John Gordon

Germinal centres in T-cell-dependent antibody responses

For more than a century follicles have been recognized as a site of intense cell proliferation and cell death. At last the significance of this activity is beginning to emerge: antigen-driven B-cell proliferation, somatic mutation, positive and negative selection, and memory and plasma cell development all appear to take place within the follicle.

The Epstein-Barr virus oncoprotein, latent membrane protein-1, reprograms germinal centre B cells towards a Hodgkin's Reed-Sternberg-like phenotype

Although the latent membrane protein‐1 (LMP1) of the Epstein–Barr virus (EBV) is believed to be important for the transformation of germinal centre (GC) B cells, the precise contribution of this viral oncogene to lymphoma development is poorly understood. In this study, we used a non‐viral vector‐based method to express LMP1 in primary human GC B cells. Gene expression profiling revealed that LMP1 induced in GC B cells transcriptional changes characteristic of Hodgkins lymphoma cell lines. Strikingly, LMP1 down‐regulated the expression of B‐cell‐specific genes including B‐cell receptor components such as CD79A, CD79B, CD19, CD20, CD22, and BLNK. LMP1 also induced the expression of ID2, a negative regulator of B‐cell differentiation. Our data suggest that in EBV‐positive cases, LMP1 is likely to be a major contributor to the altered transcriptional pattern characteristic of Hodgkin/Reed–Sternberg cells, including the loss of B‐cell identity. Copyright

Fortifying B cells with CD154: an engaging tale of many hues

Although the original antibody to what is now defined as CD40 was raised against and shown to react with bladder carcinoma cells, the major insights into the functional attributes of this important receptor molecule – now known to be found on a diverse range of cell types – have arisen from studies on B lymphocytes. This 50 000 MW glycoprotein member of the tumour necrosis factor receptor (TNF-R) family, expressed constitutively at high density throughout B-cell differentiation, provides the focus for T-cell delivered cognate help to the B cell via the capacity to engage its inducible counterstructure – CD154; or ‘CD40 ligand’ (CD40L).1–5 The aim of this article is to review the multiple stages at which the cognate CD40–CD40L pairing might serve to direct and modify the B-cell response with a focus on data generated from human in vitro studies set within an in vivo context gleaned primarily from observations in the mouse. The consequences of engaging CD40 on lymphoma B cells are discussed while, finally, we address how the quantity and quality of the CD154 being offered to its receptor may dictate the functional outcome of the cell in question.

Epitope-dependent synergism and antagonism between CD40 antibodies and soluble CD40 ligand for the regulation of CD23 expression and IgE synthesis in human B cells

Background: The induction of IgE synthesis in naïve B cells requires two T‐cell‐derived signals: one delivered through CD40 and the other via interleukin‐4 (IL‐4). The natural counterstructure to CD40 is the CD40 ligand (CD40L). We have asked about the interplay between CD40L and CD40 mAb that recognize distinct epitopes in delivering signals for regulating IL‐4‐dependent IgE synthesis and the expression of CD23, the low‐affinity IgE receptor, in resting B cells.

B‐lymphocyte subpopulations are equally susceptible to Epstein–Barr virus infection, irrespective of immunoglobulin isotype expression

While Epstein–Barr virus (EBV) is known to establish latency in the memory B‐cell compartment, there is controversy as to whether the memory or the naïve B cell is the initial target for infection. Here we have explored the infectability of the B‐cell subsets contained in peripheral blood and tonsils, as distinguished by their surface expression of the immunoglobulin isotypes that help to define naïve and memory pools. First we show that both CD21 and major histocompatibility complex (MHC) class II molecules – respectively, the major receptor and co‐receptor for EBV on B cells – are expressed at similar levels on blood and tonsillar B cells, irrespective of surface immunoglobulin class, indicating that each of the subsets demonstrate an equal potential, at least for infection. Then, following in vitro infection of total tonsillar B cells, we found that the relative frequencies of immunoglobulin (Ig)M‐, IgG‐ and IgA‐positive cells containing EBV‐encoded Epstein–Barr virus nuclear antigen 5 (EBNA5) protein at 48 hr were similar to those of the starting population. However, IgD expression was uniformly decreased, probably as a consequence of cellular activation. These data indicate that recirculating B cells have both the potential for, and susceptibility to, initial infection by EBV, irrespective of the immunoglobulin isotype expressed.

The extrafollicular-to-follicular transition of human B lymphocytes: induction of functional globotriaosylceramide (CD77) on high threshold occupancy of CD40

Amongst lymphocytes, expression of CD77 (globotriaosylceramide, Gb3) is exclusive to B cells of the germinal center (GC). Its acquisition by extrafollicular B cells may thus herald their commitment to a follicular response. Here we show that high threshold occupancy of CD40 by its cognate ligand (CD40L) promotes rapid induction of CD77 expression in non‐GC (CD38lo) B cells. The kinetics of CD77 acquisition mirrored those of GC‐related markers CD95 and CD86 but contrasted with the more delayed increase in CD38 expression. Induction of CD77 was not a simple consequence of cell cycle entry: other conditions of stimulation equally capable of driving proliferation failed to promote CD77 expression. CD77 was functional in that cells were now sensitive to Verotoxin‐1, an Escherichia coli‐derived ligand of Gb3. These data indicate that acquisition by extrafollicular B cells of CD77 results from high threshold occupancy of CD40, a situation that should be reached physiologically only once a critical level of T cell priming has been achieved.

Functional interaction between β2-adrenoceptor agonists and interleukin-4 in the regulation of CD23 expression and release and IgE production in human

Normal human peripheral blood mononuclear cells (PBMC) produced IgE when stimulated with IL-4. In the present report it was shown that beta 2-adrenoceptor agonists, salbutamol and fenoterol, potentiated the IL-4-induced IgE production without significantly affecting the expression of the low affinity receptor for IgE at the cell surface of monocytes and B lymphocytes. However, beta 2-adrenoceptor agonists were shown to enhance at day 7 the IL-4-induced release of the soluble form of CD23 (sCD23) by PBMC. This effect was specific since a beta-adrenoceptor antagonist, D,L-propranolol, inhibited the IL-4-induced IgE production by these cells. Alternatively, the beta 2-adrenoceptor agonists inhibited the production by these cells of interferon-gamma (IFN-gamma) but did not affect the production of IL-4 when stimulated with phytohemagglutinin A + a phorbol ester. These data suggest that beta 2-adrenoceptor agonists influence the IL-4-induced IgE production in humans by enhancing the release of sCD23 and inhibiting the production of endogenous IFN-gamma. In addition to the effect on the IL-4-induced IgE production it was shown that beta 2-adrenoceptor agonists potentiated the effect of IL-4 on a human promonocytic cell line, U 937, by enhancing CD23 expression and release and by inducing the differentiation of these cells into monocyte-like cells. Taken together, these data indicate that beta 2-adrenoceptor agonists potentiated the effect of IL-4 and that this functional interaction is different considering the cell-lineage and the stage of differentiation of these cells.

Phenotypic characterization of CD3–7+ cells in developing human intestine and an analysis of their ability to differentiate into T cells

We have identified a large population of CD3−7+ cells in human fetal gut. Three- and four-color flow cytometry revealed a distinct surface Ag profile on this population; the majority were negative for CD4 and CD8, whereas most of the remainder expressed the CD8αα homodimer. In contrast about half of CD3+ cells expressed CD4 and half expressed CD8α. A large proportion of CD3−7+ cells expressed CD56, CD94, and CD161, and whereas CD3+ T cells also expressed CD161, they only rarely expressed CD56 or CD94. Further studies were conducted to determine whether the CD3−7+ cells have the potential to differentiate into CD3+ cells. About half of CD3−7+ cells contain intracellular CD3ε. Rearranged TCR γ-chains were detected in highly purified CD3−7+ cells as an early molecular sign of T cell commitment, and the pattern of rearrangement with V regions spliced to the most 5′ Jγ segment is reminiscent of early thymocyte differentiation. In reaggregate thymic organ cultures, CD3−7+ cells also gave rise to CD3+ T cells. Thus, we demonstrate that the CD3−7+ cells present in the human fetal gut display a distinct phenotype and are able to develop into CD3+ T cells.

CD40 ligation downregulates EBNA‐2 and LMP‐1 expression in EBV‐transformed lymphoblastoid cell lines

Epstein‐Barr virus (EBV) drives the proliferation of human B cells in vitro and during primary infection in vivo. The transformed immunoblasts express nuclear proteins EBNA1–6, transcribed from the Cp/Wp promoter, and the membrane proteins LMP‐1, ‐2A and ‐2B (lymphoblastoid type of latency). EBV persists through life in resting memory B cells with a restricted type of latency in the absence of the Cp/Wp promoter activity. Since CD40 crosslinking can reportedly inhibit the growth of EBV‐transformed lymphoblastoid cell lines (LCLs), we have examined the effect of CD40 ligation on the expression of EBNAs and LMP‐1 and on Cp EBV promoter activity together with several phenotypic markers. CD40 crosslinking led to a partial downregulation of EBNA‐2, EBNA3–6 and LMP‐1 in LCLs, paralleled by downregulation of Cp promoter activity. It also induced upregulation of the germinal center marker CD77 on the LCL cells. Our findings suggest that the encounter of proliferating EBV‐transformed immunoblasts with CD40L, as would occur when normal B cells generate memory cells in germinal centers, may switch the viral transcription program from the full lymphoblastoid to a more restricted latency program in a proportion of cells. This would permit virus persistence in the B‐cell memory compartment.

A Combination of Calcium Ionophore and 12-O-Tetradecanoyl-Phorbol-13-Acetate (TPA) Stimulates the Growth of Purified Resting B Cells

In this study we investigated whether the calcium ionophores A23187 and ionomycin can act synergislically with the phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) to stimulate the growth of resting B lymphocytes purified from human tonsil cells, Ionomycin, A23187, and TPA added separately to cultures at doses of 0.4–1.6 μg/ml, 0.2–0.8 μ/ml, and 0.05–0.25 ng/ml respectively, did not induce DNA synthesis in resting B lymphocytes. In contrast, calcium ionophores at concentrations of 0.4–1.6 μg/ml ionomycin and 0.2–0.8 μg/ml A23187, in the presence of 0.05–4 ng/ml TPA, induced marked DNA synthesis and B‐cell proliferation, as shown by analyses of incorporation of [3H]thymidine, growth kinetics, and the percentage of cells in the S and G2+ M phases of the cell cycle. These results show that the synergistic effects of calcium ionophores and TPA can bypass the requirement for antigen and exogenous growth factors in B‐cell activation. These observations are similar to those obtained from studies of T lymphocytes by other workers.