Peripheral Blood B-cell Subsets Research Articles

Treg Cells Are Negatively Correlated with Increased Memory B Cells in Pre‐eclampsia While Maintaining Suppressive Function on Autologous B‐Cell Proliferation

The objective of this study was to determine the simultaneous changes and the correlation between circulating regulatory T (Tregs) and B-cell subsets in normal pregnancy (NP) and pre-eclampsia (PE). Meanwhile, the regulatory function of Tregs on B-cell proliferation was evaluated in vitro. A total of 32 PE and 40 NP women matched for age, gestational age, and parity were included. Using flow cytometry, the percentages of peripheral blood Tregs and B-cell subsets were determined. Moreover, the regulatory function of Tregs on autologous B-cell proliferation in PE and NP was investigated in vitro by flow cytometry and analyzed by modfit software. The proportions (%) of CD4(+) Foxp3(+) (P<0.001) and CD4(+) CD25(+) Foxp3(+) (P<0.01) cells within total CD4(+) T cells were significantly decreased in PE as compared to those of NP. However, no difference was found in CD4(+) CD25(+) cells in CD4(+) T cell population. CD19(+) CD27(+) (P<0.05) and CD19(+) CD27(+) IgD(+) (P<0.01) B cells within CD19(+) B cells (%) in PE were higher than those of NP. Negative correlations between the percentage of CD4(+) CD25(+) Foxp3(+) in CD4(+) T cells and that of CD19(+) CD27(+) (r=-0.384, P<0.05) and CD19(+) CD27(+) IgD(+) (r=-0.402, P<0.05) in CD19(+) B cells were present in PE but not in NP. Furthermore, the in vitro study demonstrated a significant increase in B-cell proliferation in PE as compared to NP. Tregs in PE were able to suppress the proliferation and precursor frequency of autologous B cells (P<0.05 each). The study indicates that a negative correlation between Tregs and memory B cells is present in women with PE, characterized by a systemic decrease in Tregs and an increase in memory B cells. Although the quantitative deficit of Tregs is present in PE, the Tregs still have a suppressive role in autologous B-cell proliferation.

409 Dynamic Changes in the Immunophenotypic Maturation Status of CD19 B Lymphocytes in Patients with Acute Cellular Rejection after Heart Transplantation

Very few studies have evaluated the potential contribution of B lymphocytes in the pathogenesis of cellular allograft rejection (AR). The dynamic changes of these antigen presenting cells have not been systemically examined in patients with AR after heart transplantation. In this study, we have studied functionally distinct CD19 B-cell subsets in peripheral blood of patients with AR.

LDOC1 mRNA is differentially expressed in chronic lymphocytic leukemia and predicts overall survival in untreated patients

AbstractWe previously identified LDOC1 as one of the most significantly differentially expressed genes in untreated chronic lymphocytic leukemia (CLL) patients with respect to the somatic mutation status of the immunoglobulin heavy-chain variable region genes. However, little is known about the normal function of LDOC1, its contribution to the pathophysiology of CLL, or its prognostic significance. In this study, we have investigated LDOC1 mRNA expression in a large cohort of untreated CLL patients, as well as in normal peripheral blood B-cell (NBC) subsets and primary B-cell lymphoma samples. We have confirmed that LDOC1 is dramatically down-regulated in mutated CLL cases compared with unmutated cases, and have identified a new splice variant, LDOC1S. We show that LDOC1 is expressed in NBC subsets (naive > memory), suggesting that it may play a role in normal B-cell development. It is also expressed in primary B-cell lymphoma samples, in which its expression is associated with somatic mutation status. In CLL, we show that high levels of LDOC1 correlate with biomarkers of poor prognosis, including cytogenetic markers, unmutated somatic mutation status, and ZAP70 expression. Finally, we demonstrate that LDOC1 mRNA expression is an excellent predictor of overall survival in untreated CLL patients.

Acute wrist and foot drop associated with hepatitis C virus related mixed cryoglobulinemia: Rapid response to treatment with rituximab

The nature of the B-cell subsets associated with chronic hepatitis C virus related type II mixed cryoglobulinemia (HCV-MC) is unclear. We report the case of a 64-year-male with acute onset wrist drop and foot drop, secondary to HCV-MC related mononeuritis multiplex, who was treated with rituximab, an anti-CD20 + antibody directed against B cells. We monitored the frequency of B-cell subsets in peripheral blood before and after rituximab, and correlated B-cell subset changes with clinical response. Significant improvements in his wrist and foot drop, as well as his vasculitic rash, depression and erectile dysfunction were evident within six days of starting rituximab and have persisted several months after B-cell recovery. More than 95% of CD20 + B cells had disappeared from peripheral blood within 1 week, returning to baseline by week 21. CD20 +CXCR3 + frequency at baseline was similar to that at week 21. CD20 +CD5 +, the human equivalent of B1 B cells and CD20 +IgM +IgD +, naïve B cells were increased. By contrast, CD20 +CD27 + memory cell frequency was reduced. These data suggest that CD27 + memory B cells, but not CD5 + and IgM +IgD + B cells may play a role in the clinical manifestations of cryoglobulinemia.

Epstein-Barr virus colonization of tonsillar and peripheral blood B-cell subsets in primary infection and persistence

AbstractEpstein-Barr virus (EBV) persists in the immune host by preferentially colonizing the isotype-switched (IgD−CD27+) memory B-cell pool. In one scenario, this is achieved through virus infection of naive (IgD+CD27−) B cells and their differentiation into memory via germinal center (GC) transit; in another, EBV avoids GC transit and infects memory B cells directly. We report 2 findings consistent with this latter view. First, we examined circulating non–isotype-switched (IgD+CD27+) memory cells, a population that much evidence suggests is GC-independent in origin. Whereas isotype-switched memory had the highest viral loads by quantitative polymerase chain reaction, EBV was detectable in the nonswitched memory pool both in infectious mononucleosis (IM) patients undergoing primary infection and in most long-term virus carriers. Second, we examined colonization by EBV of B-cell subsets sorted from a unique collection of IM tonsillar cell suspensions. Here viral loads were concentrated in B cells with the CD38 marker of GC origin but lacking other GC markers CD10 and CD77. These findings, supported by histologic evidence, suggest that EBV infection in IM tonsils involves extrafollicular B cells expressing CD38 as an activation antigen and not as a marker of ectopic GC activity.

Spi-B inhibits human plasma cell differentiation by repressing BLIMP1 and XBP-1 expression

AbstractThe terminal differentiation of B cells into antibody-secreting plasma cells is tightly regulated by a complex network of transcription factors. Here we evaluated the role of the Ets factor Spi-B during terminal differentiation of human B cells. All mature tonsil and peripheral blood B-cell subsets expressed Spi-B, with the exception of plasma cells. Overexpression of Spi-B in CD19+ B cells inhibited, similar to the known inhibitor BCL-6, the expression of plasma cell–associated surface markers and transcription factors as well as immunoglobulin production, ie, in vitro plasma cell differentiation. The arrest in B-cell differentiation enforced by Spi-B was independent of the transactivation domain, but dependent on the Ets-domain. By chromatin immunoprecipitation and assays using an inducible Spi-B construct BLIMP1 and XBP-1 were identified as direct target genes of Spi-B mediated repression. We propose a novel role for Spi-B in maintenance of germinal center and memory B cells by direct repression of major plasma cell factors and thereby plasma cell differentiation.

Characterization of phenotypically distinct B-cell subsets and receptor-stimulated mitogen-activated protein kinase activation in human cord blood B cells

Human cord blood (CB) is a valuable source of hematopoietic stem cells, but clinical reports have indicated slow recovery of B-cell development and function after CB transplantation. To investigate the basis of these B-cell defects in reconstitution, we characterized B cells purified from CB. We compared B-cell receptor activation and B-cell subsets in CB, bone marrow (BM), and peripheral blood (PB). We found that in CB B cells activation of extracellular signal-regulated kinase (ERK) and p38 following ligation of CD40 but not of the B-cell antigen receptor (BCR) was inefficient. The patterns of expression of CD5, CD34, and CD40 in the B-cell population of CB were similar to those in PB rather than in BM. The B cells in CB contained an increased proportion of B cells expressing a high level of CD24 and a low proportion of B cells expressing CD27, pointing to the presence of circulating CD24high immature transitional and CD27(-) naive B cells. CD40-mediated activation of ERK and p38 was also minimal in these B cells of CB. These findings may account for the functional defects of B cells in transplanted CB.

CHANGES IN B-CELL SUBSETS DO NOT ACCOUNT FOR DECREASED COMPLEMENT RECEPTOR 2 LEVELS IN HUMAN SYSTEMIC LUPUS ERYTHEMATOSUS.

Purpose Complement receptor 2 (CR2) levels on circulating B cells in patients with systemic lupus erythematosus (SLE) are decreased. In SLE, altered homeostasis of the circulating B-cell pool leads to increased frequency of subsets that express low CR2. We hypothesized that these shifts could account for the decreased CR2 levels demonstrated on bulk B cells of patients with SLE. Methods Peripheral blood mononuclear cells were prepared from nine subjects with SLE and eight healthy controls using a Ficoll density gradient. Cells were stained with fluorescently labeled monoclonal antibodies to CD19, CR2 (HB5 clone), IgD, CD27, and CD38. Ethidium monoazide bromide (EMA) was used as a viability marker. Previously defined peripheral blood B-cell subsets were identified using polychromatic flow cytometry: transitional (CD27−IgD+CD38++), mature naive (CD27−IgD+CD38low), marginal zone-like (CD27+IgD+), class-switched memory (CD27+IgD−CD38low), and plasmablast (CD27+/++IgD−CD38++). SimplyCellular quantification beads (Bangs Labs) were used to quantify CR2 levels. Results are expressed as mean antibody binding capacity (ABC) ± standard deviation. Results Naive B cells comprised the largest subset of circulating B cells in both healthy (43%) and lupus (43%) subjects. Transitional cells were increased in frequency in SLE subjects (17%) compared with healthy controls (3%) (p = .0047). Marginal zone-like cells were decreased in frequency in SLE subjects (6%) compared with healthy controls (20%) (p Conclusions As previously described, subjects with SLE have changes in B-cell subsets leading to increased frequency of CR2low cells. However, B cells in subjects with SLE have lower CR2 across all subsets and demonstrate a blunting of the increases in CR2 levels typically seen with maturation. Therefore, expansion of a CR2low B cell subset does not provide the mechanism for reduced B-cell CR2 levels in SLE.

Effect of Rituximab on the Peripheral Blood and Cerebrospinal Fluid B Cells in Patients With Primary Progressive Multiple Sclerosis

Rituximab, an anti-CD20 monoclonal antibody that depletes CD20(+) B cells, has demonstrated efficacy in peripheral neurological diseases. Whether this efficacy can be translated to neurological diseases of the central nervous system with possible autoimmune B-cell involvement remains unknown. To determine the effect of rituximab on cerebrospinal fluid B cells in patients with multiple sclerosis. Four patients with primary progressive multiple sclerosis were treated with rituximab. Cerebrospinal fluid and peripheral blood B-cell subsets were identified by flow cytometry from each patient before and after rituximab treatment. The B cells in cerebrospinal fluid were not as effectively depleted as their peripheral blood counterparts. Rituximab treatment temporarily suppressed the activation state of B cells in cerebrospinal fluid. The residual B cells underwent expansion after rituximab treatment. The effect(s) of rituximab on the cerebrospinal fluid B-cell compartment is limited in comparison with the effect(s) on the B cells in the periphery, but this finding will need to be confirmed in a larger group of MS patients.

Apoptotic effect of rituximab on peripheral blood B cells in rheumatoid arthritis.

Rituximab (RTX) has proven efficacious in the treatment of rheumatoid arthritis (RA). Herein, we assessed the apoptosis-inducing capability of RTX in vitro on RA peripheral blood B-cell subsets and also compared the effects of RTX on B cells from rheumatoid factor-positive (RF+) and RF- patients. The likely relevance of B cells in disease was assessed by measuring B-cell-modulating serum cytokines. Peripheral blood B cells were isolated and cultured with the presence or absence of RTX. The levels of apoptosis within the naïve, memory and IgD+CD27+ B-cell subpopulations were determined by cytofluorometric analysis and caspase 3/7 assays. Levels of serum cytokines were measured with a multiplex cytokine array system. RTX induced significant apoptosis in all B-cell subsets in both RA and controls. In naïve and memory B cells from RA patients, RTX induced significantly higher levels of apoptosis than in controls. RTX induced apoptosis of B cells in RF+ and RF- patients. Serum levels of interleukin-1beta (IL-1beta), IL-4, IL-10 and IL-13 were profoundly increased in RF+ patients compared to RF- patients and controls. Although our cohort was small (10 RA patients), the data suggest that RTX induces apoptosis in all investigated subsets of B cells from RA patients. Interestingly, memory B cells from RA patients were more sensitive to RTX than memory cells from normal controls, suggesting that the delay in treatment response to RTX observed in clinical trials may be due in part to memory cell depletion. The apoptotic effects of RTX were similar in RF+ and RF- patients, but serum levels of B-cell-activating cytokine levels were only elevated in RF+ but not RF- patients. These data suggest that RTX is less effective in RF- RA because B cells play a less significant role in RA pathogenesis in RF- patients.

Phenotypic and molecular characterization of human peripheral blood B-cell subsets with special reference to N-region addition and J kappa-usage in V kappa J kappa-joints and kappa/lambda-ratios in naive versus memory B-cell subsets to identify traces of receptor editing processes.

We identified a population of IgM+IgD+ B-cells in the peripheral blood (PB) of humans that express somatically mutated V-region genes like classical class switched or IgM-only memory B-cells and comprise around 15% of PB B-cells in adults. Mutated IgM+IgD+ cells differ from unmutated naive IgM+IgD+ cells in that they express the CD27 cell surface antigen. In addition, a very small subset of IgD-only B-cells was identified in the PB that carried rearranged VH-genes with an extremely high load of somatic mutations (up to 60 mutations per gene). A common characteristic of the four somatically mutated subsets, which altogether comprise 40% of PB B-lymphocytes in adults, is the surface expression of CD27. This antigen may thus represent a general marker for memory B-cells in the human. Somatically mutated and unmutated PB B-cell subsets were analyzed for N-region addition and J kappa-usage in V kappa J kappa-joints, and in addition for the respective kappa/lambda-ratios: N-nucleotides could be identified in a large fraction of V kappa-regions of all B-cell subsets, indicating that N-region insertion already occurs in the pre-germinal center (GC) phase of B-cell development. Both the J kappa-usage in expressed V kappa J kappa-joints and the kappa/lambda-ratio from somatically mutated B-cells do not differ substantially from those of the unmutated cells, so that in terms of these parameters, a contribution of secondary V kappa J kappa-rearrangements in shaping the memory B-cell repertoire is not detectable.

Lymphocyte activation and cytokine production in autoimmune hemolytic anaemia (AIHA).

We studied 16 patients affected by autoimmune hemolytic anaemia (AIHA), both idiopathic and associated with other diseases (B and T lymphoma, B hepatitis, gastric carcinoma, systemic lupus erythematosus) or alpha-methyldopa therapy, in order to value T- and B-cell activation. We determined the count of T- and B-cell subsets in peripheral blood, the proliferative response of peripheral blood lymphocytes (PBL) to phytohemagglutinin (PHA) and to pokeweed mitogen (PWM), the percentage of CD25+ cells in culture and interleukin (IL)-1alpha, IL-2, IL-4, tumor necrosis factor (TNF)alpha and soluble IL-2 receptor (sIL-2R) levels in sera and in culture. Except for an increase in CD4+ and CD8+ T cell number in a case of AIHA associated with a T lymphoma and an increase in the percentage of CD5+ and PCA1+ B cells in two cases of AIHA associated with B lymphoma and with SLE, no further data showed a relationship with the disease possibly associated with AIHA, so both idiopathic and secondary AIHA cases were analyzed together. CD4+ T cells were reduced in number in 9 cases, while CD8+ T cells were reduced in 6 cases. The percentage of CD5+ B cells was increased in 5 cases. The percentage of PCA1+ cells was increased in all cases (mean +/- sd: 18 +/- 22 vs 0,2 +/- 1 in controls). The average PBL proliferative response to PHA was reduced (S.I. 71 +/- 55 vs 138 +/- 45 in controls) as well as that to PWM (S.I. 27 +/- 21 vs 75 +/- 24 in controls), despite IL-2 high levels, in all cases, in both sera (mean +/- sd: 648 +/- 351 pg/ml vs 16 +/- 4 pg/ml in controls) and culture supernatants (mean +/- sd: 1045 +/- 677 pg/ml vs 195 +/- 51 pg/ml in controls). In PHA stimulated cultures the percentage of CD25+ cells was reduced (mean +/- sd: 37 +/- 18 vs 63 +/- 14 in controls), sIL-2R levels were like controls in 7 cases. In sera sIL-2R levels were increased in all cases (mean +/- sd: 1256 +/- 465 U/ml vs 256 +/- 114 U/ml in controls), IL-1alpha was increased in all cases too, while IL-4 levels were increased only in 7 cases. Linear regression analysis generally showed a low relationship between S.I. and IL-2, IL-4 and sIL-2R levels in supernatants of PHA stimulated culture as well as between S.I. and the percentage of CD25+ cells. Taken together these data suggest a state of B- and T-cell hyperactivation in AIHA. The low PBL proliferative response in vitro, explained in previous studies as a temporary functional exhaustion, might be itself a sign of the complete lymphocyte activation occurring in vivo in AIHA.

Characteristics of CD11c+CD5+ chronic B-cell leukemias and the identification of novel peripheral blood B-cell subsets with chronic lymphoid leukemia immunophenotypes [see comments

Previous studies have indicated that chronic lymphocytic leukemias (CLL) are characterized by the coexpression of CD5 and B-cell antigens, while hairy cell leukemias (HCL) typically express CD11c+CD5- B-cell immunophenotypes. In this report we describe the features of B-cell leukemias with CD11c+CD5+ immunophenotypes and the identification of novel circulating B-cell subsets defined by the expression of CD20, CD5, and CD11c antigens. Morphologic evaluation of 14CD11c+CD5+ B-cell leukemias showed that they generally had larger cellular diameters (14 to 21 microns) and lower nuclear:cytoplasm ratios than typical small lymphocyte CLL. These cases did not exhibit the well-defined nucleoli characteristic of prolymphocytic leukemia (PLL). The presenting clinical features of CD11c+CD5+ B-cell leukemias were most consistent with CLL or PLL, and none of the evaluated cases had pancytopenia, splenomegaly, and cytoplasmic villi characteristic of HCL. Examination of normal peripheral blood (n = 6) by three-color flow cytometry identified four novel B-cell subsets with the following immunophenotypes (mean percent of total CD20+ B cells +/- SE): CD20+CD5+CD11c+ (8.0 +/- 1.6); CD20+CD5-CD11c+ (12.0 +/- 2.0); CD20+CD5+CD11c- (35.0 +/- 4.9); and CD20+CD5-CD11c- (44.0 +/- 5.0). Our findings suggest that CD11c+CD5+ B-cell leukemias with atypical morphologic features represent forms of CLL or PLL rather than HCL. In addition, we have identified novel subsets of circulating B cells defined by patterns of CD20, CD5, and CD11c expression that correspond to the immunophenotypes of chronic B-cell leukemias.

Induction of human immunoglobulin secretion: III. Inhibition of induction of IgG and IgA production in spleen but not peripheral blood cells by mitogenic antibody to IgM and IgD

Human peripheral blood and spleen mononuclear cells (MNC) were induced (SI = 5–30) to proliferate in vitro after stimulation by F(ab′) 2 fragments of goat anti-IgM antibody. Lower levels of DNA synthesis (SI = 2–14) were also observed following stimulation with anti-IgD antibody, whereas no DNA synthesis was detected in cultures containing F(ab′) 2 fragments of normal goat IgG antibody. Both intact and F(ab′) 2 fragments of anti-IgM antibody had stimulatory capacity for MNC, but intact antibody was less active. Anti-immunoglobulin (Ig) antibodies alone did not induce Ig secretion in either peripheral blood or spleen cells. In spleen cultures anti-IgM completely inhibited IgM, IgG, and IgA production induced by Staphylococcus aureus bacteria strain Cowan I, pokeweed mitogen (PWM), or low concentrations of Cowan I plus PWM. Anti-IgD partially inhibited mitogen-generated Ig secretion by spleen cells. However, quite a different result was obtained with peripheral blood MNC in which anti-IgM antibody selectively abrogated the induction of IgM production by the mitogens. No inhibition was observed for induction of IgG and IgA production. Anti-IgD antibody did not show any significant suppressive effect on mitogen-induced production of the three classes of Ig using blood cells. These results suggest a difference in maturation stage of B-cell subsets in peripheral blood compared to spleen.