuals, suggesting that the early control of the viremia would maintain the normal development of B cells and the humoral response [20,21].
To better understand why MEB cells are not fully recovered during HIV-1 infection, we have investigated their phenotype, functional capability, and spontaneous cell death (SCD) susceptibility in HIV-1-infected individuals. Our results show that functional hyposplenism or apoptosis do not explain the lack of recovery of the marginal zone-like B cells in ART-HIV-1+ individuals. We also show an accumulation of MEB cells that may be originated through an early germinal center reaction: IgM-only MEB cells in ART-naive individuals and CD27−IgG+ in ART-HIV-1 study participants. In addition, our analysis of susceptibility to SCD of the main B-cell subsets suggests that the increased apoptosis of B cells observed in ART-treated individuals is because of the higher frequency of the apoptosis-prone CD27− MEB cells. Finally, B cells from HIV-1-infected individuals show a reduced proliferative capacity against BCR or toll-like receptor (TLR) stimulation that is partially recovered by ART. Interestingly, the proliferative capacity is fully recovered by the synergistic effect of BCR and TLR signaling, indicating that the response against neoantigens and vaccines might be improved by modifying vaccination protocols or vaccine formulations.
Material and methods
A cross-sectional study was designed to analyze the effect of the HIV-1 infection in the MEB repertoire. The study was approved by the Institutional Review Board of the Hospital Universitari Germans Trias i Pujol (Barcelona, Spain; EO-10-071). Aviremic adult HIV-1-infected study participants on ART (n = 51), viremic ART-naive (n = 25), and uninfected controls (n = 36) were recruited at the Hospital Universitari Germans Trías i Pujol and Hospital Universitari Vall d’Hebron. All participants gave written informed consent to participate in the study. The main characteristics of the individuals enrolled in this study are described in Table 1.
Flow cytometry analysis
Peripheral blood was collected by venipuncture in EDTA tubes and two different stainings were performed. For each one, 150 μl of whole blood were washed twice with 5 ml of phosphate-buffered saline (PBS) and then incubated for 15 min with the following antibodies: tube A: anti-CD19 allophycocyanin/cyanine 7 (APC/Cy7), anti-IgD phycoerythrin (PE), anti-IgM APC, anti-CD38 peridinin chlorophyll protein complex/cyanine 5.5 (PerCP/Cy5.5) (all from BD Pharmingen, San Jose, California, USA) anti-CD1c fluorescein isothiocyanate (FITC) (Santa Cruz Biotechnology, Dallas, Texas, USA), and anti-CD27 PE/Cy7, (eBiosciences, ThermoFisher Scientific, Waltham, Massachusetts, USA). Finally, samples were washed with 5 ml of PBS and 2.5 ml/tube of BD FACS lysing solution (BD Bioscience, San Jose, California, USA) were added and incubated for 12 min at room temperature. After washing with 5 ml of PBS, cells were resuspended in PBS.
Tube B: first, samples were extracellularly stained as above with: anti-CD19 APC/Cy7, anti-CD38 PerCP/Cy5.5 (all from BD Pharmingen), anti-CD1c FITC (Santa Cruz Biotechnology), and anti-CD27 PE/Cy7 (eBiosciences). After washing with 5 ml of PBS, cells were fixed and permeabilized (fix and perm cell fixation and cell permeabilization kit; ThermoFisher Scientific, Waltham, Massachusetts, USA) and incubated for 15 min with F(ab)2 goat antihuman IgG (Fragment crystallizable (Fc) specific) PE and goat antihuman IgA-DyLight649 (Jackson Immunoresearch, Chester County, Pennsylvania, USA). After that, cells were washed, resuspended in PBS, and analyzed in a BD LSR-II cytometer (BD Biosciences). Negative controls were performed in parallel. For tube A: anti-CD19 APC/Cy7 and anti-IgD PE and for tube B: anti-CD19 APC/Cy7, anti-IgG PE, and anti-IgA-DyLight649.
B-cells subsets were identified as follow: marginal zone B-cells: CD19+CD27+IgD+IgM+; switched MEB: CD19+IgD−IgM−; IgM-only MEB: CD19+IgD−IgM+CD27+; IgA+ MEB: CD19+IgG−IgA+, and IgG+ MEB: CD19+IgG+IgA−.
Quantification of plasma antibody isotypes
Plasma immunoglobulin (Ig) isotypes were quantified by ELISA. Briefly, 96 well MaxiSorp plates (Nunc, Roskilde, Denmark) were coated with the following antibodies (1 μg/ml in PBS): affinipure F(ab’)2 fragment goat anti-human IgG, Fcγ fragment-specific; affinity pure goat anti-human IgA antibody α-chain specific (Jackson Immunoresearch), or mouse anti-human IgM antibody (clone G20-127; BD Pharmingen). Then, plates were washed and blocked with 1% bovine serum albumin (BSA) (Sigma Aldrich, Saint Louis, Missouri, USA) in PBS. The N-Prot standard serum (Siemens Healthcare Diagnostics, Marburg, Germany) was used as standard. Plasma samples and standards were diluted in PBS/1% BSA/0.05% tween-20. Horseradish peroxidase (HRP)-goat anti-human IgG, Fc-specific, HRP-goat anti-human IgA antibody α-chain specific, or HRP-goat anti-human IgM, Fc5 μ fragment specific (all from Jackson Immunoresearch) were used as detection antibodies and plates were revealed with o-phenylenediamine dihydrochloride (Sigma Aldrich).
Absolute cell count
Absolute count of B, T, and natural killer (NK) cells was performed by flow cytometry. First, blood cells were stained with anti-CD45-V450 antibody (BD Biosciences), mixed with Perfect-Count Microspheres (Cytognos, Salamanca, Spain), and analyzed in a LSR II cytometer (BD Biosciences). Next, the frequency of the main lymphocyte populations was determined in blood stained with anti-CD45-V450, anti-CD19-V500, anti-CD3 APC/Cy7, anti-CD4 APC, anti-CD8 PerCP, anti-CD56 PE, and anti-CD16 FITC (BD Biosciences). Absolute count of each subset was determined according to the frequency of each subset and the total lymphocyte counts.
Quantification of pitted erythrocytes
The splenic function was analyzed by quantification of pitted erythrocytes as previously described . Briefly, peripheral blood from ART-HIV-1 study participants (n = 10) was collected by venipuncture in EDTA tubes. Two drops of blood were mixed with 0.5 ml of 1.25% glutaraldehyde and incubated for 2 h at 4°C. Then, samples were analyzed with an optical microscopy adapted with Nomarski optic. A total of 500 erythrocytes/sample were quantified. Results were expressed as percentage of pitted erythrocytes.
Spontaneous cell death quantification
Peripheral blood mononuclear cells (PBMCs) were purified by standard density gradient centrifugation using Ficoll–Hypaque (Atom Reactiva, Barcelona, Spain) and used immediately for ex-vivo SCD assays. B-cell death was evaluated by culturing PBMC (1 × 106 cells/ml) in Roswell Park Memorial Institute 1640 medium (RPMI-1640) supplemented with 10% of FBS (R10 medium; ThermoFisher Scientific) for 24 h. Then, PBMCs were harvested and incubated with 40 nmol/l of the potentiometric mitochondrial probe 3,3’-dihexyloxacarbocyanine Iodide (DIOC6(3)) (ThermoFisher Scientific) in R10 for 1 h at 37°C/5% CO2. After that, cells were stained with anti-CD19 APC/Cy7, anti-IgD PE, anti-CD38 PerCP-Cy5.5, anti-CD5 APC (BD Biosciences), and anti-CD27 PE/Cy7 (eBiosciences). Finally, 0.3 μm of Sytox Blue (ThermoFisher Scientific) was added and incubated for 10 min. Cells were acquired in a LSR-II flow cytometer (BD Biosciences). Live cells were identified as DIOC6(3) bright/Sytox Blue negative using FlowJo software (Tree Star Inc., Ashland, Oregon, USA).
B-cell proliferation assay
Freshly purified PBMCs were stained with 0.33 μmol/l carboxyfluorescein succinimidyl ester (CFSE) (ThermoFisher Scientific) for 5 min at room temperature in PBS 1% FBS. After extensive washes, cells were cultured in R10 medium supplemented with different stimuli: endotoxin-free CpG oligodeoxynucleotide 2006 (3 μg/ml; InvivoGen, San Diego, California, USA), R848 (1 μg/ml, Alexis Biochemicals, Lausen, Switzerland), and F(ab)2-goat anti-human Igs (5 μg/ml, Jackson Immunoresearch). After 4 days, cells were harvested and stained with anti-CD3 APC/Cy7, anti-CD19 PE/Cy7, anti-CD14 PerCP (BD Biosciences), and anti-IgD APC (Miltenyi Biotec, Bergisch Gladbach, Germany). B cells were identified as CD19+CD14−CD3−. Results were shown as the percentage of dividing cells (CFSE low).
Continuous variables were compared using nonparametric Mann–Whitney tests. Dunn's correction test was used for multiple comparisons. Spearman's correlation coefficient was calculated to assess the association between variables. Statistical analyses and plots were performed using GraphPad Prism v5 (GraphPad Software Inc., California, USA) with univariate two-tailed significance levels of 5%.
The B-cell compartment is highly affected by HIV-1 infection even though B cells are not a direct target of the virus [1,2]. To delve into our knowledge of B-cells perturbation in HIV-1 infection and their putative recovery after initiation of ART, we have designed a cross-sectional comparative study, including virologically-suppressed HIV-1-infected ART-treated individuals (ART-HIV-1), viremic-nontreated (ART naive) study participants, and uninfected controls (UC). Main characteristics of enrolled individuals are described in Table 1. Sex distribution was not uniform among study groups, the uninfected control group showed a lower frequency of men than HIV-1-infected groups. ART-naive HIV-1-infected group showed lower frequency of B cells in blood than ART-HIV-1 group (P = 0.007). As expected, and compared with uninfected controls, HIV-1-infected individuals showed a reduced CD4+ T-cell (P < 0.0001) and NK cell population (P < 0.05), and an expansion of the CD8+ T cells (P < 0.001; Table 1).
The lack of recovery of marginal zone B cells is not associated to functional hyposplenism
In agreement with previous reports [12,13], marginal zone-like B cells (CD19+IgD+IgM+CD27+) were reduced in HIV-1-infected study participants, and ART did not successfully restored the size of this compartment (Fig. 1a–c). The frequency of these cells did not correlate with the nadir of CD4+ T cells or the HIV-1 viral load and a weak negative correlation was observed with the percentage of CD4+ T cells in viremic individuals (Fig. S1, http://links.lww.com/QAD/B184). Because a reduction of marginal zone B cells has been also documented in individuals with functional hyposplenism, such as after splenectomy  or inflammatory bowel disease , we explored splenic function in ART-HIV-1 individuals. The numbers of pitted erythrocytes were determined in blood samples from a representative subset of those individuals (n = 10; Fig. 1d) and showed low values (0.78 ± 0.82%), within the normal range (<4%) , that were not correlated with the frequency of IgM+IgD+CD27+ B cells (Fig. 1e), suggesting that functional hyposplenism does not explain the lack of recovery of marginal zone-like B cells in ART-HIV-1 study participants.
CD27− and IgM-only memory B-cell subsets are expanded in HIV-1-infected individuals
HIV-1 infection induces a profound depletion of CD27+ MEB [10,15]. However, CD27+ identifies only a subset of human MEBs, which can be classically identified as switched B cells regardless of CD27 expression (Fig. 2a and b). Flow cytometry analysis of switched B cells (IgD−IgM−) (Fig. 2a) showed that this compartment was reduced in ART-naive HIV-1-infected individuals but it was rescued in ART-treated study participants (Fig. 2c and d). As expected, HIV-1-infected study participants showed lower levels of CD27 in both IgG+ or IgA+ MEB cells, indicating that despite ART restored the numbers of switched B cells, it failed in recovering the CD27+ MEB (Fig. 2b and e). Moreover, ART-HIV-1-infected study participants showed a higher IgG/IgA ratio than uninfected controls, suggesting the existence of a bias during the class switching process or a differing expansion of IgG+ and IgA+ B cells (Fig. 2f). Consistently, although the percentage of CD27+ cells was higher in IgA+ than in IgG+ B cells, the lack of CD27 was observed in both subsets (Fig. 2e). The expression of IgG and IgA could not be evaluated in samples from ART-naive HIV-1-infected individuals because of the high background observed (data not shown), maybe because the deposit of cytophilic antibodies on the surface of these cells.
Conversely, IgM-only MEB cells (Fig. 2a) were increased in ART-naive HIV-1-infected individuals (Fig. 2g and h). Given that ART restored this compartment to the levels of the uninfected controls group (Fig. 2g and h), the expansion of these cells might be driven by viral replication but in an indirect way, as no correlation with viral load was observed (data not shown). Furthermore, in viremic individuals, frequency of IgM-only MEB correlated with plasma levels of total IgM (Fig. 2i), indicating that these cells might contribute to the increased levels of IgM in viremic as compared with ART-treated individuals (Fig. S2, http://links.lww.com/QAD/B184). No correlation was observed in ART-HIV-1 or uninfected controls groups (data not shown). The level of total IgA was similar among the three study groups, whereas ART-naive individuals showed higher levels of IgGs in plasma, a characteristic of viremic individuals (Fig. S2, http://links.lww.com/QAD/B184).
In summary, HIV-1-infected individuals showed a reduced switched-MEB cell compartment that could be partially restored with ART. These cells showed lower levels of CD27 expression and a bias during the switching process, suggesting different functional properties. Furthermore, viremic HIV-1-infected study participants showed an expansion of the IgM-only B-cell population that seems to be driven by viral replication and might contribute to the plasmatic levels of total IgM.
CD27-switched memory B cells showed the highest spontaneous cell death susceptibility
B cells from HIV-1-infected study participants show a higher cell death rate that contribute to the depletion of these cells over the course of the infection [18,19]. However, little is known about the susceptibility to SCD of the different B-cell subsets. To better understand this point, SCD was determined in major B-cell subsets ex vivo by flow cytometry (Fig. 3a). The results showed that both CD27− subsets: CD27−IgD+ naive and CD27− switched MEBs, were more susceptible to SCD than CD27+ B cells (Fig. 3b). Among them, the CD27− switched B-cell population showed the highest susceptibility to SCD (Fig. 3b). When B cells from HIV-1-infected individuals were analyzed, ART-naive study participants showed higher death rate in all subsets assayed except for marginal zone-like B cells, which showed similar survival profile (Fig. 3c–f). Interestingly, B-cell subsets from ART-treated individuals showed similar degree of cell survival (Fig. 3c–f), indicating that ART normalized the susceptibility to SCD in HIV-1-infected study participants. It also highlights that viremia may be the major driving force responsible for B-cell death increase observed over the course of infection.
Proliferative capacity against B cell receptor and TLR stimulation
The proliferative capacity of B cells to BCR (anti-Ig) and/or TLR stimulation (CpG and R848) was assayed in PBMCs by a CFSE dilution assay (Fig. 4a). The results showed that B cells from ART-naive study participants had an impaired proliferative capacity, measured as the percentage of CFSE-low cells, when stimulated with an anti-Ig (Fig. 4b), CpG (TLR9), and, to a lesser extent, to the R848 (TLR7/8; Fig. 4 c and d). However, the proliferative capacity was restored when cells were stimulated through the BCR and the TLR (both 9 or 7/8), indicating that both stimuli play a synergistic role in B-cell activation and suggesting that B-cell response might be recovered by proper stimulation (Fig. 4e and f). Interestingly, ART seemed to normalize the B-cell response of HIV-1-infected individuals indicating that viral replication might be responsible for B-cell functional impairment.
HIV-1 induces a profound dysregulation of MEB cells that might negatively impact both humoral and cellular immunity. HIV-1-infected individuals show a notorious reduction of the marginal zone-like [12,13] and the CD27+ MEB cells  that correlates with the poor response against neoantigens and the drop of antibody titers against common vaccine antigens [12,15]. Although ART can restore some alterations [14,25], little is known about the mechanisms involved in the lack of recovery of marginal zone-like and CD27+ MEB cells in HIV-1 infected individuals.
Marginal zone B-cells play a major role in the T-independent response against blood-borne pathogens, and may contribute to the development of T-dependent humoral responses by delivering antigens from the marginal zone to B-cell follicles . Blood marginal zone-like B cells are reduced in HIV-1-infected study participants and are not recovered by ART [12,13]. Our results showed this fact is not because of an increased susceptibility to apoptosis in HIV-1+ individuals. Blood marginal zone-like B cells are reduced in splenectomized individuals and in those suffering from functional hyposplenism, as reported for intestinal bowel disease . As functional hyposplenism has been also described in AIDS study participants [16,27], we explored if it may account for the lack of recovery of marginal zone-like B cells in virologically-suppressed HIV-1-infected individuals. Functional hyposplenism is characterized by an increase of damaged erythrocytes in blood (pitted erythrocytes) that inversely correlates with the frequency of marginal zone-like B cells [23,24]. Our results showed that the count of pitted erythrocytes in ART-HIV-1-infected individuals was within the normal range and did not correlate with the percentage of blood marginal zone-like B cells, indicating that functional hyposplenism cannot fully explain the lack of recovery of these cells. However, it is still possible that HIV-1 infection affects more severely the marginal zone than the red pulp of the spleen, resulting in a reduction of IgM+IgD+CD27+ B cells but a normal count of pitted erythrocytes. Accordingly, atrophy of marginal zone was observed in spleen samples from HIV-1-infected individuals .
In addition to marginal zone B cells, we also analyzed other MEB cells involved in the T-dependent humoral response. Despite CD27+ MEB cells are not fully restored by ART, unless early ART initiation [20,21], we have shown that the number of the switched MEB-cell population was normalized by an expansion of CD27− subsets. These cells have been described in other diseases affecting B-cell function, such as rotavirus infection, schistosomiasis [29,30], and systemic lupus erythematosus . Moreover, we have also identified an increase in the IgM-only MEB population in ART-naive HIV-1-infected individuals whose generation or expansion might be driven by viremia as its frequency was normalized by ART. Recently, it has been described that both IgG+CD27− and IgM-only MEB may arise from an early germinal center reaction . Germinal centers are drastically affected by HIV-1 infection: T-follicular helper cells are dysfunctional [21,33] and a high apoptosis is detected within germinal center of gastrointestinal tract early after HIV-1 acquisition . Moreover, the class switch recombination process, a pivotal function of germinal center, seems to be also dysregulated in HIV-1 infection [17,35]. Accordingly, we observed a higher IgG/IgA ratio in HIV-1-infected individuals. As anti-HIV-1 humoral response is dominated by IgG1 , it is possible that viral replication is directly involved in this imbalance. Overall, all these data suggest that a deregulated germinal center reaction may hinder the development of the CD27+ MEB cells promoting the accumulation of early germinal center memory subsets (CD27−IgG+ and IgM only). Alternatively, these cells might derive from a germinal center-independent reaction or might be expanded to replenish the niche that was previously occupied by the CD27+ MEB cells. Irrespective of their origin, the fact that HIV-1-infected individuals were enriched in MEB cells that were not originated from a mature germinal center reaction may have important functional consequences impairing the development of broadly neutralizing antibodies , as these cells might show a reduced somatic hypermutation , and a limited affinity maturation.
We have also shown that CD27− subpopulations, and particularly the CD27– switched one, showed the highest susceptibility to SCD, especially in viremic individuals. However, B cells from the ART treated and uninfected control groups showed an equivalent susceptibility to SCD, indicating that once viremia is controlled, the survival of each B-cell subsets is restored. Nevertheless, a higher frequency of the apoptosis-prone CD27– switched B-cells was observed in ART-HIV-1 individuals, suggesting that the MEB compartment of these study participants might not be as long lived as the uninfected control one. Interestingly, IgG+CD27− MEB cells were enriched in anti-HIV-1 envelope-specific B cells .
HIV-1 infection impairs proliferative responses against BCR stimulation that was not fully recovered by ART. Furthermore, the TLR agonists CpG 2006 (TLR9) and R848 (TLR7/8) induced a weak stimulation of B cells from viremic individuals. Thus, the poor response observed against BCR or TLR stimulation may also contribute to the generation of a defective germinal center reaction that ultimately may result in the generation of MEB subsets with an early germinal center phenotype. Apart from, the low response against TLR agonists shown by MEB cells could also play a major role in their reduced lifespan . Interestingly, when B cells were stimulated through BCR and TLR, the proliferative capacity was restored indicating that both stimuli played a synergistic role. These results are in line with the reduced response of HIV-1-infected individuals against neo and common vaccine antigens and highlight the importance of tailoring adjuvants to improve vaccine responses in those individuals. Accordingly, the response of HIV-1-infected study participants to the hepatitis B vaccine had been notoriously improved by the administration of a doubling vaccine doses and by the addition of the TLR9 agonist CPG 7909 .
Monitoring marginal zone B cells, IgM-only MEB, IgG/IgA MEB, and the frequency of CD27 in HIV-1-infected study participants could be useful to guide clinical management as it might provide information on the immune damage prior or after ART initiation. Accordingly, the early initiation of ART after HIV acquisition seems to preserve the marginal zone B-cell compartment . Additionally, expression of IgG, IgA, and CD27 in the switched MEB-cell compartment could help to evaluate how the immune system is wearing. However, our work might be limited by the unbalanced sex distribution among the study groups. To address this point, we compared main variables between men and women observing no significant differences. Therefore, although sex must be considered as a putative confounding factor, it does not alter the conclusion of the present work.
In summary, we have shown that functional hyposplenism and SCD are unrelated to the lack of recovery of the marginal zone-like B cells in ART-HIV-1-infected individuals. Moreover, we have described a new IgM-only MEB-cell subtype dysregulated and an ex-vivo apoptosis-prone CD27− MEB subtype accumulated in HIV-1+ study participants. Both observations link B-cells dysregulation to germinal center and provide new information that might explain the poor and short humoral response observed to some antigens. Furthermore, we have shown that the poor B-cell response may be satisfactorily recovered with the proper stimulation, suggesting that HIV-1-infected individuals might benefit of new vaccination protocols or new vaccine formulations.
J.C., L.M.M.A., M.L.R.C., M.C., and M.M. performed experimental work. E.N., J.P., J.N., and M.C. selected and recruited patients and analyzed clinical data. E.V. and F.M. performed the splenic function analysis. J.C., E.N., B.C., and J.B. designed the study, analyzed the results, and wrote the manuscript. All participants have read and approved the manuscript.
The work was supported by the HIVACAT Program, the CERCA Program (Generalitat de Catalunya), the Spanish AIDS network ‘Red Temática Cooperativa de Investigación en SIDA’ (RD12/0017/0002 and RD16/0025/0041), the ‘Fondo de Investigaciones Sanitarias,’ and FEDER ‘Fondo Europeo de Desarrollo Regional’ (grant number PI14/01307, to J.B.). J.B. is a researcher from Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol supported by the Health Department of the Catalan Government (Generalitat de Catalunya). J.C. was supported by a Sara Borrell postdoctoral Fellowship from ‘Instituto the Salud Carlos III’, Spain.
Conflicts of interest
There are no conflicts of interest.
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