The rabbit CD1 and the evolutionary conservation of the CD1 gene family.

A comparison of the genes encoding the CD1 leucocyte differentiation antigens in man and mouse shows important differences which prompted us to analyze the CD1 genes of the rabbit. We have found that the rabbit genome contains multiple CD1 loci. Upon cloning and sequencing, one of these loci was found to encode the known rabbit CD1-like antigen (R-Ta) and to be closely related to the human CD1b gene, which is absent in the mouse, while a second rabbit gene is closely related to both the human R3 and the mouse CD1 genes. The data reinforce the notion of the existence of two classes of CD1 genes, one of which is conserved in all species, while the other, albeit also evolutionarily old, has been deleted in mice as well as in other rodents.

Biochemical properties of a novel rabbit thymocyte specific class I-like antigen.

Monoclonal antibody 5E2 identifies a new rabbit thymocyte specific cell surface molecule designated R-Ta. SDS-PAGE of molecules immunoprecipitated by 5E2 shows that R-Ta exists as a non-covalently associated hetero-dimer consisting of a light polypeptide chain (mol. wt approximately 12,000) and a bi-molecular species of a heavy chain (mol. wts of 45,000 and 40,000). The difference between the two forms of heavy chain can be attributed to different degrees of glycosylation. Each form of the R-Ta heavy chain has a polypeptide mol. wt of 34,000. At least three N-linked oligosaccharides and no significant O-linked sugars were found associated with R-Ta. Two dimensional electrophoresis of V8 protease peptide maps also indicate that the two forms of the heavy chains are similar, if not identical, in polypeptide primary structure. The light polypeptide was found to be serologically and structurally identical to beta-2-microglobulin. This was demonstrated in a previous study by reaction with goat anti-beta-2-microglobulin antisera. In this investigation the structural identity with beta-2-microglobulin was demonstrated by partial amino terminal sequence analysis. The partial amino acid sequence for 18 steps of the R-Ta heavy chain was also determined. A comparison of the amino acid sequence with other known sequences for the conventional Class I molecules of man, mouse and rabbit did not reveal any homology. Thus R-Ta is a new T-cell surface protein, and like human CD1, carries the unique distinction of thymocyte specificity, is beta-2-microglobulin associated, but is not Class I related.

Identification of a rabbit class I-like thymocyte-specific antigen.

The availability of a monoclonal antibody, 5E2, has made it possible to characterize a new class I-like thymocyte-specific antigen in the rabbit. Flow cytometry and cyto-fluorescent microscopy show that approximately 70 to 80% of the cells reacting with 5E2 are located throughout the thymic cortex. Structural studies reveal that the cell surface molecule recognized by 5E2 is a non-covalently associated heterodimer consisting of a 45,000 dalton glycoprotein and a low m.w. beta-2-microglobulin protein. Certain properties of the 5E2 antigen were similar to the murine TL and human T6 antigens. Therefore we have tentatively assigned to the 5E2 antigen the designation R-Ta on the premise that the structural gene(s) encoding this molecule will be associated with the rabbit major histocompatibility complex.

Simultaneous visualization of vascular permeability change and leukocyte egress in the central nervous system during autoimmune encephalomyelitis.

An unresolved issue in the study of demyelinating disease is whether blood-brain barrier damage is dependent upon the migration of inflammatory cells into the central nervous system (CNS). In a study of experimental autoimmune encephalomyelitis (EAE) in rabbits, a freeze-dried, paraffin-embedded tissue technique was exploited to enable (1) the immobilization of intravenously injected sodium fluorescein tracer, as an index of vascular permeability; and (2) an effective labeling by monoclonal antibodies of both T-lymphocytes and mononuclear phagocytes in "unfixed" neural tissue. Using these newly combined methods, evidence was found that increased vascular permeability in the CNS during EAE occurs concomitantly with, and not prior to, infiltration by mononuclear phagocytes.

Isolation and partial characterization of a fragment corresponding to the dimeric form of the CH2 domain of rabbit IgG.

A fragment corresponding to the intact dimeric form of the CH2 domain of rabbit IgG, including the hinge region disulfide linkage, was obtained by plasmin digestion of crystalline Fc derived from IgG by the action of papain. Identification and assessment of purity of the fragment was established by SDS-PAGE, amino acid composition analysis, N-terminus sequence and C-terminus amino acid analysis and SDS-urea-PAGE of the reduced fragment. The fragment retains serologic reactivity with anti-Fc specific antisera. Comparison of deglycosylation by endoglycosidase F indicates a more open special relationship between the two CH2 domains in the fragment than in Fc.

Complement C1q binding affects spin-labeled heterosaccharides of rabbit antibodies in immune but not artificial immunoglobulin G aggregates.

IgG anti-hapten antibodies were purified from the sera of rabbits homozygous for allotypic determinants d11 and d12 in the constant region of the heavy chain. Correlative with this determinant is the absence (d11) or presence (d12) of an oligosaccharide chain just below the hinge region of the IgG molecule. Both d11 and d12 molecules contain a complex heterosaccharide chain located near the carboxyl terminus of the second constant region domain. The two populations of IgG antibodies were thus selectively labeled with the spin probe Tempamine in their second constant region domains by reductive amination primarily of terminal N-acetylneuraminic acid residues. Chemical and enzymatic cleavages showed about 80% of the attached spin labels were N-acetylneuraminic acid-associated. Analysis of probe adducts by ESR spectrometry showed the presence of slower and faster moving subcomponents. Formation of immune complexes by antigen induces slight but significant restrictions of spin label mobility for both d11 and d12 IgG molecules. This restriction is qualitatively different from that seen in glutaraldehyde-, carbodiimide-, or ethanol-induced aggregates of the same IgG antibodies. The addition of purified complement C1 subcomponent C1q to immune aggregates resulted in marked immobilization of spin labels, the rotational correlation time of which was 30-40 mu s for both d11 and d12 molecules (evaluated by saturation transfer spectroscopy). A similar spin probe immobilizing effect is not seen when C1q binds to chemically aggregated IgG antibodies (which also do not activate C1). A novel model is proposed in which C1q is hypothesized to juxtapose Fc moieties in a discrete fashion required for subsequent C1 activation processes mediated by immune complexes.

Amino acid sequence of the CH2 domain from various lagomorph IgGs.

The complete amino acid sequence of a CNBr fragment (C3) corresponding to the CH2 domain of hare IgG was determined. The results when compared to analogous sequences for the domestic rabbit reveal extensive homology as expected. Similar comparisons were made for sequences of tryptic peptides isolated from the same region of the Fc from several other hares, the cottontail rabbit and pika.

Rabbit heavy chain haplotypes--allotypic determinants expressed by VH-CH recombinants.

This report summarizes our current understanding of the heavy chain haplotypes found in our laboratories' rabbits. Independently derived data from several laboratories have been synthesizes into a consistent picture of the linked inheritance of allotypic markers found on the different heavy chain classes and subclasses of rabbit immunoglobulins in pedigreed rabbits, including the families of three apparent VH-CH recombinants. In one recombinant, the entire group of CH markers (C mu, C gamma, and C alpha) recombined with the set of VH. Although in the other two recombinants all CH markers may also have recombined as a group, in one of these only IgG and IgA CH genes were informative; in the other recombinant, only the IgG allotypes were informative. Some allotypic determinants found on IgM molecules ("conformational") appear only when a specific variable region allotype (VHa) is combined with a specific mu constant region allotype (C mu). New combinations of VHa and C mu allotypes were generated in two of the genetic recombinants and led to new "conformational" determinants. The gains and losses observed lend support to the hypothesis that the determinants result from conformations generated by the combination of allotype-specific VH and C mu protein sequences. Conceivably, DNA events that join VH to diversity (D)- and joining (J)-coding sequences or mRNA processing events that splice J to C mu could be involved in generating the sequences that form allotype-specific determinants.

Control of latent allotype expression by rabbit splenocytes.

Serologic and chemical studies have suggested that immunoglobulin structural genes are under nonallelic genetic control. The transient appearance of unexpected (i.e., latent) allotypes in serum further suggests that cellular control mechanisms may be operative. We report here the results of our efforts to induce the expression of latent allotypes by cultured rabbit splenocytes. The protein A plaque assay facilitated with various anti-allotypic antisera was used to enumerate allotype-specific plaque-forming cells. When splenocytes from a b4 b4 rabbit are cultured in the presence of anti-b4 alloantisera, the number of b4 PFC (i.e., nominal) is suppressed and the number of PFC expressing the latent allotype is increased. The specificity of the induced latent PFC is dependent upon the source and allotypic mosaic of the antiserum added to the cultures. Our results suggest that lymphocytes committed to latent allotypes are normally suppressed. Activation by the appropriate alloantiserum directed against the nominal allotype may relieve a cell-mediated suppressive mechanism.

Evidence for linked expression of latent allotypes of the heavy chain constant and variable regions.

CH allotypes were determined for rabbit IgG and antibody molecules identified and isolated on the basis of their expression of latent group a (VH) allotypic determinants. Serologic and chemical typing of the purified molecules revealed that five out of 10 individual preparations studied expressed d allotypes not present in the rabbit as a nominal allotype. None of the latent VH-CH combinations represented products of a new or unusual arrangement of VH and CH structural genes. This result indicates that the molecules with latent allotypes express their VH and CH allotypes in the same combinations as do their nominal counterparts and do not result from misspairing of separate genes involved in H chain synthesis.

Amino acid sequence analysis of group e allotype-related peptides derived from lagomorph IgG.

A tryptic peptide (T 19) was isolated from a CNBr fragment of IgG from several representatives of present-day lagomorphs including domestic rabbit, hare, cottontail rabbit and pika. The amino acid sequence of this peptide revealed differences which can be correlated with serological polymorphism associated with group e IgG allotypes. The results confirm previous studies establishing the importance of Ala309 for the expression of e 15, particularly the i subspecificity. Arg315 correlates with the expression of the j subspecificity.

Serological and chemical studies of latent allotypes in the rabbit.

Genetic data has shown that antigenic variants or allotypes of variable and constant regions of the heavy and light chains of immunoglobulins represent products of allelic structural genes. New findings in several animal systems have demonstrated the non-allelic behavior of immunoglobulin allotypes. In the present study we report the results of our efforts to confirm the presence of latent allotypes in rabbit sera. Varying amounts of latent allotypes for groups a and b markers were detected in 95% of all normal and immune sera tested. Several rabbits expressed high levels of latent a and b allotypes (in excess of 200 micrograms/ml of serum). By gel diffusion and radioimmune inhibition analysis, the latent b4 marker from one b6b6 rabbit, B240, was identical to the nominal b4 allotype. Also, the L chains bearing the latent b4 marker were isolated from B240 IgG and analyzed chemically by taking advantage of an acid-labile aspartic acid-proline bond at positions 109 and 110, which is unique for b4 and b9 kappa L chains.

Serologic distinction between the rabbit kappa L chain allotype b4 and an allele b4.

Rabbit kappa light (L) chain allotypes are controlled by the allelic genes b4, b5, b6, b9 and a recently described allele b4v. The product of the b4v gene differs from that of b4 by at least two amino acid substitutions in the constant region. A serologic distinction between the b4 and b4v allotyes has now been detected by a radioimmune assay using one of six anti-b4 sera tested. The distinction was enhanced by fractionation of this antiserum on an immunoadsorbent containing cottontail rabbit IgG. IgG with L chains of the b4v type are deficient in their ability to inhibit binding of b4 IgG to the antibody preparation. L chains isolated from b4 and b4v IgG samples could not be distinguished by this assay indicating that interaction with an H chain is required for expression of the L chain determinants that differentiate b4 from b4v.

Latent expression of a Cgamma gene.

Rabbit 136 represented the first instance whereby more than two alleles for any immunoglobulin locus are detected phenotypically in a single animal. This was reported as the acquisition of the a2 and b6 markers in the animal nominally typed a1a3, b4b5 after intensive immunization. Follow-up studies indicate that another immunoglobulin marker, notably d11, also appeared in the postimmune serum. The d11 specificity was detected serologically by the hemagglutination-inhibition assay and chemically by analysis of the CNBr digestion products involving a strategic methionine.

Expression of group b light chain allotypes in cottontail rabbits.

Group b allotypic determinants (b4, b5, b6, and b9) were detected on cottontail IgG by several assays including immunodiffusion, radioimmune binding, and inhibition of radiobinding. The results indicate that cottontail IgG possess some but not all of the subspecificities present on domestic rabbit IgG. Several cottontail rabbits exhibited three of the four possible allotypic markers. In these instances, however, only two populations of IgG molecules (i.e., phenogroups) could be detected, each bearing one, two, or three of the allotypes. Five separate and distinctive phenogroups were identified. The results suggest that the phenogroups represent products of multiple allelic genes for the constant region of cottontail rabbit kappa light chain.

Surface immunolglobulin on rabbit lymphoid cells. VI. Failure to detect d and e group immunoglobulin allotypes on lymphocytes by immunoelectron microscopic labeling.

Surface immunoglobulin allotypes on rabbit peripheral blood lymphocytes (PBL) and spleen lymphocytes are detected by an immunoferritin labeling technique for electron microscopy. Human red blood cells, chemically coated with purified rabbit IgG of specific allotypes, served as test cells to assess the labeling specificity and efficiency. Immunoelectron microscopic labeling reveals that groups a, b, d and e allotypic specificities are readily detectable on passively coated test cells. However, only a and b group markers are detectable on lymphocytes. On rabbit PBL both a (VH of Fd region) and b (kappa chain constant region) group allotypes are detectable (73-78% and 68-77%, respectively); on spleen cells, 46% and 52-55% are positive for a and b locus allotypes, respectively. The d and e allotypes (i. e. gamma chain-specific) are undetectable on PBL or on spleen lymphocytes using this method. We conclude that the d and e group allotypes are either not present on the lymphocyte surface or are buried in the surface membrane and unacessible to the antisera used in this immunoferritin labeling technique.