F. Paraskevas

Macrophage—T cell interactions: I. The uptake by T cells of Fc receptors released from macrophages

Abstract Using a rosette assay for the detection of cells carrying Fc receptors (Fc+) we have been able to show that in nylon wool (NWC), separated spleen cells from different strains of mice 12 to 18% are Fc+. Within 4 hr of culture in vitro at 37 °C, 75 to 85% of the Fc+ cells lose their Fc receptors and remain Fc receptor negative even after culture for 24 hr. However the addition of 5 to 10% syngeneic (but not allogeneic) peritoneal macrophages in the NWC, resulted in the preservation of the Fc receptors on 75 to 85% of the Fc+ cells originally present. Brief exposure of NWC which have been cultured in vitro for 4 hr (lost their Fc receptors) to supernates from 3 hr cultures of peritoneal macrophages reconstituted the fc+ cells by 75 to 85%. Only the supernates from syngeneic, but not allogeneic macrophages are active. Evidence is presented which indicates that these supernates contain Fc receptor molecules of small molecular weight. These molecules can be removed by antisera directed against the I region of the major histocompatibility complex.

The double gammopathies. Clinical and immunological studies.

A retrospective review of 1135 patients with paraproteinemias recorded 28 (2.5%) as having two M components. This group included 11 patients with myeloma, 6 with lymphoproliferative disease, 5 with a nonlymphoproliferative malignancy, and 6 with a double gammopathy of undetermined significance. In 13 cases in which the M components were measured over a period of time, three distinct patterns were observed, which may reflect the cellular and subcellular origin of the two proteins: 1) In 2 cases the minor component remained relatively stable while the dominant protein changed with time and treatment, suggesting the origin to be two cell lines--the minor arising from a quiescent clone of the monoclonal gammopathy of undetermined significance, and the major M component arising from a more rapidly proliferating plasma cell line; 2) a discordant pattern was seen in 4 patients, suggesting that the two M components arose from two separate plasma cell clones; 3) seven cases in which the proteins behaved in a concordant manner probably arose from a single plasma cell clone with incomplete class switching, producing two M components with different heavy chains.

Reverse immune cytoadherence A technique for the detection of surface-associated γ-globulins on lymphoid cells☆

Abstract A technique termed reverse immune cytoadherence is described for the detection of surface-associated γ-globulins on lymphocytes. Two populations of lymphoid cells, one carrying and one lacking such γ-globulins, are distinguished by this technique in the lymphoid organs and bone marrow of mice, guinea-pig and man. Thymocytes from all three species lack these surface-associated γ-globulins. In mature normal plasma cells as well as in plasmacytoma cells such surface-associated γ-globulin is not detectable despite the abundance of γ-globulin in the process of secretion. It is probable that this technique distinguishes the surface-associated γ-globulin (permanent membrane component) from the γ-globulin in the process of secretion (soluble). A mixed rosette technique was developed using two hybrid antibodies, one specific for mouse γG (7S γ2a) with an anti-ferritin site and the other for γF (7S γ1) with an anti-egg albumin site. The two indicator systems used were ferritin-coated bacteria and egg albumin-coated sheep red blood cells. Most rosette-forming cells in mouse spleen formed mixed rosettes thus demonstrating that they carry both γG and γF surface globulins and are pluripotential. Inhibition of rosette formation has been used to demonstrate the existence of a surface component, tentatively called an ‘Fc receptor’. This receptor binds the Fc of antibodies in antigen-antibody complexes of rabbit and mouse antibodies as well as Fc from papain-digested purified rabbit antibodies. Anti-lymphocyte serum (ALS) reacting with mouse spleen cells blocks the formation of rosettes in about 30–40% of the rosette-forming cells. The blocking is observed at very high dilutions of ALS and is achieved only by the synergistic effect of the 7S and 19S fractions when both are present within certain optimal concentrations.

Macrophage-T cell interactions: III. Production from T cells, under the influence of macrophages, of a factor which complexes Ig and antigen

Abstract Soluble complexes of Ig and antigen have been detected in the serum of mice within 6 hr after immunization. Such complexes are taken up by a subpopulation of T cells. We present evidence which suggests that the complexes are formed through the mediation of a factor released from T cells, tentatively called Ig-antigen complexing factor or IACF. IACF is produced as a result of a macrophage/T-cell interaction, when macrophages are present in an optimal proportion in relation to T cells (4%). Particulate or aggregated substances stimulate macrophages to release a mediator which subsequently acts on Fc receptor-negative T cells to produce IACF. Free-SH groups are important for the activity of the macrophage mediator. Mercaptoethanol and l -cysteine can also release IACF from T cells in the absence of macrophages. Protein synthesis is necessary for the production of this factor, the activity of which is abolished by trypsin digestion. It is postulated that the complexes of Ig and antigen formed under the influence of IACF represent a mechanism of presentation of antigen to T cells.

Macrophage-T cell interactions: IV. A new receptor on T cells: Ia antigens as receptors for Ig-antigen complexes which appear early after immunization

Abstract The Fc-receptor-positive T cells have been shown to bear a new receptor which is involved in the uptake of cytophilic complexes of Ig and antigen which are detected in the serum of mice within 6 hr following immunization. It was shown that the Fc receptor on T cells is not necessary for the uptake of these complexes. This new receptor is labile during culture of the T cells in vitro and disappears together with the Fc receptors. The new receptor is taken up by the T cells from a macrophage supernate. The evidence presented suggests that the receptor for these complexes bears determinants coded by the I region of the major histocompatibility complex but is distinct from conventional Fc receptor in that it does not bind to Fc fragments of Ig.

Activation in vivo of a major antisuppressor T-cell pathway immediately after immunization: III. T-cell requirements for its induction

Immunogenic stimuli rapidly induce a potent mediator with antisuppressor activity which represents complexes of Ig and antigen. The formation of the complexes depends on the interaction of two T cells both of which bear the Ly1 phenotype. The two T cells can be separated on the basis of their sensitivity to antilymphocytic serum and dependency on the presence of thymus. T cells bearing I region coded determinants are essential for the formation of the mediator.

Liposome stimulation of anti-BSA antibody production in mice

Liposomes consisting of phosphatidylcholine, cholesterol, and dicetylphosphate in a molar ratio of 7:2:1 were prepared in the presence of an aqueous solution of bovine serum albumin (BSA). The vesicles thus formed were observed to be stable for 2 weeks under physiological conditions. The uptake of liposomes by mouse peritoneal macrophages in vitro was documented using fluorescein-labeled BSA entrapped in the liposomes. Six-week-old, female, BALB/c mice were given a single intravenous inoculation of liposomes containing BSA and the production of anti-BSA antibody was measured using an ELISA technique. Controls consisted of free BSA, empty liposomes, and a mixture of free BSA and empty liposomes. Liposome-encapsulated BSA caused a large rise in anti-BSA antibody (anti-BSA) level that peaked within 2–5 weeks and was directly related to the dose of BSA within the liposome inoculum. The free BSA doses used produced either no (10, 25 μg) or a very low (250 μg) anti-BSA response in these mice. The empty liposomes caused no nonspecific production of anti-BSA either alone or when mixed with free BSA. The stimulation of anti-BSA by liposome-encapsulated BSA lasted for over 6 months and the antibody levels continued to be dose dependent.

Induction in vivo of enhancing factors for antibody production: II. Ig-Antigen complexes formed within 6 hours after immunization markedly enhance antibody formation

Abstract Serum collected 6 hr after injection of SRBC contains helper factor(s) which enhances antibody formation. The factor which markedly enhances the 7s response is an Ig-Ag complex. The complexes contain Ia antigens as shown by the ability of proper immunoadsorbents to remove both the antibody-enhancing activity for 7s, as well as the “cytophilic Ig” which is taken up by T cells. The enhancement of 19s is due either to a different factor which contains neither Ig nor antigen, or to the same Ig-Ag complexes which act at lower concentrations. Both 19s and 7s enhancing activities show no H 2 restriction in the presence of T cells. However no antibody enhancement was obtained when allogeneic 6HS was injected in thymectomized, lethally irradiated, and bone-marrow-reconstituted Balb/c mice, which suggests that the presence of T cells overcomes the H 2 barrier which exists when the complexes act directly on B cells. Since the complexes are formed through the mediation of a T-cell factor they may be considered a T-cell replacing factor and are predominantly concerned with the regulation of 7s response.

T-cell function in B-lymphocyte-deprived mice

Previous work has identified selective defect(s) in T cells in mice deprived of B lymphocytes by the chronic administration of anti-IgM antibody. Experiments described in the present communication revealed that anti-IgM-treated mice do not possess T cells with surface Ia and FcR, and, unlike T cells from normal animals, they also fail to bind these molecules in vitro. Functional assays disclosed that an anti-suppressor pathway which relies on Ia+ donor and acceptor T cells is interrupted in these mice at both levels. These observations may provide an insight to explain the selective failure of some T cells when B lymphocytes have been deleted.

Inhibition of antisuppression by the acute murine graft-versus-host reaction

Profound suppression of both humoral and cell-mediated immunity is a significant systemic effect of graft-versus-host reactions. Although no complete explanation has been advanced for this immunosuppression suppressor cells have been implicated. The data presented in this paper indicate that acute GVH reactions in (C57BL/6J X A/J) F1-hybrid mice induced by the injection of A/J cells severely disrupts the function of the antisuppressor T-cell pathway at both its induction and effector stages. Results show that within 3 weeks of induction of the reaction, Ly1+-T antisuppressor inducer cells lose their ability to generate the serum factor that mediates antisuppression. This factor is normally taken up by and activates Ly2+ T cells which then inhibit suppressor T-cell function. The data also reveal that Ly2+ T cells collected 2 weeks after induction lose their ability to be activated by the antisuppressor factor produced in normal mice. These cells are thus unable to function as antisuppressor effector cells. The uptake of the antisuppressor factor by Ly2+ T cells depends on the expression of Ia antigens on the surface of these cells. Experiments have shown that these antigens are absent from the surface of T cells derived from mice with GVH reactions. This finding may provide an explanation for the inability of these cells to function as antisuppressor effectors. Antisuppression is an important T-cell pathway that is intimately associated with the regulation of immune function. It is possible that the immunosuppression arising in mice with GVH reactions may stem, in part, from unopposed suppressor T-cell activity that results from widespread interference by the reaction with a pathway that normally inhibits suppressor cell activity.