Quantitative Two-Dimensional Ultrastructural Distribution of Rho(D) Antigenic Sites on Human Erythrocyte Membranes*

A method is described for determining the two-dimensional distribution of specific antigens on cell surfaces, and is applied to the D antigen of the Rh antigenic system. Rh-positive human erythrocytes are allowed to react with purified (125)I-labeled human anti-Rh(o)(D) gamma-globulin antibodies, and the sensitized cells are then lysed at an air-water interface. The residual cell membranes are spread flat by surface forces, and are picked up on a carbon-strengthened collodion-coated electron microscope grid. The membranes are then stained with ferritin-conjugated goat antibodies directed against human gamma-globulins. Only Rh-positive cells sensitized with anti-Rh(o)(D) antibodies bind the ferritin-conjugated antihuman gamma-globulins. The ferritin particles are found in small clusters on the membrane surface, and the number of such clusters per unit area agrees with the number of (125)I-labeled anti-Rh(o)(D) antibodies bound per unit area. The Rh(o)(D) antigenic sites appear to be molecularly dispersed on the membrane surface, but in a random two dimensional array.     

Human monoclonal antibody against Rh(D) antigen: partial characterization of the Rh(D) polypeptide from human erythrocytes

A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29- kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS- PAGE. Amino acid composition indicated that the Rh(D) protein contained sulfhydryl groups which are essential for biological activity.     

Determination of the N-terminal sequence of human red cell Rh(D) polypeptide and demonstration that the Rh(D), (c), and (E) antigens are carried by distinct polypeptide chains

Human monoclonal antibodies (MoAbs) directed against the blood group Rh(D), (c), and (E) antigens produced by Epstein-Barr virus (EBV)- transformed lymphoblastoid cell lines have been used to characterize the Rh components of human red cell membranes and to determine whether the Rh(D), (c), and (E) epitopes are carried by distinct polypeptides. After immunoprecipitation with the anti-Rh(D) antibody and preparative gel electrophoresis, a homogenous preparation of the Rh(D) protein was obtained from two different erythrocyte samples (Blo and D--/D--), which have an increased density of Rh(D) antigen. Both preparations exhibited the same N-terminal sequence (S)-(S)-K-Y-P-R-S-V-R-R-(L)-L-P- L-X-A, indicating that different Rh(D)-positive red cells are carrying a similar Rh(D) protein. Comparative immunoprecipitation studies with the human monoclonal anti-Rh(D), (c), and (E) antibodies have also shown that Rh components from intact and papain-treated erythrocytes have similar apparent mol wt of 30 to 32 Kd and are buried in the lipid bilayer and are not readily available to the proteolytic enzyme. Further investigations by indirect affinity chromatography and one- dimensional peptide mapping of the Rh(D), (c), and (E) molecules immunopurified from a single red cell sample demonstrate that a common Rh haplotype encodes three distinct polypeptide chains carrying the Rh(D), (c), and (E) epitopes, respectively.     

Effectiveness of Four Intravenous IgG Preparations in Prevention of Phagocytosis of Anti-Rh-(D)-Coated Erythrocytes by Mononuclear Phagocytes

Four commercial intravenous immunoglobulin G (IGIV) preparations, Sandoglobulin (Sandoz), Intraglobin (Biotest), Gammonativ (Kabivitrum) and Gammaogard (Kabivitrum) showed percent inhibition of Fc receptor-mediated phagocytosis of anti-Rh(D)-coated erythrocytes as follows: 45±14 SD, 41±23 SD, 35±14 SD, 25±13 SD, respectively. Pre-incubation of Rh(D)-positive and antibody-coated Rh(D)-positive erythrocytes with IGIV did not lead to any inhibitory effects on phagocytosis. However, preincubation of mononuclear phagocytes with IGIV gave marked inhibition of phagocytosis of antibody-coated erythrocytes. In summary, all four IGIV preparations showed Fc receptor-mediated inhibition of phagocytosis of anti-Rh(D)-coated erythrocytes by mononuclear phagocytes.     

Lymphocyte-Mediated Antibody-Dependent Cytolysis: Role in Immune Hemolysis

Peripheral blood lymphocytes obtained from normal volunteers were capable of lysing Rh(D)-positive human erythrocytes in the presence of IgG anti-Rh(D) antibodies. The percent cytotoxicity produced by peripheral blood lymphocytes was approximately equivalent to that produced by unfractionated peripheral blood mononuclear cells. Neither peripheral blood mononuclear cells nor peripheral blood lymphocytes lysed Rh(D)-negative human erythrocytes in the presence of IgG anti-Rh(D) antibody.     

Expression and characterization of recombinant anti-Rh(D) antibodies on filamentous phage: a model system for isolating human red blood cell antibodies by repertoire cloning

The production of human anti-red blood cell (RBC) Igs in vitro from immunized individuals would greatly facilitate the genetic analysis of the human immune response to RBC antigens and also provide useful serologic reagents. Technical difficulties inherent in human B-cell immortalization have led to the development of molecular approaches that bypass the need for cell transformation. By cloning human Ig gene segments into bacterial expression vectors, libraries are created of filamentous phage particles displaying Fab fragments on their surfaces. Libraries have been screened with purified, soluble antigen and selected clones genetically manipulated in Escherichia coli to produce soluble Fab fragments. Our goal has been to adapt this technique to the study of RBC autoantibodies and alloantibodies that have specificities against unpurifiable membrane-bound antigens. To test the feasibility of this approach, two sets of phage were created, one set expressing a human anti-Rh(D) Ig and the other expressing a human antitetanus toxoid Ig. After verifying the presence of functional phage-displayed Fabs through biochemical, flow cytometric, and electron microscopic analyses, a model library was constructed comprising one anti-Rh(D)- expressing phage per 10(4) antitetanus toxoid-expressing phage. A method was developed for screening the library with intact Rh(D)- positive RBCs. After four rounds of panning, anti-Rh(D) specificity was enriched more than 10,000-fold to a final frequency of approximately 100%. Plasmid DNA derived from anti-Rh(D) phage was used to produce milligram quantities of soluble recombinant anti-Rh(D) Fabs purified by nitrogen cavitation and nickel-chelation affinity chromatography. The authenticity of the Fabs was confirmed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and immunoblotting, which showed bands with molecular weights of approximately 50 kD and 26 kD under nonreducing and reducing conditions, respectively. Binding of recombinant anti-Rh(D) Fabs to Rh(D)-positive RBCs was demonstrated by flow cytometry and by an agglutination assay. Our results suggest that repertoire cloning by cell-surface enrichment may have broad application to the study of the human immune response to erythroid antigens in addition to membrane-bound antigens present on other hematopoietic cells.     

Transformation of lymphocytes from immunized Rh(D)-negative subjects by Rh(D) isoantigen

Since immune memory in Rh(D)-negative isoimmunized subjects remains through life, even in the absence of measurable anti-Rh(D), we investigated the transformation of lymphocytes from such donors by Rh(D) antigen. The time lapse from the last stimulus was up to 13 years. Mononuclear cells from immunized women were stimulated by Rh(D)- positive erythrocyte stroma. Maximum transformation was observed on the sixth day of culture, with a stroma protein concentration of 8 micrograms/mL of culture medium. The stimulation index (SI) in cells from 11 immunized women was 6.8 +/- 3.1 (mean +/- SD), with a range from 3.1 to 15.0. In five different sets of control cultures, the SI ranged from 0.9 +/- 0.2 to 1.3 +/- 0.4. There was no overlap between stimulated and control cultures. No anti-D could be demonstrated in the serum of four of the 11 immunized cases studied. Also, transformation was observed in mononuclear cells from Rh(D)-negative immunized women with Rh(D)-positive erythrocytes. The findings demonstrate that lymphocytes from isoimmunized Rh(D)-negative subjects maintain the immune memory and are transformed in vitro by the Rh(D) isoantigen.     

A New Approach to the Diagnosis of Active Rho(D) Immunization in Passively Immunized Pregnant Women

An assay has been developed to distinguish active from passive Rh0(D) immunization in a patient who had recently received hyperimmune anti-Rh0(D) immunoglobulin therapy. Isolated peripheral B lymphocytes from a pregnant woman at 32 weeks gestation were co-cultured with Epstein-Barr virus in a liquid growth medium. After 7 days, anti-Rh0(D) antibodies produced in vitro by the transformed lymphocytes were detected in culture supernatants, thereby proving active immunization and indicating the potential of hemolytic disease of the newborn in the current pregnancy. This assay was also performed with peripheral B lymphocytes from three groups of individuals: mothers known to be Rh0(D) immunized and who recently delivered Rh-positive infants, women with longstanding Rh0(D) immunization, and women who were treated with anti-Rh0(D) globulin. In the first group, anti-Rh0(D) antibodies were again detected after in vitro viral stimulation. In the latter two groups, essentially no anti-Rh0(D) activity was detected.     

Polymorphism in the Mr 32,000 Rh protein purified from Rh(D)-positive and -negative erythrocytes.

A Mr 32,000 integral membrane protein has previously been identified on erythrocytes bearing the Rh(D) antigen and is thought to contain the antigenic variations responsible for the different Rh phenotypes. To study it on a biochemical level, a simple large-scale method was developed to purify the Mr 32,000 Rh protein from multiple units of Rh(D)-positive and -negative blood. Erythrocyte membrane vesicles were solubilized in NaDodSO4, and a tracer of immunoprecipitated 125I surface-labeled Rh protein was added. The Rh protein was purified to homogeneity by hydroxylapatite chromatography followed by preparative NaDodSO4/PAGE. Approximately 25 nmol of pure Rh protein was recovered from each unit of Rh(D)-positive and -negative blood. Rh protein purified from both Rh phenotypes appeared similar by one-dimensional NaDodSO4/PAGE, and the N-terminal amino acid sequences for the first 20 residues were identical. Rh proteins purified from Rh(D)-positive and -negative blood were compared by two-dimensional iodopeptide mapping after 125I-labeling and alpha-chymotrypsin digestion. The peptide maps were very similar; however, at least two additional iodopeptides were consistently noted in the Rh proteins purified from Rh(D)-positive erythrocytes. These data indicate that a similar core Rh protein (or group of related proteins) exists in both Rh(D)-positive and -negative erythrocytes, and the Rh proteins from erythrocytes with different Rh phenotypes contain distinct structural polymorphisms.     

Growth-promoting effect of Rh(D) antibody on human pancreatic islet cells

Context/Objective: Hyperinsulinism with islet cell hyperplasia is a frequent complication, of unknown cause, in hemolytic disease of the newborn, occurring in Rh(D)-positive infants of Rh-isoimmunized Rh(D)-negative mothers, but not in infants with other hemolytic disorders. We investigated the possibility that trans-placentally acquired anti-D Ig is the cause of both conditions. Design: Monolayer cultures of human islet cells were exposed to sera from Rh-isoimmunized mothers and newborns, where jaundice, hyperinsulinism, and hypoglycemia in the infant had ensued. Parallel cultures with anti-D, specific anti-D monoclonal antibodies, normal human Ig (15 mug/ml), and serum controls were also undertaken. Islet cell proliferation was determined by [(3)H]thymidine incorporation. Insulin storage and chronic and acute insulin secretion to glucose were analyzed by RIA. Rh(D) surface antigen expression was determined on islet cells by flow cytometric analysis. Results: Islet cell proliferation and insulin secretion were significantly greater in coculture with test sera (P < 0.01; n = 8) and with anti-D (P < 0.001; n = 8), compared with either controls or Ig. After 8 d of growth, the static incubation experiment showed a 3.5-fold response to glucose stimulus in all sera. Rh(D) antigen expression was detected on the islet cell surface by flow cytometry, and islet cell morphology was normal. Colocalization of the proliferation marker Ki67 with insulin by immunofluorescent staining further indicated that Rh(D) antibody promoted islet growth. Conclusions: The anti-Rh(D) islet cell proliferative effect generates neonatal hyperinsulinism in Rh isoimmunization. Anti-Rh(D) may have application for islet cell proliferation in diabetes mellitus treatment for Rh(D)-positive subjects. Further analysis is required.     

Differences between Rh(D) Negative Subjects in Response to Rh(D) Antigen

S ummary Further evidence is produced of the existence of two kinds of Rh negative subject: the first forms anti-Rh after a single injection although sometimes in such a low concentration that a clearance study with a small dose of Rh positive red cells is required to detect it; antibody usually becomes detectable serologically after a second, spaced injection; the second kind of Rh negative subject fails to form anti-Rh even after several injections and has a repeatedly normal survival of Rh positive red cells and may be incapable of becoming immunized to the Rh antigen. In the present series of 34 Rh negative subjects, approximately 50% became immunized following injections of Rh positive red cells; the number is not significantly different from the incidence of approximately 60% reported in other published series. Some further evidence is provided that the rate of clearance of Rh positive red cells is more sensitive than any serological test in indicating the presence of a low concentration of anti-Rh and that different examples of anti-Rh in presumably similar concentrations in the plasma produce very different rates of red cell clearance. Of seven subjects given at least two injections of D positive Bu^a-positive red cells, four formed anti-D, but only one formed anti-Bu^a.     

Evidence for four different polymerization sites involved in human fibrin formation.

The mechanism of association and the organization of human fibrin were studied by using affinity chromatography. Insolubilized fibrinogen, fibrin monomer, and crosslinked fibrin were used to localize the binding sites on fibrinogen and fibrin derivatives. Four different polymerization sites have been distinguished. A binding site ("a"), available without thrombin action, is present on the fibrinogen fragment D domain. The complementary ("A") is inoperative in fibrinogen and requires thrombin for activation; it is located on the fibrinogen NH2-terminal domain. A third polymerization site ("b") appears to be formed by the alignment of the fragment D domains on two fibrin monomer molecules upon polymerization; this site functions without thrombin mediation and the alignment is stabilized by the Factor XIIIa-catalyzed crosslink bonds. The "b" site is complementary to another thrombin-activated site ("B") on the fibrinogen NH2-terminal domain. The two thrombin activable sites, "A" and "B", are distinguishable, although they are located in the same fibrinogen domain.     

A Study of HL-A Types in Rh Haemolytic Disease of the Newborn

Abstract. The HL-A types of 288 women, immunised to the Rh(D) antigen as a result of pregnancy, have been compared to the HL-A types of 139 women who although they have had Rh(D)-positive children have not produced anti-Rh(D). There is an indication that an increased incidence of the HL-A3 antigen is associated with the immunised mothers. A study of the HL-A types of husbands and children of both immunised and non-immunised women did not suggest that their HL-A types played a significant role in anti-Rh(D) production. The frequencies of the HL-A antigens in each category studied have been tabulated for record purposes, as well as the frequencies in a random panel of persons living in the Newcastle environs. Full ABO, Rh and HL-A details of 50 immunised mothers and their families and of 58 non-immunised mothers and their families are available.     

The Detection and Quantification of Rh(D) Antigen Sites on Human Leukocytes

Radiolabeled eluates of human anti-D were used to measure the capacityof leukocytes to bind the D antibody in cell suspensions prepared from 16normal and 3 leukemic bloods from Rh(D) donors. The contamination of theleukocyte suspensions by D positive red cells was measured and the contribution of D antigen sites by these cells was estimated. After correction wasmade for the D antibody bound by the contaminant red cells, no specific binding of D antibody by Rh(D) leukocytes could be detected. Three pairs ofRh(D) and rh(d) leukocyte dilution curves of I¹³¹-labeled anti-D uptakewere compared with the uptake by D positive and D negative red cell dilutions. No significant differences among the D negative erythrocyte, the rh(d)leukocyte and the Rh(D) leukocyte curves were obtained. The results werecollated with previous serologic evidence concerning the presence of ABOand Rh antigens on human leukocytes.

Submitted on April 16, 1963 Accepted on June 7, 1963     

Decreased amount of the Rh antigen D in hereditary spherocytosis (HS)

This study was done to determine whether hereditary spherocytosis (HS) red blood cells (RBC) have decreased amounts of Rh antigens. Initially we studied the RBC of five members of one family, two of whom had HS. Using automated quantitative haemagglutination tests, we demonstrated that HS RBC agglutinated less with Rh antisera of four specificities than did normal RBC, indicating that Rh antigens are decreased on HS RBC. In this family, the strength of other blood group antigens on HS RBC was estimated by manual titres and agglutination scores. No appreciable differences in the agglutination of HS and normal RBC were observed with non-Rh antisera. To assess the strength of the D antigen more accurately, the number of D sites was quantitated on the RBC of 19 individuals with HS and 11 of their healthy relatives. HS RBC had 9209 +/- 4084 (mean +/- SD) D sites, whereas the normal RBC had 15 394 +/- 5763 D sites. These two means were significantly different (P less than 0.01). HS RBC were also compared to normal RBC of unrelated individuals who had the same Rh phenotype. These analyses showed that HS RBC had about half of the normal number of D sites. Our data indicate that HS red cells have decreased amount of the Rh antigen D and probably also of other Rh antigens.     

Normal Survival of Rh0 (D) Negative, LW (a+) Red Cells in a Patient with Allo-Anti LWa

The serum of an Rh0 (D)+ Caucasian female with a prior history of transfusions contained anti- LWa , reacting moderately with Rh0 (D)+ and weakly with Rh0 (D)- red cells at the antiglobulin phase. Since transfusions were required, Chromium (51Cr) survival studies and mononuclear phagocyte assays (MPA) were used to predict in vivo survival. The MPA value of 6% of the positive control predicted a low likelihood of clinically significant extravascular destruction and 51Cr survival studies indicated greater than 95% survival at 1 h and 78% survival at 20 h for rr erythrocytes. Transfusion of 4 units of Rh0 (D)-, serologically incompatible red cells increased the hemoglobin from 5.2 to 11.4 g/dl and the titer of anti- LWa against rr red cells from 4 to 1,024 7 days after the transfusion. A second 51Cr survival also indicated normal survival of rr erythrocytes. 3 additional Rh0 (D)- units were successfully transfused.     

Antibody produced against isolated Rh(D) polypeptide reacts with other Rh-related antigens

Rh(D) antigen-containing polypeptide was prepared by immune precipitation of intact cDE/cDE erythrocytes by using a high-titer preparation of polyclonal anti-D. when isolated Rh(D) polypeptide was administered to rabbits, antibody was produced that was unresponsive toward Rh-positive and -negative cells but reacted strongly with the immunogen in enzyme-linked immunosorbent assay-type immunobinding and Western blot immunostaining assays. Rabbit antibody also immunostains isolated Rh(c) polypeptide as well as the Rh antigen-containing components of sodium dodecyl sulfate-polyacrylamide gel electrophoresis- separated membrane proteins from Rh(D)-positive (cDE/cDE,CDe/CDe), Rh(D)-negative (cde/cde,Cde/Cde), and -D-/-D- cells. It does not react with any membrane protein from Rh-null regulator type cells, thus indicating a specificity for Rh-related proteins. We have also been able to demonstrate that polyclonal and monoclonal anti-D preparations that do not immunostain isolated Rh(D) polypeptide will react with it in our immunobinding assay.     

Human Monoclonal Antibodies to Rho(D)

B lymphocytes from Rh negative donors with serum anti-D antibodies were isolated and fused with the mouse-human heteromyeloma, SBC-H20, to produce hybridomas secreting IgM or IgG1 human monoclonal antibodies to D antigen. The IgM antibody in hybridoma supernatant agglutinates all normal D positive cells at the immediate spin phase of reactivity. Using concentrated IgM hybridoma supernatant of approximately 50 micrograms/ml, Du cells were also agglutinated. The IgG1 antibody reacts by indirect hemagglutination with all D and Du cells. Against Rh mosaics, different reactivity was noted for each antibody. Furthermore, D positive cells precoated with the IgG1 antibody inhibit the IgM direct hemagglutination, suggesting that the antibodies identify closely associated epitopes. These human monoclonal antibodies will be useful diagnostic reagents and, ultimately, should be useful in the prevention of Rh hemolytic disease of the newborn.     

Characteristics of Different Rhesus Alloantibodies in Lymphocyte Dependent (K Cell) Cytotoxicity to Human Erythrocytes

The property of rhesus alloantibodies to elicit antibody-dependent, cell-mediated cytotoxicity (ADCC) against target erythrocytes carrying various Rh genotypes was studied. The killer activity of normal peripheral lymphocytes on human erythrocyte target cells carrying the appropriate antigens elicited by alloantisera was measured by 51Cr release at 18 h. There was no correlation between ADCC and antibodies directed to the antigens present on the surface of different genotypes of Rh-positive red blood cells. The agglutinin titre of different Rh antibodies showed no correlation with the level of ADCC although the degree of cellular cytotoxicity was different with different anti-D sera. Anti-C + D+ E antibody caused higher ADCC than anti-C + D and the lowest cytotoxicity was observed with anti-D and anti-D+ E. This raised the possibility that ADCC was elicited by antibodies directed to other specificities. K cell lysis of human red cells by human peripheral blood lymphocytes in vitro suggests that a similar mechanism may operate in vitro in the destruction of erythrocytes coated by allo or autoantibodies.     

Quantitative Rh Typing of rGrG with Observations on the Nature of G (Rh12) and Anti-G

Abstract. Quantitative blood typing data on Mr. R. B. (r^Gr^G), his wife (R₁r), a daughter (R₁r^G), and a niece (r^Gr) strongly suggest that these Rh phenotypes are directly indicative of actual Rh genotypes. If so, the antigenic products of r^G are weak Rh2 (rh' or C), normal Rh5 (hr or e), an expression of Rh12 (rh^G) equal to that produced by R^-1,2,-3 (r' or dCe) and half of what is produced by R¹ (R or D) genes in either homozygous or heterozygous expression, very weak expression of Rh19 (hr^S), absence of Rh31 (hr^B), and slightly weakened expression of Rh17 (Hr_o or ‘not D’). The LW status of r^Gr^G cells was equivalent to that of other Rh:-1 erythrocytes. Thus r^G resembles mutant r' in which only Rh5 is expressed normally. The weak Rh2 produced by r^G reacted much better with one Rh2 antiserum than with another. Rh21 (C^G) had been used to denote such additional reactivity, but one reagent that acted as anti-Rh2 in manual tests behaved like anti-Rh21 in instrumented tests. Therefore, anti-Rh21 may only indicate a more efficiently agglutinating anti-Rh2. Mr. R. B. showed no evidence of congenital stomatocytic hemolytic anemia characteristic of Rh_null or Rh_mod. Finally, anti-Rh12 eluates, recovered either sequentially from r'r followed by R₂r or singly from r^Gr^G, agglutinated chimpanzee red cells more efficiently than did either anti-Rh1 (D) or anti-Rh4 (c), a result consistent with expectation for serological crossreactivity between Rh1 and Rh21.