bluuebunny/crossref_metadata_2025 huggingface dataset:
Mouse complement receptors type 1 (CR1;CD35) and type 2 (CR2;CD21): expression on normal B cell subpopulations and decreased levels during the development of autoimmunity in MRL/lpr mice.
Authors: Takahashi K , Kozono Y , Waldschmidt T J , Berthiaume D , Quigg R J , Baron A , Holers V M
Published: December 2022

Human complement receptors type 1 (hCR1;CD35) and type 2 (hCR2;CD21) are expressed on B lymphocytes at specific stages during differentiation and activation. These receptors play critical roles in the immune response to T-dependent Ags in addition to germinal center formation. Expression of both hCR2 and hCR1 is decreased on B lymphocytes of patients with systemic lupus erythematosus (SLE). We have studied the expression of mouse CR2 and CR1 on normal populations of mouse B lymphocytes in BALB/c mice. Our results demonstrate that expression of these receptors in the normal state closely parallels that of hCR2. During bone marrow development, expression is first detected on low B220/high IgM cells, demonstrating that complement receptors appear after central tolerance mechanisms are completed. In the splenic microenvironment the highest levels of receptor expression are found on marginal zone B lymphocytes. Mouse CR2 and CR1 are also found on peritoneal B1a and B1b cells in addition to IgA+ Peyer's patch B cells. Activation of splenic B cells under Th2 conditions results in a marked decrease in receptor expression. To determine whether the patterns of receptor expression also parallel those found in human disease, we studied the MRL lpr/lpr (MRL/lpr) model of SLE. Interestingly, we found an early decrease in complement receptor expression that is progressive and first detectable before major clinical manifestations of nephritis. We hypothesize that the early decrease in complement receptor expression such as that demonstrated by MRL/lpr mice plays an important role in the pathogenesis of murine and perhaps human SLE.
CD28-induced cytokine production and proliferation by thymocytes are differentially regulated by the p59fyn tyrosine kinase.
Authors: Ong C J , Lim A S , Teh H S
Published: December 2022

CD28 is a 44-kDa homodimeric receptor that is expressed on the majority of T cells. Engagement of the CD28 receptor by soluble anti-CD28 mAb in conjunction with phorbol ester (PMA) induces the production of cytokines and the proliferation of resting T cells via signal transduction pathways independent of the TCR. Evidence is provided herein that CD28 signals leading to cytokine production do not require the p59fyn (Fyn) tyrosine kinase, whereas CD28-mediated proliferation is dependent on the presence of the Fyn kinase in thymic, but not lymph node, cells. The defect in proliferation is not due to failure of IL-2R signaling, since addition of high concentrations of exogenous IL-2 can overcome the proliferative defect. Analysis of CD28-directed induction of the IL-2R alpha (CD25)-chain, which confers high affinity binding to IL-2, showed that Fyn-deficient thymocytes, but not lymph node cells, failed to up-regulate CD25 expression following anti-CD28 and PMA stimulation. Thus, the Fyn tyrosine kinase is critically required for thymic CD28-mediated CD25 expression and proliferation but not for CD28-mediated cytokine production.
CD40 ligand induction on T cell subsets by peptide-presenting B cells: implications for development of the primary T and B cell response.
Authors: Jaiswal A I , Croft M
Published: December 2022

Recent data suggest that CD40 ligand (CD40L)-CD40 interactions are essential for up-regulation of costimulatory activity on APC and that efficient induction of CD40L may be pivotal to the success of a CD4 T cell response. CD40L is regulated primarily by TCR signaling, but high level expression on a naive T cell appears to require additional interactions between T cell coreceptors and APC accessory molecules. The data reported here show that resting B cells presenting peptide Ag, in contrast to both dendritic cells and preactivated B cells, induce very little CD40L on naive CD4 cells, which in turn is insufficient to promote APC costimulatory activity. We also show, however, that previously activated effector T cells have enhanced responsiveness to Ag when accessory molecule help is limiting and consequently can express high levels of CD40L after interaction with resting B cells. High level CD40L expression correlated with B cell activation and up-regulation of costimulatory activity; however, blocking studies showed that CD40L was only partially responsible for these phenomena. These studies reinforce the notion that resting B cells may be tolerogenic for naive CD4 cells in part because of inefficient CD40L induction. The data also suggest that a successful primary T cell response will only occur if either the initial interaction is with a dendritic cell followed by subsequent interactions of the effector T cells with resting APC or if nonspecific inflammatory stimuli up-regulate accessory molecule expression on resting APC before an encounter with the naive T cell.
The glycosylation of IgA produced by murine B cells is altered by Th2 cytokines.
Authors: Chintalacharuvu S R , Emancipator S N
Published: December 2022

We sought to determine whether selected cytokines, known to profoundly increase proliferation and/or production of Ig by B cells and their progeny, also have as yet unrecognized effects upon IgA glycosylation. For these studies, we selected CH12LX mouse B lymphoma cells, a widely used model of B cell differentiation. Glycosylation was assessed by detection with enzyme-lectin conjugates in an immunoabsorption assay and verified by profiling and sequencing of the N-linked oligosaccharides. Stimulation of B cells with IL-4 plus IL-5 significantly alters the terminal glycosylation of secreted IgA, whereas LPS has a minor effect, despite the fact that both stimuli are equipotent at inducing Ig class switching and Ig secretion. Moreover, the alteration in terminal glycosylation was more profound on IgA secreted from surface IgM+ than from surface IgA+ CH12LX cells. These results suggest that the increased production of IL-4 and IL-5 by peripheral blood lymphocytes from IgA nephropathy patients might result in the production of abnormally glycosylated IgA. In turn, this abnormally glycosylated IgA may promote deposition of IgA in glomeruli in this disease.
T cell-independent response to Brucella-insulin identifies a preimmune repertoire for insulin.
Authors: Thomas J W , Kralick P M , Ewulonu U K
Published: December 2022

An early or preimmune repertoire for anti-insulin B cells was examined using the T cell-independent (TI) response to insulin conjugated to Brucella abortus (BA-ins). mAbs from the BA-ins response reflect a repertoire present 7 to 10 days before the first Ab-forming cells (AFC) are detected in primary T cell-dependent (TD) responses to insulin. Although 4 of 6 BA-ins mAb express IgG2 isotype, evidence for somatic mutation is limited. A total of 9 of 12 V regions are identical with known V(H) or Vkappa genes, and consensus sequences suggest two other V genes may be in germ-line configuration. The relative avidities (50% inhibition of binding) of TI anti-insulins cover a broad range and are consistent with a germ-line anti-insulin repertoire that is functionally diverse. The V(H)s of 5 mAb are from two subsets of J558 genes (205.1 and 186.3) that dominate the B cell pool of adult mice and are different from the V(H)s used by anti-insulin mAb in primary TD responses. One IgM anti-insulin (mAb 301) uses V(H)-J606 and Vkappa1, and this mAb binds beta cells. Other TI mAb use either Vkappa5 or Vkappa19.3 genes and are similar to Vkappa genes used by anti-insulin mAb from TD responses. The data show that mutations in germ-line genes are not required for measurable insulin binding by monospecific mAb from adult mice. The recurrent use of Vkappa genes in both early (TI response) and late (TD responses) suggest that these structures are important in insulin binding.
Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201-restricted Melan-A/MART-1 antigenic peptide in melanoma.
Authors: Romero P , Gervois N , Schneider J , Escobar P , Valmori D , Pannetier C , Steinle A , Wolfel T , Lienard D , Brichard V , van Pel A , Jotereau F , Cerottini J C
Published: December 2022

The Melan-A/MART-1 gene product is frequently recognized by tumor-specific HLA-A2-restricted CTL. An immunodominant nonapeptide has been localized to the region spanning residues 27-35. However, the decapeptide including residues 26-35 (the nonapeptide extended NH2 terminally by one residue) appeared to be recognized as efficiently as the nonapeptide. In this study, we show that the optimal length immunodominant peptide appears to correspond to the decapeptide 26-35, as assessed by quantitative analyses of both 4 polyclonal and 13 monoclonal populations of specific CTL. Functional assays of peptide binding to HLA-A2 indicate that the decapeptide is significantly a more efficient binder than the nonapeptide. Moreover, analogues of the decapeptide including substitutions at a secondary HLA-A2 peptide anchor further improve decapeptide binding. Finally, we show that the functional (9 CTL clones analyzed) and structural TCR repertoire (7 CTL clones) of a group of specific CTL clones is rather diverse. The findings reported here may have important implications for future peptide-based melanoma vaccination trials as well as for the monitoring of specific CTL responses in vivo.
Growth of P511 mastocytoma cells in BALB/c mouse brain elicits CTL response without tumor elimination: a new tumor model for regional central nervous system immunity.
Authors: Gordon L B , Nolan S C , Cserr H F , Knopf P M , Harling-Berg C J
Published: December 2022

We have developed a murine model to explore the tumor-specific CTL response in the immune-privileged central nervous system using P511 mastocytoma cells. Three strains with varying degrees of histocompatibility to P511 cells (CD-1, allogeneic; BALB/c, minor histoincompatible; DBA/2, syngeneic) received tumor cells (10(4)) into the putamen 7 days after cannula implantation, when the blood-brain barrier was functionally intact. Without exception, tumor formed reproducibly by day 7 in all strains. Tumor rejection occurred in CD-1 but not in BALB/c and DBA/2 mice. Using a flank injection site, both CD-1 and BALB/c, but not DBA/2 mice, ultimately rejected flank tumors. Analysis of tumor-specific CTL in BALB/c spleens revealed that P511 administration into brain or flank elicited similar responses: no fully activated CTL were detectable but a significantly expanded population of nonkilling precursors of CTL (pCTL) were present. A P511 cell-specific pCTL population was also identified at the brain tumor site 14 days post-tumor introduction, indicating that pCTL, generated in the periphery, traffic to the tumor site in brain. These data indicate that failure to reject tumor in brain is neither due to lack of afferent stimulation nor to inability of peripheral effectors (P511 cell-specific pCTL) to reach the tumor site. We hypothesize that these effector cells are prevented from developing into fully activated CTL by conditions within the central nervous system microenvironment that down-regulate CTL development.
Hydrogen peroxide-mediated transcriptional induction of macrophage colony-stimulating factor by TGF-beta1.
Authors: Hong Y H , Peng H B , La Fata V , Liao J K
Published: December 2022

TGF-beta1 and macrophages are important regulators of tissue fibrosis and remodeling. Here we show that TGF-beta1 induces the expression of macrophage-CSF (M-CSF) in vascular endothelial cells via a signaling pathway(s) involving hydrogen peroxide (H2O2). In a time-dependent manner, TGF-beta1 produced a 10- and a 6-fold increase in M-CSF mRNA and protein levels after 12 h, respectively. This increase in M-CSF expression was attenuated by a nitric oxide donor, S-nitrosoglutathione (GSNO), and by a nonspecific oxidase inhibitor, diphenylene iodonium. Furthermore, the TGF-beta1-induced M-CSF mRNA expression was inhibited by catalase, but not by superoxide dismutase, suggesting that H2O2 rather than superoxide anion (O2.-) is the primary mediator of the effects of TGF-beta1. Transient transfection studies using deletional M-CSF promoter constructs demonstrated that TGF-beta1 produced a 13-fold induction in M-CSF promoter activity that was repressed by >85% with GSNO and catalase, in part through inhibitory effects on kappaB cis-acting elements. Electrophoretic mobility shift assays revealed that the activation of nuclear factor-kappaB by TGF-beta1 was also inhibited by GSNO and catalase, but not by superoxide dismutase. In a concentration-dependent manner, treatment with exogenous H2O2 produced 14- and 4.6-fold increases in M-CSF promoter activity and mRNA expression, respectively. These results indicate that the generation of H2O2 and activation of NF-kappaB by TGF-beta1 are required for the induction of M-CSF gene transcription.
Absence of IL-1 signaling and reduced inflammatory response in IL-1 type I receptor-deficient mice.
Authors: Labow M , Shuster D , Zetterstrom M , Nunes P , Terry R , Cullinan E B , Bartfai T , Solorzano C , Moldawer L L , Chizzonite R , McIntyre K W
Published: December 2022

IL-1alpha and IL-1beta are potent inflammatory cytokines that contribute to a number of normal physiologic processes and to the development of a number of inflammatory diseases. Two IL-1R, the type I and type II receptors, have been identified. This work describes the derivation and characterization of mice deficient in expression of the type I IL-1R (IL-1RI). IL-1RI-deficient mice were viable and fertile, but failed to respond to IL-1 in a variety of assays, including IL-1-induced IL-6 and E-selectin expression and IL-1-induced fever. Similar to IL-1beta-deficient mice, IL-1RI-deficient mice had a reduced acute phase response to turpentine. In contrast, IL-1RI-deficient mice had a reduced delayed-type hypersensitivity response and were highly susceptible to infection by Listeria monocytogenes. These data demonstrate that the IL-1RI is essential for all IL-1-mediated signaling events examined, and that both IL-1alpha and IL-1beta are critical to the animals' response to injury and infection. These data also demonstrate that IL-1 function is not required for normal development or homeostasis.
Immunodominance is independent of structural constraints: each region within hen eggwhite lysozyme is potentially available upon processing of native antigen.
Authors: Moudgil K D , Sekiguchi D , Kim S Y , Sercarz E E
Published: December 2022

T cell responses to different protein Ags have been shown to focus on a few ("immunodominant") determinants. We have addressed three major, interrelated questions regarding immunodominance. First, can each area within hen eggwhite lysozyme (HEL) serve as an immunodominant focus in different inbred mouse strains or are there structural constraints that limit the utilization of certain segments of the molecule? Second, in MHC-congenic mice with identical non-MHC genes, is response to HEL restricted to one or more members of a set of HEL determinants owing to processing constraints imposed by the background genes? Third, does a truncated TCR repertoire influence the immunodominance of certain determinants of HEL? Our results in 19 strains of mice, representing 11 different MHC haplotypes, demonstrate that the immunodominant determinants within HEL are distributed all over the molecule, suggesting that there is no inherent structural constraint imposed on certain regions to be always immunorecessive. However, in different mouse strains, the emergence of identical regions of HEL as immunodominant sites strictly correlates with the identity of their MHC haplotypes but not genetic background (non-MHC) genes. We attribute this relationship to "MHC-guided processing" of native Ag. Finally, our results demonstrate that a truncated TCR repertoire can result not only in the loss of response to certain immunodominant determinants, but can also result in a gain. These results should contribute significantly to further understanding of the mechanism of immunodominance.
T cell receptor diversity in CTLs specific for the CTT-1 and CTT-2 minor histocompatibility antigens.
Authors: Johnston S L , Wettstein P J
Published: December 2022

Minor histocompatibility Ags (HA) and their associated immunogenic peptides provide a formidable barrier for successful transplantation, but there is virtually no information regarding the diversity of minor HA-specific TCRs. We have investigated the diversity of alpha- and beta-chains in TCRs specific for the CTT-1 and CTT-2 peptides that are immunodominant in CTL responses to multiple minor HA. CTLs were cloned after in vitro stimulation, and alpha- and beta-chain transcripts were amplified and sequenced to identify utilized V genes, complementarity-determining regions 3 (CDR3s), and joining region genes. Twenty-one unique CTT-2-specific TCRs were identified in 31 clones, and 22 CTT-1-specific TCRs were identified in 29 clones. A relatively limited number of V beta subfamilies were represented in these panels of TCRs, with Vbeta 5 and V beta8 genes expressed in multiple TCRs in each panel. Similar diversity was observed with V alpha usage, and V alpha4 subfamily usage was more prominent in CTT-2-specific TCRs than in CTT-1-specific TCRs. Neither alpha nor beta CDR3 regions exhibited prominent motifs or length restriction. However, CTT-1- and CTT-2-specific beta CDR3 regions included an excess of negatively over positively charged residues that were bimodally distributed among CDR3 positions. In fact, 50% of CTT-1-specific CDR3 regions included two negative charges separated by three to five amino acids. Despite the similarities in net charge of beta CDR3 regions, TCRs specific for these two minor HA peptides are relatively diverse and would expectedly withstand attempts at anti-TCR Ab-mediated immunosuppression.
IL-4-independent regulation of in vivo IL-9 expression.
Authors: Monteyne P , Renauld J C , Van Broeck J , Dunne D W , Brombacher F , Coutelier J P
Published: December 2022

We focused on the role of IL-4 in the regulation of the Th2 cytokine IL-9. In vivo, IL-9 mRNA was detected in lymph nodes after immunization with soluble Ags. IL-9 expression preceded that of IL-4, and was not affected in IL-4 knockout mice. In contrast, a significant decrease of IL-9 message was observed in IL-10-deficient mice, indicating a role for this cytokine in the induction of IL-9 production. Treatment with anti-CD4 Ab and analysis of purified CD4 cells confirmed that IL-9 was produced by CD4+ cells. Moreover, similarly to what has been reported for IL-4, IL-9 message induction was strongly decreased by infection with lactate dehydrogenase-elevating virus. IL-9 mRNA was also detected after in vivo stimulation with anti-CD3 Ab. In this model, IL-9 expression followed that of IL-4, but was not reduced in IL-4-deficient mice. This contrasts with in vitro stimulation in which, as reported in humans, IL-9 expression in lymphocytes incubated with anti-CD3 Ab and costimulatory molecules appeared as a late event, and was partly dependent on IL-4. In vitro IL-9 secretion was reduced significantly by addition of anti-IL-4 Ab, as well as in lymphocytes from IL-4 gene-deficient mice. Taken together, our results indicate that the Th2 cytokine IL-9 can be expressed by both IL-4-dependent and -independent pathways.
Functional effects of overexpression of protein kinase C-alpha, -beta, -delta, -epsilon, and -eta in the mast cell line RBL-2H3.
Authors: Chang E Y , Szallasi Z , Acs P , Raizada V , Wolfe P C , Fewtrell C , Blumberg P M , Rivera J
Published: December 2022

The rat basophilic leukemic (RBL-2H3) cell line was stably transfected with the endogenously expressed Ca2+-dependent protein kinase C-alpha (PKC-alpha) and -betaI and the Ca2+-independent delta and epsilon isoforms to study their functional roles. In addition, the Ca2+-independent PKC-eta was expressed. All transfected PKC isoforms translocated to the membrane-containing fraction in response to aggregation of the IgE-sensitized high affinity receptor for IgE (Fc epsilonRI) with the Ag dinitrophenyl(25)-BSA. All PKC transfectants, except PKC-eta, showed increased proliferative responses, and aggregation of Fc epsilonRI further enhanced the rate of proliferation. The PKC transfectants also showed increased phosphoinositide hydrolysis in response to Ag aggregation of receptors. No marked differences in the Ca2+ responses of the transfectants to Ag or thapsigargin were observed. Overexpression of PKC-alpha or -epsilon specifically inhibited receptor-dependent cytosolic phospholipase A2 (cPLA2) activity, whereas this activity was enhanced in the PKC-betaI transfectant. Analysis of the secretory response revealed that overexpression of PKC-betaI and -eta significantly enhanced secretion. A broad spectrum of cytokine mRNAs was detected in all transfectants, and overexpression of PKC-betaI significantly enhanced the receptor-dependent production of IL-2 and IL-6 mRNA. These studies identify PKC-alpha and -epsilon as negative regulators of cPLA2 activity and demonstrate the importance of PKC-beta as a positive modulator of secretion, cPLA2 activity, and cytokine production in this mast cell line.
CD154/CD40 and CD70/CD27 interactions have different and sequential functions in T cell-dependent B cell responses: enhancement of plasma cell differentiation by CD27 signaling.
Authors: Jacquot S , Kobata T , Iwata S , Morimoto C , Schlossman S F
Published: December 2022

CD40, a TNF receptor family member, plays a central role in T cell-mediated B cell activation. We have recently demonstrated that CD27, another TNF receptor family member, was also involved in B cell regulation and enhanced Ig production. In this report we compare CD27 and CD40 signals in B cell function. We selectively mimicked the effect of T cell help by addition to peripheral blood B cells activated with Staphylococcus aureus Cowan I strain and IL-2 of irradiated 300-19 cells transfected with either the CD70 (CD27 ligand) gene or the CD154 (CD40 ligand) gene, the vector alone, or both CD70 and CD154 genes. CD27 ligation induced only a slight increase in B cell proliferation compared with the dramatic enhancement induced by CD40 ligation; double ligation proved to be less efficient than CD40 ligation alone. In contrast, IgG production was increased only by CD27 ligation alone. Moreover, the CD27 signal was more efficient when it was given on day 2 of the culture rather than on day 0. Phenotypic analysis of the activated cells showed that CD27 ligation increased the percentage of cells showing a plasma cell profile (CD19-, CD38+), whereas upon CD40 ligation most of the cells still had a germinal center-like phenotype (CD19+, CD38+). Our results suggest that the CD27 and CD40 signals are not synergistic but, rather, are complementary and involve distinct steps of T cell-dependent B cell activation. CD27 may be more important in the induction of plasma cell differentiation at a time when the expansion phase has already occurred.
Introduction of exogenous class II trans-activator in MHC class II-deficient ABI fibroblasts results in incomplete rescue of MHC class II antigen expression.
Authors: Peijnenburg A , Gobin S J , van Eggermond M C , Godthelp B C , van Graafeiland N , van den Elsen P J
Published: December 2022

Previously, we have shown that fibroblasts established from type III bare lymphocyte syndrome patient ABI are characterized by the absence of MHC class II gene expression and a strongly reduced amount of MHC class I transcripts. Complementation analysis has suggested that the gene defective in these ABI fibroblasts is different from that encoding the class II trans-activator (CIITA), which has been attributed an essential role in both constitutive and inducible expression of MHC class II genes. In the present study it is shown by reverse transcriptase-PCR analysis that the amount of CIITA transcripts in ABI fibroblasts is greatly reduced compared with that in fibroblasts derived from a healthy individual. Transient cotransfection of a construct in which CIITA is under the control of a constitutive promoter with an HLA-DRA promoter-luciferase reporter plasmid resulted in enhanced luciferase expression in ABI fibroblasts. Furthermore, ABI fibroblasts stably transfected with CIITA re-express functional HLA-DR Ags, but do not express HLA-DQ and DP Ags at the cell surface. Comparison of these data with those obtained for normal fibroblasts and fibroblasts defective for CIITA indicate that the gene defect and the resulting lack of MHC class II expression in ABI fibroblasts can only partly be corrected by the introduction of CIITA. Furthermore, DNase I hypersensitivity analysis of ABI fibroblasts has revealed a closed chromatin structure in the promoter region of the MHC class II DRA gene. However, CIITA transfection resulted in an open DNA configuration, which suggests a role for CIITA in provoking changes in the chromatin structure of the DRA gene.
Activation and transdominant suppression of MHC class II and HLA-DMB promoters by a series of C-terminal class II transactivator deletion mutants.
Authors: Chin K C , Li G , Ting J P
Published: December 2022

The class II transactivator (CIITA) is a highly specific transcription factor that activates only genes known to be involved in the class II MHC processing pathway, including class II MHC, invariant chain, and HLA-DMA/B genes. In this work, we show the requirement of a new region in CIITA that is critical for its function. Deletion mutants lacking varying length of the C terminus show that the C-terminal 41 amino acids of CIITA are indispensable for the activation of both the conventional DRA and nonconventional DMB promoters. This region contains a highly charged stretch of amino acids that is homologous to a yeast transcription factor, repression activator protein-1 (RAP1)-interacting factor 1. Mutants lacking the C terminus were tested in a transdominant-negative assay to examine their capacity to block the wild-type CIITA function. Two of these deletion mutants suppressed the activity of endogenously expressed wild-type CIITA to activate both DRA and DMB promoters. It may be possible to utilize these mutants to modulate MHC class II and DM gene expression.
Effects of 5-oxo-6,8,11,14-eicosatetraenoic acid on expression of CD11b, actin polymerization, and adherence in human neutrophils.
Authors: Powell W S , Gravel S , Halwani F , Hii C S , Huang Z H , Tan A M , Ferrante A
Published: December 2022

Human neutrophils contain a highly specific dehydrogenase that converts 5-hydroxy-6,8,11,14-eicosatetraenoic acid to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). 5-Oxo-ETE is a potent stimulator of calcium mobilization, chemotaxis, and aggregation in these cells and has similar effects on eosinophils. The primary objectives of the current study were to determine whether this compound could increase the surface expression of integrins and stimulate actin polymerization in neutrophils. 5-Oxo-ETE stimulated the expression of CD11b and, to a lesser extent, CD11c, on neutrophils, but had no significant effects on the expression of CD11a, CD16 (Fc gammaRIII), or CD32 (Fc gammaRII). Surface expression of CD11b in response to 5-oxo-ETE was maximal after 12 min and remained constant thereafter. The EC50 for this response (50 nM) was lowered to 20 nM by preincubation of neutrophils with PMA. 5-Oxo-ETE (EC50, 10 nM) also rapidly stimulated actin polymerization in neutrophils, with a maximal response at 20 s. This response was blocked by pretreatment of neutrophils with the Gi protein inhibitor, pertussis toxin, and by homologous desensitization due to preincubation with 5-oxo-ETE. However, preincubation with leukotriene B4 or platelet-activating factor had no effect on the response of neutrophils to subsequent addition of 5-oxo-ETE. The adherence of neutrophils to plasma-coated plastic was also stimulated by 5-oxo-ETE with a time course similar to that for the surface expression of CD11b. Low concentrations of PMA (0.3 nM) enhanced this response. These results raise the possibility that 5-oxo-ETE could contribute to the infiltration of neutrophils into inflammatory sites.
Cerebrospinal fluid cytokines in pediatric neuropsychiatric disease.
Authors: Mittleman B B , Castellanos F X , Jacobsen L K , Rapoport J L , Swedo S E , Shearer G M
Published: December 2022

This study examines cerebrospinal fluid from patients with three neuropsychiatric diseases of childhood for the presence and levels of several cytokines relevant to cell-mediated (type 1) and humoral (type 2) immunity. The patient groups include childhood-onset schizophrenia (n = 22), obsessive-compulsive disorder (OCD) (n = 24), and attention deficit hyperactivity disorder (n = 42). The cytokines examined include IL-2, IFN-gamma, TNF-beta/LT, IL-4, IL-5, IL-10, and TNF-alpha. Patients with OCD had a preponderance of type 1 cytokines. IL-4 was detectable only in samples from patients with schizophrenia. IL-10 was rarely detected and never in patients with OCD. Few patients with schizophrenia had detectable amounts of IFN-gamma in CSFL. We conclude that there is a relative skewing of CSFL profiles toward type 1 cytokines in patients with OCD, whereas in schizophrenia the relative preponderance is toward type 2 mediators. Patients with attention deficit hyperactivity disorder exhibited profiles intermediate between OCD and schizophrenia. We infer that cell-mediated immunity may be involved in the etiopathogenesis of OCD, whereas a relative lack of cell-mediated immunity and involvement of humoral immunity may be present in schizophrenia. These data provide a rationale for immune-based strategies of study and therapeutics in childhood neuropsychiatric disease.
HIV-1 gp120 induces an association between CD4 and the chemokine receptor CXCR4.
Authors: Ugolini S , Moulard M , Mondor I , Barois N , Demandolx D , Hoxie J , Brelot A , Alizon M , Davoust J , Sattentau Q J
Published: December 2022

For efficient entry into target cells, certain T cell-tropic HIV-1 isolates require both CD4 and the coreceptor CXCR4. However, the molecular interactions among CD4, CXCR4, and the HIV-1 envelope glycoproteins are only now being elucidated. Here we show that the binding of soluble gp120 from one macrophage-tropic and four T cell-tropic viruses to a CD4+, but not to a CD4-, T cell line, decreased the binding of an mAb specific for CXCR4 to its epitope, implying an interaction among gp120, CD4, and CXCR4. To confirm such an interaction, we conducted double- and triple-color confocal laser scanning microscopy on CD4+/CXCR4+ cells and determined the extent of CD4 and CXCR4 colocalization by a semiquantitative analysis. In the absence of gp120, a low level of constitutive colocalization between CD4 and CXCR4 was observed. Treatment with T cell-tropic-derived gp120 and, to a lesser extent, macrophage-tropic-derived gp120, increased the colocalization of CD4 with CXCR4, and triple staining indicated that gp120 was associated with the CD4-CXCR4 complexes. Cocapping of the gp120-CD4-CXCR4 complexes at 37 degrees C resulted in the cointernalization of a proportion of the gp120-CXCR4 complexes into intracellular vesicles. These data demonstrate that the binding of gp120 to CD4+ T cells induces the formation of a trimolecular complex consisting of gp120, CD4, and the HIV-1 coreceptor molecule CXCR4.
Regulation of antigen-specific IgE, IgG1, and mast cell responses to ingested allergen by mucosal tolerance induction.
Authors: van Halteren A G , van der Cammen M J , Cooper D , Savelkoul H F , Kraal G , Holt P G
Published: December 2022

Mucosal administration of soluble protein Ags results in profound immunologic nonresponsiveness, characterized by reduced production of Th1 and Th2 cytokines and concomitant suppressed Ig production. It has been suggested that Th2 cells are required for the induction and maintenance of this tolerogenic state. In this study, we show that oral tolerance induction abrogates subsequent Th2-driven Ag-specific IgE and IgG1 responses, while intranasal tolerance induction only blocks the production of IgE, but not IgG1. Consistent with suppressed IgE serum levels, elevated IFN-gamma production was observed in the spleens of tolerized mice. Moreover, both oral and intranasal tolerance induction were found to inhibit intestinal mast cell responses upon subsequent priming and intragastric provocation. Transfer of total splenocytes or purified CD4+, but not CD8+, T cells from intranasally tolerized mice clearly suppressed ongoing Ag-specific IgE, but not IgG1, responses in primed recipients. In addition, coadministration of IFN-gamma-neutralizing Abs completely blocked the transfer of suppression to primed recipients. These results show that Th2 cells can be subjected to tolerance induction, by inducing cross-regulatory, IFN-gamma-producing CD4+ T cells. Moreover, our results point out differences in the regulation of T cell-dependent Ag-specific IgE and IgG1 responses.
Tolerance to a dominant T cell epitope in the acetylcholine receptor molecule induces epitope spread and suppresses murine myasthenia gravis.
Authors: Wu B , Deng C , Goluszko E , Christadoss P
Published: December 2022

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. T cells reactive to a dominant peptide alpha 146-162 of acetylcholine receptor (AChR) alpha subunit participate in murine MG pathogenesis. To suppress the autoimmune response to AChR, a high dose of alpha146-162 peptide in IFA was administered parenterally as a tolerogen, after the development of a primary T cell immune response to AChR. This form of AChR T cell peptide tolerance suppressed the in vitro T cell proliferative response to AChR and its dominant alpha146-162 and subdominant alpha182-198 peptides through epitope spread. Administration of alpha146-162 peptide in IFA after the primary immune response to AChR also significantly suppressed the serum anti-AChR Ab of the IgG2b isotype and clinical incidence of MG in C57BL/6 mice. Furthermore, the production of IFN-gamma, IL-2, and IL-10 cytokines by AChR, alpha146-162, and alpha182-198 peptide-reactive cells was suppressed by alpha146-162 peptide tolerance, and the epitope spread observed could be attributed to the reduction in the above cytokine production. Therefore, AChR T cell-dominant peptide tolerance could be adapted in the Ag-specific therapy of MG.
Long-term inhibition of murine lupus by brief simultaneous blockade of the B7/CD28 and CD40/gp39 costimulation pathways.
Authors: Daikh D I , Finck B K , Linsley P S , Hollenbaugh D , Wofsy D
Published: December 2022

Murine lupus in NZB/NZW F1 (B/W) mice can be retarded by sustained administration of CTLA4Ig and by brief treatment early in life with mAb that block CD40/gp39 interactions. We sought to determine whether brief therapy with CTLA4Ig could provide sustained benefit in B/W mice and whether a synergistic effect could be derived by blockade of both the B7/CD28 and the CD40/gp39 pathways. We found that a short course of CTLA4Ig at the onset of disease produced only short-term benefit. However, when CTLA4Ig was combined with anti-gp39, there was long-lasting inhibition of autoantibody production and renal disease. Ten months after the 2-wk course of therapy, 70% of these mice were alive, compared with only 18% and 0% of those that received only anti-gp39 or CTLA4Ig, respectively. These findings demonstrate that brief simultaneous blockade of the B7/CD28 and CD40/gp39 costimulation pathways can produce benefit that lasts long after treatment has been discontinued.
The SH3 domain of Itk/Emt binds to proline-rich sequences in the cytoplasmic domain of the T cell costimulatory receptor CD28.
Authors: Marengère L E , Okkenhaug K , Clavreul A , Couez D , Gibson S , Mills G B , Mak T W , Rottapel R
Published: December 2022

Engagement of the transmembrane receptor CD28 potentiates T cell survival, proliferation, and activation. The biochemical basis by which CD28 controls these outcomes is unclear, although early events following cross-linking of the receptor are characterized by tyrosine phosphorylation of CD28 and other cellular substrates. We demonstrate that following CD28 ligation, a CD28-associated tyrosine kinase activity is increased in parallel to activation of the T cell-specific tyrosine kinase Itk (Itk/Emt), while Lck and Fyn kinase activities are not increased. We show that Itk forms an inducible complex with CD28, mediated by the SH3 domain of Itk and the diproline motifs of CD28. Site-directed mutagenesis of the N-terminal diproline motif of CD28 abrogates the association of CD28 with the SH3 domain of Itk, while mutations within the C-terminal diproline motif have little effect. Peptides corresponding to the N-terminal diproline motif were more efficient at abrogating the interaction between CD28 and the SH3 domain of Itk, than peptides corresponding to the C-terminal diproline motif. In addition, peptides corresponding to the N-terminal diproline motif of CD28 activated the tyrosine kinase activity of Itk to levels similar to those observed following Ab-mediated cross-linking of CD28. Together, our data show that the SH3 domain of Itk binds to a proline-rich motif within the cytoplasmic tail of CD28, and define a mechanism by which CD28 couples to and activates a downstream tyrosine kinase.
Partial activation of naive CD4 T cells and tolerance induction in response to peptide presented by resting B cells.
Authors: Croft M , Joseph S B , Miner K T
Published: December 2022

Tolerance is thought to occur when Ag is presented to T cells in the absence of costimulatory interactions from APC accessory molecules. Of the professional APC, the resting B cell may be the main tolerizing cell in vivo. We have analyzed several aspects of activation of naive transgenic CD4 cells stimulated with resting or activated B cells presenting peptide Ag. Similar results were obtained with stimulation from peptide presenting fibroblast APC lacking or expressing B7-1 with intracellular adhesion molecule-1. TCR ligation with little or no accessory molecule coreceptor engagement induced efficient blastogenesis; up-regulation of CD25, CD44, CD69, CD95 and CD71; and down-regulation of CD62L over a 48-h period. Accessory molecule help enhanced the expression of CD25, CD44, CD69, and CD71, but to very modest degrees. Only two molecules, CD40 ligand and IL-2, were found to be extremely dependent on accessory molecule help, with little or no expression evident with peptide presented on resting B cells or class II-positive fibroblasts. T cells induced on resting B cells expanded minimally over 3 days, and this was followed by extensive cell death and hyporesponsiveness of the resulting cells. These studies suggest that under tolerizing conditions, such as Ag presentation by resting B cells, much of the naive CD4 response is induced efficiently. Partial activation, however, may be the overall result due to the lack of CD40 ligand expression, which may regulate costimulatory activity in APC and, in turn, may contribute to limiting the production of IL-2 required for T cell expansion and survival.
Gamma delta T cells of human early pregnancy decidua: evidence for local proliferation, phenotypic heterogeneity, and extrathymic differentiation.
Authors: Mincheva-Nilsson L , Kling M , Hammarström S , Nagaeva O , Sundqvist K G , Hammarström M L , Baranov V
Published: December 2022

The uterine mucosa in pregnancy, the decidua, allows placenta formation and survival of the fetus despite the fact that it is semiallogeneic. Decidua contains large numbers of lymphocytes, of which CD56+ cells dominate, followed by T cells expressing either alpha beta or gamma delta TCR. We have investigated the developmental relationship between the CD56- and TCR gamma delta-expressing cells in early pregnancy decidua using dual labeling immunoelectron microscopy, immunoflow cytometry, and cell fractionation. Lymphocyte subpopulations were, in addition, analyzed for expression of the cytokine receptor for IL-7 and c-kit and for mRNA expression of recombinase-activating genes 1 and 2. Four different cell populations could be distinguished: CD56+bright, CD56+dim/TCR gamma delta+low, CD56+dim/TCR gamma delta+high, and TCR gamma delta+low. Recombinase-activating genes 1 and 2 were expressed in the CD56+bright cells and to a limited degree in CD56+dim/TCR gamma delta+low cells. c-kit was preferentially expressed on the CD56+bright cells, while IL-7R was preferentially expressed on CD56+dim/TCR gamma delta+low and CD56+dim/TCR gamma delta+high cells. The CD56+dim TCR gamma delta+low and CD56+dim/TCR gamma delta+high cells displayed the characteristic morphology of large granular lymphocytes, while single positive TCR gamma delta+low cells were usually smaller and did not contain cytoplasmic granules. The gamma delta 1 gene segment was almost exclusively used in the TCR. Gamma delta T cells in mitosis were seen. We suggest that human early pregnancy decidua is a transient site for extrathymic maturation and that the progenitors of TCR gamma delta+ cells are bone marrow-derived immature cells expressing the CD56 (neural cell adhesion molecule) homing receptor.
Cooperation of binding sites for STAT6 and NF kappa B/rel in the IL-4-induced up-regulation of the human IgE germline promoter.
Authors: Messner B , Stütz A M , Albrecht B , Peiritsch S , Woisetschläger M
Published: December 2022

Ig heavy chain class switching is directed by cytokines inducing transcription from unrearranged CH genes. Subsequently, such primed cells can undergo switch recombination to express the selected new isotype. In the case of IgE class switching, IL-4 activates the IgE germline promoter by inducing the interaction of the transcription factor STAT6 (IL-4STAT) with a responsive DNA element in the proximal region of the promoter. This study describes the characterization of two additional cis-acting elements that interact with members of the NF kappa B/rel transcription factor family in an IL-4-independent fashion. Electrophoretic mobility shift assays show that the nucleoprotein complex formed on the upstream site (NF kappa B1) contains the classical p50/p65 heterodimer. The complex on the proximal site (NF kappa B2) appears to be composed of p50 and relB. IgE germline promoter reporter gene constructs carrying point mutations in the NF kappa B2 site were largely unresponsive to IL-4 stimulation in transient transfection experiments, while plasmids with similar mutations in the NF kappa B1 site responded to cytokine stimulation better than the wild-type promoter. The NF kappa B2 effect was dependent on the presence of the STAT6 binding site, demonstrating that the NF kappa B2 motif is necessary but not sufficient for mediating cytokine up-regulation. In addition, the combination of a NF kappa B/rel binding site and the STAT6 response element conferred IL-4 inducibility to a heterologous minimal promoter, while the individual sites had no effect. The available data suggest that the NF kappa B2 nucleoprotein complex may cooperate with DNA-bound STAT6 to achieve IL-4-dependent activation of the human IgE germline gene.
Abbreviated junctional sequences impoverish antibody diversity in urodele amphibians.
Authors: Patel H M , Hsu E
Published: December 2022

Of the six complementarity-determining regions (CDR) forming the structure of the Ab combining site, CDR3 of heavy chain is the most variable in length and sequence. Diversity of this loop is determined by the number of gene segments involved, extent of addition to or deletion from the joining genes, and imprecision of the site of recombination. In neonatal mice and Xenopus tadpoles, the last two factors occur less frequently than in adults, which in tadpoles result in low affinity Ab responses that do not mature. In contrast, adult urodele amphibians make larval-like responses and are notorious for lifelong poor immunocompetence. The mechanism for this is not known, and in this study we cloned germline VH genes from the axolotl and obtained rearrangements to these VH gene segments by reverse-transcriptase PCR. These sequences were analyzed for heavy chain junctional diversity and found to be even less variable than that in newborn mouse or Xenopus tadpoles, although for different reasons. Only 29% of the CDR3 loop in the axolotl consisted of somatically generated sequences, compared with 44% in tadpole, 39% in newborn mice, and 57% in both adult mice and Xenopus. This distinguishing feature of axolotl CDR3 results not only from shorter junctional sequences, but also unusually extensive integration of germline JH sequence. As the CDR3 loop is the most important portion of the Ig sequence for determining Ab combining site diversity, our data provide the molecular basis for a contributing factor in the deficient urodele amphibian Ab responses.
Induction of protective CTL responses against the Plasmodium yoelii circumsporozoite protein by immunization with peptides.
Authors: Franke E D , Corradin G , Hoffman S L
Published: December 2022

To determine the optimum combination, concentration, and formulation of synthetic peptides and adjuvants to induce protective CTL responses against the Plasmodium yoelii circumsporozoite protein (PyCSP), BALB/c mice were immunized with linear and multiple antigen peptides (MAP) including PyCSP CTL and Th epitopes in Montanide's ISA51, Lipofectin, and Lipofectamine. An H-2K(d)-restricted PyCSP CTL epitope, SYVPSAEQI (amino acids (aa) 280-288), recognized by protective CTL clones, was included in the following peptides: a 9-aa linear peptide (SYVPSAEQI; PyCSP9), a 20-aa linear peptide (aa 280-299; SYVPSAEQILEFVKQISSL; PyCSP20), a MAP containing four branches of PyCSP20 (MAP(280-299)), and a linear peptide and a MAP(MAP(280-299)p2p30) in which PyCSP20 was colinearly synthesized with two universal Th epitopes from tetanus toxin (p2p30). A MAP containing the PyCSP Th epitope (aa 57-70; KIYNRNIVNRLLGD) was included in some experiments. The highest specific lytic activity against peptide-pulsed target cells was obtained with splenocytes from mice immunized with three doses at 3-wk intervals of MAP(280-299)p2p30 in Lipofectin or Lipofectamine. Forty percent of the mice immunized with MAP(280-299)p2p30 and Lipofectin were protected against sporozoite challenge. Immunization with CTL and Th epitopes co-linearly synthesized in a MAP induced significantly better CTL than did immunization with the same sequence as a linear peptide, or immunization with a mixture of two individual MAPs, one with the CTL epitope and the second with the Th epitope.
Human tryptase fibrinogenolysis is optimal at acidic pH and generates anticoagulant fragments in the presence of the anti-tryptase monoclonal antibody B12.
Authors: Ren S , Lawson A E , Carr M , Baumgarten C M , Schwartz L B
Published: December 2022

Human tryptase is uniquely regulated by its association with heparin and resists inhibition by biological protease inhibitors. The effects of pH and B12, an IgG anti-tryptase mAb, on cleavage of the synthetic substrate tosyl-Gly-Pro-Lys-p-nitroanilide and of the biological substrate fibrinogen by tryptase were examined. Tosyl-Gly-Pro-Lys-pnitroanilide cleavage was optimal at neutral pH and was inhibited by the B12 mAb at acidic and neutral pH values. At pH 7.5, inhibition was reversible and noncompetitive. In contrast, the optimal pH for tryptase to cleave fibrinogen was acidic. B12 dramatically enhanced the rate and extent that tryptase cleaved all three fibrinogen subunits at pH 6.0 to 6.5, but inhibited these activities at neutral pH. Major fibrinogen cleavage fragments generated at acidic pH by the B12:tryptase complex were identical with those made by plasmin. Thus, at acid pH, tryptase alone destroyed the ability of fibrinogen to clot, while the B12:tryptase complex increased the rate of fibrinogenolysis and also generated the anticoagulant, fragment D. The acidic pH optimum for tryptase fibrinogenolysis may direct this activity to tissue sites of inflammation. A putative biological equivalent to B12 would limit tryptase fibrinogenolytic activity at sites of neutral pH, such as blood, but would augment activity at acidic sites.