bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of nonresponsiveness to AKR/Gross leukemia virus in AKR.H-2b:Fv-1b mice. An analysis of precursor cytotoxic T lymphocyte frequencies in young versus moderately aged mice.
Authors: Wegmann K W , McMaster J S , Green W R
Published: December 2022

As young adult AKR.H-2b:Fv-1b mice reach about 9 wk of age, they begin to develop a nonresponsiveness to AKR/Gross leukemia virus. Unlike young mice that are responders, moderately aged AKR.H-2b:Fv-1b mice, after immunization and secondary in vitro restimulation in bulk culture with AKR/Gross virus induced tumors, can not generate anti-AKR/Gross virus-specific CTL. The mechanism of conversion to nonresponsiveness in moderately aged AKR.H-2b:Fv-1b mice is not understood, but it is correlated with increased expression of endogenous ecotropic viral antigens. Our present investigation focuses on determining the frequency of anti-AKR/Gross virus precursor CTL in AKR.H-2b:Fv-1b mice as a function of age. This was achieved by performing limiting dilution cultures of immune spleen cells obtained from young and moderately aged AKR.H-2b:Fv-1b mice. Although spleen cells obtained from immune moderately aged mice can not differentiate in bulk cultures into anti-AKR/Gross virus-specific CTL, there was no evidence of substantially decreased frequencies of virus-specific precursor CTL, relative to precursor CTL frequencies observed in young responder AKR.H-2b:Fv-1b mice.
Heterogeneity of CD4+ T cells involved in anti-allo-class I H-2 immune responses. Functional discrimination between the major proliferating cells and helper cells assisting cytotoxic T cell responses.
Authors: Kitagawa S , Sato S , Azuma T , Shimizu J , Hamaoka T , Fujiwara H
Published: December 2022

MLR in various combinations with class I H-2 disparity revealed that there are three patterns of MLR in the aspect of responding T subset (CD4 vs CD8) dominance. Irrespective of the CD8 vs CD4 dominance, a single i.v. administration of class I-disparate allogeneic spleen cells resulted in almost complete abrogation of anti-class I proliferative capacity of both CD4+ and CD8+ T cells in six combinations. The suppression of proliferative responses was correlated with the striking reduction in the ability to produce IL-2 upon stimulation with the relevant class I alloantigens. In contrast, i.v. presensitized recipient mice exhibiting only marginal MLR/Il-2 production could generate comparable magnitudes of anti-allo class I CTL as well as graft rejection responses to those induced by normal unpresensitized mice. The administration in vivo of anti-CD4 antibody along with the i.v. presensitization not only suppressed the generation of CTL responses by spleen cells but also induced appreciable prolongation of allo-class I-disparate skin grafts under conditions in which neither alone did it. These results demonstrate that 1) the suppression of graft rejection responses is not necessarily reflected on the reduction of MLR; 2) CD8+ CTL precursors responsible for graft rejection can be activated by either allo-class I-reactive CD8+ or CD4+ Th cells; 3) i.v. presensitization induces functional elimination of CD8+ and CD4+ proliferative/IL-2-producing T cells but not of CD8+ CTL precursors and CD4+ Th whose capacity is expressed by assistance of CTL induction but not by their own proliferation. Thus, this study illustrates the heterogeneity of class I alloantigen-reactive CD4+ T cells in the aspect of their capacity to proliferate themselves vs contribute to CTL induction as well as graft rejection.
Circulating CD3+/T cell receptor V gamma 3+ fetal murine thymocytes home to the skin and give rise to proliferating dendritic epidermal T cells.
Authors: Payer E , Elbe A , Stingl G
Published: December 2022

The presence of CD3/TCR V gamma 3 moieties on both dendritic epidermal T cells (DETC) and fetal murine thymocytes has led to the concept that fetal thymocytes expressing this particular TCR phenotype are the actual DETC precursors. To test this assumption, we injected i.v. thymocyte suspensions prepared from day 16 and day 19 fetal mice as well as from adult animals, into syngeneic and Thy-1-disparate nude mice, the epidermis of which contains only Thy-1+/CD3- lymphocytes. Phenotypic analysis of the recipient epidermis by in situ immunolabeling revealed that injection of day 16 and day 19 fetal, but not of adult, thymocytes resulted in the appearance of distinct clusters of DETC as judged by their dendritic morphology and uniform expression of CD3/TCR V gamma 3 receptors. The presence of CD3+/TCR V gamma 3+ cells in the fetal, but not in the adult, thymocyte population(s) together with the failure to detect DETC after transfer of Thy-1+/CD3- fetal thymocytes strongly suggest that CD3+/TCR V gamma 3+ thymocytes are the DETC precursors. Kinetic studies of the DETC population from 2 to 12 wk after cell transfer revealed a substantial increase in the cell density within the DETC clusters that was not accompanied by an increase in the number of clusters. Thus, it appears that newly arriving DETC undergo proliferative activity in situ. Collectively, our results show that, under the experimental conditions chosen, CD3+/TCR V gamma 3+ fetal thymocytes are actual DETC precursors. Although it is not clear whether these experimental conditions are representative of the in vivo situation, they may serve as a useful model for studying the mechanisms underlying the homing properties of different lymphocyte subsets.
Role of glycosaminoglycans in the regulation of T cell proliferation induced by thymic stroma-derived T cell growth factor.
Authors: Kimura K , Matsubara H , Sogoh S , Kita Y , Sakata T , Nishitani Y , Watanabe S , Hamaoka T , Fujiwara H
Published: December 2022

The present study investigates the regulatory effects of glycosaminoglycans such as heparin and heparan sulfate on T cell proliferation induced by thymic stromal cell monolayer or its derived T cell growth factor (TCGF). A thymic stromal cell clone (MRL104.8a) supported the growth of Ag-specific, IL-2-dependent Th cell clone (9-16) in the absence of Ag and IL-2 by producing a unique TCGF designated as thymic stroma-derived T cell growth factor (TSTGF). The addition of heparin to cultures in which the growth of 9-16 Th cells was otherwise stimulated by the MRL104.8a monolayer or a semipurified sample of the TSTGF resulted in heparin dose-dependent inhibition of 9-16 Th proliferation. The dose of heparin required for inducing 50% reduction of TSTGF-induced proliferation of Th at a given cell number was found to be proportional to the magnitude of the TSTGF added to cultures, suggesting that heparin exerted its inhibitory effect by binding to the TSTGF rather than by acting on Th cells. A similar growth-inhibiting effect of heparin was observed in IL-7-dependent proliferation of pre-B cell line or Th, but not in IL-2-dependent T cell proliferation or IL-3-dependent myeloid cell proliferation. A strong affinity of TSTGF and IL-7 for heparin was confirmed by the fact that both TSTGF and IL-7 adhered to columns of heparin-agarose and were eluted by salt. When various glycosaminoglycans were tested for the heparin-like Th growth-regulatory capacity, heparan sulfate exhibited Th growth-inhibiting ability comparable to that observed for heparin. These results indicate that the activity of thymic and/or bone marrow stroma-derived lymphocyte growth factor (TSTGF/IL-7) but not of Th-producing TCGF (IL-2) is negatively regulated by heparin or heparan sulfate, which would represent major glycosaminoglycans in the extra-cellular matrix of stromal cells.
Pre-ligation of CR1 enhances IgG-dependent phagocytosis by cultured human monocytes.
Authors: Waytes A T , Malbran A , Bobak D A , Fries L F
Published: December 2022

Opsonization of the C3b receptor (CR1) on phagocytic cells with C3b enhances both attachment of targets to the cells and subsequent IgG-dependent ingestion of these targets. To explore mechanisms involved in this increased phagocytosis, we adhered cultured human monocytes to surfaces pre-coated with CR1 ligand or control proteins and quantitated ingestion of sheep E opsonized with IgG alone. Three ligands for CR1 resulted in markedly enhanced phagocytosis of targets when compared individually to a panel of non-ligands, as determined by both the proportion of monocytes ingesting targets (percent phagocytosis) and by the number of targets ingested per 100 monocytes (phagocytic index). The ligands included purified C3b, iC3, and Fab fragments of 1B4, a monoclonal anti-CR1, which resulted in a percent phagocytosis of 56.3 (p less than 0.01), 59.0 (p less than 0.01), and 54.4 (p less than 0.02) and a phagocytic index of 281.2 (p less than 0.01), 281.1 (p less than 0.01), and 247.1 (p less than 0.02), respectively. Control proteins including human serum albumin, hemoglobin, Fab fragments of anti-fibronectin, anti-beta 2 microglobulin, and MOPC 21, and Fc fragments of 1B4 and MOPC 21 produced no significant stimulation of phagocytosis, nor did F(ab')2 fragments of monoclonal anti-CR3, M1/70. CR1-specific augmentation of target ingestion was apparent with monocytes cultured in serum-free medium for 1 to 7 days, but was not seen with freshly elutriated cells. Phagocytosis of unopsonized or IgM-coated targets was minimal. These results suggest that the adherent monocytes are primed by CR1 cross-linking for enhanced FcR-mediated phagocytosis even when the CR1 ligand is not present on the targets. This contrasts with the behavior of CR3, and demonstrated functional divergence between these C3 fragment receptors in the phagocytic process.
Differential synthesis of 5-lipoxygenase in peripheral blood and synovial fluid neutrophils in rheumatoid arthritis.
Authors: Jobin C , Kreis C , Gauthier J , Letarte J , Beaulieu A D
Published: December 2022

In order to investigate 5-lipoxygenase enzyme regulation in neutrophils during an inflammatory reaction, we studied 5-lipoxygenase mRNA levels, as well as de novo enzyme synthesis, in resting and activated neutrophils isolated from normal individuals and patients with rheumatoid arthritis. The approach used was to analyze these activities in resting peripheral blood neutrophils of normal individuals on the one hand and in peripheral blood and matched synovial fluid neutrophils isolated from patients with rheumatoid arthritis on the other hand. Our first observation was that resting peripheral blood neutrophils of either normal individuals or patients show detectable levels of 5-lipoxygenase mRNA and are able to synthesize the enzyme de novo. Our second observation was that inflammatory activated neutrophils from synovial fluid reveal lower 5-lipoxygenase mRNA levels and enzyme synthesis than do the patient-matched peripheral blood cells. This is in spite of the fact that, for other proteins, synovial fluid neutrophils are equally or more active than their peripheral blood counterparts. We conclude that peripheral blood neutrophils are capable of synthesizing the enzyme, thus ensuring the turnover of the protein. Furthermore, complex regulatory mechanisms appear to take place in response to inflammation as it occurs in synovial fluids of patients with rheumatoid arthritis, leading to decreased mRNA levels and enzyme synthesis. Possible mechanisms of regulation are discussed and are presently under investigation.
Molecular basis of "suppressor" macrophages. Arginine metabolism via the nitric oxide synthetase pathway.
Authors: Mills C D
Published: December 2022

A molecular explanation for "suppressor" macrophage inhibition of lymphocyte proliferation is described. NG-monomethyl-L-arginine (NGMMA), a specific inhibitor of the nitric oxide synthetase pathway, markedly augments Con A-induced proliferation of rat splenic leukocytes. Macrophages are necessary and sufficient for NGMMA-releasable-suppression, as indicated by a loss of suppression after either pretreatment of isolated splenic macrophages with NGMMA or their depletion by plastic adherence or L-leucine methyl ester. L- (but not D-) arginine overrides NGMMA-releasable suppression, and suppression is blocked by RBC as would be expected if nitric oxide were the effector molecule. Unlike rats, NGMMA did not augment Con A-induced proliferation of normal mouse splenic leukocytes. However, NGMMA did augment Con A-induced proliferation of mouse splenic leukocytes induced to contain suppressor macrophages by intravenous injection of Corynebacterium parvum, which suggests a quantitative, not qualitative, difference in suppressor macrophages between rats and mice. Nitrite production, as an indicator of nitric oxide synthesis, correlated with suppressor macrophage activity in rats and mice and was inhibited by NGMMA. Finally, NGMMA also markedly enhanced proliferation with every other mitogen examined (PHA, protein A, PWM, and LPS). It is concluded that immunoregulation of lymphocyte proliferation by suppressor macrophages is mediated, in part, directly or indirectly by products of the nitric oxide synthetase pathway.
Immunologic and structural characterization of the dominant 66- to 73-kDa antigens of Borrelia burgdorferi.
Authors: Luft B J , Gorevic P D , Jiang W , Munoz P , Dattwyler R J
Published: December 2022

The 66- to 73-kDa proteins of Borrelia burgdorferi are dominant immunogens and expressed in all strains of B. burgdorferi. The humoral response to these Ag occurs relatively early during the course of infection. Two-dimensional Western blot analysis of this group of Ag revealed them to consist of a tetrad of proteins with apparent molecular mass of 66, 68, 71, and 73 kDa. Furthermore, in this study we demonstrate the 66-kDa protein to be a potent inducer of lymphoproliferation in the patient immune to B. burgdorferi. Monospecific polyclonal antibodies and mAb demonstrate that each of these proteins was immunologically distinct. However, direct amino acid sequence of the 66- and 68-kDa Ag was almost identical and had a high level of sequence similarity to the GroEL heat-shock protein (Hsp60) of Escherichia coli and the 60-kDa immunodominant protein of Treponema pallidum. The amino terminal sequence of the 71- and 73-kDa proteins of B. burgdorferi was almost identical and these proteins had remarkable sequence similarity to the DnaK heat-shock protein of E. coli (Hsp70). It appears likely, therefore, that proteins related to the heat-shock family are potent immunogens of B. burgdorferi.
The human lambda L chain Ig locus. Recharacterization of JC lambda 6 and identification of a functional JC lambda 7.
Authors: Bauer T R , Blomberg B
Published: December 2022

The human lambda L chain Ig gene complex consists of multiple JC gene segments. A seventh human lambda C region gene segment, C lambda 7, was found 2.7 kb downstream of C lambda 6 in this gene complex. A J lambda gene segment, J lambda 7, was found 1.2 kb upstream of C lambda 7 and contains potentially functional nonamer and heptamer recombination sites, an RNA splice site and J coding region. C lambda 7 maintains an open reading frame and encodes a new lambda isotype. C lambda 7 encodes Kern+ and Oz- determinants, but does not encode any of the Kern+Oz- myeloma proteins published to date. Nevertheless, we present evidence that JC lambda 7 is transcribed in normal lymphocytes and is functional. In contrast, we present new data that the C lambda 6 gene segment, reported by others to encode the Kern+Oz- protein, is non-functional due to a 4-bp insertion in our cosmid clone. The 4-bp insertion was characterized further in 32 genomic DNA samples by producing a distinctive restriction fragment length and verified by the DNA sequences of the polymerase chain reaction products of two different cell lines. We discuss the possibility that the Kern+Oz- myeloma proteins do not define an isotype and are not encoded by JC lambda 7 nor other non-allelic genes, and we discuss the level of expression of JC lamba 7 as compared to that of JC lambda 2 and JC lambda 3.
Signal transduction through the T cell antigen receptor. Activation of phospholipase C through a G protein-independent coupling mechanism.
Authors: Augustine J A , Secrist J P , Daniels J K , Leibson P J , Abraham R T
Published: December 2022

The T cell Ag (Ti-CD3) receptor complex has been proposed to regulate phosphoinositide-specific phospholipase C (PLC) through a cholera toxin (CTX)-sensitive guanine nucleotide-binding (G) protein. In this study, we have used CTX and staurosporine as pharmacologic probes to further define the linkage between the Ti-CD3 receptor and PLC activity in the human T cell line, Jurkat. CTX pretreatment inhibited Ti-CD3 receptor-dependent phosphoinositide hydrolysis and, concomitantly, protein tyrosine kinase activation in intact cells. Studies with electrically permeabilized Jurkat cells revealed that guanosine 5'-(3-O-thio) triphosphate stimulated an increase in PLC activity, that unlike the response to Ti-CD3 receptor ligation, was not affected by cellular pretreatment with CTX. In contrast, the phosphotyrosine phosphatase inhibitors, orthovanadate and molybdate anions, stimulated phosphoinositide hydrolysis in permeabilized cells through a CTX-sensitive mechanism of PLC activation. Additional studies with a known PTK inhibitor, staurosporine, supported the results obtained with CTX. Staurosporine pretreatment inhibited the phosphoinositide hydrolysis induced by anti-CD3 antibodies or phosphotyrosine phosphatase inhibitors, but failed to alter the G protein-dependent PLC activation response to guanosine 5'-(3-O-thio) triphosphate. The results of this study indicate that PLC activity(s) in Jurkat cells are regulated by both G protein- and PTK-dependent coupling mechanisms. However, the differential inhibitory effects of CTX and staurosporine on these PLC activation pathways strongly suggest that a protein tyrosine kinase activation event, rather than a G protein, mediates the functional linkage between the Ti-CD3 receptor and PLC activity in Jurkat cells.
A T helper cell hybridoma produces an antigen-specific regulatory activity. Relationship to the T cell receptor by serology and antigenic fine specificity.
Authors: Bissonnette R , Zheng H G , Kubo R T , Singh B , Green D R
Published: December 2022

We have previously shown that a T cell hybridoma, A1.1, constitutively produces an Ag-specific regulatory factor with specificity for poly-18, a synthetic polypeptide. This cell also responds to poly-18 plus I-Ad by producing lymphokines. The antigenic specificity of the factor and the T cell appeared to be the same. This suggested the possibility that some part of the TCR, responsible for antigenic specificity of the cell, also imparts specificity to the A1.1-derived factor. This was supported by the observation that the factor was bound and eluted from a monospecific anti-TCR antiserum. Further, we demonstrated that antisense oligodeoxynucleotides corresponding to the TCR V alpha of A1.1 (but not TCR V beta) block production of the Ag-specific factor. Herein, we report recent findings that strengthen the proposed relationship between the TCR and the A1.1-derived factor. The factor was bound and eluted from a monoclonal anti-TCR C alpha antibody, but not from anti-TCR beta, anti-V beta 6, nor anti-CD3 epsilon. The anti-TCR C alpha antibody bound a Mr 46-kDa protein from A1.1 supernatants, which is the same apparent size at which activity could be eluted from an SDS-PAGE gel separation of concentrated factor. Antigenic fine-specificity analysis revealed that two amino acids in poly-18 are critical for the recognition of the antigen by the Ag-specific factor. These two amino acids appear to be those recognized by the TCR. The factor that was bound and eluted from the monoclonal anti-TCR C alpha showed this fine-specificity as well. This, combined with our earlier studies, supports the view that the A1.1-derived factor is encoded, at least in part, by TCR-alpha.
Tyrosine phosphorylation is an essential event in the stimulation of B lymphocytes by Staphylococcus aureus Cowan I.
Authors: Roifman C M , Chin K , Gazit A , Mills G B , Gilon C , Levitzki A
Published: December 2022

Staphylococcus aureus Cowan I (SAC) is a potent mitogen for purified human B cells. By using Western blotting with antiphosphotyrosine antibodies, we demonstrated that the mitogenic effect of SAC is associated with rapid tyrosine phosphorylation of proteins of 45, 68, 75, 97, and 145 kDa. This tyrosine phosphorylation was detected within 30 s of the addition of SAC; it reached a maximum within 10 min, after which it declined gradually. In contrast to SAC, most soluble anti-IgM antibodies do not induce proliferation of isolated human B cells. As indicated by Western blotting, soluble anti-IgM antibodies induced a similar pattern of tyrosine phosphorylation, with the exception of the 68-kDa protein, which was the most heavily phosphorylated protein in SAC-treated cells. A similar but less intense 68-kDa band was also induced by mitogenic anti-IgM bound to beads. This suggested that tyrosine phosphorylation, especially of p68, may play an important role in B cell mitogenesis. To test this hypothesis, we determined the effect of specific tyrosine kinase inhibitors (tyrphostins) on SAC-induced tyrosine phosphorylation, oncogene expression, and B cell proliferation. The concentration dependencies of inhibition of these processes suggested that they were linked. Nonspecific toxic effects of the tyrphostins were ruled out by the demonstration that the tyrphostins did not alter cell viability and did not inhibit B cell proliferation induced by phorbol esters, which do not induce tyrosine phosphorylation. For maximal inhibition of SAC-induced cell proliferation, the tyrophostins needed to be added before or shortly after addition of SAC. Taken together, these data indicate that tyrosine phosphorylation is an obligatory early signal in B cell proliferation.
The Q7 alpha 3 domain alters T cell recognition of class I antigens.
Authors: Aldrich C J , Lowen L C , Mann D , Nishimura M , Hood L , Stroynowski I , Forman J
Published: December 2022

In this study we have analyzed the role of the alpha 3 domain of class I molecules in T cell recognition. Using the laboratory engineered molecules LLQQ (alpha 1/alpha 2 from Ld, alpha 3, and phosphatidyl inositol (PI) linked C terminus from Q7) and LLQL (alpha 1/alpha 2 from Ld, alpha 3 from Q7, transmembrane (TM) and cytoplasmic domains from Ld) we show that these molecules are not recognized by primary Ld-specific CTL. The cell membrane expression of both Ld and LLQL are upregulated by co-culture with an exogenously supplied murine cytomegalovirus-derived peptide indicating that the Q7 alpha 3 domain does not interfere with binding of Ag to alpha 1/alpha 2. However, only peptide pulsed Ld but not LLQL target cells are recognized by Ld-restricted-peptide specific CTL. In contrast to the above results, LLQL and LLQQ molecules can be recognized by bulk alloreactive anti-Ld CTL and 2/3 of CTL clones derived from in vivo primed mice. The fact that these secondary CTL recognize LLQQ indicates that a PI linkage is permissive for presentation of class I epitopes to alloreactive CTL. These secondary CTL are resistant to blocking at the effector stage by mAb against CD8 and express relatively low levels of membrane CD8 molecules compared to CTL from unprimed mice. Further, culture of unprimed CTL precursors in the presence of CD8 mAb also allows for the generation of CD8-independent CTL that recognize LLQL. Taken together, these data indicate that the alpha 3 domain of Q7 (Qa-2) prevents CD8-dependent CTL from recognizing Ld, regardless of whether the class I molecule is attached to the cell surface by a PI moiety or as a membrane spanning protein domain. We hypothesize that this defect in recognition is most likely due to an inability of CD8 to interact efficiently with the Q7 alpha 3 domain and could account for why Q7 molecules do not serve as restricting elements for virus and minor H-Ag-specific CTL.
Sequences homologous to class II MHC W, X, and Y elements mediate constitutive and IFN-gamma-induced expression of human class II-associated invariant chain gene.
Authors: Brown A M , Barr C L , Ting J P
Published: December 2022

Invariant chain (Ii) is intracellularly associated with MHC class II molecules, is implicated in class II function, and is coordinately regulated with the alpha- and beta-chains of MHC class II genes at the transcriptional level. Included among the various cis-acting elements of transcriptional control in MHC class II genes are the class II boxes, X and Y, and sequences 5' of X in the W (Z, H, S) region of class II genes. The Ii promoter region contains homologues of these elements, designated here as X, Y', and "W". This study utilized transient transfection and chloramphenicol acetyltransferase analysis to investigate the role of these elements in basal and inducible Ii gene expression. Invariant chain X, Y', and "W" all contribute to gene expression in B lymphoblastoid cell lines, making them likely candidates to mediate coordinate control of class II and Ii genes. IFN-gamma-inducible expression of the Ii gene in a glioblastoma cell line is also regulated through X, Y', and "W". Thus, the Ii class II boxes and "W" have dual roles in basal and inducible gene transcription.
NF-X, a transcription factor implicated in MHC class II gene regulation.
Authors: Kouskoff V , Mantovani R M , Candéias S M , Dorn A , Staub A , Lisowska-Grospierre B , Griscelli C , Benoist C O , Mathis D J
Published: December 2022

The X box has been shown in several assay systems to be a critical element of MHC class II gene promoters. Several X box-binding activities have been discovered in nuclear extracts from a variety of cell lines. The critical question is: which of these are responsible for mediating X box function? This report provides a further characterization of NF-X, a highly specific X box-binding activity we described previously. The cell-type distribution, structural features, and binding site characteristics of NF-X are analyzed in detail, to facilitate comparison with other reported activities. Most importantly, the functional relevance of NF-X is assessed by scanning mutagenesis, and the results indicate that this complex is indeed involved in regulating MHC class II gene expression. With these data in mind, the relationship between NF-X and RF-X, an X box-binding activity reported to be absent in patients with severe combined immunodeficiency, is discussed.
Human peripheral blood lymphocytes targeted with bispecific antibodies release cytokines that are essential for inhibiting tumor growth.
Authors: Qian J H , Titus J A , Andrew S M , Mezzanzanica D , Garrido M A , Wunderlich J R , Segal D M
Published: December 2022

We have compared the mechanisms by which human PBL targeted with bispecific antibodies either lyse tumor cells or block their growth in culture or in mice. We found that resting PBL were unable to mediate lysis, but were able to block tumor growth. Moreover, targeted PBL were unable to lyse bystander cells, whereas targeted PBL did block the growth of bystander tumor cells in culture and in nude mice. Supernatants from cultures of targeted PBL, or from PBL grown on anti-CD3-coated flasks, blocked the growth of tumor cells in the absence of added effector cells, and antibodies against TNF-alpha and IFN-gamma reversed the inhibition of tumor growth, but had no effect upon cytolysis mediated by targeted by PBL. Our results show that targeted human PBL mediate two different antitumor activities: lysis, which occurs rapidly and requires the direct attachment of the target cell to the cytotoxic cell, and tumor growth inhibition, which is mediated by cytokines released into the medium as a result of receptor cross-linking. The inhibition of bystander tumor growth in mice by targeted PBL suggests that factor release is sufficient to block tumor growth in vivo. Targeted factor release therefore provides a mechanism by which targeted PBL could block the growth of tumor cells in vivo that were not bound by the effector cells, but which were located in the vicinity of tumor cells that were bound.
Model for studying virus attachment. II. Binding of biotinylated human T cell leukemia virus type I to human blood mononuclear cells potential targets for human T cell leukemia virus type I infection.
Authors: Dhawan S , Streicher H Z , Wahl L M , Miller N , Louie A T , Goldfarb I S , Jackson W L , Casali P , Notkins A L
Published: December 2022

Purified human T cell leukemia virus type I (HTLV-I) was biotinylated and used to study its attachment to human PBMC. The use of biotinylated HTLV-I (biot-HTLV-I) in conjunction with mouse mAb specific for selected cell-surface molecules and flow cytometric analysis allowed us to positively identify virus-binding cells among a heterogeneous blood mononuclear cell population. Biot-HTLV-I efficiently bound not only to T cells, but also to B cells and monocytes. Preincubation of monocytes with excess of unlabeled HTLV-I significantly reduced the attachment of biot-HTLV-I. HTLV-I not only bound to, but also infected, B cells, as suggested by: i) in situ hybridization of a 35S-labeled full length HTLV-I DNA probe with EBV-transformed B cells, previously cocultured with HTLV-I-producing (G11MJ) T cells, and ii) hybridization of the same nick-translated 32P-labeled DNA probe with blotted DNA from similar HTLV-I-infected EBV-transformed B cells. HTLV-I infection did not affect the ability of B cells to secrete IgG. These findings suggest that HTLV-I cannot only infect cells of the T lineage, but can also infect B cells.
Elevated levels of endogenous IL-6 in systemic lupus erythematosus. A putative role in pathogenesis.
Authors: Linker-Israeli M , Deans R J , Wallace D J , Prehn J , Ozeri-Chen T , Klinenberg J R
Published: December 2022

Elevated spontaneous IgG production is characteristic of SLE. To identify the factors that support it, IL-6, a cytokine with an important role in the differentiation of IgG-secreting cells, was studied in SLE patients. Higher than normal levels of IL-6 were found, by a B9 assay, in sera of 63 of 70 patients (p less than 0.05). IL-6 was detected in 36 of 37 active SLE sera in higher titers (p = 0.009) than those for inactive SLE (n = 33), which were higher (p less than 0.05) than healthy controls (n = 15). IL-6 mRNA was detected in freshly isolated PBMC of 11 of 11 patients but not in normal PBMC, whereas IL-1 mRNA was detected only in patients with active disease. IL-6 activity was recovered from PBMC of four SLE patients, but not from four normal donors. By immunoperoxidase, IL-6 was detected in the cytoplasm of SLE monocytes and lymphocytes. When SLE PBMC were grown in short term cultures with no deliberate stimulation, expression of the IL-6 gene declined rapidly. Accordingly, the spontaneous production of IgG by SLE PBMC could be enhanced by exogenous IL-6. Spontaneous IgG production was diminished by 20 to 65% in the presence of neutralizing antibodies to IL-6, TNF-alpha, or IL-1. In contrast, neutralization of endogenous IL-4 increased production by approximately 40%. Anti-TNF-alpha treatment decreased IL-6 content of PBMC cultures, whereas anti-IL-4 augmented it, and exogenous IL-6 reversed anti-TNF-alpha effects on IgG production. Therefore, it is possible that the neutralization of TNF-alpha and IL-4 affected IgG production by modulating the synthesis/activity of IL-6. These results support the concept that SLE B cell hyperactivity is promoted by dysregulation of endogenous cytokines and suggest that IL-6, in particular, has an important pathogenic role.
FMC46, a cell protrusion-associated leukocyte adhesion molecule-1 epitope on human lymphocytes and thymocytes.
Authors: Pilarski L M , Turley E A , Shaw A R , Gallatin W M , Laderoute M P , Gillitzer R , Beckman I G , Zola H
Published: December 2022

In this report, we describe a 76-kDa glycoprotein recognized by mAb FMC46 that, by virtue of its concentration on cell protrusions involved in motility, may be important in lymphoid cell locomotion. FMC46 detects an epitope of the leukocyte adhesion molecule-1 (LAM-1), a member of the selecting family (LAM-1, Endothelial Leukocyte Adhesion Molecular-1 (ELAM-1), and Granule Membrane Protein-140 (GMP-140), that is expressed on LAM-1-transfected cell lines, is a glycosylation epitope based on its loss after culture in tunicamycin, and is closely related to the LAM-1.2 epitope. FMC46 is expressed at high density on the majority of CD45RA+ and CD45RO+ peripheral blood T cells (60 to 70%) and on a subset of thymocytes that includes the multinegative CD3- CD4- CD8- progenitor cells (100% FMC46hi) and the CD45R0- presumptive thymic generative lineage (70% FMC46hi). It appears at reduced density and frequency on CD45RA- thymocytes (50% FMC46lo), comprised mainly of death-committed thymocytes. Among thymic subsets defined by expression of CD4 and/or CD8, FMC46 is expressed at high density predominantly on a subset of single-positive cells and not on double-positive cells. These results suggest a fundamental role for LAM-1 in thymic development, with a high density preferentially expressed on cells involved in thymic generative processes and a low density on cells progressing to intrathymic death. A major subset of peripheral blood B cells and thymic B cells also express FMC46. Immunohistochemistry on frozen sections indicated strong staining in splenic follicles and around blood vessels, staining of the thymic medulla and subcapsular areas, and staining of the mantle zone of germinal centers of the lymph node. FMC46+ lymphocytes accumulated along high endothelial venules in the lymph node. On locomoting multinegative thymocytes, FMC46 is concentrated on the leading tip of extended processes, on pseudopods, and on ruffles, unlike the distribution of either CD44 or TQ1 (LAM 1.2), suggesting a role in locomotion. On dividing multinegative thymocytes, FMC46 was found almost exclusively along the cleavage furrow, implicating it in detachment processes. We conclude that the properties of the LAM-1 molecule recognized by FMC46 are consistent with a role in detachment phases of motility and of cell interactions.
Identification and characterization of the Poa p IX group of basic allergens of Kentucky bluegrass pollen.
Authors: Olsen E , Zhang L , Hill R D , Kisil F T , Sehon A H , Mohapatra S S
Published: December 2022

We reported previously the primary structure of three full-length cDNA clones that encode a new group of IgE-binding proteins of Kentucky bluegrass (KBG) pollen, designated as Poa p IX. In the present study we have further characterized the cloned Poa p IX proteins, identified the corresponding proteins in KBG pollen extract, and determined their antigenic relationships with other known grass pollen allergens. A recombinant IgE-binding polypeptide rKBG7.2 that represents the C-terminal fragment, conserved in Poa p IX proteins, appeared to contain epitopes unique to these proteins and served as an immunosorbent for the isolation of the corresponding human IgE antibodies. On two-dimensional PAGE blots these IgE antibodies bound selectively to five distinct KBG pollen proteins with molecular mass 28 to 34 kDa and isoelectric point greater than 9.5. These proteins differ in size and charge from known allergens, but are very similar to those of the recombinant Poa p IX proteins. The rKBG3.1, which represents the N-terminal region of the Poa p IX clone KBG31, as well as the corresponding natural allergens were shown to possess epitopes that crossreact with the acidic group V allergens of Timothy. Comparison of amino acid sequences of recombinant Poa p IX proteins with those of Lol p I isoallergens revealed no significant sequence similarities. In contrast, partial homology was demonstrated between the N-terminal sequences of these proteins and the Phl p V proteins. Our results confirm that the Poa p IX clones represent a distinct and major group of allergens of KBG pollen, and demonstrate structural similarities and antigenic cross-reactivities among different groups of allergenic proteins in grass pollens.
Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells.
Authors: Hestdal K , Ruscetti F W , Ihle J N , Jacobsen S E , Dubois C M , Kopp W C , Longo D L , Keller J R
Published: December 2022

Murine bone marrow cells expressing the cell surface Ag RB6-8C5 were identified by fluorescence-activated cell-sorting analysis using a rat IgG mAb. The fluorescent intensity of RB6-8C5 was variable on bone marrow cells. This made it possible to separate bone marrow cells into distinct subpopulations, RB6-8C5neg, RB6-8C5lo, and RB6-8C5hi cells. Morphologic analysis of the sorted populations demonstrated that the Ag was expressed on myeloid cells. The expression of RB6-8C5 increases with granulocyte maturation, whereas expression is transient on cells in the monocytic lineage. The RB6-8C5hi sorted cells were enriched for end-stage neutrophils (75%), whereas the RB6-8C5lo sorted cells contained more immature myeloid cells and myelocytes (75%). Lymphocytes and macrophages were less than 5% in any RB6-8C5+ population, whereas the erythroid precursors were RB6-8C5neg. The colony forming unit culture (CFU-C) (greater than 90%) were found in the RB6-8C5neg and RB6-8C5lo populations, and all the CFU-granulocyte, erythroid, megakaryocyte, and macrophage (CFU-GEMM) and burst-forming units-erythroid (BFU-E) were in the RB6-8C5neg population. Granulocyte-macrophage-CSFR (GM-CSFR) and IL-1 alpha R were expressed on RB6-8C5hi bone marrow cells, whereas no receptors could be detected on RB6-8C5neg and RB6-8C5lo cells. The expression of the RB6-8C5 Ag can be induced on RB6-8C5neg cells in liquid culture by IL-3 and granulocyte-macrophage CSF. Thus, RB6-8C5 is a myeloid differentiation Ag whose expression can be regulated by cytokines.
Modulation of mouse complement receptors 1 and 2 suppresses antibody responses in vivo.
Authors: Thyphronitis G , Kinoshita T , Inoue K , Schweinle J E , Tsokos G C , Metcalf E S , Finkelman F D , Balow J E
Published: December 2022

A mAb, 7G6, that binds to mouse CR1 and CR2 and down-modulates their expression on splenic B cells in vivo, was used to determine whether a decrease in CR1 and CR2 expression affects antibody responses to different T-dependent and T-independent Ag. Injection of mice with the mAb 7G6 prior to immunization with FITC haptenated Salmonella typhimurium (SH5771), Salmonella montevideo (SH5770), SRBC, or Ficoll dramatically decreased subsequent antibody responses to FITC. Although both IgM and IgG primary antibody responses were affected similarly, the antibody levels were most inhibited during early phases of the response. In contrast, down-modulation of the CR did not affect memory antibody responses, because injection of mice with 7G6 before a second immunization with FITC-SH5771 had no effect on subsequent anti-FITC antibody production. Moreover, polyclonal in vivo activation of the mouse immune system by anti-mouse IgD antibodies was not affected by previous administration of 7G6, because anti-IgD-induced increases in Ia expression and serum IgG1 levels were not affected. Taken together, these observations suggest that CR1 and CR2 may play an important role in enhancing primary antibody responses to many T-dependent and T-independent Ag and may contribute to a host's response to naturally occurring antigens such as bacteria.
Glucocorticoids inhibit eosinophil responses to granulocyte-macrophage colony-stimulating factor.
Authors: Lamas A M , Leon O G , Schleimer R P
Published: December 2022

The role of the eosinophil as an active proinflammatory cell in asthma and other allergic disorders has been well established. Glucocorticosteroids have long been used therapeutically as antiinflammatory agents in a variety of disease states where eosinophilia is a prominent feature. Although glucocorticoids are known to reduce tissue and circulating eosinophil numbers, the mechanisms by which they do so have not been clearly elucidated. Culture of eosinophils in vascular endothelial cell supernatants (VEC SUP) induces phenotypic and functional changes and prolongs the survival of the eosinophils. The survival-promoting activity in VEC SUP was shown to be granulocyte-macrophage CSF (GM-CSF) by neutralization with specific antibody. The potent glucocorticosteroid, dexamethasone (DEX), inhibited the prolongation of eosinophil survival caused by culture in either VEC SUP or human rGM-CSF. DEX (10(-6) M) exerted a direct survival-inhibitory effect on the eosinophil by the 4th day in culture in VEC SUP. This survival-inhibitory effect was dependent on the concentration of DEX (10(-10)-10(-6) M). Other glucocorticoids, including prednisolone (10(-7), 10(-6) M) and hydrocortisone (10(-7), 10(-6) M) also inhibited survival. The rank order of potency of the steroids indicates that this effect is mediated by a glucocorticoid receptor. This conclusion is supported by the failure of the sex steroids testosterone (10(-8)-10(-6) M) or beta-estradiol (10(-6) M) to inhibit eosinophil survival in the presence of VEC SUP. The effect of glucocorticoids on eosinophils is not a simple direct toxic effect because it was reversed by higher concentrations of GM-CSF. DEX shifted the GM-CSF dose-response curve for survival approximately fivefold to the right. GM-CSF induced a shift in eosinophil buoyant density which was partially blocked by DEX. These results suggest that glucocorticoids may inhibit elements of cytokine "priming" of eosinophils and that the eosinophilopenic effects of glucocorticoids may result in part from a direct effect on the eosinophil within a regulatory system involving cytokines.
Effect of genistein, a tyrosine kinase inhibitor, on latent EBV activation induced by cross-linkage of membrane IgG in Akata B cells.
Authors: Daibata M , Mellinghoff I , Takagi S , Humphreys R E , Sairenji T
Published: December 2022

The activation of phosphoprotein tyrosine kinases was studied in the regulation of EBV activation in Akata cells after cross-linking membrane IgG with anti-IgG. Protein tyrosine phosphorylation was induced in Akata cells after stimulation with anti-IgG, as determined by immunoblotting with the PY20 anti-phosphotyrosine mAb. The frequency of phosphotyrosine-activated cells was also measured by immunofluorescence with the PY20 antibody. Genistein, an inhibitor of tyrosine kinases, at non-cytotoxic doses blocked EBV activation, as measured in the induction of EBV Ag, EBV immediate early BZLF1 mRNA, and its protein product ZEBRA. Such inhibitions were reversed upon removing genistein from the cultures. Genistein inhibition of early Ag induction depended upon the time of addition of genistein after stimulation with anti-IgG. These findings indicate that activation of tyrosine kinase is required for EBV activation after cross-linking membrane IgG in Akata cells.
IFN-gamma- and IL-5-producing cells compartmentalize to different lymphoid organs in Trichinella spiralis-infected mice.
Authors: Kelly E A , Cruz E S , Hauda K M , Wassom D L
Published: December 2022

The differential induction of cytokines associated with Th1 and Th2 subsets has recently been described during Trichinella spiralis infection. Increased levels of resistance appear to correlate with elevated levels of the Th1-associated cytokines, IFN-gamma and IL-2. In the present report, a filter immunoplaque assay is used to quantify the actual numbers of cells that secrete IFN-gamma and IL-5. It is demonstrated that, in T. spiralis-infected B10.Q mice, Th1- and Th2-associated responses are compartmentalized to different lymphoid organs. Thus, Ag-induced IFN-gamma-producing cells predominate in the spleen, whereas IL-5-producing cells prevail in the mesenteric lymph nodes (MLN). A corresponding compartmentalization of Ag-specific IgA and IgG1 antibody-secreting cells to the MLN is also noted. The virtual absence of Th1-associated responses in the MLN appears to be an Ag-associated phenomenon. MLN from either naive or T. spiralis-infected mice do have the capacity to secrete IFN-gamma if stimulated with Con A. The striking compartmentalization of Ag-driven cytokine responses seen in this parasite system may facilitate study of the mechanisms that regulate the induction of Th1 and Th2 subsets.
Definition of an epitope and MHC class I molecule recognized by gag-specific cytotoxic T lymphocytes in SIVmac-infected rhesus monkeys.
Authors: Miller M D , Yamamoto H , Hughes A L , Watkins D I , Letvin N L
Published: December 2022

Infection of macaque monkeys with the simian immunodeficiency virus of macaques (SIVmac) results in disease similar to human AIDS. Therefore, the macaque monkey is proving to be an important model for testing the effectiveness of various AIDS vaccine approaches. A detailed analysis of the cellular immune responses is necessary for the evaluation of candidate vaccines. However, this has not been possible in macaques, due, in part, to the unknown nature of the MHC molecules that restrict their T lymphocytes. In our report we demonstrate that a particular MHC class I molecule involved in the rhesus monkey's effector T lymphocyte response to SIVmac is expressed at a high frequency in a colony of rhesus monkeys. SIVmac-infected monkeys that express this MHC class I molecule all develop CTL that are restricted by that molecule and recognize an identical nine amino acid epitope of the SIVmac gag protein. This MHC class I molecule has been defined as an HLA-A homolog by cDNA cloning and sequencing. It has also been expressed in an MHC class I-deficient cell line to demonstrate directly the cloned molecule's capacity to bind and present peptide Ag to CTL. These studies illustrate that AIDS virus-specific CTL can be characterized in detail in the rhesus monkey and lay the foundation for exploring novel approaches to AIDS virus vaccination in this species.
Complexity and sequence identification of 24 rat V beta genes.
Authors: Smith L R , Kono D H , Theofilopoulos A N
Published: December 2022

Twenty-four TCR V beta genes were cloned by anchored PCR from the Lewis rat strain and identified by nucleotide and amino acid sequence comparisons to known mouse V beta genes. Rat V beta genes exist in 17 single-member and 3 multimember subfamilies and exhibit 86 to 94 and 72 to 92% nucleotide and amino acid sequence similarities, respectively, to their mouse counterparts. A single rat gene, designated V beta 20, having no previously known mouse counterpart was identified; a closely related gene was subsequently isolated from the C57BL/6 mouse strain. Characterization of the rV beta genes provides the basis for addressing rat T cell tolerance mechanisms and V beta gene usage in immunologically mediated rat disease models.
Characterization of the proliferative response of a CD4-8- thymic T lymphoma cell line to stimulation by thymic cellular elements.
Authors: Pinto V B , Rock K L
Published: December 2022

E710.2 is a cloned T cell line that was isolated from an AKR/J thymic tumor. This clone expresses Thy-1, heat-stable Ag, and the CD3/TCR complex but does not express CD4 or CD8. When the E710.2 cell line is injected into syngeneic mice, it grows as a malignant tumor in lymphoid organs and the thymus. In contrast, this cell line does not grow in vitro under standard culture conditions. This latter property allowed us to analyze the in vitro responsiveness of this CD4-CD8- cell line to stimulation by pharmacologic agents and cellular elements from the spleen and thymus. E710.2 cells proliferate when stimulated with phorbol esters or when cocultured with thymocytes or splenocytes. We could not detect soluble stimulatory factors in cultures of E710.2 and/or lymphoid cells, suggesting that cell contact might be required for this response. The stimulatory activity in thymus and spleen appears to be broadly expressed, because all cell subsets that were examined from these tissues stimulate this cell line. The stimulation of E710.2 cells is not MHC-restricted and is not inhibited by anti-MHC mAb. Furthermore, the responsiveness of these cells is not decreased when the TCR/CD3 complex is modulated from the cell surface. Similarly, TCR/CD3-deficient E710.2 variant clones retain their responsiveness to thymic and splenic cell stimulation. These findings suggest that there is a TCR-independent pathway of activation in E710.2 that is stimulated by a broadly expressed, non-MHC-encoded molecules(s).