Louise T. Adler

Double immunocytochemical staining for the in situ study of allotype distribution during an anti-trinitrophenyl immune response in chimeric rabbits.

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.

Sequential double immunocytochemical staining for in situ identification of an auto-anti-allotype immune response in allotype-suppressed rabbits.

Immunocytochemical staining has been used to detect putative autoimmune B-cells in rabbits undergoing chronic allotype suppression. This condition is seen in heterozygous rabbits exposed perinatally to antibody against the paternal immunoglobulin allotype. Such animals develop lifelong suppression for this allotype and have been used as models for study of antibody-induced disturbance of immune regulation. Normal rabbits deliberately immunized against a heterologous allotype were used to establish the feasibility of identifying cells forming anti-allotypic antibodies in cryostat sections of rabbit lymphoid tissues. Incubation and staining of tissue sections from suppressed rabbits then revealed the presence of autoimmune B-cells, with antibody specificity for the suppressed allotype, in all chronically suppressed adult rabbits tested. Sequential incubation and staining with allotype- and anti-allotype-enzyme conjugates established that such cells were of non-suppressed origin. Auto-anti-allotype antibody-forming cells were not found in normal heterozygotes or in chimeric rabbits. The immunocytochemical techniques described here permitted simultaneous detection of specificity (i.e., anti-allotype) and origin (allotype) of antibody-forming cells involved in an autoimmune response, as well as their anatomical correlation with other B-cells of suppressed or non-suppressed origin. Since the method described can be adapted to detection of alternate cell markers, we believe it to have potential application to the study of other autoimmune phenomena.

[30] Passive hemagglutination and hemolysis for estimation of antigens and antibodies

Publisher Summary This chapter discusses the use of passive hemagglutination (PH) and hemolysis for estimation of antigens and antibodies. In passive hemagglutination (HA), erythrocytes serve as the inert carriers of suitably affixed extraneous antigens and the clumping of such indicator cells by antibody specific for the coating antigen is a sensitive test for antibody. Under appropriate conditions, the test measures antibody in concentrations of as little as 1-10 mg/ml. Because reasonably potent immune sera contain at least 1 mg of antibody per milliliter, it is apparent that their HA titer, defined as the highest dilution that will bring about agglutination, is often greater than 1:100,000. When applied to antisera obtained after intensive immunization, and thus containing mostly IgG antibodies, the HA test results generally correlate reasonably well with those of binding assays for antibody. The passive hemolytic (PH) test is an extension of HA, in which an additional reagent, complement, causes lysis of test cells that is mediated by antibody against the coating antigen. In some instances, this modification leads to further enhancement of sensitivity, but this advantage is balanced by a number of complicating factors. Dominant among these is the introduction of complement as an additional variable.

Characterization of donor-derived lymphocytes in chimeric rabbits.

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.

Immunocompetence of chimeric rabbits. I. Participation of donor-derived B lymphocytes in immunoglobulin, but not in antibody, synthesis.

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 chimeras 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.

Immunocompetence of chimeric rabbits. II: Persistence of memory and antigen-driven responses of donor cells

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 donors 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.

Selective suppressive effects of anti-allotype antibodies on spleen cells of adult rabbits

Abstract Spleen cells from adult b 4 b 4 rabbits were used to investigate the effects of anti-allotype antibodies on the immunoglobulin-synthesizing capacity of these cells in vitro . Treatment of 2 × 10 7 splenic lymphocytes with as little as 10 μg of anti-b4/ml for 2 hr caused complete loss of membrane b4, while 30 μg of anti-b4/ml was needed to bring about the same result following a 24-hr pulse. However, substantial to complete recovery of surface immunoglobulin (Ig) expression occurred on subsequent culture of cells exposed to anti-b4 concentrations of up to 300 μg/ml, while at the same time the ability of treated cells to secrete b4 Ig into the culture fluid and to mount a primary response against a bacteriophage antigen was severely impaired. Inhibition of Ig secretion and of antibody formation was dependent on the concentration of anti-b4 used and was more pronounced following a 24-hr antibody pulse than one for 2 hr. Such cells showing selective suppression of Ig secretion but not of membrane Ig expression may provide an in vitro analog for mIg + lymphocytes demonstrable in allotype-suppressed rabbits at a time when secretion of that allotype is still severely impaired.

Comparative suppressive effects of anti-allotype antibodies on spleen cells of immature and adult rabbits

Abstract The suppressive effects of anti-allotype antibody on splenic lymphocytes of adult (>3 months of age) and newborn (1-week-old) rabbits were compared in vitro . An approximately 10-fold lower concentration of anti-b4 sufficed to modulate completely membrane-bound b4 immunoglobulin (Ig) when newborn cells were pulse-treated for 2 or 24 hr than when adult cells were tested. In contrast to splenic lymphocytes from adults, which regenerated most of the original proportion of b4 + cells in culture following treatment with up to 300 μg of anti-b4 for 24 hr (4), immature lymphocytes were susceptible to irreversible modulation of membrane-bound Ig even when lower concentrations of anti-b4 were used. However, under conditions permitting reversible modulation of membrane b4, lymphocytes of newborns regenerated the membrane product more rapidly than did adult cells. Both mature and immature splenocytes were shown to be capable of some level of b4 synthesis even in the continuing presence of anti-b4. No evidence for susceptibility to complement-mediated killing of newborn spleen cells by anti-allotypic antibodies was obtained. The observations reported here support the concept that the greater sensitivity of B lymphocytes from the newborn to succumb to irreversible suppressive effects by anti-allotype antibody plays a significant role in the restriction for induction of allotype suppression to the perinatal period.

Allotype suppression in the rabbit: Persistence of passive antibody and the establishment or abrogation of chronic suppression

Abstract Lasting allotype suppression of the rabbit, induced by perinatal treatment with the appropriate antibodies, can be alleviated by the administration of immunoglobulin of the suppressed type during the first few weeks of life but not later. Experiments described here sought to determine the relative roles of the age of the treated rabbit and of the level of residual passive immunity in this apparently age-dependent phenomenon. The data show that spontaneous termination of effective passive immunity prior to 4 weeks of age results in significant though incomplete suppression which is insensitive to the modifying maneuver, and that prolonging the state of passive immunity beyond the second month of life similarly renders the rabbit resistant to induced accelerated release from suppression. Thus neither age-dependent characteristics of the rabbit or its lymphoid cells nor the duration of passively acquired immunity alone are sufficient explanations for the critical period during which modification of suppression can be achieved.

Immunocompetence of chimeric rabbits: III. Serial passage and persistence of B-Lymphocyte memory

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.