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Speedy restoration of immune responsiveness in bone marrow recipients has been the objective of studies in which the donor was immunized so that specific immunologic memory could be transferred adoptively and selectively. Using unrelated rabbits, matched for major histocompatibility antigens but mismatched for their immunoglobulin allotypes, it could be shown that recipients of lymphoid cells from naive donors became B cell chimeras but did not use donor-derived B cells for their antibody responses to test antigens. In contrast, cells from donors primed for such antigens dominated antibody production in recipients in response to specific challenge. Clonal restriction in such adoptive responses was demonstrated. We now show that the induction of effective memory in cells from naive donors can be achieved in vitro during the preparation of donor cells for transfer to the recipient. Early challenge of the recipient enhances expression of the transferred immune response quantitatively and also results in the establishment or preservation of a larger diversity of clones from the donor.

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The proliferative potential of membrane Ig (mIg)-bearing B lymphocytes was assessed in an adoptive transfer system based on the use of non-inbred rabbits matched for major histocompatibility (MHC) antigens and mismatched for immunoglobulin (Ig) allotypes. Cell suspensions made from spleens (SP), mesenteric lymph nodes (LN), or bone marrow (BM) of allotype b4b5 rabbits were deprived of B cells with mIg of the b4 type by adherence to plastic dishes coated with affinity-purified anti-b4. When such b4-depleted cell populations were injected into newborn hosts of allotype b6b6, stable and lasting chimerism promptly resulted, in which donor-derived products were almost entirely of the b5 allotype. Chimeras formed by transfer of unfractionated cells from b4b5 donors, on the other hand, exhibited a predominance of the b4 allotype, as seen in the living donors. BM but not SP or LN contained precursors capable of differentiating into mIg+ lymphocytes in culture, but no evidence was obtained for engraftment and differentiation by such B-cell precursors or more primitive stem cells in vivo. These studies suggest a potentially significant role for mature B cells in reconstituting the immune system of human transplant recipients.

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The expression of individual clonal products during long-term in vivo culture was investigated using a rabbit model system of bone marrow transplantation. RLA (MHC) matched rabbits were deliberately mismatched for kappa light chain immunoglobulin allotype to facilitate identification of antibodies as being of donor or recipient origin. Recipients of cells from antigen-primed donors responded to antigen stimulation with antibody of donor origin, showing that cells were effectively triggered for antibody production in the recipient. Isoelectric focusing followed by affinity immunoblotting of the expressed antibodies showed that the responding B-cell clonotype repertoire remained virtually unchanged throughout the extensive cell transfer protocol used. These results suggest that B-memory-cell stimulation, rather than stem cell differentiation, was responsible for the observed response patterns. There was no detectable increase in the heterogeneity of the donor-derived antibody response with time and no new clonotypes appeared which were not present in the cell donor. Unlike previous studies, early stimulation with antigen was not required for successful engraftment and memory cell establishment. However, our data suggest that the timing of antigenic challenge may determine which of the donor-derived clones will dominate a response after antigen challenge of the recipient.

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The adoptive transfer of immunity by means of spleen and lymph node cells has been analysed in rabbits of defined major histocompatibility (RLA) types and immunoglobulin (Ig) allotypes. Previous results had shown that chimaeras formed by transplanting adult cells into RLA-matched newborn hosts become stable B-lymphocyte chimaeras, as determined by their continuous production of donor type Ig. However, specific antibodies made by adult chimaeras were demonstrably only of recipient origin, unless the donor had been primed. The present report describes early events after the transfer of cells from donors primed with trinitrophenylated keyhole limpet haemocyanin (TNP-KLH). Double-staining with enzyme-labelled antigen or antibody conjugates was applied to identify lymphocytes of donor or recipient origin in spleen sections of transplanted rabbits and also to identify cells producing anti-TNP. Lymphocytes with membrane-bound Ig (mIg+ cells) of donor allotype were readily identified scattered singly or in small clusters throughout the recipients' B-lymphocyte areas within 5 days after transfer. Groups of donor-derived cells producing anti-TNP were demonstrable only in animals that had been specifically stimulated. Serological analysis and lymphocyte marker and function studies correlated well with results obtained by the histocytochemical approach. These results show that a systematic study of the role and importance of mature B lymphocytes in transplantation is now feasible.

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After incubation of tissue sections with anti-allotype-enzyme conjugates, the localization of immunoglobulin-allotype-bearing cells in the lymphoid tissues of conventional and chimeric rabbits could be established. The use of anti-allotype sera bearing distinct enzyme labels allowed simultaneous recognition of B cells producing immunoglobulin of one or the other parental types in heterozygous rabbits, or of B cells from the donor and recipient in chimeras. After immunization of chimeric rabbits with trinitrophenyl-keyhole limpet hemocyanin, anti-trinitrophenyl antibody-forming cells could be demonstrated through the use of a trinitrophenyl-alkaline phosphatase conjugate. Simultaneous incubation of sections with this reagent and with horseradish peroxidase coupled to (donor or recipient) anti-allotype sera made possible the determination of the origin (donor or recipient) of the antibody-forming cells. In agreement with the results of plaque assays and analyses of serum antibodies, all the anti-TNP producing cells were of donor origin when the chimeras had been created through injection of spleen or lymph node cells from trinitrophenyl primed donors. With this study we introduce a simple, direct method for the simultaneous identification of cells that produce antibody of a given allotype and a given specificity, applicable to appropriate studies in heterozygous or chimeric rabbits. The procedure has various advantages over previously reported methods.

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In a model consisting of noninbred rabbits matched for major histocompatibility antigens and mismatched for immunoglobulin allotypes, using cell donors and recipients unrelated to each other, B-cell memory has been demonstrated to persist through three successive transfers for a period approaching 2 years. Memory cells from the original donor are shown to dominate specific antibody responses of the primary and secondary recipients. Vigorous antibody responses by donor-derived cells are obtained even when antigenic stimulation is delayed by several months. The data suggest that B memory cells may be particularly efficient in the colonization of recipients, and the potential significance of these findings for adoptive immunization of human bone marrow recipients is discussed.

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Stable and lasting B lymphocyte chimerism induced in newborn rabbits through the introduction of spleen or lymph node cells from adult donors matched with the recipients for major histocompatibility antigens, is characterized by an apparent immunodeficiency of donor-derived cells. However, priming of the donor with an antigen that is subsequently used to immunize the recipients results in the selective and effective participation of donor cells in the chimera's antibody response to this antigen. These findings are ascribed to limitations in the repertoire of cells from the unprimed donor that colonize the recipients. Polyclonal stimulation secondary to allogeneic effects has been suggested as an explanation for the participation of donor-derived B cells noted in occasional recipients of cells from unprimed donors matched with recipients with respect to major but not minor histocompatibility antigens, and seen more regularly in surviving recipients of unmatched or mismatched donor cells.

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Chimeras established by transferring spleen or lymph node cells from primed donors use donor-derived B lymphocytes in responses to the antigen used in priming of the donor, whereas cells of the recipient are used in responses to other antigens. Clonal dominance by donor cells lasts for at least 9 months. Treatment of newborn rabbits with the antigen used in priming the donors elicits copious production of antibody bearing the donor's allotypic markers in chimeras, but tolerance is induced in nonchimeric controls. Lasting and effective memory is also established in chimeras in the absence of immediate antigenic stimulation. This model for transplantation of allogeneic lymphoid cells into recipients matched with the donor for major histocompatibility antigens shows that priming of the donor facilitates the specific, effective, and enduring acquisition of immunocompetence in the recipient for the antigen used in priming the donor.

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An IgG1 monoclonal antibody, termed ACM-1, has been shown to react with rabbit T cells, but not Ig+ cells or macrophages. This antibody appears to recognize the same epitope as the previously described 9AE10 antibody and, together with 9AE10, has been used to obtain highly pure and fully functional T- and B-cell populations. However, the relevant epitope does not appear to be homologous to rodent Thy-1 since quantitative absorptions failed to show reactivity with rabbit brain. Furthermore, attempts to obtain in vivo T-cell depletion resulted in larger decreases in white blood cells than would be expected for simple T-cell removal. In vitro assays on enriched neutrophil preparations revealed that 80-95% of these cells were reactive with ACM-1 and 9AE10. Thus, it appears that in the rabbit, T cells and neutrophils share a major epitope.

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This study was designed to determine whether natural immune responses could elicit immunoregulatory auto-antiidiotypic antibodies. Female rabbits heterozygous at the a and b Ig loci were bred to homozygous males. Offspring of one such breeding were studied for natural production of antibodies specific for the noninherited allotypes and for the production of immunoregulatory auto-antiidiotypic antibodies. All offspring mounted natural antiallotype responses. The anti-a1 responses cycled as a function of time whereas the anti-b5 responses were invariant. Anti-a1 responses from two offspring were shown to change specificity for different a1 subsets as they cycled. Anti-a1 was purified from the first cycle and was used to assay for auto-antiidiotypic responses. Auto-antiidiotypic antibodies were detected and were found to cycle in an inverse way with the anti-a1 cycles. The idiotopes detected using the natural auto-antiidiotypic antisera were strongly cross-reactive. Subsequent deliberate immunization showed that antibodies specific for all a1 subsets could be elicited after auto-antiidiotypic regulation had functioned. The data support the interpretation that idiotype network interactions indeed function in naturally occurring immunologic situations and are not merely laboratory curiosities or artifacts.

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Transfer of adult spleen, lymph node, or bone marrow cells to newborn recipients matched with the donor with respect to the major histocompatibility antigen or antigens, but mismatched with regard to immunoglobulin allotypes results in lasting B cell chimerism. Using such chimeras as donors for secondary recipients, the persistence of B cells from the original donor and the ability of such cells to propagate in the secondary recipient have been demonstrated. In contrast to the effective establishment of donor B cells in primary and secondary recipients, functional T cells of donor origin were not demonstrable among lymphocytes of primary recipients.

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Noninbred rabbits, matched with regard to the major histocompatibility complex (RLA-A and RLA-D loci) but mismatched for Ig allotypes, served as donors (adult) and recipients (newborn) of lymphoid cells. Lasting chimerism regularly followed the transfer of 1 x 10(8)-3 x 10(8) spleen, lymph node, or bone marrow cells, as indicated by the continued production of Ig with allotypic determinants of both donor and recipient. Typically, Ig of donor allotype accounted for 25-50% of total allotypic Ig at 4 wk of age and the amount of donor Ig produced remained stable for up to 20 mo. Total allotypic Ig levels remained normal in the chimeric rabbits. "Chimeric drift" or a gradual diminution of donor products over a period of several months, occurred in some individuals. Transfer of lymphoid cells from allotype-suppressed adult donors to newborns of appropriate allotypes did not result in specific suppression of the target allotype in the recipients. Other experiments showed that lymphoid cells from suppressed donors adoptively transferred to histocompatible recipients continued to synthesize Ig of the nonsuppressed type only. The suitability of using an outbred population of histocompatible but allotype-mismatched rabbits for analyzing allotype suppression and other immunoregulatory phenomena is demonstrated by the results presented here.

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