Chemical camouflage of antigenic determinants: Stealth erythrocytes

Abstract

In a number of clinical circumstances it would be desirable to artificially conceal cellular antigenic determinants to permit survival of heterologous donor cells. A case in point is the problem encountered in transfusions of patients with rare blood types or chronically transfused patients who become allosensitized to minor blood group determinants. We have tested the possibility that chemical modification of the red blood cell (RBC) membrane might serve to occlude antigenic determinants, thereby minimizing transfusion reactions. To this end, we have covalently bound methoxy(polyethylene glycol) (mPEG) to the surface of mammalian RBC via cyanuric chloride coupling. Human RBC treated with this technique lose ABO blood group reactivity as assessed by solution-phase antisera agglutination. In accord with this, we also find a profound decrease in anti-blood group antibody binding. Furthermore, whereas human monocytes avidly phagocytose untreated sheep RBC, mPEG-derivatized sheep RBC are ineffectively phagocytosed. Surprisingly, human and mouse RBC appear unaffected by this covalent modification of the cell membrane. Thus, mPEG-treated RBC are morphologically normal, have normal osmotic fragility, and mPEG-derivatized murine RBC have normal in vivo survival, even following repeated infusions. Finally, in preliminary experiments, mPEG-modified sheep RBC intraperitoneally transfused into mice show significantly improved (up to 360-fold) survival when compared with untreated sheep RBC. We speculate that similar chemical camouf lage of intact cells may have significant clinical applications in both transfusion (e.g., allosensitization and autoimmune hemolytic disease) and transplantation (e.g., endothelial cells and pancreatic ␤ cells) medicine.The transfusion of red blood cells (RBC) is the most common, and best tolerated, form of tissue transplantation. Indeed, in 1993, over 14 million units of blood were donated for transfusion in the United States alone (1). In most transfusions, simple blood typing (ABO͞Rh-D) is sufficient to identify appropriate donors. Occasionally, however, appropriate donors for patients with rare blood types cannot quickly be found; a situation that may become life-threatening. More often, problems are encountered in individuals who receive chronic transfusions, such as patients with sickle cell anemia and thalassemia. In such patients, alloimmunization against minor RBC antigens can make it nearly impossible to identify appropriate blood donors (2-4). Previous studies in which purified proteins were covalently modified with poly(ethylene glycol) (PEG) provided a possible solution to this problem. PEG-modified proteins have been shown to have increased in vivo survival and to be nonimmunogenic, even with repeated infusions (5, 6). We therefore explored the hypothesis that the covalent binding of PEG to intact RBC might mask RBC surface antigens and thereby permit the survival of heterologous or even xenogeneic RBC.To experimentally test this hypothesis, human, mouse, and sheep RBC were...

Microfluidic analysis of cellular deformability of normal and oxidatively damaged red blood cells

“…The control and oxidized RBC were analyzed via the microfluidic device to obtain deformability profiles. For the in vivo survival studies, murine RBC were labeled using a fluorescent, membrane anchored marker, PKH-26 (Sigma, St. Louis, MO) [45,46]. Equal numbers of control or oxidized RBC (40% hematocrit) were transfused as previously described [46,47].…”

“…For the in vivo survival studies, murine RBC were labeled using a fluorescent, membrane anchored marker, PKH-26 (Sigma, St. Louis, MO) [45,46]. Equal numbers of control or oxidized RBC (40% hematocrit) were transfused as previously described [46,47]. Blood samples from the recipient mice were followed until the labeled RBC were cleared from circulation (approximately 40-50 days for allotransfusions).…”

“…Blood samples from the recipient mice were followed until the labeled RBC were cleared from circulation (approximately 40-50 days for allotransfusions). Survival of fluorescently labeled control and PMS-oxidized RBC was monitored by analyzing the percent of fluorescently labeled RBC by flow cytometry (FACSCalibur Flow Cytometer, BD Biosciences, San Jose, CA) [45,46]. To better correlate PMS-treatment with a known hematological abnormality, model murine ß thalassemic cells were prepared via osmotic lysis and resealing as previously described [3,17,48,49].…”

Immunocamouflage of latex surfaces by grafted methoxypoly(ethylene glycol) (mPEG): Proteomic analysis of plasma protein adsorption

“…Previous studies have shown that hydrophilic surfaces have decreased total protein adsorption and are also more biocompatible [12]. Thus, mPEG grafting generates a steric barrier while simultaneously biophysically camouflaging surface charge thus reducing immune recognition of implantable biomaterials, allogeneic red blood cells [5,[13][14][15][16][17][18], lymphocytes [3,4,[19][20][21] and viruses ( Figure 6BD) [22]. As shown in this study, consequent to PEGylation, latex particles demonstrated significant inhibition of protein adsorption in a polymer doseand length-dependent manner, as measured quantitatively with colorimetric protein assay and flow cytometry ( Figure 2A and B) and qualitatively by SDS-PAGE, fluorescent microscopy and iTRAQ-MS analysis (Figures 3-5).…”

“…A similar concept of immunomodulation can be applied to biological products. Immunocamouflage of cells and tissues refers to the biophysical and biological camouflage of cell surfaces from interaction with other cells, macromolecules and viruses [3][4][5][6]. This "biomaterials" approach for tissue engineering has emerged as a promising technique for preventing allorecognition of foreign cells, particularly in the context of transfusion and transplantation medicine.…”