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Induction of immune tolerance to a transplantation carbohydrate antigen by gene therapy with autologous lymphocytes transduced with adenovirus containing the corresponding glycosyltransferase gene.

Induction of tolerance to transplantation carbohydrate antigens is of clinical significance in recipients of ABO-incompatible allografts, or of xenografts. The experimental animal model used for studying such tolerance was that of alpha1,3galactosyltransferase (alpha1,3GT) knockout (KO) mice, which lacks the alpha-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) and which can produce the anti-Gal antibody against it. In contrast, wild-type (WT) mice synthesize the alpha-gal epitope and are immunotolerant to it. KO lymphocytes transduced in vitro with adenovirus containing the alpha1,3GT gene (AdalphaGT) express alpha-gal epitopes. Administration of such lymphocytes into KO mice resulted in tolerization of naïve and memory anti-Gal B cells. Mice tolerized by AdalphaGT transduced lymphocytes failed to produce anti-Gal following immunizations with pig kidney membranes (PKM) expressing multiple alpha-gal epitopes. This tolerance was perpetuated by transplanted syngeneic WT mouse hearts expressing alpha-gal epitopes. Transplanted WT hearts survived in the tolerized KO mice for at least 100 days, despite repeated PKM immunizations. Control mice receiving lymphocytes transduced with adenovirus lacking the alpha1,3GT gene were not tolerized, but produced anti-Gal and rejected transplanted WT hearts. This study suggests that autologous lymphocytes transduced with adenovirus containing A or B transferase genes may induce a similar tolerance to blood group antigens in humans.

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Role of different B-cell subsets in the specific and polyclonal immune response to T-independent antigens type 2.

Role of different B-cell subsets in the immune response to T-independent antigen type 2 (TI-2) was studied. BALB/c and C57BL/6 mice were immunized by polyvinylpyrrolidon (PVP), and the numbers of antibody- and Ig-forming cells (AFC and IFC, respectively) were determined by ELISPOT method. The number of cells producing non-specific Ig (nIFC) was calculated as the difference between the number of IFC and AFC; the number of nIFC induced by PVP was calculated as the difference between the number of nIFC in immune and control splenocytes. Immunization by PVP induced not only the AFC appearance, but also the increase in the number of the antigen-induced nIFC. The treatment of splenocytes by anti-CD5 antibodies and guinea pig complement reduced the increase in the numbers of newly formed AFC and nIFC to approximately 40% of control level. It means that CD5+ cells play an important role not only in the specific, but also in polyclonal immune response to non-self TI-2. To be sure that the decrease of AFC and nIFC numbers is due to depletion for CD5+ B-cells, but not CD5+ T-cells, splenocytes were separated to B-1 and B-2 subsets, and the numbers of AFC, IFC and nIFC were determined in each B-cell subpopulation separately. The overwhelming majority of newly formed AFC and nIFC was detected in B-1 subset. The numbers of AFC and nIFC in B-1 compartment was approximately 10-fold greater than in B-2 cells. A close parallelism between AFC and nIFC formation was observed. It is concluded that specific and polyclonal immune response to non-self TI-2-PVP-depends mainly on CD5+ B-1 subset.

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Characterization of monoclonal antibodies recognizing immunoglobulin kappa and lambda chains in pigs by flow cytometry.

The existence of two types of the immunoglobulin (Ig) light chain in pigs was documented>30 years ago and has been confirmed by the cloning of porcine light chain genes homologous to human and murine Ig kappa (Igkappa) and Ig lambda (Iglambda). However, immunochemical reagents defining these two light chain isotypes have not been characterized. Here, we show that rabbit antisera specific for human Igkappa and Iglambda and certain anti-porcine light chain monoclonal antibodies (mAb) are useful in distinguishing light chain isotypes by flow cytometry (FCM). Porcine B cell lines L23 and L35 stained positive only with anti-human Iglambda antiserum and were negative when tested using anti-human Igkappa antiserum. While mAbs K139.3E1, 1G6 and 27.7.1 also tested positive on these cell lines, mAb 27.2.1 did not. Therefore, FCM was used to examine the hypothesis that K139.3E1, 1G6 and 27.7.1 are Iglambda-specific whereas mAb 27.2.1 recognizes the Igkappa chain in pigs. Double staining of peripheral blood mononuclear cells (PBMC) with pairs of anti-light chain mAbs and using cocktails of anti-light chain mAbs and anti-human polyclonal antiserum, confirmed this hypothesis with the exception that mAb K139.3E1 appears to recognize only a subset of Iglambda(+) B cells in most pigs. In summary, we identified two pan-specific anti-pig Iglambda mAbs, one anti-lambda mAb that recognizes a lambda-light chain subset and one anti-pig Igkappa mAb.

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Characterization of porcine CD5 and CD5+ B cells.

Evidence strongly supports a role for the lymphocyte transmembrane glycoprotein CD5 in intracellular signalling events, whereby antigen-dependent growth and differentiation signals are augmented. Apart from its role in activation-related signalling, CD5 has been regarded as a possible B cell lineage marker differentiating subsets, CD5+ B cells (also termed B1 cells) and conventional B cells (or B2 cells). To extend these investigations to the study of pigs, porcine B cells were examined for evidence of CD5 expression. The influence of cellular activation on CD5 expression and CD5's role in signal transduction events and lymphocyte proliferation were examined. Using an anti-porcine CD5 MoAb (b53b7), porcine B cells were shown to be heterogeneous for CD5 expression. As in other species, B lymphocyte CD5 expression is low (dull), while IgM is high (bright). Ten to 30% of pig blood B lymphocytes are CD5+, with the highest frequency in neonates. Anti-CD5 antibody treatment was sufficient to induce rapid but transient calcium ion flux in porcine peripheral blood lymphocytes (PBL). CD5 expression increased on PBL following treatment with phorbol myristate acetate (PMA), lipopolysaccharide (LPS), or immobilized anti-IgM. LPS, PMA, and concanavalin A (Con A) but not anti-CD5, anti-IgM, or combinations of these antibodies induced lymphocyte 3H-thymidine uptake. CD5+B cells are a common constituent of porcine circulating lymphocytes and resemble B1 cells of mice, man and other species in CD5 expression, frequency and lymphoid organ distribution. Porcine CD5, like CD5 in other species, mediates signal transduction, leading to changes in intracellular calcium concentration, but this signal alone is insufficient to promote cell division. A subset of porcine B cells up-regulates CD5 expression following phorbol ester activation.

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Presentation of antigen by B cells subsets. I. Lyb-5+ and Lyb-5- B cells differ in ability to stimulate antigen specific T cells.

We have examined the antigen presenting cell (APC) function of different B cells. Resident, peritoneal B cells from normal mice were more efficient than splenic B cells in presenting antigen to CD4+ T cell lines. Peritoneal B cells from X-linked immunodeficient (Xid) mice, by contrast, stimulated no detectable responses. Xid splenic B cells were much less efficient APC than normal splenic B cells. B cells from neonatal mice also were very poor APC until the mice were 3 to 4 weeks old. Xid B cells presented antigen to T cell hybridomas as well as normal B cells showing that they process antigen normally. Thus, the defect is most likely in providing secondary signals. The ability of B cells to present antigen efficiently correlates with the percentage of B cells reported to express the Lyb-5 antigen. Anti-Lyb-5 serum and complement abrogated the APC activity of B cells suggesting that Lyb-5+, but not Lyb-5- cells are efficient APC. We also found that activated and resting normal splenic B cells, separated by buoyant density, presented antigen equally. Both populations also contained Lyb-5+ B cells although they were a larger fraction of the activated cells. Lyb-5 is now thought to be an activation antigen rather than a differentiation antigen. If this idea is correct, then our data indicate that anti-Lyb-5 more cleanly separates activated and resting B cells than buoyant density techniques.

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