Selective deficiency of IgG and IgA as a first stage defect in B cell differentiation.

A case of selective deficiency of IgG and IgA, in a 13-year old girl, is described. Immunologic investigations, showing an almost complete absence of IgG- and IgA-bearing lymphocytes and significant amounts of IgD and IgM positive cells, suggest the possibility of a block in the shift from IgM to IgG synthesis at the B lymphocyte level.     

The murine B cell response to TNP-polyacrylamide beads: the relationship between the epitope density of the antigen and the requirements for T cell help and surface IgD

Spleen cells were cultured with high or low epitope TNP-polyacrylamide beads (TNP-PAB) in order to investigate the effect of epitope density on the requirements for T cell help and surface IgD on responding B cells. The response to low epitope density TNP-PAB was abolished by treatment with anti-Thy-1.2 and complement, whereas approximately 50% of the response to high epitope density TNP-PAB was retained after similar treatment. Thus, an increase in epitope density resulted in a decreased requirement for T cell help. An increase in epitope density was also associated with a decreased requirement for interaction of antigen and surface IgD as determined by "blocking" studies with anti-delta; further, the majority of the T-independent portion of this response was not blocked by anti-delta antibodies. This finding indicate that the T-independent portion of the anti-TNP response does not require interaction of antigen with surface IgD on B cells. These results are discussed in terms of differential cross-linking of IgM and IgD receptors on B cells by multivalent antigens.     

Polyclonal activation of the murine immune system by an antibody to IgD. VIII. Stimulation of IgD secretion

The low levels of serum IgD found in mice and the lack of a typical DNA switch sequence between C delta and C mu raise the possibility that the generation of murine IgD-secreting cells results from a chance "mistake" rather than a controlled process. The recent observation that injection of mice with purified IgD upregulates IgD receptor expression on helper T cells and enhances the ability of these T cells to induce B cells to differentiate into antibody secreting cells led us to look for evidence of controlled differentiation of B cells into IgD-secreting cells. To do this, we injected mice with a goat antibody to IgD (GaM delta), because this antibody stimulates large increases in IgM, IgG1, IgG2a, and IgE secretion. Mice injected with GaM delta demonstrated a large increase in splenic content of mRNA specific for the secreted form of delta-chain, as well as a greater than 100-fold increase in the percentage of splenic IgD-containing plasmablasts. The secretory IgD response was totally T-dependent. Production of the secretory form of IgD was not seen until 7 days after GaM delta injection, and peaked sharply on day 8, whereas by day 6 IgM secretion had already peaked and IgG1 and IgG2 secretion had attained substantial levels. This observation suggests that: 1) either cells that synthesize large quantities of the secretory form of delta-chain, unlike cells that synthesize large quantities of the secretory forms of gamma-, epsilon-, or alpha-chains, do this without deleting C mu, or, despite the absence of a typical DNA switch sequence between C mu and C delta, controls must exist to effect the C mu deletion and VDJ-C delta joining; and 2) if secreted IgD has a role in the regulation of a humoral immune response it most likely is involved in later processes, such as memory cell generation or response termination, rather than in relatively early processes, such as helper T cell activation.     

Characterization of the immunodeficiency of RIIS/J [corrected] mice. I. Association with the CD5 (LY-1) [corrected] B cell lineage

RIIIS/J mice produce low antibody responses to several polysaccharide Ag of bacterial origin. They have low levels of serum IgM and IgG3 and high levels of serum IgG2a and IgG2b. Low serum IgM and IgG3 have been attributed to a low frequency of CD5 (Ly-1) B cells, which play an important role in the production of natural antibodies. Indeed, RIIIS/J mice have a low frequency of CD5 (Ly-1)+, IgM bright+, Ly-5 (B220)dull+ (i.e., CD5 (Ly-1) B) cells in their peritoneum. RIIIS/J mice treated with LPS produce a low anti-bromelain-treated mouse RBC splenic plaque-forming cell response and a normal anti-mouse transferrin splenic PFC response. Those data are compatible with the fact that CD5 (Ly-1) B cells contain the precursors of B lymphocytes secreting anti-bromelain-treated mouse RBC antibody. However, they have a higher frequency of IgM bright+, Mac-1+ cells in their peritoneum. These cells represent the CD5 (Ly-1) "sister population" of CD5 (Ly-1) B cells described by others. This suggests that characteristics usually associated with the CD5 (Ly-1) lineage are applicable only to the CD5 (Ly-1)+ Mac-1+ IgM+ population, but not the related CD5 (Ly-1)- Mac-1+ IgM+ population. RIIIS/J mice should thus prove a valuable model to study the CD5 (Ly-1) B cell lineage.     

Human B cell development. II. Subpopulations in the human fetus

In man, during fetal development the B cell populations show distinct phenotypes at different tissue sites. The pre-B and B lymphocytes of the fetal liver and bone marrow express IgM and B cell markers, B1 (CD20) and BA-1 (CD24). These "early" cells are negative with a number of other reagents, anti-IgD, RFB4 (CD22), RFB6 (CD21), and RFA-2, which on the other hand recognize peripheral B cells. These peripheral B lymphocytes in the developing fetus are heterogeneous. The diffusely distributed B cells in the earliest lymph node samples, 16 to 17 wk of gestational age, and from 16 to 21 wk in the spleen, are strongly IgM+ (IgD+,RFB4+,RFB6+, and RFA-2+) but lack T cell-associated markers such as T1 (CD5, p 67,000 dalton equivalent of murine Ly-1) and Tü-33. In fetal lymph nodes, primary nodules develop around the follicular dendritic (FD) cells from 17 wk onward, and contain a virtually pure population of B cells; B1+,BA1+,RFB4+,RFB6+,RFA-2+, which simultaneously express IgM,IgD together with T1 (CD5), a T cell-associated antigen. A sizeable subpopulation of these IgM+,T1+ cells are also positive for Tü-33, another T cell-associated marker. In the spleen, the B cells of the IgM+,IgD+,T1+ type appear in smaller numbers and only relatively late around wk 22. These cells are diffusely distributed at first, and start accumulating around the small FD cell clusters as soon as these emerge about the 23rd gestational wk. At that time, the IgM+,T1+B cells can also be washed out from the peritoneal and pleural cavities. The T1+,IgM+B cells may represent the normal equivalent cells of B chronic lymphoid leukemia and centrocytic lymphoma, and appear to be the counterpart of Ly-1+,IgM+B cells in the mouse.     

Ig isotype switching in B lymphocytes. Isolation and characterization of clonal variants of the murine Ly-1+ B cell lymphoma, CH12, expressing isotypes other than IgM

We have selected and cloned variant cells from the murine B cell lymphoma, CH12, that produce a variety of other Ig isotypes in addition to or in place of the original IgM and IgD. Variants were selected by flow cytometry and automated cloning and isotype production was analyzed by membrane immunofluorescence and ELISA of culture fluids. Variants have been isolated that produce the single isotypes IgA, IgG2b, and IgG3, as well as variants that produce more than one isotype simultaneously, i.e., IgM, IgD, and IgA; IgG2b and IgA; IgG3 and IgA. All isotypes have been seen as cell surface proteins and all except IgD have been found in culture supernatants. All isotypes display the same idiotype and Ag-binding specificity for phosphatidyl choline as the original IgM and all are translated from the same VDJH and VJ kappa gene assemblies. Production of more than one isotype by a variant clone is due to simultaneous production of all the isotypes by each cell within the clone. The finding that the variants producing more than one isotype are all tetraploid suggests the interesting possibility that each isotype is derived from an independently switching chromosome. All isotype variants can be stimulated by LPS to secrete the appropriate Ig isotype at an increased rate similar to the IgM expressing parent. The variants differ in stability; some have remained stable for more than 9 months in culture, whereas other have undergone further isotype switching. The facts that some isotypes have not been seen, that multistep switching has occurred, and that many variants produce IgA in addition to another isotype are discussed in relation to current notions of isotype switching mechanisms.     

Natural cytotoxicity against mouse hepatitis virus-infected cells. II. A cytotoxic effector cell with a B lymphocyte phenotype

The effector cell in mouse spleen which mediates natural cytotoxicity against mouse hepatitis virus (MHV)-infected target cells was characterized. The target cells were MHV-infected BALB/c 3T3, and the assay time was 3 hr. The effector cell, designated virus killer (VK) cell for the purpose of discussion, had the following phenotype: lymphocyte morphology, plastic-nonadherent, nylon wool-adherent, nonphagocytic, cyclophosphamide-sensitive; by antibody plus complement (C) depletion studies, it was asialo GM1-, NK 1.2 alloantigen-negative, Thy-1.2-, Lyt-5-, and macrophage antigen-negative; by rosetting techniques, it was Fc receptor-positive and surface Fab+; by flow cytometry (FACS) analysis, it was Lyt-2-, MAC-1-, Ia+, IgG (gamma)+, IgM (mu)+, IgD (delta)+, and B cell lineage antibody B-220+. NK cells, measured for cytotoxicity on YAC-1 cells, were similarly tested and were found to differ from the VK cell in the following properties: nylon wool-nonadherent, asialo GM1+, NK alloantigen-positive, Lyt-5+, surface Fab-, MAC-1+, Ia-, IgG-, IgM-, IgD-, and B-220-. The VK effector cell had a phenotype highly distinguishable from NK cells, effectors most commonly associated with antiviral natural cytotoxicity. The VK cell had a phenotype identical to that of a B lymphocyte and was identified as such. Although the effector cells displayed cell surface antibody, the antibody did not appear to be involved in lysis, because lysis could not be blocked by F(ab)'2 directed against Fab, mu, or delta. Cytotoxicity was more likely associated with recognition of the B lymphocyte surface by the MHV glycoprotein E2, as shown in the accompanying companion paper. This is the first demonstration that natural cytotoxicity can be mediated by B lymphocytes.     

Inappropriate expression of IgD from a transgene inhibits the function of antigen-specific memory B cells

IgD expression has been shown to be downmodulated upon mitogenic or antigenic activation of B cells. To investigate whether this decrease is of functional significance we studied a mouse strain that expresses transgenic IgD on all B cells. The rearranged gene encoding the heavy chain of this IgD requires endogenous gene rearrangement before it can be expressed; therefore, normal B cell development is not affected. As a result, both transgenic IgD and endogenous IgM and IgD are expressed on all peripheral B cells. We show that the presence of extraneous IgD does not affect normal B cell activation by polyclonal stimulators, nor does it affect the primary IgM or IgG responses to TI or TD antigens. However, the secondary memory response is significantly diminished. The decrease is not attributable to a defective generation of memory B cells; instead the activation of memory cells appears to be compromised. Since the depressed response can be overcome by prior aggregation of the transgenic IgD with allotype-specific anti-IgD antibodies, it appears that persistence of the transgenic IgD on memory cells may influence their ability to be activated. Thus, the decrease in IgD expression on normal B cells after activation may be necessary for optimal activation of memory cells.     

The class of surface immunoglobulin on virgin and memory B lymphocytes.

The class of surface immunoglobulin receptors for antigen on B cell precursors of different classes of antibody-forming cells was determined by utilizing a technique of class-specific antigen suicide. Spleen cells are first treated with a class-specific antiserum under conditions that result in the stripping of that class from the cell surface. The cells are then permitted to bind a highly radioactive trinitrophenyl (TNP)-conjugated protein, which leads to lethal irradiation of all TNP-specific B cells except those whose TNP receptors had been removed by the class-specific stripping of surface immunoglobulin. In this way, the class of antibody-forming cells resulting from TNP stimulation of B cells with different classes of surface immunoglobulin can be examined. It was found that the virgin B cell precursors of IgM-producing cells are two types: cells bearing IgM receptors only and those bearing both IgM and IgD receptors. All virgin B cells that gave rise to IgG1 antibody-forming cells had both IgM and IgD on their surfaces, demonstrating that an antigen-dependent switch from IgM and IgD to IgG1 production is a common feature of B cell maturation. In contrast, memory B cell precursors of IgG1 antibody-forming cells had predominantly IgG1 as their surface antigen receptor. The implications of these findings on current models of B cell maturation are analyzed.     

The type-specific capsular carbohydrate of Hemophilus influenzae B is a potent mitogen for murine B lymphocytes

In this study we investigated the in vitro mitogenic properties of the capsular carbohydrate of Hemophilus influenzae b, polyribosylribitolphosphate (PRP). PRP was found to be a potent polyclonal activator of murine B lymphocytes. PRP induced normal B cells to undergo blastogenesis, DNA synthesis, and differentiation to IgM and IgG secretion. IgG3 accounted for the majority of the IgG. No PRP-specific antibody was detectable, indicating the polyclonal origin of the secreted immunoglobulin (Ig). T lymphocytes were neither activated by PRP nor required for B cell proliferation or Ig secretion. In addition, T cell-depleted spleen cells also depleted of accessory (A) cells by passage through Sephadex G-10 retained responsiveness to PRP. Trace lipopolysaccharide (LPS) contamination was not responsible for the mitogenic effect, as shown by the ability of C3H/HeJ spleen cells to proliferate in response to PRP and by the failure of polymyxin B to inhibit PRP-induced DNA synthesis. The B cell responses induced by PRP and LPS were similar with respect to T cell and A cell independence, to the magnitude of DNA synthesis, and to Ig secretion and the Ig isotypes expressed. These data, taken with the finding that the combination of optimal doses of PRP and LPS did not give an additive DNA synthetic response, indicate that PRP and LPS were activating similar B cell populations. However, in contrast to LPS, PRP was capable of inducing significant DNA synthesis in cultures containing as few as 1,000 B cells, suggesting that PRP-driven proliferation was less dependent on cellular interactions than the response to LPS. The differential ability of PRP and LPS to stimulate C3H/HeJ B cells and to stimulate B cell proliferation at low density indicates basic differences between these two mitogens in their mechanisms of B cell activation.     

Isolation, characterization, and expression of cDNA clones encoding the mouse Fc receptor for IgE (Fc epsilon RII)1

We have isolated cDNA clones encoding a mouse low affinity receptor for IgE (Fc epsilon RII) from a cDNA library of BALB/c splenic B cells activated with LPS and IL-4. The 2.2-kb cDNA clone encodes a 331 amino acid membrane glycoprotein that is homologous to human Fc epsilon RII (CD23) and a family of carbohydrate-binding proteins. COS7 cells transfected with the cDNA clones expressed a 45,000 m.w. protein that bound IgE and the anti-Fc epsilon RII mAb, B3B4. Fc epsilon RII mRNA was up-regulated in mouse B cells by culture with IL-4, but not in B cells cultured with IgE. Fc epsilon RII mRNA was detected in IgM+/IgD+ B cell lines, but not in pre-B cell lines or in B cell lines which have undergone differentiation to secrete Ig. The monocyte line P388D1, mast cell lines MC/9 and PT18, and peritoneal macrophages stimulated with IL-4 lacked detectable Fc epsilon RII mRNA, as did Thy-1.2+, CD4+, and CD8+ normal T cells and Thy-1.2+ T cells from Nippostrongylus brasiliensis-infected mice.     

Cholera toxin promotes B cell isotype differentiation

Cholera toxin (CT) is a powerful oral immunogen and adjuvant that elicits strong IgG and IgA antibody responses. In our study we investigated whether this property of CT was associated with an effect on B cell isotype differentiation. Initially, we determined the effect of CT on normal LPS-induced Peyer's patch B cells and found that whereas CT is strongly inhibitory of IgM production, it increases by approximately three-fold the number and frequency of IgG- and IgA-producing cells. Subsequently, using cell sorting technology, we demonstrated that CT acts on membrane (m)IgM+, mIgG/mIgA- B cells rather than mIgG/mIgA+ B cells. In addition, we showed that CT does not cause selective inhibition of mIgM, or enhancement of mIgG/mIgA B cell proliferation. In parallel studies we determined the effect of CT on the differentiation of a clonal B cell population, CH12.LX cells, i.e., a population comprised mainly of mIgM+ cells (98%) admixed with a small subpopulation of mIgA+ cells (2%). Here we found that CT (in the absence of LPS) causes a rapid decrease (24 h) in the intensity of mIgM expression as well as a marked increase in the size of the subpopulation expressing mIgA. In addition, we found that CT (in the presence of LPS), inhibits CH12.LX IgM production while increasing the absolute number and frequency of IgA-producing cells. In contrast, CT inhibits IgA production by CH12.LX.A2 cells, a subclone of CH12.LX cells that bears only IgA. Finally, we demonstrated that CT is equally inhibitory of the proliferation of CH12.LX cells and CH12.LX.A2 cells. Taken together, these effects of CT on normal B cells and a clonal B cell line indicate that CT induces substantial numbers of mIgM+ cells to undergo isotype differentiation into mIgG+ or mIgA+ B cells. In a final series of studies we showed that the effect of CT on isotype differentiation was mimicked by the B subunit of CT, i.e., the subunit that does not activate intracellular adenylate cyclase; thus the induction of isotype differentiation by CT is not mediated by a perturbation in cAMP level.     

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.     

Regulation of isotype production by IL-4 and IL-5. Effects of lymphokines on Ig production depend on the state of activation of the responding B cells

The effects of IL-4 and IL-5 on the production of Ig of different isotypes was investigated. We compared B cells from spleen and from Peyer's patches either stimulated with LPS or without added polyclonal stimulation. We also compared high density (small) and low density (large) B cells. The effect of lymphokines depended on the size and source of the B cells as well as on whether LPS was added. As expected, small B cells from either lymphoid compartment responded to LPS alone and IL-4 suppressed IgM and IgG3 production and enhanced IgG1. In contrast, when large B cells were examined, the suppressive effects of IL-4 were much less apparent but the enhancement of IgG1 was still marked. IL-5 alone had only minimal effects in LPS-stimulated cultures but the combination of IL-4 plus IL-5 appeared to overcome much of the IL-4-mediated suppression of IgM, and IgA production was enhanced. In the absence of LPS, a quite different profile is seen. First, small B cells make little if any response. Second, there is dramatic synergy between IL-4 and IL-5 in the response of large B cells, which is independent of isotype. Third, IL-4 does not suppress any isotype in the absence of LPS. Fourth, IL-4 plus IL-5 stimulate large Peyer's patch B cells to produce 10 times more IgA but three times less IgM than large spleen B cells. Fifth, Th2 cells directly stimulate both large and small B cells.     

Extinction of B-cell surface differentiation markers in hybrids between murine B-lymphoma and myeloma cells

Fusion between murine B-lymphoma cells bearing membrane IgM with either IgG or light chain secreting myeloma, resulted in cell hybrids synthesizing and secreting large quantities of IgM. In contrast, the hybrids did not secrete IgD even though it is also present on the surface of the B-lymphoma cells. B-Cell surface markers such as the IgM, IgD, Ia and the Fc receptor, which were present on the B-lymphoma cells, but not the myeloma cells were not expressed on the surface of the hybrids. Hybrids which secrete IgM and retain the B-cell membrane differentiation antigens were not detected, even when selection was done under conditions which favor the growth of the lymphoma parental cells.     

Regulation of T15 idiotype dominance. II. Genes unlinked to the Igh locus regulate T15 dominance of the secondary adoptive transfer response to phosphocholine

We have studied the idiotype and fine specificity of the secondary immune response to phosphocholine (PC) in C57BL (B10, B10.D2, and B.C8) and BALB (BALB/c, BAB-14, and C.B20) congenic strains of mice. In vivo IgM responses of mice from these two genetic backgrounds differed in their T15 idiotypic representation. BALB strains expressed the T15 idiotype on greater than 90% of their IgM, PC-specific plaque-forming cells (PFC), whereas C57BL strains expressed the T15 idiotype on approximately 50% of their IgM PFC. All strains examined expressed greater than 75% PC-inhibitable, VHPC idiotype-positive, IgM PFC. The IgG3 and IgA memory responses were similar to the IgM memory response; BALB strains produced a higher proportion of T15+ PFC than C57BL strains; however, the majority of IgG3 and IgA PFC in all strains were VHPC+, and PC-inhibitable. In contrast, the IgG1 memory response was not dominated by T15+, VHPC+, PC-inhibitable PFC in any of the strains tested. The IgG1 PFC required nitrophenylphosphocholine (NPPC) for efficient inhibition. The IgG2 memory response generally mimicked the IgG1 response with respect to idiotype and specificity. These data demonstrate that the representation of the T15 idiotype in the anti-PC immune response is determined by genes outside both the MHC and Igh genetic loci. Control of T15 expression in secondary IgM, IgG3, and IgA anti-PC responses was examined by using a cell-mixing protocol with primed T and B cells from BALB/c and B10.D2 mice. T15 representation in these responses was determined by the genotype of the B cell, not by the genotype of the helper T cell. Similarly, the B cell genotype was responsible for the idiotypic profile of a primary, in vitro, T-dependent, anti-PC response.     

Polyclonal activation of the murine immune system by an antibody to IgD. XI. Contribution of membrane IgD cross-linking to the generation of an in vivo polyclonal antibody response

The injection of mice with a foreign, polyclonal antibody to IgD sequentially induces: 1) activation of B cells by cross-linking of their cell membrane (m) IgD; 2) B cell processing and presentation of the bound anti-IgD antibody to T cells; 3) activation of these T cells; and 4) T-dependent stimulation of B cell differentiation into IgG1 secreting cells. To determine whether the cross-linking of B cell membrane IgD and/or the resulting B cell activation that follows contribute to the generation of the polyclonal IgG1 response, we examined the abilities of three sets of anti-delta mAb or mAb fragments to stimulate polyclonal IgG1 production. Within each set mAb were matched for species and Ig isotypic determinants, but differed in avidity for IgD or in ability to cross-link IgD. In addition, experiments were performed to determine whether the anti-delta mAb had to be foreign to the immunized mouse to stimulate an IgG1 response. Results of these experiments indicate that: 1) recognition of the injected anti-delta antibody as foreign is required for the induction of a polyclonal IgG1 response; 2) the cross-linking of B cell membrane Ig, which directly activates B cells, can contribute considerably to the generation of in vivo IgG1 production; and 3) that even relatively weak cross-linking of membrane Ig by ligands that bind it with low avidity can make this contribution.     

Regulation of IgM and IgD synthesis in B lymphocytes. I. Changes in biosynthesis of mRNA for mu- and delta-chains

Although IgD is expressed on the surface of resting B cells at a higher density than IgM, we determined that both the steady-state level and the biosynthetic rate of mu m-mRNA is higher than that of delta m-mRNA, suggesting that translational or post-translational processing of the Ig heavy chains may modulate the expression of cell surface Ig. After B cell activation by LPS, delta m-mRNA decreases drastically, accounting for the observed decrease in expression of IgD. Concomitantly, a dramatic increase in the level of mu s- and gamma s-mRNA corresponds to the observed increase in secretion of these Ig isotypes in LPS-stimulated cells.