The subcellular distribution of antigen in macrophages.

The intracellular fate of phagocytosed antigens in cells from peritoneal exudate in CBA mice has been studied by using (126)I and (131)I labeled antigens. After uptake of labeled antigen, cells were homogenized and the subcellular fractions were analyzed by isopycnic centrifugation in a sucrose gradient. The uptake of heat-denatured BSA (c BSA) by these cells in vivo is 3.5 microg/mg c BSA injected/10(8) cells. The uptake by cells in animals which were exposed 2 days earlier to 900 r whole body irradiation is slightly lower but does not differ significantly. 90% of the phagocytosed material is degraded within 2-3 hr, the residual 10% is retained at least over an 8 hr periods. Using a pulse and chase technique, with (125)I and (131)I c BSA in vitro and in vivo it was shown that newly phagocytosed antigen is found mainly in a lysosomal turnover compartment of a density 1.19 g cm(-3). Antigen which has been in the cells for longer was found in a denser fraction (1.26 g cm(-3)). In a comparison of nhrmal and X-irradiated cells it can be shown that after irradiation with 900 r less c BSA is found in this storage compartment. Binding of the antigen to the subcellular fractions, and its behavior towards several detergents has been studied. Subcellular fractions do not have the increased immunogenic capacity of antigen enclosed in living macrophages. Two synthetic polypeptide antigens, poly(D-Tyr, D-Glu, D-Ala) and poly-(L-Tyr, L-Glu) have a different subcellular distribution from c BSA, BSA, or bovine gamma globulin. Apart from also being found in the 1.26 storage compartment the polypeptide antigens are mainly located in a 1.15 compartment and only to a small extent in the 1.19 compartment. The half-life of these antigens in the cells is much longer than the half-life of the protein antigens studied. The finding of several subcellular compartments is discussed in connection with the functions possibly performed by macrophages.

Influence of different antigen doses on number and properties of antigen binding cells.

The number of antigen binding cells, as revealed by capping experiments, is strongly influenced by the concentration of antigen used. At high concentrations of antigen an additional population of cells of the size of lymphocytes can be detected, which is not stained after incubation with low doses of antigen. These cells bear immunoglobulin on their surface and can be removed from the spleen by carbonyl iron treatment.

Modulation of the antigraft response by preimmunization. Lack of correlation between regulatory events and graft survival.

Using two H-2 congeneic mouse strain combinations, the effect of donor specific i.v. preimmunizations with allogeneic spleen cells was studied. Parameters analyzed were survival time of the subsequent skin graft, graft-induced delayed-type hypersensitivity (DTH) reactions, and humoral immune responses evoked by immunization and grafting. Depending on the dose of allogeneic cells used for preimmunization, a suppression of the DTH response was observed, commonly accompanied by a similarly dose-dependent antibody formation that was inversely related to the DTH response. Neither DTH reactivity nor the antibody response were correlated in this system with the survival time of the skin graft. The only modulation of the survival time to be observed was an accelerated rejection of the graft caused by nonirradiated, as compared with irradiated, donor spleen cells. The data are discussed with regard to the clinically observed effect of blood transfusions on the survival of renal transplants and the relationship between DTH and graft rejection.

Differences in the induction of tumorspecific T suppressor lymphocytes among the peritoneal exudate cells in histocompatible BALB/cAnPt and BALB/cJ mouse sublines.

The BALB/c inbred strain of mice exists in several sublines, two of which are of special interest to tumorimmunologists. BALB/cAnPt mice develop plasmacytomas upon intraperitoneal injection of mineral oil in 61% of the animals. BALB/cJ mice on the other hand are relatively resistant and only a few animals develop tumors. The cellular immune response of BALB/cAnPt mice towards the transplantable plasmacytoma ADJ-PC-5 is characterized by the dominance of specific T suppressor (Ts) lymphocytes, when an immunization protocol is used which mimics some aspects of early stages of tumorigenesis by increasing exponentially the antigenic tumor load. Application of this in vivo induction protocol for Ts cells reveals that ADJ-PC-5-specific Ts cells capable of suppressing the generation of an in vitro cytotoxic response can be induced in BALB/cAnPt but not in BALB/cJ mice. No subline difference could be found when specific Ts cells were induced in vitro. The data point towards a subline difference of in vivo cell interactions rather than T cell repertoire composition.

Regulation of immune responses against the syngeneic ADJ-PC-5 plasmacytoma in BALB/c mice. IV. Tumor-specific T suppressor cells, induced at early stages of tumorigenesis, act on the induction phase of the tumor-specific cytotoxic T cell response.

The BALB/c plasmacytoma ADJ-PC-5 induces specific non-cytolytic T suppressor (Ts) cells under experimental conditions which closely resemble early stages of tumorigenesis. The phenotype of these Ts cells is Thy-1.2+, Lyt-1.2-, Lyt-2.2+, I-Ad-. They inhibit the induction of the cytotoxic T cells in a primary mixed lymphocyte tumor culture (MLTC) against the ADJ-PC-5 plasmacytoma. Specificity of suppression can be demonstrated by testing them in MLTC against a panel of syngeneic control tumor. Only the induction phase of a primary MLTC can be suppressed, and within 24 h of MLTC, cytotoxic T (Tc) cells become resistant towards suppression. ADJ-PC-5-specific Tc cell clones are also resistant to suppression. The fact that Ts cell-mediated specific suppression can be overcome by addition of semipurified IL2 to the MLTC suggests that Th cells, and not Tc cell precursors, are the target. The findings are discussed with respect to the potential use of Tc effector cells in cancer therapy.

Characterization of idiotype-specific I-Ed-restricted T suppressor lymphocytes which confine immunoglobulin class expression to IgM in the anti-alpha (1- > 3) Dextran B 1355 S response of BALB/c mice.

The humoral immune response of conventionally raised BALB/c mice to the so-called "thymus independent" antigen alpha (1- > 3) Dextran B 1355 S (Dex) is predominantly of the IgM class. The response is further characterized by Igha allotype linkage and the dominance of the public idiotypes (Id) J558 and MOPC 104. In germfree raised BALB/c and in BALB/c nu/nu mice immunized with the same antigen an additional IgG response of the public Id is observed. Analysis of the regulation of the class expression reveals existence of specific Ts cells in euthymic mice which must have been activated pre- or perinatally by exposure to environmental bacterial antigens. They permit or enforce differentiation of Dex-specific B cells into B gamma memory cells without allowing further development into IgG producing plasma cells. An analogue of these splenic Ts cells has now been cloned and identified as an I-Ed restricted Id-specific T cell with exactly the properties ascribed above to the splenic Ts cells. This paper describes phenotypical and functional properties of the Ts cell clone 178-4. It evaluates this clone's role in controlling efficient anti-bacterial IgM-mediated immunity under conditions where a class switch to IgG antibody production is actively suppressed; possibly as a measure to avoid hazardous autoimmune reactions on the basis of crossreaction and antigenic mimicry between polysaccharide antigens.

An evaluation of the existence of soluble suppressor factors in cloned antigen-specific T suppressor lymphocytes.

The suppressive activity of two bovine serum albumin-specific class II-restricted T suppressor cell clones (BVI/5 and 83/2-D11) was compared to that of a feeder cell-independent, IL 2-dependent subline (HF1.IL-2) of an originally antigen-dependent class II-restricted Ts cell clone (HF1). No soluble suppressor factors can be found in BVI/5 and 83/2-D11 Ts cell extracts or culture supernatants under conditions where an unspecific factor can be derived from HF1.IL-2 cells. This factor, when isolated from cell extracts in the presence of n-octyl-beta-D-glycopyranoside, has a molecular weight of 70-80 kD. In absence of this non-ionic detergent, it has a high affinity to membrane fragments and is associated with a molecular weight of 300 kD or more. The data are discussed in connection with recent findings of direct T suppressor to T helper interaction by cell-cell contact.

CD8+ tumor-specific Tc cells primed in vivo or in vitro against the BALB/c plasmacytoma ADJ-PC-5 use the same TcR V beta families but display distinct TC1 or TC2 characteristics.

The involvement of counteractive CD8+ T cell subsets in tumor-specific unresponsiveness was analyzed in a syngeneic murine tumor model. CD8+ cytotoxic T cells against the IL-10 producing BALB/c plasmacytoma ADJ-PC-5 can be easily induced in vitro, in a primary syngeneic mixed lymphocyte tumor cell culture (MLTC), or in vivo, by repeated immunization of syngeneic BALB/c mice with high doses of X-irradiated ADJ-PC-5 tumor cells. Long term cultivated CD8+ ADJ-PC-5-specific Tc lines use either TcR of the V beta 6 or V beta 8.1/8.2 type, irrespective if the lines were derived from a primary MLTC or from immunized mice. While most of the Tc lines produce type-1 cytokines (IFN-gamma, no IL-4) upon stimulation, at least two of them, which were derived from a primary MLTC, display a type-2 cytokine spectrum (IL-4, no IFN-gamma). The primary in vitro Tc response against ADJ-PC-5 cells shows characteristics of a TC2 response: CD8+ Tc cells which are induced in a primary MLTC do not produce IFN-gamma, and the tumor-specific Tc response is enhanced by IL-4 but suppressed by IFN-gamma or IL-12. In contrast, ADJ-PC-5-specific CD8+ Tc cells from immunized mice are IFN-gamma producing TC1 cells. Since the primary in vitro Tc response against the tumor is suppressed even by lowest numbers of irradiated ADJ-PC-5-specific TC1 cells via IFN-gamma, these TC1 cells behave similar to a previously described regulatory subset of IFN-gamma producing CD8+ T cells, which are induced during early stages of ADJ-PC-5 tumorigenesis and inhibit the induction of a tumor-specific Tc response from naive BALB/c spleen cells in vitro.

The thymus as primary site for antigen-specific T suppressor cells in neonatally induced tolerance to bovine serum albumin.

The ontogeny of antigen-specific T suppressor cells in thymus and spleen was analyzed in CBA/Ca mice which were rendered tolerant as neonates by subimmunogenic doses of bovine serum albumin (low-zone tolerance). Activity of T suppressor cells from those mice was assessed by an assay in which spleen cells from animals primed with fluorescein-conjugated human gamma globulin can be stimulated in vitro to produce IgG anti-fluorescein antibodies when cultured in the presence of fluorescein-conjugated bovine serum albumin. Carrier-specific T suppressor cells appear first in the thymus (day 10), and much later (day 30) in the spleen. The data are discussed in connection with the possible role of T suppressor cells during induction of tolerance in newborn mice.

Multidirectional interactions in vitro between lymphoid cells from Mls-disparate strains.

Chromosome 1 of the mouse carries a number of genes for alloantigens which can be recognized by T cells, among those the mixed lymphocyte stimulation (Mls) locus and the lymphocyte stimulating determinant (Lsd) locus have been described. The Relationship between the antigens coded for by these two loci has been further analyzed using a B10.BR anti C3H/Tif T cell clone E11. Segregation analysis was done with 167 individuals from backcrosses of (BALB/c (Mlsb, Lsd-) x DBA/2 (Mlsa, Lsd+] F1 animals into BALB/c mice. The analysis of the backcross data and of a limited number of recombinant inbred (RI) strains did not allow the segregated of the two loci, despite the fact that stable clusters were obtained in cluster analysis of the segregation data. The reasons for this failure are discussed. Parallel to the genetic analysis, we have studied the functional properties of the E11 T cell clone. First, there is the proliferation of the E11 T cells upon stimulation by macrophages or B cells of Mls-disparate stimulator strains. Second, there is an induction of differentiation of B cells of stimulator strains, requiring the direct interaction between E11 T and stimulator B cells. Third, there is an inhibitory influence of E11 T cells on the production of a mediator by Mls-disparate spleen cells presumably by their macrophages. We propose that the Mls antigen is a receptor regulating macrophage differentiation and transmitting a signal influencing the production of a mediator. This signal could be either the down regulation of the production of a stimulatory monokine or the induction of the production of an inhibitory monokine. Our presented data demonstrate that the anti Mls response is more complex than hitherto anticipated. It involves a multidirectional interaction between the Mls-disparate cell partners.

Involvement of CD28 cosignalling in the T cell-mediated suppression of the IgG antibody response against the TI-2 antigen alpha(1-->3) dextran.

The humoral immune response against alpha(1-->3) dextran (Dex) in BALB/c mice is characterized by the formation of predominantly IgM antibodies bearing the J558 idiotype. IgG antibodies do not appear in euthymic mice. In athymic animals, however, the response proceeds to a vigorous IgG production. In euthymic mice formation of IgG is suppressed by J558 idiotype specific regulatory T cells recognizing in association with I-Ed and in cognate T/B interaction the V(H) CDR3 derived peptide of the J558 idiotype. Only B-2 lymphocytes produce IgG whereas B-1 cells do not participate in the production of this Ig class. Using novel synthetic all alpha(1-->3)-D-gluco configured tetrasaccharide the Dex-specific B cells can for the first time be analyzed in FACS. In experiments using this newly designed low molecular Dex no signs of B cell apoptosis can be found. This demonstrates a true silencing of persisting Bgamma memory cells as previously suggested by adoptive transfer experiments. In this suppression a further involvement of CD28 and B7-1 interaction can be demonstrated which delivers a necessary costimulatory suppression signal in addition to the cognate TCR/peptide-I-Ed interaction between J558 specific T cells and J558 idiotype bearing B cells.

The genetic control of T cell-mediated immunity against the DBA/2 mastocytoma P-815. II. Low responsiveness in T cell-mediated cytotoxicity accompanied by the inability to produce antibodies in a secondary response.

Low responsiveness of DBA/1 mice in T cell-mediated cytotoxicity against the DBA/2 mastocytoma P-815 (H-2d) is genetically controlled and inherited as a recessive trait. For an analysis of the humoral anti-H-2d response of DBA/1 mice after immunization with P-815 mastocytoma cells both complement-dependent lysis (CDL) and the cytotoxicity inhibition assay (CIA) were used to demonstrate antibodies. Both assays show that DBA/1 mice are unable to produce anti-H-2d antibodies in a secondary response. Even high doses of immunizing cells give only a weak response. The response is fully recovered upon allogeneic interaction. A transient primary anti-H-2d activity can be found in the CIA but not by CDL. The data show that the genetically controlled determinant-specific defect in T cell-mediated cytotoxicity among DBA/1 mice has its correlate in a blockade of memory formation or expression in the humoral anti-H-2d response.

Selective elimination through a cytolytic mechanism of bovine serum albumin-specific T helper lymphocytes by T suppressor cells with the same antigen specificity.

An antigen-specific T suppressor cell clone isolated from a CBA/J mouse tolerized to low doses of bovine serum albumin (BSA) has previously been analyzed with regard to its effector functions. The T suppressor cell clone HF1 specifically inhibits T helper cell responses to the antigen. It also has characteristic cytolytic activity which can neither be classified as cytotoxic T cell nor as natural killer cell activity. Since this lytic capacity might be of relevance in immunoregulation, it has now been studied in more detail. For that purpose BSA-specific T cell lines have been isolated from immune CBA/J mice in order to test them in 51Cr-release assays as possible targets for HF1 T suppressor cells. Two T cell lines, both BSA specific and restricted to recognition of I-Ek major histocompatibility complex determinants, have been selected for the studies because one is a helper cell (83/1), the other a suppressor cell type (83/2). HF1 T cells are able to lyse cells of line 83/1 but not those of line 83/2. Control experiments show that 83/1 cells are not a natural killer cell target and that on the other hand 83/2 cells are susceptible to lysis in an alloreactive BALB/c anti-CBA/J cytotoxic T cell response. The extent of lysis of 83/1 T cells by HF1 T cells changes with time after antigenic stimulation. The lysis is based on direct effector: target cell interaction and not caused by soluble mediators. The data are discussed with regard to the effector function of a type of T suppressor cells which expresses I-A and I-E molecules and whose proliferation is restricted to the recognition of I-A or I-E determinants.