The inheritance of individual antigenic specificities of rabbit antibodies to streptococcal carbohydrates.

The inheritance of individual antigenic specificities (IAS) of rabbit antibodies to the Group C streptococcal carbohydrate was demonstrated in a selectively bred rabbit family. The IAS of the antibodies from 3 proband rabbits were also observed in the Group C antibodies in as many as 7 out of 42 related rabbits, but in none of the Group C antibodies from 48 unrelated rabbits, Immunodiffusion analyses and quantitative radioprecipitin experiments revealed that this cross-specificity may be either partial or complete. Quantitative inhibition of the precipitin reaction between the proband antibody and its antiserum by preimmune IgG revealed 30-fold differences in the proportion of molecules with cross-specificity for the proband antibody. This proportion is higher in the preimmune IgG of the proband rabbit and of those relatives which produced cross-precipitating antibodies than it is in the IgG of rabbits which had the same group a allotype, but did not produce cross-precipitating antibodies. The proportion is much lower in the IgG of rabbits with a group a allotype different from that of the proband antibody. These data suggest that serologically detected individual antigenic specificities are inherited markers of immunoglobulins.

Isolation and characterization of IgG molecules expressing latent group b allotypes from pedigreed b4b4 rabbits.

Latent group b markers were detected in sera, in IgG preparations, and on isolated L chains from rabbits bred for homozygosity at the b locus. Serologic analysis of sera from an extended family of homozygous b4 rabbits revealed the presence of latent b allotypes in 5 of 37 sera tested. Latent b5 and b9 markers were identified; none of the sera tested contained latent b6. In two instances, the level of latent b9 allotypes was sufficiently high to permit isolation and detailed serologic characterization of the immunoglobulin population bearing this allotype. The fact that latent allotypes were detected in pedigreed homozygous rabbits minimizes the possibility that lymphoid cell chimerism is involved in latent allotype expression. Furthermore, characterization of the b9 IgG population indicates that the latent allotypic determinants do not reside on a subset of molecules with dual allotypic reactivity.

Heavy and light chain allotypic markers on rabbit homocytotropic antibody.

Evidence has been presented that rabbit homocytotropic antibody prepared against ovalbumin bears allotypic markers of both group a (heavy chain) and group b (light chain). This was shown by specific removal of activity in passive cutaneous anaphylaxis assays by anti-allotype immunoadsorbents. The homocytotropic antibody has properties which indicate that it belongs to a new class of rabbit immunoglobulin. These results demonstrate that this newly described class possesses the allotypes of both groups a and b, as has been previously shown for other rabbit immunoglobulin classes.

Nonallelic behavior of rabbit variable-region allotypes.

Group a allotypes not detected by qualitative typing or anticipated from breeding data (latent allotypes) were detected at low levels in 50% of normal rabbit sera tested. The latent allotypes, which were serologically identical to allotypes of pooled IgG, were detected in sera from rabbits with all possible combinations of group a allotypes and their occurrence in individual rabbits was transitory and sporadic. These findings give reason to question the assumption that the synthesis of immunoglobulin allotypes is directed by allelic structural genes.

A genetic polymorphism in the constant region of rabbit b4 kappa chains.

Amino acid sequence analysis of a b4 light chain from a rabbit homogeneous antistreptococcal antibody revealed the presence of two amino acid substitutions in the constant region not previously reported for these positions. These interchanges, consisting of serine for alanine at position 121 and leucine for glutamine at position 124, were also present in about 30% of the pooled b4 light chains isolated from pooled IgG from the rabbit (4539) that produced the homogeneous antibody. In addition, these interchanges (b4var) were found, always at the same levels, in varying percentages in nonimmune or early immune bleedings from related rabbits in this pedigreed family and could be traced for five generations. The inheritance pattern of b4var was consistent with autosomal codominant inheritance.

Human T lymphocyte virus 1 from a leukemogenic cell line mediates in vivo and in vitro lymphocyte apoptosis.

HTLV-1 is implicated in the development of diverse diseases. However, most HTLV-1-infected individuals remain asymptomatic. How HTLV-1 infection leads to disparate consequences remains a mystery, despite extensive investigation of HTLV-1 isolates from infected individuals. As in human infection, experimental HTLV-1 infection in rabbits is generally benign, although HTLV-1-infected rabbit T cell lines that mediate lethal leukemia-like disease have been reported. We report here that thymuses from mature outbred rabbits inoculated with a lethal leukemia-like disease have been reported. We report here that thymuses from mature outbred rabbits inoculated with a lethal HTLV-1 T cell line (RH/K34) showed morphological and biochemical evidence of apoptosis, whereas thymuses from rabbits inoculated with nonlethal HTLV-1 T cell lines showed no signs of apoptosis. Exposure of rabbit or human lymphocytes to purified virus from RH/K34 caused rapid induction of apoptosis, providing an in vitro correlate to the pathogenic effects. By contrast, virus isolated from a nonlethal cell line mediated dose-dependent lymphocyte proliferation. These data implicate lymphocyte apoptosis as a potential mechanism by which the lethal HTLV-1 cell line causes fulminant disease and provide a means to identify factors contributing to HTLV-1 disease. Results from this HTLV-1 infection model can provide insight into variations in HTLV-1 pathogenicity in human infection.

Allotype exclusion in uniform rabbit antibody to streptococcal carbohydrate.

Rabbit antibodies to streptococcal polysaccharide are described which show selectivity of expression of the allotypic specificities on both the heavy (H) and light (L) chains. One of these antibodies binds weakly to Sephadex. A purification method based on this binding has yielded antibody completely lacking any group a allotypic marker on its H chains.

An idiotypic cross-reaction between allotype a3 and allotype a negative rabbit antibodies to streptococcal carbohydrate.

Two antibodies to Group C streptococcal carbohydrate isolated from an individual rabbit had similar relative binding affinities for a Group C immuno-adsorbent column. Their light chains were similar, if not identical, as were the constant regions of their heavy chains. Differences in the variable regions of the H chains of the two antibodies were detected by chemical analysis. The two antibodies had serologically identical idiotypic determinants although one antibody possessed the a3 allotype and the other had no detectable group a marker. The occurrence of such antibodies indicates the absence of obligatory associations between group a allotypes and idiotypic specificities, despite the fact that both determinants have antigenic components in the V(H) region of the H chain.

The inheritance of antibody V regions in the rabbit: linkage of an H-chain-specific idiotype to immunoglobulin allotypes.

Anti-idiotype antibodies specific for the H chain of an homogeneous antistreptococcal antibody (4135 Ab) were prepared by injection of recombinant molecules consisting of the H chains from 4135 Ab and L chains isolated from the injected rabbit. The antibodies prepared in this fashion (anti-HId) were specific for the VH region of 4135 Ab. Using this preparation in an inhibition of binding assay, sera from rabbits related and unrelated to 4135 were screened for the presence of the 4135 HId. It was found that about 45% of the related rabbits, when immunized with streptococcal Group C vaccine, produced antibodies with a cross-reactive idiotype, while less than 10% of similarly immunized unrelated rabbits produced molecules bearing HId. The expression of HId was linked to the a3 allotype present in the H chain allogroup J. Antibody molecules bearing the HId determinant were isolated from heterozygous (a1a3 and a2a3) rabbits and shown to express the a3 allotype. One rabbit lacking the a3 allotype produced significant amounts of antibodies expressing HId. These antibodies were found to express both VH and CH allo-types characteristic of the J allogroup, although neither allotype was found in a preimmune IgG sample from this rabbit.

Insertion/deletion-related polymorphisms in the human T cell receptor beta gene complex.

Insertion/deletion related polymorphisms (IDRP) involving stretches of 15-30 kb within the human TCR-beta gene complex were revealed by pulse-field gel electrophoresis. Two independent IDRP systems were detected by analysis of Sfi I- and Sal I-digested human DNA samples using probes for TCR C and V region gene segments. The allelic nature of these systems was verified in family studies, and mapping data allowed localization of one area of insertion/deletion among the V gene segments and the other near the C region genes. All but one of 50 individuals tested could be typed for the two allelic systems, and gene frequencies for the two allelic forms were 0.37/0.61 and 0.46/0.54, indicating that these polymorphisms are widespread.

Infection of rabbits with human immunodeficiency virus 1. A small animal model for acquired immunodeficiency syndrome.

Injection of rabbits with a human T cell line infected with HIV-1 caused seroconversion within 6 wk, and HIV-1 could be isolated from PBL cultures of infected rabbits. Identity of the isolated virus with HIV-1 was shown by analysis of products amplified by the polymerase chain reaction. HIV-1 infection was seen in rabbits injected with HIV-1-infected cells alone as well as in those that were first infected with HTLV-1 and subsequently with HIV-1. There were no consistent signs of disease in the rabbits infected with HIV-1 alone but HTLV-1/HIV-1-infected rabbits showed signs of illness including diarrhea and weight loss, transient neurologic impairment and, in one animal, a rapidly progressing mammary adenocarcinoma. Examination of organs taken at necropsy from both HIV-1- and HTLV-1/HIV-1-infected animals showed splenic hyperplasia and lymphocyte infiltration of the lungs, as well as moderate damage to liver and kidney in some cases.

Differentiation antigens identify subpopulations of rabbit T and B lymphocytes. Definition by flow cytometry.

A panel of six monoclonal antibodies produced against cell surface glycoproteins of a rabbit T lymphocyte line was used with flow cytometry to define rabbit lymphocyte subpopulations. Four thymocyte populations were characterized by size and expression of cell surface antigens and appear to represent stages in thymocyte differentiation. Rabbit spleen contained five subpopulations: two of T lineage, two of B, and a null cell subset. Bimodal distribution of staining of thymocytes and peripheral T cells was observed using an antibody (9AE10) directed against a Thy-1 analogue in the rabbit, suggesting two separate T cell lineages. One of the monoclonal reagents, L11/135, reacted strongly with peripheral rabbit T cells as shown by two-color immunofluorescence. In functional studies, only the L11/135-bearing cells responded to the T cell mitogens concanavalin A and phytohemagglutinin and to allogeneic splenocytes. The thymocyte subpopulations and the peripheral T and B cell subsets differ from those described in mouse and man.

Role of immune recognition in latent allotype induction and clearance. Evidence for an allotypic network.

The role of allotype recognition in the regulation of the expression of latent allotypes has been investigated in two series of experiments. The first experiments were designed to investigate the apparent instability of latent allotypes in circulation. In these experiments, clearance rates of IgG preparations bearing allotypes matched and unmatched to the recipient were examined. In all cases, iodinated IgG matched in allotype to the recipient was cleared at a normal rate from the serum. However, in several cases, iodinated IgG of an unmatched allotype was cleared at a rate and in a manner suggesting prior sensitization of the recipient to IgG of that allotype. Such apparent sensitization correlated with the presence of the foreign allotypes as a latent allotype in several bleedings taken both before and after the clearance experiment. In the second series of experiments, designed to test the ability of antiallotype antibodies to affect the expression of latent allotypes, five rabbits were immunized first with purified antiallotype antibodies and then after 3-4 mo, with streptococcal vaccine. Examination of the antistreptococcal antibodies for latent allotype revealed, in all cases, that the allotype against which the antiallotype antibodies were directed was present in levels 8- to 20-fold greater than were observed before the antiallotype injections. These results indicate that recognition of allotypic determinants is an important element in the control of latent allotype expression and suggest the existence of a regulatory network involving antiallotype antibodies.

Diverse expression of group a allotype specificities on the heavy chains of homogeneous rabbit antibodies.

Variations in the ability of homogeneous antibodies to absorb group a allotype antisera suggest that these V(H) region allotypes comprise a spectrum of specificities. While no homogeneous antibody tested would completely absorb antisera to the group a specificities, the total IgG preparations from individual rabbits was capable of doing so, including preimmune IgG from the rabbits that produced the homogeneous antibodies. Differences in the serologic reactivities of homogeneous antibodies indicate that each possesses only a portion of the spectrum of the allotypic specificities expressed by the total IgG.

Detection of idiotypic cross-reactions among streptococcal antisera from related rabbits.

Idiotypic cross-reactions among antibodies to Group C streptococcal carbohydrate were studied using idiotypic antisera prepared in allotypically matched rabbits. Antibodies with idiotypic cross-specificity to one proband antibody were detected in 58% of the antisera from related rabbits, while approximately 1% of nonrelated rabbits produced antibody with this specificity. The cross-specificity was related to the group a (V(H)) allotype of 133 rabbits tested with only one exception. Studies utilizing antisera against a second proband antibody failed to detect antibodies with idiotypic cross-reactivity among the same group of related rabbits. This result emphasizes the variation in expression of idiotypic determinants of antibodies. It was further shown that the presence of anti-IgG's in the streptococcal antisera interfere with the detection of idiotypic cross-reactions. These anti-IgG's masked the presence of antibodies with idiotypic cross-specificity when inhibition of precipitation tests were used for their detection.

Selection of rabbit CD4- CD8- T cell receptor-gamma/delta cells by in vitro transformation with human T lymphotropic virus-I.

In vitro transformation of rabbit peripheral blood mononuclear cells (PBMC) with human T lymphotropic virus-I (HTLV)-infected human or rabbit cells resulted in CD4- CD8- cell lines, some of which caused acute leukemia when injected into rabbits. Structural analyses of the proviruses from cell lines with diverse pathogenic effects provided no clear correlation with lethality. The rabbit lines were provisionally designated T cells because they express interleukin 2R (IL-2R) and CD5 and lack surface immunoglobulin, but none express functional T cell receptor (TCR) alpha or beta transcripts. A more detailed characterization of the HTLV-I-infected cells was required to determine cell lineage and its potential influence on pathogenic consequences. Probes for rabbit TCR gamma and delta genes were derived and used to detect gamma and delta TCR RNA transcripts, identifying the in vitro transformed lines as gamma/delta T cells. CD4+ and CD8+ lines were derived from PBMC of HTLV-I-infected rabbits and CD4+ TCR-alpha/beta HTLV-I lines were derived from rabbit thymus, eliminating the possibility that the HTLV-I isolates used here transform only CD4- CD8- TCR-gamma/delta cells. The percentage of gamma/delta cells in rabbit PBMC is relatively high (23% in adult rabbits); this with diminution of CD4+ and CD8+ cells in IL-2-supplemented PBMC or thymocyte cultures may account for selection of rabbit HTLV-I-infected gamma/delta T cell lines in vitro. The availability of well-characterized T cell lines with diverse in vivo effects in the rabbit HTLV-I disease model allows evaluation of roles played by cell type in HTLV-I-mediated disease.

Recombination within the HLA-D region. Correlation of molecular genotyping with functional data.

Molecular genotyping of the HLA-D/DR region in a family correlated with serologic and cellular typing data. It was further possible to predict a subtle difference in SB region-related functions from such molecular studies. A family that included an individual who inherited an HLA haplotype with a paternal recombination between HLA-B and the HLA-D/DR region was identified by classic HLA typing techniques. Segregation of HLA-D/DR region genes in this family was studied by Southern blot analysis using cDNA probes for DR alpha, DR beta, DC alpha, DC beta, and SB beta. Restriction enzyme fragment polymorphisms observed for every gene tested were in concordance with assigned HLA haplotypes (including the individual known to have inherited a paternal recombinant haplotype) with one exception: two HLA identical siblings were observed to have different SB beta restriction fragment patterns. Further testing revealed that one individual inherited a maternal HLA haplotype recombinant between the HLA-D/DR region and SB beta. Although both maternal SB alleles typed as SB4, allelic differences could be detected cellularly by primed lymphocytes and by the differential expression of a class II cell surface antigen using monoclonal antibody. Therefore, predicted and nonpredicted recombinant haplotypes were detected in a family by molecular genotyping.

A novel HLA-D/DR-like antigen specific for human B lymphoid cells. Biochemical evidence for similarity to but nonidentity with known HLA-D/DR antigens.

The polymorphic human B cell-specific antigen, 33.1, detected by a murine monoclonal antibody, was compared by genetics and structural analysis with known human Ia antigens from a panel of DR homozygous Epstein-Barr virus-transformed B lymphoblastoid cell lines. Cells homozygous for DR 1, 2, 4, 5, and w6 were positive, while cells that are DR3,3 or DR7,7 usually failed to express this antigen. Mutant DR null, DC/MB-positive cells were 33.1 positive while DR null, DC/MB-negative cells failed to express this antigen, suggesting the segregation of 33.1 with the DC antigen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that 33.1 alpha and beta chains were of lower molecular weights than the DR alpha and beta chains isolated from the same cell line. Partial N-terminal amino acid sequence analyses were carried out for the heavy and light chains of the 33.1 antigen radiolabeled with [3H] phenylalanine. The results of these analyses, in conjunction with previous data on tissue distribution, indicate that the 33.1 antigen is a non-DR but Ia-like antigen closely related to the previously defined I-A homologues, DC and DS.

Phagocytic rabbit cell lines expressing class II MHC products: establishment of cell lines by viral transformation.

Continuous rabbit macrophage-like cell lines were established after in vitro infection of spleen cells with either Simian virus 40, lymphotropic papovavirus or herpesvirus sylvilagus. These cell lines are characterized as morphologically similar to mature macrophages, esterase positive, and have unrearranged immunoglobulin genes. They possess macrophage functionality because they are highly phagocytic and are able to mediate an antibody dependent cell mediated cytotoxicity assay on chicken red blood cells. Northern blot analysis of total cellular RNA with a DQ alpha probe indicates that the cell lines constitutively express message for class II gene products. In addition, cell sorter analysis indicates that two of these lines display class II antigen at the cell surface.

Partial amino acid sequence analysis of rabbit MHC class II molecules isolated from two-dimensional polyacrylamide gels.

Partial N-terminal amino acid sequence analyses were performed on rabbit MHC class II molecules eluted from 2-D electrophoretic gels. Rabbit spleen cells were biosynthetically labelled with 3H-phenylalanine, 3H-tyrosine and 35S-methionine and class II molecules were immunoprecipitated with a monoclonal antibody, 2C4. The immunoprecipitates were electrophoresed on 2-D gels and as many as 15 spots were observed. Individual spots corresponding to alpha and beta chains were eluted from unfixed gels following visualization of the spots by autoradiography of 35S-Met labelled polypeptides. Ten eluted polypeptides were subjected to amino acid sequence analysis to locate Phe and Tyr residues. Comparison of these partial sequences with sequences of human class II molecules indicated that each of six beta chains and three of four alpha chains were homologous to human DQ molecules; one of the alpha chains appeared homologous to DR alpha or DP alpha. The assignment of alpha or beta chain to the polypeptides was confirmed by radiosequence of molecules labelled with 35S-Cys residues. Thus, by a relatively simple procedure, individual MHC class II polypeptides in spleen cell lysates have been separated from each other and partial amino acid sequences have been obtained.