bluuebunny/crossref_metadata_2025 huggingface dataset:
Sm and DNA binding by dual reactive B cells requires distinct VH, V kappa, and VH CDR3 structures.
Authors: Retter M W , Eisenberg R A , Cohen P L , Clarke S H
Published: December 2022

We have previously demonstrated an overlap of the anti-Sm and anti-DNA responses in MRL/Mp-lpr/lpr mice. The Ab produced by many anti-Sm hybridomas bind DNA and are encoded by Ig V genes used by anti-DNA hybridomas. In addition, some anti-Sm Ab that bind DNA have acquired mutations that improve DNA binding, indicating that DNA is a selecting Ag in the anti-Sm response. To gain insight into the basis for the dual binding ability of these Ab, we coexpressed the H chain from the anti-Sm hybridoma 2-12 with nine different L chains. Hybridoma 2-12 binds Sm but not DNA, yet expresses the same J558 VH gene as three anti-Sm hybridomas that bind ssDNA and at least one anti-DNA hybridoma that does not bind Sm. We found that most of the transfectoma Ab bind Sm, but their avidities vary over more than 3 orders of magnitude. Five of the nine transfectoma Ab bind ssDNA, and none bind dsDNA. In general, the ability to bind each Ag follows the binding ability of the hybridoma from which the L chain is derived. H Chain swapping experiments indicate that the H chain, VH CDR3 in particular, contributes to the binding of both Sm and DNA. We conclude that Sm and DNA select for distinct features of VH, V kappa, and VH CDR3, suggesting selection by both Ag in the anti-Sm response.
Regulation of human B cell function by recombinant CD40 ligand and other TNF-related ligands.
Authors: Jumper M D , Nishioka Y , Davis L S , Lipsky P E , Meek K
Published: December 2022

To assess the potential of CD40 ligand (CD40L) and the related molecules CD27 ligand (CD27), CD30 ligand (CD30L), and membrane TNF-alpha to stimulate B cell responses, expression of these proteins in the baculovirus system was performed. Sf9 cells expressing these membrane molecules were cultured with normal human B cells and a variety of B cell lines to assess the functional outcome. The signal provided by CD40L promotes aggregation of B cells, stimulates vigorous proliferation, and induces germ-line transcription of downstream heavy chain constant region genes in the absence of cytokine costimulation. In contrast, CD27L, CD30L, and TNF-alpha had no effects on B cell proliferation. CD27L and TNF-alpha had no effect on the induction of germ-line transcripts, whereas CD30L consistently inhibited constitutive and CD40L-induced germ-line transcription of the epsilon gene by B cell lines that express CD30. These results demonstrate the various members of the TNF family exert specific effects on human B cell function, with CD40L and CD30L providing powerful, but opposing, effects on l epsilon transcription.
IL-12 increases resistance of BALB/c mice to Mycobacterium tuberculosis infection.
Authors: Flynn J L , Goldstein M M , Triebold K J , Sypek J , Wolf S , Bloom B R
Published: December 2022

IL-12, a cytokine produced by macrophages and B cells, has recently been found to exert pleiotropic effects on the immune system. When BALB/c mice, a strain highly susceptible to virulent Mycobacterium tuberculosis infection, were given IL-12 at the initiation of infection with M. tuberculosis, their mean survival time doubled from 58 to 112 days. IL-12-treated mice had diminished bacterial burdens, whereas treatment with exogenous IFN-gamma had no effect on survival or bacterial burden. IL-12 treatment also delayed lung pathology in BALB/c mice. In contrast with the findings in the BALB/c model, IL-12 did not increase survival of M. tuberculosis-infected gko mice, transgenic mice in which the IFN-gamma gene has been disrupted, indicating that IL-12 does not induce protection against tuberculosis in mice in the absence of IFN-gamma.
Clearance of adenovirus-infected hepatocytes by MHC class I-restricted CD4+ CTLs in vivo.
Authors: Yang Y , Wilson J M
Published: December 2022

E1-deleted recombinant adenoviruses have been developed for liver-directed gene therapy because efficient gene transfer to hepatocytes can be achieved in vivo. However, these viruses also express viral proteins in hepatocytes, leading to the development of destructive immune responses. Our previous studies indicated that MHC class I-restricted C D8+CTLs are major effectors in eliminating virus-infected cells, and CD4+ cells are also necessary in developing a fully competent CTL response by secretion of IFN-gamma, which sensitizes the virus-infected hepatocytes to CTLs through up-regulation of MHC class I expression. In this study, we have used adoptive transfer techniques in combination with mice deficient in immune functions to further define the role of CD4+ cells in the primary response to adenovirus-mediated gene transfer to the liver. Our studies indicate that CD4+ cells alone are capable of destroying virus-infected hepatocytes. Adoptive transfer experiments with beta 2m- mice along with in vitro CTL assays suggest that these CD4+ can act as CTL effectors, which are MHC class I-restricted. Depletion of these CD4+ effectors in vivo leads to prolongation of adenovirus-mediated transgene expression in hepatocytes. These results suggest that class I-restricted CD4+ CTLs contribute to elimination of adenovirus-transduced hepatocytes and extend our understanding of functional importance of CD4+ cells in viral pathogenesis.
IL-3-induced histamine release from human basophils. Dissociation from cationic dye binding and down-regulation by Na+ and K+.
Authors: Tedeschi A , Palella M , Milazzo N , Lorini M , Miadonna A
Published: December 2022

The effect of recombinant human IL-3 on human basophils from normal subjects was evaluated by the decrease of toluidine blue-positive basophils (TB+) and by histamine release. IL-3 (0.001 to 100 ng) caused the decrease of TB+ without concomitant histamine release, whereas anti-IgE-induced TB+ decrease was accompanied by histamine release. IL-3 enhanced both anti-IgE-induced TB+ decrease and histamine release. IL-3-induced TB+ decrease was dose- and Ca2+-dependent and related to the time of incubation (detectable after an incubation of 5 min and maximal after incubation of 2 h). When extracellular Na+ was replaced isosmotically with choline, N-methyl-D-glucamine, or glucose, histamine release also was observed after direct stimulation with IL-3. The effect was dose dependent, related to the time of incubation (detectable after 30 min and maximal after 60 min), and required extracellular Ca2+. The increase in extracellular Na+ or K+ concentrations was accompanied by a reduction of histamine release, with a more marked effect on IL-3- than on anti-IgE-induced histamine release. In line with these results, the Na+ ionophore gramicidin D, which increases Na+ influx, and the K+ channel blocker 4-aminopyridine, which decreases K+ efflux, dose-dependently inhibited anti-IgE- and IL-3-induced histamine release. The inhibitory effects on Na+ and K+ were slightly additive, suggesting an action via the same pathway, which most probably is membrane potential. These results indicate that: 1) IL-3 can induce TB+ decrease without concomitant histamine release, 2) basophils from normal subjects can release histamine on challenge with IL-3 once the inhibitory effect of Na+ is removed, and 3) Na+ and K+ down-regulate IL-3 as well as anti-IgE-induced histamine release.
A zona pellucida 3 peptide vaccine induces antibodies and reversible infertility without ovarian pathology.
Authors: Lou Y , Ang J , Thai H , McElveen F , Tung K S
Published: December 2022

Zona pellucida 3 (ZP3) is a major glycoprotein of the zona pellucida that possesses the sperm receptor function. ZP3 induces autoantibody that can block sperm/oocyte interaction. However, the feasibility of a ZP3 contraceptive vaccine has been marred by the finding that ZP3-specific T cells mediate ovarian autoimmune disease. Moreover, as reported in this work, only some inbred mouse strains respond to the ZP3 peptide. We now describe a chimeric peptide that induces Abs to native ZP3 regardless of the MHC haplotype of the inbred mice tested. Study in one mouse strain resulted in reduction in fertility that correlates well with zona pellucida Ab titer, and most importantly, the mice do not develop concomitant autoimmune oophoritis. Moreover, the infertility was completely reversible. The design of the vaccine chimeric peptide is governed by the inclusion of two essential components: 1) a promiscuous foreign T cell peptide capable of eliciting a Th cell response regardless of the MHC haplotype of the animals, and 2) the native B cell peptide of ZP3 that has been modified by substitution of residue(s) critical for T cell but not B cell response to ZP3.
Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokines.
Authors: Sozzani S , Sallusto F , Luini W , Zhou D , Piemonti L , Allavena P , Van Damme J , Valitutti S , Lanzavecchia A , Mantovani A
Published: December 2022

Trafficking to tissues and then to lymph nodes is a crucial aspect of the immunobiology of dendritic cells. The present study was designed to identify molecules able to direct the migration of human blood-derived dendritic cells. fMLP (representative of formyl peptides of bacterial origin), C5a, and the C-C chemokines monocyte chemotactic protein (MCP)-3, MIP-1 alpha/LD78, and RANTES elicited chemotactic migration and a rise of intracellular free calcium in dendritic cells. In contrast, the C-X-C chemokines IL-8 and IP-10 and the C-C chemokines MCP-1 and MCP-2 were inactive as chemoattractants. Thus, dendritic cells respond to classical chemotactic signals and to a set of chemokines distinct from that active on monocytes and neutrophils. Chemoattractants are likely to contribute to localization and trafficking of dendritic cells and provide tools to recruit these cells in the design of immunization strategies.
Opposing roles of CD95 (Fas/APO-1) and CD40 in the death and rescue of human low density tonsillar B cells.
Authors: Cleary A M , Fortune S M , Yellin M J , Chess L , Lederman S
Published: December 2022

The regulation of B cell death plays roles in the selection of Ag-specific B cells in humoral immune responses, controlling B cell homeostasis and perhaps limiting transformation. The present work addresses whether CD95 induces tonsillar B cells to undergo apoptosis and, if so, whether contact-dependent CD40-L:CD40 signaling can rescue tonsillar B cells from CD95-induced apoptosis. CD95 triggering by anti-CD95 mAb (APO-1) was studied in human tonsillar B cell populations that were separated by density centrifugation into fractions enriched for either low density, CD38+ B cells or high density, resting B cells. Low density tonsillar B cells express CD95 and undergo anti-CD95-mediated apoptosis by analysis of cellular morphology or DNA fragmentation by TUNEL assay. The induction of apoptosis in low density tonsillar B cells by anti-CD95 mAb is inhibited by CD40 signals provided by stably transfected CD40-L+ 293 cells, but not by control transfected 293 cells (expressing CD8). In addition, the rescuing effect of CD40-L+ cells is inhibited specifically by anti-CD40-L (mAb 5c8). The counteracting effects of CD95 and CD40 signaling were also studied in Ramos 2G6, a homogeneous B cell tumor line of germinal center phenotype that expresses CD95 and CD40. Similar to the behavior of low density tonsillar B cells, Ramos 2G6 undergoes anti-CD95-mediated apoptosis, which is prevented by CD40-mediated rescue. These data show that CD95 induces apoptosis in low density tonsillar B cells and that CD40-L:CD40 interactions rescue low density tonsillar B cells or the B cell tumor Ramos 2G6 from CD95-induced apoptosis, and suggest roles for CD95 and CD40 in B cell death and selection, respectively.
Secondary response to Listeria infection requires IFN-gamma but is partially independent of IL-12.
Authors: Tripp C S , Kanagawa O , Unanue E R
Published: December 2022

During a secondary immune response to Listeria monocytogenes (LM), the production of IFN-gamma was still required for resistance, but it was considerably less dependent on IL-12 production. When IL-12 was neutralized in vivo using specific hamster antimurine IL-12 mAbs, there was a dramatically increased susceptibility to infection during primary listeriosis but much less during a secondary infection. However, neutralization of IFN-gamma in vivo resulted in a similar increased susceptibility during both primary and secondary listeriosis. In culture, splenocytes isolated from unimmunized mice produced IFN-gamma in response to heat-killed L. monocytogenes (hk-LM) that was absolutely dependent upon IL-12 production. However, directly stimulating the TCR with anti-CD3-epsilon mAbs resulted in IFN-gamma production that was unaffected by neutralizing IL-12 in vitro. In contrast, splenocytes isolated from LM-immune mice produced IFN-gamma in response to hk-LM, part of which was independent on IL-12 production. However, anti-CD3-epsilon Ab-stimulated IFN-gamma production remained independent of IL-12 production. The source of hk-LM-induced, IL-12-independent IFN-gamma production was the T cell because anti-Thy1.2 Ab plus complement treatment in vitro completely abolished it. Together, these data support a model of memory T cells being produced during the primary infection with LM that can be stimulated to produce IFN-gamma during the secondary response to LM, partially independent of macrophage IL-12 production.
Defective regional immunity in the respiratory tract of neonates is attributable to hyporesponsiveness of local dendritic cells to activation signals.
Authors: Nelson D J , Holt P G
Published: December 2022

A variety of studies suggest that the increased susceptibility of neonates to allergic and infectious respiratory diseases is due to delayed postnatal maturation of local mucosal immune function. We have recently demonstrated that the postnatal development of the major resident APC population in the respiratory tract (RT), class II MHC (Ia)-bearing dendritic cells (DC), is delayed relative to that in other tissues, and that both the intensity of Ia expression on these RTDC and their density within respiratory epithelia remain low until after weaning. The present study focuses on the functional capacity of neonatal RTDC and their responses to exogenous stimuli, and demonstrates that 1) infant Ia+ RTDC respond poorly to GM-CSF, under conditions that stimulate high levels of Ia expression and concomitant APC activity in adult cells; 2) both infant and adult RTDC contain a subpopulation of Ia- cells recognized by mAb OX62 that also respond poorly to GM-CSF; 3) inhalation of microbial stimuli or parenteral administration of IFN-gamma triggers rapid recruitment of DC into the airway epithelium and lung parenchyma of adults; this response is markedly attenuated in newborns and does not attain levels of competence until after weaning; and 4) endogenous macrophage-mediated suppression of the RTDC response to GM-CSF, the principal mechanism limiting in situ DC functional maturation in the adult lung, is highly active in the neonates. Taken together with earlier evidence of the relatively rapid postnatal development of T and B cell function in these animals, the present findings suggest that the sluggish performance of respiratory mucosal immune function(s) during infancy is attributable primarily to delayed maturation of local DC populations.
In vivo regulation of macrophage IL-12 production during type 1 and type 2 cytokine-mediated granuloma formation.
Authors: Chensue S W , Ruth J H , Warmington K , Lincoln P , Kunkel S L
Published: December 2022

IL-12 is a pivitol cytokine that promotes NK cell activity and Th1 (type 1)-mediated immune responses. This study analyzed the cytokines that regulate macrophage (M phi) IL-12 production in vitro and in vivo. IL-12 was produced by elicited but not resident peritoneal M phi stimulated with endotoxin. Addition of graded doses of cytokines (0.1 to 10 ng/ml) indicated that the Th1-related (type 1) cytokine, IFN-gamma, augmented endotoxin-stimulated IL-12 production by nearly sixfold in oil-elicited M phi. TNF-alpha also increased production but only at the 10 ng/ml concentration. In contrast, the Th2-related (type 2) cytokines, IL-4 and especially IL-10, were profoundly inhibitory. IL-1 beta and IL-2 had no effect. For in vivo analysis, type 1 and type 2 cytokine-mediated lung granulomas (GR) were induced in presensitized mice by embolization of beads coupled to purified protein derivative of Mycobacteria tuberculosis or soluble Ags derived from Schistosoma mansoni eggs. Analysis of M phi isolated from type 1, type 2, or control pulmonary GR revealed that M phi of type 2 GR develop impaired IL-12-producing capacity. Depletion studies using anti-IFN, anti-IL-12, anti-IL-10, and anti-IL-4 neutralizing polyclonal Abs corroborated the in vitro studies. Anti-IFN or anti-IL-12 reduced IL-12 production by M phi from type 1 GR (70 to 80%) as well as IFN and IL-12 production by draining lymph nodes (75 to 90%). Conversely, anti-IL-10 and anti-IL-4 reversed the impaired IL-12 production observed in type 2 GR M phi. These data indicate a positive feedback stimulation of IL-12 production by IFN that is regulated by IL-10 and IL-4 in vivo.
Characteristics of ATP-dependent peptide transport in isolated microsomes.
Authors: Yang B , Braciale T J
Published: December 2022

This report examines the transport properties and specificity of ATP-dependent peptide transport by the murine transporter for Ag presentation (TAP) complex in isolated microsome preparations from H-2d haplotype mice. The murine TAP complex has a Km of 661 nM and a maximum velocity of 2.9 fmol/min.micrograms microsome protein for a modified peptide corresponding to a defined MHC class I binding epitope from influenza nucleoprotein recognized by CD8+ CTL in association with the Kd molecule. This high Km value for peptide transport suggests that the rate and efficiency of peptide transport of the TAP complex are influenced by the concentration of processed peptides derived from self and foreign proteins in the cell cytoplasm. Furthermore, these findings imply that competition among peptides for TAP-dependent transport is unlikely to be an important factor in determining the immunodominance of certain peptide epitopes within a foreign protein recognized by CD8+ T lymphocytes. We also examined the specificity of TAP transport for peptides containing bona fide murine MHC class I binding epitopes and provide evidence that certain flanking residues can affect the efficiency of peptide epitope transport by the TAP complex.
Analysis of CD2 and TCR-beta gene expression in Jurkat cell mutants suggests a cis regulation of gene transcription.
Authors: Kamoun M , Woods J S , Sano N , Makni H , Smith R , de Lau W B , van Oers A , Wotton D , Owen M J , Hashimoto Y , Clevers H C
Published: December 2022

Thirty CD2- J32 stable clones, derived by mutagenesis and subsequent immunoselection with anti-CD2 Ab, were used to study the regulation of CD2 and TCR gene expression. Analysis of RNA expression revealed that the loss of surface expression of CD2 was due to a lack of expression of CD2 mRNA and was associated with a lack of expression of VDJ TCR-beta transcripts in 12 of these mutants, sparing the expression of DJ TCR-beta, TCR-alpha, CD3 gamma, delta, epsilon, and zeta RNA. The expression of other differentiation molecules was unaffected, except for CD1, CD4, and CD5, which were either decreased or absent in most of these mutants. A gain in the expression of TCR-gamma transcripts was observed in each of these mutants, while, as expected, no TCR-gamma transcripts were detected in wild-type J32 cells. Several mutants were able to use the human CD2 enhancer and the murine TCR-beta enhancer and promoter to activate transcription from reporter genes in the context of heterologous promoters, indicating that the mutation(s) does not affect transcription pathways. Consistent with this finding is the adequate expression in these mutants of several lineage-specific transcription factors. The expression of CD2 in several of these mutants was rescued by gene transfer using a genomic 28.5-kb CD2 fragment, suggesting that the enhancer function of this gene may be dependent on the enhancer site. These observations suggest that the coordinate expressions of CD2 and TCR-beta genes share common regulatory mechanisms involving factors regulating chromatin structure and accessibility.
Cytokine and nitric oxide regulation of the immunosuppression in Trypanosoma cruzi infection.
Authors: Abrahamsohn I A , Coffman R L
Published: December 2022

An intense suppression of splenic T cell proliferation to mitogens and to Ags from the parasite is characteristic of the acute phase of Trypanosoma cruzi infection in mice. The impairment of proliferation is coincident with high levels of IFN-gamma and nitrite and decreased production of IL-2 in the supernatants of spleen cell cultures from infected mice. Previous work demonstrated that suppression of proliferation is largely mediated by the population of adherent cells in the infected spleen. In this study we confirmed the active suppression exerted by these cells on Con A, anti-CD3, and parasite Ag-stimulated proliferation of CD4+ splenic T cells. Inasmuch as the high production of IFN-gamma and of nitrite were compatible with intense macrophage activation and nitric oxide (NO) production, we determined the effects of cytokines that regulate macrophage activation and of NO on the proliferation of spleen cells from infected mice. We show that spleen cell proliferation to Ag and to T cell polyclonal stimuli is increased by neutralizing mAbs to IFN-gamma, TNF-alpha and -beta, or by the inhibitor of NO synthase, NG-monomethyl-L-arginine, added to the cultures. The addition of rIL-2 or rIL-4 also contributed to suppression reversal, and the combined addition of rIL-2 and anti-IFN-gamma mAb further increased lymphocyte proliferation. Anti-IL-4, anti-IL-10, or anti-TGF-beta neutralizing mAbs did not modify suppressed proliferative responses, and the addition of rIL-10 or of rTGF-beta also did not recover cell proliferation. Thus, the suppression of proliferative responses in T. cruzi-infected mice resulted largely from increased NO production by macrophages activated by IFN-gamma and TNF allied to insufficient IL-2 to fully support in vitro growth of T lymphocytes.
Differential responsiveness of purified mouse c-kit+ mast cells and their progenitors to IL-3 and stem cell factor.
Authors: Lantz C S , Huff T F
Published: December 2022

Murine mast cell development is regulated by a number of T cell- and fibroblast-derived growth factors, of which IL-3 and stem cell factor (SCF) appear to be the most important. To determine the relative effects of these two growth factors on different stages of developing mast cells, we isolated highly purified populations of c-kit+ mast cells and their progenitors and examined their ability to respond to SCF and/or IL-3. We show that purified c-kit+ cells isolated from bone marrow, mesenteric lymph nodes of Nippostrongylus brasiliensis-infected mice, and the peritoneal cavity do not form mast cell colonies in response to SCF as a single agent. Although unpurified populations of lymph nodes of Nippostrongylus brasiliensis-infected mice or peritoneal cells were able to form mast cell colonies in response to SCF alone, this probably reflects paracrine factors produced by accessory cells. IL-3 alone did not promote mast cell colony formation from either population. Within bone marrow, we detected both multipotential and unipotential mast cell progenitors. Interestingly, IL-3 alone promoted only the development of multipotential progenitors, while SCF plus IL-3 promoted both multipotential and unipotential mast cell progenitors. Together, these results indicate that the developmental/proliferative effects observed with a particular mast cell population is determined in part by the stage of maturation of mast cells at the time they are exposed to SCF and IL-3, and suggest that SCF primarily influences mast cell development only in the context of other cytokines.
Self-antigen-induced Th2 responses in experimental allergic encephalomyelitis (EAE)-resistant mice. Th2-mediated suppression of autoimmune disease.
Authors: Cua D J , Hinton D R , Stohlman S A
Published: December 2022

Immunization of a limited number of rodent strains with central nervous system-derived Ags induces experimental allergic encephalomyelitis (EAE). In contrast to susceptible female SJL mice, age-matched males are resistant to actively induced EAE. The ability of immunization with neuroAg to induce Ag-specific T cell activation in resistant male mice was examined. Ag-specific T cell proliferation was found following immunization of both male and female SJL mice. Draining lymph node cytokine mRNA patterns demonstrated that immunization of EAE-resistant male mice resulted in a Th2-type pattern. By contrast, immunization of EAE-susceptible female mice resulted in a Th1-type pattern. Priming of Th1- and Th2-type responses was confirmed by analysis of cytokines secreted following Ag-specific proliferation. In contrast to the transfer of myelin basic protein (MBP)-specific Th1-type T cells derived from female mice, which induced acute and relapse EAE, transfer of MBP-specific Th2-type T cells derived from male mice resulted in no clinical or histologic evidence of EAE. A mixture of MBP-specific Th1 and Th2 type cells was transferred to naive recipients to determine if the neuroAg-specific Th2-type cells exerted a regulatory influence on EAE. Acute disease was partially eliminated and relapses were completely eliminated in these recipients. Analysis of spinal cords showed the presence of both Th1 and Th2 cytokine mRNAs. These data are consistent with both the ability of Th2-type cells to suppress autoimmunity and a homeostatic mechanism of T cell regulation based on the cross-regulation of Th1 and Th2 cells in the maintenance of peripheral tolerance.
Relationship between beta cell injury and antigen presentation in NOD mice.
Authors: Shimizu J , Carrasco-Marin E , Kanagawa O , Unanue E R
Published: December 2022

We have made three observations that are important for our understanding of the dynamics of presentation of diabetogenic Ags in immunologically induced diabetes. First, the APC in the islets of Langerhans were found normally to contain diabetogenic peptides on their I-Ag7 molecules. This was found in freshly harvested islet cells from prediabetic NOD mice and, importantly, from NOD.SCID mice. Second, the presence of diabetogenic lymphocytes improved the presenting function of intra-islet APC. We interpret this to mean that lymphocytes can regulate intra-islet APC function before the development of diabetes. Finally, spleen APC can be found bearing diabetogenic Ag after acute injury to islets. These APC may be important in lymphocyte stimulation outside the environment of the pancreas.
High avidity state of leukocyte function-associated antigen-1 on rheumatoid synovial fluid T lymphocytes.
Authors: Yokota A , Murata N , Saiki O , Shimizu M , Springer T A , Kishimoto T
Published: December 2022

The integrin LFA-1 (CD11a/CD18) is a cell surface adhesion molecule required for leukocyte extravasation and subsequent immune and inflammatory responses. Rapid transition between nonadherent and adherent states of LFA-1 is of key importance to Ag-specific recognition of T lymphocytes. In this paper, LFA-1-mediated adhesiveness of peripheral blood (PB) and synovial fluid (SF) T lymphocytes to affinity-purified ICAM-1-coated plates was studied in patients with rheumatoid arthritis (RA) and in patients with non-RA panels, including osteoarthritis, ankylosing spondylitis, erythema nodosum, pseudogout, and pustulosis. LFA-1-mediated adhesiveness of SF T lymphocytes was not observed in any of the 10 non-RA patients studied, although cross-linking of the TCR on lymphocytes from these patients rapidly converted LFA-1 to an adhesive state. In contrast, SF T lymphocytes from 10 of 12 RA patients exhibited LFA-1-mediated adhesiveness without a requirement for cross-linking of the TCR. No difference was seen in the cell surface density of LFA-1 between non-RA and RA T lymphocytes, suggesting that the difference in adhesiveness was due to a high avidity state of LFA-1 on SF T lymphocytes in RA. Furthermore, exposure of PB T lymphocytes, which showed a low avidity state of LFA-1, to whole SF from RA patients that was depleted of T lymphocytes could induce a high avidity state of LFA-1 in vitro. Cellfree SF from RA patients also could stimulate adhesiveness, although to a lesser extent. These data suggest the existence of a LFA-1-activating environment that is selectively found in SF from RA patients.
Identification of epitopes within a highly immunogenic region of acetylcholine receptor by a phage epitope library.
Authors: Barchan D , Balass M , Souroujon M C , Katchalski-Katzir E , Fuchs S
Published: December 2022

We have employed a hexapeptide phage-epitope library to identify epitopes for a mAb (mAb 5.14), which is directed to a determinant within a highly immunogenic, cytoplasmic region of the alpha-subunit of acetylcholine receptor (AChR). We have selected two different peptide-presenting phages (SWDDIR-phage and LWILTR-phage) which interact specifically with mAb 5.14. This interaction is specifically inhibited by AChR and by synthetic peptides corresponding to the hexapeptides presented by the selected phages. Although mAb 5.14 binds to AChR in its native as well as its denatured form, the selected hexapeptides do not exist as such in the AChR molecule. However, three amino acid sequence homologies with these hexapeptides were shown to be present in the cytoplasmic region of Torpedo AChR. By extending the selected hexapeptides, at one or both ends, with amino acid residues flanking the hexapeptides in the phage, we obtained mimotopes with an up to two order of magnitude higher affinity to the Ab. These extended peptides were able to efficiently block the binding of mAb 5.14 to both peptide-presenting phages, and to AChR.
IgE-dependent activation of cytokine-primed mouse cultured mast cells induces a delayed phase of prostaglandin D2 generation via prostaglandin endoperoxide synthase-2.
Authors: Murakami M , Bingham C O , Matsumoto R , Austen K F , Arm J P
Published: December 2022

Mouse bone marrow-derived mast cells (BMMC) developed with IL-3 generate prostaglandin D2 (PGD2) through the utilization of prostaglandin endoperoxide synthase (PGHS)-1 within several minutes of cross-linking the high affinity Fc receptor for IgE (Fc epsilon RI) by hapten-specific IgE and Ag. We now report that this immediate generation of PGD2 is followed by a 15-fold induction of steady-state transcripts for PGHS-2, with a maximum at 30 min, accompanied by transient expression of PGHS-2 protein. When BMMC were pretreated with c-kit ligand (KL) in combination with IL-10 for 2 h, sensitized with IgE, and activated with Ag, their expression of steady-state transcripts for PGHS-2 increased 111-fold and their expression of PGHS-2 protein was markedly enhanced, with maximal expression at 1 h and 5 h, respectively, after activation. These events were accompanied by PGD2 generation from 1 to 10 h after activation that accounted for approximately 50% of total PGD2 generation. The expression of PGHS-1 protein did not change during this period. The optimal priming interval for the effect of KL plus IL-10 on the IgE-dependent induction of PGHS-2 was 2 h, at which time only this particular cytokine combination acted synergistically with activation by IgE and Ag. In contrast, at 2 days the accessory cytokines that could provide priming with KL included IL-3 and IL-9 in addition to IL-10. Dexamethasone, which inhibited the expression of PGHS-2 but not PGHS-1, and NS-398, a selective inhibitor of PGHS-2, each suppressed the delayed phase but not the immediate phase of PGD2 generation. Conversely, valeryl salicylate, a selective inhibitor of PGHS-1, suppressed the immediate but not the delayed phase of PGD2 generation after cell priming and IgE-dependent activation.
Staphylococcal enterotoxin D functions as a human B cell superantigen by rescuing VH4-expressing B cells from apoptosis.
Authors: Domiati-Saad R , Attrep J F , Brezinschek H P , Cherrie A H , Karp D R , Lipsky P E
Published: December 2022

Staphylococcal enterotoxins are potent superantigens, in that they activate T cells bearing specific V beta-chain gene segments. In this study, we analyzed the capacity of staphylococcal enterotoxin D (SED) to function as a B cell superantigen. SED induced T cell-dependent polyclonal proliferation and differentiation of B cells. In the absence of T cells, SED induced survival of B cells uniquely expressing VH4 containing IgM. The mechanism of survival of VH4-expressing B cells appeared to relate to the countering of apoptosis initiated by the engagement of HLA-DR by SED. Analysis of the VH4 gene products expressed by SED-stimulated B cells revealed the usage of six of the known functional VH4 genes with a variety of different CDR3 regions, employing different DH and JH gene segments. Moreover, the sequence analysis identified a possible site for SED binding of VH4 that includes the solvent-exposed surfaces of 3' CDR2/FR3 and/or FR1. Thus, SED appears to function as a unique B cell superantigen by inducing survival of VH4-expressing B cells.
Aberrant regulation of cytokines in HIV-1 TAT72-transgenic mice.
Authors: Garza H H , Prakash O , Carr D J
Published: December 2022

The trans-activator of transcription or TAT gene from HIV-1 encodes a protein that increases the processivity of transcription from the HIV-1 genome. TAT protein can also affect cellular processes in the absence of its ribonucleic HIV target sequence trans activation response element and may be responsible for some aspects of HIV pathogenesis apart from infectious virus or other viral gene products. We have previously shown that TAT72 decreases CTL activity in TAT72-transgenic mice, and we now demonstrate aberrant regulation of mitogen-elicited IL-2 at both transcriptional and translational levels. In contrast, alloantigen stimulation resulted in increased IL-6 and IL-10 production in the TAT72-transgenic mice. Con A-stimulated cultures of splenic lymphocytes from TAT72-transgenic mice do not undergo clonal proliferation of CD4+ cells as compared with CD8+ cells monitored over 72 h. These results suggest that TAT is sufficient to induce some pathology associated with AIDS and is a potent immunologic manipulator apart from its function as trans-activator.
Generation of a new gamma delta T cell-specific monoclonal antibody (GD3.5). Biochemical comparisons of GD3.5 antigen with the previously described Workshop Cluster 1 (WC1) family.
Authors: Jones W M , Walcheck B , Jutila M A
Published: December 2022

In this report, we describe GD3.5, a new lineage-specific gamma delta T cell marker that is distinct from TCR and known Workshop Cluster 1 (WC1). FACS analysis indicated that GD3.5Ag is expressed on approximately 90% of the peripheral blood gamma delta T cell population and GD3.5 specifically stained gamma delta T cells and not alpha beta T cells, B cells, neutrophils, or monocytes. Also, a significant portion of the GD3.5-positive population was WC1-negative. Nonreducing Western blot analysis and immunoprecipitation experiments revealed a single 220- to 240-kDa glycoprotein recognized by GD3.5 compared with two WC1 bands at 200 kDa and 300 kDa recognized by the IL-A29 Ab. Cross-immunoprecipitation experiments demonstrated that GD3.5 could be immunoprecipitated from lysates cleared of IL-A29/WC1 complexes. Reciprocally, WC1 could be immunoprecipitated from lysates cleared of GD3.5Ab/GD3.5Ag complexes. Digestion of WC1 and GD3.5 Ag with V-8 protease resulted in digestion profiles that clearly distinguished the glycoproteins. Additionally, GD3.5 Ag and WC1 possess disparate sensitivity to PNGase F, O-sialoglycoprotease, and neuraminidase, indicating differences in N- and O-linked sugars and the presence of sialic acid residues. Both GD3.5 Ag and WC1 appeared to be sialomucin-like molecules that share similar O-sialoglycoprotein endopeptidase sensitivity with other cell surface molecules, such as PSGL-1. Lastly, GD3.5 Ag, but not WC1, was exquisitely sensitive to very low-dose chymotrypsin treatment. Therefore, our data suggest that GD3.5 Ag is a previously uncharacterized, lineage-specific gamma delta T cell Ag. Furthermore, we show that GD3.5 and WC1 are sialomucins, which provides important clues to their function.
Early B cell factor interacts with a subset of kappa promoters.
Authors: Sigvardsson M , Akerblad P , Leanderson T
Published: December 2022

A conserved sequence element situated between the decamer and TATA box in V kappa II and V kappa V promoters has a high homology to the binding site for early B cell factor (EBF). The kappa promoter element was shown to bind EBF specifically using both in vitro-translated protein and nuclear extract. Concomitant binding of EBF and Oct proteins to a wild-type kappa promoter template was observed at low efficiency, and such dual occupancy was dependent on an intact amino terminus of the Oct protein. When the two binding sites were separated by a 10-bp spacer, this dependency disappeared. A single kappa promoter EBF site together with a TATA box and an Ig heavy chain enhancer showed marginal transcriptional stimulatory activity. In contrast, the EBF site acted synergistically with a decamer element in EBF-negative plasmacytoma cells, but not in B cells of an earlier differentiation stage. In these cells, a distinct protein was observed that interacted with the EBF binding motif, while overexpression of EBF down-regulated the expression of a reporter construct containing Ig control elements.
Oxidative killing of Cryptococcus neoformans by human neutrophils. Evidence that fungal mannitol protects by scavenging reactive oxygen intermediates.
Authors: Chaturvedi V , Wong B , Newman S L
Published: December 2022

Polymorphonuclear neutrophils (PMN) kill Cryptococcus neoformans (Cn) by oxidative mechanisms, but the roles of various reactive oxygen intermediates (ROIs) are not known. We used a mannitol low-producing Cn mutant (Cn MLP) and its wild-type parent (Cn H99) to examine the role of ROIs distal to H2O2 in PMN killing and to determine whether mannitol produced by Cn protects the fungus against ROIs. At PMN:Cn cell ratios of 1:1, 10:1, and 100:1, PMN killed significantly more Cn MLP than Cn H99 cells after 2 and 4 h (p less than 0.05). Superoxide dismutase and the hydroxyl radical (OH.) scavengers mannitol and DMSO inhibited killing of both strains (p less than 0.05), but catalase did not. Cn H99 and Cn MLP stimulated PMN to produce similar amounts of O2- and H2O2. In contrast, Cn MLP stimulated greater luminol-dependent chemiluminescence than did Cn H99 (p less than 0.05). Finally, H2O2 alone killed similar numbers of Cn H99 and Cn MLP cells, but oxidants generated by FeSO4 (1 microM), H2O2 (10 microM), and iodide (1 to 3 microM) killed significantly more Cn MLP than Cn H99 cells in 1 h (p less than 0.05). Mannitol, DMSO, and catalase completely inhibited killing of both Cn strains by this cellfree system, but superoxide dismutase did not. These results suggest that 1) distal ROIs such as OH. and HOCI are key effector molecules against Cn, and 2) mannitol produced by Cn may protect against oxidative killing by scavenging distal ROIs.
Immunization with granulocyte-macrophage colony-stimulating factor-transduced, but not B7-1-transduced, lymphoma cells primes idiotype-specific T cells and generates potent systemic antitumor immunity.
Authors: Levitsky H I , Montgomery J , Ahmadzadeh M , Staveley-O'Carroll K , Guarnieri F , Longo D L , Kwak L W
Published: December 2022

Recently, genetically modified tumor cell vaccines have been described for nonhematopoietic cancers in which the relevant Ags are unknown. Several of these cell-based vaccine strategies have been shown to induce T cell-mediated systemic antitumor immunity, either by enhancing the processing and presentation of tumor Ags by host APCs or by facilitating effective Ag presentation by the tumor vaccine itself. These strategies were compared in a model B cell lymphoma, a tumor derived from APCs, which have the inherent capacity to activate Ag-specific T cells. Eradication of pre-established systemic lymphoma was achieved following immunization with lymphoma cells engineered to produce granulocyte-macrophage (GM)-CSF, and to a lesser extent cells producing IL-4, whereas vaccination with lymphoma cells transfected with the genes encoding IL-2 or B7-1 had no effect. The systemic immunity generated by GM-CSF- or IL-4-transfected lymphoma required both CD4+ and CD8+ T cells. Previous immunotherapeutic strategies for the treatment of lymphoma have focused on the generation of Ab responses targeted to the unique Ig Id as a tumor-specific Ag. Anti-idiotypic Abs were undetectable in animals vaccinated with GM-CSF-transduced lymphoma cells. In contrast, such immunization did result in the induction of Id-specific T cell responses. This is the first demonstration that T cell responses specific for a native tumor Ag are generated by GM-CSF-transduced tumor cell-based vaccination, suggesting that B cell lymphoma may be a suitable disease for genetically modified tumor vaccine strategies.
Acceleration of influenza virus clearance by Th1 cells in the nasal site of mice immunized intranasally with adjuvant-combined recombinant nucleoprotein.
Authors: Tamura S , Miyata K , Matsuo K , Asanuma H , Takahashi H , Nakajima K , Suzuki Y , Aizawa C , Kurata T
Published: December 2022

The protective roles of influenza viral nucleoprotein (NP), together with the cellular mechanism of the protection in the nasal site, were examined in BALB/c mice immunized intranasally with an adjuvant (cholera toxin B subunit containing 0.2% of the whole toxin)-combined A or B virus recombinant NP. The NP-immune mice, when challenged intranasally with a sublethal dose of the virus 3 wk after immunization, had accelerated virus clearance from the nasal site in both an influenza type-specific and a nonspecific manner, as shown by the protection from high morbidity from the second day after challenge. Both type-specific and nonspecific acceleration of recovery was confirmed by the increased survival rate after challenge with a lethal dose of virus in mice immunized and boosted with adjuvant-combined NP. The acceleration of nasal virus clearance was accompanied with acceleration of type-specific systemic delayed-type hypersensitivity (DTH) and with IFN-gamma production by nasal lymphocytes. The nasal lymphocytes from the immunized and challenged mice generated a significantly high level of DTH when transferred locally, but no class I MHC-restricted CTL response. Moreover, nasal CD4+ T cells, induced by NP immunization and increased in number by the subsequent challenge, were involved in the accelerated IFN-gamma production. These results suggest that nasal Th1 cells, capable of producing IFN-gamma and mediating DTH, are involved in the type-specific acceleration of recovery from influenza after challenge in mice immunized intranasally with adjuvant-combined NP, although the nonspecific mechanism of accelerated recovery remains to be solved.
Human herpes simplex virus (HSV)-specific CD8+ CTL clones recognize HSV-2-infected fibroblasts after treatment with IFN-gamma or when virion host shutoff functions are disabled.
Authors: Tigges M A , Leng S , Johnson D C , Burke R L
Published: December 2022

Herpes simplex virus (HSV)-specific CD8+ CTL cloned from individuals infected with HSV-2 efficiently lyse HSV-infected EBV-transformed B lymphoblastoid cells; however, these same CTL fail to lyse infected dermal fibroblasts. By 3 h after infection (early), class I MHC expression is reduced to less than 20% of that in uninfected fibroblasts, and expression is further reduced to less than 1% of the level in uninfected cells between 6 and 18 h after infection (late). We used an HSV-2 mutant that lacked the virion host shutoff (vhs) function to demonstrate that vhs plays a role in the loss of class I expression. While fibroblasts infected with this mutant are lysed by CTL that recognize virion proteins presented early as a consequence of introduction into the cytoplasm by the infecting virus, they are resistant to lysis by CTL that recognize viral proteins that must be synthesized de novo to be presented as class I Ags. Fibroblasts infected with a mutant that lacks the transporter-associated protein inhibitor ICP47 and is partially vhs defective are sensitive to CTL lysis. Pretreatment of fibroblasts with IFN-gamma prior to HSV infection sustained the level of class I expression for longer periods after infection, and these fibroblasts, infected with wild-type HSV-2, were partially sensitive to lysis by HSV-specific CTL. Taken together, these results suggest that the combined effects of the HSV-2 vhs and ICP47 gene products are to block Ag presentation by class I MHC. However, this effect can be transiently counteracted by IFN-gamma providing an early role for CD8+ CTL in the cellular immune response to HSV-2.
Eosinophil degranulation in the presence of bronchial epithelial cells. Effect of cytokines and role of adhesion.
Authors: Takafuji S , Ohtoshi T , Takizawa H , Tadokoro K , Ito K
Published: December 2022

It has been suggested that eosinophils (Eos) are responsible for damage to bronchial epithelial cells by releasing toxic eosinophil granule proteins in bronchial asthma. We examined eosinophil cationic protein (ECP) release from human Eos cultured in the presence of human bronchial epithelial cell line BEAS-2B (Ep). ECP release was potentiated only when both Eos and Ep were activated by IL-5 and TNF, respectively, while it was not potentiated when either Eos or Ep were activated. ECP release from Eos activated by IL-5 was also enhanced when Ep was stimulated by IFN-gamma. Paraformaldehyde fixation of Ep had no effect on ECP enhancement, excluding the possibility that soluble factors from Ep contribute to ECP potentiation. Coculture of Eos and Ep with cytokine treatment resulted in the enhancement of eosinophil adhesion and ECP release, and eosinophil adhesion preceded ECP release in the kinetic study. The enhancement of ECP release was partially inhibited by anti-CD18 mAb, which caused partial and comparable inhibition on the potentiation of eosinophil adhesion. These results suggest that the activation of Ep may profoundly affect the ability of cocultured Eos to release ECP and that CD18-dependent adhesion of Eos to Ep may be considered as one of the mechanisms of ECP enhancement.
CD95 (Fas/Apo-1)-induced apoptosis results in loss of glucose transporter function.
Authors: Berridge M V , Tan A S , McCoy K D , Kansara M , Rudert F
Published: December 2022

Treatment of activated human T cells with CD95 (Fas/Apo-1) ligand or Abs against CD95 results in apoptotic cell death. Although cellular responses to CD95 ligation have been described in some detail, the early molecular events that result in T cell death are only now beginning to be elucidated. Using Jurkat cells as a model of activated human T cells, we have investigated the effects of CD95 ligation on glucose transport and on glucose transporter function. We show that within minutes of CD95 activation, the ability to transport glucose across the plasma membrane is compromised and that transient exposure to Abs against CD95 for as little as 3 min results in reduced glucose transport and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) responses measured at 16 h. The effects of CD95 ligation on glucose transport are shown to be associated with loss of affinity of glucose transporters for glucose without altered maximum velocity and without changes in the cell surface expression of Glut 1, the predominant glucose transporter isotype on Jurkat cells. These results support a model of CD95 induced cell death that, at least in its early stages, does not depend on signaling to the nucleus or on macromolecular synthesis. Acute regulation of glucose transport is proposed to be an early effector mechanism in CD95-induced apoptotic cell death.