ORIGINAL RESEARCH published: 15 January 2021 doi: 10.3389/fimmu.2020.614319 A Longitudinal Analysis Reveals Early Activation and Late Alterations in B Cells During Primary HIV Infection in Mozambican Adults Montse Jiménez 1†, Lucía Pastor 1,2,3†, Victor Urrea 1, Marı́a Luisa Rodrı́guez de la Concepción 1, Erica Parker 4, Laura Fuente-Soro 2,3, Chenjerai Jairoce 3, Inacio Mandomando 3, Jorge Carrillo 1, Denise Naniche 2,3 and Julià Blanco 1,5* Edited by: Veerle Somers, 1 AIDS Research Institute-IrsiCaixa, Institut Germans Trias i Pujol (IGTP), Hospital Germans Trias i Pujol, Universitat Autonoma University of Hasselt, Belgium de Barcelona, Badalona, Spain, 2 ISGlobal, Barcelona Institute for Global Health, Hospital Clı´nic–Universitat de Barcelona, Reviewed by: Barcelona, Spain, 3 Centro de Investigação em Saúde da Manhiça (CISM), Maputo, Mozambique, 4 Faculty of Health and Christopher Sundling, Medical Sciences, University of Western Australia, Perth, WA, Australia, 5 Universitat de Vic—Universitat Central de Catalunya Karolinska Institutet (KI), Sweden (UVIC-UCC), Vic, Spain Philip Cohen, Temple University, United States Primary HIV infection (PHI) and subsequent chronic infection alter B-cell compartment. *Correspondence: Julià Blanco However, longitudinal analysis defining the dynamics of B-cell alterations are still limited. jblanco@irsicaixa.es We longitudinally studied B-cell subsets in individuals followed for 1 year after PHI (n = 40). † These authors have contributed Treated and untreated chronic HIV infected (n = 56) and HIV-uninfected individuals (n = 58) equally to this work were recruited as reference groups at the Manhiç a District in Mozambique. B cells were Specialty section: analyzed by multicolor flow-cytometry. Anti-HIV humoral response and plasma cytokines This article was submitted to were assessed by ELISA or Luminex-based technology. A generalized activation of B cells B Cell Biology, a section of the journal induced by HIV occurs early after infection and is characterized by increases in Activated Frontiers in Immunology and Tissue-like memory cells, decreases in IgM-IgD- (switched) and IgM-only B cells. Received: 05 October 2020 These alterations remain mostly stable until chronic infection and are reverted in part by Accepted: 01 December 2020 ART. In contrast, other parameters followed particular dynamics: PD-1 expression in Published: 15 January 2021 memory cells decreases progressively during the first year of infection, Transitional B cells Citation: Jiménez M, Pastor L, Urrea V, expand at month 3–4 after infection, and Marginal zone-like B cells show a late depletion. Rodrı´guez de la Concepción ML, Plasmablasts expand 2 months after infection linked to plasma viral load and anti-p24 Parker E, Fuente-Soro L, Jairoce C, Mandomando I, Carrillo J, IgG3 responses. Most of well-defined changes induced by HIV in B-cell activation and Naniche D and Blanco J (2021) A memory subsets are readily observed after PHI, lasting until ART initiation. However, Longitudinal Analysis Reveals subsequent changes occur after sustained viral infection. These data indicate that HIV Early Activation and Late Alterations in B Cells During Primary HIV infection impacts B cells in several waves over time, and highlight that early treatment Infection in Mozambican Adults. would result in beneficial effects on the B-cell compartment. Front. Immunol. 11:614319. doi: 10.3389/fimmu.2020.614319 Keywords: B-cell activation, plasmablasts, marginal zone (MZ) B cell, transitional B cell, PD-1 Frontiers in Immunology | www.frontiersin.org 1 January 2021 | Volume 11 | Article 614319 Jiménez et al. B-Cells in Acute HIV Infection INTRODUCTION alterations with cytokine and humoral responses elicited against HIV. Our data suggest the occurrence of a multi-wave HIV infection mainly targets myeloid and CD4+ T cells (1); immunological B-cell damage, characterized by early activation however, the damage infringed by chronic viral replication associated with antiviral responses and subsequent events that spreads to the whole immune system, including CD8 T cells extend several months after viral setpoint has been achieved. and B cells (2, 3). For T and B-cell compartments, a generalized hyper-activation status has been widely described, that specifically in B cells is characterized by an increased frequency of activated memory cells and hypergammaglobulinemia (4). A MATERIAL AND METHODS large number of studies have provided a detailed description of the impact of chronic HIV infection (CHI) on the phenotype and Study Population function of B cells [excellently reviewed in (5)], demonstrating The present study is a sub-analysis of a prospective cohort of that the homeostasis of B-cell subsets is drastically affected by primary HIV-infected adults enrolled and followed up for 12 CHI resulting in expansion of immature/transitional cells (6), months in the Gastro-intestinal biomarkers in Acute-HIV activated memory cells, and differentiated plasmablasts (4). infected Mozambican Adults (GAMA) study (15, 21–23). The Perturbations of mature B cells include an impact on naïve study population was enrolled between 2013 and 2014 at the cells, that are reduced in number and function early after HIV Manhiça District Hospital (MDH) in Southern Mozambique infection (7), and memory B cells, which seem to be the most (15, 22). affected subset, showing lower numbers of switched IgM-IgD- Participants (>18 years of age) were screened at the outpatient memory cells, expansion of IgM-only memory cells (8) and an clinic of MDH for non-specific febrile symptoms or voluntary aberrant Tissue-like memory (TLM) repertoire (9). Besides HIV testing. PHI participants (n = 57) were identified by altering B-cell subsets, HIV infection also impairs B-cell negative or indeterminate rapid serology test and positive function. The expression of the check-point receptor HIV-RNA test. A control population was established by programmed cell-death protein 1 (PD-1) has been widely randomly selecting HIV-uninfected individuals at the associated with B-cell dysfunction and increased levels of this outpatient clinic (n = 58). Additionally, adults with marker have been described in primary HIV infection (PHI) (7) documented HIV diagnosis for more than 12 months and CHI (10, 11). PD-1 may impact general (12) and specific attending routine scheduled visits were enrolled as CHI anti-HIV humoral responses by affecting different subsets, patients. CHI patients were included as ART-naïve or treated, including TLM cells, which appear to encompass an important depending whether they had previously initiated treatment fraction of HIV specific B cells (13). B-cell dysfunction affects according to the national guidelines at recruitment (ART was both T-dependent and independent responses. The latter is provided to individuals with a CD4 T-cell count ≤350 cells/mm3 controlled by the Marginal Zone-like (MZ-like) B-cell or presenting AIDS-associated diseases). A subset of PHI population, which is reduced in CHI individuals, although the individuals (n = 44) were followed up for 12 months as splenic function seem to be maintained (8). previously described (15). Briefly, only plasma samples were As for other viral infections, PHI induces a rapid immune available at PHI recruitment, while follow-up samples response including sequentially: a prominent response of from month 2 (M2) to month 12 post infection (M12), CHI inflammatory cytokines (the “cytokine storm”) (14), followed and HIV-uninfected individuals included stool and by a rapid expansion and activation of CD8 T cells and antibody cryopreserved PBMC samples, which were used for B-cell secreting cells (plasmablast) (7, 14, 15). However, the challenges immunophenotyping. Sample timepoints were adjusted by of identifying PHI and the lack of longitudinal studies addressing Fiebig stage as described (15). B-cell phenotypes and function in recently HIV infected patients After recruitment, demographic and clinical data was have resulted in a lack of information on the timing of events collected and medical consultation and HIV counseling was leading to chronic B-cell damage after HIV-infection, especially provided to all participants. CD4 and CD8 T-cell counts in in Sub-Saharan African populations (16). Studies addressing fresh blood was determined using Trucount tubes and a early/acute or hyperacute HIV-infected PHI individuals FACScalibur flow cytometer. PBMCs were isolated by Ficoll indicate early alterations of B cells similar to those reported in density gradient and were stored in liquid nitrogen. Plasma chronic infection (7, 17); however, the impact of such early Viral Load (VL) determination was performed as previously alterations on humoral responses to HIV (17) and on chronic B- described (15). Additional testing for most prevalent infections cell dysfunction are still poorly defined (18, 19). Interestingly, in the area, including malaria, hepatitis B virus (HBV), syphilis, antiretroviral treatment (ART) fails to fully normalize B-cell gastro-intestinal protozoa, bacteria, and parasites was also dysfunction (8), including lack of response to vaccines which performed (15). remain impaired in treated HIV-infected individuals (20). Multiplex biomarker profiling was performed for a total of 61 In this study, we provide a longitudinal characterization of immune response inflammatory biomarkers in plasma samples different B-cell subsets over the first year after PHI in a cohort of (22, 23). In parallel, humoral responses against HIV proteins Mozambican adults and we compare these changes with CHI Env, Gag, and Integrase (In) were determined by Luminex based individuals, either ART-naïve or treated, and HIV-uninfected technology in plasma samples (22). The phenotype of peripheral subjects. Additionally, we explore the association of B-cell T cells was previously described (15). Frontiers in Immunology | www.frontiersin.org 2 January 2021 | Volume 11 | Article 614319 Jiménez et al. B-Cells in Acute HIV Infection B-Cell Immunephenotyping described previously (15). Fifty-eight HIV-uninfected individuals Cryopreserved PBMCs were thawed at 37°C, washed in RPMI/60% were randomly selected from screened negative individuals and 56 and RPMI/20% of fetal bovine serum (FBS), and incubated for 1 h at CHI individuals were recruited at the Manhiça District Hospital, 26 37°C in RPMI/10%FBS. PBMCs were then stained with the Fixable of them were untreated (CHI-naïve) and 30 were on ART (CHI- Viability Stain-FVS780r (APC-H7 detect, BD Biosciences) for 15 ART). The demographic and clinical characteristics of the 40 PHI mins. After PBMCs washing in PBS/1%FBS, cells were incubated in individuals who started follow-up, the HIV-uninfected and the CHI a U-bottom 96-well plate at a density of 1.5 million/well and stained groups, have been previously described (15). Briefly, PHI with selected 14-color panel including: anti-CD19-AF700 (Clone individuals were mostly young (mean 27-year-old), females HIB19), anti-IgD-Pe-Cy7 (Clone IA6-2), anti-IgM-BB515 (Clone (60%), and most prevalent co-infections were HBV, intestinal G-20-127), polyclonal goat anti-IgA-Dylight®649 (from Jackson infections, syphilis, and malaria [<20% of recruited individuals Immunoresearch), CD10-BV650 (Clone H10a), CD21-PE-CF594 presented some of these infections, Supplementary Table 1 (15)]. (Clone B-LY4), CD27-BV510 (Clone L128), CD38-BV786 (Clone After adjusting for time since infection according to Fiebig HIT2), CD45-RB-PE (Clone MT4), CD86-PerCP-Cy5.5 (Clone stage, peak VL was identified at 1 month after infection, M1) 2331), CD279 (PD-1)-BV421 (Clone EH12.1), all from BD with a median VL value of 6.9 RNA Log10 copies/ml (IQR 6.2– Biosciences unless indicated, for 15 mins. After washing twice in 7.5), rapidly stabilizing afterwards (Figure 1A, gray line) (15). PBS/1% FBS, cells were fixed in PBS/1% formaldehyde, acquired in CD4 T-cell counts were significantly lower in PHI as compared a BD LSRFortessa (BD Biosciences) cytometer using a plate HTS to HIV-uninfected individuals (median 565 cells/µl at M2 after loader (BD Biosciences) and analyzed with FlowJo software (Tree infection and 855 cells/µl, respectively, p < 0.001) and remained Star). Gating strategy is described in Supplementary Figure 1. stable over the first year of infection (15). CD8 T cells showed Lymphocyte gate was defined manually by morphological maximal expansion at M2 and slowly decreased until M5-6 to parameters excluding non-viable cells. B cells were identified as achieve the immunological setpoint (15). CD19+ CD21+/− cells and gated according to the expression of different markers to identify B-cell maturation stages as described in Dynamics of B-Cell Subsets and Supplementary Figure 1. Association With Viral Pathogenesis We initially analyzed the frequency of B cells (CD19+) and their Statistical Analysis functionally distinct subsets defined by CD21 and CD27 Intergroup comparisons were performed using the Fisher exact test expression: Naïve cells (CD21+CD27−), resting memory cells for categorical variables and the non-parametric Kruskal-Wallis test (CD21+CD27+), activate memory cells (CD21–CD27+), and for continuous variables, using Dunn’s test for post-hoc pairwise TLM cells (CD21−CD27−). The dynamics of the different comparisons. Spearman’s correlation was used to assess correlations populations along the first year of HIV-infection was analyzed between continuous variables and multiple testing was further using non-parametric and linear regression modeling as adjusted by False Discovery Rate (FDR). Relative changes (Z- described (15). score) with respect to the control (HIV-uninfected) group have No significant changes in the frequency of B cells were been represented by a transformation of the fitted longitudinal observed over PHI (Figures 1A, B). However, frequencies of models by subtracting the mean and dividing by the standard the main B-cell subsets rapidly changed after infection compared deviation of Control group distribution (logarithmic transformation to HIV-uninfected individuals, with a significant reduction in was used for non-normal distributions). Longitudinal models for naïve and resting memory cells at M2 (p < 0.01), concomitant to the different immunological variables were modeled by fitting expansions of activated and TLM cells (p < 0.005). Most changes smoothing-splines mixed-effects models using the “sme” package remained stable over the first year of infection with no significant of R. To infer if there was a significant association of selected differences between M2 and M12 after infection (Figure 1). biomarkers with the time variable, polynomial time effects Consistently, similar alterations remained in CHI-naïve, being approximation until third degree were fitted using linear mixed- partially reverted in CHI-ART individuals (Figures 1C–J). effects regression models. Best model was selected based on The initial alterations of B cell were associated with clinical likelihood-ratio tests under maximum likelihood model and immunological parameters. The expansion of activated estimations. Statistical analyses were performed using R-3.3.1 and memory B cells observed at M2 negatively correlated with CD4 Stata14 software. T-cell frequency (p = 0.022), positively correlated with CD8 T- cell activation (HLA-DR+CD38+ cells, p = 0.033), and showed a positive trend with VL that did not reach statistical significance. RESULTS A similar observation was noticed for the early increase of TLM cells, although in this case only the negative association with CD4 Characteristics of the Study Population T cells was significant (p = 0.043, Figure 2). From the 57 individuals identified as PHI, 44 attended the follow-up visits and provided blood samples for PBMC isolation and Dynamics of PD-1 Expression subsequent B-cell subset analysis. Among them 21, 5, 7, and 11 The expression dynamics of PD-1 was characterized in the were categorized into Fiebig I-III, Fiebig IV, Fiebig V, and Fiebig VI above-defined B-cell subsets. As compared to HIV-uninfected stages, respectively, and were adjusted for time since infection as individuals, naïve and resting memory cells in PHI showed an Frontiers in Immunology | www.frontiersin.org 3 January 2021 | Volume 11 | Article 614319 Jiménez et al. B-Cells in Acute HIV Infection A B C D E F G H I J FIGURE 1 | Dynamics of main B-cell subsets after HIV infection. Left panels show individual data observations, median, IQR (box), and min/max values (bars, except for outliers) across the different timepoints and study groups: HIV-uninfected (green), primary HIV infected (red, M indicates months after infection), and chronically naïve (CHI-naïve) or treated (CHI-ART) HIV infected individuals (light and dark blue respectively). Gray line represents the profile of VL dynamics for clinical reference. Right panels show the modeled dynamics as Z-score values for PHI individuals over HIV-uninfected individuals. Solid lines show non-parametric models, while dotted lines indicate the best fitting for polynomial time effects regression approximation. The frequency of total B cells (A, B) and the different subsets defined by CD21 and CD27 markers (C–J) is shown as percentage. ns non significant; *p < 0.05; **p < 0.01; ***p < 0.005. Frontiers in Immunology | www.frontiersin.org 4 January 2021 | Volume 11 | Article 614319 Jiménez et al. B-Cells in Acute HIV Infection A B FIGURE 2 | Association of B-cell activation with viral and immunological parameters. The alterations in Activated Memory (A) and TLM (B) B cells at month 2 after infection was correlated with virological and immunological parameters at the same timepoint: Plasma viral load (VL) as RNA Log10 copies/ ml, CD4 depletion (frequency of CD4 T cells), and CD8 activation (frequency of HLA-DR+CD38+ CD8 T cells). Spearman r and p values are shown in each graph. early (M2) increase of PD-1 expression at M2 that was only individuals (p < 0.05 and p < 0.001, respectively), and significant for resting memory cells (p < 0.01), while this remained stable over PHI and CHI, being also recovered in expression was normalized in CHI-ART (Figures 3A–D). In CHI-ART individuals (Figures 4E–H). contrast, the PD-1 expression in activated and TLM B cells was not significantly different at M2 as compared to HIV-uninfected Dynamics of Transitional and Marginal individuals, showing a progressive decrease during PHI. Also, the Zone-Like B Cells PD-1 expression in these two subsets was significantly lower in In addition to the classical memory/naïve B-cell classification, we CHI-naïve (p < 0.01) and seemed to be not normalized in CHI- also explored the dynamics of other relevant subsets in B-cell ART individuals, as compared to HIV-uninfected individuals function, known to be altered after HIV infection (7, 24). First, (Figures 3E–H). we analyzed the frequency of Transitional B cells, which are defined as CD19+CD10+CD38+CD27− cells (Supplementary Analysis of IgM/IgD Subsets Figure 1) and identify an intermediate state between immature To further define the dynamics of memory B cells, we next and mature B cells. The frequency of Transitional B cells showed analyzed the changes in B-cell subsets defined by the expression a complex behavior over PHI, with a significant increase after 3 of IgM and IgD. The IgM+IgD+ subpopulation remained stable months of HIV infection compared to M2 and uninfected over PHI showing values similar to HIV-uninfected or CHI individuals (p < 0.05 in both cases). This delayed expansion individuals (Figures 4A, B). In contrast, double negative was followed by a stepwise return to values of uninfected switched, IgD-only and IgM-only B-cell subsets, were rapidly individuals at M12. Despite this observation, transitional cells altered after HIV infection. The frequency of IgM-only B cells were significantly increased in CHI-naïve and normalized in significantly increased at M2, as compared to HIV-uninfected CHI-ART, as compared to HIV-uninfected individuals (Figures individuals (p < 0.001 and p < 0.05, respectively), and remained 5A, B). stable over PHI and CHI, being partly recovered in CHI-ART A second relevant subset is defined by CD19+CD27+IgD (Figures 4C, D). An opposite behavior was observed for IgD- +CD21+ and identifies innate-like or MZ-like B cells, which are only and switched B cells, whose frequency significantly involved in T-cell independent B-cell responses. The frequency decreased after infection as compared to HIV-uninfected of MZ-like-B cells showed a unique behavior among B-cell Frontiers in Immunology | www.frontiersin.org 5 January 2021 | Volume 11 | Article 614319 Jiménez et al. B-Cells in Acute HIV Infection A B C D E F G H FIGURE 3 | Dynamics of PD-1 expression in main B-cell subsets after HIV infection. As in Figure 1, left panels show individual data, median, IQR (box) and min/ max values (bars, except for outliers) across the different timepoints and study groups: HIV uninfected (green), acutely HIV infected (red, M indicates month after primoinfection), and chronic untreated or treated HIV infected individuals (dark and light blue respectively). Gray line represents the profile of VL dynamics for reference. Right panels show the modeled dynamics as Z-score values for PHI individuals over HIV-uninfected individuals. Solid lines show non-parametric models, while dotted lines indicate the best fitting for polynomial time effects regression approximation. The frequency of PD-1+ cells in the indicated subsets defined by CD21 and CD27 markers (A–H) is shown. ns non significant; *p < 0.05; **p < 0.01. subsets, with a remarkable stability over the first year of HIV Dynamics of Plasmablast Expansion infection and a significant decline in untreated CHI, as and Association With Cytokine and compared to HIV-uninfected individuals. Notably, this late Humoral Responses depletion of MZ-like B cells seemed to be irreversible since it Finally, we addressed the dynamics of antibody secreting cells was not recovered in CHI-ART individuals (Figures 5C, D). (plasmablast, CD38++CD27++ CD19+ cells). These cells showed Frontiers in Immunology | www.frontiersin.org 6 January 2021 | Volume 11 | Article 614319 Jiménez et al. B-Cells in Acute HIV Infection A B C D E F G H FIGURE 4 | Early loss of switched B cells and IgD-only B cells. As in Figure 1, left panels show individual data, median, IQR (box), and min/max values (bars, except for outliers) across the different timepoints and study groups: HIV uninfected (green), acutely HIV infected (red, M indicates month after primoinfection), and chronic untreated or treated HIV infected individuals (dark and light blue respectively). Gray line represents the profile of VL dynamics for reference. Right panels show the modeled dynamics as Z-score values for PHI individuals over HIV-uninfected individuals. Solid lines show non-parametric models, while dotted lines indicate the best fitting for polynomial time effects regression approximation. The frequency of the different B-cell subsets defined by IgD and IgM expression (A–H) is shown. ns non significant; *p < 0.05; **p < 0.01; ***p < 0.005. an early significant expansion at M2 compared to uninfected levels against three different viral antigens (the envelope individuals (p < 0.0005), expansion was transient and glycoprotein subunit gp41, the gag Capsid protein p24, and the progressively declined until month 7–8, remaining stable pol p31 integrase, Figure 6C). The IgG3 response against p24 afterwards (Figures 6A, B). To better describe the association showed the highest correlation with the plasmablast frequency of early plasmablast expansion and the elicitation of anti-HIV (r = 0.54, p = 0.0085), while there was no significant association humoral responses, we analyzed the correlation of the frequency with any other of HIV-specific responses. Then, the association of plasmablasts at the peak (M2) with the IgG and IgA expression of plasmablast frequencies with the VL and cytokine levels was Frontiers in Immunology | www.frontiersin.org 7 January 2021 | Volume 11 | Article 614319 Jiménez et al. B-Cells in Acute HIV Infection A B C D FIGURE 5 | Late impact on Transitional and Marginal Zone-like B cells. As in Figure 1, left panels show individual data, median, IQR (box), and min/max values (bars, except for outliers) across the different timepoints and study groups: HIV uninfected (green), acutely HIV infected (red, M indicates month after primoinfection), and chronic untreated or treated HIV infected individuals (dark and light blue respectively). Gray line represents the profile of VL dynamics for reference. Right panels show the modeled dynamics as Z-score values for PHI individuals over HIV-uninfected individuals. Solid lines show non-parametric models, while dotted lines indicate the best fitting for polynomial time effects regression approximation. The frequency of Transitional B cells (CD21+CD10+CD38+CD27−, A, B) and Marginal Zone-like B cells (CD27+IgD+CD21+, C, D) is shown. ns non significant; **p < 0.01; ***p < 0.005. analyzed at M1 and M2 after infection. The frequency of additionally show that these changes are commonly sustained plasmablasts positively correlated with VL at M2 (r = 0.513, p over the first year of HIV infection, and maintained in chronic = 0.0146) after infection, but poorly with peak VL at M1 (r = untreated individuals. The early effect of HIV infection on these 0.148, p = 0.509). From the wide range of analyzed cytokines, subsets shows some association with CD4 T-cell frequencies and negative correlations were only significant for CXCL16 and activated CD8 T cells, suggesting that the earliest changes in B sCD23 at M1 (r = −0.43, p = 0.0434), while the most cells may be associated with the massive viral replication and significant positive correlation with plasmablast frequencies CD4 T-cell depletion. In addition to these expected data, changes was found for GCSF at M1 (r = −0.57, p = 0.0054),. Additional in PD-1 expression in these subsets were observed over the significant positive correlations at M1 were observed for MCP-1, follow-up of PHI individuals. Lower PD-1 expression in Naïve IFN-alpha, TNF-alpha, and IL-8 (p < 0.05). At M2, only IL-12, and Resting memory B cells compared to Activated and TLM IL-8, and MCP-1 were positively associated with plasmablast cells has been previously reported (25, 26), along with a partial frequencies (p < 0.05), being IL-12 the most significant (r = decrease in PD-1 expressing memory cells in chronic infection −0.55, p = 0.0087, Figure 6D). (26). Consistently, our data indicate a gradual loss of PD-1 expression during PHI. This observation is particularly interesting, given the prominent role of PD-1 in B-cell DISCUSSION depletion during acute HIV infection (27). However, the specific role of PD-1 may be modulated by the expression of a We conducted a wide analysis of B-cell populations in peripheral wide range of other inhibitory receptors (LAIR-1, CD22. CD32b, blood of PHI individuals over the first year of infection. Our FCRL4, CD85), which are also perturbed by HIV infection (11), main objective was to characterize the dynamics of B-cell subsets and may therefore contribute to the unresponsive state of B cells during early HIV infection and evaluate their association with and to reduce the lifespan of these cells (28). humoral and inflammatory responses. The analysis of IgM and IgD expression in B cells showed an Previous studies reported changes in the distribution of increase in IgM only cells and a decrease in IgD only and CD21and CD27 expression by B cells in chronic or early HIV switched (IgM-IgD-) cells, as previously described (8). These infection (5, 8, 9). Our data indicate similar changes in AHI, changes showed a stable kinetics over PHI and chronic infection mainly characterized by a decrease in naïve and resting memory with a slight recovery in individuals that initiated ART, cells and an increase in activated and TLM B cells. We suggesting again that early events in HIV infection lead to Frontiers in Immunology | www.frontiersin.org 8 January 2021 | Volume 11 | Article 614319 Jiménez et al. B-Cells in Acute HIV Infection A B C D FIGURE 6 | Dynamics of plasmablasts and association with immune responses. As in Figure 1, (A) shows individual data, median, IQR (box) and min/max values (bars, except for outliers) across the different timepoints and study groups: HIV uninfected (green), primary HIV infected (red, M indicates months after infection), and chronically naive and ART-treated HIV infected individuals (dark and light blue respectively). Gray line represents the profile of VL dynamics for clinical reference. (B) shows the modeled dynamics as Z-score values for PHI individuals over HIV-uninfected individuals. Solid lines show non-parametric models, while dotted lines indicate the best fitting for polynomial time effects regression approximation. (C, D) show the correlation of the plasmablasts’ frequency at month 2 after infection with the specific humoral responses against HIV proteins and with the levels of cytokines at 1 or 2 months after infection. Color code for correlation coefficients are shown in each panel (upper left corner). ns non significant; **p < 0.01; ***p < 0.005. long-lasting immune perturbations. In contrast, Transitional and Transitional B cells, a heterogeneous pool of pre-mature B cells MZ-like B cells showed no significant differences at M2 early (29), peaked at month 3–4 post infection and normalized after HIV infection but later changes over the course of the afterwards, while levels were significantly increased in CHI- infection, as compared to levels in HIV-uninfected individuals. naïve individuals, as reported by others (6). Although Frontiers in Immunology | www.frontiersin.org 9 January 2021 | Volume 11 | Article 614319 Jiménez et al. B-Cells in Acute HIV Infection homeostatic mechanisms could be behind this observation, it is chronic infection. Transient changes were observed in unclear whether these mechanisms are triggered by rapid effects plasmablasts and transitional B cells (peaking at month 2 and of HIV infection on B-cell survival or by the cytokine storm month 3–4, respectively), while a late wave of effects impact associated with PHI. Indeed, the frequency of Transitional B cells specifically MZ-like B cells, which are stable over the first year of has been associated with plasma levels of IL-7 (30), a cytokine infection but drop in chronic infection. Our data also showed that is upregulated in acute (31) and chronic HIV infection. complex dynamics of check point receptor PD-1, whose changes Unfortunately, we could not evaluate such an association in our extend beyond virological and immunological setpoint cohort because IL-7 was not quantifiable in more than 75% of the (occurring at M3 and 6, respectively). Further detrimental samples and was therefore excluded from the analysis. A second events for B cells were associated with sustained viral particular dynamics was observed for MZ-like B cells. These cells replication, in particular MZ-like B-cell depletion and are involved in T-cell independent responses in spleen (32) and progressive damage of resting and switched memory B cells. its depletion in HIV infected individuals has been largely Therefore, we postulate that several waves of cytopathic events documented (7, 8, 33). However, our data show that MZ-like B affect B cells over the course of HIV infection, leading to the cells remain stable over early infection, at least until 1 year, and impaired responses to vaccines or antigens (either T-dependent they only irreversibly decline in chronic phases as compared to or independent) in chronic infection. These observations further levels in HIV-uninfected individuals. highlight the need for early ART initiation, which is known to Finally, plasmablast dynamics showed early expansion until minimize immune B-cell damage and recover B-cell function month 6 after infection (34). An in-depth analysis at the peak of (40, 41). plasmablast expansion showed a positive correlation with the levels of IgG3 anti-p24 antibodies, suggesting a predominant response to this antigen. Consistently, previous studies have shown that HIV-specific IgG3 responses, in particular against DATA AVAILABILITY STATEMENT p55Gag, peak during acute HIV infection (22, 35). In addition, The raw data supporting the conclusions of this article will be levels of some cytokines showed certain association with made available by the authors, without undue reservation. plasmablast expansion. While positive associations were observed for most inflammatory cytokines, in particular IL-12, which is involved in B-cell differentiation (36), this could be explained by the parallel association with VL, suggesting antigen ETHICS STATEMENT driven expansion. Of particular interest is the negative The studies involving human participants were reviewed and association observed for the levels of sCD23 and to a lesser approved by the Ministry of Health of Mozambique (461/CNBS/ extent CXCL6. Previous data in this cohort showed that plasma 12). The patients/participants provided their written informed sCD23 levels were significantly decreased prior to seroconversion consent to participate in this study. (22), which could explain the negative correlation. However, the functional implications are unclear and maybe related to the complex modulation of Th2 responses in which IgE and its receptor CD23 have been recently involved (37). AUTHOR CONTRIBUTIONS Although our study provides new insights on the dynamics of B cells during PHI, it has several limitations. First, due to study DN, JC, and JB designed the study and the analysis. LP, EP, LF-S, design, no data on B-cell subsets was available at the peak of IM, and CJ performed laboratory analysis. LP, MJ, VU, and JC viremia (M1), compromising the identification of earliest B-cell performed PBMCs phenotyping and validation of the data. MJ changes, such as the peak of plasmablast expansion, reported at and VU performed statistical analyses. MJ, VU, JC, DN, and JB day 7 post infection (17) or the rapid changes in PD-1 expression interpreted the data. MJ and JB drafted the paper. MJ, LP, VU, (27). Furthermore, differences found in the CHI groups could be DN, and JC provided relevant critical data review and reviewed influenced by older age and the national guidelines at the the manuscript writing. All authors contributed to the article and moment of the study (2013–2014) that recommended ART approved the submitted version. initiation in patients with CD4 T-cell counts ≤350 cells/mm3 or presenting with an AIDS-defining event. Late ART initiation and age are known to impact immune status and immune FUNDING recovery in HIV infected individuals (38, 39), thus potentially biasing the naïve and ART-treated CHI groups. A final limitation This work was supported by The Spanish Ministry of Science is associated with the loss to follow-up of PHI individuals. (Mineco) (SAF-2011-27901) to DN; Bill and Melinda Gates Although common in rural settings (56), low retention rate Foundation (OPP1068252) to DN and The Spanish Ministry of was further affected in our study by migration and pregnancy. Health through the Institute of Health Carlos III (ISCIII) (FI12/ In conclusion, our data suggest that the effects of HIV 00096) to LP and (DTS15/00185) to JB. The funders had no role infection on B cells occur in different waves. Early changes in study design, data collection and analysis, decision to publish, involve B-cell activation and are maintained over early and or preparation of the manuscript. Frontiers in Immunology | www.frontiersin.org 10 January 2021 | Volume 11 | Article 614319 Jiménez et al. B-Cells in Acute HIV Infection ACKNOWLEDGMENTS laboratory coordination between partner institutions. The authors are particularly grateful to all study participants. The authors are grateful for the continued support of the clinical ISGlobal, IrsiCaixa, and IGTP are members of the CERCA staff at the Manhiça District Hospital, as well as the study staff Program, Generalitat de Catalunya. working exhaustively at the field and laboratory at the Centro de Investigaçao de Saú de de Manhiça (CISM). The authors are particularly grateful to Marco A. Ferná ndez from the IGTP Flow Cytometry Core Facility for his technical support and advice SUPPLEMENTARY MATERIAL designing the cytometry panels. The authors thank Diana Barrios for her contribution to biomarker quantification, together with The Supplementary Material for this article can be found online Aina Casellas for her statistical guidance. The authors also thank at: https://www.frontiersin.org/articles/10.3389/fimmu.2020. Laura Puyol and Helder Bulo for their contribution to study and 614319/full#supplementary-material during hyperacute HIV-1 infection. 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