bluuebunny/crossref_metadata_2025 huggingface dataset:
Age-dependent V region expression in the human antibody response to the Haemophilus influenzae type b polysaccharide.
Authors: Lucas A H , Azmi F H , Mink C M , Granoff D M
Published: December 2022

The human L chain antibody repertoire specific for the Haemophilus influenzae type b (Hib) polysaccharide (PS) is composed of kappa I, kappa II, kappa III, kappa IV, and lambda L chain V regions, but the most commonly occurring VL is encoded by the unmutated V kappa II-A2 gene. To determine whether this VL repertoire is influenced by age, we used idiotypic probes to monitor V kappa II-A2, V lambda, and V kappa III usage in the antibody response to an Hib PS-protein conjugate vaccine. A single dose of a vaccine consisting of Hib PS coupled to an outer membrane protein complex of Neisseria meningitidis was administered. Adults (n = 35), 18-month-old infants (n = 35), and 2-month-old infants (n = 46), all with > or = 0.9 microgram/ml anti-Hib PS antibodies, were tested for VL region markers in postvaccination sera. V kappa III anti-Hib PS antibodies were not detected in any of the 2-month-old infants but were detected in 29% of the 18-month-old infants and 69% of the adults (p < 0.001). The lack of kappa III antibodies in 2-month-old infants could not be accounted for by lack of a kappa response, because kappa antibodies to Hib PS were present (> 0.15 microgram/ml) in 45% of these infants. Hibld-1, an idiotope expressed by anti-Hib PS antibodies having the kappa II-A2 V region, was present in postvaccination sera of 66% of the adults and 80% of the 18-month-old infants but was less frequent in the 2-month-old infants (35%, p < 0.001). In contrast, Hibld-2, which is an idiotope expressed by a subset of V lambda VII anti-Hib PS antibodies, was rare or infrequent in adults and 18-month-old infants (0% and 6%, respectively) but was present in 43% of 2-month-old infants (p < 0.001). Our findings demonstrate that dramatic changes in VL region utilization occur in the human antibody response to this Hib PS conjugate vaccine as a function of age. Because previous studies have shown that V region usage correlates with antibody fine specificity and avidity for Hib PS, these age-related differences in V region expression may affect the ability of vaccines to confer protective immunity at different ages.
Inhibition of IL-2-dependent proliferation by a prostaglandin-dependent suppressor factor.
Authors: Ferreri N R , Herzog W R , Askenase P W
Published: December 2022

In the picryl chloride contact sensitivity system in mice, i.v. injections of trinitrobenzene sulfonic acid (TNBSA) prevents elicitation of delayed-type hypersensitivity reactions. This suppression is due in part to a non-specific, PG-dependent factor (TNBSA-F) that is induced by i.v. injection of TNBSA and is produced by pooled spleen and lymph node cells in vitro. Inasmuch as a role for lymphokines such as IL-2 has been postulated in delayed-type hypersensitivity, we determined the in vitro effects of TNBSA-F on the responsiveness of HT-2 target cells to IL-2. TNBSA-F induced a dose-dependent unresponsiveness of HT-2 cells to IL-2. The inhibitory activity was not present in supernatants from lymphoid cells of sham-treated mice. In the presence of indomethacin, spleen, and lymph node cells from TNBSA-immunized mice produced a factor whose activity was much reduced compared to TNBSA-F. This suggested that PG were required for TNBSA-F activity. However, PG alone did not induce the unresponsiveness because TNBSA-F but not sham-treated mice had inhibitory activity despite containing similar levels of PGE2. Rather, the combination of i.v. TNBSA injections and PG synthesis during production of TNBSA-F were required to produce a suppressive TNBSA-F. The inhibitory effect of TNBSA-F was not due to the presence of transforming growth factor-beta, soluble immune-response suppressor, INF-gamma, or JE in the factor preparation. Partial characterization showed a single peak of in vitro TNBSA-F activity (molecular mass approximately 35-55 kDa) by Sephadex G-200 gel filtration chromatography and by HPLC. In addition, TNBSA-F retained its activity after multiple cycles of freeze-thaw and heating for 1 h at 56 degrees C. The inhibitory effects of TNBSA-F on IL-2-induced proliferation suggest that suppression of delayed type hypersensitivity after i.v. administration of TNBSA may, in part, be due to a PG-dependent suppressor factor that inhibits the responsiveness of target cells to IL-2.
Dextran-conjugated anti-IgD antibodies inhibit T cell-mediated IgE production but augment the synthesis of IgM and IgG.
Authors: Peçanha L M , Yamaguchi H , Lees A , Noelle R J , Mond J J , Snapper C M
Published: December 2022

We previously demonstrated that anti-IgD antibodies conjugated to dextran (alpha delta-dex) were a potent co-stimulus for Ig secretion by resting murine B cells in the presence of cytokines. However, although alpha delta-dex stimulated the secretion of most Ig isotypes it selectively failed to costimulate IgE production even in the presence of high concentrations of IL-4. Earlier reports indicated that unconjugated anti-IgM, which was not an effective costimulus for Ig secretion, in fact inhibited Ig production induced by LPS. We determined the effect of alpha delta-dex, at concentrations that costimulated cytokine-induced Ig secretion, on Ig production by LPS- or T cell-activated B cells, and whether IgE production was affected in a selective manner. We observed that alpha delta-dex inhibited Ig isotype production (IgE > IgG > IgM) by LPS-activated B cells, while further stimulating their proliferation. This effect of alpha delta-dex was mediated directly at the level of the B cell and was accompanied by a comparable inhibition in Ig class switching, as assessed by flow cytometric analysis of membrane Ig isotype-positive cells. The inhibitory effects of alpha delta-dex on LPS-induced Ig secretion and class switching occurred at 1000-fold lower concentrations of anti-IgD than that reported necessary for inhibition by unconjugated anti-IgM. Whereas IL-4 + IL-5 costimulated Ig isotype production by alpha delta-dex-activated cells, the further addition of LPS led to a marked ablation of the Ig secretory response indicating the cross-inhibitory effects of these two modes of B cell activation. By contrast, alpha delta-dex augmented IgM and IgG1 secretion by resting B cells stimulated with either an anti-CD3-activated CD4+ Th2 clone or with activated T cell membranes in combination with IL-4 + IL-5. However, alpha delta-dex potently inhibited T cell-mediated IgE secretion. These findings underscore the existence of, and demonstrate a number of novel interrelationships between, three distinct pathways of B cell differentiation induced by different modes of activation. Further, the observation that pg/ml quantities of alpha delta-dex selectively inhibits T cell-induced IgE production in vitro suggests a novel strategy to down-regulate this Ig isotype in vivo.
Induction of costimulatory molecule B7 in M12 B lymphomas by cAMP or MHC-restricted T cell interaction.
Authors: Watts T H , Alaverdi N , Wade W F , Linsley P S
Published: December 2022

M12 B lymphomas expressing transfected Ak molecules with truncated cytoplasmic domains have a defect in Ag presentation to some autoreactive T cell hybrids. This defect in Ag presentation is corrected by pretreatment of the B cells with agents that elevate intracellular cAMP. Here we show that dibutyryl-cAMP treatment of M12 B lymphomas leads to cell surface expression of the costimulatory molecule B7. Furthermore, CTLA4Ig, a ligand for B7, inhibits activation of an accessory signal-dependent T hybrid. B7 is also inducible in M12 B lymphomas upon MHC-restricted interaction with T cells that can be activated by the APC, but not by T cells that fail to respond to truncated MHC-bearing M12 cells. Activation of the unresponsive T hybrids with immobilized anti-CD3 confers on them the ability to induce B7 in the APC. Direct engagement by immobilized antibodies of MHC class II on M12 B lymphomas did not induce B7 expression. Taken together, these results imply that during T-B interaction, initial T cell activation events lead to the ability of the T cell to induce costimulatory activity in the B cell, which in turn further activates the T cell. Activated T cell supernatants induced a small amount of B7 but were not nearly as effective as cAMP or as coincubation of T and B cells. These results suggest a role for T-B contact or localized cytokine secretion in the induction of B7 during T-B interaction.
Stimulation of human T cells by streptococcal "superantigen" erythrogenic toxins (scarlet fever toxins).
Authors: Braun M A , Gerlach D , Hartwig U F , Ozegowski J H , Romagné F , Carrel S , Köhler W , Fleischer B
Published: December 2022

The pyrogenic (erythrogenic) exotoxins A and C (SPEA and SPEC) of Streptococcus pyogenes belong to the family of mitogenic toxins of which the staphylococcal enterotoxins are the prototypes. The erythrogenic toxin B (SPEB) is a proteinase precursor. All SPE have been reported to be superantigens. Here we have analyzed the human T cell response to these toxins. We used highly purified preparations of SPEA, SPEB, and SPEC from different S. pyogenes strains. These toxins were apparently homogenous in SDS-PAGE, IEF, and HPLC. In addition, recombinant SPEA and SPEC were produced in Escherichia coli. In cultures of PBMC, all three toxins expanded preferentially a fraction of T cells. Using mAb against V beta 2, -5, -6, -8, and -12, we investigated the phenotype of the stimulated cells. Natural SPEA, SPEB, and SPEC strongly stimulated V beta 8+ T cells, whereas recombinant SPEA and SPEC did not. Both natural and recombinant SPEA stimulated V beta 12+ cells and both natural and recombinant SPEC stimulated V beta 2+ cells. In accordance with these findings, a human V beta 8+ line responded to all three toxins derived from S. pyogenes but not to the recombinant proteins. An antiserum against natural SPEC neutralized specifically the V beta 2-stimulating activity of SPEC and the V beta 8-stimulating activity of all three toxins, but had no effect on the response to other superantigens. This shows that trace amounts of a potent novel V beta 8-stimulating activity not identical to SPEA and SPEC are responsible for the stimulation of V beta 8+ T cells by natural SPEA and SPEC reported previously. In a preliminary screening of S. pyogenes strains from patients, we found that this novel superantigen appears to be more widely distributed than SPEA and SPEC. Furthermore, we present evidence that also the superantigenic properties of SPEB are due to contaminations with this V beta 8 stimulator. The response to SPEB usually required 1000 times higher concentrations than to SPEA or SPEC. Antisera to SPEC but not to SPEB inhibited the response of PBMC and V beta 8+ Jurkat cells to SPEB. Furthermore, more stringent purification of SPEB yielded SPEB preparations devoid of mitogenic activity. These results indicate that the mitogenicity that is commonly attributed to SPEB is due to minute contaminations of the V beta 8 stimulator. These results raise two important caveats for the work with these highly potent T cell mitogens.(ABSTRACT TRUNCATED AT 400 WORDS)
Comparison of the requirements for cognate T cell help for IgG anti-double-stranded DNA antibody production in vitro: T helper-derived lymphokines replace T cell cloned lines for B cells from NZB.H-2bm12 but not B6.H-2bm12 mice.
Authors: Cawley D , Chiang B L , Naiki M , Ansari A A , Gershwin M E
Published: December 2022

We have isolated, from NZB.H-2bm12 mice, several autoreactive cloned T cell lines that provide help for anti-dsDNA IgG antibody production in vitro. The purpose of the work described herein was to examine the requirement for cognate help for the production of anti-DNA antibodies in vitro. Thus, the ability of cloned T cell lines or lymphokines derived from them to provide help for T-depleted spleen cells from both normal B6.H-2bm12 mice and SLE-prone NZB.H-2bm12 mice was examined. Two autoreactive cloned T cell lines were selected for detailed study. 410F T cells respond to APC from both I-Ab and I-Abm12 mice, whereas 410H T cells are restricted to I-Abm12. By using Percoll gradients, B cells from both low density and high density fractions were cultured with autoreactive cloned T cell lines or lymphokines secreted by such cloned T cell lines, and anti-DNA antibody production was determined. Lymphokines elicited IgM anti-ssDNA antibody production from cells in all Percoll fractions from both B6.H-2bm12 and NZB.H-2bm12 mice. Lymphokines did not elicit production of IgG anti-dsDNA antibody production by cells from 2-month-old B6.H-2bm12 mice. In contrast, substantial production of IgG anti-dsDNA antibody was observed for NZB.H-2bm12 cells in response to lymphokines alone. Thus, B cells from NZB.H-2bm12 mice, because of previous activation in vivo, can proceed to IgG anti-dsDNA antibody production in vitro without direct T cell interaction. When we examined direct T cell help for the IgG anti-dsDNA antibody response, we found that we could distinguish the actions of the two cloned T cell lines studied. 410F T cells provided help predominantly for cells from low density Percoll fractions whether the cells were derived from B6.H-2bm12 or NZB.H-2bm12 mice. 410H T cells were capable of providing help for cells from both the low and high density fractions, and this help accounted for more than half of the antibody production in vitro by cells from B6.H-2bm12 mice.
Promoter of the human NF-kappa B p50/p105 gene. Regulation by NF-kappa B subunits and by c-REL.
Authors: Cogswell P C , Scheinman R I , Baldwin A S
Published: December 2022

NF-kappa B is a transcription factor involved in the regulation of numerous genes encoding proteins involved in immune function, in inflammation or in cellular growth control. NF-kappa B is typically characterized as a heterodimer of a 50-kDa subunit (p50) and a 65-kDa (p65) subunit. Interestingly, the p50 subunit is derived by processing of a 105-kDa precursor. Induction of NF-kappa B DNA-binding activity involves both the release of cytoplasmically stored factor from its inhibitor known as I kappa B and the induction of NF-kappa B gene expression. We report here the cloning and functional analysis of the promoter of the p50/p105 NF-kappa B gene. Our data suggest the existence of multiple transcription initiation sites for this gene in the B cell line Raji, Jurkat T cells, and HeLa cells. The promoter is constitutively active in these cells and is inducible by phorbol ester and mitogen stimulation of Jurkat T cells. Expression of I kappa B inhibits this inducible activation of the p50/p105 promoter. Furthermore, we have shown that co-transfection of a p50/p105 promoter-reporter plasmid with expression vectors encoding the p50 or p65 subunits of NF-kappa B or c-Rel results in stimulation of gene expression. Supportive of the transfection data, we have identified a DNA-binding site for NF-kappa B in the promoter of the p50/p105 gene that is responsive only to a combination of p50 and p65. The data demonstrate that the p50/p105 NF-kappa B gene is regulated by members of the NF-kappa B/Rel family and likely by other important transcription factors.
Transcriptional control of MHC class I and beta 2-microglobulin genes in vivo.
Authors: Drezen J M , Babinet C , Morello D
Published: December 2022

The expression of class I and beta 2-microglobulin (beta 2-m) genes, which encode the H and L chains of the H-2 histocompatibility Ag, respectively, is complex both in the adult mouse and during development. Although they are ubiquitously expressed in the adult, the mRNA levels of these genes are variable from one organ to another, being high in liver, lung, and lymphoid organs and low in brain and testis. During development, both class I and beta 2-m mRNA are poorly expressed. To determine the molecular mechanism, either transcriptional or post-transcriptional, controlling class I and beta 2-m mRNA levels, we have compared their transcriptional activities by performing run-on experiments with nuclei extracted from several embryonic and adult organs. We show that most of the differences observed in H-2 and beta 2-m mRNA steady state levels are the reflection of their different transcriptional activities. These results demonstrate that MHC class I and beta 2-m gene expression in adult organs, as well as during development, is mainly controlled at the transcriptional level.
Chromosomal organization of the human VH4 gene family. Location of individual gene segments.
Authors: van der Maarel S , van Dijk K W , Alexander C M , Sasso E H , Bull A , Milner E C
Published: December 2022

To investigate the organization and evolution of VH gene segments, we characterized the elements belonging to the VH4 gene family from the germline of a single subject. One hundred sixty VH4-carrying lambda-phage clones were isolated from a genomic library. A combination of hybridization and sequence analysis yielded 13 distinct VH4 clones. Six of these elements had one or more nucleotide substitutions that distinguished them from previously identified VH4 genes, whereas seven elements were identical to previously described VH4 genes. In four of the six new sequences, nucleotide substitutions resulted in amino acid replacements. One pseudogene was identified. On the basis of sequence-specific hybridization using oligonucleotide probes corresponding to these sequences, each of the elements could be assigned to a specific band in a BglII digest. Since the VH4-carrying BglII bands have been mapped in genomic DNA, it was also possible to assign chromosomal locations to the specific VH4 elements. The results indicate that the majority of VH4 elements are located in a region of approximately 500 kb, extending from approximately 500 to 1000 kb 5' of the JH locus. The distribution of shared structural motifs among the VH4 elements indicates that the VH4 gene family has evolved through repeated duplication and gene conversion events.
Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-alpha, IL-6 and IL-8 release by human monocytes and alveolar macrophages.
Authors: Dentener M A , Bazil V , Von Asmuth E J , Ceska M , Buurman W A
Published: December 2022

CD14 has been reported to function as a receptor for bacterial LPS complexed with serum proteins, transducing an activation signal for TNF-alpha production. We found that the anti-CD14 mAb MEM-18 inhibited not only LPS-induced release of TNF-alpha, but also LPS-induced, TNF-alpha independent release of IL-6 and IL-8 by human monocytes and alveolar macrophages. Inhibitory effect of MEM-18 was detected both in the presence of human or bovine calf serum and under serum-free conditions. In contrast, MEM-18 did not block release of these cytokines induced by IL-1 beta, TNF-alpha, PMA, and zymosan. We conclude that CD14 is involved in LPS-induced release of TNF-alpha, IL-6, and IL-8 by monocytes and alveolar macrophages and that this receptor appears to be able to recognize LPS directly in the absence of serum.
Characterization of T helper cell responses to the trypanosome variant surface glycoprotein.
Authors: Schleifer K W , Filutowicz H , Schopf L R , Mansfield J M
Published: December 2022

T cell responses to the variant surface glycoprotein (VSG) previously have not been detected in animals infected with the African trypanosomes despite the fact that such animals make strong T-dependent B cell responses to VSG molecules displayed by the parasites. In the present study, we have examined B 10.BR mice for VSG-specific Th cell responses at different times after infection with Trypanosoma brucei rhodesiense clone LouTat 1. T cell populations derived from different tissues were tested for their ability to proliferate and secrete cytokines when stimulated with purified LouTat 1 VSG. Furthermore, VSG-specific T cell lines and clones were derived from immunized mice and examined for their phenotypic and functional profiles in comparison with T cell responses of infected mice. The results of this study show that VSG-specific T cells were not consistently detected in the peripheral lymphoid tissues such as spleen or lymph nodes of infected animals. In contrast, VSG Ag-specific T cells were detectable principally in the peritoneal T cell populations of infected mice. Peritoneal T cells did not proliferate in response to VSG, yet produced substantial cytokine responses when stimulated; the cytokines produced were IFN-gamma and IL-2, without detectable IL-4. The cellular phenotype of VSG-responsive T cells was that of classical Th cells in that all cells were CD4-positive and expressed the CD3 alpha/beta TCR membrane complex. Thus, the VSG appears to preferentially stimulate a Th1 cell subset response during infection. Intrinsic molecular characteristics of the VSG molecule did not induce mice to make this response, however, since VSG-specific T cell lines derived from VSG-immunized mice displayed cytokine profiles characteristic of both Th1 and Th2 cells. Isolation of Th1 clones from selected lines demonstrated that these cells displayed the same membrane-phenotypic characteristics and cytokine profiles as the T cells from infected mice. Furthermore, all Th clones were VSG type-specific, APC-dependent, and I-Ak-restricted in their responses. In summary, these experiments provide the first direct evidence for VSG-specific responses at the T cell level. T cell responses to the VSG molecule during infection appear to be anatomically compartmentalized and exhibit evidence of clonal maturation (cytokine production) but not clonal expansion (proliferation) after antigenic stimulation. The cellular phenotype and cytokine profiles predict that infection predisposes the animals to mount Th1 cell subset responses to VSG. The results of this study, including the T clones generated, provide an experimental basis for examining the regulation of VSG-specific immune responses during infection.
Colonial morphotype as a determinant of cytokine expression by human monocytes infected with Mycobacterium avium.
Authors: Shiratsuchi H , Toossi Z , Mettler M A , Ellner J J
Published: December 2022

Mycobacterium avium is an intracellular pathogen that causes disseminated infection in patients with AIDS. Colonial morphotype (smooth-transparent (SmT) vs smooth-domed (SmD)) is a key determinant of virulence in mice and the capacity for replication in human monocytes. Some cytokines (IL-1 and IL-6) promote, whereas others (IFN-gamma and TNF) inhibit intracellular M. avium growth. The specific factors that determine virulence of M. avium, however, are not clear. In this study, we examined cytokine expression by human monocytes stimulated with isogeneic cloned isolates of M. avium. Monocytes were prepared from healthy donors and cultured with or without isogeneic M. avium for up to 7 days. Cytokine levels (IL-1, IL-6, and TNF-alpha) in monocyte supernatants and cell lysates were measured by immunoassay using an ELISA. The expression of cytokine mRNA by monocytes infected with M. avium also was determined by extracting total RNA and subjecting it to Northern blot analysis. Optimal cytokine release occurred at 24 h. SmD induced higher levels of the following cytokines in supernatants than SmT: IL-1 alpha (140 +/- 32 (mean +/- SE) vs 47 +/- 16 pg/ml, p < 0.02), IL-1 beta (4.0 +/- 0.9 vs 1.7 +/- 0.5 ng/ml, p < 0.01), and TNF-alpha (2725 +/- 546 vs 1464 +/- 409 pg/ml, p < 0.01). IL-6 production was comparable for both strains. SmD and SmT isolates induced comparable levels of steady state mRNA for IL-1 beta, TNF, and IL-6. Pulse-chase analysis indicated that differences in cytokine expression between SmT and SmD occurred in monocyte lysates at the earliest time point (immediately after pulse-labeling). The dissociation of the expression of specific mRNA from production of IL-1 and TNF suggests that translational capacity for the expression of certain cytokines was reduced by the more virulent SmT. Differential induction of cytokine may be a factor in the pathogenicity of M. avium strains isolated from patients with AIDS.
The regulation of T cell-dependent antibody formation in vitro by CD40 ligand and IL-2.
Authors: Grabstein K H , Maliszewski C R , Shanebeck K , Sato T A , Spriggs M K , Fanslow W C , Armitage R J
Published: December 2022

Our study demonstrates the central role for the murine CD40 ligand and IL-2 in contact-dependent T cell help for Ag-specific primary antibody responses in vitro. Helper T cell clones were found to express CD40 ligand after activation with CD3 mAb. Membrane-bound recombinant CD40 ligand expressed on fixed CV1/EBNA cells had similar B cell-activating properties as T cell clones that had been activated and then fixed. These activities include the induction of B cell proliferation, induction of polyclonal secretion of multiple Ig isotypes in a cytokine-dependent manner, and induction of Ag-specific antibody responses by purified B cells. The induction of polyclonal Ig secretion by the recombinant CD40 ligand required IL-4 and IL-5 although optimal Ag-specific antibody formation required IL-2. Finally, soluble CD40.Fc inhibited the induction of Ag-specific antibody responses by fixed, activated Th cell clones. The requirement for both CD40 ligand and IL-2 for induction of Ag-specific antibody responses was mediated, in part, by the induction of B cell IL-2R expression by CD40 ligand. We conclude that the interaction of CD40 on B cells with its ligand on activated T cells is an integral event in the early activation of B cells to grow and differentiate to antibody formation.
Costimulation of peripheral blood T cell activation by human endothelial cells. Enhanced IL-2 transcription correlates with increased c-fos synthesis and increased Fos content of AP-1.
Authors: Hughes C C , Pober J S
Published: December 2022

Endothelial cells (EC) act as APC for resting PBL in vitro, and may have important roles in vivo in the pathogenesis of allograft rejection and delayed hypersensitivity. We previously reported that human umbilical vein EC provide costimulatory signals to PHA-stimulated PBL via CD2:lymphocyte function-associated Ag-3 and an unidentified ligand pair, resulting in a three- to eight-fold enhancement of IL-2 production. The physiologic relevance of this increase was demonstrated by the proliferative advantage provided by EC to PBL suboptimally stimulated with mAb OKT3. We now report that EC costimulation causes increased levels of IL-2 mRNA as a result of increased IL-2 transcription in PBL. We therefore examined the effects of EC on T cell nuclear factors known to regulate IL-2 transcription, including c-jun and c-fos-two components of the transcription factor AP-1, NFAT, and others. PBL constitutively express c-jun transcripts, and the level of c-jun mRNA is not altered by PHA activation in the absence or presence of EC. In contrast, c-fos mRNA is absent from resting T cells and is induced on PHA activation. EC alone do not induce c-fos mRNA but augment the level of c-fos mRNA in PHA-activated T cells by 3- to 10-fold. This effect is largely independent of the CD2:lymphocyte function-associated Ag-3 pathway. Gel-shift analysis reveals the constitutive presence of nuclear factors in resting PBL that bind to the proximal AP-1 site of the IL-2 promoter and that contain immunoreactive c-Jun but not c-Fos protein. In contrast, AP-1 from PHA-activated cells contains c-Jun and low levels of c-Fos. Strikingly, costimulation with EC results in a dramatic increase (up to 15-fold) in the c-Fos content of AP-1. Levels of other nuclear factors involved in IL-2 regulation were not altered by EC, although NFAT-DNA complexes migrated at a slightly different mobility. In summary, our data suggest that changes in the composition of transcription factor AP-1 is a key molecular mechanism for increasing IL-2 transcription and may underlie the phenomenon of costimulation by EC.
Modulation of the intracellular Ca2+ and inositol trisphosphate concentrations in murine T lymphocytes by the glycosylphosphatidylinositol-anchored protein sgp-60.
Authors: Maschek B J , Zhang W , Rosoff P M , Reiser H
Published: December 2022

Stimulation of T lymphocytes via the TCR/CD3 complex initiates a series of intracellular biochemical events. Among the earliest cellular responses are the stimulation of the turnover of phosphatidylinositol and an increase in the intracellular Ca2+ concentration. The significance of these second messengers for distal cellular responses such as lymphokine production or cell proliferation is not understood. We have previously shown that hamster mAb to the murine glycosylphosphatidylinositol-anchored protein sgp-60 can inhibit IL-2 production, IL-2R expression, and cell proliferation, events normally observed after stimulation of T cells with an antibody to the TCR/CD3 complex or with the lectin Con A. We now show that the anti-sgp-60 mAb inhibits the activation-induced increase in intracellular Ca2+ concentration. Additional analysis demonstrates that the mAb interferes with the release of Ca2+ from intracellular stores and with the generation of inositol 1,4,5-trisphosphate. Our findings suggest a role for this glycosylphosphatidylinositol-anchored protein in the generation of second messengers in signal transduction through the TCR/CD3 complex in murine T lymphocytes.
Staphylococcus aureus Wood 46 strain activates human B cells without affecting DNA synthesis or tyrosine phosphorylation.
Authors: Saiki O , Kawamoto M , Fukuzumi M , Kanou M , Utsumi S
Published: December 2022

Induction of Ig secretion in human tonsilar B cell by protein A-deficient Staphylococcus aureus WOOD 46 strain (SAW) was examined. SAW induced as much Ig secretion as protein A-rich S. aureus Cowan I strain (SAC) when IL-2 was present in the culture. Activated and resting B cells are separated by Percoll gradient to determine whether SAW stimulates either resting or activated B cells. In resting B cells, SAW plus IL-2 induced IgM secretion significantly, but neither IL-2 nor SAW alone induced IgM secretion. In activated B cells, however, IL-2 induced IgM secretion by itself and SAW plus IL-2 did not induce additional IgM secretion. These results suggest that SAW activates small resting B cells rather than preactivated B cells. Subsequently, mechanisms of B cell activation by SAW and SAC were compared. SAW did not induce [3H]-TdR incorporation through day 1 to 5, and the number of viable cells was not increased by SAW stimulation. Moreover, SAW did not induce significant tyrosine phosphorylation at any concentration tested, when tyrosine phosphorylation of B cells was examined. However, SAC induced both [3H]-TdR incorporation and tyrosine phosphorylation of B cell efficiently. In further experiments, induction of IL-2R and IgM mRNA expressions were examined. SAW by itself induced IL-2R and IgM mRNA expressions without affecting expression of membrane-type IgM mRNA. These results show that SAW activates human B cells in quite a different manner from SAC by up-regulating the expression of secretory type IgM mRNA without affecting cell proliferation nor tyrosine phosphorylation, proposing a distinct B cell activation model from SAC.
Inhibition of murine T cell activation by cholera toxin B subunit is not mediated through the phosphatidylinositol second messenger system.
Authors: Woogen S D , Turo K , Dieleman L A , Beagley K W , Elson C O
Published: December 2022

Although cholera toxin B subunit is a potent mucosal immunogen in vivo, its predominant effect in vitro is inhibition of T cell and B cell activation. We reported earlier that this inhibition was not mediated through activation of adenylate cyclase and increases in intracellular cAMP. There is increasing evidence that T cell activation is initiated through the phosphatidyl inositol second messenger system in which phosphatidyl inositol bisphosphate is hydrolyzed by phospholipase C, producing inositol trisphosphate (IP3) and diacylglycerol. IP3 increases cytosolic calcium and diacylglycerol binds, translocates, and activates protein kinase C (PKC). These signals lead to a complex series of events eventuating in activation of a number of genes important in cell proliferation. In this study, we asked whether the mechanism of T cell inhibition by B subunit of cholera toxin (CT-B) was due to interference with the phosphatidyl inositol second messenger system. We found that substitution of ionomycin and PMA for IP3 and diacylglycerol, respectively, in culture induced T cell proliferation but only if both were present simultaneously. Such proliferation was inhibited by CT-B even if added hours after the start of culture. An assay for cytosolic PKC activity demonstrated that PMA translocation of PKC from cytosol to membrane was not inhibited by CT-B, indicating that CT-B does not inhibit activation of PKC. There was no inhibition of Con A-stimulated T cell phosphoinositol turnover. Moreover, Con A added to Fura-2 AM-loaded cells caused a rapid rise in cytosolic calcium, which CT-B preincubation did not alter. These results indicate that CT-B did not inhibit IP3 generation or action. We next looked at expression of genes involved in T cell proliferation. CT-B inhibited the production of IL-2 by mitogen-activated T cells; Northern analysis showed that this inhibition was associated with decreased levels of IL-2 mRNA. Expression of IL-2R and of transferrin receptors was only modestly reduced. Despite the presence of IL-2R on the T cells exposed to CT-B, the addition of exogenous IL-2 to the cultures did not reverse the CT-B-induced T cell inhibition. We conclude that the T cell inhibition by CT-B is not mediated by interference with the activation of the phosphatidylinositol second messenger system but occurs at a later stage of T cell activation.
Regulation of MHC class I and beta 2-microglobulin gene expression in human neuronal cells. Factor binding to conserved cis-acting regulatory sequences correlates with expression of the genes.
Authors: Drew P D , Lonergan M , Goldstein M E , Lampson L A , Ozato K , McFarlin D E
Published: December 2022

MHC class I molecules are coexpressed with beta 2-microglobulin (beta 2-M) on many somatic cells. However, these proteins are normally not present on cells of the central nervous system (CNS). Cells derived from human neuroblastomas were used as a model for investigating the molecular basis for the paucity of MHC class I and beta 2-M gene expression in neural cells and for the induction of these genes by two cytokines, IFN-gamma, and TNF-alpha. These cytokines independently increased MHC class I and beta 2-M cell surface expression on the neuroblastoma cell lines. IFN-gamma or TNF-alpha also increased MHC class I and beta 2-M steady-state RNA levels and the expression of MHC class I and beta 2-M CAT reporter constructs transiently transfected into the neuroblastoma cell lines, indicating that the cytokines acted by increasing the transcription of these genes. MHC class I and beta 2-M genes share two conserved regulatory elements, an NF kappa B-like site and the IFN consensus sequence, that act as a constitutive enhancer and an IFN-responsive element, respectively. Low MHC class I and beta 2-M gene expression in these cells was accounted for by undetectable to low factor binding activity specific for the above regulatory elements of these genes. TNF-alpha increased factor binding activity specific for the NF kappa B-like elements and IFN-gamma increased factor binding activity specific for the IFN consensus sequence elements of the MHC class I and beta 2-M genes, but not vice versa. Taken together, our results indicated that IFN-gamma and TNF-alpha increased MHC class I and beta 2-M gene expression in the neuroblastoma cell lines by inducing factor binding to the regulatory elements present in both genes.
Endogenous Ig production in mu transgenic mice. II. Anti-Ig reactivity and apparent double allotype expression.
Authors: Imanishi-Kari T , Huang C A , Iacomini J , Yannoutsos N
Published: December 2022

In 17.2.25 mu transgenic mice (M54, M95), many of the expressed Ig, whether encoded by the transgene or endogenous H chain genes, react with Ig. IgM antibodies encoded by the 17.2.25 mu transgene transfected into J558L myeloma cells are also Ig reactive. In addition, anti-Ig reactivity was manifested by antibodies of the IgM, IgG, and IgA isotypes from the transgenic mice, suggesting that this characteristic reactivity is associated with VH and VL domains of these antibodies. These antibodies bind the (Fab')2 fragment of mouse IgG1 mAb known to be directed against C mu allotypic determinants. This finding could account for the so called "double producer" B cells found in mu transgenic mice and previously identified in serologic assays conducted with two different anti-mu allotypic reagents. In transgenic mice, a high frequency of the antibodies encoded by the transgene or endogenous H chain genes react with polyclonal and monoclonal antiidiotypic antibodies raised against the 17.2.25 Id. The idiotypic and/or antiidiotypic reactivity displayed by antibodies derived from these transgenic mice is similar to that of antibodies expressed by neonatal B cells of normal mice. Thus, our data suggest that transgene expression can play an important role in shaping the endogenous repertoire of antibody specificities.
A monoclonal antibody produced during infection which recognizes an epitope of influenza hemagglutinin only in the context of H-2k MHC class I antigen.
Authors: McLain L , Dimmock N J
Published: December 2022

A hybridoma prepared from cells isolated from the lungs of H-2k mice infected with influenza virus (A/WSN) produces a monoclonal IgM (LM41) which recognizes an epitope of the A/WSN hemagglutinin (HA) Ag only when it is expressed in H-2k cells, as shown by Ag binding and complement-mediated lysis. However, it did not bind to the native HA of virus particles or any form of HA protein present on A/WSN-infected H-2d cells. LM41 inhibited Ag-specific, MHC class I-restricted lysis of H-2k target cells infected with a vaccinia recombinant virus expressing the HA of A/WSN by CTL from A/WSN virus-infected H-2k mice. We conclude that LM41 has a similar Ag specificity to the HA-specific TCR.
Changes in mechanisms of monocyte/macrophage-mediated cytotoxicity during culture. Reactive oxygen intermediates are involved in monocyte-mediated cytotoxicity, whereas reactive nitrogen intermediates are employed by macrophages in tumor cell killing.
Authors: Martin J H , Edwards S W
Published: December 2022

Freshly isolated human blood monocytes were spontaneously cytotoxic toward K562 tumor cells. During culture of the monocytes in vitro cytotoxicity decreased during the first 48 h but tumoricidal competence was restored after 3 to 4 days in vitro. These changes were accompanied by changes in both reactive oxygen intermediate generating capacity and reactive nitrogen intermediate production. Lucigenin-dependent chemiluminescence stimulated with either FMLP or PMA declined during the first 2 days in culture and was negligible during the later days in culture. Superoxide radical production in response to either FMLP or PMA remained fairly constant for the first few days in vitro and then declined. NO2- concentration in monocyte-conditioned medium was fairly constant during the first few days in vitro but increased after 6 days. The return to tumoricidal competence after 3 to 4 days in culture was decreased by the addition of NG-monomethyl-L-arginine. These results indicate that reactive oxygen intermediates are employed by monocytes in the killing of tumor cells. However, after maturation of monocytes to macrophages, this mechanism becomes less important and reactive nitrogen intermediates are employed in mediating macrophage cytotoxicity.
Agonist-dependent phosphorylation of N-formylpeptide and activation peptide from the fifth component of C (C5a) chemoattractant receptors in differentiated HL60 cells.
Authors: Tardif M , Mery L , Brouchon L , Boulay F
Published: December 2022

Attenuation of signaling is a key step in controlling the cytotoxic potential of leukocyte responses to chemotactic factors. Antipeptide antibodies, directed against the N-formyl chemotactic peptide receptor (FPR) and the activation peptide from the fifth component of C (C5a) anaphylatoxin receptor (C5aR) of human neutrophils, were used to analyze the ability of these receptors to be phosphorylated. Our data show that, in granulocyte-like differentiated HL-60 cells, both FPR and C5aR undergo an agonist dose-dependent phosphorylation that reaches completion in less than 2 to 3 min, consistent with the rate and the dose-dependent attenuation of signaling in phagocytes. Therefore, phosphorylation might be one of the possible mechanisms involved in the desensitization process of FPR and C5aR. Addition of either C5a or the protein kinase C activator (PMA) did not appear to induce the phosphorylation of FPR in the absence of FMLP or to modulate the phosphorylation of the latter at low concentrations of agonist. In contrast, although FMLP at a saturating concentration barely stimulated the phosphorylation of unoccupied C5aR, it markedly potentiated C5aR phosphorylation in cells exposed to low concentrations of C5a. Moreover, PMA was able to induce C5aR phosphorylation in the absence of agonist, indicating that protein kinase C or protein kinase C-activated kinase(s) could be involved in the phosphorylation of C5aR. Pretreatment of cells with staurosporine, a potent but nonspecific inhibitor of protein kinase C, resulted in the partial inhibition of both FPR and C5aR phosphorylation induced by saturating concentrations of agonist, suggesting that a kinase different from protein kinase C might be mainly responsible for the phosphorylation of these chemotactic receptors.
Detection of HIV-specific cell-mediated cytotoxicity in the peripheral blood from infected children.
Authors: Buseyne F , Blanche S , Schmitt D , Griscelli C , Rivière Y
Published: December 2022

Cytotoxic T lymphocytes may play a significant role in containing the spread of HIV in infected individuals. Although HIV-infection is associated with immune suppression, a vigorous T lymphocyte response has been detected in infected adults. HIV can be transmitted from mother to child, either during pregnancy, when differentiation of the T lymphoid compartment is ongoing, or at birth when the neonate immune system is partially competent. The shorter asymptomatic period of pediatric infection could be related to differences in the host immune control of viral replication. HIV-specific cell-mediated cytotoxicity (CMC) from fresh and in vitro stimulated PBMC of HIV-infected children was measured. CD8+CD3+ T lymphocytes were found to be the major effector population. The vast majority of children examined had detectable HIV-specific CMC. A cross-sectional analysis of CMC responses as a function of clinical status revealed that 71% of asymptomatic children (CDC stage P1) recognized the Env protein, 14% the Gag protein, but none of them recognized the Pol protein. Cytolytic activities directed against these three proteins were detected in two thirds of paucisymptomatic children (P2A). In contrast, symptomatic children (P2B-F) did not show cytolytic activities toward the Gag and Pol proteins, and only 20% recognized the Env protein. In contrast in vitro generated secondary CTL were consistently detected at all stages of disease, even in children with low CD4+ cells counts.
Patterns of cytokine gene expression by CD4+ T cells from young and old mice.
Authors: Hobbs M V , Weigle W O , Noonan D J , Torbett B E , McEvilly R J , Koch R J , Cardenas G J , Ernst D N
Published: December 2022

We have analyzed the patterns of induced cytokine gene expression and cell cycle activity by CD4+ cells from mice, and have examined how these response patterns change during the aging process. CD4+ cells were isolated from spleens of young adult and old C57BL/6NNia mice and were stimulated in vitro with plate-bound anti-CD3 epsilon mAb. The cells were then assessed over time for the capacity to accumulate transcripts for IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, and TNF-beta; to secrete IL-2, IL-3, IL-4, IL-5, IL-6, and IFN-gamma; and to progress through S phase. Before the first major cell division in culture (< 32 h), stimulated CD4+ cells of the old group contained similar peak levels of IL-2, TNF-alpha, and TNF-beta transcripts relative to young adult controls, whereas IL-3, IL-4, IL-5, and IFN-gamma transcripts accumulated to significantly higher peak levels in the old group. These findings were consistent with the patterns of cytokine secretion later in culture (24 to 72 h): the peak IL-2 levels were similar between age groups, but the old group exhibited an enhanced capacity to release IL-3, IL-4, IL-5, and IFN-gamma. In contrast, CD4+ cells of the young group were superior in the hyper-expression of the housekeeping gene, rpL32, before cell division and in the levels of S phase activity throughout 3-day cultures. Similar analyses of CD4+ cells from mice of intermediate ages showed that the alterations in cytokine profiles occurred gradually from young adulthood to old age, whereas the reductions in proliferative capacity were late life changes. Consistent with previous reports, we found that the splenic CD4+ cell group also underwent a progressive, age-dependent increase in the proportions of cells expressing high levels of membrane CD44 (a phenotype associated with memory or effector cells). Moreover, the analysis of IL-3, IL-5, and IFN-gamma production by isolated CD4+CD44lo and CD4+CD44hi cells revealed that the capacity to produce these cytokines segregated predominantly with the CD44hi subset, regardless of donor age. Taken together, our data suggest that gradual age-associated shifts in the subset composition of the splenic CD4+ cell pool underlie progressive changes in the patterns of cytokine gene expression by this cell group.
Serum-IgE-facilitated allergen presentation in atopic disease.
Authors: van der Heijden F L , Joost van Neerven R J , van Katwijk M , Bos J D , Kapsenberg M L
Published: December 2022

The occurrence of facilitated Ag presentation (FAP) was investigated in atopic disease using an in vitro model in which the ability of CD23-expressing EBV-B cells was tested to present Der p II, a major allergen of housedust mite Dermatophagoides pteronyssinus, to cells of autologous Der p II-specific CD4+ T lymphocyte clones. Purified Der p II protein was immune complexed with Ig by preincubation in sera from atopic patients containing Der p II-specific IgE. Incubation of EBV-B cells with these complexes before using the cells as APC results in proliferation of the Der p II-specific T cells at a 1000-fold lower Der p II concentration than required for T cell activation by presentation of uncomplexed Der p II. FAP is not evident using sera from allergic and nonallergic control donors not containing Der p II-specific IgE. FAP is mediated by IgE and not by IgG, because it can be blocked completely by preincubating the serum from atopics with polyclonal anti-IgE and not with polyclonal anti-IgG. After pulsing EBV-B with precomplexed Der p II at 4 degrees C, FAP is as strong as after incubation at 37 degrees C, suggesting that FAP is the result of facilitated trapping of IgE-complexed allergen. CD23 was involved in facilitated trapping of the complexes, because FAP is blocked by preincubation of EBV-B with anti-CD23. In contrast to FAP of Der p II, which was mediated by all Der p II-specific sera tested, only one of four sera containing cat allergen-specific IgE was appropriate to mediate FAP to cat allergen-specific T cell clones. Inasmuch as FAP enables functional presentation of allergen in vivo at doses as low as several nanograms, FAP may contribute to the continuing of chronic allergic reactions in response to minute doses of aeroallergens, especially housedust mite allergens, in the environment.