Skewed B cell differentiation affects lymphoid organogenesis but not T cell-mediated autoimmunity.

B cell receptor (BCR) signalling determines B cell differentiation and may potentially alter T cell-mediated immune responses. In this study we used two transgenic strains of BCR-deficient mice expressing Epstein-Barr virus latent membrane protein (LMP)2A in B cells, where either follicular and marginal zone differentiation (D(H)LMP2A mice) or B-1 cell development (V(H)LMP2A mice) were supported, and evaluated the effects of skewed B lymphocyte differentiation on lymphoid organogenesis and T cell responses in vivo. Compared to wild-type animals, both transgenic strains displayed alterations in the composition of lymphoid organs and in the dynamics of distinct immune cell subsets following immunization with the self-antigen PLP185₋206. However, ex-vivo T cell proliferation to PLP185₋206 peptide measured in immunized D(H)LMP2A and V(H)LMP2A mice was similar to that detected in immunized control mice. Further, clinical expression of experimental autoimmune encephalitis in both LMP2A strains was identical to that of wild-type mice. In conclusion, mice with skewed B cell differentiation driven by LMP2A expression in BCR-negative B cells do not show changes in the development of a T cell mediated disease model of autoimmunity, suggesting that compensatory mechanisms support the generation of T cell responses.

Natural occurrence and origin of somatically mutated memory B cells in mice.

While most murine peripheral B cells express germline-encoded antibodies of classes M and D (mu+ delta+ cells), small numbers of memory B cells expressing somatically mutated immunoglobulin G antibodies are generated upon T cell-dependent immunization. Analyzing the antibody repertoire of the mu-delta- B cell pool in unimmunized mice, we show that these cells express somatically mutated VH genes and that most of these genes derive from a set of germline VH genes dominantly expressed by mu+delta+ B cells. Thus, class-switched memory B cells are generated in the absence of intentional immunization, presumably in response to environmental antigens. These cells are either recruited from mu+delta+ B cells or selected from newly arising B cells in parallel to the latter, by the same antigens.

Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells.

To assess the role of immunoglobulin D (IgD) in vivo we generated IgD-deficient mice by gene targeting and studied B cell development and function in the absence of IgD expression. In the mutant animals, conventional and CD5-positive (B1) B cells are present in normal numbers, and the expression of the surface markers CD22 and CD23 in the compartment of conventional B cells indicates acquisition of a mature phenotype. As in wild-type animals, most of the peripheral B cells are resting cells. The IgD-deficient mice respond well to T cell-independent and -dependent antigens. However, in heterozygous mutant animals, B cells expressing the wild type IgH locus are overrepresented in the peripheral B cell pool, and T cell-dependent IgG1 responses are further dominated by B cells expressing the wild-type allele. Similarly, in homozygous mutant (IgD-deficient) animals, affinity maturation is delayed in the early primary response compared to control animals, although the mutants are capable of generating high affinity B cell memory. Thus, rather than being involved in major regulatory processes as had been suggested, IgD seems to function as an antigen receptor optimized for efficient recruitment of B cells into antigen-driven responses. The IgD-mediated acceleration of affinity maturation in the early phase of the T cell-dependent primary response may confer to the animal a critical advantage in the defense against pathogens.

Determination of antibody class in a system of cooperating antigenic determinants.

19S and 7S memory is analyzed in a system of cooperating antigenic determinants. Cooperation occurs in the induction of both 19S and 7S secondary antibodies, and for both responses carrier specificity can be entirely accounted for by presensitization of the animal to carrier determinants. The class distribution of secondary anti-hapten antibody depends on the dose of the hapten primary-carrier conjugate used for priming, and on the time interval between priming with the hapten primary-carrier conjugate and secondary injection. The conditions of priming with the secondary carrier influence the extent of the secondary response but not the class distribution of secondary antibody. The data confirm the cooperation hypothesis of antibody induction. Specifically, we interpret them to mean that in hapten-carrier cooperation, the hapten-specific memory cells are predetermined for the class of the emerging antibodies. Together with the hapten-specific memory cells, the carrier-specific helpers are responsible for the extent of the secondary response.

Helper T cells reacting to idiotype on IgG but not IgM.

Immunization with an IgG1 but not an IgM monoclonal anti-NP (4-hydroxy-3-nitrophenyl acetyl) antibody induced idiotype-recognizing T helper cells, although these two antibodies carry the same variable regions. The T cells appear to react to an idiotype on the IgG1 but not the IgM antibody. They selectively enhance the expression of that idiotype in the IgG1 fraction of an in vitro anti-NP response.

Idiotope regulation by isotype switch variants of two monoclonal antiidiotope antibodies.

Previous work has shown that the injection of antiidiotope antibodies specific for idiotopes of the germline-encoded anti-(4-hydroxy-3-nitro-phenyl) acetyl (NP) antibody B1-8 enhanced or suppressed the expression of B1-8 idiotopes in subsequent humoral anti-NP responses, depending on the dose and perhaps also the isotype of the injected antibody. To formally answer the question of whether the isotype of an antiidiotope determines its effector function in this type of idiotypic control, we have performed regulatory experiments with isotype switch variants selected from two hybridomas secreting anti-B1-8 idiotopes of CBA (Ighj) and C57BL/6 (Ighb) origin. The antibodies of each variant family differ from each other only in the constant region of the heavy chain. The results show that, irrespective of whether an antiidiotope antibody belongs to the IgG1, IgG2b, IgG2a, or IgE class, a 10-ng dose enhances idiotope expression whereas a dose of 10 micrograms exerts a suppressive effect. It emerges from the present and parallel data that the expression of antibody V regions resembling idiotypically that of antibody B1-8 can be enhanced and suppressed by any of four antiidiotope antibodies that recognize distinct idiotopes on those V regions. This suggests that the initial step in the regulatory process induced by an antiidiotope is its binding to antibody V regions carrying the target idiotope. The antiidiotopes preferentially regulate the expression of antibodies that coexpress with the target idiotope other B1-8 idiotopes, despite the fact that some B1-8 idiotopes are also expressed independently of each other in anti-NP responses of idiotypically unmanipulated mice. This finding may reflect high affinity binding of the antiidiotopes to the target against which they were originally raised (i.e., antibody B1-8) or, more likely, a preferential recognition of B1-8-like V regions by regulatory T cells.

B cell antigen receptor specificity and surface density together determine B-1 versus B-2 cell development.

Mice expressing the immunoglobulin (Ig) heavy (H) chain variable (V) region from a rearranged V(H)12 gene inserted into the IgH locus generate predominantly B-1 cells, whereas expression of two other V(H) region transgenes (V(H)B1-8 and V(H)glD42) leads to the almost exclusive generation of conventional, or B-2, cells. To determine the developmental potential of B cells bearing two distinct B cell antigen receptors (BCRs), one favoring B-1 and the other favoring B-2 cell development, we crossed V(H)12 insertion mice with mice bearing either V(H)B1-8 or V(H)glD42. B cells coexpressing V(H)12 and one of the other V(H) genes are readily detected in the double IgH insertion mice, and are of the B-2 phenotype. In mice coexpressing V(H)12, V(H)B1-8 and a transgenic kappa chain able to pair with both H chains, double H chain-expressing B-2 cells, and B-1 cells that have lost V(H)B1-8 are generated, whereas V(H)B1-8 single producers are undetectable. These data suggest that B-1 but not B-2 cells are selected by antigenic stimuli in whose delivery BCR specificity and surface density are of critical importance.

The repertoire of somatic antibody mutants accumulating in the memory compartment after primary immunization is restricted through affinity maturation and mirrors that expressed in the secondary response.

The anti-(4-hydroxy-3-nitro-phenyl)acetyl (NP) response is dominated by lambda 1 chain-bearing antibodies expressing the VH gene V186.2 in combination with the D element DFL16.1. lambda 1-positive B cells were isolated from the spleens of mice immunized with NP-chicken gamma globulin 6 wk earlier. Rearranged V186.2 genes were amplified from the genomic DNA of these cells and sequenced. In cases where the rearrangement was typical for secondary anti-NP antibodies, the VHDJH sequences were generally heavily mutated. The frequency and the nature of the nucleotide exchanges mirrored those of secondary response antibodies. V186.2 genes with other rearrangements and V186.2-related genes isolated concomitantly were essentially unmutated. These results demonstrate: (a) that somatic antibody mutants are largely restricted to a small compartment of peripheral B cells, namely, that of memory cells; (b) that the memory compartment is strongly selected for high affinity precursors and largely purged from antigen-binding loss mutants; and (c) that the repertoire of binding specificities expressed in the secondary response is established in its final form before secondary immunization.

Homeostasis of peripheral B cells in the absence of B cell influx from the bone marrow.

To study homeostasis of peripheral B lymphocytes in the absence of B cell influx from the bone marrow, we generated a mouse mutant in which the recombination-activating gene (RAG)-2 can be inducibly deleted. When RAG-2 was deleted at the age of 8-10 wk, splenic naive follicular B cells were gradually lost over a year of observation, with a half-life of approximately 4.5 mo. By contrast, the pool of marginal zone B cells in the spleen and of B-1 cells in the peritoneal cavity were kept at normal level. In lymph nodes, approximately 90% of the B cells were lost within 4 mo, and B cell numbers remained constant thereafter. Mice in which RAG-2 was deleted at birth maintained a small population of activated B cells with an increased proportion of marginal zone B cells. Additionally, an increase of the pool of IgM secreting cells and B-1a cells was observed.

Transfected plasmacytoma cells do not transport the membrane form of IgM to the cell surface.

Expression vectors coding for membrane-bound IgM antibodies were introduced into myeloma and B lymphoma cells. Only the lymphoma but not the myeloma cells were able to express the antibodies on the cell surface, although in both cases, complete antibodies were assembled intracellularly. In myeloma cells, the Ig molecules did not reach the Golgi compartment. Thus, the intracellular transport of membrane-bound antibodies is controlled in the B cell lineages in a developmentally ordered fashion.

Variable region gene analysis of B cell subsets derived from a 4-year-old child: somatically mutated memory B cells accumulate in the peripheral blood already at young age.

Tonsillar germinal center and immunoglobulin M+ (IgM+)IgD+ B cells as well as peripheral blood (PB) CD5+ and CD5- (conventional) B cells from a 4-yr-old child were isolated and nucleotide sequences of expressed Ig heavy chain variable regions encoded by VH4 gene family members were determined from amplified cDNA. Whereas both tonsillar IgM+IgD+ cells and the majority of IgM-expressing CD5+ and CD5- PB B cells showed no or little somatic mutation, tonsillar germinal center (GC) B cells and IgG-expressing PB B cells carried a high load of somatic mutations in their V region genes. This suggests that somatically mutated memory B cells which have switched isotype accumulate in the PB already at young age. Their frequency seems to increase with age. On the other hand, the antibody repertoire of tonsillar IgM+IgD+ B cells and the majority of IgM-expressing PB B cells is determined by germline-encoded specificities and by generation of variability in the complementary determining region III through VH-DH-JH recombination. A fraction of IgM-bearing PB B cells carries somatically mutated V region genes and probably represents GC-derived B cells which have left the GC at an early stage of the GC reaction without undergoing isotype switching. 10 VH4 germline genes were found to be expressed. Three gene segments were overrepresented in the sequence collection (35 of 50 clones): VH4.21 (30%), V71-4 (20%), and 3D279D (20%). It appears that most potentially functional VH4 germline genes are expressed in peripheral B cells. Some members of this VH gene family are clearly overrepresented over others.

The immune response to a hybrid protein molecule; specificity of secondary stimulation and of tolerance induction.

Upon immunization with LDH-III (subunit composition AABB) rabbits produce anti-A and anti-B antibodies in comparable amounts. These antibodies fit equally well to the hybrid enzyme and to LDH-V (AAAA) or LDH-I (BBBB) respectively, as tested by passive hemagglutination inhibition. No antibodies reacting with both LDH-I and LDH-V were detected. A minority of hybrid-specific antibodies was, however, present in the sera. Animals primed with LDH-III respond regularly to a boosting injection of LDH-V with the production of large amounts of anti-A (but not anti-B) antibodies. A similar injection of LDH-I stimulates (if it has any effect at all) the production of anti-B antibodies only. Stimulation with one of the pure types does not impair a subsequent response to the other. The majority of the animals primed with LDH-III responded not at all or weakly to a boosting injection of LDH-I, though antibodies to LDH-I were present in the sera at the time of stimulation. This effect can hardly be explained on the basis of serological sepcificity. Hyporesponsiveness to LDH-III can be induced by injection of LDH-V into the newborn. Both anti-A and anti-B titers are equally depressed. Within the dose range tested, LDH-I does not exert any tolerogenic action with respect to LDH-III. The carrier property of subunit A is evident in the induction of both immunity and tolerance to LDH-III. The early phase of the immune response to the hybrid enzyme may be carrier-specific, and receptors for the haptenic subunit B may not exist at that stage.

Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells.

Immunoglobulin (Ig)M+IgD+ B cells are generally assumed to represent antigen-inexperienced, naive B cells expressing variable (V) region genes without somatic mutations. We report here that human IgM+IgD+ peripheral blood (PB) B cells expressing the CD27 cell surface antigen carry mutated V genes, in contrast to CD27-negative IgM+IgD+ B cells. IgM+IgD+CD27(+) B cells resemble class-switched and IgM-only memory cells in terms of cell phenotype, and comprise approximately 15% of PB B lymphocytes in healthy adults. Moreover, a very small population (<1% of PB B cells) of highly mutated IgD-only B cells was detected, which likely represent the PB counterpart of IgD-only tonsillar germinal center and plasma cells. Overall, the B cell pool in the PB of adults consists of approximately 40% mutated memory B cells and 60% unmutated, naive IgD+CD27(-) B cells (including CD5(+) B cells). In the somatically mutated B cells, VH region genes carry a two- to threefold higher load of somatic mutation than rearranged Vkappa genes. This might be due to an intrinsically lower mutation rate in kappa light chain genes compared with heavy chain genes and/or result from kappa light chain gene rearrangements in GC B cells. A common feature of the somatically mutated B cell subsets is the expression of the CD27 cell surface antigen which therefore may represent a general marker for memory B cells in humans.

Most peripheral B cells in mice are ligand selected.

Using amplified cDNA and genomic libraries, we have analyzed the VH gene repertoire of pre-B cells and various B cell subsets of conventional mice at the level of VH genes belonging to the J558 VH gene family. The sequence data were evaluated on the basis of a newly established list of 67 J558 VH genes that comprise approximately two-thirds of the J558 VH genes of the murine IgHb haplotype. The results of the analysis demonstrate that VH gene utilization in pre-B cells, although biased to some extent by B cell autonomous VH gene selection, scatters over the whole range of J558 VH genes present in the germline. In contrast, in mature, peripheral B cells comprising long-lived mu + delta high B cells as well as Ly-1 B cells, small overlapping sets of germline VH genes are dominantly expressed. The data indicate that the recruitment of newly generated B cells into the long-lived peripheral B cell pool is mediated through positive selection by internal and/or external antigens. Because of the absence of immunoglobulin class switching and somatic hypermutation, this process is different from the selection of memory B cells in T cell-dependent immune responses.

Hodgkin and Reed-Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells.

In Hodgkin's disease (HD), the Hodgkin and Reed-Sternberg (HRS) cells represent only a minute population in the diseased tissue. The investigation of lineage derivation and clonal origin of these cells has yielded conflicting results. We have analyzed HRS cells micromanipulated from infiltrated tissue sections of 10 primary HD patients for rearranged V genes, extending a previous study. Clonally related rearrangements were found in nine cases, indicating that HRS cells represent a dominant clone of B lineage-derived cells in at least a large fraction of cases of HD. Rearranged VH genes from HRS cells carried a high load of somatic mutation, indicating that HRS cells are derived from germinal center (GC) cells or their progeny. Stop codons in some in-frame V gene rearrangements suggest that the HRS cell precursors reside inside GCs, have acquired crippling mutations that prevent antigenic selection, but escape apoptosis through some transforming event.

The requirement of more than one antigenic determinant for immunogenicity.

Rabbits primarily stimulated with a BSA (bovine serum albumin)-sulfanilic acid complex will produce a good secondary response to the sulfanilic acid hapten if the carrier used in the secondary stimulus is again BSA, and not if the secondary carrier is HGG (human gamma globulin). In the latter situation, a good secondary response is obtained, however, if the rabbits are pretreated a few weeks earlier with free HGG. We conclude that the immune stimulus involves the recognition of carrier determinants unrelated to the hapten. As the receptors for recognition of unrelated determinants are probably situated on different cells, we suggest that the immune stimulus leading to antibody formation requires the interaction of two antigen-bridged cells.

Isolated hapten-binding receptors of sensitized lymphocytes. III. Evidence for idiotypic restriction of T-cell receptors.

The primary antibody response of C57BL/6 mice to the 4-hydroxy-3-nitrophenylacetyl (NP) hapten is restricted to antibody molecules expressing the NPb idiotype. This idiotype is a genetic marker for V genes in the heavy chain linkage group. In the secondary response, the frequency of NPb idiotype-positive molecules within the antibody population drops to very low values. Accordingly, isolated NP binding receptors from NP-sensitized B lymphocytes are largely devoid of this idiotype. In contrast, the NPb idiotype is expressed on the majority of the receptor fractions which we consider T-cell derived. This finding suggests that the antigen receptors of T lymphocytes may be restricted to the expression of major (germ-line encoded?) heavy chain idiotypes.

B cells are essential for murine mammary tumor virus transmission, but not for presentation of endogenous superantigens.

Murine mammary tumor viruses (MMTVs) are retroviruses that encode superantigens capable of stimulating T cells via superantigen-reactive T cell receptor V beta chains. MMTVs are transmitted to the suckling offspring through milk. Here we show that B cell-deficient mice foster nursed by virus-secreting mice do not transfer infectious MMTVs to their offspring. No MMTV proviruses could be detected in the spleen and mammary tissue of these mice, and no deletion of MMTV superantigen-reactive T cells occurred. By contrast, T cell deletion and positive selection due to endogenous MMTV superantigens occurred in B cell-deficient mice. We conclude that B cells are essential for the completion of the viral life cycle in vivo, but that endogenous MMTV superantigens can be presented by cell types other than B cells.

Somatic mutation of the CD95 gene in human B cells as a side-effect of the germinal center reaction.

Somatic hypermutation specifically modifies rearranged immunoglobulin (Ig) genes in germinal center (GC) B cells. However, the bcl-6 gene can also acquire somatic mutations during the GC reaction, indicating that certain non-Ig genes can be targeted by the somatic hypermutation machinery. The CD95 gene, implicated in negative selection of B lymphocytes in GCs, is specifically expressed by GC B cells and was recently identified as a tumor suppressor gene being frequently mutated in (post) GC B cell lymphomas. In this study, the 5' region (5'R) and/or the last exon coding for the death domain (DD) of the CD95 gene were investigated in naive, GC, and memory B cells from seven healthy donors. About 15% of GC and memory, but not naive, B cells carried mutations within the 5'R (mutation frequency 2.5 x 10(-4) per basepair). Mutations within the DD were very rare but could be efficiently selected by inducing CD95-mediated apoptosis: in 22 apoptosis-resistant cells, 12 DD mutations were found. These results indicate that human B cells can acquire somatic mutations of the CD95 gene during the GC reaction, which potentially confers apoptosis resistance and may counteract negative selection through the CD95 pathway.

Idiotypic analysis of lymphocytes in vitro. II. Genetic control of T-helper cell responsiveness to anti-idiotypic antibody.

When the IgG1 fraction of anti-idiotypic antibodies raised in guinea pigs is injected into mice, sensitization of idiotypic T and B lymphocytes occurs (1-3). In the present study we analyze the genetic requirements for T-helper cell sensitization by anti-idiotypic antibody. This was done by measuring, in a suitable panel of mouse strains, helper cell responsiveness to two anti-idiotypic reagents which recognize distinct, strain-specific idiotypes, namely the A5A and the S117 marker. Whenever helper cell sensitization by anti-idiotypic antibody was successful, helper function could be specifically inhibited by the same and only the same anti-idiotype. This indicates that helper cells induced by anti-idiotypic antibody express idiotypic determinants on their receptors for antigen. Helper cell sensitization by anti-idiotypic antibody was found in all strains expressing the corresponding or a cross-reactive idiotype at the immunoglobulin level. Idiotype-negative strains were always unresponsive to anti-idiotypic stimulation. In addition, responsiveness did not depend on the H-2 haplotype. Since the A5A and the S117 idiotype are markers for V genes in the heavy-chain linkage group, the present results support the view that the same genes in the Ig-1 complex code for variable portions of immunoglobulins and T-helper cell receptors.