Antigen-induced acceleration of shedding and replacement of B cell antigen receptors requires mature T cells

To determine which early and intermediate events in the response of antigen-binding B cells to a T-dependent antigen (sheep erythrocytes [SRC]) require T help, the antigen-induced changes in receptor turnover and surface IgD loss in BALB/c athymic nu/nu mice were compared with that of nu/+ littermates and +/+ BALB/c mice. Nonimmune SRC antigen-binding spleen B cells (ABC) from +/+, nu/+, and nu/nu BALB/c mice coexpressed IgM and IgD, and 85 to 95% retained receptors well when incubated for 2.5 hr in 100 micrograms/ml cycloheximide (which prevents receptor replacement). Also they were able to regain their ability to bind antigen by 18 hr after pronase treatment, but not by 2 hr. However, 5 days after in vivo immunization, 1) the proportion of ABC expressing surface IgD declined from around 90% to less than 50% in +/+ mice and nu/+ mice but not in nu/nu mice; 2) substantial recovery of antigen-binding occurred by 2 hr after pronase treatment in +/+ and nu/+ ABC but not in nu/nu ABC; and 3) when spleen cells were incubated in cycloheximide, uncompensated receptor shedding reduced +/+ and nu/+ ABC by around 80% but produced only about a 10% reduction in nu/nu ABC. Thus, although the ABC in nonimmune nu/nu mice appeared normal with respect to their surface Ig turnover and expression, they failed to undergo the normal antigen-induced loss of IgD or acceleration of surface Ig shedding and replacement, suggesting that these intermediate activation events require interaction with mature T cells. To determine whether this interaction had to occur during B cell development, during the development of the immune response, or during receptor shedding or replacement itself, cell transfer experiments were carried our wherein nu/+ T cells were transferred i.v. to nu/nu littermates 1 day before immunization with SRC. In the transfer recipients, pronase-treated day 5 ABC were then able to replace and shed their receptors at the accelerated rate, like ABC from +/+ and nu/+ mice. In contrast, the co-incubation of 5-day immune nu/+ T cells with nu/nu B cells did not alter the rate of shedding or replacement.     

Lymphocyte function in human bone marrow. I. Characterization of two T cell populations regulating immunoglobulin secretion

We analyzed the regulation of immunoglobulin (Ig) production in short-term cultures of human (rib) bone marrow cells. In contrast to blood or tonsil cell cultures, large quantities of IgG and IgA, but not IgM, were secreted by unstimulated marrow cells. The addition of pokeweed mitogen or phytohemagglutinin resulted in the suppression of this Ig secretion. Both mitogens induced the production of high levels of interleukin 2 (IL 2) in marrow cultures, and the addition of IL 2 alone mimicked the suppressive effect of mitogens. Incubation of marrow cells with Epstein Barr virus resulted in enhanced Ig secretion, primarily of the IgM isotype. The addition of mitogen or IL 2 suppressed Ig production in these cultures as well. The mitogen-induced suppression of Ig secretion in stimulated or unstimulated marrow cultures was inhibited by the monoclonal anti-TAC (IL 2 receptor) antibody. Cell separation experiments indicated that the induction of suppressor activity in marrow cultures involved two distinct populations of marrow-resident T lineage cells. The first population responds to activation by mitogens with the production of IL 2. This population has a surface phenotype appropriate for helper T cells. The second T cell population expresses T8 and TAC determinants. These cells acquire suppressor cell activity after exposure to IL 2. The expression of suppressor function does not require additional (e.g., mitogenic) activation signals. The IL 2-dependent marrow suppressor T cells represent a newly recognized T lymphocyte subset. The regulatory pathway delineated may be important for the regulation of antibody formation in bone marrow, the major site of Ig production in man.     

Differentiation of B lineage cells from liver of neonatal mice: generation of immunoglobulin isotype diversity in vitro

The development and differentiation of B cells expressing different immunoglobulin (Ig) isotypes was studied in cultures of murine neonatal liver cells. Before culture, 5 to 15% of the liver cells were mu + pre-B cells; 1 to 3% had surface IgM and less than 0.1% had slgG. During 4 days in culture the number of pre-B cells declined, whereas the number of IgM B cells increased greater than 20-fold; IgG B cells also increased in number. Of the four subclasses, IgG3+ and IgG2b+ cells predominated, each representing 3 to 10% of the total B cells at day 4. IgG1+ and IgG2a+ cells were present in lower numbers, representing 1 to 5% and 0.3 to 2.5% of B cells, respectively. Most IgG+ cells also expressed sIgM. Only a minority (less than 10%) of the sIgM+ cells were sIgD+, and most sIgG+ cells were sIgD-. Few T cells were present in these cultures (less than 0.5% in newborn liver), and sIgG+ cells were generated in normal frequencies in cultures of cells from nude mice. The numbers of B cells expressing each IgG subclass were similar in cultures from athymic nu/nu mice, nu/+ heterozygous littermates, and normal BALB/c mice. Plasmablasts and plasma cells appeared over a 14-day culture interval, and these expressed cytoplasmic IgM, IgG3, IgG1, IgG2b, IgG2a, and IgA. Measurable amounts of the first four isotypes were detected in the culture supernatants by radioimmunoassay. These results indicate that neonatal B cells can undergo isotype switching in the absence of T cell help, and that the expression of sIgD may not be a prerequisite for cells to switch Ig isotypes.     

A cell population in nu/nu spleen can prevent generation of cytotoxic lymphocytes by normal spleen cells against self antigens of the nu/nu spleen.

Spleen cells from athymic nu/nu mice contain two kinds of physically separable active cells that can have very different effects on the generation of CL (cytotoxic lymphocytes) by normal LN cells in an in vitro response against allogeneic stimulator cells. They can provide an accessory cell required for the activation of CLP (cytotoxic lymphocyte precursor cells) which need not be H-2 identical to the CLP and will function normally even when H-2 identical to the stimulator cells. They can also provide a suppressor cell that prevents the activation of CLP that can recognize the H-2 of the nu/nu mouse. Thus, with A, B, and C to represent three H-2 differnt mouse strains, a culture containing CLP from strain A and nu/nu spleen cells from strain B or strain (A x B)F1 will produce CL against strain C or (A x C)F1 stimulator cells but not against strain B or strain (A x B)F1 stimulator cells unless the suppressor cell is first removed. It is proposed that the in vivo role of the suppressor cell in a normal mouse is to prevent the activation of CLP reactive against self.     

Analysis of T cell and B cell function in Peyer's patch and lamina propria of New Zealand Black and DBA/2 mice

We have analyzed gastrointestinal immune function in both DBA/2 and spontaneously autoimmune New Zealand Black (NZB) mice. We have studied both in vitro proliferation and differentiation of Peyer's patch cells and have measured immunoglobulin (Ig) secretion by cultured jejunal segments. Peyer's patch B cells and T cells from both DBA/2 and NZB mice showed similar proliferative responses to Con A and lipopolysaccharide (LPS), respectively. Unlike NZB splenic B cells, isolated Peyer's patch B cells from NZB mice did not spontaneously secrete Ig of any isotype. Seven-day cultures of equal numbers of Peyer's patch T cells and B cells resulted in similar patterns of secretion of IgA, IgG, and IgM in both strains. The addition of Con A to cultures of DBA/2 Peyer's patch cells consistently resulted in a onefold to threefold increase in IgA secretion after 7 days. Con A stimulation of NZB Peyer's patch cells did not produce any increment in IgA secretion. LPS stimulation of Peyer's patch cells from either strain resulted in a similar increase in IgG secretion with little effect on IgA secretion. The in vivo correlate of this finding was seen in the IgA to IgG ratio of Ig secreted by cultured jejunal fragments. In DBA/2 mice the rates of IgA/IgG varied from 2.36 to 4.85, whereas in NZB mice the ratio never exceeded 0.5. These experiments show that defects on the T cell compartment of NZB mice encompass gut-associated lymphoid tissue. The possible relationship of these findings and previously observed defects in oral tolerance is discussed.     

A role for isotype-specific binding factors in the regulation of IgA- and IgG-specific responses by the anti/contrasuppressor T cell circuit

Previous studies have shown that the isotype of an antibody response is selected, in part, by the inhibition of isotype-specific suppression. The antisuppressor model predicts that isotype selection is initiated through an interaction between Ag, Ig, and a T cell-derived factor within 6 h of immunization. This report characterizes some of these molecules and their contribution to isotype regulation. Cultures of murine spleen cells stimulated with the T cell-dependent Ag SRBC led to Ag-specific IgG and IgA responses that could be suppressed and then antisuppressed by a molecular complex produced by mixing purified serum Ig with the supernatant of Ag-pulsed macrophages co-cultured with T cells. The supernatants from separate cultures of Ag-pulsed macrophages and rIL-1 alpha stimulated CD4+ T cells, could be pooled and mixed with Ig to produce functional antisuppressive complexes thereby allowing the factors from the different cell types to be studied separately. Adsorption of the co-culture or the rIL-1 alpha stimulated T cell supernatants against monoclonal IgG or IgA, removed IgG and IgA binding factors, respectively, and abrogated the ability to enhance the corresponding isotype. The adherent material could be recovered and used to reconstitute enhancement by the supernatants depleted of the binding factors. When affinity purified IgG or IgA was used as the source of Ig within the antisuppressive complexes, the enhancement of the antibody response was limited to the isotype of the regulatory Ig used to form the complex. Thus, manipulation of the antisuppressive molecules has a predictable effect on isotype selection. Release of isotype-specific binding factors by CD4+ cells by rIL-1 alpha supports the hypothesis that T cell circuits play a role in initiating isotype regulation.     

Surface Ig isotypes on cells responding to lipopolysaccharide by IgM and IgG secretion.

Using the fluorescence-activated cell sorter (FACS), we have investigated the Ig isotypes present on murine B cells, which can be polyclonally activated by lipopolysaccharide (LPS) in low cell density cultures. The LPS response was partly inhibited as a result of staining with anti-IgD and anti-IgM reagents, but not with anti-IgG reagents. The IgM+, IgD+, or IgG- fractionated cell populations gave both an IgM and an IgG response comparable to controls, whereas the response of the IgM-, IgD- cells was 5- to 20-fold lower. IgG- cells separated 1 day after LPS stimulation could still mount an IgM and IgG response indistinguishable from controls at the peak of the response. It is concluded that IgM+, IgD+, IgG- cells constitute the major LPS-sensitive cell population in the low cell density culture system and that IgG is not a necessary cell surface isotype for precursors of IgG-secreting cells.     

The use of specific helper T cell clones to study the regulation of isotype expression by antigen-stimulated B cell clones

In order to determine the mechanism by which helper T cells regulate the production of the various immunoglobulin (Ig) classes, a number of helper T cell clones specific for keyhole limpet hemocyanin (KLH) were generated. These helper T cell clones then were used in a modified splenic fragment system whereby cloned helper T cells and a source of B cells were limit-diluted into naive, lethally irradiated recipients. The B cell clones that were subsequently stimulated in such an assay system by the addition of the antigen 2,4-dinitrophenol (DNP)-KLH then were tested for the various isotypes produced. The results of these studies indicate that the use of a single helper T cell clone could result in the production of all known Ig isotypes including IgE. Moreover, the use of a single helper T cell clone could result in multiple isotype production by a single B cell clone. However, a comparison of the isotypes secreted by a number of different B cell clones that were stimulated with the same helper T cell clone indicated that a variety of isotypic patterns could be obtained. In addition, it was found that the majority of B cell clones produced in the presence of T cell clones secrete fewer numbers of different isotypes compared with B cell clones generated with a heterogeneous population of T cells. Finally, no evidence could be found for isotype-specific helper T cell clones, although a few of the T cell clones appeared to induce a somewhat restricted isotype pattern in which only two or three different isotypes were observed.     

Changes in proliferation activity and relative distributions of lymphoid cell subpopulations in congenitally athymic nu/nu mice and lurcher mice with spontaneous olivopontocerebellar degeneration

Changes observed in mice with congenital damage of some part of the CNS-neuroendocrine-immune regulatory system are described. nu/nu mice with congenital absence of thymus and Lurcher mice with spontaneous olivopontocerebellar degeneration displayed changes in the histoarchitecture of adrenal gland, immune organs (thymus, spleen, axillar lymph nodes) and intestine. Changes were also observed in IgM+, IgG+, CD4+ and CD8+ lymphoid cell subpopulations in the main lymphoid organs--the spleen and axillar lymph nodes and in the proliferative ability of whole lymphoid cell populations. The extreme decrease of lymphoid T-cell subpopulations in athymic nu/nu mice is the consequence of the absence of thymus, the organ of their maturation. On the other hand, a relative increase of B-cell subpopulations was found in this mouse strain. A relative decrease of CD4+ lymphocytes and a different influence of immunization on B-cell subpopulations were found in the spleen in neurodeficient Lurcher mice. The high percentage of apoptotic cells, cells in the S-phase of cell cycle and increased proliferation index in nu/nu mice suggest that the turnover and renewal of lymphoid cells in the spleen in nu/nu mice is more rapid than in control immunocompetent BALB/c mice.     

Thymus differentiation and T-cell specificity in nu/nu +/+ mouse aggregation chimaeras

Thymus development and T cell differentiation were studied in mouse chimaeras produced by aggregating pre-implantation embryos of thymus-deficient nude BALB/c (nu/nu) and wild-type C57BL/6 (+/+) mice and vice versa. Chimaeras showed mosaic distribution of skin and coat pigmentation, of hair follicles, of glucosephosphate isomerase within all tested organs and of lymphocytes expressing the different major transplantation antigens (H-2). When tested for their capacity to generate vaccinia virus-specific and self-H-2 specific cytotoxic T cells, all chimaeras of BALB/c (nu/nu) H-2d in equilibrium C57BL/6 (+/+) H-2b type generated T cells of one or both parental origins that were specific for virus and for self-H-2 of the +/+ (H-2b) type only. In contrast, some BALB/c (+/+) H-2d in equilibrium C57BL/6 (nu/nu) H-2b chimaeras generated vaccinia virus-specific cytotoxic T cells specific for either H-2d (+/+) type or for H-2b (nu/nu) type. These asymmetrical results can be interpreted to indicate the following: (i) The +/+ thymus part alone is functional, but because of asymmetrical cross-reactivities of anti-self-H-2 specificities, the observed T cell restriction phenotypes differ. (ii) Both nu/nu and +/+ thymus parts are functional but immune response defects may be exaggerated in such chimaeras producing unexpected non-responsiveness to vaccinia virus linked to H-2d in H-2b (+/+) in equilibrium H-2d (nu/nu).     

In vivo polysaccharide-specific IgG isotype responses to intact Streptococcus pneumoniae are T cell dependent and require CD40- and B7-ligand interactions.

In vivo Ig responses to soluble, haptenated polysaccharide (PS) Ags are T cell independent and do not require CD40 ligand (CD40L). However, little is known regarding the regulation of in vivo PS-specific Ig responses to intact bacteria. We immunized mice with a nonencapsulated, type 2 Streptococcus pneumoniae (R36A) and compared the parameters that regulated in vivo Ig isotype responses to the bacterial cell wall C-PS determinant, phosphorylcholine (PC), relative to Ig responses to the cell wall protein, pneumococcal surface protein A. Consistent with previous reports using soluble PS and protein Ags, the anti-PC and anti-pneumococcal surface protein A responses differed in that the anti-PC response was induced more rapidly, had a distinctive Ig isotype profile, and failed to demonstrate boosting upon secondary challenge with R36A. However, in contrast to previous studies, the IgG anti-PC response was TCR-alphabeta+ T cell dependent, required CD40L, and was blocked by administration of CTLA4 Ig. The nature of the T cell help for the anti-PC response had distinct features in that it was only partially blocked by CTLA4 Ig and was dependent upon both CD4+ and CD8+ T cells. Surprisingly, whereas the IgM anti-PC response was largely T cell independent, a strong requirement for CD40L was still observed, suggesting the possibility of an in vivo T cell-independent source for CD40L-dependent help. These data suggest that the regulatory parameters that govern in vivo Ig responses to purified, soluble PS Ags may not adequately account for PS-specific Ig responses to intact bacteria.     

Physiology of receptor capping induced by erythrocyte surface antigens

The capping of antigen-binding cell receptors by bound sheep erythrocytes (SRC) demonstrates that antigen mounted on a cell surface can generate a signal leading to the capping reaction. SRC-induced capping of ABC is: (a) highly dependent on both aerobic and anaerobic glycolysis, (b) unaffected by agents altering intracellular cyclic nucleotide concentrations, (c) slightly more vigorous in strain A than in CBA mice, (d) inhibited by calcium ionophore, (e) inhibited by the local anesthetic dibucaine and the tranquillizer chlorpromazine, (f) dependent on cytoskeletal activity (i.e., inhibited by the simultaneous presence of colchicine and cytochalasin B), (g) not dependent on the membrane ATPases inhibited by ouabain, (h) not dependent on motility, in that agents which inhibit motility (cytochalasin B alone) or stimulate motility (carbachol) do not alter capping behavior. These properties represent similarities between the capping of surface Ig by the cellular antigens on SRC and by proteins such as anti-Ig. SRC-induced capping is much slower than anti-Ig-induced capping, and only engages 30–40% of ABC, indicating that the nature of the crosslinking agent can influence the kinetics and extent of capping. But SRC cap with the rapid kinetics typical of anti-Ig-induced capping if the surface membrane of the ABC is first cleared of other glycoproteins with trypsin. The removal of negatively charged sialic acid residues by neuraminidase has no such effect. It is probable that the compression of bound SRC into a small area of the membrane requires more energy than does the capping of protein ligands, and that some cells cannot muster enough energy to achieve it.     

Identification and separation of pre T-cells from nu/nu mice: differentiation by preculture with thymic reticuloepithelial cells.

Spleens and lymph nodes of nu/nu, congenitally athymic, mice have 16–32% and 72–76%, respectively, of large, brain-associated-T (BAT) antigen positive cells. These BAT positive large cells were isolated from nu/nu spleens with a fluorescence-activated cell sorter (FACS-II) and then cultured on thymic reticuloepithelial cell (TRC) monolayers. Both concanavalin A and mixed lymphocyte reactivity was induced during this culture period. We concluded that these BAT positive cells are pre-T cells which can he induced to at least some T cell functions by an inductive factor produced by TRC.     

Interleukin 2 production by lymphoid cells from congenitally athymic (nu/nu) mice

The ability of lymphoid cells from congenitally athymic (nu/nu) mice to produce interleukin 2 (IL 2) was investigated. Spleen or lymph node cells (superficial or mesenteric) from nude mice on an N:NIH(S)II or BALB/c genetic background were stimulated with concanavalin A (Con A) or with irradiated allogeneic (DBA/2) spleen cells that had been depleted of T cells by treatment with monoclonal anti-Thy-1.2 antibody plus complement. After 24 hr, supernatants were harvested and assayed for their ability to support the proliferation of a cloned IL 2-dependent cytolytic T cell line. With this quantitative microassay, IL 2 production was not detectable in spleen and lymph nodes of 6-wk-old N:NIH(S)II nude mice; however, by 12 mo of age, IL 2 production increased more than 100-fold to reach levels comparable to control (nu/+) animals. Con A was more potent than alloantigen in the induction of IL 2 in either nude or control (nu/+) animals. Furthermore, differences in the genetic background of nude mice resulted in corresponding differences in both numbers of T cells (defined by monoclonal anti-Thy-1 antibody) and IL 2 production. By using negative selection with monoclonal antibodies plus complement, IL 2 production in aged nude mice was shown to depend upon a subpopulation of cells that expressed Thy-1 but not Lyt-2. These data thus demonstrate that a subpopulation of IL 2-producing cells with a Thy-1+ Lyt-2- surface phenotype can develop in the apparent absence of thymic influence.     

Lymphocyte function in human bone marrow. III. Isotype commitment, metabolic and secretory characteristics of immunoglobulin producing cells

We have examined the functional and metabolic properties of immunoglobulin (Ig)-secreting cells in adult (rib) bone marrow, the tissue which provides the major proportion of serum Igs. In the absence of polyclonal activators, high rate Ig production (1-2 micrograms/day/10(6) marrow mononuclear cells) was sustained from the beginning of culture throughout 2 weeks and then declined. Ten percent of the Ig secreted was of the IgM isotype and IgG/A made up the remainder at equal proportions. Infection of marrow cells with Epstein-Barr virus (EBV) induced the production of large amounts of IgM, but virtually all IgG/A-committed cells were refractory to stimulation with EBV. Both EBV-induced and the "spontaneous" Ig production was inhibited by cycloheximide, but only EBV-induced IgM production was blocked by hydroxyurea and gamma-irradiation. The polyclonal activators PHA and PWM induce suppressor-T-cell activity in marrow cultures. This suppressor function involves nonproliferating cells which acquire suppressive activity 3-4 days after mitogenic activation. Prednisolone and cyclosporine A modulate Ig production in cultures of peripheral lymphocytes but had no effect on Ig secretion in marrow cell cultures. This observation was reminiscent of the absent or at best marginal short-term effects on in vivo serum Ig levels which is typical for these drugs. Our observations suggest that the marrow Ig-producing B-lymphoid cell compartment shows major differences to other tissue sites with respect to properties of the Ig-secreting cells the immunoregulatory activities able to control their function, and the response of these cells to clinically important drugs.     

Regional variations in the distributions of small intestinal intraepithelial lymphocytes (IELs) in BALB/c +/+, nu/+, and nu/nu mice

Regional variations in intraepithelial lymphocytes (IELs) in the small intestine were examined in BALB/c +/+, nu/+, and nu/nu mice. The small intestine was obtained from 11- to 12-week-old mice and divided equally into three (proximal, middle, and distal) parts. The IELs were isolated from each part of the intestine, and the total numbers of IELs in nu/+ and nu/nu mice were about a fifth of those in +/+ mice. Regional variations in the distribution of the IEL alphabeta, but not the gammadelta T-cell subset were found by use of flow cytometry in +/+ and nu/+ mice. On the other hand, such differences were not found in nu/nu mice, suggesting that thymus-independent development of T cells is not different among regions. Different local expansion of thymus-dependent alphabeta T cells may cause the regional variations seen in the distribution of alphabeta T cell IELs in +/+ and nu/+ mice.     

Human peripheral blood B lymphocyte subpopulations: functional and phenotypic analysis of surface IgD positive and negative subsets

Highly purified human peripheral blood B cells stimulated with Cowan I Staphylococcus aureus (SA) and mitogen-activated T cell supernatants (T supt) generated large numbers of immunoglobulin (Ig)-secreting cells (ISC), whereas fewer ISC developed in cultures containing T supt in the absence of SA. To determine whether surface Ig isotype expression defined responsive B cell subsets, IgD+ and IgD- B cells were prepared with the fluorescence-activated cell sorter. Whereas both the IgD+ and IgD- B cells responded to SA + T supt, only the IgD- subset generated substantial numbers of ISC in response to T supt alone. Analysis of secreted Ig revealed that IgG and IgA were the predominant isotypes secreted by IgD- B cells in response to T supt or SA + T supt. By contrast, the IgD+ cells secreted predominantly IgM in response to SA + T supt but not to T supt alone. When responsiveness to pokeweed mitogen (PWM) was examined in the presence of supplemental T cells, the IgD- subset was found to be greatly enriched for responsive cells, and again, IgG and IgA were the predominant isotypes secreted, although these cells were also capable of secreting some IgM. The magnitude of the response induced by PWM from IgD- B cells was usually greater than that induced by SA + T supt. Although IgD+ B cells responded poorly to PWM, the differentiation of a small number of IgM-secreting cells was routinely stimulated by this polyclonal activator in the presence of T cells. The magnitude of the PWM response by IgD+ B cells was always greatly diminished compared with that stimulated by SA + T supt. Cell cycle analysis after acridine orange staining, cell volume measurement, and staining for expression of activation antigens (transferrin receptor and 4F2) indicated that the IgD- B cells were largely resting, but did contain a population of activated cells. Removal of activated 4F2+ cells from the IgD- subset diminished but did not abolish their capacity to generate ISC in response to SA + T supt or PWM in the presence of T cells. These results suggest that the IgD- population contains both an activated 4F2+ and a resting 4F2- subset. The data emphasize that multiple subpopulations of peripheral blood B cells contain precursors of ISC. Moreover, the responsiveness of the subsets to various stimuli and the Ig isotype subsequently secreted appear to be intrinsic features of each subset.     

Isotype switching by human B cells is division-associated and regulated by cytokines

Isotype switching by murine B cells follows a pattern whereby the proportion of cells undergoing switching increases with division number and is regulated by cytokines. Here we explored whether human B cells behaved in a similar manner. The effect of IL-4, IL-10, and IL-13, alone or in combination, on Ig isotype switching by highly purified naive human CD40 ligand (CD40L)-activated B cells was measured against division number over various harvest times. Switching to IgG was induced by IL-4 and, to a lesser extent, IL-13 and IL-10. The combination of IL-10 with IL-4, but not IL-13, induced a higher percentage of cells to undergo switching. Isotype switching to IgG by human CD40L-activated naive B cells was found to be linked to the division history of the cells: IgG(+) cells appeared in cultures of B cells stimulated with CD40L and IL-4 after approximately the third cell division, with the majority expressing IgG1, thus revealing a predictable pattern of IgG isotype switching. These results reveal a useful quantitative framework for monitoring the effects of cytokines on proliferation and isotype switching that should prove valuable for screening Ig immunodeficiencies and polymorphisms in the population for a better understanding of the regulation of human humoral immune responses.     

Epstein-Barr Virus Infection of Na?ve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology

Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth-transforming infections yet establishes persistence only in IgD⁺ CD27⁺ non-switched memory (NSM) and IgD⁻ CD27⁺ switched memory (SM) B cells, not in IgD⁺ CD27⁻ naïve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV-driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively-harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naïve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCL''s evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes.