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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-07-2151. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-07-2151v1 101/6/2318    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ogawa, H. Articles by Galili, U. Search for Related Content PubMed PubMed Citation Articles by Ogawa, H. Articles by Galili, U. Related Collections Immunobiology Transplantation Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 March 2003, Vol. 101, No. 6, pp. 2318-2320 IMMUNOBIOLOGY Brief report Tolerance induction to a mammalian blood group-like carbohydrate antigen by syngeneic lymphocytes expressing the antigen Haruko Ogawa, Deng-Ping Yin, Jikun Shen, and Uri Galili From the Departments of Cardiovascular-Thoracic Surgery, Surgery and Immunology, Rush University, Chicago, IL.     Abstract Top Abstract Introduction Study design Results and discussion References Tolerance induction to transplantation-associated carbohydrate antigens, such as blood group A or B and the -gal epitope (Gal1-3Gal1-4GlcNAc-R), is of clinical significance. This study demonstrates tolerance induction to the -gal epitope in the experimental animal model of 1,3galactosyltransferanse knockout mice (KO mice) lacking -gal epitopes by administering syngeneic lymphocytes expressing -gal epitopes. Repeated immunization of control KO mice with pig kidney membranes (PKM) expressing many -gal epitopes induces an extensive anti-Gal antibody response against this epitope. In contrast, KO mice that received as few as 2 × 106 wild-type (WT) lymphocytes were tolerized and failed to produce anti-Gal following PKM immunizations. Accordingly, control mice producing anti-Gal rapidly rejected transplanted WT hearts, whereas tolerized mice did not reject WT hearts. These findings suggest that autologous blood lymphocytes processed to express a carbohydrate antigen may induce a similar tolerance to such an antigen upon administration into humans. (Blood. 2003;101:2318-2320) © 2003 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References The tolerance mechanism to mammalian carbohydrate antigens, such as ABO blood group antigens, is poorly understood. Understanding this tolerance may lead to novel methods for the induction of tolerance to incompatible carbohydrate antigens in transplantation. Carbohydrate antigens on glycoproteins differ from peptide antigens in that they cannot activate T cells directly because of their protrusion from the major histocompatibility complex (MHC) groove.1 Therefore, incompatible carbohydrate antigens on syngeneic cells cannot activate T cells.2 In the present study we determined in 1,3galactosyltransferase (1,3GT) knockout mice (KO mice) whether syngeneic lymphocytes expressing an incompatible carbohydrate antigen can affect the immune response to that antigen. KO mice are syngeneic to the C57BL/6 strain but lack the -gal epitope (Gal1-3Gal1-4GlcNAc-R).3 This epitope, which resembles the structure of blood groups A and B,4 is produced in wild-type (WT) mice and in other nonprimate mammals and New World monkeys by 1,3GT.5 Humans, apes, and Old World monkeys lack -gal epitopes and produce the natural anti-Gal antibody against it.4-8 The interaction between anti-Gal and -gal epitopes prevents xenotransplantation of pig organs in humans and monkeys.9-12 Lymphocytes of the C57BL/6 WT mice differ from lymphocytes of KO mice only in that they express ~1.5 × 105 -gal epitopes/cell.13 Our study indicates that WT lymphocytes, introduced into KO mice, tolerize these mice to the -gal epitope.     Study design Top Abstract Introduction Study design Results and discussion References Immunization and antibody measurements KO mice3 on H-2b background received syngeneic C57BL/6 WT spleen lymphocytes in the tail vein and after 14 days were immunized intraperitoneally with 50 mg pig kidney membrane (PKM) homogenates.2 PKM immunization was repeated 3 additional times at one-week intervals, and anti-Gal IgG production was measured by enzyme-linked immunosorbent assay (ELISA) one week later, using -gal epitopes linked to bovine serum albumin (BSA) (-gal-BSA; Dextra, Reading, United Kingdom) as previously described.2 Anti-Gal B cells secreting the antibody in the spleen were identified by enzyme-linked immunospot assay (ELISPOT) in wells coated with -gal-BSA, as previously described.14 Heart transplantation KO mice received heterotopical transplants of C57BL/6 mouse WT hearts in the abdominal cavity. The WT pulmonary artery was connected to KO inferior vena cava and WT aorta to KO aorta. Heart function was assessed by palpation. WT hearts grafted into tolerized KO mice were removed 2 months after transplantation and subjected to immunohistology studies with peroxidase-conjugated antibodies to mouse IgM, IgG, C3, and C5 (Pharmingen, San Diego, CA). Identification of WT lymphocytes WT lymphocytes were identified by flow cytometry after binding of fluoresceinated Bandeiraea (Griffonia) simplicifolia IB4 (fluoroscein isothiocyanate [FITC]-BS lectin) to -gal epitopes on these cells.5,8     Results and discussion Top Abstract Introduction Study design Results and discussion References KO mice produce no detectable amounts of natural anti-Gal IgG.2 However, 4 PKM immunizations resulted in effective stimulation of anti-Gal B cells by the -gal epitopes on pig membranes,15 resulting in extensive anti-Gal production in the control group (Figure 1A). Mice in experimental groups received intravenously 50 × 106, 20 × 106, or 2 × 106 live C57BL/6 WT spleen lymphocytes (ie, syngeneic lymphocytes expressing -gal epitopes). Starting 14 days later, the mice received 4 weekly PKM immunizations. All mice receiving WT lymphocytes, even at the low number of 2 × 106 per mouse, were tolerized to the -gal epitope, as indicated by the complete lack of anti-Gal after 4 PKM immunizations (Figure 1A [data for recipients of 50 × 106 WT lymphocytes not shown]). This tolerance was found to be specific to anti-Gal B cells, since antibodies to pig peptide antigens were produced at similar titers in control and experimental mice (not shown). View larger version (21K): [in this window] [in a new window]   Figure 1. Induction of tolerance to -gal epitopes by WT lymphocytes. (A) Production of anti-Gal IgG in KO mice immunized 4 times with pig kidney membranes (PKM) () and in KO mice that received 20 × 106 () or 2 × 106 () WT lymphocytes prior to the 4 PKM immunizations. Data are from 8 mice in the first group, 8 in the second group, and 4 in the third group. Mean of data in the () group is presented as (). In the () and () groups no mean is presented since all mice displayed a complete lack of anti-Gal response. Statistical analysis by t tests indicated P < .05 between tolerized and control mice in all points. (B) ELISPOT analysis of anti-Gal secretion by spleen lymphocytes obtained from KO mice receiving 2 × 106 WT lymphocytes and immunized 4 times with PKM () (mean presented as ), or from control mice undergoing the same PKM immunization (). Mean presented as . Data are from 4 mice in each group. Statistical analysis by t tests indicated P < .05 between tolerized and control mice in all points. Tolerized mice lack anti-Gal B cells that secrete the antibody, as demonstrated by an ELISPOT assay developed with anti-mouse IgM and anti-mouse IgG (Figure 1B). In contrast, lymphocytes from PKM-immunized control mice displayed many specific anti-Gal spots, implying the presence of multiple B cells producing anti-Gal (Figure 1B). The absence of anti-Gal in tolerized mice was not the result of adsorption on WT lymphocytes, which may excessively proliferate following PKM immunizations. Staining of WT lymphocyte by BS lectin indicated that in KO mice receiving 20 × 106 WT lymphocytes, these cells comprised less than 1.0% of the lymphocytes in various lymphoid organs after 4 weekly PKM immunizations (Table 1). Since the total number of lymphocytes in each lymphoid organ of PKM-immunized mice did not increase by more than 15%, in comparison to these organs in age-matched nonimmunized mice (not shown), it is probable that the number of WT lymphocytes did not change by more than 2-fold following 4 PKM immunizations. In vitro adsorption of sera from control mice producing anti-Gal with a much higher number of WT lymphocytes (1 × 108 WT lymphocytes/mL) did not affect significantly this antibody activity (not shown).                                View this table: [in this window] [in a new window]   Table 1. Analysis of WT lymphocyte proportion (%) in lymphoid organs of mice receiving 20 × 106 WT lymphocytes and immunized with PKM Anti-Gal IgM response could not be determined, since PKM-immunized mice produce IgM antibodies that bind nonspecifically in ELISA.14 Lack of anti-Gal IgM in tolerized mice was demonstrated indirectly by transplantation of WT hearts. Three KO mice tolerized by 20 × 106 WT lymphocytes received heterotopical transplants of WT hearts one week after the fourth PKM immunization. The hearts were not rejected for 2 months, despite 3 additional PKM immunizations after transplantation. Immunohistology analysis of heart muscle revealed no abnormal features (Figure 2B) and no IgM, IgG, or complement deposits within the blood vessels (Figure 2D). Since endothelial cells in WT hearts express -gal epitopes, these findings suggest that the tolerized mice produce no anti-Gal IgM. Similar transplantation into 3 anti-Gal-producing control mice resulted in WT heart rejection within 1 hour. This rejection displayed characteristics of hyperacute rejection,12 including red cell and platelet aggregates in blood vessels (Figure 2A) and deposits of IgM, IgG, and complement (Figure 2C). This rejection is mediated by anti-Gal IgM and IgG, since donor cells differ from recipient cells only in -gal epitope expression. View larger version (178K): [in this window] [in a new window]   Figure 2. Histology of WT hearts transplanted into PKM-immunized control mice and into tolerized mice. Histology of WT hearts transplanted into PKM-immunized control mice, which were rejected after 30 minutes (A,C), or into tolerized mice and then removed 2 months after transplantation (B,D). Immunostaining of transplanted WT hearts with anti-IgM antibodies (C-D). (A) Hematoxylin-eosin (H&E) staining of WT heart undergoing hyperacute rejection in a PKM-immunized control mouse. Note the blood clot within the artery and the edema among the myocytes. (B) H&E staining of WT heart in a tolerized mouse. The tissue displays no abnormal features. The small spaces within and between the myocytes are an artifact of the staining procedure. (C) Immunostaining with anti-IgM antibodies, demonstrating IgM deposits (counterstaining with hematoxylin) in the WT heart undergoing rejection as in panel A. Similar deposits were observed in sections stained with antibodies to IgG, C3, and C5, using the corresponding antibodies. (D) Immunostaining for IgM deposits in WT heart transplanted into a tolerized mouse as in panel B. Note that no deposits of IgM are observed. Similarly, no distinct deposits of IgG, C3, or C5 were detected (not shown). Sections are from a representative mouse, out of 3 in each group. Original magnification, × 200. Since mice in the present study were not irradiated, it is probable that WT lymphocytes induced tolerance, not only on newly formed anti-Gal B cells in the bone marrow, but also on circulating naive anti-Gal B cells. Studies in progress16 suggest that primed (ie, memory) anti-Gal B cells also are tolerized by syngeneic lymphocytes expressing the -gal epitope. The mechanism of this tolerance is currently under study. The present study is the first to demonstrate tolerance induction by lymphocytes expressing an incompatible carbohydrate antigen. Our study differs in 2 aspects from previous studies demonstrating tolerance induction by chimerism with WT bone marrow (BM) cells expressing -gal epitopes17-19: (1) our study indicates that induction of tolerance to the -gal epitope is not a characteristic limited to BM cells, but is a more basic phenomenon as it also can be induced by other cells expressing this epitope; and (2) tolerance induction by BM cells requires complete or partial myeloablation of the recipient,17-19 whereas tolerance induction by WT lymphocytes requires no myeloablation. Our observations may be of potential significance in allotransplantation and xenotransplantation. In vitro manipulation of autologous blood lymphocytes to express blood group A or B or -gal epitopes and administration of such lymphocytes back into the patient may result in similar tolerance induction. Expression of carbohydrate antigens may be achieved by transduction with replication defective adenovirus containing the corresponding glycosyltransferase gene, as we have recently demonstrated.20 Studies on tolerance induction by KO lymphocytes transduced in vitro to express -gal epitope are currently in progress.     Footnotes Submitted July 18, 2002; accepted October 21, 2002. Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood- 2002-07-2151. Supported by National Institutes of Health grant AI45849. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Uri Galili, Department of Cardiovascular-Thoracic Surgery, Rush University, 1653 West Congress Parkway, Chicago, IL 60612; e-mail: uri_galili{at}rush.edu.     References Top Abstract Introduction Study design Results and discussion References 1. Speir JA, Abdel-Motal UM, Jondal M, Wilson IA. Crystal structure of a MHC class I presented glycopeptide that generates carbohydrate-specific CTL. Immunity. 1999;10:51-61[Medline] [Order article via Infotrieve]. 2. Tanemura M, Yin D, Chong AS, Galili U. Differential immune response to -gal epitopes on xenografts and allografts: implications for accommodation in xenotransplantation. J Clin Invest. 2000;105:301-310[Abstract/Free Full Text]. 3. Thall AD, Maly P, Lowe JB. Oocyte Gal1-3Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse. J Biol Chem. 1995;270:21437-21440[Abstract/Free Full Text]. 4. Galili U, Buehler J, Shohet SB, Macher BA. The human natural anti-Gal IgG, III: the subtlety of immune tolerance in man as demonstrated by crossreactivity between natural anti-Gal and anti-B antibodies. J Exp Med. 1987;165:693-704[Abstract]. 5. Galili U, Shohet SB, Kobrin E, Stults CL, Macher BA. Man, apes, and Old World monkeys differ from other mammals in the expression of -galactosyl epitopes on nucleated cells. J Biol Chem. 1988;263:17755-17762[Abstract/Free Full Text]. 6. Galili U, Rachmilewitz EA, Peleg A, Flechner I. A unique natural human IgG antibody with anti--galactosyl specificity. J Exp Med. 1984;160:1519-1531[Abstract]. 7. Galili U, Macher BA, Buehler J, Shohet SB. Human natural anti--galactosyl IgG, II: the specific recognition of (1-3)-linked galactose residues. J Exp Med. 1985;162:573-582[Abstract]. 8. Galili U, Clark MR, Shohet SB, Buehler J, Macher BA. Evolutionary relationship between the natural anti-Gal antibody and the Gal1-3Gal epitope in primates. Proc Natl Acad Sci U S A. 1987;84:1369-1373[Medline] [Order article via Infotrieve]. 9. Galili U. Interaction of the natural anti-Gal antibody with -galactosyl epitopes: a major obstacle for xenotransplantation in humans. Immunol Today. 1993;14:480-482[Medline] [Order article via Infotrieve]. 10. Good H, Cooper DKC, Malcolm AJK, et al. Identification of carbohydrate structures which bind human anti-porcine antibodies: implications for discordant grafting in man. Transplant Proc. 1992;24:559-562[Medline] [Order article via Infotrieve]. 11. Sandrin MS, Vaughan HA, Dabkowski PL, McKenzie IF. Anti-pig IgM antibodies in human serum react predominantly with Gal1-3Gal epitopes. Proc Natl Acad Sci U S A. 1993;90:11391-11395[Abstract]. 12. Collins BH, Cotterell AH, McCurry KR, et al. Cardiac xenografts between primate species provide evidence of the -galactosyl determinant in hyperacute rejection. J Immunol. 1994;154:5500-5510. 13. Galili U, LaTemple DC, Radic MZ. A sensitive assay for measuring -gal epitope expression on cells by a monoclonal anti-Gal antibody. Transplantation. 1998;65:1129-1132[Medline] [Order article via Infotrieve]. 14. Tanemura M, Ogawa H, Yin D, Chen Z, DiSesa VJ, Galili U. Elimination of anti-Gal B cells by -gal ricin. Transplantation. 2002;73:1859-1868[Medline] [Order article via Infotrieve]. 15. Tanemura M, Maruyama S, Galili U. Differential expression of -gal epitopes (Gal1-3Gal14GlcNAc-R) on pig and mouse organs. Transplantation. 2000;69:187-190[Medline] [Order article via Infotrieve]. 16. Mohiuddin M, Ogawa H, Yin D-P, Galili U. Tolerance induction on memory anti-Gal B cells by syngeneic cells expressing the cognate antigen. Am J Transplant. 2002;2(suppl 3):162. 17. Yang YG, deGoma E, Odhan H, et al. Tolerization of anti-Gal1-3Gal natural antibody-forming B cells by induction of mixed chimerism. J Exp Med. 1998;187:1335-1342[Abstract/Free Full Text]. 18. Ohdan H, Yang YG, Shimizu A, Swenson KG, Sykes M. Mixed chimerism induced without lethal conditioning prevents T cell- and anti-Gal1,3Gal-mediated graft rejection. J Clin Invest. 1999;104:281-290[Abstract/Free Full Text]. 19. Bracy JL, Sachs DH, Iacomini J. Inhibition of xenoreactive natural antibody production by retroviral gene therapy. Science. 1998;281:1845-1847[Abstract/Free Full Text]. 20. Deriy L, Chen Z, Gao G, Galili U. Expression of -gal epitopes on HeLa cells transduced with adenovirus containing 1,3galactosyltransferase cDNA. Glycobiology. 2002;12:135-144[Abstract/Free Full Text]. © 2003 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: U. Abdel-Motal, S. Wang, S. Lu, K. Wigglesworth, and U. Galili Increased Immunogenicity of Human Immunodeficiency Virus gp120 Engineered To Express Gal{alpha}1-3Gal{beta}1-4GlcNAc-R Epitopes J. Virol., July 15, 2006; 80(14): 6943 - 6951. [Abstract] [Full Text] [PDF] N. Mitsuhashi, J. Fischer-Lougheed, I. Shulkin, A. Kleihauer, D. B. Kohn, K. I. Weinberg, V. A. Starnes, and M. Kearns-Jonker Tolerance induction by lentiviral gene therapy with a nonmyeloablative regimen Blood, March 15, 2006; 107(6): 2286 - 2293. [Abstract] [Full Text] [PDF] M. M. Mohiuddin, H. Ogawa, D.-P. Yin, and U. Galili Tolerance induction to a mammalian blood group--like carbohydrate antigen by syngeneic lymphocytes expressing the antigen, II: tolerance induction on memory B cells Blood, July 1, 2003; 102(1): 229 - 236. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2002-07-2151v1 101/6/2318    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ogawa, H. Articles by Galili, U. Search for Related Content PubMed PubMed Citation Articles by Ogawa, H. Articles by Galili, U. Related Collections Immunobiology Transplantation Brief Reports Social Bookmarking                   What's this?

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