Relationship between lectin binding properties and the expression of blood group ABH antigens in vascular endothelia and red blood cells from 18 primate species

Summary The reactivity was examined of horseradish peroxidase labelledUlex europaeus agglutinin-I (UEA-I) andGriffonia simplicifolia agglutinin I-B₄ (GSAI-B₄) with red blood cells and vascular endothelium in formalin-fixed, paraffin embedded tissues from 18 primate species. The expression of blood group ABH antigens in these cells as well as secretions from other tissues was also examined by the indirect immunoperoxidase method using monoclonal anti-ABH antibodies as primary antibodies. In Prosimians and New World monkeys which lack ABH antigens on both red blood cells and endothelial cells, but produce these antigens in other tissue secretions, GSAI-B₄ always reacted with both red blood cells and endothelial cells. In Old World monkeys, which express blood group antigens on endothelial cells but not on red blood cells, neither GSAI-B₄ nor UEA-I reactivity were observed, except the endothelial cells from blood group B or O individuals occasionally reacted with GSAI-B₄ or UEA-I, respectively. Although UEA-I reactivity was not observed in the endothelial cells of gibbon, it reacted with these cells from chimpanzees. In these two anthropoid apes, both endothelial cells and red blood cells expressed ABH antigens as in humans. These results suggest the close evolutionary relationship between the expression of blood group ABH antigens and lectin binding properties of red blood cells and endothelial cells in primate species.     

Immunization with recombinant HLA classes I and II, HIV-1 gp140, and SIV p27 elicits protection against heterologous SHIV infection in rhesus macaques

Major histocompatibility complex (MHC) molecules expressed on the surface of human immunodeficiency virus (HIV) are potential targets for neutralizing antibodies. Since MHC molecules are polymorphic, nonself MHC can also be immunogenic. We have used combinations of novel recombinant HLA class I and II and HIV/simian immunodeficiency virus (SIV) antigens, all linked to dextran, to investigate whether they can elicit protective immunity against heterologous simian/human immunodeficiency virus (SHIV) challenge in rhesus macaques. Three groups of animals were immunized with HLA (group 1, n = 8), trimeric YU2 HIV type 1 (HIV-1) gp140 and SIV p27 (HIV/SIV antigens; group 2, n = 8), or HLA plus HIV/SIV antigens (group 3, n = 8), all with Hsp70 and TiterMax Gold adjuvant. Another group (group 4, n = 6) received the same vaccine as group 3 without TiterMax Gold. Two of eight macaques in group 3 were completely protected against intravenous challenge with 18 50% animal infective doses (AID₅₀) of SHIV-SF162P4/C grown in human cells expressing HLA class I and II lineages represented in the vaccine, while the remaining six macaques showed decreased viral loads compared to those in unimmunized animals. Complement-dependent neutralizing activity in serum and high levels of anti-HLA antibodies were elicited in groups 1 and 3, and both were inversely correlated with the plasma viral load at 2 weeks postchallenge. Antibody-mediated protection was strongly supported by the fact that transfer of pooled serum from the two challenged but uninfected animals protected two naïve animals against repeated low-dose challenge with the same SHIV stock. This study demonstrates that immunization with recombinant HLA in combination with HIV-1 antigens might be developed into an alternative strategy for a future AIDS vaccine.     

In Vivo Transfer of cellular immunity to primates with transfer factor prepared from human or primate leucocytes

Dialyzable transfer factor (TF_d) prepared from a human donor and transfer factor (TF) from baboon whole cell lysates was administered to 3 species of nonhuman primates: baboons, cebus monkeys and marmosets. In vivo transfer was evaluated with in vivo skin test and in vitro blastogenic responses to multiple antigens. Transfer of cellular reactivity in all three nonhuman primate species was demonstrated with both human TF_d and baboon TF. A cumulative conversion rate of 45% for skin test responses and 65% for lymphocyte blastogenesis was demonstrated following human TF_d injection while conversion was 17% and 33% respectively following baboon TF. Specificity was supported by the absence of conversion to TF donor negative antigens. There were no signficant differences observed between the 3 recipient primate species.     

Monoclonal antibodies against two separate alloantigenic sites of HLA-1340

Two monoclonal antibodies (MB40.2 and MB40.3) which are highly specific for HLA-B40 and HLA-B7 were made. They appear to be directed against two separate alloantigenic sites of these HLA molecules. Semiquantitative analysis of the kinetics of antibody binding show that MB40.2 recognizes a site which shows a degree of cross-reactivity with B7 and is specific to some B40 molecules. This antibody also distinguishes between different molecules typed as B40. In contrast, MB40.3 recognizes an antigenic determinant which is less variable between B7 and B40 and more closely approximates a public antigen or common antigenic site. This study suggests that the introduction of monoclonal antibodies into MHC serology not only permits but demands a quantitative analysis of these complex systems of homologous but highly polymorphic molecules.Abbreviations used in this paper MHC major histocompatibility complex - IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - BSA bovine serum albumin - FCS fetal calf serum - RAM rabbit anti-mouse IgG F(ab)₂ fragments For convenience the terms HLA antigen and H-2 antigen will refer only to the ₂-microglobulin-associated, class I molecules coded for by the major histocompatibility complex of man and the mouse, respectively.     

Surface antigen changes during B-lymphocyte activation in primates

It is shown that B-cell-specific surface antigens are conserved on lymphocytes from phylogenetically distant primate species. Characterization of the expression of those antigens on the surface of simian B lymphocytes has led to two observations with important implications for human B-cell physiology. First, lectin stimulation in vitro or antigen stimulation in situ in lymph nodes drives a population of human B lymphocytes to express the B2 but not the B1 antigen on its surface. Second, under pathologic circumstances, this activated B cell can be found in the peripheral blood of monkeys. Thus, the “B2 only” cell defines an activated B lymphocyte whose presence may provide useful diagnostic information concerning pathologic processes.     

Correlation between classification of human urothelial cell lines and HLA-A,B,C expression

Summary Quantitative changes in major histocompatibility class I antigen expression in tumour cells are believed to affect the host immune response against the tumour. In tumourigenic (TGrIII) human urothelial cell lines the apparent loss of polymorphic HLA-A,B epitopes has previously been demonstrated. In the present study, 3 non-tumourigenic (TGrII) and 6 tumourigenic (TGrIII) human urothelial cell lines have been investigated for their quantitative expression of monomorphic HLA-A,B,C and B₂-microglobulin. Evidence is provided that an inverse correlation exists between tumourigenicity and HLA-A,B,C and B₂-microglobulin expression. Furthermore, treatment of the cells with neuraminidase partly restored the expression of monomorphic HLA-A,B,C suggesting that at least some of the observed quantitative differences could be due to masking of the membrane bound HLA antigens by sialic acid-containing glycoconjugates.     

An antiviral T-Cell clone defines a functional supertypic specificity shared by different HLA-DR molecules from DR2-short,DRw11, and DRwl3 Haplotypes

An influenza virus-specific HLA class IIrestricted human T4⁺ clone (Ij) allows us to define a new functional supertypic HLA class II specificity shared by three different haplotypes. Influenza A virus-infected antigen-presenting cells of these three haplotypes, HLA-DR2 short, DRw11, and DRw13, are able to stimulate Ij cells. The same precise viral specificity is seen in all three cases. Proliferation inhibition experiments using HLA-specific monoclonal antibodies demonstrate that HLA-DR products are involved in all cases. However, according to the DR specificity of the antigen-presenting cell, differential blockings by a series of DR-specific monoclonal antibodies suggest that the functional epitope is shared by different HLA DR molecules. This is confirmed by two-dimensional gel analysis of the HLA DR chains expressed in the three haplotypes.Abbreviations used in this paper APC antigen-presenting cell - EBV Epstein-Barr virus - HA hemagglutinin - HLA human leukocyte antigens - IL-2 interleukin-2 - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - mAb monoclonal antibody - PAGE polyacrylamide gel electrophoresis - PBM peripheral blood mononuclear cells - % RR relative response percent     

Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2K^k. In the absence of exogenous IL-2 the clones require the presence of Ia⁺, Thy-1⁻ accessory cells and of Thy-1⁺, Lyt-1⁺2⁻ cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A_αA_β and/or E_αE_β had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2⁺ cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E_αE_β when B10.A(4R) spleen cells, which do not express E_αE_β, or when B10.A(4R) accessory cells, which were reconstituted with (BALB/c X B10.A(4R)) F1 T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2K^d). These and previous results suggest that H-2K-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A_αA_β and E_αE_β molecules with syngeneic accessory cells. This interaction results in IL-2 production by the stimulator T cells and thus in the proliferation of the clones.     

HLA antibody responses in HLA class I transgenic mice

In a previous report we described how cross-immunizations of pairs of transgenic mice expressing different HLA class I antigens led to the production of antibodies directed exclusively at polymorphic epitopes. This was ascribed to self-tolerance of HLA that prevents immune responses to monomorphic epitopes and focuses responses on polymorphic ones. In the present report we extend our findings and demonstrate that immunizations of class I transgenic mice with HLA transfected mouse fibrosarcoma as well as with human lymphoblastoid cells also preferentially yield antibodies to polymorphic epitopes. This was the case whether or not immunizations were carried out across locus barriers [e.g., Tg (HLA-A *0201) or Tg (HLA-Cw*0301) transgenic mice immunized with HLA-B27 transfectants] or within the same locus [e.g., Tg (HLA-B*1302) transgenic mice immunized with HLA-B27 transfectants or B27-expressing lympho-blastoid cell]. Use of an extended immunization protocol with four or more booster injections favored antibodies of IgG isotype with affinities high enough to lyse normal peripheral blood lymphocytes (PBLs) in complement-dependent cytotoxicity assays and to immunoprecipitate HLA antigens. The specificities covered by the monoclonal antibodies (mAbs) could be either broad or narrow, depending on the genetic distance of the HLA antigens or alleles involved. For instance, a Tg(HLA-B*1302) transgenic mouse immunized with B27 produced both broad B7/B27-specific antibodies, Bw4-specific antibodies, and one antibody reacting with all B alleles except B13 and with some C alleles. On the other hand, a Tg(HLA-B*1302) transgenic mouse immunized with Bw47 transfectants responded narrowly with an antibody to Bw60 and Bw47. Thus it appears that by choosing appropriate recipient mice and closely related or more distant HLA antigens, antibodies of a programmed specificity can be generated. Address correspondence and offprint requests to: U. Hämmerling.     

Influence of HLA genotype on birth weight of patients with Turner syndrome

Summary Growth failure starting before birth is a common characteristic in Turner syndrome, and its pathogenesis is still not completely explained. Experiments performed in mice and rats to test whether a genetic disparity between mothers and offspring and maternal immunological status have any influence on litter size have demonstrated that allogenic litters are significantly larger in size than genetically compatible ones. Studies in humans have given contrasting results, but some authors have found that heterozygosity at enzyme loci and in blood groups is positively correlated with intrauterine growth. HLA class I and II polymorphisms were defined in 53 patients with Turner syndrome and in their parents, and lymphocytotoxic antibody detection was performed in 36 mothers. These data were related to the patients' birth weight. The frequency of the HLA-B16 allele in patients with a birth weight > 10th centile was significantly higher in comparison with those < 10th centile. HLA antigen sharing was present in 43 couples (81.1%). Mean birth weight was 2934 ± 472 g in patients without HLA antigen parental sharing and 2721 ± 529 g in those whose parents shared HLA antigens. The mean birth weight of the 10 patients whose parents do not share HLA antigens was significantly higher than that of the patients with parental HLA–B+DR sharing (P < 0.05) and not significantly higher than in those patients with parental HLA sharing at other HLA loci. Patients whose parents shared B+DR antigens also had significantly smaller birth weights than those with B and A+B+DR sharing (P < 0.025 and P < 0.025). No significant difference in mean birth weight was found in relation to other parameters, such as mother-child histocompatibility, HLA homozygosity and lymphocytotoxic production in the mothers.     

Receptor-like role of HLA-class I antigens: regulation of T cell activation

Class I major histocompatibility antigens are known to restrict the cytotoxic activity of T lymphocytes. However, experiments using monoclonal antibodies against class I antigens showed that these antigens also play some role in the regulation of T cell activation. Three monoclonal antibodies, namely W6/32 (anti-class I HLA-A, B, C, antigens), 4E (anti-class I HLA-B antigens), and BBM.1 (anti-beta 2-microglobulin) significantly suppressed the phytohemagglutinin-induced T cell proliferation. The inhibitory effect of anti-class I antibody was found to depend on the presence of monocyte/macrophage-type adherent cells. In the presence of antibody, adherent cells released a factor that suppressed T cell proliferation. These results suggest that HLA class I antigens on Mo1+ monocyte/macrophage cells function like ligand-receptor molecules, and regulate the secretion of suppressor factor(s).     

Erythrocyte Sensitization by Blood Group-Specific Bacterial Antigens

Human and chicken erythrocytes are readily coated in vitro by blood group active protein-lipopolysaccharides and lipopolysaccharides from E. coli O₈₆ and E. coli O₁₂₈. Serum albumin, α₂- and β-lipoproteins inhibit this sensitization. Blood group B specific agglutination of erythrocytes with B or B-like antigens was obtained with antibodies purified by adsorption on and elution from B erythrocytes. Anti-blood group B and E. coli O₈₆-specific antibodies could be eluted from E. coli O₈₆-coated O erythrocytes. Eel anti-H(O) serum agglutinated O erythrocytes and only those A₁B red cells which were coated with blood group H(O) active E. coli products. Blood group active substances specifically inhibited agglutination of lipopolysaccharide-coated erythrocytes by anti-B and anti-H(O) agglutinins. Demonstrable amounts of lipopolysaccharide could only be removed from coated erythrocytes by washing them at elevated temperatures (58°C) in physiological solutions. Red cell sensitization with B active E. coli O₈₆ substances was achieved in vivo in a minority of severely diseased infants and in germ-free and ordinary chicks which were in tourniquet shock after treatment with cathartics. Therefore, a possible mode by which erythrocytes of patients with severe intestinal disorders acquire antigens is the fixation of bacterial substances to their surfaces, if there are not enough of the normally interfering plasma factors present.     

Immune phagocytosis inhibition by commercial immunoglobulins

Sixteen commercially available immunoglobulins (Ig) and 5 anti-Rho (D) hyperimmune globulins were investigated for immune phagocytosis inhibition (IPI) factors as well as for T, B lymphocytotoxic and monocytotoxic antibodies. All Ig contained IPI factors with lowest inhibitory IgG concentrations ranging from 0.08 to 50 mg/ml. Pepsin-digested Ig was noninhibitory. IPI factors in anti-D preparations were uniformly high (inhibitory IgG concentrations 0.6-2.5 mg/ml). Cytotoxic antibodies against T, B lymphocytes and monocytes were found in 2,2 and 7 products, respectively. Since we have recently shown that IPI is caused by antibodies against major histocompatibility complex antigens, most likely HLA, the hypothesis is put forward that IPI factors in Ig are HLA-related, cytotoxic as well as noncytotoxic antibodies which act via Fc receptor blockade of human monocytes.     

Lateral diffusion of human histocompatibility antigens in isolated plasma membranes

We have prepared large (5–10 μm) plasma membrane fragments by lysis of VA-2, human, cells adherent to Sephadex beads. The membrane fragments may be removed from beads by sonication and stained with fluorescent antibodies to human histocompatibility antigens, HLA antigens. Lateral diffusion of labelled antigens is followed by the method of fluorescence photobleaching recovery (FPR). HLA antigens of isolated membranes diffuse at the same rate, approx. (2–4) · 10^?10 cm² · s^?1 as they do in intact cells. This rate may be modified by incubating membranes in a variety of media. Buffers of slightly acid pH (6.5 or less) enhance lateral diffusion, while the presence of divalent ions slightly reduces diffusion rates. Our major finding is that incubation of 37° in 0.10 M phosphate buffer increases lateral diffusion 3–5-fold.     

CDP7657, an anti-CD40L antibody lacking an Fc domain,inhibits CD40L-dependent immune responses without thrombotic complications: an in vivo study

IntroductionCD40 ligand (CD40L) blockade has demonstrated efficacy in experimental autoimmune models. However, clinical trials of hu5c8, an anti-human CD40L IgG₁ antibody, in systemic lupus erythematosus (SLE) were halted due to an increased incidence of thrombotic events. This study evaluated CDP7657, a high affinity PEGylated monovalent Fab'' anti-CD40L antibody fragment, to assess whether an Fc-deficient molecule retains efficacy while avoiding the increased risk of thrombotic events observed with hu5c8.MethodsThe potency and cross-reactivity of CDP7657 was assessed in in vitro assays employing human and non-human primate leukocytes, and the capacity of different antibody formats to activate platelets in vitro was assessed using aggregometry and dense granule release assays. Given the important role CD40L plays in regulating humoral immunity, in vivo efficacy was assessed by investigating the capacity of Cynomolgus monkeys to generate immune responses to the tetanus toxoid antigen while the potential to induce thrombotic events in vivo was evaluated after repeat dosing of antibodies to Rhesus monkeys. A PEGylated anti-mouse CD40L was generated to assess efficacy in the New Zealand Black/White (NZB/W) mouse model of SLE.ResultsCDP7657 dose-dependently inhibited antigen-specific immune responses to tetanus toxoid in Cynomolgus monkeys, and in contrast to hu5c8, there was no evidence of pulmonary thrombovasculopathy in Rhesus monkeys. Aglycosyl hu5c8, which lacks Fc receptor binding function, also failed to induce thrombotic events in Rhesus monkeys. In vitro experiments confirmed that antibody constructs lacking an Fc, including CDP7657, did not induce human or monkey platelet activation. A PEGylated monovalent Fab'' anti-mouse CD40L antibody also inhibited disease activity in the NZB/W mouse model of SLE after administration using a therapeutic dosing regimen where mice received antibodies only after they had displayed severe proteinuria.ConclusionsThese findings demonstrate for the first time that anti-CD40L antibodies lacking a functional Fc region do not induce thrombotic events in Rhesus monkeys and fail to activate platelets in vitro but, nevertheless retain pharmacological activity and support the investigation of CDP7657 as a potential therapy for systemic lupus erythematosus and other autoimmune diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0757-4) contains supplementary material, which is available to authorized users.     

Demonstration and isolation of murine melanoma-associated antigenic surface proteins

Melanomas are antigenic and induce the formation of antibodies in both syngeneic and xenogeneic species. The nature of melanoma-associated antigens remains problematic, however. We found that xenogeneic (goat) antiserum to the mouse (C57BL/6) B16 melanoma, following appropriate absorptions with nonmelanoma cells, showed specificity for melanoma-associated surface antigens of B16 and one other murine melanoma. The antibody to B16 did not react with histocompatibility antigens, mouse-specific xenoantigens, viral antigens or melanin. The IgG fraction of the goat antibody was cross-linked covalently to protein A-Sepharose using dimethylsuberimidate. This immunoadsorbent was used to isolate shed antigens from cultures in which B16 cells had been grown and from detergent extracts of biosynthetically labeled (³H-leucine) B16 cells. The immune-affinity purified antigen preparation contained two major components of apparent molecular weight 60,000 and 50,000 daltons as assessed by SDS-polyacrylamide gel electrophoresis. Immunization of rabbits with immune-affinity purified B16 antigens induced antibodies which bound specifically to B16 cells.     

Idiotypic network regulations of immune responses to HLA

The development of antiidiotypic autoimmunity with respect to HLA alloantigens provides an attractive explanation for the phenomenon of maternal tolerance to the fetus and for the tandem selection of two antagonistic traits, major histocompatibility complex polymorphism and alloreactivity. We have demonstrated that both T and B lymphocyte responses to allogeneic HLA antigens are subjected to feedback regulation by autologous antiidiotypic immunity. Idiotypic receptors for the alloantigen expressed by T lymphocytes induce antiidiotypic antibodies that are readily detectable in serum during pregnancy, and antiidiotypic T cells that can be revealed in the autologous mixed lymphocyte culture system. Such antiidiotypic T cells and antibodies inhibit specifically the alloimmune function of autologous T cell lines. Similarly, antiidiotypic antibodies (Ab2) to HLA antibody molecules (Ab1) block the binding of the latter to the immunizing HLA antigen. The prevalence of Ab2 over Ab1 during pregnancy may explain the maternal tolerance to the fetus.     

Probable crossing-over in theB blood group system of chickens

Erythrocytes of two chickens from among 1,206 hybrids obtained from a (CB x CC)F₁xWB cross reacted with antisera against B antigens of all three lines involved in the cross. The possibility that the two birds had not originated from these crosses was excluded. The irregularity observed in cock 744 is accounted for by assuming a recombinant chromosome, B^R1, transmitting part of the B1 and part of the B2 antigenic specificities. Inheritance of B antigens is assumed to be effected by alleles of at least two loci,B-F andB-G. We found that the alleles of theB-F locus determined the serologically defined (SD) and histocompatibility (H) antigens, and the alleles of theB-G locus determined only the SD antigens. The irregularity in theB system of cock 2,349 has not yet been satisfactorily explained.     

Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three V_H gene segments. Panels of somatically mutated antibodies encoded by a common V_H gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the V_H gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.