bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a monoclonal antibody-defined human melanoma-associated antigen susceptible to induction by immune interferon.
Authors: Matsui M , Temponi M , Ferrone S
Published: December 2022

To develop monoclonal antibodies (mAb) recognizing human melanoma-associated antigens (MAA) susceptible to modulation by immune interferon (IFN-gamma), hybridomas were constructed with splenocytes from a BALB/c mouse immunized with IFN-gamma-treated melanoma cells Colo 38. Screening of supernatants with control and IFN-gamma-treated melanoma cells showed that the mAb CL203 and CL207 display preferential reactivity with IFN-gamma-treated melanoma cells. The two mAb recognize the same (or spatially close) determinant on a 96,000 MAA which has a density of 0.36 X 10(6) antigenic sites/cell on untreated melanoma cells Colo 38 and of 1.39 X 10(6) and 1.54 X 10(6) on melanoma cells Colo 38 treated with IFN-gamma (final concentration, 200 U/ml) for 24 and 48 hr, respectively. The effect of IFN-gamma on the 96,000 MAA is dose- and time-dependent, reversible, and blocked by inhibitors of RNA and protein synthesis. Furthermore, the effect of IFN-gamma on the induction of the 96,000 MAA appears to be specific, inasmuch as IFN-alpha and IFN-beta do not induce the expression of the 96,000 MAA. The latter is also induced by IFN-gamma in a variety of carcinoma cell lines, but its level is markedly lower than on melanoma cells. Furthermore, the apparent m.w. of the antigen synthesized by the carcinoma cell lines in the presence of IFN-gamma ranges between 93,000 and 96,000. This molecular heterogeneity appears to reflect differences in the degree of glycosylation of the polypeptide moiety because the antigen synthesized by a variety of cell lines in the presence of tunicamycin has an apparent m.w. of 51,000.
Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. IV. Partial tissue distribution and mapping of immunodominant antigens.
Authors: Wettstein P J , Colombo M P
Published: December 2022

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed.
Subpopulations of B lymphocytes in germinal centers.
Authors: Fyfe G , Cebra-Thomas J A , Mustain E , Davie J M , Alley C D , Nahm M H
Published: December 2022

With two new monoclonal antibodies and flow cytometry, we defined three subpopulations among B cells expressing binding sites for peanut agglutinin (i.e., B cells of the germinal center). On monoclonal antibody (5B5) binds globotriaosyl ceramide. The B lymphocytes binding 5B5 have binding sites for peanut agglutinin on the surface and express only small amounts of sIgD and sIgM. When tested against a panel of B cell lines, only Burkitt's lymphoma cells were 5B5+. Moreover, the 5B5+ cells have larger average sizes and a large fraction of proliferating cells. The other monoclonal antibody (HK23) binds a 90,000 protein. Lymphocytes binding HK23 are 5B5- and include T cells and a subpopulation of B cells. In contrast to 5B5+ cells, the HK23+ and peanut agglutinin positive B cells express a large amount of sIgM. These two subpopulations of germinal centers are distinct from the germinal center B cell subpopulation expressing the CD23 (Blast-2) antigen. The CD23+ B cells are 5B5- and express an intermediate level of HK23 antigen. In addition, CD23+ B cells are highly variable in number, whereas the proportions of HK23+ and 5B5+ cells are relatively stable.
Tolerance to class II major histocompatibility complex molecules is maintained in the presence of endogenous, interleukin-2-producing, tolerogen-specific T lymphocytes.
Authors: Mohler K M , Streilein J W
Published: December 2022

We have examined the requirement for clonal reductions of tolerogen-reactive lymphocytes in mice of the A strain background rendered neonatally tolerant of class II major histocompatibility complex molecules. Tolerogen-specific mixed lymphocyte reactivity of lymphocytes obtained from 130 adult, class II tolerant mice, bearing a healthy skin allograft, was examined. Lymphocytes obtained from 86 mice responded to the tolerogen, in vitro, with a positive mixed lymphocyte response (MLR) indicating that a large proportion (75%) of adult class II tolerant mice on the A strain background are not clonally deleted for tolerogen-reactive lymphocytes. In addition, lymphocytes from 29 mice were MLR-negative to the tolerogen, and lymphocytes from 15 mice demonstrated such high amounts of proliferation to syngeneic stimulators that their specific response to the tolerogen could not be determined. In view of the discordance between the in vivo and in vitro expressions of tolerance in the MLR-positive mice, lymphocytes from these mice were compared with normal lymphocytes by several assays. 1) Tolerogen-specific proliferative responses obtained from both normal and tolerant lymphocytes could be inhibited by the addition of monoclonal antibodies specific for the relevant class II antigens; 2) quantitative differences in the ability of normal, as compared with tolerant cells, to respond to the tolerogen in the MLR were not apparent; 3) no evidence of qualitative differences in the cell-surface phenotype of the proliferating cell was observed, (i.e., the cells were Thy-1+, L3T4+, Lyt-2-); and 4) lymphocytes from both normal and MLR-positive tolerant mice produced substantial amounts of interleukin-2 in response to the tolerogen. Thus, clonal deletion of helper cells is not required for tolerance to class II major histocompatibility complex antigens and we propose that tolerance may be maintained by either 1) in vivo suppression of the tolerogen-specific helper cells or 2) selective deletion or suppression of class II specific effector cells.
Antibody-dependent cellular cytotoxicity-inducing antibodies against human immunodeficiency virus. Presence at different clinical stages.
Authors: Ljunggren K , Böttiger B , Biberfeld G , Karlson A , Fenyö E M , Jondal M
Published: December 2022

The presence of antibodies mediating antibody-dependent cellular cytotoxicity (ADCC) against human immunodeficiency virus (HIV)-infected target cells was investigated with 170 sera from patients with varying severity of HIV infection. Approximately 40% of sera from individuals representing all stages of infection were ADCC-positive when tested against HTLV-IIIB infected 0937 clone 2 target cells. The positive sera had higher HIV antibody titers as measured by enzyme-linked immunosorbent assay compared with ADCC-negative sera. ADCC titers were lower in patients with acquired immune deficiency syndrome than in asymptomatic carriers. This decline in ADCC titer was not correlated with a general decrease of HIV antibodies. No correlation between the CD4:CD8 lymphocyte ratio and ADCC activity was found. The possible beneficial effect of ADCC-inducing antibodies early in infection is discussed in relation to the effect of ADCC-inducing antibodies in other retrovirus systems and to the nature of lentivirus infections.
Similar in vivo expansion of B cells from normal DBA/2 and autoimmune NZB mice in xid recipients.
Authors: Klinman D M , Steinberg A D
Published: December 2022

B cells from normal DBA/2 and autoimmune NZB mice were transferred into H-2-compatible xid recipients where they engrafted without irradiation or other manipulation of the host. The properties of these cells and their interaction with the host environment were analyzed at the single cell level with a splenic focus assay. When similar numbers of NZB and DBA/2 anti-DNA-producing B cell precursors were transferred, they expanded at similar rates in xid recipients. The rate of expansion varied with the strain of the recipient: it was fastest in autoimmune-prone NZB . xid and slowest in DBA/2 . xid hosts. Cells producing antibodies reactive with the autoantigen DNA proliferated substantially faster than those reactive with the non-autoantigen trinitrophenylated keyhole limpet hemocyanin. These results suggest that 1) B cells from NZB mice do not behave differently from DBA/2 B cells, 2) the internal milieu of the recipient into which the cells are transferred has an important effect on B cell proliferation, and 3) B cells capable of autoantibody production may have a selective growth and/or differentiation advantage relative to other B cells.
Interleukin 1 production by human lung tissue. II. Inhibition by anti-inflammatory steroids.
Authors: Bochner B S , Rutledge B K , Schleimer R P
Published: December 2022

Glucocorticoids block the localized accumulation of leukocytes as sites of inflammation by preventing their adherence to vascular endothelium. This implies that glucocorticoids are acting either on the leukocytes, endothelium, or cells which produce adherence-promoting factors (such as interleukin 1 (IL-1)). Previous studies have shown that dexamethasone (DEX) treatment of either polymorphonuclear leukocytes (PMN) or human umbilical vein vascular endothelial cells (VEC) or both in vitro does not prevent adherence induced by thrombin or formylmethionyl-leucyl-phenylalanine (f-met peptide). We now show that pretreatment of PMN and/or VEC for 24 hr with 0.1 microM DEX had no effect on adherence of PMN to VEC activated with IL-1 (2 U/ml), lipopolysaccharide (1 microgram/ml), or 12-O-tetradecanoylphorbol-13-acetate (30 ng/ml) suggesting that glucocorticoids may inhibit adherence in vivo by blocking formation of IL-1 and other adherence-inducing stimuli. We have recently established that cultured human lung fragments produce IL-1 in vitro. To investigate whether glucocorticoids could inhibit the production of adherence-inducing factors, we examined the effect of glucocorticoids on IL-1 production from human lung tissue. Treatment of human lung fragments in vitro for 18 hr with glucocorticoids such as DEX and hydrocortisone resulted in dose dependent inhibition of IL-1 production; these and other glucocorticoids, at concentrations ranging between 0.1 and 1 microM, produced greater than 50% inhibition of IL-1 release. Nonglucocorticoid steroids including testosterone and beta-estradiol (1 microM) had no effect. Inhibition of IL-1 production occurred after a lag period 5 of 16 hr, and the relative glucocorticoid potencies agreed with their known anti-inflammatory potencies in vivo (beta-methasone approximately triamcinolone acetonide greater than DEX greater than fludrocortisone greater than prednisolone greater than hydrocortisone). Inhibition of IL-1 production in vivo may, in part, explain the remarkable ability of glucocorticoids to prevent the adherence of leukocytes to endothelium and their accumulation at an inflammatory site.
Membrane interleukin 1 induction on human endothelial cells and dermal fibroblasts.
Authors: Kurt-Jones E A , Fiers W , Pober J S
Published: December 2022

Human endothelial cells and dermal fibroblasts both expressed a membrane-associated interleukin 1 (IL-1) activity when stimulated with either recombinant tumor necrosis factor (TNF) or recombinant lymphotoxin but stimulated endothelial cells expressed significantly more membrane IL-1 per cell than did fibroblasts. Lipopolysaccharide induced membrane IL-1 activity on endothelial cells but not fibroblasts. Interferon-gamma treatment of endothelial cells and fibroblasts had no direct effect on membrane IL-1 expression and little effect when used as a pretreatment for TNF or lipopolysaccharide stimulation. Endothelial cell membrane IL-1 activity was induced within 24 hr of culture with TNF or lipopolysaccharide, and increased up to 72 hr of incubation. Antibodies raised against human monocyte-derived IL-1 species neutralized the membrane IL-1 activity of TNF-stimulated endothelial cells. Both absorption studies and neutralization with specific sera indicated that endothelial cell membrane IL-1 is structurally related to IL-1 alpha. Endothelial cells expressed both IL-1 beta mRNA in response to TNF, lymphotoxin, and recombinant IL-1 species, as detected by Northern blot analysis. These studies demonstrate that endothelial cells can be activated to express a cell-surface IL-1 activity which is structurally, as well as functionally, related to the secreted form of IL-1.
Sequential effect of a high molecular weight B cell growth factor and of interleukin 2 on activated human B cells.
Authors: Vazquez A , Auffredou M T , Gerard J P , Delfraissy J F , Galanaud P
Published: December 2022

A high m.w. B cell growth factor (50,000 BCGF) prepared from lectin-activated human peripheral blood lymphocyte culture supernatants acts only on B cells preactivated by a first signal. This first signal can be delivered in vitro (with anti-mu antibody (Ab)) or in vivo. Upon costimulation with anti-mu Ab the 50,000 BCGF induces an early and transient proliferative response, whereas the response to interleukin 2 (IL-2) develops more progressively. To determine the respective targets of the 50,000 BCGF and of IL-2, B cells were activated with anti-mu Ab and separated according to the expression of the IL-2 receptor (cluster designation (CD)25 antigen). CD25+ B cells do not respond to the 50,000 BCGF and do not acquire this responsiveness after an additional culture with IL-2. CD25- B cells respond to the 50,000 BCGF and not to IL-2. However, when CD25- B cells are cultured for 3 days with the 50,000 BCGF they become responsive to IL-2. These results demonstrate a pathway of B cell activation based on the ordered and sequential action of anti-mu Ab, the 50,000 BCGF, and IL-2.
Interleukin 4 enhances the ability of antigen-specific B cells to form conjugates with T cells.
Authors: Sanders V M , Fernandez-Botran R , Uhr J W , Vitetta E S
Published: December 2022

T cell-B cell conjugates are formed when trinitrophenyl-specific B cells are exposed to trinitrophenyl-ovalbumin and ovalbumin-specific T hybridoma cells. The proportion of conjugates was increased two- to threefold when antigen-pulsed trinitrophenyl-specific B cells, but not T cells, were pre-exposed to interleukin 4. Antigen-specific B cells pretreated with antigen and interleukin 4 and cultured in the presence of specific T helper cells also produced a larger proportion of antibody-secreting cells as compared to cells pretreated with antigen alone. The interleukin 4-induced enhancement of T/B conjugate formation occurred over a wide range of antigen concentrations, was dependent on the concentration of interleukin 4, and was inhibited by the monoclonal anti-interleukin 4 antibody, 11B11. The importance of Ia antigens in the enhancement of conjugate formation and generation of antibody-secreting cells is suggested by a) the fact that the interleukin 4-mediated increase in the density of Ia antigens on the antigen-specific B cells correlated with their enhanced ability to form T/B conjugates, b) the kinetics of the interleukin 4-mediated increase in conjugate formation and surface Ia expression were similar, c) 10- to 20-fold higher concentrations of anti-I-A antibody were required to inhibit T/B conjugate formation by 50% with interleukin 4-treated antigen-specific B cells compared with untreated antigen-specific B cells, and d) interferon-gamma, which inhibits the interleukin 4-mediated increase in Ia antigens, inhibited the interleukin 4-induced enhancement of T/B conjugate formation. These results indicate that the interleukin 4-induced increase in the expression of Ia antigens on B cells plays an important role in the enhancement of T/B cell interactions and the subsequent differentiation of antigen-specific B cells into antibody-secreting cells.
Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes.
Authors: Henkart P A , Berrebi G A , Takayama H , Munger W E , Sitkovsky M V
Published: December 2022

Percoll gradient fractions of homogenates of murine cloned cytotoxic T lymphocytes (CTL) were analyzed for the trypsin-like enzyme alpha-N-benzyloxy-carbonyl-L-lysinethiobenzyl ester (BLT) esterase recently described in CTL homogenates. Enzymatic activity was found in three areas of the gradient: the dense cytolysin containing granules; a light granule fraction; and a variable amount in the soluble fraction at the top of the gradient. Gel filtration columns showed a major peak of BLT esterase activity eluted at the position of a 60-kDa protein, and an additional, minor BLT esterase peak eluting at about 27 kDa. The separated enzymes were both significantly inhibited by the serine protease inhibitors diisopropylfluorophosphate and phenylmethyl sulfonyl fluoride (PMSF), indicating they are both serine proteases, but showed different patterns of inhibition by a series of inhibitors, suggesting the larger enzyme is not a simple dimer of the smaller. pH activity profiles of both CTL BLT esterases showed an optimum at about pH 8. PMSF inactivation of BLT esterase in detergent extracts of CTL diminished sharply as the pH was dropped below 7. Agents which raise the pH of acidic intracellular compartments were found to markedly enhance the PMSF inactivation of BLT esterase in intact CTL, showing that the granules have a low internal pH. Similarly, [3H]diisopropylfluorophosphate labeling of intact CTL gave four protein bands on non-reduced gels, of which two were labeled threefold more effectively in the presence of chloroquine. In parallel studies of inactivation of CTL lytic activity, PMSF pretreatment caused a 50% reduction of the lytic activity under conditions where greater than 90% of the BLT esterase activity was inactivated. Addition of agents raising the intragranular pH dramatically enhanced the BLT esterase inactivation but did not concomitantly reduce CTL lytic activity. These results indicate that inactivation of lytic function by PMSF is unlikely to be due to its reaction with protease in acidic granules, and suggest that the activity of these enzymes may not be required for cytotoxicity.
Pertussis toxin triggers rapid second messenger production in human T lymphocytes.
Authors: Rosoff P M , Walker R , Winberry L
Published: December 2022

Pertussis toxin (PT) is a known mitogen for T lymphocytes. The mechanism by which the toxin stimulates proliferation has remained obscure and paradoxical because, in some types of cells, the toxin also inhibits growth factor-mediated signal transduction. It has previously been shown that the adenosine-diphosphate ribosyltransferase activity of the toxin is not required to produce the mitogenic effect. A biochemical explanation for the mitogenic activity has therefore remained obscure. We investigated the biochemical basis for the mitogenic activity of PT by using the transformed human T cell line, Jurkat. PT stimulated a rapid rise in cytosolic-free [Ca2+] from both intra- and extracellular sources. This was associated with an increase in the cellular diacylglycerol and inositol triphosphate levels with a concomitant decrease in the levels of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. The half-maximal effective dose of PT was 1.7 nM. PT also stimulated the production of interleukin 2. Only the holotoxin or B-oligomer (the presumptive membrane-binding subunit) was capable of stimulating an increase in [Ca2+] in these cells. This activity of PT mimicked that of some anti-T3-T cell antigen receptor complex monoclonal antibodies that also stimulate increases in the second messengers, diacylglycerol and Ca2+. The effects of PT and anti-T3 complex antibody were identical and not additive in Jurkat cells, suggesting that both agents were activating the same signal transduction pathway. These data provide a mechanistic explanation for the mitogenic effects of PT and suggest that the toxin may be interacting with a specific receptor in the T lymphocyte plasma membrane.
Anti-basement membrane glomerulopathy in experimental trypanosomiasis.
Authors: Bruijn J A , Oemar B S , Ehrich J H , Foidart J M , Fleuren G J
Published: December 2022

The nature of kidney lesions in BD IX rats infected with Trypanosoma brucei was investigated. Proteinuria developed and increased up to 236 +/- 35 mg/24 hr at 7 wk after the infection. Antibodies were found to be deposited along the glomerular basement membrane (GBM) predominantly in a linear fashion, which changed to a more granular pattern 7 wk after the infection. At this stage of the disease, electron-dense deposits were found subendothelially along the GBM. In the sera and kidney eluates of diseased rats, anti-GBM antibodies were present. Enzyme-linked immunosorbent assay (ELISA) studies showed antibodies which reacted with GBM components laminin and type IV collagen and not with fibronectin. The antibody specificity was confirmed by using competitive and cross-absorption ELISA techniques, as well as immunoblotting. With the use of indirect immunofluorescence, no common antigenic sites were found on trypanosomes and GBM components. The observed linear immunofluorescence pattern seems to be caused by glomerular binding of antibodies directed against laminin and type IV collagen, which are known to be able to induce renal disease. Subendothelial complex formation in later stages of the disease might result from a molecular rearrangement of GBM components after in situ binding of the antibodies. The formation of auto-antibodies directed against laminin and type IV collagen is probably caused by restricted polyclonal B cell stimulation, a well known feature of trypanosomiasis.
Relationship of human variable region heavy chain germ-line genes to genes encoding anti-DNA autoantibodies.
Authors: Dersimonian H , Schwartz R S , Barrett K J , Stollar B D
Published: December 2022

In order to identify the V region genes encoding systemic lupus erythematosus (SLE)-derived anti-DNA autoantibodies, we have determined the nucleotide sequence of heavy chain mRNA from several DNA-binding immunoglobulins secreted by human hybridomas. We used the technique of cDNA primer extension for determining sequences of the VH, D, and JH gene segments of anti-DNA autoantibodies from three different primary hybridoma growths from an SLE patient and one hybridoma from a leprosy patient. Immunoglobulins from two of the SLE hybridomas expressed the same idiotype, Id-16/6, which is also expressed on immunoglobulins in sera of patients with active SLE. Their mRNA sequences showed complete homology to each other in the V, D, and J genes and more than 99% homology to the VH26 germ-line gene sequence, a member of the human VHIII gene family. The VH mRNA sequence of the third SLE hybridoma, 21/28, which was idiotypically unrelated to the other two, was 93% homologous to a different VH germ-line gene sequence, HA2, a member of the human VHI gene family. The fourth anti-DNA-producing hybridoma, 8E10, was derived from a leprosy patient of different ethnic origin than the SLE patient. It was idiotypically related to 21/28 and expressed a VH segment gene identical to that of 21/28. Hybridomas 21/28 and 8E10 shared sequence homology with the VH26 anti-DNA antibodies in the first complementarity-determining region. In addition, 21/28 shared sequence homology with the Id-16/6+ group in the region encoded by the D and J gene segments. Our findings indicate that some SLE autoantibodies are encoded by unmodified or scarcely modified VH germ-line genes that are conserved in the human population and identify two distinct VH germ-line genes that can encode segments of anti-DNA immunoglobulins.
In vivo priming of helper and suppressor T cells by alloantigens. Frequency analysis with the use of an in vitro limiting dilution assay.
Authors: Bianchi A T , Schilham M W , Benner R , Young P , Lefkovits I
Published: December 2022

Earlier studies have demonstrated that T cells activated in mixed lymphocyte reactions can exert positive as well as negative allogeneic effects on B cells expressing the appropriate alloantigens on their surface. We investigated the effect of in vivo priming of T cells with alloantigens on their capacity to help or suppress allogeneic B cell cultures against sheep erythrocytes. We used immunization protocols that have been shown to be optimal for induction of alloantigen-specific delayed-type hypersensitivity (DTH) and alloantigen-specific suppressor T (Ts) cells for DTH. The results show that in vivo stimulation with alloantigens, depending on the immunization route and the lymphoid organ studied, can be as effective as in vitro stimulation in increasing the frequency of alloantigen-specific helper T (Th) cells and Ts cells. Subcutaneous immunization induced a 10-fold frequency raise of Th cells as well as of Ts cells in the lymph nodes. In the spleen the Th cell population was hardly affected by s.c. immunization, whereas the Ts cell population increased by at least a factor 20. Intravenous immunization, on the other hand, selectively expanded the Th cell population in the spleen, whereas the splenic Ts cell population and the Th and Ts cells in the lymph nodes were not affected. Comparison of these results with our previous data concerning characteristics and the requirements of in vivo activation of alloantigen-specific DTH reactive T cells and of alloantigen-specific Ts cells suggest that different Ts cell populations are involved in suppression of alloantigen-specific DTH in vivo and of allogeneic suppression of in vitro induced sheep erythrocytes specific antibody formation.
Mechanism of inhibition and induction of cytolytic activity in cytotoxic T lymphocytes by CD3 monoclonal antibodies.
Authors: van Seventer G A , Kuijpers K C , van Lier R A , de Groot E R , Aarden L A , Melief C J
Published: December 2022

The objective of this study was to elucidate the mechanism responsible for inhibition as well as induction of cytolytic activity in cytotoxic T lymphocytes (CTL) by cluster-defined 3 (T3) (CD3) monoclonal antibodies (mAb). A series of isotype heavy chain switch variants (murine IgG1, IgG2a, IgG2b, IgA, and IgE) of a single CD3 mAb was used in the analysis of effects of CD3 mAb on the cytolytic activity of an allospecific CTL clone. The results obtained indicate that the inhibition of cytolytic activity of CTL by CD3 mAb is not caused by interference with events that occur after conjugate formation, such as programming for lysis and the delivery of the lethal hit, but by inhibition of specific recognition of the target cell by the CTL. We also present evidence that induction of nonspecific CTL activity by CD3 mAb is only achieved by direct binding of the Fc receptor to the CD3 mAb. This observation allowed us to use the CD3 mAb-induced nonspecific cytolytic activity of CTL as a sensitive assay for detection of Fc receptors for various heavy chain isotypes. So far, evidence was found for human Fc receptors reactive with murine IgG1, IgG2a, and IgG2b, but not for murine IgA or IgE. The combined results indicate a similar role for Fc receptors in induction of cytolytic activity of CTL by CD3 mAb as well as in CD3 mAb-induced proliferative responses of T cells.
Establishment of an antigen-specific B cell clone by somatic hybridization.
Authors: Hamano T , Murata Y , Yamasaki T , Yasuda Y , Iwasaki T , Nagai K
Published: December 2022

Splenic B cells of A/J mice immunized with 2,4,6-trinitrophenyl (TNP)-lipopolysaccharide were fused with 2.52M, a mutant of a B cell line, in the presence of polyethylene glycol and dimethyl sulfoxide. TP67.21, a subclone of a resulting hybridoma, expresses IAk, IEk, IgM, B220, P50, and receptors for C3 fragment of complement, the Fc portion of IgG, and interleukin 2 receptor on the cell membrane; it also possesses receptor molecules for TNP on its surface, derived from TNP-reactive B cells of A/J mice primed with TNP-lipopolysaccharide used for somatic hybridization, by a rosette-forming assay with TNP-sheep erythrocytes. In contrast, parental 2.52M lacks IAk and IEk on the cell membrane and does not bind to TNP-sheep erythrocytes under the same conditions. Thus, it is likely that TP67.21 is an antigen-specific B cell clone directed against TNP. The antigen binding of cells was markedly inhibited by the specific free hapten or anti-IgM antibodies. Interestingly, TP67.21 was induced to generate a significant amount of anti-TNP antibody when treated with TNP conjugates including T cell-independent and -dependent antigens, such as TNP-lipopolysaccharide, TNP-bovine serum albumin, TNP-ovalbumin, and TNP-keyhole limpet hemocyanine in the absence of T cell help, as well as polyclonal activators; this was followed by a marked decrease in the expression of B cell surface markers on the cell membrane. This suggests that the cross-linkage of receptor molecules on TP67.21 by antigen may directly provide a differentiative signal for maturation to a lineage of B cells, and consequently results in the generation of antigen-specific antibodies without T cell involvement.
Monoclonal antibodies to CD2 and lymphocyte function-associated antigen 3 inhibit human thymic epithelial cell-dependent mature thymocyte activation.
Authors: Denning S M , Tuck D T , Vollger L W , Springer T A , Singer K H , Haynes B F
Published: December 2022

Recent study of human thymocyte-thymic epithelial (TE) cell interactions has demonstrated that thymocytes bind to TE cells, and a consequence of this binding is the provision of accessory cell signals by TE cells for phytohemagglutinin (PHA)-induced mature thymocyte activation. In this paper we report on studies of the molecules involved in TE cell-dependent mature thymocyte activation. TE-thymocyte interactions necessary for PHA-induced thymocyte activation were inhibited by monoclonal antibodies against the cluster of differentiation (CD)2 antigen on thymocytes and lymphocyte function-associated (LFA)-3 antigen on TE cells. Inhibition of TE accessory cell signals by antibodies against CD2 (alpha CD2) and LFA-3 (alpha LFA-3) antigens occurred early on during thymocyte activation and prevented thymocyte interleukin 2 receptor expression. Further, alpha CD2 and alpha LFA-3 inhibited PHA-induced thymocyte activation in whole thymic explant cultures suggesting a significant role of the CD2 and LFA-3 antigens in thymocyte activation when accessory cell signals for PHA-induced thymocyte triggering were delivered by cells within an intact thymic microenvironment.
A monoclonal antibody raised to tumor-specific T cell-derived suppressor factors also recognizes T suppressor inducer factors of the 4-hydroxy-3-nitrophenyl acetyl hapten suppressor network.
Authors: Steele J K , Kawasaki H , Kuchroo V K , Minami M , Levy J G , Dorf M E
Published: December 2022

A monoclonal antibody (mAb), B16G, was raised from BALB/c mice immunized with affinity-purified T suppressor factors (TsF) specific for the murine mastocytoma P815. This mAb was found to bind to polyclonal TsF isolated from the spleens of tumor-bearing animals, and to the TsF released from a P815-specific T cell hybridoma. In this study, B16G was tested for its reactivity with TsF produced in the 4-hydroxy-3-nitrophenyl acetyl hapten system. The factors from three types of suppressor T cell hybridomas, each representing the immortalized analogues of the inducer T suppressor cell (Ts1), transducer suppressor cell (Ts2), and effector suppressor cell (Ts3) network populations, were tested. B16G was found to be reactive with two sources of TsF1 as assayed by enzyme-linked immunosorbent assay and delayed-type hypersensitivity bioassay. By contrast, TsF2 and TsF3 were nonreactive with B16G. These results indicate that B16G recognizes class-specific suppressor factor determinants, and that the transducer/effector factors of the network are apparently serologically distinct. Because the B16G mAb fails to recognize 4-hydroxy-3-nitro-phenyl acetyl-specific TsF3 that share idiotype-related determinants with TsF1 yet binds to TsF1 molecules that have interacted with antigen, the binding is apparently independent of the site of antigen recognition. Additionally, the results show that the tumor-specific TsF1 raised in one suppressor system share serologic determinants with anti-hapten TsF1 raised in another.
T cell antigen receptor phosphorylation induced by an anti-receptor antibody.
Authors: Samelson L E , O'Shea J J , Luong H , Ross P , Urdahl K B , Klausner R D , Bluestone J
Published: December 2022

In previous studies we demonstrated that the antigen receptor complex on murine T cells is phosphorylated after antigen or mitogen activation. After the clonotypic structures bind antigen, the invariant subunits or CD3 molecules are the target of dual kinase activation. The antigen receptor CD3-gamma-chain subunit is phosphorylated on serine residues by activated protein kinase C and the p21 subunit is phosphorylated by a tyrosine kinase. Herein we demonstrate that another mechanism of receptor activation by the stimulatory monoclonal antibody 145-2C11, which binds the CD3-epsilon chain, results in a similar pattern of kinase activation and receptor phosphorylation.
Calcium ionophore A23187 does not stimulate lipopolysaccharide nonresponsive C3H/HeJ peritoneal macrophages to produce interleukin 1.
Authors: Shinomiya H , Nakano M
Published: December 2022

C3H/HeJ mice are hyporesponsive to the biologic effects of bacterial lipopolysaccharide (LPS). The defect in the strain of mice is believed to be due to the expression of a mutant allele designated Lpsd at the chromosome four locus. The molecular basis of this hyporesponsiveness is not known, but it may result from some defective membrane signal transductions. To examine this possibility, we compared the abilities of interleukin 1 (IL-1) production by C3H/HeJ macrophages with those by C3H/He macrophages (LPS responsive) after stimulation with the calcium ionophore A23187 or phorbol myristate acetate (PMA). A23187 induced IL-1 production by C3H/He macrophages, but it did not induce IL-1 production by C3H/HeJ macrophages and neither did LPS. However, it had the ability to increase intracellular free Ca2+ in C3H/HeJ macrophages as well as in C3H/He macrophages, this being examined by the changes in cytosolic Ca2+ in the macrophages by using Quin 2. In contrast, PMA was able to induce IL-1 production by both C3H/He and C3H/HeJ macrophages without increasing intracellular Ca2+. Since polymyxin B did not inhibit A23187- or PMA-induced IL-1 production by C3H/He macrophages, these results are not due to the little amount of LPS in culture medium, but due to their own characteristics. A calmodulin antagonist W-7 effectively inhibited A23187-induced IL-1 production by C3H/He macrophages. However, it hardly inhibited LPS-induced IL-1 production except at high concentration, and it caused no inhibition of the PMA-stimulated one. These results suggest that the blocking sites expressed phenotypically by the Lpsd are shared by LPS- and A23187-stimulated cellular processes, although the actions of LPS and A23187 are different from each other. In addition to the direct study with LPS or lipid A, A23187 should provide another useful approach to clarify the molecular mechanisms of Lpsd defect in C3H/HeJ macrophages.
Chemotactic effect of human recombinant interleukin 2 on mouse activated large granular lymphocytes.
Authors: Natuk R J , Welsh R M
Published: December 2022

Human recombinant interleukin 2 (hrIL-2) was demonstrated in vitro to be chemotactic for mouse large granular lymphocytes (LGL) activated in vivo by virus infection. Peritoneal exudate cells harvested from virus-infected mice were used as a source of LGL. LGL collected from mouse hepatitis virus-infected mice at 3 days postinfection were a source for NK 1.1 positive natural killer (NK)/LGL. LGL collected from mice treated with antiserum to gangliotetraosylceramide and infected with lymphocytic choriomeningitis virus for 7 days were used as a source for Lyt-2 positive cytotoxic T lymphocytes (CTL)/LGL. Both NK/LGL and CTL/LGL responded chemotactically to hrIL-2, purified IFN-beta, and to crude cell-free washout fluids collected from the peritoneal cavity of virus-infected mice. hrIL-2 had chemotactic activity for virus-elicited granular and agranular lymphocytes but did not attract the contaminating macrophages, in contrast to IFN-beta, which displayed chemotactic activity for virus-elicited granular and agranular lymphocytes as well as macrophages. The migration to hrIL-2 was inhibited by a monoclonal antibody (7D4) to the IL-2 receptor, but treatment with 7D4 did not affect migration in response to IFN-beta. Microscopic examination of Wright's-Giemsa-stained migrated NK/LGL and CTL/LGL revealed that the majority of migrated LGL in either LGL population had a blast cell morphology (enlarged cells with rich basophilic cytoplasm). The frequency of cells bearing the LGL morphology within the virus-elicited nonadherent peritoneal exudate cell population was on incubation in vitro, stabilized by either hrIL-2 or IFN-beta. These data suggest that another important immunomodulating function of IL-2 may be to attract activated NK/LGL and CTL/LGL to sites of inflammation.
The role of protein kinase C in transmembrane signaling by the T cell antigen receptor complex. Effects of stimulation with soluble or immobilized CD3 antibodies.
Authors: Manger B , Weiss A , Imboden J , Laing T , Stobo J D
Published: December 2022

Phorbol esters, such as phorbol myristate acetate (PMA), are known to be potent co-stimulants with calcium ionophores for activation of T lymphocytes. The most extensively studied intracellular effect of PMA is its ability to activate the cytoplasmic enzyme protein kinase C (pkC). Herein, we examined the role of pkC activation during T cell activation. During physiologic activation, this enzyme is activated by diacylglycerol which is generated through the hydrolysis of polyphosphoinositides. Therefore, we studied the activation of T lymphocytes induced by a synthetic diacylglycerol, dioctanoylglycerol. In contrast to PMA, this compound can be metabolized in T cells and presumably more closely mimics physiologic activation of pkC. Dioctanoylglycerol together with reagents that induce increases in intracellular free Ca2+ concentration, Ca2+ ionophores, or anti-cluster designation (CD)3 monoclonal antibodies (mAb) were able to induce interleukin 2 receptor expression and proliferation of T lymphocytes. Previous studies have demonstrated that the stimulation of T cells via the CD3/T cell antigen receptor complex by mAb against CD3 leads to an increase in cytoplasmic free Ca2+ and to an activation of pkC. Paradoxically, however, soluble CD3 antibodies do not cause proliferation of resting purified T cells. Inasmuch as immobilization of CD3 mAb has been shown to influence the agonist properties of such antibodies, we compared the ability of soluble and immobilized CD3 mAb to activate pkC. We demonstrated herein that soluble CD3 mAb cause only a very transient activation of pkC in the T cell leukemic line Jurkat. This pkC activation is markedly prolonged when Jurkat cells are stimulated with immobilized rather than soluble CD3 antibodies. These studies suggest that activation of pkC plays a major role in T cell activation and that the activation of pkC is influenced by the form in which CD3 mAb is presented to T cells.
Molecular evidence that SJL reticulum cell sarcomas are derived from pre-B cell. Clonal rearrangement of heavy chain but not of light chain immunoglobulin genes.
Authors: Nakauchi H , Osada H , Yagita H , Okumura K
Published: December 2022

In order to investigate the clonal origin of SJL reticulum cell sarcoma (RCS), two-color cell membrane-staining and molecular biologic analyses were performed. Flow cytometric analysis revealed that the SJL RCS consists of about 50 to 60% Thy-1-positive and 20 to 30% B220-positive cells and that the majority of the Thy-1-positive cells are L3T4-positive, whereas a few are Lyt-2-positive. In spite of this pleomorphic nature of SJL RCS shown by cell membrane analysis, when the immunoglobulin heavy chain J segment (JH) was used to probe the DNA obtained from the tumor, we observed clonal rearrangements of the gene. Results of the cell-sorting experiment combined with Southern hybridization using the JH gene probe confirmed that those clonally expanding cells are Ia and B220 antigen-positive. Furthermore, all tumors derived from a given mouse showed the same rearrangement pattern. However, no clonal rearrangement was observed when the DNA was probed with the T cell receptor beta-chain gene or with immunoglobulin kappa- or lambda-light chain genes. From these data, we conclude that SJL RCS is a tumor of B cells and that the neoplastic event takes place at an immature B cell stage.
Idiotype vaccination against murine B cell lymphoma. Humoral and cellular responses elicited by tumor-derived immunoglobulin M and its molecular subunits.
Authors: Campbell M J , Carroll W , Kon S , Thielemans K , Rothbard J B , Levy S , Levy R
Published: December 2022

C3H/HeN mice were immunized with idiotypic immunoglobulin M (IgM) and its molecular subunits from the syngeneic 38C13 lymphoma. Immunization with idiotypic IgM (38C-Id) resulted in idiotype-specific humoral and cellular immunity and protection against a lethal tumor cell challenge. Heavy (H38C) and light (L38C) chains were isolated by electroelution from preparative polyacrylamide gels. Both of these immunogens induced significant resistance to a subsequent tumor challenge. Variable region immunogens, in the form of trpE-fusion proteins, were obtained by cloning heavy and light chain variable region genes into the expression plasmid pATH-11. Of these, only the trpE-VH38C immunogen yielded immune resistance to tumor challenge. Finally, the nucleic acid sequence of 38C-Id light chain was determined and, based on the corresponding amino acid sequence and an analysis of predicted secondary structure, a region of potential antigenicity in complementarity-determining region 3 was chosen for the production of a synthetic peptide. Vaccination with this synthetic peptide resulted in significant suppression of tumor growth. Analysis of the humoral and cellular immunity generated by these vaccines revealed the presence of antibodies reactive with native idiotypic IgM only in 38C-Id, H38C, and trpE-VH38C immune sera, although the latter two were not idiotype-specific. Idiotype-specific lymphocytes, which proliferated in response to native 38C-Id, were observed in all immune animals. With the exception of the fusion protein immunogens, conjugation to an immunogenic carrier protein (keyhole limpet hemocyanin or thyroglobulin) was required for optimal humoral and cellular responses.
The T11 (CD2) molecule is functionally linked to the T3/Ti T cell receptor in the majority of T cells.
Authors: Breitmeyer J B , Daley J F , Levine H B , Schlossman S F
Published: December 2022

Most mature human T lymphocytes express both the multichain T3 (CD3)/Ti T cell receptor for antigen (TCR), and the biochemically distinct 55-kDa T11 (CD2) glycoprotein. Stimulating the T11 molecule causes profound T cell proliferation and functional activation in vitro, but the relationship of T11-mediated activation to antigenic stimulation of T lymphocytes in vivo remains unknown. We now present evidence that T11 function is directly linked to TCR components in T3/Ti+ T11+ human T cells. First, we found that stimulating peripheral blood T cells with the mitogenic combination of anti-T11(2) cells with the mitogenic combination of anti-T11(2) plus anti-T11(3) monoclonal antibodies caused the phosphorylation of TCR T3 chains. The predominance of T3-gamma-phosphorylation that occurred in anti-T11(2) plus anti-T11(3)-treated T cells is similar to the pattern previously observed in antigen-stimulated T cell clones. Second, T11 function depended upon concurrent cell-surface expression of the TCR. Thus, when peripheral blood T cells were deprived of cell surface T3/Ti by anti-T3 modulation, anti-T11(2) plus anti-T11(3)-induced mitogenesis and transmembrane signal generation in the form of calcium mobilization were inhibited. The mechanism of TCR-T11 interdependence was investigated in a series of TCR-deficient variants of a T cell lymphoblastoid cell line. T3/Ti negative variants expressed cell surface T11, but anti-T11(2) plus anti-T11(3) failed to cause detectable calcium mobilization. The TCR-deficient variants also failed to express T11(3) activation epitopes after incubation with anti-T11(2) antibodies, suggesting that T11(3) expression is an essential and TCR-dependent intermediate in the T11 activation mechanism in these cells. Taken together, our results suggest that T11 function depends upon cell-surface expression of TCR in many T3/Ti+ T11+ T lymphocytes, and T11-mediated activation is intimately interconnected with TCR activation mechanisms. A model in which stimulating signals delivered via T11 may be a part of antigenic activation of T lymphocytes is presented.
Heymann antibodies induce complement-dependent injury of rat glomerular visceral epithelial cells.
Authors: Camussi G , Salvidio G , Biesecker G , Brentjens J , Andres G
Published: December 2022

The aim of this study was to investigate the in vitro role of the complement membrane attack complex (MAC) in the injury induced by nephritogenic anti-brush border vesicle (Fx1A) antibodies on rat glomerular visceral epithelial cells (GEC). Both sheep and rabbit anti-rat brush border vesicle IgG-induced complement-dependent lysis of cultured GEC. Fab fragments of sheep anti-rat brush border vesicles and polyclonal or monoclonal gp330 IgG were devoid of lytic activity. Shedding of cell-surface antigens induced by sheep or rabbit anti-rat brush border vesicle IgG protected GEC from subsequent exposure to lytic antibodies and complement, an effect that was not obtained with Fab fragments. When GEC were incubated with sheep or rabbit anti-rat brush border vesicle IgG in capping conditions, the C3 component was co-redistributed with Heymann immune complexes; in contrast, the MAC remained diffusely bound to the cell surface, indicating that it was not associated with the antigen-antibody complexes. The MAC was demonstrated on the surface of GEC by immunofluorescence staining with anti-MAC neoantigen and by electron microscopy of negatively stained membranes showing focal clusters of 110 A MAC lesions. When GEC were treated with sheep IgG or rabbit IgG plus C6-deficient sera, the cells were not lysed and MAC was not demonstrable on the surface; however, lytic activity was restored when C6-deficient sera were reconstituted with purified C6. The results are consistent with the interpretation that injury induced by Heymann antibodies on GEC is MAC-dependent.