Genetic studies on Cochin Jews in Israel: 2. Gm and Inv data — polymorphism for Gm3 and for Gm1,17,21 without Gm(26)

Serum samples from 223 Jews from Cochin, India were tested for Gm(1,2,3,5,6,13,14,17,21,26) and for Inv(1). Certain samples were also tested for Gm(15) and Gm(16). The Cochin Jews are polymorphic for: 1) Gm³, a haplotype that does not lead to the formation of γ³, as was shown by tests of the serum of a homozygote, and 2) Gm^1,17,21, a haplotype lacking Gm(26), which is ordinarily present in this haplotype. The Gm data indicate considerable admixture with southern Indians. There is no evidence for African admixture, such as has been found for all other Jewish populations studied thus far. The Inv data are similar to those for other Jewish populations.     

Qualitative and quantitative studies on IgG2 globulins in individual human sera with an antiserum capable of differentiating between Gm(n+) and Gm(n-) proteins

A monkey antiserum was obtained by immunization with Gm(n+) IgG2 proteins. After it had been rendered specific for the IgG2 subclass, it was shown to contain antibodies of two specificities, i.e. anti-γ2 and anti-Gm(n). Precipitation and haemagglutination experiments, using unabsorbed antiserum and antiserum absorbed with Gm(n+) or Gm(n−) proteins, made it clear that the anti-Gm(n) was unable to form precipitates with Gm(n+) proteins without the assistance of anti-γ2 antibodies. Results obtained in radial immunodiffusion with serum samples of known Gm genotype showed that the antiserum differentiates three types of individuals: homozygous Gm(n− /n−), heterozygous Gm(n+ /n−) and homozygous Gm(n+ /n+).The amount of IgG2 present in serum samples from seventy-four individuals of variable Gm types was determined by comparison with a standard serum. It was found that the mean level of female sera (4·2 mg/ml) was higher than that of male sera (3·3 mg/ml). It was further shown that the IgG2 level was correlated with Gm(n). The total IgG2 content of individuals possessing the Gm(n) marker was higher than that of individuals of otherwise identical Gm type but lacking this factor. In individuals heterozygous for Gm(n) the level of Gm(n+) protein was significantly higher than that of Gm(n−) protein.     

Alpha-globulin-like antigens in bacteria.

Seventy-two strains of bacteria of the families Bacillaceae, Enterobacteriaceae and Micrococcaceae were investigated for antigens resembling Gm (1), Gm (2) and Inv (1) human immunoglobulin group factors. Antigens similar to these factors were encountered in some strains of the genus Bacillus. Staph. epidermidis strains had antigenic structures resembling Gm (1) and Inv (1) factors, and strains of Enterobacteriaceae structures resembling Gm (2) and Inv (1). Occurrence among various species of bacteria of antigens resembling human gamma-globulin group factors suggests immunization by bacterial infection as one of the causes of presence of anti-Gm and anti-Inv antibodies in human beings.     

A quantitative method for determining gamma G allotype antigens. 3. Gm genes and gene complexes in different human populations

The sera expression of human immunoglobulin heavy chain genes and gene complexes was analyzed in different ethnic groups by quantitative determination of Gm allotype antigens. In all populations tested, IgG3 subclass heavy chains of Gm(b+) allotype had greater sera expression than the allele Gm(g). The percentage of IgG3 allotype molecules was higher in Chinese and Easter Islanders than in Caucasians due to higher Gm(b) values. In general, IgG1 allotypes did not show quantitative differences correlated with Gm genotype. A possible exception was the Gm^{za, g} gene complex which had a lower Gm(a) concentration than comparable Chinese sera of different genotype. Certain similarities in the regulation of Gm genes were consistently observed among the populations tested; these included the restricted range of percentage of IgG1 allotype antigens, a gene dosage effect, and differential gene activity of Gm^b and Gm^g IgG3 Gm genes.     

Surface immunoglobulin of rabbit peripheral blood lymphocytes: detection of allotypes by fluorescence and rosetting

Specific anti-allotype reagents were prepared to detect heavy and light chain allotypes on surface immunoglobulin-positive (sIg⁺) cells from a³ a³ b⁴ b⁴, a² a² b⁵ b⁵ homozygous and a² a³ b⁴ b⁵ heterozygous rabbits. Total sIg⁺ peripheral blood lymphocytes (PBL) were detected with fluorescein-labeled goat anti-light chain and sheep anti-rabbit Ig reagents. The total sIg⁺ cells detected with these reagents and the sum of a and b allotypes individually measured with specific fluorescent anti-allotype reagents (a2 + a3 or b4 + b5) were more or less the same when measured by fluorescence-activated cell sorter (FACS) II flow microfluorometry. The data provided no evidence for single cells expressing both allelic allotypes (allelic inclusion). We found that FACS and rosetting techniques were generally equally sensitive. However, we detected a greater proportion of total b4⁺ plus b5⁺ cells by rosetting than by fluorescence in some PBL preparations from heterozygous b⁴ b⁵ rabbits. This was not seen with artificial mixtures of b⁴ b⁴ and b⁵ b⁵ cells. The nature of these cells is not yet known. Conceivably, they were not scored as lymphocytes by light-scatter analysis on the FACS and hence were not counted, but were indistinguishable from lymphocytes by microscopy.     

Localization of Gm markers to different molecular regions of the Fc fragment

pFc' fragments (corresponding to homology region Cγ3) and smaller subfragments produced by trypsin and papain have been studied by haemagglutination inhibition techniques for the presence of a range of Fcγ allotypic markers. The markers which were identified in the pFc' fragment included Gm(a), (x), (b⁰), (b³), (b⁴), (b⁵), (c³), (c⁵) and `non a'. In addition, an IgG 4 marker which possibly occupies the same molecular region as the Gm(a) and `non a' antigens was detected in pFc' fragments from IgG 4 proteins.     

ON THE ZYGOSITY OF Gm(a)

A family study on Gm determinants is reported starting from the pair: mother Gm(a - x - f + b +)/child Gm(a + x - f - b -). The genotypes derived from the results show the probable influence of the inactive allele Gm^- described earlier. The importance of this and similar findings for our knowledge of the Gm system and for its use in practical medico legal work is stressed.     

Serological specificity of IgG and IgM antiglobulin antibodies in anti-Gm(a) antisera

Serological study of a chromatographed Ragg anti-Gm(a) serum has shown differences in specificity of the IgG serum agglutinators and the IgM rheumatoid factor monospecific for Gm(a). The specificity of the IgG serum agglutinators is determined by the enzymatic modification of the globulin molecules and is unrelated to the Gm type.

Both serum agglutinators and anti-Gm(a) antibodies were demonstrable in a (heterologous) baboon anti-Gm(a) serum; however, both types of antibodies in this animal were IgG.

     

Ein Beitrag zur Populationsgenetik des Gm- und Inv-Systems

Zusammenfassung 2000 Seren gesunder Blutspender aus dem Raume Hessen wurden auf die Gamma-Globulin-Serumgruppen Gm(1) [Gm(a)], Gm(2) [Gm(x)], Gm(4) [Gm(f)], Gm(12) [Gm(b)] und Inv(1) [Inv(1)] hin untersucht. Drei miteinander nicht verwandte Probanden wiesen den sehr seltenen Phänotyp Gm(-1, 2, 4, 12) auf. In 15 Fällen fand sich ein diskordantes Verhalten der Faktoren Gm(4) und Gm(12). Die Ergebnisse werden kurz diskutiert.

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Summary 2000 sera of healthy blood donors of the region Hessen habe been examined for the Gamma-Globulin serum-groups Gm(1) [Gm(a)], Gm(2) [Gm(x)], Gm(4) [Gm(f)], Gm(12) [Gm(b)] and Inv(1) [Inv(1)]. Three non related probands showed the extremly rare phenotype Gm(-1. 2, 4, 12). In 15 cases Gm(4) and Gm(12) appeared discordantly.The results are shortly discussed.

     

The physiochemical and immunological characterization of Gm (1) antibodies from normal human serum.

The mol. wt and charge characteristics of Gm(1) antibodies from normal human serum were studied by gel filtration and DEAE- anion exchange chromatography. The effect on anti-Gm(1) activity, of incubating individual antisera with disulphide reducing agents, and with anti-IgG or anti-IgM immunoabsorbents were also studied. The results demonstrate the existence of a low molecular weight IgM protein with anti-Gm(1) activity.     

Gm40600 promotes CD4+ T‐cell responses by interacting with Ahnak

It is important to characterize novel proteins involved in T‐ and B‐cell responses. Our previous study demonstrated that a novel protein, Mus musculus Gm40600, reduced the proliferation of Mus musculus plasmablast (PB)‐like SP 2/0 cells and B‐cell responses induced in vitro by LPS. In the present study, we revealed that Gm40600 directly promoted CD4⁺ T‐cell responses to indirectly up‐regulate B‐cell responses. Importantly, we found that CD4⁺ T‐cell responses, including T‐cell activation and differentiation and cytokine production, were increased in Gm40600 transgenic (Tg) mice and were reduced in Gm40600 knockout (KO) mice. Finally, we demonstrated that Gm40600 promoted the Ahnak‐mediated calcium signalling pathway by interacting with Ahnak to maintain a cytoplasmic lateral location of Ahnak in CD4⁺ T cells. Collectively, our data suggest that Gm40600 promotes CD4⁺ T‐cell activation to up‐regulate the B‐cell response via interacting with Ahnak to promote the calcium signalling pathway. The results suggest that targeting Gm40600 may be a means to control CD4⁺ T‐cell‐related diseases.     

IgG1 SUBCLASS PROTEIN WITH THE GENETIC Gm MARKERS Gm(z + non-a +): ANOTHER EXAMPLE OF INTRAGENIC HYBRIDIZATION AMONG IgG SUBCLASS GENES

A new type of Gm genetic marker combination in an IgG1 immunoglobulin, Gm(z) together with a non-a marker, is here described. This indicates the presence of a new gene, Gm^z,non-a, which most likely originated on the basis of an intragenic recombination at the IgG1 cistron. Immunochemical and immunogenetic studies showed that no other IgG1 Gm markers were expressed by the unusual gene, and there was no evidence for deletions or duplications at the IgG1 cistron.     

Enzyme linked immunosorbent assay (ELISA) for human IgG (Gm) allotype determination

An inhibition enzyme-linked immunosorbent assay (inhibition-ELISA) was developed for the quantitative determination of human IgG (Gm) allotypes using rabbit anti-Gm antisera, alkaline-phosphatase-conjugated goat anti-rabbit IgG and, as the calibrant, purified human myeloma proteins possessing the relevant Gm allotype. The assay is reproducible and can detect as little as 10 ng/ml of G1m(a), G2m(n) or G3m(st), and 100 ng/ml of G1m(f) or G3m(g). Using this assay, the "gene dosage effect" and "allelic balance" in healthy Japanese were studied.     

Studies of the antibody nature of the rheumatoid factor: Reaction of the rheumatoid factor with sheep erythrocytes sensitized with human anti-sheep cell antibodies and with O Rh positive cells sensitized with incomplete anti-Rh antibodies

The reaction of the rheumatoid factor (RF) with 7S γ-globulin antibodies of nine persons immunized with sheep erythrocytes was studied. All of a panel of rheumatoid sera with high Waaler-Rose titres agglutinated sheep cells sensitized with the human anti-sheep cell antibodies and O Rh positive cells sensitized with the `diagnostic' anti-CD serum Ripley. The RF measurable with these systems could be absorbed to diphtheria toxoid—human antitoxin precipitate, whereas the absorption with egg albumin—rabbit anti-egg albumin precipitate did not result in any appreciable titre reduction. However, the eluate from the rabbit precipitate was highly active in these systems, whereas Rh positive cells sensitized with anti-Rh sera suitable for Gm(a) typing were not regularly agglutinated.

A markedly greater concentration of native than of heat-aggregated γ-globulin was needed for inhibition of the agglutination by the RF of cells heavily sensitized with the human anti-sheep cell antibodies. No appreciable difference in this respect was seen when using lightly sensitized cells. Only the heavily sensitized cells fixed complement. The `natural' 7S γ-globulin antibodies against sheep cells were neither suited for demonstration of RF nor did they fix complement.

Sheep cells coated with 7S γ-globulin antibodies of a Gm(a+) person were agglutinated by a non-rheumatoid anti-Gm(a) serum, and this system was well suited for Gm(a) typing, whereas cells coated with antibodies of a Gm(a-) person were not agglutinated. Rheumatoid anti-Gm(a) sera agglutinated cells sensitized with antibodies of both Gm(a+) and Gm(a-) persons. Using cells coated with Gm(a+) antibodies, some distinction between Gm(a+) and Gm(a-) sera could be obtained under carefully controlled conditions. The use of a Gm(a-) coat resulted in a slight difference in the inhibiting capacity, which had no relation to the serum's Gm(a) type.

The results suggest that the reaction of the RF with sheep cells heavily sensitized with human anti-sheep cell antibodies is essentially an interaction of the RF with immune aggregated γ-globulin, whereas when using lightly sensitized cells the individual γ-globulin molecules play a prominent role.

     

Isolated hapten-binding receptors of sensitized lymphocytes. V. Cellular origin of receptor molecules

Chimeric mice were constructed by injection of thymus cells into nu/nu mice. The thymus cells carried a different immunoglobulin (Ig) heavy chain linkage group than the cells of the recipients. The chimeric animals were then immunized with the hapten (4-hydroxy-3-nitro-phenyl)acetyl (NP) on an appropriate carrier, and NP-binding receptor molecules were isolated from the spleen cells of the animals. Receptor molecules and antibodies were analyzed for the presence of two Ig heavy chain variable region (V_H) markers which are specific for the anti-NP response of mice carrying the Ig^b allogroup, namely the NP^b idiotpye and the heteroclitic fine specificity of hapten binding. Two types of receptor molecules are obtained by our method, those carrying determinants of Ig constant domains (anti-Ig⁺ receptors) and others lacking such determinants (anti-Ig^? receptors). The two V_H region markers were found on anti-Ig^? receptors but not on anti-Ig⁺ receptors and humoral antibodies when the T cells in the chimeric mice carried the Ig^b allogroup, whereas the B cells were Ig^a/a. Conversely, anti-Ig⁺ receptors and humoral antibodies but not anti-Ig^?receptors, expressed the two markers when the B cells of the chimeras carried the Ig^band the T cells the Ig^a allogroup. From these results and previous data showing that anti-Ig^? receptors are enriched together with T lymphocytes and anti-Ig⁺ receptors together with B cells, we conclude that the V_H-bearing anti-Ig^? receptors are not only present on T cells, but are indeed synthesized by these cells.     

The Reaction of Rheumatoid Anti-Gm Antibodies with Native and Aggregated Gm-Negative IgG

Rheumatoid anti-Gm(a) antibodies were shown to react with native Gm(a-) IgG. This reaction takes place with the C_H3 domain (pFc' fragment) of IgG and is not due to anti-isotype specificities contaminating the test system. The reactivity of the Gm(a-) IgG is greatly increased by heat aggregation, and this increase is clearly related to the amount of aggregates formed. Rheumatoid anti-Gm(a) reacts with aggregated IgG1. IgG2. and IgG3 Gm(g+) proteins but not with IgG3 Gm(b+) or IgG4 proteins. Thus there is a clear specificity in the reactions with aggregated IgG of different subclasses. Quantitative hemagglutination inhibition studies in the AutoAnalyzer suggested that the antigenic determinants of native and aggregated Gm(a+) or Gm(a-) IgG involved in the reaction with rheumatoid anti-Gm(a) were very similar. Furthermore, immunosorbent studies showed that the saint population of rheumatoid anti-Gm(a) antibodies is reacting with the native and aggregated Gm(a+) and Gm(a-) IgG Analogous findings were obtained using rheumatoid anti-Gm(b¹) and anti-Gm(g).     

A biased Gm haplotype and Gm paraprotein allotype in multiple myeloma suggests a role for the Gm system in myeloma development

The association between a particular Gm haplotype and susceptibility to multiple myeloma (MM) is not clear. The reason is probably because no investigations have so far been carried out on the relationship between the Gm haplotype, which represents the inherited combination of IgG Gm allotypes, and the Gm allotype expressed at the IgG paraprotein (M-component), which reflects the enhanced gene expression within the haplotype in MM. We studied the incidence of Gm allotypic markers present in IgG subclasses in the serum from 52 patients with MM and in parallel with the isolated IgG paraproteins. The results showed that 84.6% of the patients were heterozygous for haplotypes Gm(a; z; n-; g;)/Gm(f; n+/n-; b1; b0; b5) and 15.3% were homozygous for Gm(f; n/n-; b1; b0; b5), while no homozygous Gm(a; z; n-; g) individuals were found among the studied patients. The incidence of these combinations in the healthy population in Serbia is 34%, 66% and < 1%, respectively. Subjects with Gm(a; z; n-; g)/Gm(f; n+/n-; b1; b0; b5) combination are over 10 times [odds ratio (OR) = 10.69; 95% confidence interval 1.67-68] as likely to be affected by the disease as the subjects with homozygous Gm(f; n+/n-; b1; b0; b5) combination (OR = 0.35, 95% confidence interval 0.06-2.23). However, despite the Gm heterozygosity, most of the Gm(a; z; n-; g;)/Gm(f; n+/n-; b1; b0; b5) positive patients with MM (86.3%) had IgG paraprotein with the allotypic marker from the Gm(f; n+/n-; b1; b0; b5) haplotype. Together with patients homozygous for this haplotype, the relative number of patients with serum IgG paraprotein carrying allotypic marker from the Gm(f; n/n-; b1; b0; b5) haplotype was 88.5%. These results suggest that the development of an M-component could be related to a disturbance on chromosome 14q32 carrying the Gm (f; n+/n-; b1; b0; b5) set of genes.     

Quantitation of anti-NP (4-hydroxy-3-nitrophenyl)-acetyl idiotype expression on spleen and thymus cells

Direct binding of ¹²⁵I-labeled rabbit anti-NP^b idiotype antibodies (RaId) was used to quantitate the expression by immune spleen and thymus cells of NP^bId, the characteristic Id of the λ1-containing antibodies made by C57BL/6 (B6) mice to the (4-hydroxy-3-nitrophenyl)acetyl (NP) group. Direct binding of RaId by B and T cell preparations reached a maximum of 12 ng RaId per 10⁸ cells at 7 days after immunization. Spleen T cell preparations maintained similar levels of binding after positive selection for Thy-1.2⁺ cells and overnight culture. RaId binding was also demonstrated for immune B6 thymus cells and for spleen and thymus cells of immune SJL mice, which have the appropriate heavy chain allotype for NP^bId expression but have only barely detectable serum Id. However, the NP^bId of T and B cell preparations were indistinguishable by (a) the susceptibility of RaId binding by the cells to inhibition by hapten or by antibodies to the variable regions of λ light chains (anti-V_λ) and by (b) the ability of anti-V_λ and of monoclonal antibodies to the constant region of λ1 chains (anti-C_λ1) to immunoprecipitate antigen (NP₁₀-bovine serum albumin)-binding proteins from detergent extracts of isotopically labeled cells. The results strongly imply that virtually all of the NP^bId of T cell preparations is due to conventional NP^bId antibody that is tightly bound to T cells. The results do not, however, exclude the possibility that the T cell preparations contain a trace amount (≤ 1 ng/10⁸ cells) of unusual NP^bId-like molecules that lack λ chains.     

The T/B cell interaction involved in induction of the mouse IgG2ah suppression is restricted by major histocompatibility complex class I,but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2a^b suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igh^a haplotype, when neonatally transferred into histocompatible Igh^a/b heterozygotes, are able to induce a specific and total suppression of the IgG2a^b allotype. Nevertheless, only transfer of IgG2a^b-primed Igh^a T splenocytes induces this suppression in Igh^b/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8⁺ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4⁺CD8⁺ T cell cooperation and operated via the recognition by the involved TCR of Cγ2a^b-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Igh^b mice genetically deficient for MHC class I (β2-microglobulin^%, or β2m^%) or class II (I-Aβ^%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2a^b T cells transferred into Igh^b H-2^b I-Aβ^% mice carry out the suppression process normally, while in Igh^b H-2^b β2m^% recipients, their suppression induction capacity is significantly inhibited. Moreover, the Cγ2a^b 103–118 peptide, identified as the sole Cγ2a^b-derived peptide able to amplify the anti-IgG2a^b T cell reactivity in Igh^a H-2^b mice, is also able to stabilize the H-2D^b, but not the H-2K^b class I molecules at the surface of RMA-S (TAP2^?, H-2^b) cells. These results indicate that, despite the CD4⁺/CD8⁺ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2a^b+ B cells for suppression establishment.     

Heterogeneity of Igh-linked allotypic determinants expressed on functional T cell subsets as detected by monoclonal antibodies

Hybridomas producing monoclonal antibodies (mAb) specific for immunoglobulin heavy chain (Igh) allotype-linked gene products expressed only on functional T cells but not on B cells and macrophages were established by fusion of allotype congenic SJL (Igh-1^b) and SJA/9 (Igh-1^a) B cells immunized reciprocally with partner spleen cells with a myeloma P3-X63-Ag8-653 of BALB/c origin. Nine mAb have been selected on the criteria that they can specifically block various antigen-dependent functions of known T cell subsets in in vitro immune responses of mouse strains having the corresponding Igh allotype, but not the other one. These included (a) four mAb that augment the in vitro secondary antibody response of either Igh-1^a or Igh-1^b strains and thus are considered to react with the Igh-linked allotypic determinant expressed on suppressor T cells, (b) one mAb that inhibits the helper T cell activity of Igh-1^b but not of Igh-1^a strains, (c) two mAb that inhibit the antigen-induced pro-liferative response of Igh-1^a but not Igh-1^b strains, and (d) two mAb that block the cytotoxicity of alloreactive cytotoxic T cells of Igh-1^a strains. The linkage to Igh-1 allotype of the T cell products was established by testing with Igh-1-congenic strains with different backgrounds including the H-2 complex. Some of the mAb were able to react with cloned hybridomas and a continuous cell line of the given allotype and functions. Each mAb was able to block one of the known functions of T cell subsets, but not others, indicating the existence of the heterogeneity and multiplicity of the Igh allotype-linked products on T cells.