Expression of kappa chain allotypes at the cell surface and in serum immunoglobulins of normal and allotype-suppressed heterozygous rabbits

The kappa light chain allotypes b4 and b5 were measured in the serum and on the surfaces of peripheral blood lymphocytes of normal and allotype-suppressed heterozygous rabbits. Surface immunoglobulins (sIg) were detected by fluorescence microscopy and additional quantitative data were obtained by flow microfluorometry. Although a few b5-suppressed animals had no b5 in serum or on cell surfaces for years, most b5-suppressed and all b4-suppressed animals studied had some cells with sIg of the suppressed type by 1 year of age. In suppressed animals the level of serum Ig remained depressed throughout life but cells with sIg appeared in disproportionately large numbers. The effect was particularly striking in those animals suppressed for the b4 type.     

Immunofluorescence studies on the expression of VH a allotypes by pre-B and B cells of homozygous and heterozygous rabbits

Fluorochrome-conjugated antibodies specific for C mu determinants and VH a allotypes were used to examine pre-B cells and B lymphocytes for expression of these markers. The majority of mu+ bone marrow pre-B cells were shown to express a allotypic determinants in conjunction with C mu. Both pre-B and B cells from a2 a3 heterozygous rabbits showed allelic exclusion of these allotypes. Pre-B cells expressing the a2 or a3 specificities appeared to be generated in approximately equal numbers in heterozygotes, while B lymphocytes expressing a3 appeared to undergo preferential clonal expansion very early in development. It was also observed that rabbit bone marrow and blood contained a population of myeloid cells which, in a2 a3 heterozygotes, stained for both a2 and a3 determinants. The frequencies of this cell type, which exhibited bright immunofluorescence staining for a allotypes, could not be reduced by prolonged incubation at 37 degrees C but were readily reduced after cell suspensions were treated with low pH buffer. It is concluded that these myeloid cells bear high avidity Fc receptors for serum immunoglobulin and may be the culprits in studies which have found production of two a or b allotypes by individual B lymphocytes.     

The structural correlates of the rabbit light chain b allotypes: Sequence studies of b5 and b6 chains

Rabbit kappa light chains of allotype b5 and b6 were prepared from antibodies of restricted heterogeneity made by animals hyperimmunized with, respectively, strain III and strain II pneumococcal vaccines. The amino-acid sequence of several tryptic peptides were determined. The variable region fragments of the b5 and b6 chains appear to be quite similar to the corresponding fragments of b4 and b9 chains, albeit some residues seem to be allotype associated. In contrast the chains of different allotypes vary right from the start of the constant region in a number of positions, suggesting that b allotypes correlate with amino-acid substitutions in this region. The number of substitutions between the b5 and b6 and the previously determined b4 and b9 constant regions sequences ranges from 20 to 35%. Serological studies suggest that Leporidae b allotypes diverged no more than 2 × 10⁶ years ago. By this time only 1% of the substitutions could be generated by ‘conventional evolution’. Duplication and mutation of the individual C_K genes could account for the high level of divergence observed. The data reported here support the notion that the structural genes encoding the light chain constant regions of the various b allotypes coexist on the same chromosome and that the allelism is controlled by a regulatory mechanism.     

Rearrangement and expression of {chi} light chain genes can occur without {micro} heavy chain expression during differentiation of pre-B cells

The kinetics of light (L) chain gene rearrangement and expressionon mRNA and protein level has been studied with four stromalcell/IL-7 reactive, long-term in vitro proliferating pre-B celllines and clones, two from fetal liver of normal mice and twofrom fetal liver of E_µH-bcl-2 transgenic (bcl-2-tg) mice.These pre-B cell lines and clones are DJ_H-rearranged on bothH chain alleles. Two of the clones harbor H chain rearrangementswhich do not allow the expression of V_HDJ_H rearranged H chaingenes as _µH chain proteins. Upon removal of IL-7 fromthe pre-B cell cultures all four cell lines rearrange V_H-DJ_Hand V_L-J_L gene segments, loose the surface expression of c-kit,CD43, and surrogate light chain, as well as the capacity tobe clonable on stromal cells in the presence of IL-7. Pre-Bcells from normal mice die by apoptosis during differentiation,while those from bcl-2-tg mice do not. All four lines and clonesexpress comparable levels of mRNA for _µH and _µLchains with the same time kinetics during 3 days of differentiation.However, only two of the four pre-B cell lines and clones express_µH chain protein, whereas all four pre-B cell lines andclones express _µL chain protein at comparable levels between2x10⁵ and 1.40x10⁶ _µL chain molecules per cell. Theseresults suggest that _µH chain expression is not mandatoryfor rearrangement and normal expression of _µL chain geneswhen pre-B cells differentiate to B cells.     

A subpopulation of small pre-B cells in rabbit bone marrow expresses x light chains and exhibits allelic exclusion of b locus allotypes

The expression of immunoglobulin heavy and light chains by pre-B cells and B lymphocytes was examined by two-color immunofluorescence in heterozygous b⁵b⁹ rabbits. Allelic exclusion of b5 and b9 x light chain allotypes was observed for both surface immunoglobulin-negative pre-B cells and surface immunoglobulin-positive B lymphocytes. In newborn bone marrow, pre-B cells and immature B lymphocytes expressing b9 were as numerous as those expressing b5. In contrast, circulating B cells and bone marrow plasma cells expressing the b5 marker outnumber b9⁺ cells by 2 to 1 in adult b⁵b⁹ animals. Whereas most B lymphocytes expressed x light chain b allotypes, approximately 80% of the μ heavy chain-positive pre-B cells did not. The pre-B cells that expressed detectable light chains were relatively small lymphocytes. A model is presented which includes a “transitional” pre-B cell that expresses both p chains and x chains.     

Exclusion of VHa and VHy loci expression on individual B cells from normal and VH allotype-suppressed rabbits.

The distribution of two heavy chain subgroups, VHa and VHy, on rabbit peripheral blood lymphocytes was examined by double membrane immunofluorescence. Fluroescent anti-a1 and anti-y33 were found to react with separate B cell populations; no doubly stained cells were observed. Further evidence for the independent expression of genes controlling the VHa and VHy subgroups were obtained by neonatal suppression of a 2 or y33 in a2y33/a3y- heterozygous rabbits. Suppression of VHa did not affect the expression of VHy, nor did the suppression of VHy affect the expression of VHa. The expression of a single VH gene per B cell is in marked contrast to the simultaneous expression of multiple CH genes.     

Regulation of allotype expression in heterozygous rabbits. II. Concomitant suppression of b4 and b6 allotypes in the same cell.

Cells from heterozygous b4b6 rabbits were treated at 4 degrees with anti-b4 or anti-b6 antibodies and then warmed at 37 degrees. A disappearance of both b4 and b6 allotypes (concomitant modulation) ensued. When cells which had undergone extensive comodulation were cultured overnight we noted that those cells were unable to re-express either allotype at pre-modulation levels. This suppression was likely linked to the initial events which culminated in comodulation. Those cells were not further suppressible when suppressive antibodies were added to the cultures whereas cell cultures which had undergone little or no previous modulation or comodulation were readily suppressed for both allotypes after anti-allotype antibodies had been added to the cultures overnight (concomitant suppression). This indicated that in vitro suppression of allotype may depend on cell surface allotype being present at a sufficiently high density. We present data which show that events at the cell surface may play a role in the regulation of cell surface allotype expression and propose that concomitant suppression may have bearing on cellular mechanisms which control allotype expression and also allotype suppression.     

Regulation of allotype expression in heterozygous rabbits. I. Concomitant modulation of cell surface allotypes on peripheral blood lymphocytes from b4b6 rabbits.

Treatment of b4b6 rabbit peripheral blood lymphocytes with b5b5 anti-b4 antibodies at 4 degrees resulted in the modulation (disappearance) ob b4 and b6 cell surface allotype after subsequent incubation in serum-free medium for 1 h at 37 degrees. A clear dose dependence on the sensitizing anti-b4 antibody was observed. Similarly, b5b5 anti-b6 treatment demonstrated a dose dependence for b6 modulation and a threshold dose effect for b4 comodulation. Cells which formed rosettes with anti-b4-coupled SRBC (anti-b4 direct antiglobulin (DAG) rosettes) also demonstrated concomitant modulation of b4 and b6 allotype when incubated at 37 degrees. When cells formed anti-b6 DAG rosettes, subsequent b6 modulation could also be demonstrated, but no b4 comodulation occurred. Concomitant modulation did not occur when cells were incubated with anti-allotype antibodies at 37 degrees. Blocking studies disclosed that the two allotypes are not contiguous in the membrane since uptake of one antiallotype antibody did not block the uptake of another at 4 degrees. We therefore propose that concomitant modulation might occur during a process similar to patch formation.     

Kinetics of escape from suppression of Ig heavy chain allotypes in multiheterozygous rabbits.

Three rabbits of genotype a1n81f73g74/a2n82f71g75 which had been injected at birth with anti-a l (VH) antiserum and which were previously shown to be suppressed for the paternal allotypes a 1, n81, f73 and g74 at 8 weeks of age, were monitored over a 2-year period for the concentration of suppressed and nonsuppressed allotypes in their sera. In all three suppressed animals, the f73 (C alpha) and g 74 (C alpha) allotypes were expressed again at a much greater rate than the a1 (VH) and n81 (C mu) allotypes. In one suppressed animal, the a l (VH) allotype was re-expressed at a much greater rate than the n81 (C mu) allotype and reflected primarily the reappearance of a l IgG. Thus, the escape from allotype suppression in this animal was in the order IgA, IgG, IgM which is the reverse of the order of appearance of these Ig classes during ontogeny. While the al(VH) and n81 (C mu) allotyes remained suppressed, the f73 (C alpha) and g74 (C alpha) allotypes were re-expressed to the same concentration as in the unsuppressed controls, and no compensatory decrease of the f71 and g75 allotypes occurred. During the re-expression of the f73 and g74 allotypes, the ratio of the concentrations of f73/g74 remained approximately constant.     

Partial sequence of the variable region of IgG light chains of b5 allotype from A B4B5 rabbit.

The partial sequence (positions 29–71) of the variable region of light chains of predominately b5 allotype from the IgG of a single allotype-suppressed rabbit was obtained by traditional sequencing methods on isolated tryptic and chymotryptic peptides. The peptides from this region were isolated in relatively high yields and probably represent a dominant sequence. The framework sequence between positions 35 and 49 (FR 2) is identical to an FR 2 sequence commonly found in light chains from antibodies produced by b4 rabbits as well as murine and human myeloma light chains, with the exception of an interchange of threonine for proline at position 43 or 44. This may be b5 allotype-related since, to date, all the b4 light chains have had proline, and a b9 light chain was found with arginine at position 43. The fact that a dominant sequence could also be found for positions corresponding to the second complementarity determining region (CDR 2, 50–56) in other species, confirms previous observations that this portion of the light chain is not extremely variable in the rabbit.     

Surface immunoglobulin receptors of rabbit lymphoid cells. Evaluation of fluorescent staining with antibodies to immunoglobulin light chain allotypes

Rabbit lymphoid cells were stained with fluorescein-labelled antiallotype antibodies. The double layer technique was found to be more sensitive than the direct staining. Rabbit B cells are stained only via their surface immunoglobulin (sIg) receptors and not via the receptors for the Fc portion of the IgG. Peritoneal exudate macrophages do not carry sIg receptors and are stained via their Fc receptors. Removal of protein aggregates from the system and use of reagents prepared from F(ab')2 immunoglobulin fragments prevent staining of the macrophages through their Fc receptors. There was a good agreement between the percent of RABELA-positive and sIg-containing spleen cells. In appendix there were approximately 20% more RABELA-positive than sIg-positive cells.     

Synthesis of lambda light chain subtypes by stimulated and unstimulated mouse B cells

Inbred mouse make 3 lambda chain subtypes. The lambda 1 and lambda 3 chains have similar variable (V) regions (in both the same V gene segment [V lambda 1] is used), whereas lambda 2 and lambda 3 have similar constant (C) regions. Despite the lambda 1 and lambda 3 V region similarity, lambda 1 occurs much more frequently than lambda 3 (and lambda 2) in the serum immunoglobulins and antibody responses of most inbred strains of mice. To explore the basis for the lambda 1 predominance, we compared the rates of synthesis of the 3 subtypes and the frequencies of the B cells that synthesize them, focussing on "resting" (i.e., unstimulated) and on polyclonally stimulated B cells from spleens of unimmunized BALB/c mice. In resting cells the relative rates of synthesis and the relative frequencies of the respective B cells were in accord, indicating that the rate of lambda chain synthesis is approximately the same per resting B cell, regardless of the lambda subtype it produces. However, in the polyclonally stimulated cells, lambda 1 was made 7 times faster than lambda 2 and 10 times faster than lambda 3; normalizing these rates by the frequencies of the respective stimulated cells suggests that in stimulated B cells lambda 1 chains are made 5 times faster per cell than lambda 2 or lambda 3, while the latter are made at about the same rate per cell. In view of the marked structural homology between lambda 2 and lambda 3 genes in segments other than the V-gene segment, we suggest that the pronounced differences among polyclonally stimulated B cells in expression of the genes for the various lambda subtypes may be due to the presence of less potent enhancer-like sequences in the lambda 2 and lambda 3 genes than in the lambda 1 gene.     

Localization by immunofluorescence of gamma-globulin allotypes in lymph node cells of homozygous and heterozygous rabbits

The cellular production of two rabbit γ-globulin allotypic specificities, A4 and A5, determined by allelic genes was investigated by the fluorescent antibody method. The 7S γ-globulin fractions of precipitating antisera were conjugated to fluorescein isothiocyanate and to lissamine rhodamine B sulphonyl chloride. Frozen sections of lymph nodes from eighteen rabbits, A4—A4 and A5—A5 homozygotes and A4—A5 heterozygotes, were studied after exposure to the fluorescent antibody conjugates. The conjugates, each specific for antigenic determinants of 7S γ-globulin, reacted specifically with the cytoplasm of plasma cells and intrinsic cells of the germinal centres. The rabbit anti-A4 conjugate reacted only with lymph node cells of A4—A4 and A4—A5 rabbits; the rabbit anti-A5 conjugates reacted only with cells of A5—A5 and A4—A5 rabbits; the horse anti-rabbit γ-globulin conjugates reacted with cells of all three genotypes. By a variety of techniques, identical cellular localization of the two allotypes, A4 and A5, was found in the A4—A5 heterozygotes. Less than 1 per cent of the cells in any heterozygous lymph node section contained one allotype without the other.     

Human B cell colony growth from pre-B cells in vitro.

We describe a simple one-step technique for the growth of human B cell colonies in semi-solid agar in vitro. This method used conditioned medium from the human plasmacytoma cell line LICR-LON-H My 2 as a source of stimulating activity. A linear relationship exists between the number of B cells seeded and the number of colonies formed (r = 0.95). Most colony forming cells, approximately 1 in 500 of B cells seeded, lack surface immunoglobulin, possess Fc receptors and mark with the Leu 12 monoclonal antibody. Cells within developing colonies are found to have cytoplasmic IgM, IgA and IgG depending on the length of time in culture.     

Modulation of 5-HT3 receptor desensitization by the light chain of microtubule-associated protein 1B expressed in HEK 293 cells

Regulation of ligand-gated ion channel (LGIC) function and trafficking by cytoskeleton proteins has been the topic of recent research. Here, we report that the light chain (LC1) of microtubule-associated protein 1B (MAP1B) specifically interacted with the 5-HT_3A receptor, a predominant serotonin-gated ion channel in the brain. LC1 and 5-HT_3A receptors were colocalized in central neurons and in HEK 293 cells expressing 5-HT_3A receptors. LC1 reduced the steady-state density of 5-HT_3A receptors at the membrane surface of HEK 293 cells and significantly accelerated receptor desensitization time constants from 3.8 ± 0.3 s to 0.8 ± 0.1 s. However, LC1 did not significantly alter agonist binding affinity and single-channel conductance of 5-HT_3A receptors. On the other hand, application of specific LC1 antisense oligonucleotides and nocodazole, a microtubule disruptor, significantly prolonged the desensitization time of the recombinant and native neuronal 5-HT₃ receptors by 3- to 6-fold. This kinetic change induced by nocodazole was completely rescued by addition of LC1 but not GABA_A receptor-associated protein (GABARAP), suggesting that LC1 can specifically interact with 5-HT_3A receptors. These observations suggest that the LC1–5-HT_3A receptor interaction contributes to a mechanism that regulates receptor desensitization kinetics. Such dynamic regulation may play a role in reshaping the efficacy of 5-HT₃ receptor-mediated synaptic transmission.     

The quaternary Gs3 and Gs7 allotypes of the rabbit: their association with the b4.1 and b4.2 alleles of the kappa light chain

Several allotypic determinants of the rabbit immunoglobulin are present only on intact immunoglobulin molecules but not on isolated heavy or light chains. Some of these allotypes, although genetically segregating with the κ light chain, are nevertheless restricted to a single heavy chain class of Ig. It is shown that two IgG allotypes, Gs3 and Gs7, are linked to the presence of b4.1 and b4.2 light chains and that these allotypes can be regenerated by recombining an appropriate light chain with a rabbit γ chain. They are also linked to the equivalent IgM markers Ms3 and Ms7.