Rabbit Anti-Human β2 Microglobulin Antibodies: A T-Cell Mitogen

Rabbit anti-beta 2 microglobulin antibodies have been described as a potent mitogen for human B lymphocytes. However, when fractionated after activation, only the T-cell enriched population is actively dividing. The induction of proliferation in purified T cells requires the presence of non-T cells. Daudi cells (which do not express beta 2 microglobulin on their cell surface) were shown to produce 'macrophage replacing factors' which supported anti-beta 2-induced T cell division. Non-T cells could be effectively replaced by addition of interleukin 2 containing cell supernatants, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or teleocidin B. Thus, anti-beta 2 appears to selectively activate T cells. In contrast to other T cell mitogens, anti-beta 2 microglobulin antibodies did not induce secondary immunoglobulin production in B cells from peripheral blood or spleen.     

The Genetic Control of HLA-A and B Antigens in Somatic Cell Hybrids: Requirement for β2 Microglobulin

The lymphoblastoid cell line Daudi lacks both HLA—A and B antigens and β₂ microglobulin. Somatic cell hybrids derived from a fusion between this line and D98/AH-2 were shown to express four HLA antigens not detectable on either parent cell, A1, A10(Aw26), Bw16(Bw38, Bw17. The initial definition by direct cytotoxicity assay was confirmed by absorption of reactions against target T lymphocytes, thus avoiding problems due to contaminating Ia antibodies, and by blocking the reactions by pretreatment with a chicken anti-human β₂ microglobulin serum. That the new specificities were due to the Daudi HLA region was confirmed by the finding that interspecific hybrids between Daudi and A9L, containing a single human chromosome 6, expressed A10 and Bw17. This also defined the haplotypes of Daudi as A10(Aw26), Bw17 and A1, Bw16(Bw38).
The re-expression of the Daudi HLA—A and B antigens in two independent sets of hybrids indicates that it does not carry a mutation in the HLA region. It has previously been reported that somatic cell hybrids with Daudi, which contain chromosome 15, do not express human β₂ microglobulin. These results suggest that the reason for the lack of HLA—A and B antigens on Daudi is a secondary effect due to the mutation(s) in the β₂ microglobulin gene.     

Quantitation of β2-Microglobulin and HLA on the Surface of Human Cells

Relative amounts of beta2-microglobulin (beta2m) and of HLA specificities were analysed on the surface of resting unfractionated peripheral human lymphocytes, enriched B and T cells, and on in-vivo- and in-vitro-stimulated lymphoblasts. Single-cell cytofluorometry and a very sensitive radioimmunoassay were used to determine as closely as possible the absolute amounts of membrane-bound beta2m/HLA antigens. B and T "resting" and "stimulated" lymphoid cells express very similar numbers of beta2m and HLA antigenic determinants, respectively, per unit of surface area when compared with each group, although beta2m was found to exist in two- to three-fold excess of HLA.     

Quantitation of β2-Microglobulin and HLA on the Surface of Human Cells

Beta2-microglobulin (beta2m) participates as an integral part in molecules of the major histocompatibility complex (MHC) type. Absence of beta2m makes the residual heavy MHC chain largely inactive as antigen. Striking reductions in the density per unit surface area of beta2m in seven out of nine malignant lymphoid tumour lines in comparison with normal lymphocytes or "immortalized" Epstein-Barr-virus-transformed lymphoblastoid lines were found is this study. This would seemingly represent a specific reduction in the ability of the malignant cells to express actively produced beta2m, since their HLA antigenic determinants were not reduced to the same extent and no indications were obtained suggesting that free beta2m could transfer from one cell to another. However, that beta2m is important in conveying serological specificities of MHC type to cells was shown by fusion of beta2m-negative and beta2m-positive cells, yielding hybrid cells with synergistically increased numbers of detectable, HLA-related determinants. Whether the reduction of beta2m on malignant versus nonmalignant lymphoid cells bears any relevance as to emergence of the malignant clones and resistance to possible anti-tumour reactions would now be an issue for study.     

Increased expression of collagen receptors: α1β1 and α2β1 integrins on blood eosinophils in bronchial asthma

BACKGROUND: Eosinophils are one of the major effector cells in bronchial asthma. Their infiltration of airways correlates with the asthma severity. Recruitment and activation of eosinophils are partially mediated by integrins alpha4beta1 and alpha4beta7. Collagens type I and IV constitute important components of extracellular matrix and vascular basement membrane, respectively. Therefore, collagen-binding integrins (alpha1beta1 and alpha2beta1) may also play a role in eosinophil lung infiltration. OBJECTIVE: To evaluate the possible presence of alpha1beta1 and alpha2beta1 integrins on peripheral blood eosinophils from asthmatic subjects. METHODS: Collagen receptors were studied on eosinophils separated by immunomagnetic CD16-negative method from healthy donors (n=13) and patients with moderate persistent atopic bronchial asthma (n=15). Surface receptor identification was performed by flow cytometry and cell adhesion assay. RESULTS: Eosinophils isolated from the patients showed increased expression of both alpha1beta1 and alpha2beta1 integrins as compared with healthy controls. Moreover, adhesive function of eosinophils to collagen type IV was inhibited by snake venom disintegrins: viperistatin and obtustatin. These disintegrins contain KTS active motif and are specific inhibitors of alpha1beta1 integrin. CONCLUSION: We demonstrated for the first time that collagen receptors: alpha1beta1 and alpha2beta1 integrins are overexpressed on the surface of peripheral blood eosinophils of asthmatic subjects. Further studies may reveal potential application of KTS-disintegrins or their structural analogs for therapy of bronchial asthma.     

Vaccination with β2-Microglobulin-Deficient Dendritic Cells Protects Against Growth of β2-Microglobulin-Deficient Tumours

Defects in cell surface expression of major histocompatibility complex class I antigen molecules are common in tumour cells. We have previously described the generation of adaptive immunity to tumour cells deficient in the transporter associated with antigen processing molecule. In this study, we demonstrate enhanced in vivo protection against growth of β₂-microglobulin-deficient tumour cells in syngeneic C57Bl/6 mice, following vaccination with β₂-microglobulin-deficient dendritic cells. In vitro analysis suggested that vaccinated mice produced CD3⁺ cells, which could induce apoptosis in syngeneic β₂-microglobulin-deficient tumour and non-malignant cells. Further investigation of target cell recognition suggested that also tumour cells lacking expression of classical major histocompatibility complex class I heavy chains and functional transporter associated with antigen processing molecules were recognized by CD3⁺ effector cells from vaccinated mice. Histopathological examination of organs from vaccinated mice showed no significant vaccination-induced pathology. The present findings point to a new possible strategy to counteract the growth of major histocompatibility complex class I-deficient tumour cells.     

Studies on the Cytophilic Properties of Human β2-Microglobulin

The mechanism by which exogenously added beta2m binds to lymphoid cells has been explored. In the mouse it has been shown that beta2m remains associated with plasma membrane macromolecules following solubilization with NP-40 and that approximately 25-30% of the binding could be accounted for by binding to H-2 antigens. No binding to mouse immunoglobulin or Ia antigens could be detected. The sites for binding of the remainder of the cell-bound beta2m were not determined. Whereas normal human lymphocytes showed little or no capacity to bind exogenously added beta2m, it was found that phytohaemagglutinin (PHA)-stimulated cells could bind beta2m. This binding occurred optimally 2 days after PHA stimulation. Approximately half of the binding could be accounted for by binding to HLA antigens. The possible significance of these findings with respect to cellular interactions involving major histocompatibility complex gene products in the immune response is discussed.     

Mitogenic Properties of Fab and F(ab'')2 Fragments of Rabbit Anti-Human β2-Microglobulin for Human Lymphocytes

Bivalent F(ab')2 fragments and monovalent Fab fragments of rabbit anti-human beta2-microglobulin (anti-beta2m) stimulated DNA synthesis in human lymphocytes. Mitogenicity of anti-beta2m antibodies can therefore be ascribed to the antigen-binding site and not to the Fc portion of the molecule. The mitogenic response to F(ab')2, and sometimes Fab, fragments of anti-beta2m IgG was comparable to that obtained with original IgG antibodies when tested at the same protein concentration. Since Fab monomers of anti-beta2m can cause lymphocyte activation, 'cross-linking' of hypothetical beta2-microblobulin-containing lymphocyte receptors does not seem necessary for activation. F(ab')2, as well as Fab, fragments of anti-beta2m blocked the cytotoxic effect of anti-beta2m IgG, showing that the fragments did indeed react with beta2-microblobulin on the cell surface. F(ab')2 dimers, but not Fab monomers, of anti-beta2m were capable of inhibiting the cytotoxic effect of an anti-HLA-A2 antiserum. The mitogenic activity of both anti-beta2m IgG and Fab monomers of such antibodies disappeared after absorption with highly purified beta2-microblobulin. The mitogenic effect of anti-beta2m IgG was inhibited to a minor extent by exposure of cells to high concentrations of pooled multispecific anti-HLA antibodies. This effect was probably nonspecific.     

Affinity Chromatography of β2-Microglobulin from Human Lymphocytes on Concanavalin-A-Sepharose

Beta2-microglobulin was extracted from human lymphocytes with nonionic detergent and separated by affinity chromatography on concanavalin-A-Sepharose. The retarded part of beta2-microglobulin is assumed to be associated with HLA antigens. Using a radioimmunoassay for beta2-microglobulin, the average number of presumably free beta2-microglobulin molecules and of presumably HLA-associated beta2-microglobulin molecules per lymphocyte was estimated to be 3.8 x 10(5) and 1.4 x 10(5), respectively.     

Serial Measurements of Pregnancy-specific β1-glycoprotein (β1SP1) in Patients with Trophoblastic Disease

A radioimmunoassay for pregnancy-specific-β₁-glycoprotein (Bohn. 1971) has been established with a limit of detection of 2 μMg/litre in serum. The assay has been used to measure serial levels of pregnancy-specific-β₁-glycoprotein (β₁SP₁) in the serum of patients with choriocarcinoma and teratoma for comparison with measurements of the β-subunit of human chorionic gonadotrophin. The value of the assay for β₁SP₁, in the management of these patients is discussed.     

Primate HLA Antibodies are Specifically Mitogenic for Human Lymphocytes and Act Synergistically with β2m Antibodies

A monkey antiserum against HLA-A28 was raised by immunization with papain-solubilized antigens. The titre of this serum was 1/370 for beta2-microglobulin (beta2m) 1/750 for A28 and 1/200 for the cross-reacting HLA-A2 determinant. This serum was highly mitogenic for human blood lymphocytes. Mitogenicity was reduced or abolished after absorption of the serum on a beta2m-spharose column. Column-eluted anti-beta2m antibodies were not mitogenic. However, mitogenicity was restored by reconstitution with unabsorbed and absorbed antibodies. Mitogenicity was target cell specific. Tuhs, cells from all individuals were activated by the unabsorbed serum. The absorbed anti-A28 serum was mitogenic only for target cells carrying A28 and/or the cross-reacting determinant A2, but not for cells lacking either of these antigens. A definite gene-dose effect was recorded, since target cells homozygous for A2 responded better to polyclonal activation by the primate serum than cells which were heterozygous.     

B-Cell Mitogenic Effects on Human Lymphocytes of Rabbit Anti-Human β2-Microglobulin

Rabbit Ig against human beta2-microglobulin was found to be mitogenic for human peripheral lymphocytes, tonsil lymphocytes, and appendix and spleen cells. Anti-beta2-m gave increased DNA synthesis, with peak responses on day 3 or 4 and showed a dose-response effect when diluted. The effect was seen in both serum-free and serum-containing culture medium. Anti-beta2-m, as well as lipopolysaccharide, induced polyclonal antibody production and intracellular immunoglobulin synthesis in blast cells, which is taken as evidence that these substances are human B-cell mitogens. Anti-beta2-m, but not lipopolysaccharide, could, in these experiments, activate peripheral blood lymphocytes, in addition to lymphoid cells from other sources. Thus, anti-beta2-m can serve as a functional marker for peripheral human B lymphocytes.     

Binding of β2-Microglobulin by Heterologous Sera

Purified human, rat or guinea-pig beta 2-microglobulin (beta 2m) was mixed with sera from guinea-pig, rat, mouse, rabbit, horse, goat, cow, rhesus monkey or man. The mixtures were incubated at 37 degrees C for various lengths of time. When the sera were separated by gel-chromatography on Sephadex G-200, beta 2m was traced not only in 'free' form but also in fractions with higher molecular weights. Evidence is presented suggesting that heterologous beta 2m binds to beta 2m-containing molecules in sera by exchange with the homologous counterpart.