Karine Bollore

Characterization of a Transitional Preplasmablast Population in the Process of Human B Cell to Plasma Cell Differentiation

The early steps of differentiation of human B cells into plasma cells are poorly known. We report a transitional population of CD20low/−CD38− preplasmablasts along differentiation of human memory B cells into plasma cells in vitro. Preplasmablasts lack documented B cell or plasma cell (CD20, CD38, and CD138) markers, express CD30 and IL-6R, and secrete Igs at a weaker level than do plasmablasts or plasma cells. These preplasmablasts further differentiate into CD20−CD38highCD138− plasmablasts and then CD20−CD38highCD138+ plasma cells. Preplasmablasts were fully characterized in terms of whole genome transcriptome profiling and phenotype. Preplasmablasts coexpress B and plasma cell transcription factors, but at a reduced level compared with B cells, plasmablasts, or plasma cells. They express the unspliced form of XBP1 mRNA mainly, whereas plasmablasts and plasma cells express essentially the spliced form. An in vivo counterpart (CD19+CD20low/−CD38−IL-6R+ cells) of in vitro-generated preplasmablasts could be detected in human lymph nodes (0.06% of CD19+ cells) and tonsils (0.05% of CD19+ cells). An open access “B to Plasma Cell Atlas,” which makes it possible to interrogate gene expression in the process of B cell to plasma cell differentiation, is provided. Taken together, our findings show the existence of a transitional preplasmablast population using an in vitro model of plasma cell generation and of its in vivo counterpart in various lymphoid tissues.

Human Milk-Derived B Cells: A Highly Activated Switched Memory Cell Population Primed to Secrete Antibodies

While secretory Abs have been extensively explored in human breast milk, the existence, features, and functions of B lymphocytes remain largely unexplored in this compartment. We analyzed breast milk and blood lymphocytes from 21 lactating women, including 12 HIV-1-infected mothers. Breast milk B cells displayed a phenotype of class-switched memory B cells, with few IgD+ memory and naive B cells. We observed that breast milk B lymphocytes bore a unique profile of adhesion molecules (CD44+, CD62L−, α4β7+/−, α4β1+). Higher percentages of activated B cells (CD38+), large-sized B cells, plasmablasts, and plasma cells (CD19+, CD20low/−, CD27high, CD138+) were found as compared with blood. This indicates that a significant proportion of breast milk B cells underwent terminal plasma cell differentiation. We also observed a higher frequency of cells secreting Ig spontaneously in breast milk. Among these cells, IgG-secreting cells predominated over IgA-secreting cells as measured by Ig ELISPOT assays. Specific Ab-secreting cells were investigated following polyclonal activation using the CD40L ligation. Finally, the detection of anti-HIV-1-secreting cells demonstrates the existence of B cells specific to HIV-1 Ag in breast milk from HIV-1-infected women. Breast milk B cells display a phenotype strikingly different from blood, are primed to secrete Abs, and have a mucosal homing profile similar to B cells located in gut-associated lymphoid tissue.

IL-6 supports the generation of human long-lived plasma cells in combination with either APRIL or stromal cell-soluble factors

The recent understanding of plasma cell (PC) biology has been obtained mainly from murine models. The current concept is that plasmablasts home to the BM and further differentiate into long-lived PCs (LLPCs). These LLPCs survive for months in contact with a complex niche comprising stromal cells (SCs) and hematopoietic cells, both producing recruitment and survival factors. Using a multi-step culture system, we show here the possibility to differentiate human memory B cells into LLPCs surviving for at least 4 months in vitro and producing immunoglobulins continuously. A remarkable feature is that IL-6 is mandatory to generate LLPCs in vitro together with either APRIL or soluble factors produced by SCs, unrelated to APRIL/BAFF, SDF-1, or IGF-1. These LLPCs are out of the cell cycle, express highly PC transcription factors and surface markers. This model shows a remarkable robustness of human LLPCs, which can survive and produce highly immunoglobulins for months in vitro without the contact with niche cells, providing the presence of a minimal cocktail of growth factors and nutrients. This model should be useful to understand further normal PC biology and its deregulation in premalignant or malignant PC disorders.

CD4 + T cells spontaneously producing human immunodeficiency virus type I in breast milk from women with or without antiretroviral drugs

BackgroundTransmission of human immunodeficiency virus type 1 (HIV-1) through breast-feeding may involve both cell-free and cell-associated virus. This latter viral reservoir remains, however, to be fully explored. CD4+ T cell-associated virus production in breast milk was therefore investigated.MethodsThe ex vivo spontaneous production of HIV-1 antigen and HIV-1 RNA by CD4+ T cells was measured in paired blood and breast milk samples from 15 HIV-1 infected women treated or not with antiretroviral drugs. Spontaneous antigen secreting cells (HIV-1-AgSCs) from breast milk and blood were enumerated by an ELISpot assay, and cell-associated HIV-1 RNA was quantified by real-time PCR in supernatants of CD4+ T cells cultured for 18 hours without addition of polyclonal activators.ResultsAmong the CD4+ T cells present in breast milk, memory cells expressing high levels of cell-surface activation markers were predominant. Spontaneous HIV-1-AgSCs were detected and enumerated in the breast milk of all 15 women, with a median number of 13.0 and 9.5 HIV-1- AgSCs/106 CD4+ T cells in aviremic (n = 7) and viremic (n = 8) women, respectively. Cell- associated HIV-1 RNA was detected in cell-free supernatants from 4/7 aviremic and 5/8 viremic individuals at median levels of 190 and 245 copies/ml, respectively.ConclusionsActivated CD4+ T cells producing HIV-1 are detected in the breast milk of untreated individuals as well as those receiving highly active antiretroviral therapy. This finding strongly suggests that HIV-1 replication occurs in latently infected CD4+ T cells that, upon spontaneous activation, revert to productively infected cells. These cells might be responsible for a residual breast milk transmission despite maternal highly active antiretroviral therapy.

Functional Epstein-Barr virus reservoir in plasma cells derived from infected peripheral blood memory B cells

The Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes latency in resting memory B lymphocytes, and is involved in oncogenesis through poorly understood mechanisms. The EBV lytic cycle is initiated during plasma cell differentiation by mRNAs transcripts encoded by BZLF1, which induce the synthesis of EBV proteins such as the immediate-early antigen ZEBRA and the late membrane antigen gp350. Therefore, we assessed the capacity of circulating EBV-infected B lymphocytes from healthy EBV-seropositive subjects to enter and complete the EBV lytic cycle. Purified B lymphocytes were polyclonally stimulated and BZLF1- or gp350-secreting cells (BZLF1-SCs or gp350-SCs) were enumerated by ELISpot assays. The number of BZLF1-SCs ranged from 50 to 480/107 lymphocytes (median, 80; 25th-75th percentiles, 70-150) and gp350-SCs from 10 to 40/107 lymphocytes (median, 17; 25th-75th percentiles, 10-20). gp350-SCs represented only 7.7% to 28.6% of BZLF1-SCs (median, 15%; 25th-75th percentiles, 10.5%-20%). This EBV functional reservoir was preferentially restricted to plasma cells derived from CD27(+) IgD(-) memory B lymphocytes. In 9 of 13 subjects, EBV DNA quantification in B-cell culture supernatants gave evidence of completion of EBV lytic cycle. These results demonstrate that EBV proteins can be secreted by EBV-infected B lymphocytes from healthy carriers, a majority generating an abortive EBV lytic cycle and a minority completing the cycle.

Multicytokine Detection Improves Latent Tuberculosis Diagnosis in Health Care Workers

ABSTRACT In a low-incidence setting, health care workers (HCW) are at a higher risk of tuberculosis than the general population. The suboptimal sensitivity of the QuantiFERON-TB Gold In-Tube (QFT) test remains a critical issue when identifying occupational latent tuberculosis infection (LTBI) in HCW. The aim of this study was to identify additional biomarkers in order to overcome the limits of gamma interferon (IFN-γ) release assays (IGRAs) and improve the performance of LTBI diagnosis within this population. Seventy Bacille Calmette-Guérin-vaccinated HCW regularly exposed to Mycobacterium tuberculosis were grouped according to QFT results into an LTBI-positive group (positive QFT, n = 8), an LTBI-negative group (normal QFT and negative tuberculin skin test [TST], n = 21), and an undetermined group (subpositive QFT and/or positive TST, n = 41). The secretion of 22 cytokines in response to QFT-specific stimulation was quantified using a multiparameter-based immunoassay. As a result, thresholds discriminating LTBI-positive from LTBI-negative HCW were established by comparing areas under the receiver operating characteristic curves for interleukin-2 (IL-2), IL-15, IFN-γ-induced protein 10 (IP-10), and the monokine induced by IFN-γ (MIG), which are biomarkers differentially secreted by the two groups. The combination of IL-15 and MIG provided a sensitivity of 100% and a specificity of 94.1% in distinguishing LTBI-positive from LTBI-negative HCW. When using IL-15 and MIG among the undetermined group, 6/45 HCW could be classified in the LTBI-positive group. The use of additional biomarkers after IGRA screening could improve the diagnosis of LTBI. The performance of these biomarkers and their use in combination with TST and/or QFT, as well as the cost-effectiveness of such a diagnostic strategy, should be evaluated in further larger clinical trials.

B-cell polyclonal activation and Epstein-Barr viral abortive lytic cycle are two key features in acute infectious mononucleosis.

BACKGROUND Acute infectious mononucleosis (AIM) is generally associated with a large EBV B cell reservoir cells and an intense B-cell polyclonal activation whereas the number of quiescent EBV-infected memory B cells in chronically EBV-infected healthy controls is very low. OBJECTIVES To evaluate the extent and functionality of ex vivo B-cell polyclonal activation, quantify the EBV DNA integrated in B cells, enumerate the functional EBV DNA reservoir in B cells and circulating B cells spontaneously secreting EBV antigens in AIM. STUDY DESIGN Circulating B cells and B cells differentiating into plamablasts and plasma cells, early (BZLF1)- and late viral antigen (gp350)-secreting-cells (SCs) were enumerated in six AIM patients and seven healthy EBV carriers. RESULTS In vitro B-cell polyclonal activation induced 8000-24,000 BZLF1- and 1000-3000gp350-SCs/10(6) B cells, respectively. These data suggest that only 11.1-19.5% of cells expressing BZLF1 synthesized gp350 and so completed the EBV-lytic cycle. Furthermore, circulating spontaneous BZLF1- and gp350-SCs that reflect ongoing viral replication were rare (20-120 and 10-30/10(6) B cells, respectively), and their low numbers contrasted with the high levels of circulating plasma cells (1.1-10.2% of CD19(+) B cells). CONCLUSION The in vivo terminal-B-cell differentiation into plasma cells could unmask EBV B-cell reservoir to specific cytotoxic T-cell response and combined with a predominant abortive functional-EBV-reservoir, strongly contribute to rapid decay of cellular EBV reservoir in AIM.

HIV-1 infection impairs HSV-specific CD4(+) and CD8(+) T-cell response by reducing Th1 cytokines and CCR5 ligand secretion.

Background:The concept of HIV-1/HSV-negative immunosynergy has recently come to light, which leads us to explore the impact of HIV-1 infection on HSV-specific T-cell immunity. Methods:A combination of interferon (IFN)-γ ELISpot and Luminex-based multicytokine profiling assays was used to compare, in a cross-sectional study, the HSV-specific CD4+ and CD8+ T-cell responses between 20 HIV-1/HSV-coinfected and 12 HIV-1-uninfected/HSV-infected individuals after in vitro restimulation with HSV glycoprotein D (gD) peptide epitopes. Results:In response to CD4+ and CD8+ gD peptide epitopes, mean value (±standard errors of the mean) of the different IFN-γ-secreting T cells (ISC) means was significantly reduced in HIV-1/HSV-coinfected individuals (70 ISC ± 10 and 60 ISC ± 8/106 cells) compared with HIV-1-uninfected/HSV-infected individuals (280 ISC ± 25 and 234 ISC ± 23/106 cells, both P < 0.001). After stimulation with the immunodominant CD4+ gD228-258 and CD8+ gD53-61 peptide epitopes, the Th1 cytokine and CCR5 ligand secretions were decreased in the HIV-1-infected group although Th17 cytokines increased. The mean concentration of interleukin (IL)-2, IFN-γ, the IFN-γ-induced protein 10 kDa, and the monokine induced by IFN-γ was correlated to the mean concentration of macrophage inflammatory proteins (MIP-1α, MIP-1β), RANTES and Eotaxin (ρ = 0.56, P = 0.02 and ρ = 0.52, P = 0.03). Conclusions:HIV-1 infection impairs both the number and function of HSV-specific T cells. The downregulation of Th1 cytokines and CCR5 ligands in HIV-1/HSV-coinfected individuals may further facilitate both HSV reactivations and HIV-1 replication.

Comparison of EBV DNA viral load in whole blood, plasma, B‐cells and B‐cell culture supernatant

Epstein–Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV‐associated disorders in immunosuppressed individuals and in patients with EBV‐associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B‐cells, and B‐cell short‐term culture supernatant using quantitative real‐time PCR. Samples were collected from 33 subjects with either HIV infection or B‐cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B‐cell samples, in 82% of B‐cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B‐cell and B‐cell culture supernatant material (ρ = 0.92; P < 0.0001), but no significant correlation existed between EBV DNA levels in whole blood and enriched B‐cells (ρ = −0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B‐cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B‐cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B‐cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host–pathogen interactions in various clinical scenarios. J. Med. Virol. 86:851–856, 2014.

The potential impact of CD4+ T cell activation and enhanced Th1/Th2 cytokine ratio on HIV-1 secretion in the lungs of individuals with advanced AIDS and active pulmonary infection

Bronchoalveolar lavage fluid (BALF) provides a source of mucosal CD4(+) T cells. We investigated the physiological properties of T lymphocytes from BALF and blood and their role on the dynamic of HIV-1 replication among AIDS patients with active lung infections. Pulmonary CD4(+) T cells consist mainly of effector memory cells (CD45RO(+) and CCR7(-)) with increased expression of activation markers (HLA-DR(+) and CD69(+)) when compared to the blood counterpart. We observed a high frequency of BALF cells capable of secreting HIV-1-Ags suggesting that the local lung environment may support favorable conditions for CD4(+) T lymphocytes harboring HIV-1 DNA to initiate the viral cycle. Nevertheless, the high number of IFN-γ-producing cells and the predominance of Th1 immune response in the lung could limit the secretion of HIV-1 RNA. In conclusion, the capacity of activated CD4(+) T cells to produce HIV-1 is driven by both the level and quality of cellular activation in the lung.