An immunoferritin labeling study of H-2 antigens on dissociated epithelial cells.

This report describes an immunoferritin labeling study of mouse H-2 histocompatibility antigens on epithelial cells dissociated from stomach, duodenum-jejunum, ileum, trachea, diestrus uterus, gall bladder, and vas deferens. Before cell dissociation, most of the organs were prefixed in periodate-lysine-paraformaldehyde to preserve the shape of the cells and to immobilize H-2 antigens in their native positions. Five kinds of epithelial cells expressed H-2 antigens on lateral and basal membranes but not on apical membranes. These were the lining cells of the upper intestine, ileum, gall gladder, uterus, and the tracheal brush cell. The antigens were continuously distributed on the lateral and basal membranes of these cells and appeared to be absent from the apical membranes, rather than masked by the fuzzy coat. On four other epithelial cell types H-2 antigens could not be detected. These were the lining cells of the vas deferens, parietal and chief cells from the stomach, and ciliated tracheal cells. It does not seem to be uncommon for normal nucleated cells to lack H-2 antigens. On fixed and labeled epithelial cells from the upper intestine the zonula occludens membranes were unlabeled, while the zonula adherens and desmosome membranes were labeled as densely as the remainder of the lateral membranes. The zonula occludens membrane thus constituted the boundary betewen the unlabeled apical membrane and the labeled lateral membrane of these cells. Intestinal epithelial cells dissociated without prefixation showed a patchy distribution of H-2 antigens on their lateral membranes after indirect labeling, indicating antigen mobility in this membrane. On the same unfixed dissociated cells the antigens were able to migrate from lateral to apical membranes, a movement which appears to be prevented in the intact epithelial layer by the occluding junction. The absence of H-2 antigens from apical membranes and their inability to migrate through an intact zonula occludens suggest that these molecules must reach the lateral membranes of epithelial cells by a pathway which is distinct from that followed by apical membrane components.     

The occurrence and distribution of H-2 antigens on mouse intestinal epithelial cells.

This report describes an immunoferritin labeling study of mouse H-2 histocompatibility antigens on epithelial cells dissociated from the small intestine by EDTA and trypsin. Before cell dissociation, the intestine was prefixed in paraformaldehyde or periodate-lysine-paraformaldehyde in order to preserve the shape of the cells and to immobilize H-2 antigens in their native positions. The results demonstrated the presence of H-2 antigens on the lateral and basal cell membranes at about the same high density that was observed at the surface of mouse monocytes. No H-2 antigens could be detected at the apical surface of dissociated or undissociated epithelial cells. It is unlikely that the fuzzy coat masked H-2 antigens at the apical surface because it was essentially absent from the apical membranes of dissociated cells. These observations extend our knowledge of the cellular distribution of transplantation antigens, and provide further evidence of a discontinuity in the expression of membrane components at the junctional complex of epithelial cells.     

Demonstration of Ia antigens on mouse intestinal epithelial cells by immunoferritin labeling

Several kinds of epithelial cells that express H-2 antigens were studied by immunoferritin labeling with an antiserum reacting only with antigens of theI region of theH-2 complex. Spleen lymphocytes were used to test the labeling system and the effect of the epithelial cell dissociation procedure on Ia antigens. Immunoglobulin-positive B10.BR lymphocytes were labeled with an anti-la^k serum (A.TH anti-A.TL serum absorbed with BALB/c and B10.D2 cells), while congenic B10.D2 lymphocytes were unlabeled. The distribution of labeled Ia antigens on living B10.BR lymphocytes was patchy, while on cells fixed in periodate-lysine-paraformaldehyde before labeling, the distribution of label was continuous. Fixation evidently immobilized Ia antigens in the lymphocyte membrane. Trypsin and collagenase, as used in the epithelial cell dissociation procedure, had no discernible effect on the Ia antigens of lymphocytes. The epithelial cells studied included the columnar absorptive cells of the small intestine, uterine lining epithelium, tracheal brush cells, and pancreatic exocrine and duct cells. These cells were fixed before dissociation from their respective tissues. Ia antigens were detected only on the columnar absorptive cells of the small intestine. These cells labeled equally well with an antiserum reacting only with theK -end of theH-2 complex. In both cases, congenic control intestinal cells were unlabeled. Thus, intestinal epithelial cells appear to express theIa, K, and presumablyD regions of theH-2 complex, while the other epithelial cell types express only the K and D antigens. On fixed intestinal epithelial cells, Ia and H-2K antigens were continuously distributed on the lateral and basal cell membranes including the zonula adherens, but the antigens were absent from the apical microvillous membrane and the zonula occludens.     

Synergistic interactions between lymph node and thymus cells in response to antigenic differences limited to selected regions of the H-2 complex.

Thymus and lymph nodes from the A.TL recombinant line were utilized as sources of responding cells in MLR (mixed lymphocyte response) assays to MHC-determined (major histocompatibility complex) antigenic differences. Cells from both sources were stimulated to proliferate by antigenic determinants controlled by the H-2K region alone, H-2D region and the H-2I-H-2S regions. Nylon-fiber-adherent splenic cells from each of the stimulating cell strains stimulated T-cell-dependent responses. Synergistic interactions between A.TL thymus and lymph node cells were initiated by antigenic products limited to single H-2 regions. Antigenic differences determined within the H-2I region were not required for synergistic responses to H-2K-controlled products or for the generation of cytotoxic killer cells to H-2D-associated antigens. The H-2I-region-associated products also were very effective in stimulating T-cell synergy. These data demonstrate that the two responsive T-cell subpopulations can both be stimulated by alloantigens coded within a single known H-2 region.     

Ia antigens: markers of specific T suppressors immune to H-2 complex antigens

Two antisera to Ia antigens, products of the H-2 complex I-Cd and I-JkEk subregions, respectively, have been obtained by immunisation of the F1 hybrids of recombinant strains of mice. These antisera are shown to display the 50 per cent cytotoxic effect in vitro in the presence of complement upon lymphocyte populations immune to the H-2 complex antigens and enriched for specific suppressor T cells (SSC) by fractionation on the monolayer of target cells. The specificity of anti-Ia cytotoxins is shown by the cross antibody absorption with T- and B-cells of mice originated from the recombinant H-2 haplotypes and bearing either particular I-Cd, I-Jk and I-Ek antigens, or their combinations. Anti-I-Cd cytotoxins are found to react with both B and T cells at a different rate, and the anti-I-JkEk serum contains two antibody types directed to I-Ek and I-Jk products, respectively, the latter being able to react preferently with T cells. Although both antisera do inactivate the in vitro SSC function in the presence of complement at a similar degree, the inactivating action of the anti-I-Cd serum, but not that of the anti-I-JkEk serum, occurs without complement. SSC are established to bear both Ia-antigens, I-J and I-C on the same cell, as demonstrated by the cross antibody absorption and variation of the H-2 origin of SSC. These two markers are suggested to function differently in the SSC immune to the H-2 antigens and the I-C antigen expression on the SSC surface is presumed to be required for their interaction with the inhibited responder T cells proliferating in MLC.     

The primary pulmonary lymph node complex in paracoccidioidomycosis

A case of primary pulmonary lymph node complex in paracoccidioidomycosis is reported.     

Narrow specificity of T-suppressor receptors immune to H-2 complex antigens

Specific d anti-b and b anti-a suppressor T cells induced by intravenous injection of mice with gamma-irradiated allogenic lymphoid cells, are not a homogenous population of cells as shown by their selective absorption on macrophage monolayers of various H-2 haplotypes. This is proved by separation of suppressor T cells to two subpopulations, at least, each of them being able to react with the products of only one (K or D) end of the H-2 complex. Moreover, the fine specificity study of d anti-b suppressor T cells enriched by elution from macrophage monolayers of different H-2 haplotypes, demonstrated these suppressors to represent a set of narrow-specific clones, each of them carried receptors reactive in a selective fashion with a particular determinant of the H-2 molecule irrespective of this linkage with other products of the H-2 complex. Two such clones reactive with H-2a and H-2f third-party antigens, respectively, were isolated by elution from the corresponding cell monolayers, each of them accounted for about 1.5% of the total d anti-b suppressor population. These data are discussed in the light of differences of suppressor T cells from other T cell subclasses and their resemblance to B cells with respect to the clonal structure and the receptor specificity.     

The temporal organization of the complex nodule in the lymph node

The aim of the work is to study rhythmic processes at tissue level in the lymph nodule and in the T-territory adjacent to it in order to reveal temporal interrelations in functioning the germinative center, crown and T-territory. The lymph nodule sections are stained with methylene green-pyronine. Small and middle lymphocytes, immunoblasts, plasmoblasts, immature and mature plasma cells, mitotic figures are taken into account. Spectral composition and rhythmic parameters are determined for each type of the cells in the ultradian, circadian and infradian ranges. Periods, approaching the circadian one, are revealed in the germinative centers for small lymphocytes and immature plasma cells. Practically, for all plasma cells the functional period near to 7 h is found; this attests the presence of the common synchronous rhythm driver. Phase difference of immunoblast, plasmablast, immature plasma cell fluctuation in the germinative centers makes it possible to suppose the time, necessary to transfer the immunoblast into the plasmablast (1.6-2.6 h) and the plasmablast into the immature plasma cell (3 h). Owing to the knowledge of the spectral composition of the rhythmic and parameters of certain components it is possible to approximate the total course of the process. Combination of fluctuations with various periods results in their recurrence in more prolonged time intervals.     

Relationship between trinitrophenyl and H-2 antigens on trinitrophenyl-modified spleen cells. I. H-2 antigens on cells treated with trinitrobenzene sulfonic acid are derivatized.

Spleen cells were treated with TNBS in order to determine if cell surface H-2 antigens are derivatized with TNP. By labeling the cell membrane of the TNP-modified cells with 125I, followed by detergent lysis and immune precipitation with anti-TNP, it was determined that no H-2 antigenic activity remained in the supernatant. Further, by the use of an antibody-induced antigen redistribution assay it was found that previous exposure to TNP-modified cells to anti-TNP in the absence of complement rendered these cells resistant to lysis by anti-H-2 in the presence of complement. Together these data indicate that at the concentration of TNBS used for modification, H-2 antigens are derivatized with TNP. However, in addition to H-2, other proteins including immunoglobulin were also derivatized with TNP. Anti-TNP cytotoxic effector cells were blocked from their cytotoxic activity by anti-TNP antiserum. These data indicate that TNP directly couples to H-2 antigens on the cell surface of TNP-modified cells and that TNP is associated with the antigenic determinant that the cytotoxic T cell recognizes.     

Spleen cells from animals neonatally tolerant to H-2Kk antigens recognize trinitrophenyl-modified H-2Kk spleen cells

A.TL mice injected with (A.AL × A.TL)F₁ cells within 24 hours after birth were rendered tolerant to H-2K^k antigens, as evidenced by acceptance of A.TL skin grafts. When spleen cells from these tolerant animals were cocultured with A.AL stimulator cells, no cytotoxic effector cells were generated in a cell-mediated lympholysis assay. However, when the A.AL stimulator cells were derivatized with trinitrophenol, effector cells that displayed a cytotoxic effect against trinitrophenyl-modified H-2K^k target cells were generated. These data indicate that animals tolerant to H-2 determinants but chimeric to only a minor extent possess cytotoxic precursor cells in sufficient frequency to mount a primary in vitro response against trinitrophenol in the context of an allogeneicH-2K region.     

Growth inhibition of Candida albicans by interleukin-2-induced lymph node cells

Previous reports have demonstrated natural killer cells (NK) to exert growth inhibitory effects against certain fungi, but not against Candida albicans. In this investigation, interleukin-2 (IL-2)-induced lymph node cells with phenotypic and functional characteristics of NK were shown to inhibit the growth of C. albicans. Growth inhibition was evaluated by both the release of 51Cr by the fungus and the inhibition of microcolony growth of the fungus on Sabouraud's dextrose agar. Lymphoid cells derived from C57Bl/6 mice and immediately assessed for hyphal growth inhibition showed little or no activity. However, significant hyphal growth inhibition was produced by lymph node cells cultured with recombinant IL-2. Growth inhibitory activity was dependent upon the concentration of IL-2 and was mediated by nonadherent lymphocytes which lysed an NK-susceptible and to a lesser extent an NK-resistant cell line. Treatment of the IL-2-induced cells with anti-asialo GM1 but not anti-Thy-1 and complement abrogated growth inhibition of C. albicans. These results suggest that IL-2-induced lymph node cells with functional and phenotypic characteristics similar to those of activated NK, mediate in vitro growth inhibition of the hyphal form of C. albicans.     

Detection of the localization of C1. botulinum antigens in regional lymph node cells by the immunofluorescence technic]

As a result of the study of lacalization of the C1. botulinum toxoids of the A, B, and E types in cells of the regional lymph nodes of rabbits by the indirect Coons' method and by the smear-print method it was revealed that different types of lymphoid tissue cells took part in the ingestion and digestion of these antigens; the antigens of the A and B types were at first revealed in the cytoplasm of pseudoeosinophils, and then in the macrophages. The antigen of the E type was revealed in the course of the whole experiment in the macro phages of the regional lymph node, although the number of pseudoeosinophils also increased. Apparently there was a different activity of pseudoeosinophils against the C1. botulinum toxoids, the types A, B and E.     

Synthesis of H-2 antigens by preimplantation mouse embryos

An enzyme-linked immunosorbent assay (ELISA), which utilized anti-H-2 monoclonal antibody, was used to detect H-2 antigens on preimplantation mouse embryos. All embryonic stages studied, including unfertilized eggs and 1-cell, 2-cell, 8-cell, and blastocyst-stage embryos, showed the presence of H-2 antigens. To prove that the H-2 antigens were not cytophilically adsorbed to the embryos, blastocysts were treated with papain to strip off the H-2 antigens, and then the embryos were further incubated to allow the H-2 antigens to regenerate. After a 3-h incubation time, 60% of the H-2 antigens on the embryos had reappeared, proving that the H-2 antigens were synthesized by the embryos themselves.     

Immunoprecipitation of insulin receptors by antibodies against Class 1 antigens of the murine H-2 major histocompatibility complex

In rat lacrimal gland, cholinergic, α- or β-adrenergic or methylxanthine stimulations of protein secretion are extracellular calcium dependent. 10 μM trifluoperazine (TFP) inhibited only cholinergic and α-adrenergic stimulations. Half maximal effect was observed at 30 μM, with all inducers except norepinephrine (3 μM). 10 or 30 μM TFP also suppressed the decrease of L-[³H]leucine incorporation into protein due to carbamylcholine. 100 μM TFP inhibited protein secretion and L-[³H]leucine incorporation. 500 μM TFP promoted cell lysis. It is suggested that: (a) at 100 μM TFP, inhibition is not specific for protein secretion; (b) at 30 μM TFP, inhibition could be related to a role of calmodulin in the secretory regulation process.     

Dimethyl sulfoxide induces expression of H-2 antigens on mouse lung carcinoma cells

The addition of dimethyl sulfoxide (DMSO) to cultures of line 1 carcinoma cells can increase the surface expression of H-2K and H-2D antigens at least 100-fold from barely detectable initial levels, as determined by using specific monoclonal antibodies and flow cytometry. H-2 values stabilize approximately 1 wk after exposure to maximally inducing concentrations of DMSO (3% vol) at densities found on normal spleen cells. Increased expression of H-2 antigens is not the result of cell selection, it is specific in that expression of an unrelated surface protein decreases, and it is associated with increased synthesis of these antigens as measured by incorporation of [35S]methionine. Additional DMSO-induced changes in the growth, cycling, lectin binding, and antigenic properties of line 1 cells are consistent with increased cell maturation. All changes are reversed when DMSO is removed. This system may facilitate study of products associated with differentiation that influence tumor cell malignancy.     

Association between H-2 and vaccinia virus-induced antigens on the surface of infected cells.

The relationship between H-2 molecules and vaccinia virus-induced antigens on the surface of H-2d infected cells was investigated by the differential redistribution method and by the blocking capacity of monospecific anti-H-2 sera on an anti-vaccinia cell-mediated cytotoxicity (CMC). Capping of either H-2K or H-2D molecules upon addition of monospecific and anti-H-2 sera was followed by the complete redistribution of viral antigens, suggesting the formation, on the cel membrane, of complexes of H-2K, H-2D molecules and vaccinia virus-induced antigens. However, not all H-2 molecules were involved in this association since i) free H-2K and H-2D molecules still moved independently on the cell surface, and ii) capping of vaccinia virus-induced antigens failed to induce the redistribution of all the H-2K and H-2D molecules. In addition, either monospecific anti-H-2K or anti-H-2D antiserum was found to exert potent blocking activity on anti-vaccinia CMC, indicating also a close topographical relationship between H-2K, H-2D molecules and vaccinia virus-induced antigens.     

The spatial relationship of the viral and H-2 antigens recognized by anti-viral CTLs

With the use of monospecific rabbit anti-G protein and mouse monoclonal anti-H-2K^k, we have analyzed the spatial relationship of the serologically defined H-2K^k antigens and the major surface glycoprotein (G protein) of vesicular stomatitis virus (VSV) to those antigens recognized by B10.A (k, d) anti-VSV cytotoxic T lymphocytes (CTLs). The ability of monoclonal anti-H-2K^k or rabbit anti-G protein to inhibit specifically the cytolytic activity of B10.A anti-VSV CTLs indicates that the G protein and the H-2K^k molecules are in close proximity to the viral and H-2K^k antigens recognized by the anti-VSV (CTLs. By the method of sequential immunoprecipitation, we also demonstrated that only 10–30 percent of the serologically defined G and H-2K^k molecules are in theG-H-2K ^k complexes.Abbreviations used in this paper Con A Concanavalin A - cpm counts per minure - CTLs cytotoxic T lymphocytes - E: T ratio effector: target ratio - G major surface glycoprotein of VSV - MHC major histocompatibility complex - MOI multiplicity of infection - NP40 Nonidet-P40 - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - SaCI Staphylococcus aureus, Cowan I strain - SDS sodium dodecyl sulfate - UV ultraviolet light