Erwin Diener

Mechanisms at the cellular level during induction of high zone tolerance in vitro

Abstract The in vitro induction of high zone tolerance to polymerized flagellin of S. adelaide in CBA mouse spleen cells consists of two stages: (a) Attachment of tolerance inducing concentrations of antigen to the surface of immunocompetent cells. (b) Inactivation of the immunocompetent cells by the surface-bound antigen. The definition of these two stages is based on the finding that trypsin treatment of spleen cells within 16 hr after tolerance induction in vitro completely reverses the state of unresponsiveness. However, reversibility of the tolerant state could not be observed by trypsinization of cells after 3 days of culture with antigen. Trypsin treatment of spleen cells before exposure to antigen prevented the induction of tolerance in vitro . This suggests that antigen binding was abolished by removal of recognition sites by the proteolytic enzyme.

The cellular basis of cortisone-induced immunosuppression of the antibody response studied by its reversal in vitro.

Spleen cells from mice pretreated with cortisone were impaired in their ability to support an immune response in vitro to two antigens: sheep erythrocytes (SRBC) and polymeric flagellar protein of Salmonella adelaide (POL). We conclude that this immunosuppression is consistent with cortisone causing a dysfunction in accessory cells (macrophages?) and thymus-derived (T) helper cells without seriously affecting antibody forming cell precursors. Evidence for this comes from reconstitution of the response with additions of peritoneal exudate cells (PEC, irradiated, and anti- θ -treated), activated T cells, and 2-mercaptoethanol (2-Me) in culture. Additions of PEC or 2-Me restored the T cell-independent anti-POL response not only in cortisone-treated spleen cell cultures, but, contrary to previous reports, also in cultures of normal spleen cells depleted of adherent (accessory) cells by carbonyl iron treatment. The anti-SRBC response (which is dependent on T cells and accessory cells) in cortisone-treated spleen cell cultures was fully restored only by a combination of activated T cells and PEC or activated T cells and 2-Me. However, with lower doses of cortisone pretreatment, activated T cells or 2-Me alone was effective.

Antigen recognition, i. Immunological significance of antigen-binding cells.

Abstract This communication reports an attempt to correlate the quantitative assessment of antigen-binding cells with the sedimentation velocity distribution profile of immunocompetence within a given population of CBA-mouse spleen cells before and during the early immune response. Combining autoradiography with cell separation techniques, it was found that the distribution profile of antigen-binding cells from unimmunized mice corresponds entirely with that of in vitro and in vivo immunological activity. Antigenic stimulation induced a significant proportion of antigen binding cells to differentiate into larger cells. These differentiated cells followed the distribution profile of immunocompetence within a cell population.

A New Perspective on B Cell Triggering Control of the Immune Response by Organizational Changes in the Lipid Bilayer

Immunological triggering of bone marrow derived (B) lymphocytes can be defined as the molecular process which drives the cell from its initial resting state into a metabolically active phase, resulting in clonal proliferation, differentiation and subsequent secretion of immunoglobulin (Ig). Contact with antigen plays a major role in the triggering process, as is evident from the selectivity of the antibody response. However, it is clear that other stimuli also are involved since the immune response of B cells is known to be infiuenced by the presence of (a) antigen-specific thymu:; derived (T) cells (Miller & Mitchell 1969), (b) antigen-specific T cell secretory products (Feldmann & Basten 1972a, Taussig 1974), (c) nonspecific T cell secretory produts (Dutton et al. 1971, Schimpl & Wecker 1972, Watson 1973, Armerding & Katz 1974, Dutton 1974), (d) nonspecific mitogenic substances (Sjoberg et al. 1972, Dutton & Hunter 1974), (e) histocompatibility-linked gene products present in the T or B cell population (Katz & Benacerraf 1974), (f) accessory cells such as macrophages (Unanue 1972), (g) antigen-specific suppressor T cells (Gershon & Kondo 1971).

MHC control of T lymphocyte-macrophage interactions

Identity at the major histocompatibility complex (MHC) of primed T cells and macrophages was essential for the development of a T cell proliferative response to Purified Protein Derivative of tuberculin (PPD) in the presence of macrophage-associated antigen and potential allogeneic effects were eliminated by the use of one-way fetal liver chimeras as a T cell source. By contrast, such MHC restriction could not be shown for the T cell—macrophage interaction when antigen was present in soluble form.It was found that the proliferative response of primed (responder × nonresponder) F1 T cells to the Ir-gene controlled antigen, TNP-18 [Glu-Tyr-Lys (TNP) (Glu-Tyr-Ala)5], could only be restored by responder macrophages with bound antigen, while both responder and nonresponder macrophages reconstituted the response to soluble TNP-18. Supernatants from cultured responder or nonresponder macrophages could at least partially replace viable macrophages in the latter case.These results argue for two distinct antigen presentation mechanisms, depending on the physical state of the antigen rather than its chemical nature: one involves recognition of antigen in association with MHC-coded determinants and shows H-2 restriction, while the other, mediated by soluble factors and antigen, does not.

Facilitation of allogeneic bone marrow transplantation by a T cell-specific immunotoxin containing daunomycin

Daunomycin coupled via an acid-sensitive spacer to monoclonal Thy-1.2-specific antibody was used to purge T lymphocytes from a 1:1 mixture of murine C57BL/6J bone marrow and spleen cells prior to engraftment in fully allogeneic, irradiated BALB/c recipients. Treatment of bone marrow with the immunotoxin at a concentration used for purging had no effect on the viability of committed hematopoietic progenitor or multipotent stem cells. All of the recipients of purged bone marrow were at least 80% chimeric for donor peripheral blood cells and none developed graft-versus-host disease. Out of 50 chimeras, 49 were still alive more than 200 days posttransplantation. The chimeras were shown to be tolerant to donor tissue as tested by mixed lymphocyte reactivity, cell-mediated cytotoxicity, and skin grafting. The same tests revealed full immunocompetence of chimeras to third-party alloantigens. In vivo IgM and IgG antibody responses to sheep red blood cells were similar in magnitude in allogeneically and syngeneically reconstituted mice.

Immunological competence and host-specific tolerance of antibody-facilitated bone marrow chimeras.

We have generated murine antibody-facilitated (AF) bone marrow chimeras in the genetic combination P1----(P1 X P2)F1 by the simultaneous injection of P1 bone marrow cells and anti-P2 MHC monoclonal antibody into normal (unirradiated) adult (P1 X P2)F1 recipients. These mice have normal life spans and appear to be healthy, with no overt signs of graft-versus-host disease. We have undertaken an extensive survey of the ability of stable, long-term AF chimeras to generate immune responses in vitro and in vivo. Both T and B lymphocyte functions have been analyzed in proliferative and effector cell assays. The AF chimeras respond normally to mitogenic as well as antigenic stimuli, and exhibit normal capacities for cellular collaboration in the generation of immune responses. However, splenic lymphocytes from AF chimeras are substantially and specifically hyporesponsive or nonresponsive to host, P2-encoded, alloantigens in in vitro assays of cell-mediated immunity. This host-specific tolerance is exhibited by the cytotoxic T lymphocyte lineage; T helper cells necessary for the generation of a cytotoxic response may also have decreased reactivity to host determinants. We conclude that our protocol for the production of AF chimeras does not compromise the immune system of chimeric animals but does allow the maintenance of host-specific tolerance, after stable equilibrium has been attained.

CAN EXPERIMENTAL B CELL TOLERANCE SERVE AS A MODEL FOR SELF TOLERANCE

We have examined the abilities of the mature and immature immune systems to discriminate between tolerogenic and nontolerogenic forms of a hapten-carrier conjugate; both forms are multivalent nonimmunogenic polymers of the same molecular weight, and have the same avidity for free, hapten-specific antibody and hapten-binding B cells. Hapten-specific tolerance was induced in adult B cells by nonimmunogenic dinitrophenylated carboxymethyl cellulose or methyl cellulose. Oxidation and subsequent reduction of the vicinal hydroxyl groups of both carriers aborgated tolerogenicity, although they remained nonimmunogenic. This chemical modification did not affect the carriers molecular weight, and it did not reduce the binding avidity of their hapten derivatives to hapten-specific antibody or to antigen-binding B cells. The same experiments, when carried out in either neonatal mice or mice that had been lethally irradiated and given the above compounds during treatment with 13-day-old fetal liver cells, invariably yielded the same results. Like adult mice, these immunologically immature animals were capable of distinguishing between the tolerogenic and the nontolerogenic form of each antigen. It has also been shown (C. A. Waters et al., in preparation) that neonatally induced tolerance to TNP-HGG is irreversible, whereas tolerance to TNP-BSA is reversible by challenge with TNP-LPS. These results are in conflict with the clonal abortion hypothesis.

Ontogeny of Hemopoietic Colony-Forming Units in the Chick Embryo Spleen

Inoculation of spleen cells of chicken embryos onto the chorioallantoic membrane (CAM) of other chicken embryos led to the formation of white (granulocytic) and pink (mixed erythrocytic and granulocytic) hemopoietic colonies. The number of CAM colonies formed was a linear function of the number of cells inoculated indicating that each colony originated from a single colony-forming unit (CAM-CFU). CAM-CFU’s consist of an aggregate of primitive undifferentiated cells which penetrate the ectodermal layer of the CAM, then proliferate and differentiate into mature granulocytes or erythrocytes. Deaggregated CAM-CFU’s failed to form colonies indicating that cell interaction may be required for colony formation. The genetic constitution of the host embryo has no effect on colony formation but mixtures of donor cells from inbred embryo spleen were found to be more efficient colony formers than mixtures of donor cells from non-inbred embryo spleens. Ontogeny studies demonstrated that the chick embryo spleen contains peak hemopoietic activity between fifteen and seventeen days of incubation declining thereafter to undetectable levels two days post hatching. At this time the inoculation of spleen cell suspensions leads to the formation of lymphoid colonies which increase in number with age of the donor.

The role of macrophages in B cell tolerance: I. Antigen-specific failure of Thy-1, Ly-2 negative adherent cells from tolerant mice to reconstitute immunocompetence in adherent cell deficient spleen cells☆

T-Cell-independent B-cell tolerance to the hapten derivatives of carboxymethyl cellulose (CMC) or methyl cellulose (MC) appears to be controlled by Thy-1-, Ly-2- adherent (A) cells contained in the spleen or peritoneal fluid. Immunocompetence in nonadherent (NA) normal spleen cells could be restored in vitro by irradiated A cells from normal mice. However, NA cells reconstituted with irradiated A cells derived from hapten specifically tolerant mice failed to respond to the same hapten, but responded normally to an immunogenic challenge with another unrelated antigen. A cells that had been preincubated at 4 degrees C with hapten derivatized MC also failed to restore immunocompetence. While preincubation of unfractionated spleen cells with the tolerogen under the same conditions resulted in B-cell unresponsiveness, such treatment of NA cells failed to render B cells tolerant. Treatment of A cells from tolerant mice with the reducing agent potassium iodide (KI) in vitro restored their capacity to render cultures of NA cells immunocompetent to the relevant hapten. Moreover, treatment with KI of spleen cells from mice injected with the tolerogen was shown to render them responsive. We suggest that B-cell tolerance induced by hapten derivatives of CMC and MC is mediated by suppressive macrophages contained among A cells. Certain subpopulations of macrophages are known to exert cytotoxic effects upon target cells by the release at close range of oxidating agents. We postulate that hapten derivatized CMC and MC, through unique properties of the carrier, bind to and possibly activate macrophages rendering them specifically suppressive for hapten binding B cells.