bluuebunny/crossref_metadata_2025 huggingface dataset:
Response to and production of interleukin 2 by peripheral blood and cerebrospinal fluid lymphocytes of patients with multiple sclerosis.
Authors: Merrill J E , Mohlstrom C , Uittenbogaart C , Kermaniarab V , Ellison G W , Myers L W
Published: December 2022

In an effort to further characterize the defective proliferative response of T lymphocytes to mitogens in multiple sclerosis (MS) patients, we examined the response to and production of interleukin 2 (IL 2) by both peripheral blood lymphocytes (PBL) and cerebrospinal fluid mononuclear cells. We also examined the proportion of cells bearing receptors for IL 2 and transferrin. Chronic progressive MS patients have an abnormally low response to exogenous IL 2 as compared to controls. Whereas acute relapse patients' PBL demonstrated a normal IL 2 response during an exacerbation, they showed reduced responsiveness during remission. These abnormalities could not be explained by different dose or kinetic response optima to PHA or IL 2, nor could they be explained by depressed numbers of IL 2 or transferrin receptor-bearing lymphocytes. Production of IL 2 by PBL was also abnormal in MS patients. Chronic progressive patients produced elevated levels of IL 2, whereas acute relapse patients undergoing an exacerbation produced diminished levels of IL 2. During remission, these levels returned to that of controls'. The effect of 1200 rad x-irradiation or nylon wool removal of adherent cells was a significantly greater augmentation of IL 2 production in MS patients than in other neurologic disease or normal controls. Cerebrospinal fluid lymphocytes from MS patients had normal proportions of IL 2 receptor-bearing cells, but were deficient in their IL 2 response and production as compared to autochthonous or control PBL. The inability of some MS patients' lymphocytes to clonally expand in response to IL 2 might contribute to the pathogenicity of the disease.
Experimental allergic encephalomyelitis: successful treatment in vivo with a monoclonal antibody that recognizes T helper cells.
Authors: Brostoff S W , Mason D W
Published: December 2022

Monoclonal antibody W3/25, which recognizes rat T helper cells, was injected i.p. or i.v. into Lewis rats with clinical signs of experimental allergic encephalomyelitis (EAE). This treatment, which was given 12 or 13 days after inducing EAE by the injection of purified myelin basic protein, significantly affected the course of the disease in that treated animals recovered, on the average, in 2 days, whereas control animals exhibited signs of EAE for a minimum of 4 days. Two pan-T cell monoclonal antibodies given in similar dosage failed to affect the course of the disease.
A permanent rat T cell line that mediates experimental allergic neuritis in the Lewis rat in vivo.
Authors: Linington C , Izumo S , Suzuki M , Uyemura K , Meyermann R , Wekerle H
Published: December 2022

A rat T cell line of the "helper" phenotype (W3/25-positive, OX 8-negative) has been derived from Lewis rats inoculated with P2 protein isolated from bovine PNS myelin. The line LiP2/A is exquisitely specific for P2 protein, exhibiting no reactivity to bovine basic protein or to PPD. In addition to responding strongly to the intact P2 protein, the line cells show some response to a synthetic peptide containing the neuritogenic amino acid sequence of P2 protein (SP-B, residues 66-78). Intravenous inoculation of naive rats with as few as 10(4) activated LiP2/A cells leads to the onset of mild clinical signs of experimental allergic neuritis. Higher doses of cells lead to more severe clinical disease. Histologic examination of clinically ill animals confirmed the disease as EAN. The pathologic lesions were confined to the PNS and spared the central nervous system. The lesions consisted of marked perivascular cuffs and infiltrates of inflammatory cells associated with marked degenerative changes--demyelination and some axonal degeneration.
The murine IL 2 receptor. I. Monoclonal antibodies that define distinct functional epitopes on activated T cells and react with activated B cells.
Authors: Ortega G , Robb R J , Shevach E M , Malek T R
Published: December 2022

The properties of three distinct rat monoclonal antibodies, designated 3C7, 7D4, and 2E4, to the murine IL 2 receptor have been compared in binding, biochemical, and functional assays. 3C7 appears to define an epitope near or identical to the IL 2-binding site of the receptor, because 3C7 inhibited the binding of radiolabeled IL 2 to CTL-L cells and because unlabeled IL 2 inhibited the binding of FITC-3C7 to CTL-L cells. 7D4 and 2E4 had no effect on IL 2 binding. Competitive antibody-binding studies confirmed that the epitope seen by 3C7 was distinct from the epitope(s) seen by 7D4 and 2E4. Sequential immunoprecipitation studies demonstrated that all three antibodies were reactive with the same molecular species, and that each precipitated identical components of 20,000 to 25,000 daltons, 50,000 to 60,000 daltons, and 100,000 to 120,000 daltons from the surface of CTL-L. FACS studies demonstrated a quantitatively and qualitatively identical cell distribution for the antigen defined by each antibody. They failed to stain more than 95% of resting lymphocytes, but were strongly reactive with Con A T blasts and substantially less reactive with LPS B blasts. Unlabeled IL 2 was also able to inhibit the binding of FITC-3C7 to LPS B cell blasts, suggesting the presence of IL 2-binding sites on activated B cells. Each antibody inhibited IL 2-driven proliferation of HT2 or CTL-L cells. 3C7 and 7D4 were more potent inhibitors of proliferation than was 2E4, and the combined use of 3C7 and 7D4 resulted in greater levels of inhibition of proliferation than that shown from the use of either antibody alone. Collectively, the results support the hypothesis that these antibodies detect two distinct functional regions of the IL 2 receptor.
High-level secretion of interleukin 2 by a subset of proliferating thymic lymphoblasts.
Authors: Caplan B , Rothenberg E
Published: December 2022

We have characterized the thymocytes that can be induced to secrete interleukin 2 (IL 2) after polyclonal stimulation with Con A. For maximal activation, an important adjunct to the Con A is the phorbol ester TPA. In the presence of TPA, IL 2 production by thymocytes is relatively independent of adherent accessory cells; this allows us to compare the abilities of different thymic subpopulations to make IL 2. The most numerous class that includes IL 2 producers is made up of cells with a typical "medullary" population, the phenotype: moderately small, postmitotic cells that fail to bind peanut agglutinin. In addition, however, a population of large, proliferating lymphoblasts is competent in IL 2 production directly as isolated. Relative to the total "medullary" population, the lymphoblasts are enriched for the ability to make IL 2. They account for a significant proportion of the total IL 2 produced by thymocytes, and demonstrate that this aspect of immunocompetence is not restricted to cells that have finished their intrathymic proliferation. The IL 2-producing lymphoblasts do not bind peanut agglutinin or express thymus-leukemia antigen, but they do express high levels of Lyt-1. Although distinct from most medullary thymocytes, therefore, they are also distinct from the majority of cortical blast cells for which a direct precursor role has been established. They may be a subset of the rare proliferating blast cells in the medulla. Further heterogeneity in the thymic IL 2 producers is demonstrated by their expression of the Lyt-2 glycoprotein. The majority of IL 2 producers are Lyt-2- as are the majority of peripheral T "helper" cells. However, a distinct minority of the thymic IL 2 producers express Lyt-2. Therefore, the ability of some peripheral Lyt-2+ cells to secrete IL 2 may be determined at the time of their initial programming in the thymus.
Comparison of allogeneic and self-restricted stimulation of lymphokine production by dual-reactive cloned T cells.
Authors: Wilde D B , Prystowsky M B , Beller D I , Goldwasser E , Ihle J N , Vogel S N , Fitch F W
Published: December 2022

Murine T cell clones were derived that proliferated in response to stimulation by alloantigen or by ovalbumin (OVA) in the presence of irradiated syngeneic spleen cells. Two cloned cell lines of strain B10.BR (H-2k) proliferated in response to alloantigen encoded by I-Ab, whereas the response to OVA was restricted by an element encoded by I-Ak. A cloned cell line of strain B10.A (H-2a) proliferated in response to alloantigen encoded by I-As, whereas the response to OVA was restricted by an element encoded by I-Ak. Cloned cells were stimulated by alloantigen or by OVA to produce lymphokines and to incorporate thymidine. Culture supernatants were collected 24 hr later and were assayed for interleukin 2, colony stimulating factor, interferon, Ia-inducing activity, and interleukin 3; thymidine incorporation was measured 72 hr after stimulation. For each clone tested, stimulation by alloantigen or by OVA led to the production of an identical array of lymphokines. Likewise, the strength of stimulation by alloantigen was approximately equal in magnitude to the strength of stimulation by a particular concentration of OVA. Lymphokine production and thymidine incorporation were co-variant measures of the intensity of stimulation. These data, in combination with data linking OVA reactivity and alloreactivity to identical regions of the major histocompatibility complex, suggest that dual reactivity represents a cross-reaction between alloantigen and self determinants associated with nominal antigen.
Induction of cytotoxic lymphocyte responses by antigen-specific helper factors.
Authors: Krowka J F , Shiozawa C , Diener E , Paetkau V , Pilarski L M
Published: December 2022

Antigen-specific helper factors (ASHF) were purified by antigen-affinity chromatography from supernatants of long-term helper T lymphocyte (TH) lines. We have modified an established helper-dependent assay system to demonstrate the antigen specificity and H-2 restriction properties of ASHF in the induction of cytotoxic T lymphocyte precursors (CTLp). Antigen specificity is demonstrated by the binding of ASHF molecules only to nominal antigen, both during purification and in tests of functional activity. Our ASHF preparations do not contain any interleukin 2 (IL 2) activity. The ASHF, purified by antigen-affinity chromatography in the presence of Ca++, is defined as Ca++-sufficient ASHF, whereas ASHF purified on antigen-affinity columns in the absence of Ca++ is defined to be Ca++ deficient. Ca++-sufficient ASHF is not H-2 restricted (as defined by the phenotype of the ASHF-producing cells) in the recognition of nominal antigen or in its interactions with CTLp or adherent stimulator cells. In contrast, when the "complete" (Ca++-sufficient) ASHF is functionally dissociated into subunits by removal of Ca++, the "incomplete" antigen-specific subunit of ASHF (Ca++-deficient ASHF) is still H-2-unrestricted in its ability to bind nominal antigen, but requires products from syngeneic adherent cells to trigger CTLp. When adherent cells that are H-2 identical to the ASHF are provided in culture, the "incomplete" ASHF is able to trigger either syngeneic or allogeneic CTLp in an antigen-specific manner. We interpret the results of our experiments to suggest that an H-2-restricted molecular interaction occurs in CTLp induction by ASHF. An antigen-specific, TH-derived receptor appears to require association with Ca++ and self major histocompatibility complex (MHC)-encoded molecules to form a "complete" ASHF that is able to trigger CTLp in an apparently H-2-unrestricted manner.
Soluble immune response suppressor (SIRS) inhibits microtubule function in vivo and microtubule assembly in vitro.
Authors: Irons R D , Pfeifer R W , Aune T M , Pierce C W
Published: December 2022

Soluble immune response suppressor (SIRS) is a product of concanavalin A-stimulated murine T cells that, when activated or oxidized by macrophages or H2O2 (SIRSox), suppresses in vitro immune responses and inhibits cell division by normal and neoplastic cells. SIRSox is inactivated by a variety of electron donors, which suggests that SIRSox may be an oxidizing agent. Incubation of lymphocytes with SIRSox, but not with SIRS, partially reversed concanavalin A-mediated inhibition of capping of membrane immunoglobulin on B cells, and disrupted the cytoplasmic array of microtubules visualized by fluorescence microscopy. SIRSox also inhibited microtubule assembly in vitro in a concentration-dependent manner. Inactivation of SIRSox by dithiothreitol prevented SIRSox-mediated reversal of inhibition of capping and inhibition of microtubule assembly. These results reveal a pattern of SIRSox activity similar to sulfhydryl-dependent cytoskeletal disrupting agents (e.g., N-ethylmaleimide, cytochalasin A, p-benzoquinone), and suggest that SIRSox-mediated suppression of proliferation may involve interference with sulfhydryl-dependent cytoskeletal events critical for cell division.
Purification and N-terminal amino acid sequence of the human T cell surface antigen T8.
Authors: Snow P M , Keizer G , Coligan J E , Terhorst C
Published: December 2022

Monoclonal antibodies allow for the detection of structures on the cell surface of human cytotoxic T lymphocytes (CTL) that are involved in their effector function. Among these cell surface components, T8 is of particular interest because it is required during the recognition of target cells by a subset of CTL. An understanding of its role during CTL:target adhesion requires detailed biochemical structural analysis of the T8 molecule. This has been hindered by the small amounts of protein currently available. Here we describe the development of a purification scheme that will permit the accumulation of larger quantities of T8. We studied the binding of T8 to several lectins and determined that one of these, wheat germ agglutinin, bound T8 quantitatively. Experiments designed to test the properties of T8 in a phase separation system with the use of Triton X-114 were performed. These indicated that T8 partitions into the aqueous phase rather than the detergent phase during this procedure. With this in mind, we developed a protocol that resulted in a significant purification of T8 after affinity chromatography. Upon preparative SDS-PAGE followed by electroelution, the T8 antigen was purified to homogeneity and used for N-terminal acid acid sequencing. This analysis yielded the amino terminal 22 amino acids of T8. On purification, it was observed that the protein existed as two bands of Mr 33 and 34 kilodaltons after SDS-PAGE analysis. The relationship between these chains was investigated by limited radio-sequencing and tryptic peptide map analysis; our results indicated that the two polypeptides were identical. The two chains were treated with trifluoromethane sulfonic acid to determine whether carbohydrates accounted for the difference in m.w. This reagent, which cleaves both N-linked and O-linked sugars, cleaved approximately 2000 daltons of oligosaccharides from the T8 molecule. These oligosaccharides are most likely of the O-linked rather than the N-linked variety.
A limited region within hen egg-white lysozyme serves as the focus for a diversity of T cell clones.
Authors: Manca F , Clarke J A , Miller A , Sercarz E E , Shastri N
Published: December 2022

The C57BL/6 (H-2b) mouse is a nonresponder to hen egg-white lysozyme (HEL) injected i.p., owing to a T suppressor cell-inducing determinant at the amino-terminal region. After immunization with a 93-amino acid fragment (a.a. 13-105) of HEL lacking this determinant, all clones from two independently derived C57BL/6 T cell lines were found to be specific for epitopes within a subregion of peptide 74-96. Three specificity patterns for the clones could be defined on the basis of cross-reactivities with only two other species variant lysozymes. Reactivities of all three specificity groups was consistent with the serine to threonine substitution at position 91, although reactivity of one of the groups could be affected by substitutions at position 84. The results confirm at the clonal level that even for distantly related antigens, only limited regions are recognized by T cells. They are consistent with the notion that specific sites on the antigen capable of interaction with Ia molecules lead to dominance of certain regions for T cell reactivity. Moreover, the diversity in specificity among clones suggests that the limiting feature of T cell responsiveness is not a lack of available T cells in the repertoire directed against a single antigenic site.
Analysis of the reactivity of four anti-mouse IgM allotype antibodies with mu+ B lineage cells at various stages of differentiation.
Authors: Velardi A , Kubagawa H , Kearney J F
Published: December 2022

We have used two rat monoclonal antibodies (Mab) (Bet-1 and 331.12), a mouse Mab (AF6-78.25), and an alloantiserum (SJA anti-BAB/14) in two-color immunofluorescence analysis and enzyme-linked immunosorbent assays to examine the expression of IgM allotypes at various stages of mouse B cell development, especially at the pre-B cell stage. In agreement with findings previously reported by others, these antibodies displayed the patterns of allotypic reactivity with IgM+ B cells and plasma cells from the appropriate strains of mice: Bet-1 antibody is specific for a allotype, AF6-78.25 and SJA anti-BAB/14 for b allotype, and 331.12 for both a and b allotypes but not e allotype. These antibodies, however, did not react with isolated mu-heavy chains prepared from secreted molecules or synthesized in situ by normal pre-B cells or pre-B cell-derived hybridomas. The requirement of light chain participation in the expression of IgM allotypic determinant(s) was additionally suggested by analysis of a fetal liver-derived hybridoma (F1-26-11) that has been shown to secrete IgM heteromolecules composed of C57BL/6-derived mu-chain and BALB/c-derived kappa-chains. In contrast to the nonreactivity with C57BL/6 mu-only pre-B cell-derived hybridomas, all three antibodies with specificity for the b allotype (AF6-78.25, SJA anti-BAB/14, and 331.12) reacted with this particular hybridoma. After treating surface IgM molecules on B lymphocytes with papain to cleave Fab fragments, the b allotype reactivity of AF6-78.25 mouse Mab, but not 331.12 rat Mab, disappeared, whereas the a allotype reactivities of two rat Mab (Bet-1, 331.12) were not altered. These results suggest that the IgM allotypic epitopes defined by these antibodies appear to be sterically dependent on the assembly of the whole IgM molecules, and rat Mab 331.12 and mouse Mab AF6-78.25 define two separate b allotype specificities, one on the Fc portion and the other on the Fab portion of the IgM molecules, respectively.
Species-restricted target cell lysis by human complement: complement-lysed erythrocytes from heterologous and homologous species differ in their ratio of bound to inserted C9.
Authors: Hu V W , Shin M L
Published: December 2022

The cytolytic efficiency of the terminal complement complex (C5b-9) against erythrocytes of different species is, in part, dependent on the species of C9 origin. In the present study, we have examined the interaction of C9 with erythrocytes in terms of the binding, dimerization, and insertion of C9 into the membranes of sheep and human erythrocytes lysed by human complement (C). The membranes of these C-lysed erythrocytes were analyzed for bound, dimerized, and inserted C9 by a combination of photolabeling, SDS-PAGE, electroblotting, and immunostaining techniques. We found that neither binding nor dimerization of C9 could be correlated with the relative hemolytic efficiency of human C on these erythrocytes, but that C9 insertion into the membranes of these cells varied in direct relation to the extent of lysis. Interestingly, the binding of C3 to these cells under conditions of equivalent C1 fixation also correlated with lytic efficiency. These data indicate that the C9-related differences in the cytolytic efficiency of C against erythrocytes from different species is primarily due to the efficiency of C9 insertion into these cells. Moreover, these data emphasize that neither the binding of C9 to a target membrane nor the formation of C9 dimers necessarily leads to the insertion of C9 into the membrane, suggesting the presence of membrane-bound but inactive C5b-9 complexes. Because the extent of C3 binding also correlated with the relative degree of lysis of sheep vs human erythrocytes, the possibility exists that surface-bound C3 may regulate hemolysis by directing the insertion of C9 in terminal complexes into cells.
In vivo transfer of persisting (P) cells; further evidence for their identity with T-dependent mast cells.
Authors: Crapper R M , Thomas W R , Schrader J W
Published: December 2022

Previously we described the persistent in vitro growth of lines of cells (persisting [P] cells) that shared many cytochemical, biochemical, and functional characteristics with mast cells and depended for their survival and growth on a specific T cell-derived factor, P cell-stimulating factor (PSF). Here we present further evidence for their identity with the T-dependent or atypical subset of mast cells and show that they retain characteristics of T-dependent mast cells when transferred in vivo. One week after the injection of P cells into the dermis of mutant Wf/Wf mice, which have a genetically determined deficiency in mast cells, large numbers of mast cells were present at the injection site, although by 2 wk or later these had disappeared. These mast cells resembled T-dependent mast cells rather than connective tissue mast cells in terms of their size and staining characteristics. Further evidence that these mast cells belonged to the T-dependent subset was that they retained their sensitivity to PSF. Thus, if P cells were injected into the dermis of Wf/Wf mice that bore in one groin a subcutaneous tumor (WEHI-3B) that produced PSF, increased numbers of mast cells were still evident at the injection site 4 wk later; this was not the case in mice bearing a non-PSF-producing variant of the same tumor. Experiments with cloned P cells generated from mice bearing the beige (bgJ/bgJ) mutation and with the giant granules of cells of this genotype used as a marker showed conclusively that the mast cells at the injection sites were derived from the injected P cells. P cells sensitized in vitro with monoclonal antigen-specific IgE or IgG1 antibodies and then injected intracutaneously into W/Wv mice transferred local cutaneous anaphylactic responses. P cells sensitized with IgG1 transferred local cutaneous anaphylactic responses to rats. These results support the view that P cell lines are cognate with the atypical or T-dependent subset of mast cells and that these cells retain their functional capabilities when injected in vivo.
LPS regulation of the immune response: separate mechanisms for murine B cell activation by lipid A (direct) and polysaccharide (macrophage-dependent) derived from Bacteroides LPS.
Authors: Williamson S I , Wannemuehler M J , Jirillo E , Pritchard D G , Michalek S M , McGhee J R
Published: December 2022

Lipopolysaccharide (LPS) derived from Bacteroides fragilis has been reported to stimulate mitogenic responses in spleen cell cultures from the classical LPS-hyporesponsive C3H/HeJ mouse strain; however, we have shown that purified splenic B cells from C3H/HeJ mice are hyporesponsive to phenol-water extracted LPS from B. fragilis ATCC 25285 (B-LPS). In the present study, B-LPS and its purified lipid A and polysaccharide components were tested for their ability to induce mitogenic and polyclonal IgM synthesis in spleen cell and purified splenic B cell cultures from classical LPS-responsive and -hyporesponsive mice. Mitogenic responses to B-LPS and E. coli K235 LPS(Ph) of whole spleen cells (2 X 10(5) cells/culture) or purified B cells (5 X 10(5) cells/culture) from classical LPS-responsive mouse strains (C3H/HeN, BALB/c, C57BL/6J, C57BL/10Sn, and DBA/2), F1 mice (derived from crosses between LPS responsive and C3H/HeJ mice), and classical LPS-hyporesponsive mice (C3H/HeJ and C57BL/10ScN) were high, intermediate, and low, respectively. When a higher number of whole spleen cells (5 X 10(5) cells/well) were cultured, B-LPS induced high mitogenic responses in C3H/HeN, intermediate responses in F1, and lower but significant responses in C3H/HeJ cultures. Similar results were obtained when polyclonal IgM synthesis was assessed in cultures containing 1 X 10(6) cells/culture. In contrast, the purified lipid A component of B-LPS failed to induce mitogenic responses in either whole spleen or purified B cell cultures. The addition of purified splenic B cells from C3H/HeJ mice to C3H/HeN or C3H/HeJ splenic adherent cells resulted in mitogenic responses to B-LPS, implying that the hyporesponsiveness to B-LPS seen in whole spleen cell cultures from C3H/HeJ mice at the lower cell concentration was due to limiting numbers of M phi. When splenic B cells and M phi from either C3H/HeN or C3H/HeJ mice were incubated with the lipid A or the polysaccharide moiety of B-LPS, lipid A induced mitogenic responses only in C3H/HeN cultures, whereas the polysaccharide moiety induced similar responses in both C3H/HeN and C3H/HeJ cultures. These results suggest that Bacteroides lipid A does not stimulate B cells from the classical LPS-hyporesponsive C3H/HeJ mouse strain, whereas the polysaccharide moiety of B-LPS is biologically active and mediates B cell stimulation via M phi.
Expression of Lyb-5 and Mls determinants by Peyer's patch B lymphocytes of X-linked immunodeficient mice.
Authors: Eldridge J H , Yaffe L J , Ryan J J , Kiyono H , Scher I , McGhee J R
Published: December 2022

Treatment of the Peyer's patch (PP) B cells from X-linked immunodeficient (xid) (CBA/N X DBA/2)F1 male mice with anti-Lyb-5 plus complement kills approximately 75% of these cells, although xid spleen B cells are unaffected. Cytotoxic depletion of Lyb-5+ cells renders the xid PP B cell population unable to generate an in vitro direct plaque-forming cell response to the TI-2 antigen TNP-Ficoll. In addition, the B cell-enriched population from the PP of xid (CBA/N X CBA/J)F1 male mice were strong stimulators of proliferation in an M1s-defined MLR, thereby demonstrating that they also express the M1s antigen(s). Two cell surface antigens associated with mature B cells are therefore expressed by xid PP B cells, suggesting that the TNP-Ficoll responsive cells in this population are mature B cell.
Size-dependent B lymphocyte subpopulations: relationship of cell volume to surface phenotype, cell cycle, proliferative response, and requirements for antibody production to TNP-Ficoll and TNP-BA.
Authors: Thompson C B , Scher I , Schaefer M E , Lindsten T , Finkelman F D , Mond J J
Published: December 2022

Murine splenic B lymphocytes were separated into size-dependent subpopulations by using counterflow centrifugation. Spleen cells were rigorously depleted of T lymphocytes to yield a population of cells that were greater than 90% surface immunoglobulin (Ig)-positive and that had a mean cell volume of 136.6 +/- 3.3 microns. From this population, five fractions of cells were obtained with mean cell volumes that ranged from 115.8 +/- 3.7 microns in fraction 1 to 168.0 +/- 6 microns in fraction 5. The cells in these five subpopulations were characterized by analysis on a fluorescence-activated cell sorter after staining with acridine orange to evaluate RNA and DNA content, and with fluorescein-conjugated anti-mu, anti-delta, and anti-Ia antibodies to evaluate their surface membrane phenotypes. DNA analysis revealed that virtually all of the cells in fractions 1 to 4 had 2 N DNA. Between 7 and 21% of fraction 5 cells were either in S-phase or contained 4 N DNA. In contrast, RNA content increased through the fractions, suggesting a transition from G0 to G1 in the subpopulations with increasing B cell size. As another measure of cell activation seen with increasing cell size, we observed a progressive increase in the expression of surface Ia and a decrease in the expression of surface IgD. In the absence of in vitro stimulation, the larger cells showed significantly higher levels of thymidine incorporation. When polyclonal B cell activators such as LPS or anti-Ig antibody were added, peak proliferative responses were similar in all of the fractions, but the time necessary to achieve a maximal response was shorter for the larger-sized cell subpopulations than it was for the smaller-sized cell subpopulations. Unprimed, size-dependent B lymphocyte subpopulations exhibited spontaneous or "background" antibody formation that occurred primarily in the subpopulations containing the largest cells. T cell factors present in EL4 supernatant enhanced the efficiency of in vitro differentiation of these same subpopulations. When cultured in the absence of T cell help, the thymus-independent type 1 (TI-1) antigen TNP-Brucella abortus (TNP-BA) or the thymus-independent type 2 (TI-2) antigen TNP-Ficoll induced the largest anti-TNP plaque-forming cell (PFC) responses in the fractions containing intermediate-sized cells, suggesting that in vitro, antigen-specific responses came primarily from B cells that have been influenced in vivo to leave their small resting state. The subpopulations containing the smallest size B cells required the presence of both a TI antigen and EL4 supernatant for efficient differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)
Peripheral (somatic) expansion of the murine cytotoxic T lymphocyte repertoire. II. Comparison of diversity in recognition repertoire of alloreactive T cells in spleen and thymus of young or aged DBA/2J mice transplanted with bone marrow cells from young or aged donors.
Authors: Gorczynski R M , Chang M P
Published: December 2022

Lethally irradiated (1000 R whole body) DBA/2J mice of 10 wk or 20 mo of age were repopulated with anti-Thy-1.2-treated DBA/2J bone marrow cells of 10-wk- or 20-mo-old donors. Sixty days post-transplant, limiting dilution cultures of the spleen and thymus cell population of individual mice (for each group) were examined to assess the within-group and between-group diversity in the anti-H-2Kb allo-recognition repertoire. Our data are consistent with a significant expansion of the CTLp repertoire taking place in the periphery, beyond the early appearing specificities present in the thymus. Moreover, comparison of the repertoires in young recipients of young or aged marrow, or in aged recipients of young or aged marrow, support the notion that there is a defect in the peripheral environment of aged mice that results in altered expansion of the thymic CTLp repertoire. In addition, there is an intrinsic difference in bone marrow precursor cells of CTLp in aged mice that is revealed only in an aged environment.
Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. XIV. Carrier requirement for suppressor cell induction.
Authors: Sherr D H , Vietor H E , Liu Y N , Dorf M E
Published: December 2022

The carrier requirements for the induction of helper and suppressor T (Ts) cells were compared. Although H-2-linked Ir genes control the development of helper T cells and hapten-specific B cells, they do not influence Ts3 generation. That is, GL phi nonresponder C57BL/6 mice can generate NP-specific Ts3 cells after priming with NP-GL phi. The Ts3 cells generated under these conditions are functionally and phenotypically identical to the NP-specific Ts3 cells previously characterized. Furthermore, these Ts3 populations can be specifically depleted with a monoclonal anti-idiotope antibody prepared against monoclonal anti-NP antibodies. By using related polymers, carrier effects on Ts3 induction were noted. NP-D-GL and NP-Ficoll failed to induce Ts3 cells, whereas NP-L-GL induced this suppressor subset. The data demonstrate that Ts3 induction is independent of the carrier requirements involved in helper T cell induction and is not dependent upon B cell priming. The implications of these results with regard to the mechanisms of Ts3 induction are discussed.
Continuous cultivation of equine lymphocytes: evidence for occasional T cell-like maturation events in horses with hereditary severe combined immunodeficiency.
Authors: Magnuson N S , Perryman L E , Wyatt C R , Ishizaka T , Mason P H , Namen A E , Banks K L , Magnuson J A
Published: December 2022

Peripheral blood mononuclear cells (PBMC) from 14 foals with hereditary severe combined immunodeficiency (SCID) were studied to determine the extent of lymphocyte differentiation that occurs in this disorder. PBMC from all 14 horses had the morphologic characteristics of large granular lymphocytes (LGL). Cells from only one of 14 horses were responsive to phytolectin stimulation in a standard blastogenesis assay; however, PBMC from all 14 horses proliferated in continuous culture in the presence of partially purified interleukin 2. Furthermore, there were differences in the growth patterns of these cultured cells that correlated with their ability to respond to phytolectin stimulation. PBMC obtained from the 13 phytolectin-unresponsive foals survived in culture for only 4 to 5 wk, divided very slowly, developed large granules composed primarily of calcium phosphate, and accumulated high concentrations of histamine. In contrast, PBMC from the phytolectin-responsive SCID foal proliferated in continuous culture for over 100 days, divided as rapidly as normal equine PBMC under identical culture conditions, and did not accumulate granules or histamine. These observations indicate that lymphoid cell differentiation occurs in some horses with SCID even though the identity of the LGL is unresolved. Two possibilities are that LGL are products of a pathway separate from that of lymphocytes or that LGL are precursors of mature lymphocytes.
Specific killing of lymphocytes that cause experimental autoimmune myasthenia gravis by ricin toxin-acetylcholine receptor conjugates.
Authors: Killen J A , Lindstrom J M
Published: December 2022

Experimental autoimmune myasthenia gravis (EAMG) is an autoimmune disease in which antibodies to acetylcholine receptor (AChR) cause loss of AChR from muscle, thereby impairing neuromuscular transmission. Here we report the use of a hybrid molecule that contains ricin toxin, irreversibly coupled to AChR to specifically suppress the immune response to AChR in vitro. Lymph node cell cultures from rats with EAMG pretreated with ricin toxin-AChR conjugates exhibited suppressed T helper cell proliferation and B cell antibody synthesis in response to the subsequent addition of AChR. Nonspecific toxicity of the conjugates was measured by suppression of the T cell proliferative response to the mitogen concanavalin A and the antigen keyhole limpet hemocyanin (KLH), and B cell antibody production to KLH. We have evaluated different pretreatment conditions and ricin toxin covalently coupled to AChR in different molar ratios to optimize specific immunosuppression. By varying the number of ricin molecules covalently bound to AChR in the immunotoxin, we were able to minimize the nonspecific toxicity while still maintaining specific killing of AChR-reactive lymphocytes. Furthermore, B cells were more susceptible to specific killing than were the T cells. The specific immunosuppression was potentiated by performing the pretreatment with immunotoxin in the presence of chloroquine. Chloroquine raises lysosomal pH and probably delays the degradation of immunotoxin in the cell. It should be noted that ricin toxin was covalently coupled to AChR by using a novel, non-reducible reaction. These in vitro results suggest that it may be feasible to use immunotoxin molecules to specifically suppress this autoimmune response in vivo.
Differential binding of chemoattractant peptide to subpopulations of human neutrophils.
Authors: Seligmann B , Chused T M , Gallin J I
Published: December 2022

Binding of the fluoresceinated chemoattractant N-formylmethionylleucylphenylalanyllysine to neutrophils was measured simultaneously with cell membrane potential by flow cytometry to determine how chemoattractant was bound by different populations of responding neutrophils. Cells exhibiting an apparent depolarization of membrane potential bound more chemoattractant than cells which did not respond or which exhibited a small hyperpolarization. Approximately 45% of the peptide bound to depolarized cells was displaced by unlabeled peptide, whereas only 3% was displaced from the nonresponding cells. By utilizing the well-known observation that extracellular fluorescein fluorescence is quenched by acid pH, it was determined that approximately one-half of the nondisplaceable peptide of both cell types was internalized, but a significant amount remained tightly bound to the extracellular membrane surface and accessible to lowering of the extracellular pH to 5.5. Treatment of cells with cytochalasin B to convert nonresponding cells to depolarizing cells also resulted in expression of displaceable binding by these cells, suggesting a close correlation between displaceable peptide binding and depolarization. Differential binding of chemoattractant among neutrophils and its modulation may account for observations of functional heterogeneity of neutrophils.
The requirement for proteinase activity for human lymphocyte-mediated natural cytotoxicity (NK): evidence that the proteinase is serine dependent and has aromatic amino acid specificity of cleavage.
Authors: Hudig D , Redelman D , Minning L L
Published: December 2022

We used reagents specific for serine-dependent proteinases to verify that a proteinase of this class is necessary for natural cytotoxicity (NK). NK was inhibited by phenylmethylsulfonylfluoride (PMSF), by diisopropylfluorophosphate (DFP), and by the plasma antiproteinase alpha-1-antichymotrypsin (alpha-1-X), all of which are specific for serine-dependent proteinases. Substrate specificity was then determined on the basis of the specificity of the plasma and fungal anti-proteinases and synthetic alternate substrates that affected NK. alpha-1-X, which inhibits only serine proteinases with aromatic amino acid specificity, blocked NK. Chymostatin, but not other fungal inhibitors, also blocked NK activity. Furthermore, the only synthetic substrates that effectively reduced NK were those derived from aromatic amino acids. The ester derivatives of these substrates inhibited NK better than the amides. NK inhibition with these alternate substrates was also stereospecific, with the L forms twofold more active than the D forms. These reagents did not block initial lymphocyte-target cell binding. Therefore we propose that the "NK-proteinase" is involved in either the initiation of cytolysis, perhaps as part of stimulus and secretion of cytolytic molecules, or in the cascade of events that may lead to the formation of final lytic substance.
Capsular polysaccharides of nongroupable streptococci that cross-react with pneumococcal group 19.
Authors: Lee C J , Koizumi K , Henrichsen J , Perch B , Lin C S , Egan W
Published: December 2022

The cross-reactivity and chemical characterization of the nongroupable streptococcal and pneumococcal group 19 polysaccharides (PS) have been studied. Extensive cross-reactions were observed between capsular PSs of streptococcal strains 14636/74, 4907, 4731 and pneumococcal type 19F and 19A antisera. Streptococcal 14636/74 PS had an identical composition to that of pneumococcal 19F PS. Type 19F and 14636/74 PS were composed of equimolar amounts of rhamnose, glucose, N-acetyl mannosamine, and phosphorus. The capsular PS of strains 4731 and 4907 contained rhamnose, glucose, ribose, N-acetyl mannosamine, and N-acetyl glucosamine in different molar ratios. Extensive immunologic reactivity was observed between the 19F and 14636/74 PS, as determined by light scattering rate nephelometry, passive immune hemolysis, and precipitin reaction. There was an identity reaction by immunodiffusion between type 19F and 14636/74 PS when reacted with rabbit antiserum against either organism. Biochemical studies showed that strain 14636/74 was not a pneumococcus, because it was optochin resistant, was bile insoluble, did not possess the C-carbohydrate antigen common to all pneumococci, and produced neither pneumolysin nor IgA protease. Furthermore, it grew in comparatively simple media in contrast to the complex nutritional requirements of pneumococci. The 13C-NMR spectra of the 19F and 14636/74 PS were identical. These two capsular PS can, therefore, be considered identical.
Mechanisms of resistance to the intracellular protozoan Encephalitozoon cuniculi in mice.
Authors: Schmidt E C , Shadduck J A
Published: December 2022

Mechanisms of resistance to the obligate intracellular protozoan Encephalitozoon cuniculi were studied in BALB/c mice. Resistance to lethal disease was T cell-dependent because transfer of T-enriched, but not T-depleted, spleen cells from sensitized BALB/c donors would protect infected BALB/c-nu mice. A modified focus-forming assay was utilized to measure effects on E. cuniculi infectivity in vitro. The results show that antibodies exert an opsonization effect and may block parasite entry into nonphagocytic cells. No cytotoxic T cells were demonstrated. Supernatants from E. cuniculi-sensitized spleen cells incubated with E. cuniculi in vitro could induce adherent PEC to kill E. cuniculi.
Cryptic epitope explains the failure of a monoclonal antibody to bind to certain isolates of Trypanosoma cruzi.
Authors: Kirchhoff L V , Hieny S , Shiver G M , Snary D , Sher A
Published: December 2022

A mouse monoclonal antibody, WIC 29.26 Ab, has previously been characterized as recognizing a carbohydrate epitope on a 72,000 m.w. glycoprotein (GP72) expressed on the surface of Trypanosoma cruzi epimastigotes and metacyclic trypomastigotes. This molecule has been implicated as a receptor in the control of parasite transformation, and when used as an immunogen in mice, partially protects against T. cruzi infection. In previous experiments in which a radioimmunoassay was used, WIC 29.26 Ab was found to react with approximately 50% of T. cruzi strains and clones derived from a variety of sources. In this study, we attempted to determine whether the WIC 29.26 Ab-nonreactive isolates lack the entire GP72 or merely lack the epitope recognized by this monoclonal antibody. WIC 226.4 Ab, a monoclonal antibody raised against periodate-treated GP72, reacted in an immunofluorescence assay with all strains and clones studied, including those which had not reacted with WIC 29.26 Ab. Likewise, two polyvalent rabbit sera, directed specifically against GP72, bound to all T. cruzi isolates tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of detergent lysates of surface-labeled epimastigotes immunoprecipitated with WIC 29.26 Ab showed that the epitope bound by this antibody was present in all but one of the parasites that were surface-nonreactive, as well as in all those that were surface-reactive. WIC 29.26 Ab precipitated a single 72K Mr band from most strains and clones, but in several cases 79K Mr and 66K Mr bands were seen. Isolates from both the surface-reactive and the surface-nonreactive groups showed the latter pattern. These results demonstrate that GP72, or similar electrophoretic variants--and with one exception, the carbohydrate epitope bound by WIC 29.26 Ab--are present in the surface membrane of all strains and clones tested. This observation suggests that in intact epimastigotes of the surface-nonreactive isolates, the epitope is not accessible because of structural changes in the molecule itself or because of differences in the membrane environment of GP72.
In vivo significance of NK cell on resistance against virus (HSV-1) infections in mice.
Authors: Habu S , Akamatsu K , Tamaoki N , Okumura K
Published: December 2022

Susceptibility to infection with herpes simplex virus type 1 (HSV-1) was examined in euthymic as well as athymic nude mice which were continuously depleted of natural killer (NK) cell activity by i.v. injection of anti-asialo GM1. In those NK cell activity-depleted mice, the mortality rate of infection with HSV-1 and the virus titers in the brain, liver, and spleen were notably higher than in the control mice. The enhanced susceptibility was demonstrated only in the mice receiving anti-asialo GM1 and HSV-1 simultaneously, but not in the mice in which NK cell deletion was postponed by injecting the antisera 5 days after the virus inoculation. Interferon (IFN) production of peritoneal exudate cells was also reduced in the anti-asialo GM1-injected mice. The decline of resistance against HSV-1 infection proved to be primarily due to deletion of NK cells, but not due to the inability to produce IFN, because repeated injections of IFN increased the NK cell activity and prolonged the life of HSV-1-infected mice with an intact NK cell activity. In the NK cell activity-depleted mice, however, neither the NK cell activity nor the life span was improved by the administration of IFN.
Structure of a germline rabbit immunoglobulin V kappa-region gene: implications for rabbit V kappa-J kappa recombination.
Authors: Lieberman R , Emorine L , Max E E
Published: December 2022

Rabbit kappa-immunoglobulin chains exhibit diversity in the number of amino acids between the invariant residues Cys 88 and Phe 98; this length diversity is formally similar to that found in the human and mouse heavy chain systems, in which it results from interposition of the D element between V and J. To explore the molecular basis for this length diversity in rabbit kappa-chains we have determined the nucleotide sequence of a rabbit germline V kappa immunoglobulin gene. The spacing between the 7-mer and 9-mer signal elements of this gene suggest that it could recombine with J kappa without a D element. We discuss alternative explanations for the length diversity of rabbit kappa-chains.
Reorientation of the microtubule-organizing center and the Golgi apparatus in cloned cytotoxic lymphocytes triggered by binding to lysable target cells.
Authors: Kupfer A , Dennert G
Published: December 2022

By immunofluorescence observations with cell couples of cloned murine cytotoxic T lymphocytes (CTL) and target cells, evidence is presented for a rapid reorientation of the microtubule-organizing center (MTOC) and the Golgi apparatus (GA) in the effector cell (but not in the target cell) toward the contact area with the target. The reorientation of the MTOC/GA and the cytotoxic activity of the CTL were inhibited reversibly by nocodazole, a microtubule-disrupting agent. In lectin-formed cell couples of CTL and neuraminidase-treated target cells, the MTOC in essentially all of the CTL was oriented toward the effector-target contact area of a lysable target cell, but was left randomly oriented with a nonlysable target cell. A similar random orientation of the effector-MTOC was also observed in cell couples of cloned natural killer cells and nonlysable targets. These findings indicate that the repositioning of the MTOC and the GA, which is shared by CTL and natural killer cells, is an essential and early event in the onset of the cytolytic mechanism. It is suggested that this reorientation serves the purpose of directing to the bound target cell secretory vesicles derived from the GA that contain cytotoxic substances.