Gregory R. Halverson

Isolation and functional characterization of human erythroblasts at distinct stages: implications for understanding of normal and disordered erythropoiesis in vivo

Terminal erythroid differentiation starts from morphologically recognizable proerythroblasts that proliferate and differentiate to generate red cells. Although this process has been extensively studied in mice, its characterization in humans is limited. By examining the dynamic changes of expression of membrane proteins during in vitro human terminal erythroid differentiation, we identified band 3 and α4 integrin as optimal surface markers for isolating 5 morphologically distinct populations at successive developmental stages. Functional analysis revealed that these purified cell populations have distinct mitotic capacity. Use of band 3 and α4 integrin enabled us to isolate erythroblasts at specific developmental stages from primary human bone marrow. The ratio of erythroblasts at successive stages followed the predicted 1:2:4:8:16 pattern. In contrast, bone marrows from myelodysplastic syndrome patients exhibited altered terminal erythroid differentiation profiles. Thus, our findings not only provide new insights into the genesis of the red cell membrane during human terminal erythroid differentiation but also offer a means of isolating and quantifying each developmental stage during terminal erythropoiesis in vivo. Our findings should facilitate a comprehensive cellular and molecular characterization of each specific developmental stage of human erythroblasts and should provide a powerful means of identifying stage-specific defects in diseases associated with pathological erythropoiesis.

A human monoclonal anti‐D antibody which detects a nonconformation‐dependent epitope on the RhD protein by immunoblot

We describe the first human monoclonal anti‐D (LOR‐15C9) which reacts with a D‐specific motif exposed either on a native form on intact D‐positive red cells or on a denatured form of the RhD protein (33 kD), and detected by immunoblotting. LOR‐15C9 was able to precipitate RhD but not RhcE proteins produced by in vitro transcription‐translation assays. The reactivity of the antibody, using panels of red cells with various partial D phenotypes known to lack some D epitopes and corresponding in RHD gene variants, suggested that LOR‐15C9 reactivity depends on the portion of the RhD polypeptide encoded by the exon 7 (amino acids 314–358). These findings correlate well with the reactivity of LOR‐15C9 with erythrocytes of some nonhuman primates (Dgor‐positive gorillas), but not of chimpanzee and Old or New World monkeys.

Molecular basis for Rhnull syndrome: Identification of three new missense mutations in the Rh50 glycoprotein gene

Rhnull is a rare autosomal recessive disorder characterized by an absence of Rh antigens and a varying degree of hemolytic anemia and spherostomatocytosis. We report studies of two Japanese Rhnull cases and describe three new missense mutations of RHAG, the locus that encodes Rh50 glycoprotein and modulates Rh antigen expression. In Rhnull(HT), RHAG harbored in exon 6 two G→A transitions, GTT→ATT and GGA→AGA, which cause Val270→Ile and Gly280→Arg substitutions, respectively. These missense mutations were cotransmitted from the propositus to the children and were predicted to reside in endoloop 5 and transmembrane (TM) segment 9, respectively. In Rhnull(WO), RHAG contained in exon 9 a single G→T transversion, GGT→GTT, which caused a Gly380→Val missense change in TM12 segment. The G→T transversion, which is located at the +1 position of exon 9, had also affected pre‐mRNA splicing and caused partial exon skipping. Although both Rhnull cases had a structurally normal RH antigen locus, hemagglutination and immunoblotting showed no expression of Rh antigens or proteins. These results correlate each mutation with a structural defect in the respective TM domain of Rh50 glycoprotein. Am. J. Hematol. 62:25–32, 1999.

The production of red blood cell alloantibodies in mice transfused with blood from transgenic Fyb‐expressing mice

BACKGROUND: A murine model would be useful to identify which immune mechanisms could be manipulated to treat or prevent red blood cell (RBC) alloimmunization in patients who become sensitized to multiple or widely expressed antigens.

Cytoskeletal protein 4.1R negatively regulates T-cell activation by inhibiting the phosphorylation of LAT

Protein 4.1R (4.1R) was first identified in red cells where it plays an important role in maintaining mechanical stability of red cell membrane. 4.1R has also been shown to be expressed in T cells, but its function has been unclear. In the present study, we use 4.1R-deficient mice to explore the role of 4.1R in T cells. We show that 4.1R is recruited to the immunologic synapse after T cell-antigen receptor (TCR) stimulation. We show further that CD4+ T cells of 4.1R-/- mice are hyperactivated and that they displayed hyperproliferation and increased production of interleukin-2 (IL-2) and interferon gamma (IFNgamma). The hyperactivation results from enhanced phosphorylation of LAT and its downstream signaling molecule ERK. The 4.1R exerts its effect by binding directly to LAT, and thereby inhibiting its phosphorylation by ZAP-70. Moreover, mice deficient in 4.1R display an elevated humoral response to immunization with T cell-dependent antigen. Thus, we have defined a hitherto unrecognized role for 4.1R in negatively regulating T-cell activation by modulating intracellular signal transduction.

The Fya, Fy6 and Fy3 epitopes of the Duffy blood group system recognized by new monoclonal antibodies: identification of a linear Fy3 epitope.

Four new anti‐Duffy murine monoclonal antibodies (MAbs): two anti‐Fy6 (MIMA‐107 and MIMA‐108), one anti‐Fya (MIMA‐19) and one anti‐Fy3 (MIMA‐29) were characterized. Identification of epitopes by means of synthetic peptides (Pepscan) showed that the anti‐Fy6 reacted most strongly with peptides containing the sequence 19QLDFEDV25 of the Duffy glycoprotein, and less strongly with peptides containing LDFEDV (MIMA‐107) or LDF only (MIMA‐108). The anti‐Fya recognized epitope 38DGDYGA43 containing the Gly42 residue, which defines the Fya blood group antigen. MIMA‐29 is the first anti‐Fy3 reactive with a linear epitope 281ALDLL285 located in the fourth extracellular domain (ECD4, loop 3) of the Duffy glycoprotein. The four new antibodies extend the list of six anti‐Fy MAbs formerly characterized by Pepscan analysis that allow some general conclusions. Fine specificities of various anti‐Fya, or anti‐Fy6 are not identical, but all of them recognize linear epitopes located around, respectively, Gly42 or between two potential N‐glycosylation sites at Asn16 and Asn27. Anti‐Fy3 recognize either a linear epitope located in ECD4, or a conformational epitope that includes amino acid residues of ECD4 and of other ECDs.

A DNA‐based immunization protocol to produce monoclonal antibodies to blood group antigens

A major challenge facing transfusion medicine is the establishment of immunological methods to produce specific and avid blood group typing reagents to the many polymorphic blood group antigens. This is especially true when sources of human antibody are limited. Based on the knowledge that inoculation with plasmid DNA can induce a humoral response in the host animal, we inoculated mice with plasmid DNA followed by a single boost injection with plasmid‐transfected cells that have a high level of expression of the same target protein. Using this method, several hybridoma clones that produced strongly reactive antibodies specific for the Kell polymorphic antigens (anti‐K, anti‐k, anti‐Kpa) were isolated. The monoclonal antibodies that were produced with this method have potential clinical utility for identifying a patients blood type and for screening for antigen‐negative donor blood.

Immunization of transgenic mice for production of MoAbs directed at polymorphic blood group antigens

BACKGROUND: Antibodies of human origin for blood typing are increasingly difficult to obtain, and, despite aggressive efforts, MoAbs with specificities to several blood group polymorphisms have eluded production. As an approach for the generation of MoAbs with defined specificities, the feasibility of immunizing mice that are transgenic for the target polymorphism, Fya/Fyb of the Duffy blood group system, was tested with a source of the antithetical antigen.

Direct evidence for the existence of Miltenbergera antigen

The Miltenberger (Mi) subsystem, which originally consisted of four phenotypes, now has 11 phenotypes. The antigens of this subsystem belong to the MNS blood group system. The Mia antigen has been reported to be present on red blood cells with several Miltenberger phenotypes, namely: Mi.I, Mi.II, Mi.III, Mi.IV, Mi.VI and Mi.X. However, the existence of the Mia antigen as a separate entity has been in question and difficult to prove with polyclonal reagents. We report the first monoclonal anti‐Mia (GAMA210), whose epitope is TNDKHKRD or QTNDMHKR, and thereby confirm the existence of the Mia antigen.

A DOB allele encoding an amino acid substitution (Phe62Ser) resulting in a Dombrock null phenotype

BACKGROUND: The gene polymorphisms responsible for the antigens Doa, Dob, Hy, and Joa in the Dombrock (Do) blood group system have been identified. Four different mutations have been reported to cause the Dombrock null [Gy(a–)] phenotype. These include splice mutations, an eight‐nucleotide deletion, and insertion of a stop codon. Here a Dombrock null caused by a single‐amino‐acid substitution in the full‐length protein is reported.