Generation of transgenic mice with antithetical KEL1 and KEL2 human blood group antigens on red blood cells

Abstract

BACKGROUND KEL1, also known as “K,” is one of the most immunogenic red blood cell (RBC) antigens. KEL2, also known as “k,” differs from KEL1 by a single amino acid. Anti-Kell system antibodies can lead to significant adverse clinical outcomes in humans, including hemolytic complications in alloimmunized transfusion recipients or in infants of alloimmunized mothers. To provide a platform for in-depth immunologic studies of alloimmunization and subsequent sequelae, we generated transgenic mice expressing the human KEL1 or KEL2 antigens. STUDY DESIGN AND METHODS Vectors were created in which cDNAs encoding either KEL1 or KEL2 were regulated by an erythroid specific β-globin promoter and enhancer. Pronuclear microinjections were carried out into a C57BL6 background, and founder pups were identified by polymerase chain reaction and screened for expression by flow cytometry. RBC life span and antigen stability were assessed by dye labeling RBCs, transfusing into agammaglobulinemic (μMT) recipients, and tracking by flow cytometry. RESULTS The expression of either KEL1 or KEL2 is RBC specific and first occurs on early RBC precursors. Both KEL1 and KEL2 RBCs have a normal circulatory life span and stable antigen expression. Expression of KEL1 or KEL2 does not result in altered levels of murine Kell, and resulting RBCs have normal hematologic variables. CONCLUSION The KEL1 and KEL2 mice represent the first murine system of RBC immunity with antithetical antigens, allowing a more precise modeling of human RBC immunology in general and also a platform for development of novel therapeutics to prevent or minimize the dangers of RBC alloimmunization to the KEL1 and KEL2 antigens in particular.

Alloantibodies to a paternally derived RBC KEL antigen lead to hemolytic disease of the fetus/newborn in a murine model

“…After engraftment and after confirming a lack of circulating KEL RBCs, organ reverse transcription (transcriptase)-PCR was completed. As expected, the KEL transgene, which is driven by the human Bglobin promoter cassette, 19,26 was RBC-specific with a lack of KEL expression on the organs or in the bone marrow of transplanted animals.…”

“…We have recently described a mouse model in which transgenic animals have RBC specific expression of the clinically significant human KEL glycoprotein antigen. 19 Herein, we demonstrate that paternally derived KEL antigens on fetal RBCs lead to maternal anti-KEL RBC alloimmunization through pregnancy/ delivery, with subsequent pregnancies and transfusions being adversely affected by these boostable anti-KEL glycoprotein alloantibodies. This model thus lays the groundwork to investigate pregnancyassociated alloantibody formation and subsequent RBC clearance mechanisms, as well as targeted therapeutic strategies to prevent or mitigate the dangers of pregnancy-associated alloantibodies.…”

“…Mice transgenic for the KEL human RBC antigen (specifically the KEL2 or Cellano antigen with mid-levels of expression near 1300 copies/ RBC, previously described as "KEL2B") were generated as previously described, 19 using a B-globin promoter. C57BL/6 mice were purchased from the National Cancer Institute (Fredricksburg, MD); MuMT, ubiquitin C green fluorescence protein (uGFP), and triple congenic IgHa (IgHa 3 Thy1.1 3 GPI) mice were purchased from The Jackson Laboratory (Bar Harbor, ME).…”

“…The KEL transgenic animals were made using a B-globin specific promoter, 19 and thus expression of the KEL antigen would be predicted to be RBC specific. This B-globin promoter has been shown to be expressed on approximately day 14 26 ; thus, KEL expression was investigated on RBC precursors from postconception (p.c.)…”

IgG-Mediated Immune Suppression to Erythrocytes by Polyclonal Antibodies Can Occur in the Absence of Activating or Inhibitory Fcγ Receptors in a Full Mouse Model

“…These models include human blood group Ags (glycophorin A, Fy b , or Kell) (31)(32)(33) and also model Ags that facilitate the analysis of immune mechanisms (mHEL or HOD) (34,35). HOD transgenic mice contain an RBC-specific recombinant protein composed of the hen egg lysozyme (HEL) in tandem sequence with chicken OVA and the human transmembrane Duffy Ag (34).…”