Cutting edge: anti-tumor necrosis factor therapy in rheumatoid arthritis inhibits memory B lymphocytes via effects on lymphoid germinal centers and follicular dendritic cell networks

Rheumatoid arthritis (RA) is mediated by a proinflammatory cytokine network with TNF at its apex. Accordingly, drugs that block TNF have demonstrated significant efficacy in the treatment of RA. A great deal of experimental evidence also strongly implicates B cells in the pathogenesis of RA. Yet, it remains unclear whether these two important players and the therapies that target them are mechanistically linked. In this study we demonstrate that RA patients on anti-TNF (etanercept) display a paucity of follicular dendritic cell networks and germinal center (GC) structures accompanied by a reduction in CD38+ GC B cells and peripheral blood memory B cell lymphopenia compared with healthy controls and RA patients on methotrexate. This study provides initial evidence in humans to support the notion that anti-TNF treatment disrupts GC reactions at least in part via effects on follicular dendritic cells.     

CD31, a novel cell surface marker for CD4 cells of suppressor lineage, unaltered by state of activation.

In this study, we examined the role of CD31 as a cell surface marker for subsets of human CD4 cells. CD31, as defined by a newly developed mAb termed anti-1F11, can divide activated as well as resting CD4 cells into distinct functional subpopulations, based on its surface expression. Among CD4 cells freshly isolated from peripheral blood, anti-1F11 preferentially reacts with the CD45RA+ subset. The majority of helper activity for B cell IgG synthesis and memory function to recall Ag such as tetanus toxoid or mumps was found within the CD31- CD4 cell population, whereas CD31+ CD4 cells provided poor helper function for B cell IgG synthesis and were more responsive to Con A and autologous MHC (autologous MLR). The expression of CD31 on CD45RA+ CD4 cells did not change after activation, despite the loss of CD45RA from the cell surface. Conversely, CD31 was not acquired after activation of CD45RO+ CD45RA- CD4 cells. Furthermore, activated CD4 cells expressing CD31 can induce suppressor function for B cell IgG synthesis, whereas the reciprocal population of activated CD4 cells (CD31-) provide strong helper function for B cell IgG production. Finally, IL-4 production could only be induced by stimulation with PMA and ionomycin in either resting or activated CD31- CD4 cells. Thus, CD31 may prove useful in defining CD4 populations with reciprocal functional programs. Moreover, unlike other markers used for this purpose, the expression of CD31 does not change after activation and may serve as a more useful marker for identification of cells of suppressor or helper lineage.     

Expansion of functionally immature transitional B cells is associated with human-immunodeficient states characterized by impaired humoral immunity

X-linked lymphoproliferative disease (XLP) is a severe immunodeficiency associated with a marked reduction in circulating memory B cells. Our investigation of the B cell compartment of XLP patients revealed an increase in the frequency of a population of B cells distinct from those previously defined. This population displayed increased expression of CD10, CD24, and CD38, indicating that it could consist of circulating immature/transitional B cells. Supporting this possibility, CD10+CD24highCD38high B cells displayed other immature characteristics, including unmutated Ig V genes and elevated levels of surface IgM; they also lacked expression of Bcl-2 and a panel of activation molecules. The capacity of CD24highCD38high B cells to proliferate, secrete Ig, and migrate in vitro was greatly reduced compared with mature B cell populations. Moreover, CD24highCD38high B cells were increased in the peripheral blood of neonates, patients with common variable immunodeficiency, and patients recovering from hemopoietic stem cell transplant. Thus, an expansion of functionally immature B cells may contribute to the humoral immunodeficient state that is characteristic of neonates, as well as patients with XLP or common variable immunodeficiency, and those recovering from a stem cell transplant. Further investigation of transitional B cells will improve our understanding of human B cell development and how alterations to this process may precipitate immunodeficiency or autoimmunity.     

EBV-specific CD8+ T cell memory: relationships between epitope specificity, cell phenotype, and immediate effector function

EBV infection in humans induces CD8+ T cell memory to viral epitopes derived from both lytic and latent cycle Ags. We have analyzed the relationship between the phenotype and function of the memory pool of T cells specific for these Ags. Lytic epitope-specific populations were heterogeneous in terms of CD45RO/RA and CD28 expression, whereas latent epitope-specific populations were uniformly CD45RO+ and CD28+, consistent with the higher antigenic challenge from lytic epitopes driving some memory cells toward a CD45RA+, CD28- phenotype. However, both types of memory population showed immediate epitope-specific cytotoxicity and type 1 cytokine production in ex vivo assays. Cytotoxic function was not associated with preactivated T cells, as EBV-specific populations were negative for activation markers such as CD69 or CD38, nor could cytotoxic function be ascribed to CD27- or CD56+ subsets, as such cells were not detected in EBV-specific memory. Furthermore, cytotoxicity was not limited to CD45RA+ and/or CD28- fractions, but also was observed in CD45RO+, CD28+ populations in lytic and latent epitope-specific memory. Cytokine (IFN-gamma, TNF-alpha) responses, measured by intracytoplasmic staining after peptide stimulation, also were detectable in CD45RO+ and RA+ subsets as well as CD28+ and CD28- subsets. Of other markers that were heterogeneous in both lytic and latent epitope populations, CCR7 gave the best discrimination of functionality; thus, CCR7+ cells consistently failed to give an IFN-gamma or TNF-alpha response, whereas many CCR7- cells were responsive. Our data are consistent with effector functions having a broad distribution among phenotypically distinct subsets of "effector memory" cells that have lost the CCR7 marker.     

Decreased influenza-specific B cell responses in rheumatoid arthritis patients treated with anti-tumor necrosis factor

Introduction

As a group, rheumatoid arthritis (RA) patients exhibit increased risk of infection, and those treated with anti-tumor necrosis factor (TNF) therapy are at further risk. This increased susceptibility may result from a compromised humoral immune response. Therefore, we asked if short-term effector (d5-d10) and memory (1 month or later) B cell responses to antigen were compromised in RA patients treated with anti-TNF therapy.

Methods

Peripheral blood samples were obtained from RA patients, including a subset treated with anti-TNF, and from healthy controls to examine influenza-specific responses following seasonal influenza vaccination. Serum antibody was measured by hemagglutination inhibition assay. The frequency of influenza vaccine-specific antibody secreting cells and memory B cells was measured by EliSpot. Plasmablast (CD19+IgD-CD27hiCD38hi) induction was measured by flow cytometry.

Results

Compared with healthy controls, RA patients treated with anti-TNF exhibited significantly decreased influenza-specific serum antibody and memory B cell responses throughout multiple years of the study. The short-term influenza-specific effector B cell response was also significantly decreased in RA patients treated with anti-TNF as compared with healthy controls, and correlated with decreased influenza-specific memory B cells and serum antibody present at one month following vaccination.

Conclusions

RA patients treated with anti-TNF exhibit a compromised immune response to influenza vaccine, consisting of impaired effector and consequently memory B cell and antibody responses. The results suggest that the increased incidence and severity of infection observed in this patient population could be a consequence of diminished antigen-responsiveness. Therefore, this patient population would likely benefit from repeat vaccination and from vaccines with enhanced immunogenicity.     

Turnover rates of B cells,T cells,and NK cells in simian immunodeficiency virus-infected and uninfected rhesus macaques

We determined average cellular turnover rates by fitting mathematical models to 5-bromo-2'-deoxyuridine measurements in SIV-infected and uninfected rhesus macaques. The daily turnover rates of CD4(+) T cells, CD4(-) T cells, CD20(+) B cells, and CD16(+) NK cells in normal uninfected rhesus macaques were 1, 1, 2, and 2%, respectively. Daily turnover rates of CD45RA(-) memory T cells were 1%, and those of CD45RA(+) naive T cells were 0.5% for CD4(+) T cells and approximately 1% for CD4(-)CD45RA(+) T cells. In SIV-infected monkeys with high viral loads, the turnover rates of T cells were increased approximately 2-fold, and that of memory T cells approximately 3-fold. The turnover of CD4(+)CD45RA(+) naive T cells was increased 2-fold, whereas that of CD4(-)CD45RA(+) naive T cells was marginally increased. B cells and NK cells also had increased turnover in SIV-infected macaques, averaging 3 and 2.5% per day, respectively. For all cell types studied here the daily turnover rate increased with the decrease of the CD4 count that accompanied SIV infection. As a consequence, the turnover rates of CD4(+) T cells, CD4(-) T cells, B cells, and NK cells within each monkey are strongly correlated. This suggests that the cellular turnover of different lymphocyte populations is governed by a similar process which one could summarize as "generalized immune activation." Because the viral load and the CD4 T cell count are negatively correlated we cannot determine which of the two plays the most important role in this generalized immune activation.     

Expanded population of activated antigen-engaged cells within the naive B cell compartment of patients with systemic lupus erythematosus

Polyclonal B cell activation is a well-described feature of systemic lupus erythematosus (SLE), but the immune mechanisms leading to this activation are unclear. To gain insight into these processes, we extensively characterized the activated peripheral blood B cell populations in SLE. PBMC from lupus patients and healthy controls were stained with various combinations of conjugated Ab to identify distinct peripheral B cell subsets, and activation was assessed by measurement of forward scatter and CD80 or CD86 expression using flow cytometry. SLE patients had altered proportions of several B cell subsets, many of which demonstrated increased activation as assessed by forward scatter. This activation occurred at an early developmental stage, as B cells in the transitional (T2) stage were already significantly larger than those seen in controls. Increased proportions of CD80- or CD86-expressing cells were also seen in multiple B cell subsets, with the most striking differences observed in the naive CD27-CD23+ population. Within the CD23+ subset, increased costimulatory molecule expression was most pronounced in an IgD+IgMlow population, suggesting that activation follows Ag engagement. Although controls also had IgD+IgMlowCD23+ cells, they were reduced in number and not activated. Thus, there is an altered response to Ig receptor engagement with self-Ags in lupus.     

Differential recovery of polymorphonuclear neutrophils,B and T cell subpopulations in the thymus,bone marrow,spleen and blood of mice following split-dose polychemotherapy

In these studies, we examined the effect of a maximum-tolerated, split-dose chemotherapy protocol of cyclophosphamide, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea carmustine on neutrophil and lymphocyte subpopulations in the peripheral blood (PBL), thymus, bone marrow and spleen. It was found that this protocol of polychemotherapy, modeled after the induction protocol used with autologous bone marrow transplantation for breast cancer, suppressed both B and T cell populations and T cell function at times when the absolute neutrophil count had returned to normal or supernormal numbers. In the peripheral blood, 7 days following initiation of chemotherapy, there was a twofold increase in the percentage of granulocytes as compared to the level in control animals on the basis of a differential count. The polymorphonuclear neutrophil (PMN) frequency in the bone marrow was increased on day 14 and statistically identical to that in control mice on all other days analyzed. In contrast to the bone marrow cells and PBL on day 7, the frequency of PMN in the spleen and thymus was depressed. B cells (B220+) were depressed in the PBL, spleen and bone marrow and took 18–32 days to return to their normal frequency, while the frequency of B cells in the thymus was increased owing to a loss of immature T cells. The percentage of CD3+ cells in the thymus, spleen and bone marrow was significantly increased and required 10–18 days to return to normal levels, while the absolute number of CD3+ cells in the blood varied around the normal value. The ratio of CD4+ to CD8+ cells in all the organs studied varied only slightly owing to a similar reconstitution of CD4+ and CD8+ cells. In contrast to the phenotypic recovery of the CD3+, CD4+ and CD8+ cells, the ability of the splenic lymphocytes to respond to concanavalin-A was depressed and remained depressed, despite the phenotypic reconstitution of the T cell subsets, on the basis of both percentage and absolute cell number. These results show a selective T and B cell depression following multi-drug, split-dose chemotherapy in tissue and blood leukocyte populations and a chronic depression in T cell function.     

ICOS and B7 costimulatory molecule expression identifies activated cellular subsets in rheumatoid arthritis.

To better define important cell subsets expressing activation markers in rheumatoid arthritis (RA), we compared selective lymphocyte and monocyte B7H1, B7H2, B7RP.1, B7RP.2, and inducible costimulatory molecule (ICOS) expression from normal peripheral blood (NL PB), RA PB, and RA synovial fluid (SF) by multicolor flow cytometry and immunohistochemistry. RA SF memory lymphocytes expressed B7RP.1 and B7RP.2, suggesting that T-cells may function as antigen presenting cells (APCs) in RA joints. We found similar results for ICOS expression. RA SF CD14+ monocytes also expressed B7RP.1 (an ICOS ligand) and the homologous ligand B7RP.2, identifying monocytes as potential mediators of antigen processing and lymphocyte activation in RA. Furthermore, we found an increased population of RA SF CD14+ monocytes expressing B7H1 and B7H2. [The FACS analysis was supported by immunohistochemistry, showing intense lymphocyte and APC (macrophages with dendritic morphology) ICOS staining in RA synovial tissue (ST). Overall, these results define elevated populations of memoryT-lymphocytes expressing proinflammatory B7 molecules in RA SF that either stimulate T cells through ICOS (via ICOS ligands B7RP.1 and B7RP.2), or down-regulate RA ST T-lymphocytes through B7H1 and B7H2.] Therefore, in the same joint, there may exist positive and negative influences on the inflammatory response, and perhaps, the negative signals dominate as joint inflammation resolves.     

Heterogeneity and function of human B lymphocytes

B lymphocytes represent an important arm of the immune system. Besides their main function of providing antibodies protecting against pathogens, they also exert some regulatory functions, in particular for secondary lymphoid tissue differentiation. Human B cells can be divided in various subsets representing different maturation stages and different pathways of humoral immune responses. Na?ve IgMlow IgDhigh CD27- B cells can participate in T-cell dependent immune responses leading to germinal center formation in follicles of secondary lymphoid organs. Interactions with follicular helper T cells, a recently identified CD185+ T cell population providing help to follicular B cell, involve costimulatory molecules including CD40, CD27, CD278 and SAP-recruiting receptors. B cell interaction with follicular helper T cells represents a critical step controlling the generation of plama cells that ultimately produce high affinity, somatically mutated, class-switched antibodies or of their memory B cell counterpart (identified as CD27+ Ig switched or IgMonly B cells). IgMhigh IgDlow CD27+ B cells are a puzzling population apparently specialized in T-independent responses to bacterial capsular polysaccharides. The extra-follicular, probably antigen-independent, differentiation pathway of these cells, allowing pre-immune repertoire diversification by somatic hypermutation, is not yet characterized. However, circulating IgMhigh IgDlow CD27+ B cells are similar to splenic marginal zone B cells. In addition to these subsets, minor populations can also be identified in peripheral blood, such as transitional B cells and plasma blasts. All together, deciphering human B cell heterogeneity provides tools for investigations of humoral immunodeficiencies and auto-immune diseases, that will in return shed more light on B cell biology.     

Functions of rat CD4+ T cell subsets defined by CD45RB: CD45RB- cells have a much stronger response to recall antigens, whereas polyclonally activated cells of both subsets are equally efficient producers of IFN in the presence of exogenous IL-2.

CD4+45RB- rat T cells were shown to respond strongly to recall antigens and produce IFN and TNF after polyclonal activation. Compared to CD4+45RB- cells, CD4+45RB+ cells showed a very weak response to recall antigens but produced higher amounts of IFN and TNF after polyclonal activation. Addition of rIL-2 reduced the difference between the subsets with respect to the level of IFN produced at 48 and 72 hr after activation, but did not influence the level of TNF production. The CD4+45RB- cells clearly showed a faster response to polyclonal activation than that of CD4+45RB+ cells detected as an earlier IFN production and CD25 expression. The earlier IFN production by the CD45RB- population could not only be explained by their faster production of IL-2, since the difference persisted when rIL-2 was added to both populations at the beginning of culture. We conclude that the CD4+45RB- rat T cell population resemble the CD4+45RA-0+ human T cell subset with respect to a good responsiveness to recall antigen and efficient production of IFN. However, the CD4+45RB+ rat T cell subset functionally differs from the CD4+45RA+0- human T cell subset. We suggest that the CD4+45RB+ subset comprises a major CD4+45RA+B+0- and a minor CD4+4+45A-B+0+ T cell subpopulation, the latter possibly mediating a response to recall antigen and the production of IFN.     

Incomplete activation of CD4 T cells by antigen-presenting transitional immature B cells: implications for peripheral B and T cell responsiveness

B cells leave the bone marrow as transitional B cells. Transitional B cells represent a target of negative selection and peripheral tolerance, both of which are abrogated in vitro by mediators of T cell help. In vitro, transitional and mature B cells differ in their responses to B cell receptor ligation. Whereas mature B cells up-regulate the T cell costimulatory molecule CD86 (B7.2) and are activated, transitional B cells do not and undergo apoptosis. The ability of transitional B cells to process and present Ag to CD4 T cells and to elicit protective signals in the absence of CD86 up-regulation was investigated. We report that transitional B cells can process and present Ag as peptide:MHC class II complexes. However, their ability to activate T cells and elicit help signals from CD4-expressing Th cells was compromised compared with mature B cells, unless exogenous T cell costimulation was provided. A stringent requirement for CD28 costimulation was not evident in interactions between transitional B cells and preactivated CD4-expressing T cells, indicating that T cells involved in vivo in an ongoing immune response might rescue Ag-specific transitional B cells from negative selection. These data suggest that during an immune response, immature B cells may be able to sustain the responses of preactivated CD4(+) T cells, while being unable to initiate activation of naive T cells. Furthermore, the ability of preactivated, but not naive T cells to provide survival signals to B cell receptor-engaged transitional immature B cells argues that these B cells may be directed toward activation rather than negative selection when encountering Ag in the context of a pre-existing immune response.     

Heterogeneity of CD4 and CD8+ memory T cells in localized and generalized Wegener's granulomatosis

Memory T cells display phenotypic heterogeneity. Surface antigens previously regarded as exclusive markers of naive T cells, such as L-selectin (CD62L), can also be detected on some memory T cells. Moreover, a fraction of CD45RO+ (positive for the short human isoform of CD45) memory T cells reverts to the CD45RA+ (positive for the long human isoform of CD45) phenotype. We analyzed patients with biopsy-proven localized Wegener's granulomatosis (WG) (n = 5), generalized WG (n = 16) and age- and sex-matched healthy controls (n = 13) to further characterize memory T cells in WG. The cell-surface expression of CD45RO, CD45RA, CD62L, CCR3, CCR5 and CXCR3 was determined on blood-derived T cells by four-color flow cytometric analysis. The fractions of CCR5+ and CCR3+ cells within the CD4+CD45RO+ and CD8+CD45RO+ memory T cell populations were significantly expanded in localized and generalized WG. The mean percentage of Th1-type CCR5 expression was higher in localized WG. Upregulated CCR5 and CCR3 expression could also be detected on a fraction of CD45RA+ T cells. CD62L expression was seen on approximately half of the memory T cell populations expressing chemokine receptors. This study demonstrates for the first time that expression of the inducible inflammatory chemokine receptors CCR5 and CCR3 on CD45RO+ memory T cells, as well as on CD45RA+ T cells ('revertants'), contributes to phenotypic heterogeneity in an autoimmune disease, namely WG. Upregulated CCR5 and CCR3 expression suggests that the cells belong to the effector memory T cell population. CCR5 and CCR3 expression on CD4+ and CD8+ memory T cells indicates a potential to respond to chemotactic gradients and might be important in T cell migration contributing to granuloma formation and vasculitis in WG.     

Maintenance of long-lived plasma cells and serological memory despite mature and memory B cell depletion during CD20 immunotherapy in mice

CD20 mAb-mediated B cell depletion is an effective treatment for B cell malignancies and some autoimmune diseases. However, the full effects of B cell depletion on natural, primary, and secondary Ab responses and the maintenance of Ag-specific serum Ig levels are largely unknown. The relationship between memory B cells, long-lived plasma cells, and long-lived humoral immunity also remains controversial. To address the roles of B cell subsets in the longevity of humoral responses, mature B cells were depleted in mice using CD20 mAb. Peritoneal B cell depletion reduced natural and Ag-induced IgM responses. Otherwise, CD20+ B cell depletion prevented humoral immune responses and class switching and depleted existing and adoptively transferred B cell memory. Nonetheless, B cell depletion did not affect serum Ig levels, Ag-specific Ab titers, or bone marrow Ab-secreting plasma cell numbers. Coblockade of LFA-1 and VLA-4 adhesion molecules temporarily depleted long-lived plasma cells from the bone marrow. CD20+ B cell depletion plus LFA-1/VLA-4 mAb treatment significantly prolonged Ag-specific plasma cell depletion from the bone marrow, with a significant decrease in Ag-specific serum IgG. Collectively, these results support previous claims that bone marrow plasma cells are intrinsically long-lived. Furthermore, these studies now demonstrate that mature and memory B cells are not required for maintaining bone marrow plasma cell numbers, but are required for repopulation of plasma cell-deficient bone marrow. Thereby, depleting mature and memory B cells does not have a dramatic negative effect on preexisting Ab levels.     

Changes in B Cell Populations and Merozoite Surface Protein-1-Specific Memory B Cell Responses after Prolonged Absence of Detectable P. falciparum Infection

Clinical immunity to malaria declines in the absence of repeated parasite exposure. However, little is known about how B cell populations and antigen-specific memory B cells change in the absence of P. falciparum infection. A successful indoor residual insecticide spraying campaign in a highland area of western Kenya, led to an absence of blood-stage P. falciparum infection between March 2007 and April 2008. We assessed memory B cell responses in 45 adults at the beginning (April 2008) and end (April 2009) of a subsequent 12-month period during which none of the adults had evidence of asymptomatic parasitemia or clinical disease. Antibodies and memory B cells to the 42-kDa portion of the merozoite surface protein-1 (MSP-1₄₂) were measured using ELISA and ELISPOT assays, respectively. B cell populations were characterized by flow cytometry. From 2008 to 2009, the prevalence of MSP-1₄₂-specific memory B cells (45% vs. 55%, respectively, P = 0.32) or antibodies (91% vs. 82%, respectively, P = 0.32) did not differ significantly, although specific individuals did change from positive to negative and vice versa, particularly for memory B cells, suggesting possible low-level undetected parasitemia may have occurred in some individuals. The magnitude of MSP-1₄₂-specific memory B cells and levels of antibodies to MSP-1₄₂ also did not differ from 2008 to 2009 (P>0.10 for both). However, from 2008 to 2009 the proportions of both class-switched atypical (CD19+IgD-CD27-CD21-IgM-) and class-switched activated (CD19+IgD-CD27+CD21-IgM-) memory B cells decreased (both P<0.001). In contrast, class-switched resting classical memory B cells (CD19+IgD-CD27+CD21+IgM-) increased (P<0.001). In this area of seasonal malaria transmission, a one- year absence of detectable P. falciparum infection was not associated with changes in the prevalence or level of MSP-1₄₂ specific memory B cells, but was associated with major changes in overall memory B cell subsets.     

Alterations in peripheral blood B-cell populations in SHIV89.6P-infected macaques (Macacca fascicularis)

In addition to CD4+ T cell depletion, the B cell compartment of HIV-infected patients exhibits abnormalities, including deficits and diminished responses to ex vivo antigenic stimulation and in vivo vaccination. We used chimeric simian-human immunodeficiency virus (SHIV) infection of cynomolgus macaques to determine the dynamics of peripheral blood B cell alterations in this model of HIV infection. During the course of infection, we observed progressive loss of total and memory (CD27+) B cells, increased percentages of activated (CD95+) B cells, hypergammaglobulinemia, and deficits in the CD21+ B cell population. In addition, we noted declines in subsets of memory B cells, including both IgM+ and class-switched (IgD-IgM- CD27+) cells, with sustained deficits in the IgM+ memory (IgM+CD27+) B cell population. The similarity of the B cell alterations in these studies to those observed in HIV+ subjects supports the utility of the SHIV macaque model for examination of HIV-related B cell dysfunction.     

Expression, regulation, and function of B cell-expressed CD154 in germinal centers.

Activated B cells and T cells express CD154/CD40 ligand in vitro. The in vivo expression and function of B cell CD154 remain unclear and therefore were examined. Tonsillar B and T cells expressed CD154 at a similar density both in situ and immediately ex vivo, whereas a significantly higher percentage of the former expressed CD154. CD154-expressing B cells were most frequent in the CD38positiveIgD+ pre-germinal center (GC)/GC founder, CD38positive GC and CD38-IgD- memory populations, and were also found in the CD38-IgD+ naive and CD38brightIgD+ plasmablast subsets, but not in the CD38brightIgD- plasma cell subset. B cell expression of CD154 was induced by engaging surface Ig or CD40 by signals that predominantly involved activation of AP-1/NF-AT and NF-kappaB, respectively. The functional importance of CD154-mediated homotypic B cell interactions in vivo was indicated by the finding that mAb to CD154 inhibited differentiation of CD38positiveIgD- GC B cells to CD38-IgD- memory cells. In addition, mAb to CD154 inhibited proliferation induced by engaging sIg or CD40, indicating the role of up-regulation of this molecule in facilitating B cell responsiveness. Of note, CD154 itself not only functioned as a ligand but also as a direct signaling molecule as anti-CD154-conjugated Sepharose beads costimulated B cell responses induced by engaging surface Ig. These results indicate that CD154 is expressed by human B cells in vivo and plays an important role in mediating B cell responses.     

Promotion of activated human B cell apoptosis and inhibition of Ig production by soluble CD95 ligand: CD95-based downregulation of Ig production need not culminate in activated B cell death

CD95/CD95L interactions are vital to normal lymphoid homeostasis and in the protection against autoimmunity. To directly assess the effects of CD95L on activated B cell survival and Ig responses, purified human peripheral blood B cells, activated in vitro with SAC + rIL2, were incubated with a soluble CD95L fusion protein (fp) and assayed for apoptosis and IgG/IgM production. CD95L fp reproducibly increased apoptosis of these activated B cells and inhibited their Ig production. However, CD95L fp-mediated effects on activated B cell survival could be uncoupled from those on Ig production in that a soluble CD40L fp was incapable of reversing CD95L fp-mediated downregulation of Ig responses despite inhibiting CD95L fp-mediated apoptosis. Moreover, despite the specific caspase-8 inhibitor z-IETD-fmk substantially protecting transformed CL-01 B cells from CD95L fp-mediated apoptosis and permitting their ongoing proliferation, caspase-8 inhibition had no protective effects on CD95L fp-mediated inhibition of constitutive IgM production by CL-01 B cells. Collectively, these results point to a CD95-based downregulatory pathway in activated B cells that need not necessarily culminate in their death.     

The coexpression of CD45RA and CD45RO isoforms on T cells during the S/G2/M stages of cell cycle.

The tyrosine phosphatase CD45 is alternatively spliced to generate isoforms of different molecular weights (180-220 kDa) which are differentially expressed on hematopoietic cells. Monoclonal antibodies reacting with either the 180-kDa (UCHL-1, CD45RO) or the 200- to 220-kDa (2H4, CD45RA) isoform have been used to subdivide T cell populations based on their expression of one or the other of these two epitopes. CD45RA T cells have "naive" characteristics of unresponsiveness to recall antigens and prominence in cord blood, while CD45RO T cells are considered "memory" T cells because they proliferate to recall antigens and increase following PHA activation of cord blood. However, we have recently demonstrated the expression of the CD45RA isoform on a subpopulation of CD45RO+ T cell clones, suggesting that CD45RA is not a universal marker for naive T cells. Using propidium iodide staining of the DNA to determine cell cycle stage, we now show that CD45RA expression is significantly higher on T cell clones during the S, G2, and M stages of cell cycle when compared to CD45RA expression on cells in Go and G1. Furthermore, CD45RA expression on cells undergoing mitosis is not limited to long-term activated T cell clones, as uncultured peripheral blood T cells in the S/G2/M phase express significantly more CD45RA. The percentage of T cells coexpressing CD45RA and CD45RO also increases following PHA activation, indicating that T cells in the process of division express both isoforms. These results suggest a potential role of the CD45RA isoform during the stages of cell cycle leading to mitosis.