bluuebunny/crossref_metadata_2025 huggingface dataset:
Circumvention of defective CD4 T helper cell function in HIV-infected individuals by stimulation with HLA alloantigens.
Authors: Clerici M , Stocks N I , Zajac R A , Boswell R N , Via C S , Shearer G M
Published: January 2023

PBL from approximately 50% of asymptomatic individuals infected with HIV have been previously demonstrated to exhibit defective in vitro Th function that is selective for influenza A virus (FLU), but not for HLA alloantigens (ALLO). In this report, we have further studied HIV+ individuals with this selective Th defect, and demonstrate that defective in vitro CTL responses to FLU can be restored by costimulation with FLU + ALLO. In contrast, HIV+ patients who have lost Th responses to ALLO were unable to correct CTL responses to FLU by this costimulation procedure. These findings indicate that intact Th responses to ALLO can be used in vitro to provide Th signals necessary to activate the T effector cell response to a potential pathogenic virus. Our results raise the possibility that a program of in vivo coimmunization with ALLO plus antigens of potential pathogens (including HIV) can be useful in HIV+ patients exhibiting selective defects in Th function. Furthermore, this approach could be incorporated in vaccine trials aimed at enhancing immunity to HIV in patients who have been infected previously with this virus.
Functional and ontogenetic analysis of murine CD45Rhi and CD45Rlo CD4+ T cells.
Authors: Lee W T , Yin X M , Vitetta E S
Published: January 2023

CD4+ murine T cell clones, TH1 and TH2, can be distinguished by both functional responses and by their patterns of lymphokine secretion. Recently, a mAb, 23G2, which reacts with a subset of CD45 molecules (CD45R), has been reported to bind differentially to clones of TH1 and TH2 cells. In the present study, normal splenic T cells were analyzed for differences in 23G2-reactivity and were separated into two populations based on their density of CD45R (CD45Rhi and CD45Rlo). The CD45Rhi cells secrete more IL-2 than IL-4 after stimulation in vitro; the reverse is true for the CD45Rlo cells. Because neither population secretes only IL-2 or IL-4, we were unable to classify cells as TH1 or TH2. In vivo and in vitro analyses of the CD45Rhi and CD45Rlo cells suggest a lineage relationship between the two subsets that correlates with the degree of Ag exposure and the state of maturation of the mice. In newborn mice and mice raised under sterile conditions, splenic CD4+ T cells are predominantly CD45Rhi. Under conditions of increased antigenic exposure and maturation of the mice, CD45Rlo cells develop; after long term priming in vivo, the majority of specific Ag-reactive cells are CD45Rlo. Adoptive transfer studies using BALB/c nu/nu recipients demonstrate that CD45Rhi cells become CD45Rlo cells and that the recall response (IgG) to specific Ag is mediated by CD45Rlo cells. Taken together, these data indicate that the level of expression of CD45R on CD4+ T cells distinguishes virgin (CD45Rhi) from primed/memory (CD45Rlo) T cells in normal mice.
An absence of T cells in murine bone marrow allografts leads to an increased susceptibility to rejection by natural killer cells and T cells.
Authors: Murphy W J , Kumar V , Cope J C , Bennett M
Published: January 2023

The mechanisms behind the increased incidence of marrow graft failure in recipients that receive allogeneic marrow depleted of T cells were studied. Recipient mice were lethally irradiated and challenged with bone marrow cells (BMC) from C.B-17 +/+ (+/+) donors. Radioisotope 125IUdR incorporation was assessed 5 to 7 days after transfer to determine the extent of engraftment. Some groups received BMC in which the T cells were removed by treatment with antibody and C. In addition, some groups received BMC from T cell-deficient C.B-17 scid/scid (SCID) mice to determine the postulated need for donor T cells in hematopoiesis and engraftment. In a model system that distinguishes between possible host NK cell and radioresistant T cell-mediated rejection of marrow allografts, it was determined that the absence of donor T cells in a marrow graft does not affect engraftment in syngeneic recipients. However, both host NK cell and radioresistant T cell rejection was markedly enhanced when SCID BMC or BMC from C.B-17 +/+ donors that had T cells removed by antibody and complement were infused into irradiated allogeneic recipients. Furthermore, the addition of alloreactive thymocytes as a source of T cells could abrogate this increased susceptibility of the BMC to host rejection mechanisms. As determined by histology and 59Fe uptake, the addition of thymocytes resulted in enhanced erythropoiesis. These results suggest that the increased incidence of marrow graft failure when BMC depleted of T cells are used is a result of active rejection by host effector cells and that the adverse effect of marrow T cell depletion can be reversed by the addition of thymocytes.
IL-4 blocks the up-regulation of IL-2 receptors induced by IL-2 in normal human B cells.
Authors: Lee H K , Xia X , Choi Y S
Published: January 2023

A negative influence of IL-4 on the IL-2-induced B cell proliferation and differentiation has recently been reported. In this study, we have further investigated a role of IL-4 on human tonsillar B cell proliferation and IL-2R expression. IL-4 enhanced Staphylococcus aureus Cowan 1 strain (SAC)-induced B cell proliferation, reaching the peak on day 3. However, from day 4, IL-4 inhibited IL-2-induced proliferation. In the cross-linking study, IL-4 enhanced the density of 125I-IL-2-binding protein at low affinity binding condition (2 nM of 125I-IL-2) in SAC-activated B cells. However, IL-4 blocked the enhancement in the density of 125I-IL-2-binding proteins induced by IL-2, from day 3, in both high (50 pM of 125I-IL-2) and low affinity binding conditions, suggesting that IL-4 is able to block IL-2-induced IL-2R up-regulation. This was confirmed by a binding study: B cells that cultured for 3 days with SAC plus IL-2 expressed an average of 180 +/- 20 high affinity receptors/cell with a Kd of 12 pM and 5800 +/- 500 low affinity receptors/cell with a Kd of 980 pM. By coculturing with IL-4, high affinity receptors were almost undetectable and the expression of low affinity receptors was reduced by more than 80%. IL-4-mediated inhibition of IL-2-induced IL-2R expression does not seem to be due to the direct interaction between IL-4 and cell surface receptors, inasmuch as preincubation of cells with IL-4 for 60 min at 37 degrees C did not alter the binding of 125I-IL-2 to cells previously cultured for 3 days with SAC plus IL-2. These data suggest that IL-4 has a capacity to block the up-regulation of the high as well as low affinity IL-2R-induced by IL-2 in normal human B cells, and could provide a possible explanation for the decreased responsiveness of B cells to IL-2 in the presence of IL-4.
On the mechanism of action of dexamethasone in a rat mast cell line (RBL-2H3 cells). Evidence for altered coupling of receptors and G-proteins.
Authors: Collado-Escobar D , Ali H , Beaven M A
Published: January 2023

Prolonged exposure of rat basophilic leukemia (RBL-2H3) cells, a cultured analog of rat mast cells, to 0.1 microM dexamethasone resulted in global suppression of various stimulatory events in response to Ag and a global enhancement of the same stimulatory events to the adenosine analog, N-(ethylcarboxamide)adenosine (NECA). We had previously shown that Ag and NECA both activate phospholipase C but by different mechanisms; cells that had been treated with cholera or pertussis toxin, for example, responded to Ag but not to NECA with the release of inositol phosphates, increase in levels of cytosolic Ca2+, and secretion. Because the toxins still inhibited the responses to NECA in dexamethasone-treated cells, the effects of dexamethasone may have been exerted at the level of receptor/G-protein coupling rather than at the level of effector systems. Additional evidence for this was the following: 1) NECA-induced hydrolysis of the inositol phospholipids was still enhanced after permeabilizing (with streptolysin O or Staphylococcus alpha-toxin) and washing the cells; 2) the response to the G-protein stimulant, guanosine 5'-(3-O-thio)triphosphate was also enhanced in permeabilized, dexamethasone-treated cells and 3) binding and kinetic studies suggested that the enhanced responsiveness to NECA was attributable in part to an increase in receptor number. The suppressive action of dexamethasone on Ag-induced hydrolysis of inositol phospholipids, however, was readily lost by permeabilizing RBL-2H3 cells. The results indicate, therefore, that treatment with dexamethasone leads to changes in receptor-coupling mechanisms that are either resistant to (i.e., NECA-mediated responses) or reversed by (i.e., Ag-mediated responses) cell permeabilization.
Importance of subtype in the immune response to the pre-S(2) region of the hepatitis B surface antigen. I. T cell fine specificity.
Authors: Milich D R , Hughes J L , McLachlan A , Langley K E , Thornton G B , Jones J E
Published: January 2023

Previous studies of murine T cell recognition of the pre-S(2) region of the hepatitis B surface Ag (HBsAg) identified high (H-2b,d,q), intermediate (H-2s,k), and low to nonresponder (H-2f) haplotypes. However, these studies utilized the y subtype of HBsAg. The purpose of this study was to examine the influence of viral subtype on T cell recognition of the pre-S(2) region and to identify specific T cell recognition sites in a panel of H-2 congenic strains. Immunization with pre-S(2) containing HBsAg particles of the d and y subtypes indicated that T cell recognition of the pre-S(2) region is predominantly subtype-specific in murine strains of eight different H-2 haplotypes. Furthermore, the B10.M strain (H-2f) classified as a T cell nonresponder to the y subtype of the pre-S(2) region responds efficiently to the d subtype, indicating that pre-S(2) responder status can be subtype-dependent as well as subtype-specific. Studies using a truncated pre-S(2) polypeptide and synthetic peptides illustrated that the C-terminal sequence (p148-174) of the pre-S(2) region is the dominant focus of T cell recognition in multiple murine strains. Specifically, 17 distinct T cell recognition sites were defined within the C-terminal half of the pre-S(2) region. The fine specificity of T cell recognition of the pre-S(2) region was dependent on the H-2 haplotype of the responding strain. T cell recognition of all 17 sites was subtype specific, which is consistent with the fact that the C-terminal sequence is highly polymorphic between the d and y subtypes of the pre-S(2) region. Lastly, it was shown that the ability of synthetic peptides to elicit T cells cross-reactive with the native pre-S(2) region was variable and depended on the nature of the immunizing peptide. The pre-S(2)-containing HBsAg vaccines currently in clinical trials are composed of ra single subtype, either d or y. The results of this study suggest that both subtypes should be incorporated to increase the frequency of T cell responders to the pre-S(2) region, and to insure Th cell memory relevant to infection with hepatitis B virus of either the d or y subtypes.
Expression of IgE from a nonrearranged epsilon locus in cloned B-lymphoblastoid cells that also express IgM.
Authors: Chan M A , Benedict S H , Dosch H M , Hui M F , Stein L D
Published: January 2023

During development, B lymphocytes have the ability to switch from synthesis of IgM to immunoglobulins of another isotype such as IgG, IgA, or IgE. This class switching mechanism has been shown to involve DNA rearrangement and concomitant deletion of intervening CH genes. In our report, an EBV-transformed B lymphoblastoid cloned cell line is described that simultaneously expressed and secreted both IgM and IgE. DNA analysis showed the (nonproductive) rearrangement of one allele to gamma and (productive) rearrangement of the other allele to mu. Germ-line arrangement of the C epsilon gene was preserved on both alleles.
Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines.
Authors: Nuñez G , London L , Hockenbery D , Alexander M , McKearn J P , Korsmeyer S J
Published: January 2023

The t(14;18) of human follicular B cell lymphoma translocates the Bcl-2 gene into the Ig H chain locus and markedly deregulates Bcl-2 expression. We sought to determine if Bcl-2 could be directly implicated in a growth-factor pathway. Consequently, we introduced a retrovirus containing the murine Bcl-2 gene (N2-M-Bcl-2) or the parental retrovirus (N2) into a series of factor-dependent hemopoietic cell lines. Overexpressed Bcl-2 resulted in no long term IL-2, IL-3, or IL-6 independent clones, indicating that Bcl-2 could not spare the need for a specific ligand-receptor interaction. However, Bcl-2 did extend the short term survival of IL-3-dependent cell lines after factor deprivation. Although viable, IL-3-deprived pro B lymphocytes (FL5.12) bearing N2-M-Bcl-2 were in Go, and deregulated Bcl-2 did not obviously influence cell-cycle progression. Bcl-2 predominant effects were to delay the onset of cell death and to modestly augment viable cell growth in the first 48 h after IL-3 deprivation. This death sparing was associated with increased levels of Bcl-2 RNA and protein in factor-deprived cells possessing N2-M-Bcl-2. This result was not restricted to prolymphocytes because an IL-3-dependent mast cell line (32D) as well as a promyeloid line (FDC-P1) demonstrated the same response to Bcl-2. Moreover, the effect was not limited to the IL-3/IL-3R signal transduction pathway in that promyeloid cells maintained in granulocyte-macrophage-CSF or IL-4 displayed a similar response. Yet, Bcl-2-enhanced cell survival was not universal as an IL-2-dependent T cell line, and an IL-6-dependent myeloma line demonstrated no consistent effect upon IL withdrawal. Thus, Bcl-2 appears to interfere with cell death but in a cell type and/or factor-restricted fashion.
Hybridomas derived from lipopolysaccharide-activated anti-mu-treated B cells have active suppression of IgM secretion, demonstrable by secondary fusion.
Authors: Lawton A R , Summerlin V S , Dooley J S
Published: January 2023

Treatment of LPS-stimulated mouse B cells with bivalent anti-mu antibodies suppresses differentiation to Ig-secreting cells without interfering with proliferation. This treatment selectively inhibits up-regulation of transcription of differentiation-related genes. Induction of anti-mu suppression requires RNA and protein synthesis, suggesting involvement of a transcriptional repressor. We describe experiments designed to capture the repressed phenotype of anti-mu-treated cells. Fusions of anti-mu-treated cells with the B lymphoma line M12.4.5, but not plasmacytoma Ag8.653, yielded a significantly lower frequency of secretory hybridomas than did parallel fusion of cells treated with LPS only. To test for active repression, nonsecreting cloned hybridomas were secondarily fused to LPS-activated normal B cells. Secondary hybridomas with anti-mu parentage had a very low frequency of IgM secretion. Active suppression could only be demonstrated by secondary fusion. Neither supernatants nor extracts of repressor hybridomas influenced LPS-driven differentiation of normal B cells. These results confirm that this form of differentiation suppression is an active process, probably mediated by transcriptional controls. Repressor hybridomas should prove useful for studies of molecular mechanisms.
Aberrant regulation of IL-1 expression in macrophages from young autoimmune-prone mice.
Authors: Donnelly R P , Levine J , Hartwell D Q , Frendl G , Fenton M J , Beller D I
Published: January 2023

IL-1 is a multifunctional, immunoregulatory polypeptide produced by many cell types. Because activated macrophages are a major source of IL-1 and have also been implicated in the pathogenesis of autoimmune disease, we investigated the regulation of IL-1 expression in several autoimmune-prone strains of mice. Peritoneal macrophages derived from the autoimmune-prone strains MRL/lpr, MRL/+, NZB, and NZB/W F1, as well as NZW, displayed transient expression of IL-1 in contrast to the stable expression characteristic of control normal strains including A. Thy, A/J, B10, B10.A, B10.D2, C57BL/6, BALB/c, and C3H/HeN. The down-regulation of IL-1 by macrophages from the autoimmune-prone mice was not attributable to inherently defective signal transduction because macrophages from both the normal and autoimmune-prone strains displayed substantial initial levels of cell-associated and secreted IL-1. However, during the first 2 to 3 days in culture, macrophages from autoimmune-prone mice became progressively refractory to both induction and maintenance of IL-1, a pattern that correlated with changes in the levels of IL-1 alpha and beta mRNA. The progressive reduction in IL-1 expression by macrophages from these autoimmune-prone strains was not due to a reduction in general metabolism or viability, because expression of cell surface antigens, including MHC class I and II Ag and LFA-1, was comparable to that of control macrophages. Because IL-1 plays a critical role in the homeostasis of a variety of cell lineages, defective expression, and maintenance of IL-1 (and perhaps other cytokines) by macrophages from the autoimmune-prone strains may contribute to the immune dysregulation that develops in these mice. Alternatively, cytokine dysregulation might not contribute directly to disease, but rather reflect a more basic defect related to specific signal transducing or gene regulatory pathways.
Identification of sites within gp41 that serve as targets for antibody-dependent cellular cytotoxicity by using human monoclonal antibodies.
Authors: Tyler D S , Stanley S D , Zolla-Pazner S , Gorny M K , Shadduck P P , Langlois A J , Matthews T J , Bolognesi D P , Palker T J , Weinhold K J
Published: January 2023

In an effort to determine the functional activity of anti-HIV-1 human mAb and to define the epitopes against which they are directed, supernatants from 10 EBV-transformed lymphoblastoid cell lines producing mAb to HIV were tested. Five clones producing mAb to gp41 and five producing mAb to p24 were identified. The anti-HIV-1 human mAb were tested in neutralization and cell fusion assays in the form of cell culture supernatants at concentrations ranging from 1.7 to 22.0 micrograms/ml. None of the human mAb were found either to inhibit HIV-1-(IIIB or RF) associated cell fusion or to neutralize HIV-1 (IIIB) infection of AA5 cells. All human mAb were additionally tested in 6 h 51Cr release assays for their ability to direct HIV-1 specific antibody-dependent cellular cytotoxicity (ADCC). For ADCC assays, PBMC were isolated from healthy seronegative donors and used as effector cells. HIV-1 infected (IIIB, RF, and MN) CEM.NKR cells as well as CEM.NKR cells with purified gp120 adsorbed onto their surface served as targets. None of the anti-p24 mAb mediated ADCC. In contrast, three of the anti-gp41 mAb were able to direct a significant level of ADCC against each of the infected targets, but as expected, failed to lyse gp120 adsorbed cells. To define the specific epitopes against which the anti-gp41 mAb were directed, seven small peptides homologous to regions within the extracellular domain of gp41 were synthesized. Using RIA, two of the mAb could be mapped. The most effective ADCC-directing human mAb bound to a peptide comprising amino acids 644-663, whereas the least effective ADCC directing anti-gp41 human mAb bound to a region within the immunodominant portion of gp41 outlined by amino acids 579-604. Together, these results for the first time assign a functional activity to human mAb directed at specific regions within gp41 by demonstrating that areas within this molecule can serve as targets for ADCC.
cAMP differentially regulates expression of mRNA encoding IL-1 alpha and IL-1 beta in murine peritoneal macrophages.
Authors: Ohmori Y , Strassman G , Hamilton T A
Published: January 2023

The influence of PGE2 and the consequent rise in intracellular cAMP on LPS-induced IL-1 alpha and IL-1 beta mRNA levels has been examined in murine peritoneal macrophages. As has been previously reported, neither PGE2 nor dibutyryl cAMP modulated the levels of LPS-induced IL-1 alpha mRNA. In contrast, the levels of IL-1 beta mRNA were markedly potentiated (greater than 10 fold) under the same experimental conditions. This effect was due to elevation of intracellular cAMP because other agents known to elevate cAMP levels (e.g., cholera toxin, forskolin, 1-isomethyl-3-isobutylxanthine) had the same selective effect on IL-1 beta mRNA levels. PGE2 and dBcAMP not only potentiated LPS-induced IL-1 beta mRNA levels but were also able to stimulate modest accumulation of IL-1 beta mRNA in the absence of LPS. Although measurement of IL-1 activity by bioassay suggested that dBcAMP and PGE2 could suppress LPS-induced IL-1 expression, levels of IL-1 protein, determined by radioreceptor assay, were markedly elevated. cAMP did not appreciably alter the stability of either IL-1 alpha or IL-1 beta mRNA. Instead dBcAMP independently stimulated the transcriptional activity of the IL-1 beta gene. In concert the results demonstrate that cAMP can modulate the response of mononuclear phagocytes to LPS in a complex pattern; gene expression may be unaltered, suppressed, or potentiated and will thereby affect both the quality and magnitude of the ensuing inflammatory response.
A small proportion of cationic antibodies in immune complexes is sufficient to mediate their deposition in glomeruli.
Authors: Gauthier V J , Mannik M
Published: January 2023

Positively charged antibodies mediate enhanced deposition of circulating immune complexes at the glomerular basement membrane. The presented experiments demonstrate that when soluble immune complexes were prepared with a mixture of antibodies containing 10 to 25% cationic antibodies, then noncationic antibodies in the complexes were deposited in mouse glomeruli. One or two cationic antibodies in each immune complex sufficed for deposition of the complexes. Proof for this was obtained by two kinds of experiments. First, the injected immune complexes were prepared in Ag excess from mixtures of radiolabeled noncationic rabbit antibodies to human serum albumin (HSA) and unlabeled cationized rabbit antibodies to HSA, thus permitting the specific quantitation of the deposition of noncationic antibodies in glomeruli because of the presence of cationized antibodies within the same complexes. As a control experiment, immune complexes prepared only with noncationic antibodies resulted in very little deposition in kidneys over the same time period. Second, detection of the localization of the noncationic antibody in deposits in glomeruli by immunofluorescence microscopy was accomplished using immune complexes prepared with mixtures of noncationic goat antibodies to HSA and cationized rabbit antibodies to HSA. Thus, the synthesis of a small population of cationic antibodies during the immune response may lead to the formation of circulating immune complexes with enhanced propensity for deposition in glomeruli in patients with SLE or other immune complex diseases.
Ultraviolet cross-linking of helical oligonucleotides to two monoclonal MRL-1pr/1pr anti-DNA autoantibodies. Variations in H and L chain binding to DNA.
Authors: Jang Y J , Stollar B D
Published: January 2023

Experiments were performed to determine whether both H and L chains of different anti-native DNA autoantibodies are uniformly involved in binding to DNA. Two purified monoclonal mouse (MRL-1pr/1pr) IgG autoantibodies, H241 and 2C10, were tested. They both bound synthetic helical oligonucleotides of 10 to 20 base pairs in a gel electrophoresis retardation assay but differed in their preferences for given base sequences. Exposure of antibody-radiolabeled oligonucleotide mixtures to UV light (254 nm) for 10 min led to specific covalent cross-linking of oligonucleotide to both the H and the L chains of H241 but only to the H chain of 2C10. Single labeling events were detected without higher aggregation. The oligonucleotides were not cross-linked to unrelated IgG, even after 2 h of irradiation. Cross-linked (radioactively labeled) H and L chains of H241 and 2C10 were isolated from denaturing electrophoresis gels and digested with lysyl endopeptidase and/or staphylococcal V8 protease. H241 and 2C10 H chains each yielded a major labeled peptide fragment, but the peptides from the two antibodies were different. These experiments measured only some of the antibody-DNA interactions, probably with bases in the major groove of the DNA. They indicated that two MRL-1pr/1pr IgG anti-native DNA antibodies differ in their H and L chain contacts with DNA and provide an approach to identifying affinity-labeled binding sites in the antibodies.
Proteolytic fragmentation of sialophorin (CD43). Localization of the activation-inducing site and examination of the role of sialic acid.
Authors: Remold-O'Donnell E , Rosen F S
Published: January 2023

Sialophorin (CD43) is the major surface mucin on many hematopoietic cells. It has been implicated in regulating the survival of T lymphocytes in the circulation, and its functions in vitro as the receptor of a T lymphocyte and monocyte activation pathway. The structure of CD43 was examined by protease treatment of lymphoblastoid cells bearing surface CD43. Trypsin treatment converts CD43 (apparent Mr 115,000) to species of apparent Mr 100,000 called T-100, which remains cell-associated; however, the mechanism of trypsin action was not clarified. Pancreatic elastase and Staphylococcus aureus V8 protease cleave CD43 at discrete extracellular sites. V8 protease generates two fragments, which together account for all properties and mass of the parent molecule. The COOH-terminal fragment V-90 (apparent Mr 90,000) consists of the intracellular and transmembrane regions and part of the extracellular region. The fragment V-30 (apparent Mr 30,000), which is released from the cell, comprises the NH2-terminal approximately 78 amino acids with attached oligosaccharides. V-30 contains the binding sites for the antibodies L2 and L10; the latter is the antibody that activates lymphocytes and monocytes. These findings subdivide the extracellular region of CD43 and indicate that the activation-inducing epitope is located in the most distal portion of the molecule. It is shown that CD43 is insensitive to all but very high concentrations of three proteases. Pretreatment with sialidase enhances sensitivity 13-fold for trypsin, 40-fold for S. aureus V8 protease, and 400-fold for elastase, suggesting that sialic acid influences the survival of surface CD43 molecules when cells are exposed to protease.
Insulin-like growth factor-I supports proliferation of autocrine thymic lymphoma cells with a pre-T cell phenotype.
Authors: Gjerset R A , Yeargin J , Volkman S K , Vila V , Arya J , Haas M
Published: January 2023

We have studied the phenotypic characteristics and growth properties of murine T lymphoma cell lines derived from primary x-ray-induced thymic lymphomas at the earliest stage at which they can be detected, and well before spreading to other organs has occurred. These cell lines serve as model systems for the earliest events in T cell lymphoma induction, before tumor cell progression and spreading to other organs. We find that primary x-ray-induced T cell lymphoma lines have phenotypic characteristics of thymic pre-T cells and show no proliferative response to any of the IL tested nor to other hematopoietic growth factors. However, they do proliferate in response to insulin-like growth factor I (IGF-I) and to a small autocrine peptide distinct from IGF-I, which we term lymphoma growth factor. One of the earliest lesions in T cell lymphoma induction may therefore be an inhibition of differentiation at one of several specific points. In its early stages, T lymphoma cell growth may be restricted to an environment where local concentrations of specific growth factors such as IGF-I or lymphoma growth factor are sufficiently high.
Immunologic memory to phosphocholine keyhole limpet hemocyanin. Recurrent mutations in the lambda 1 light chain increase affinity for antigen.
Authors: Brown M , Stenzel-Poore M , Stevens S , Kondoleon S K , Ng J , Bächinger H P , Rittenberg M B
Published: January 2023

Anti-phosphocholine (PC)-keyhole limpet hemacyanin hybridomas representative of a memory response that express the lambda 1 L chain isotype have a high reactivity to PC-protein. A common feature of these hybridomas possessing high affinity for PC-protein is the occurrence of somatic mutations resulting in replacement changes in three CDR2 positions of the lambda 1 L chain. The influence of each of these three positions on the Ag binding properties of these antibodies was examined by site-specific mutagenesis and expression of recombinant antibody molecules by transfected cells. Affinity measurements and fine specificity profile determinations demonstrated the importance of the three lambda 1 CDR2 positions in Ag binding. Compared to antibodies expressing germline lambda 1, including one with an additional junctional serine that is not encoded by V or J, those antibodies possessing critical changes in CDR2 would have a strong selective advantage based on affinity differences for Ag. Sequence analysis of a group of clonally related hybridomas expressing mutated lambda 1 genes allowed construction of a hypothetical genealogic tree that suggests selection based on changes in CDR2 of lambda 1 in the absence of H chain mutations. The results are consistent with stepwise acquisition of mutations and selection based on affinity constraints.
Modification of proinflammatory cytokine production by the antirheumatic agents tenidap and naproxen. A possible correlate with clinical acute phase response.
Authors: Sipe J D , Bartle L M , Loose L D
Published: January 2023

The cytokines IL-6, IL-1, and TNF play a key role in the pathogenesis of rheumatoid arthritis (RA) and initiate hepatic serum amyloid A (SAA) expression after injury. To provide a possible mechanistic explanation for the previous observation that plasma SAA concentrations decreased during treatment of RA patients with tenidap, but increased during treatment with naproxen, the present study compared the effects of tenidap and naproxen on the two stages of SAA expression: cytokine production by human PBMC and cytokine-stimulated SAA synthesis by human Hep3B hepatoma cells. Tenidap inhibited production of IL-6 greater than TNF greater than IL-1; the effect of naproxen on production of all three cytokines was lesser and least on IL-6. Indeed, an increase in IL-6 production was observed after exposure to naproxen. PBMC beta-2-microglobulin production and total protein synthesis were unaffected at concentrations and times at which effects on cytokine production were observed. Cell density was a significant factor in the extent to which cytokines were stimulated by LPS. Approximately physiologic cell densities, 0.5 to 1 x 10(6) cells/ml, were optimal for stimulation of IL-1-beta and IL-6 production by LPS; however, greater amounts of TNF were produced at lower cell densities. Because neither tenidap nor naproxen inhibited SAA synthesis by cytokine-stimulated Hep3B cells and because they differ most significantly in their effect on IL-6 production, the results support a role for IL-6 in the continued stimulation of SAA production during RA.
Thymus-independent expression of a truncated T cell receptor-alpha mRNA in murine kidney.
Authors: Madrenas J , Pazderka F , Parfrey N A , Halloran P F
Published: January 2023

During studies on gene expression in the kidney, we unexpectedly observed that murine kidney expresses a truncated form of TCR-alpha mRNA (1.3-1.4 kb). This transcript was not associated with the presence of complete TCR-alpha mRNA (1.7 kb) or detectable TCR-beta or -delta transcripts, thus indicating that the truncated TCR-alpha mRNA could not be attributed to blood contamination of the kidney RNA preparation. The truncated TCR-alpha message appeared to contain at least the C alpha region, as suggested by hybridization with an intra-C alpha 24 oligonucleotide probe, by amplification of the C alpha region with the polymerase chain reaction from total kidney mRNA, and by sequencing of, and hybridization with, the amplified products. In situ hybridization of kidney sections indicated that the transcript was expressed in interstitial cells. Northern blots of cortex and medulla RNA showed that the cells expressing the truncated TCR-alpha mRNA were predominantly located in the medulla. To investigate the possibility that the transcript was not produced by T cells or NK cells, fractionation of renal cell suspensions were performed. The truncated TCR-alpha mRNA was detected in a fraction containing large (low buoyant density) cells in which no expression of CD3, Thy 1, or NK-1.1 was detected, indicating that these cells are not mature T cells, do not express a functional TCR, and are not NK cells. The cells expressing the truncated TCR-alpha mRNA were radiosensitive, and were not thymus dependent, because this transcript was as abundant in nude mice as in normal mice. The transcript was not detected in bone marrow. Expression of the truncated TCR-alpha mRNA was not dependent on an intact recombinase activity as its expression was not affected by the severe combined immunodeficiency mutation. Our results show that murine kidney contains a population of radiosensitive thymus-independent large interstitial cells that express a truncated TCR-alpha mRNA that is not associated with surface expression of functional TCR. These cells may have attempted to rearrange TCR-alpha genes, suggesting that they may be related to the lymphoid lineage.
Requirement for extrathymic class I histocompatibility antigens for positive selection of thymus-derived T lymphocytes.
Authors: Fuller B , Lefrançois L
Published: January 2023

We have examined whether the thymus can produce immature T cell precursors for subsequent positive selection in the periphery. Using the intestine as a model system, we demonstrate that extrathymic MHC class I molecules positively select thymus-derived TCR-alpha beta CD8+ intestinal intraepithelial lymphocytes. Grafting of MHC class I+ thymus onto MHC class I- hosts resulted in the generation of donor and host-derived CD4-CD8+ T cells in lymph node, but few TCR-alpha beta CD4-CD8+ intraepithelial lymphocytes. The results indicate that the intestine can act as a site of positive selection for thymus-derived T cells. Bone marrow reconstitution studies demonstrated that non-hematopoietic, radiation-resistant cells, perhaps intestinal epithelial cells, were responsible for extrathymic positive selection of thymus-derived T cells. These findings demonstrate that the thymus can support organ-specific immunity via provision of targeted precursor populations.
Nitric oxide induces apoptosis in mouse thymocytes.
Authors: Fehsel K , Kröncke K D , Meyer K L , Huber H , Wahn V , Kolb-Bachofen V
Published: January 2023

Nitric oxide (NO) produced at high concentrations by the inducible NO synthase is an important effector molecule involved in immune regulation and defense. We have examined whether NO represents a signal for triggering apoptosis in thymocytes. Freshly isolated thymocytes were incubated with different chemical NO donors for various intervals. Apoptosis was determined by detection of DNA strand breaks with in situ nick translation. All NO donors induced thymocyte apoptosis with 30% positive thymocytes vs 10% in controls after 8 h. Apoptosis was prevented by addition of ZnSO4. Short-term pre-exposure to NO resulted in protection from apoptosis induced by glucocorticoids comparable with the protective effect of heat shock. Flow cytometry revealed that NO treatment as well as heat shock or dexamethasone incubation is accompanied by reduction in the CD4+ CD8+ thymocyte subpopulation. Apoptosis induction was accompanied by increased expression of p53, as detected by PCR analysis 2 h after NO donor addition. In vivo treatment of mice with endotoxin results in increased thymic apoptosis. Focal apoptosis was found to occur in close proximity to blood vessels 18 h after LPS treatment. Capillary endothelium and dendritic cells adjacent to apoptotic foci were found to stain strongly for inducible NO synthase expression. Furthermore, in an in vitro experiment using cocultures of thymocytes with LPS/cytokine-activated endothelial cells expressing inducible NO synthase, a significantly increased rate of thymocyte apoptosis was found, and this could be prevented completely by inhibiting NO production. Addition of dexamethasone to these cocultures did not lead to a further increase in the percentage of apoptotic thymocytes, underlining the protective effect of NO on dexamethasone-induced apoptosis.
T cell precursors in the spleen give rise to complex T cell repertoires in the thymus and the intestine.
Authors: Hamad M , Whetsell M , Klein J R
Published: January 2023

T cell precursors located in peripheral immune tissues have been studied according to the potential to repopulate the thymus and the gut of lethally irradiated mice. T cell repopulation could be achieved with spleen cells from athymic or euthymic mice thoroughly devoid of mature T cells. Repopulation did not occur with lymph node lymphocytes as determined from studies in congenic mice. The kinetics of T cell repopulation differed in the gut and thymus in that donor-derived T cells appeared in the gut by day 7 after cell transfer, and in the thymus by day 14 after cell transfer. The multipotent nature of splenic T cell precursors was evident from the finding that all major phenotypic subsets of T cells in the thymus and the gut developed after spleen cell transfer. T cell repopulation of the intraepithelial lymphocytes also occurred efficiently in athymic radiation chimeras injected with spleen cells from congenitally athymic nude mice, demonstrating that gut T cell repopulation by those cells does not require a functional thymus. PCR-spectratype analyses of twenty-four V beta TCR genes in thymocytes and intraepithelial lymphocytes revealed extensive TCR-beta repertoires in both tissues 1 to 2 wk after cell transfer, although T cells with rearranged TCR were undetectable in the donor spleen cell population. The minimal phenotype of the splenic T cell precursor was determined to be CD3-, CD4-, CD8-, HSA+.
Multiple isoforms of guinea pig decay-accelerating factor (DAF) generated by alternative splicing.
Authors: Nonaka M , Miwa T , Okada N , Nonaka M , Okada H
Published: January 2023

Human decay-accelerating factor (DAF, CD55), which is one of the regulators of complement activation (RCA), prevents complement activation on homologous cell membranes, resulting in a functional discrimination between self cells and invading microorganisms. DAF is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein and has been identified at the molecular level only in primates. We have isolated guinea pig DAF cDNA clones from a spleen library and identified six different classes. All encode the same four short consensus repeat domains with 58% amino acid sequence identity to human DAF, but show variability in the C-terminal region. Alternative splicing of two optional exons generates transmembrane, GPI-anchored, and secreted forms of guinea pig DAF, and differential usage of splice sites in the single exon composed of internally quintuplicated sequences generates variable Ser/Thr-rich regions. Multiple isoforms were expressed ubiquitously in all tissues and cells tested. Similar variability in the Ser/Thr-rich region has been reported in human membrane cofactor protein (MCP), another membrane inhibitor of the RCA family. There seems to be a common ancestral sequence in DAF and MCP consisting of a Ser/Thr-rich region, but obviously multiplication occurred independently, indicating the importance of variability of the Ser/Thr-rich region for the effective regulation of complement activation.
Molecular characterization of bovine V lambda regions.
Authors: Sinclair M C , Gilchrist J , Aitken R
Published: January 2023

Using an inverse PCR technique with primers directed against constant region sequences, 18 V lambda regions have been isolated at random from bovine splenic cDNA for detailed analysis. On the basis of protein sequences in FR1 and their homologies to the human lambda chains NEWM and MCG, the bovine determinants can be separated into two families that have been designated V lambda 1 (94% of all clones isolated) and V lambda 2 (6% of all clones isolated). Scrutiny of the nucleotide sequences has allowed subdivision of the dominant family into V lambda 1a, V lambda 1b and V lambda 1c groupings, which represent 29, 65, and 6% of all V lambda 1 clones respectively. It appears that the size of CDR1 is V lambda group-specific--the region comprises 14 amino acids in V lambda 1a determinants and 13 residues in V lambda 1b and V lambda 1c,--whereas with a single exception, seven amino acids make up CDR2. The size of CDR3 is variable, ranging from 10 to 15 amino acids in length. Conservation of those nucleotide sequences that encode the signal peptide and FR3 has enabled us to amplify and characterize 14 V lambda 1 genes from kidney genomic DNA. Interestingly, half of the germline sequences were not represented in the data gathered from splenic cDNA, while the remaining genes could be identified as members of the V lambda 1a and V lambda 1b groups. The findings indicate that the V lambda 1 family dominates the expressed bovine light chain repertoire but that a large proportion of the germline segments from which this repertoire is derived may be nonfunctional.
IL-6 stimulates vitronectin gene expression in vivo.
Authors: Seiffert D , Geisterfer M , Gauldie J , Young E , Podor T J
Published: January 2023

We tested the hypothesis that vitronectin (Vn) is regulated as an acute phase reactant in response to inflammatory stimuli. In initial experiments, Vn levels were measured during the surgically induced acute phase response in humans. The plasma concentration of Vn increased approximately twofold following elective orthopedic surgery and remained elevated up to 5 days. To examine the mechanism(s) of increased Vn synthesis, hepatic Vn mRNA expression and serum levels were examined in three rat models of acute inflammation: LPS (i.v.), CFA (i.p.), or turpentine (s.c.) injection. The serum concentration of Vn increased approximately twofold 24 h following treatment with turpentine. The expression of Vn mRNA in the liver increased markedly as early as 3 h after treatment in these models and remained elevated up to 18 h. Northern blot analysis of RNA isolated from fractionated liver cells derived from rats treated with LPS indicated that Vn was mainly expressed in hepatocytes, but not in the endothelial or nonparenchymal cell fractions. To analyze the individual effects of raised corticosterone and IL-6 levels on the expression of hepatic Vn mRNA, rats were injected (i.p.) with either dexamethasone or purified recombinant rat IL-6. Vn mRNA expression was elevated within 1 h after IL-6 injection, whereas dexamethasone-injected rats showed unchanged Vn expression. Vn mRNA also was increased in rats chronically injected with IL-6. These results indicate that the Vn gene is up-regulated in acute and chronic inflammation, and this induction is primarily mediated by IL-6.
Constitutive secretion of transgene-encoded IgG2b autoantibodies leads to symptoms of autoimmune disease.
Authors: Radic M Z , Ibrahim S M , Rauch J , Camper S A , Weigert M
Published: January 2023

Anti-DNA Ab are strictly regulated, except in autoimmunity, where they are expressed and may contribute to pathogenicity. To study constitutive anti-DNA Ab secretion in nonautoimmune mice, two anti-dsDNA H/L chain transgene combinations were constructed using an IgG2b C region with secretory but no transmembrane domain exons. One H/L combination, consisting of the VH3H9 H and V kappa 4 L chain transgenes, was chosen to recreate 3H9, an autoantibody that originally arose in an autoimmune MRL/lpr mouse; the other paired a higher affinity variant of VH3H9, 56R, with the same V kappa 4 L chain. Elevated titers of IgG2b along with normal levels of other isotypes were observed in transgene-positive mice, indicating that constitutive transgene-directed Ab secretion was achieved. Sera and hybridoma supernatants from VH3H9 gamma transgene-positive animals exhibited binding to dsDNA, ssDNA, and cardiolipin. Mice expressing the 56R gamma H chain and the V kappa 4 L chain showed enhanced binding. Expression of the transgenes correlated with signs of autoimmune disease, including prolonged plasma clotting in vitro, and reduced litter size. The results suggest that even a single autoreactive H chain that escapes tolerance may suffice to induce features of autoimmune disease.
LPS induces CD14 association with complement receptor type 3, which is reversed by neutrophil adhesion.
Authors: Zarewych D M , Kindzelskii A L , Todd R F , Petty H R
Published: January 2023

CD14, a glycosylphosphatidyl inositol (GPI)-linked membrane protein, is a key membrane binding site for LPS (endotoxin). Although CD14 lacks transmembrane and cytoplasmic sequences, it activates CR3-mediated leukocyte adhesion and cytokine release. Since CR3 has been shown to interact with other GPI-linked membrane proteins, we tested the hypothesis that CD14 can physically associate with CR3. Using qualitative and quantitative resonance energy transfer microscopy, we show that LPS in the presence of serum or LPS binding protein triggers formation of CD14-CR3 complexes. Kinetic studies show that CD14-CR3 complexes dissociate as neutrophils attach to substrates. We speculate that LPS-charged CD14 enhances CR3-mediated adhesion by directly binding to CR3.
Fas- and activation-induced apoptosis are reduced in human T cells preactivated in the presence of TGF-beta 1.
Authors: Cerwenka A , Kovar H , Majdic O , Holter W
Published: January 2023

The elimination of activated but not resting T cells involves apoptosis induced either by restimulation via the TCR/CD3 complex, CD2, or by signaling through the Fas Ag. The factors regulating the shift of an apoptosis-resistant to an apoptosis-sensitive phenotype and vice versa have not so far been clarified. Here we report that TGF-beta 1, when present during a PHA activation course, significantly increases viability of human T cells upon reculture in medium alone, following restimulation via CD2, CD3, or after triggering the Fas Ag. Using DNA gel electrophoresis and an in situ nick translation technique we further show that activation-induced and Fas-mediated apoptosis are reduced in T cells that were prestimulated with PHA plus TGF-beta 1, compared with control cells prestimulated with PHA alone. Moreover, when PHA-preactivated T cells are further expanded in IL-2, inclusion of TGF-beta 1 results in higher cell yields at any timepoint from day 30 to 75 of cell culture compared with control cultures without TGF-beta 1. However, no differences in Fas or bcl-2 protein expression are found between cells stimulated in the absence or presence of TGF-beta 1. Together, our data identify TGF-beta 1, when present during an activation course, as an important viability factor possibly of importance for the generation of effector and/or long-lived memory T cells.