bluuebunny/crossref_metadata_2025 huggingface dataset:
IgH gene rearrangements on the unexpressed allele in rabbit B cells.
Authors: Tunyaplin C , Knight K L
Published: December 2022

VDJ genes are assembled in two sequential DNA recombination steps, rearrangement of D and J(H) gene segments followed by rearrangement of a V(H) gene segment. In mouse and human, VDJ gene rearrangements occur in a regulated step in which D rearranges to J(H) on both alleles before V(H) rearranges to DJ. To determine how VDJ gene rearrangements are regulated in the rabbit, we studied the IgH gene rearrangements in rabbit B cells by examining the gene rearrangement on the unexpressed allele in rabbit hybridomas. By Southern blot analysis with probes from the J(H) and 3'V(H)1 regions, we found that approximately 50% of the hybridomas had DJ gene rearrangements on the unexpressed allele, whereas the remaining 50% did not rearrange either J(H) or V(H) genes. Unexpectedly, we found a VD gene rearrangement on the unexpressed allele of a rabbit B cell line, so we PCR-amplified the VD gene rearrangements from splenocyte DNA and found a few VD gene rearrangements in normal B cells as well. The data taken together show that IgH gene rearrangements in rabbit B cells are regulated differently than those in the mouse, and we hypothesize that in some B cells, VDJ gene rearrangements proceed through a VD intermediate rather than through a DJ intermediate.
Structurally similar TCRs differ in their efficiency of positive selection.
Authors: Yelon D , Berg L J
Published: December 2022

Studies of TCR transgenic mice have demonstrated that in these systems positive selection is not an efficient process. The capacity of the thymus to produce mature T cells is limited, even when all immature thymocytes express appropriate Ag receptors. Analysis of TCR transgenic mice expressing reduced levels of MHC molecules have shown that MHC surface density can be a limiting factor during development. Whether peptide availability in the thymus also limits the efficiency of positive selection remains controversial. Here, we examine the efficiency of positive selection in three similar lines of TCR transgenic mice, all of which express V alpha11/beta3+ TCRs specific for cytochrome c peptides bound to I-Ek. We demonstrate that thymocytes expressing these similar TCRs mature with very different efficiencies in H-2k mice. Furthermore, efficient positive selection of thymocytes expressing these three TCRs varies in its dependence on MHC density. These data suggest that similar TCRs can differ in their degree of specificity for peptide/MHC complexes during positive selection; some TCRs may require specific complexes for selection while other TCRs are more promiscuous in their thymic interactions. Alternatively, these three TCRs may differ in their affinities for positively selecting ligands.
Perforin, Fas/Fas ligand, and TNF-alpha pathways as specific and bystander killing mechanisms of hepatitis C virus-specific human CTL.
Authors: Ando K , Hiroishi K , Kaneko T , Moriyama T , Muto Y , Kayagaki N , Yagita H , Okumura K , Imawari M
Published: December 2022

In chronic hepatitis C, Fas expression is up-regulated in the hepatocytes, especially near liver-infiltrating lymphocytes, and Fas ligand is expressed on the lymphocytes. The presence of hepatitis C virus (HCV)-specific CTLs has been demonstrated both in peripheral blood and among liver-infiltrating lymphocytes of patients with chronic hepatitis C. We studied the killing mechanisms of HCV-specific human CTLs using target cells that were sensitive or resistant to agonistic anti-Fas Abs and TNF-alpha. We show that HCV-specific CTL clones kill non-Ag-bearing bystander cells as well as Ag-bearing cells, although the bystander killing is less efficient than the specific target cell killing, and the efficacy of the bystander killing of anti-Fas- and soluble TNF-alpha-sensitive cells is greater than that of resistant cells. We also show that the killing of Ag-presenting, sensitive cells is mediated by Fas ligand and TNF-alpha as well as perforin, although the latter plays a major role in the killing at a low E:T ratio, and that the killing of sensitive bystander cells is primarily mediated by Fas ligand and TNF-alpha on CTLs expressed upon specific Ag stimulation, which may be relevant to the bystander lysis by HCV-specific CTLs of uninfected hepatocytes, in which Fas expression is up-regulated. Activated CTLs also kill bystander cells by the perforin-based mechanism, although it requires a high E:T ratio. The effective bystander killing requires a close intercellular contact between CTLs and target cells, although TNF-alpha released from the CTLs mediates lysis of the bystander cells without a close cell-cell contact.
Role of CD19 tyrosine 391 in synergistic activation of B lymphocytes by coligation of CD19 and membrane Ig.
Authors: Li X , Sandoval D , Freeberg L , Carter R H
Published: December 2022

CD19 and CD21, which form a complex on B lymphocytes, are required for normal Ab responses to T-dependent Ags. Coligation of the CD21/CD19 complex with membrane IgM (mIgM) powerfully enhances B cell activation in vitro and in vivo. To determine how CD19-mIgM synergy is produced, we examined immediate and downstream signaling events after ligation of either complex alone or after CD19-mIgM coligation in normal and lymphoblastoid B cells. Ligation of mIgM alone is known to result in tyrosine phosphorylation of CD19 and association of CD19 with phosphatidylinositol 3-kinase and Vav, and these events are not enhanced by coligation. In contrast, tyrosine phosphorylation of phosphatidylinositol 3-kinase and Vav is markedly enhanced after CD19-mIgM coligation. Coligation also results in synergistic, prolonged enhancement of mitogen-activated protein kinase activity, relative to ligation of either receptor complex alone. Mutation of CD19 Y391, the site at which Vav binds, but not mutation of CD19 Y482 and Y513, the sites of association with phosphatidylinositol 3-kinase, blocks the enhancement of mitogen-activated protein kinase activation. These findings suggest that the synergistic enhancement of B cell activation following CD19-mIgM coligation results from greater tyrosine phosphorylation of Vav and Vav-dependent enhanced activation of mitogen-activated protein kinases.
IFN-gamma-deficient mice develop experimental autoimmune uveitis in the context of a deviant effector response.
Authors: Jones L S , Rizzo L V , Agarwal R K , Tarrant T K , Chan C C , Wiggert B , Caspi R R
Published: December 2022

Experimental autoimmune uveitis (EAU) is a T cell-mediated disease that targets the neural retina and serves as a model of human uveitis. Uveitogenic effector T cells have a Th1-like phenotype (high IFN-gamma, low IL-4), and genetic susceptibility to EAU is associated with an elevated Th1 response. Here we investigate whether the ability to produce IFN-gamma is necessary for the development of EAU by immunizing IFN-gamma-deficient (GKO) mice with the uveitogenic protein interphotoreceptor retinoid binding protein (IRBP) and characterize the associated immunologic responses. GKO mice developed EAU comparable in severity and incidence to that of their wild-type littermates. However, the cytokine profile in their uveitic eyes as well as the cytokines produced by primed lymph node cells in response to IRBP showed a distinct profile: undiminished TNF-alpha and elevated IL-5, IL-6, IL-10, and lymphotoxin (but not IL-4) responses. The inflammatory infiltrate in GKO eyes contained an excess of granulocytes and IL-5- and IL-6-producing cells, but uveitic GKO mice did not up-regulate inducible nitric oxide synthase. GKOs had enhanced lymphocyte proliferation and delayed-type hypersensitivity responses to IRBP. Histology of the delayed-type hypersensitivity lesion in GKO had superimposed elements of an allergic-like response. Anti-IRBP Ab isotypes of GKO mice showed a reduction of IgG2a, but no enhancement of IgG1. Comparison of responses in +/+ and +/- wild-type mice revealed some limited evidence of a gene-dose effect. We conclude that IFN-gamma is not required for priming of pathogenic T cells or for effecting the retinal damage and photoreceptor loss typical of EAU. However, what appears to be a grossly similar disease is caused in the GKO by a deviant type of effector response.
Conformational transitions in CD4 due to complexation with HIV envelope glycoprotein gp120.
Authors: Denisova G , Raviv D , Mondor I , Sattentau Q J , Gershoni J M
Published: December 2022

The binding of the surface envelope glycoprotein gp120 to its receptor, CD4, has been well characterized and is the primary basis for the cell tropism of HIV. In this study, the interaction between recombinant soluble CD4 and native membrane-associated CD4 with gp120 is probed by the use of mAbs. Complexation of gp120 with both forms of CD4 induces conformational epitopes that can be defined with specific mAbs. CG1, CG7, and CG8 are three novel mAbs that have a distinct preference for CD4 complexed over noncomplexed with gp120. The epitopes of these unique mAbs were mapped by cross-inhibition with previously characterized mAbs to a region encompassing the CDR2 and CDR3 loops in domain 1 of CD4. Systematic analysis of CG mAbs binding to CD4 and CD4/gp120 complex delineates a region in the D1 domain of CD4 that undergoes conformational rearrangements upon gp120 binding to its receptor.
Virus-specific IgD in acute viral infection of mice.
Authors: Moskophidis D , Moskophidis M , Löhler J
Published: December 2022

In phylogenetically diverse species with the help of T lymphocytes or soluble factors, viral infections induce the Ag-specific B lymphocytes to proliferate and terminally differentiate into IgM, IgG, IgA, IgD, or IgE Ab-secreting cells. Based on previous studies searching for IgD, it was inferred that serum IgD in the mouse is nearly undetectable, although in other species, e.g., humans, IgD is a measurable component of serum Ig. More recently, new information has been obtained indicating that IgD is secreted in minute quantities during normal B cell differentiation. We observed that IgD is secreted in significantly increased quantities in mice undergoing an acute infection with lymphocytic choriomeningitis virus or vesicular stomatitis virus compared with uninfected animals. A substantial fraction of the observed IgD was found to be virus specific. Using a solid-phase immunoenzymatic technique, virus-specific IgD Ab-forming cells were detected in the spleen; their numerical increase correlated with the level of secreted antiviral IgD. In addition, immunohistochemical staining revealed IgD+ plasma cells that occurred with a similar kinetic profile as the virus-specific IgD Ab-forming cells. These findings provide direct evidence that synthesis of IgD is a physiologic event in the mouse. Its precise function in the immune response to pathogens, however, remains to be determined.
Protection against autoimmune diabetes in mixed bone marrow chimeras: mechanisms involved.
Authors: Mathieu C , Casteels K , Bouillon R , Waer M
Published: December 2022

After bone marrow transplantation, complete nonobese diabetic (NOD) into (NOD x C57Bl/10)F1 chimeras developed insulitis in 100% (7/7) and diabetes in 58% (7/12) of cases. In contrast, when mixed chimerism was induced, a near complete protection against insulitis (3/23, p < 0.0001 vs complete chimeras) and diabetes (0/25, p < 0.001 vs complete chimeras) was achieved even in chimeras with as few as 5% F1 cells. In cotransfer experiments, splenocytes from mixed chimeras failed to prevent diabetes, thus overruling a role for suppressor cells in protection. Whereas splenocytes from complete chimeras transferred diabetes into young irradiated NOD mice in all cases, NOD splenocytes from mixed chimeras only rarely transferred diabetes (3/16, p < 0.01 vs complete chimeras), suggesting that the latter cells had developed tolerance toward beta cells. To achieve this tolerance, the presence of an F1 thymus was not needed. Indeed, whereas splenocytes isolated from complete chimeras were diabetogenic when transferred into neonatally thymectomized irradiated NOD recipients (3/4), they failed to induce diabetes when transferred into neonatally thymectomized irradiated F1 recipients in case again a state of mixed chimerism was induced (0/4, p < 0.001 vs NOD recipients). Finally, a role for non-MHC Ags was demonstrated by experiments in F1 mice crossed between NOD and C57Bl/6, a strain congenic to C57Bl/10. Here protection against disease was only seen in the presence of high levels of F1 cells (>25%). In conclusion, mixed bone marrow chimerism can induce self-tolerance in autoimmunity prone NOD immune cells. This can be achieved extrathymically but may depend on non-MHC Ags and the level of chimerism.
Polymorphism in the alpha 1 helix of the HLA-B heavy chain can have an overriding influence on peptide-binding specificity.
Authors: Barber L D , Percival L , Arnett K L , Gumperz J E , Chen L , Parham P
Published: December 2022

Previously, we reported overlap in the repertoires of peptides endogenously bound by a group of HLA-B allotypes related to HLA-B7. Extending such analysis to four members of the B17 family and seven members of the B15 family shows that allotypes that share sequence identity in the alpha 1 helix of the class I heavy chain possess markedly similar peptide-binding specificities. Members of the B17 family share a preference for peptides with serine, threonine, or alanine at position 2 and aromatic residues at the carboxyl terminus. Strikingly, the presence of a segment of the B17 alpha 1 helix in B*1516 and B*1517 confers the B17-like peptide-binding motif. The strong influence of natural variation in the alpha 1 helix is exemplified by the differences in peptide-binding specificity of B15 allotypes related by conversion events that replaced segments of the alpha 1 helix. In contrast, evolutionary changes that are confined to the alpha 2 domain confer less dramatic change. They do not perturb the primary anchors of the peptide-binding motif but can modulate the specificity through development and diversification of secondary anchors. Our results, in combination with those obtained previously for other HLA-B allotypes, suggest a general trend whereby polymorphism in the alpha 1 helix is the overriding influence on peptide-binding specificity of HLA-B allotypes, while amino acid substitutions in the alpha 2 domain play a more modulatory role.
Purification and characterization of the pig analogue of human membrane cofactor protein (CD46/MCP).
Authors: van den Berg C W , Pérez de la Lastra J M , Llanes D , Morgan B P
Published: December 2022

A panel of mAbs were raised against pig lymphocytes. Seven mAbs immunoprecipitated a 50- to 60-kDa membrane-bound protein. This protein, termed JM4C8-Ag, was expressed on a wide variety of cells, including all circulating cells and cells of fibroblast, epithelial, and endothelial origin. The JM4C8-Ag was transmembrane-anchored and glycosylated. One of the Abs was used in immunoaffinity chromatography to isolate JM4C8-Ag from erythrocyte membranes. N-terminal amino acid analysis through the first 28 residues showed a 43% homology with the human complement regulatory molecule membrane cofactor protein (MCP; CD46). The purified protein had cofactor activity for factor I-mediated cleavage of human and pig C3b, confirming its identity as the pig analogue of human MCP. The purified protein also strongly inhibited lysis of rabbit erythrocytes by human and pig complement after activation of the classical or alternative pathway. This is the first report of a nonprimate analogue of MCP. The presence of a resident MCP on pig cells capable of acting as a cofactor in the control of human complement activation has consequences for the use of pig organs in xenotransplantation.
Aggregation of low affinity IgG receptors induces mast cell adherence to fibronectin: requirement for the common FcR gamma-chain.
Authors: Dastych J , Hardison M C , Metcalfe D D
Published: December 2022

Mast cells have been reported to increase at sites of immune complex-induced inflammation where these cells appear to potentiate the inflammatory response. The mechanism by which mast cells accumulate at these sites is unknown. One possibility is that aggregation of low affinity IgG receptors could signal mast cells to adhere to components of the connective tissue matrix. To test this hypothesis, we first added aggregated IgG to a mast cell adhesion assay employing fibronectin as a matrix component and observed an increase in cell adhesion. Even a small amount of aggregated IgG (< 60 ng/ml) demonstrated by fast protein liquid chromatography in untreated IgG preparations was sufficient to increase mast cell adhesion by 100%. We next explored the Fc gamma receptors involved. Fc gammaRII/III, which are receptors for oligomeric IgG and were first verified as present on these mast cells by FACS analysis and immunoprecipitation, signaled mast cells to rapidly adhere to fibronectin when aggregated with the anti-receptor Ab2.4G2. The adhesion process mediated by Fc gammaRII/III was not associated with beta-hexosaminidase release. Bone marrow-cultured mast cells from common gamma-chain deficient mice, unlike mast cells cultured from +/+ mice, did not respond to Fc gammaRII/III aggregation. This demonstrated requirement for a gamma-chain implicates oligomeric Fc gammaRIII in the adhesion process. Thus, aggregation of Fc gammaRIII on mast cells leads to mast cell adhesion, demonstrating a previously unknown biological function for this receptor on mast cells and providing a mechanism for mast cell accumulation in immune complex-dependent inflammation.
Human anti-nicotinic acetylcholine receptor recombinant Fab fragments isolated from thymus-derived phage display libraries from myasthenia gravis patients reflect predominant specificities in serum and block the action of pathogenic serum antibodies.
Authors: Graus Y F , de Baets M H , Parren P W , Berrih-Aknin S , Wokke J , van Breda Vriesman P J , Burton D R
Published: December 2022

Myasthenia gravis (MG) is a prototype Ab-mediated autoimmune disease in which Abs against nicotinic acetylcholine receptors (AChR) induce loss of functional receptors at the neuromuscular junction. Germinal centers present in MG hyperplastic thymus contain activated B cells spontaneously producing anti-human AChR (anti-huAChR) Ab in vitro. To access the anti-huAChR repertoire, phage display Fab libraries of thymic lymphocytes were constructed from two MG patients. Four Fabs highly specific for huAChR were isolated that bind to determinants in or near the main immunogenic region. These anti-huAChR Fabs showed evidence of significant somatic mutations, supporting the idea that the anti-huAChR Ab response in MG patients is driven by Ag. Two Fabs were able to inhibit up to 90% of donor serum anti-huAChR Abs. Competition with serum anti-huAChR Ab was also observed in unrelated MG patients and indicate that anti-huAChR Fabs bind to epitopes on huAChR recognized by the majority of MG patients. In vitro antigenic modulation studies demonstrated that anti-huAChR Fabs were able to induce AChR loss when cross-linked by an anti-Fab Ab but not as monovalent Fab. Moreover, anti-huAChR Fabs were able to protect against AChR loss by antigenic modulation induced by MG serum Abs, suggesting a potential therapeutic role for these recombinant Fabs in patients with a myasthenic crisis.
Construction and characterization of human CD7-specific single-chain Fv immunotoxins.
Authors: Pauza M E , Doumbia S O , Pennell C A
Published: December 2022

To develop novel therapeutic agents for treatment of human T cell malignancies, we constructed two single-chain Fv (sFv) immunotoxins specific for the T cell-associated Ag CD7. The sFv fragments were derived from the murine hybridomas 3A1e and 3A1f and were expressed as soluble proteins in Escherichia coli. Surface plasmon resonance analyses demonstrated that the purified 3A1e and 3A1f sFv fragments specifically bound CD7 with high affinity, 8.1 and 1.8 nM, respectively. The difference in affinity is chiefly due to a slower dissociation rate for the 3A1f sFv fragment. Despite this difference, both monovalent sFv fragments were comparably internalized by CD7+ human T leukemic cells within 30 min. These data support findings of previous studies suggesting that CD7 internalization does not require cross-linking. The sFv immunotoxins were assembled by linking ricin toxin A chain to the C termini of the sFv fragments via disulfide bonds. Both sFv immunotoxins were comparably potent in their ability to inhibit protein synthesis in vitro in CD7+ Jurkat cells (50% inhibiting concentration = 15 pM). Further preclinical studies on the use of the 3A1e and 3A1f sFv immunotoxins to treat human T cell diseases therefore appear warranted.
Endothelial cell tube formation depends on cadherin 5 and CD31 interactions with filamentous actin.
Authors: Matsumura T , Wolff K , Petzelbauer P
Published: December 2022

Capillary tube formation represents a specialized endothelial cell function and is a prerequisite for the establishment of a continuous vessel lumen. Tube formation is thought to depend on cell-cell adhesion molecules, but whereas cadherins, Ig superfamily proteins, and integrins have been considered as potential candidates, the specific proteins subserving this function are unknown. We identified cadherin 5 and CD31 as two molecules critical for endothelial cell tube formation. Using human dermal microvascular endothelial cells or human umbilical vein endothelial cells for an in vitro capillary tube formation assay or, for comparison, an in vivo wound healing model in SCID mice, we show that tube formation in vivo and in vitro were prevented by adhesion-blocking mAbs to both proteins when used in conjunction but not by either mAb alone. In addition, both mAbs but not each individual mAb blocked cytoplasmic filamentous actin (F-actin) reorganization that resulted in cytoplasmic F-actin clumps similar to those induced by cytochalasin D, suggesting that cadherin 5 and CD31 tune tube formation in concert with F-actin. Indeed, cytochalasin D blocked capillary tube formation but colchicine did not, the latter inhibiting microtubule but not F-actin assembly. Using immunoprecipitation methods, we show for the first time that not only cadherin 5 but also a portion of CD31 connects to beta-catenin, which is part of the adherens junction complex known to be linked to F-actin. In conclusion, we provide evidence that cadherin 5, CD31, beta-catenin, and F-actin shape a functional complex that controls endothelial cell tube formation.
Alternate splicing of mouse fusin/CXC chemokine receptor-4: stromal cell-derived factor-1alpha is a ligand for both CXC chemokine receptor-4 isoforms.
Authors: Heesen M , Berman M A , Höpken U E , Gerard N P , Dorf M E
Published: December 2022

Inspection of the intron splice junction of the mouse chemokine receptor fusin/CXC chemokine receptor R-4 (CXCR-4) revealed a potential in-frame alternative splice site in the region encoding the N-terminal ectodomain of the receptor. Both predicted splice products were detected by reverse transcriptase-PCR. Cell lines of T lymphocyte, B lymphocyte, and macrophage lineage, plus populations of elicited peritoneal exudate cells, thymocytes, and astrocytes coexpressed mRNA for both isoforms. The full length cDNA of the shorter alternate splice product, termed CXCR-4B, was cloned from peritoneal exudate cell RNA. Chinese hamster ovary cells transfected with either isoform, CXCR-4A or CXCR-4B, responded to stromal cell-derived factor-1alpha with a rise in intracellular calcium. Both alternate splice products are therefore functional stromal cell-derived factor-1 alpha receptors.
Human glomerular mesangial cell phagocytosis of apoptotic neutrophils: mediation by a novel CD36-independent vitronectin receptor/thrombospondin recognition mechanism that is uncoupled from chemokine secretion.
Authors: Hughes J , Liu Y , Van Damme J , Savill J
Published: December 2022

In this study we examined the mechanisms by which glomerular mesangial cells ingest apoptotic cells and the mesangial cell response to this event, since there is in vivo evidence that such semiprofessional phagocytes participate in phagocytic clearance of both apoptotic leukocytes and apoptotic resident cells from inflamed glomeruli, thereby promoting resolution of glomerulonephritis. Mesangial cell phagocytosis of apoptotic neutrophils in vitro was not affected by inhibitors of lectin-like receptors, phosphatidylserine receptors, the 61D3 Ag, and beta1 and beta2 integrins, receptors which have been implicated in phagocytosis of apoptotic cells by particular populations of semiprofessional and professional phagocytes. However, the specific inhibitory effects of cationic aminosugars, Arg-Gly-Asp-Ser (RGDS) peptide, and mAbs to phagocyte alpha(v)beta3 vitronectin receptor integrin and "bridging" thrombospondin 1 (TSP1) indicated that mesangial cell phagocytosis of apoptotic cells involved an alpha(v)beta3/TSP mechanism akin to that described for human monocyte-derived macrophages (Mphi) in which Mphi CD36 plays an important role in binding "bridging" TSP1. However, mesangial cells did not express CD36 and there was no evidence for involvement of alternative phagocyte receptors for TSP1, heparan sulfate proteoglycan and sulfatides. Nevertheless, phagocytosis of apoptotic neutrophils by either mesangial cells or Mphi failed to elicit secretion of IL-8 and MCP-1, representatives of each major class of proinflammatory chemotactic cytokines. We conclude that mesangial cell phagocytosis of apoptotic neutrophils involves a novel CD36-independent, alpha(v)beta3/TSP-mediated mechanism that is uncoupled from chemokine secretion, emphasizing the injury-limiting potential of mesangial cell phagocytosis of apoptotic cells.
Outside-in signaling by lipopolysaccharide through a tailless integrin.
Authors: Ingalls R R , Arnaout M A , Golenbock D T
Published: December 2022

Ligand binding to integrins activates intracellular signaling pathways that coordinate and regulate a variety of cellular responses. There is evidence to suggest that the cytoplasmic tails play a key role in several of these signaling events. We sought to determine whether the beta2 integrin complement receptor type 3 (CR3; CD11b/CD18), a receptor for LPS, could initiate an intracellular signal in the absence of its cytoplasmic domains. Expression of full length CR3 in a Chinese hamster ovary-K1 fibroblast line enabled serum-independent translocation of nuclear factor-kappaB in response to binding LPS. Unexpectedly, a cell line expressing a mutated form of CR3 deficient in the cytoplasmic domains was also competent for transmitting a signal in response to LPS. In contrast, phagocytosis of whole Gram-negative bacteria and iC3b-coated erythrocytes took place only with a full length receptor. Thus, while full length CR3 is necessary for productive phagocytic signals, LPS activation does not require the cytoplasmic domains. CR3 may function to activate cells by presenting LPS to a downstream signal transducer.
Activated T helper 1 and T helper 2 cells differentially express the beta-2-adrenergic receptor: a mechanism for selective modulation of T helper 1 cell cytokine production.
Authors: Ramer-Quinn D S , Baker R A , Sanders V M
Published: December 2022

We recently reported that resting clones of murine Th1 cells, but not resting Th2 cells, expressed a detectable level of the beta-2-adrenergic receptor (beta 2AR). In the present study, we proposed that the level of beta 2AR expression on anti-CD3 mAb-activated CD4+ effector Th cells may differ from the level on resting cells, and that a change in receptor expression may alter the functional responsiveness of these cells to either the beta 2AR-selective ligand terbutaline or the sympathetic neurotransmitter norepinephrine. Following anti-CD3 activation, the beta 2AR was expressed on Th1 cells, but not Th2 cells. The number of binding sites on Th1 cells was maintained, with no change in affinity, over a 24-h activation period. When Th clones were exposed to terbutaline following anti-CD3 activation, Th1 cell, but not Th2 cell, cytokine production was modulated. IL-2 production by Th1 cells was decreased, while IFN-gamma production was not significantly altered. The decrease in IL-2 production was concentration dependent and was blocked by an antagonist. In comparison with control supernatants, the lower level of IL-2 present in terbutaline-exposed culture supernatants supported the proliferation of an IL-2-dependent Th1 clone to a lesser degree. Additionally, norepinephrine down-modulates IL-2, but not IFN-gamma, production by binding specifically to the beta-adrenergic receptor. Thus, a detectable level of the beta 2AR is expressed on activated Th1 cells, but not activated Th2 cells, thereby providing a mechanism by which IL-2 production is preferentially modulated by an endogenous and therapeutic ligand following Th1 cell activation.
Mercuric chloride-induced vasculitis in the Brown Norway rat: alpha beta T cell-dependent and -independent phases: role of the mast cell.
Authors: Kiely P D , Pecht I , Oliveira D B
Published: December 2022

Mercuric chloride induces a necrotizing vasculitis in the Brown Norway (BN) rat. This occurs in two phases, between 1 and 5 days (early) and between 12 and 20 days (late) after initiation of HgCl2. One outbred and four inbred rat strains were found to be susceptible to early vasculitis, but only the BN strain developed late vasculitis. In the BN strain, treatment with the mAb R73 (anti-alpha beta TCR) inhibited T cell function, completely prevented the late vasculitis, but had no effect against early vasculitis, indicating that early and late vasculitis is controlled by different genetic and cellular mechanisms. The role of the mast cell in the alpha beta T cell-independent early phase was studied. Serum concentrations of rat mast cell protease II rose following HgCl2 treatment, indicating mast cell degranulation. The reagents Doxantrazole and the mAb G63, which suppress mast cell secretory responses, also prevented the rise in rat mast cell protease II and significantly reduced the early vasculitis. The demonstration of an alpha beta T cell-dependent phase supports previous experimental data that T cells play an important role in the pathogenesis of vasculitis. The presence of an earlier alpha beta T cell-independent phase is a unique observation. The data support a role for the mast cell in the early vasculitis.
Inhibitory effects of chronic ethanol consumption on cellular immune responses to hepatitis C virus core protein are reversed by genetic immunizations augmented with cytokine-expressing plasmids.
Authors: Geissler M , Gesien A , Wands J R
Published: December 2022

Chronic hepatitis C viral (HCV) infection is a major clinical problem in alcoholics with liver disease and may result from ethanol effects on the host immune response. To experimentally assess such effects, the DNA-based immunization approach was used to produce humoral and cellular immune responses against the HCV core protein in mice. Mice were fed an ethanol or isocaloric pair-fed control liquid diet followed by immunizations with HCV core DNA-expressing constructs. Chronic ethanol feeding was found to inhibit Th cell and CTL activities and substantially reduced cytokine secretion as well. In addition, a switch from Th1 to Th0 subtype was observed in proliferating CD4+ T cells derived from chronic ethanol-fed mice. These immunosuppressive effects were directly due to ethanol since crossover experiments to an isocaloric control diet restored the defects in cellular immunity. Furthermore, we determined if coadministration of an IL-2 or GM-CSF DNA expression plasmid with a plasmid expressing the HCV core protein (pHCV2-2) would reverse the inhibitory effects of chronic ethanol feeding on cellular immune responses. Coimmunization of chronic ethanol-fed mice with either IL-2 or GM-CSF expression plasmids restored cellular immunity and induced CD4+ inflammatory T cell and CD8+ CTL responses comparable with control mice immunized with pHCV2-2 alone. These studies provide evidence of how chronic ethanol feeding may effect cellular immune responses to a viral structural protein in the context of genetic immunization.
oct-2 gene disruption eliminates the peritoneal B-1 lymphocyte lineage and attenuates B-2 cell maturation and function.
Authors: Humbert P O , Corcoran L M
Published: December 2022

Targeted mutation of the gene for the Oct-2 transcription factor in mice caused neonatal lethality and abrogated mitogen-induced proliferation and differentiation of mature B lymphocytes in vitro. Here we show that Oct-2 is required for normal humoral responses upon immunization with T cell-dependent as well as T-independent Ags. oct-2-null T cell behavior was normal, implying a B cell-restricted lesion. oct-2-/- B cells displayed aberrant behavior during activation in vitro: both acquisition of markers of cellular activation and cell survival were diminished. Production of early B lineage cells in the bone marrow was normal, yet mature B cells were under-represented in blood and lymphoid organs. Furthermore, peritoneal B-1 lymphocytes were not detected in animals with a reconstituted oct-2-/- lymphoid system. We conclude that Oct-2 is required for B-1 cell maintenance and for normal Ag-driven maturation of conventional B cells in vivo.
Cloning and analysis of the promoter region of CCR5, a coreceptor for HIV-1 entry.
Authors: Moriuchi H , Moriuchi M , Fauci A S
Published: December 2022

The chemokine receptor CCR5 is a cofactor for cellular entry of macrophage-tropic strains of HIV-1. Expression of CCR5 is restricted to T cells, macrophages, and certain cell lines; however, the mechanisms controlling its expression remain largely unknown. To delineate these mechanisms, approximately 1.0 kb of DNA from the immediate 5' upstream region of CCR5 was cloned and characterized. CCR5 promoter activity was up-regulated by PMA, and a region spanning -417 to +61 relative to the transcription start site was sufficient for the basal and induced activity. DNase I footprinting assays demonstrated several protected areas within this region, and gel shift assays determined binding sites for transcriptional factors Oct-1, Oct-2, T cell factor 1alpha, and GATA1. CCR5 promoter activity was also induced by IL-2 or anti-CD3 Ab, while stimulation with anti-CD28 Ab markedly reduced CD3-mediated up-regulation of the CCR5 promoter. Flow cytometry confirmed the findings at the level of cell surface expression. Further delineation of the regulation of the CCR5 promoter will be important for a more comprehensive understanding of the pathogenesis of HIV disease.
Involvement of mitogen-activated protein kinase pathways in the nuclear responses and cytokine production induced by Salmonella typhimurium in cultured intestinal epithelial cells.
Authors: Hobbie S , Chen L M , Davis R J , Galán J E
Published: December 2022

Central to the pathogenesis of Salmonella typhimurium is its ability to engage the host cell in a two-way biochemical interaction. As a consequence of this interaction, a dedicated protein secretion system, termed type III, is activated in these bacteria and directs the translocation of signaling proteins into the host cell. Secretion of these proteins stimulates host cell signal transduction pathways that lead to a variety of cellular responses. An important feature of S. typhimurium pathogenesis is the induction of a profound inflammatory response in the intestinal epithelium. In this report, we show that S. typhimurium induces host cell signal transduction pathways that lead to the activation of the transcription factors NF-kappaB and AP-1, resulting in the production of proinflammatory cytokines such as IL-8. We also show that S. typhimurium infection of cultured intestinal epithelial cells results in the activation of the mitogen-activated protein (MAP) kinases ERK, JNK, and p38. Induction of these signaling pathways and the synthesis of IL-8 was strictly dependent on the function of the invasion-associated type III protein secretion system encoded by S. typhimurium. Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB 203580 prevented S. typhimurium-induced IL-8 production. These results indicate that the inflammatory response induced by S. typhimurium may be due to the specific stimulation of MAP kinase signaling pathways leading to nuclear responses.
C1q augments platelet activation in response to aggregated Ig.
Authors: Peerschke E I , Ghebrehiwet B
Published: December 2022

Immune complexes and aggregated IgG (agg-IgG) induce platelet aggregation and the release reaction. Immune complexes also activate the complement system and interact with the complement component C1q. Since platelets possess both Fc and C1q receptors capable of signal transduction, the present study focused on the interaction between these binding sites and platelet activation. Subaggregating doses of agg-IgG (20-400 microg/ml) were identified for washed platelets from each of 11 healthy donors, and platelet aggregation was monitored in the presence or the absence of increasing concentrations of C1q (5-100 microg/ml). C1q produced a dose-dependent potentiation of platelet alphaIIb/beta3 integrin activation, platelet aggregation, and granule secretion when combined with low doses of agg-IgG. C1q alone was without effect. Maximal enhancement of agg-IgG-induced platelet activation was noted at C1q concentrations ranging from 50 to 100 microg/ml. The observed C1q-induced potentiation of platelet aggregation in response to agg-IgG was blocked by polyclonal antibody F(ab')2 directed against platelet binding sites recognizing the collagen-like domain of C1q (cC1qR) or by mAb Fab (IV.3) directed against platelet FcgammaRII receptors. These data suggest a cooperative interaction between platelet FcgammaRII and cC1q receptors and support a potential role for platelet cC1q receptors in pathologic platelet activation by circulating immune complexes often associated with in vivo thrombosis and thrombocytopenia.
Suppression of collagen-induced arthritis by continuous administration of IL-4.
Authors: Horsfall A C , Butler D M , Marinova L , Warden P J , Williams R O , Maini R N , Feldmann M
Published: December 2022

The onset of collagen-induced arthritis in DBA/1 mice is accompanied by a predominantly Th1 response, characterized by production of the proinflammatory cytokines IFN-gamma and TNF-alpha, and a predominance of IgG2a anti-collagen Abs. This study has primarily addressed the effects of continuous administration of exogenous IL-4, a Th2 cytokine, on collagen-induced arthritis in terms of time of onset, clinical symptoms, and histologic changes compared with those in untreated controls. The contributions of Th1 and Th2 cell responses were studied by examining anti-CII IgG subclasses, serum IgE levels, and cytokine production by synovial membrane and lymph node cell cultures. Continuous exposure to IL-4 for 28 days significantly delayed the onset of arthritis from 19 to 37 days and suppressed clinical symptoms. Arthritis occurred approximately 13 to 24 days after treatment ceased. Thereafter, the severity and duration of clinical symptoms were similar to those in control animals, although both joint damage and inflammation at the histologic and cellular levels were less severe than those in untreated controls. During IL-4 treatment, anti-collagen Ab levels were reduced (most significantly those of the IgG2a subclass), histology scores were lower, and the most striking effect was a 1000-fold decrease in TNF-alpha secretion by synovial cells. No significant differences in IgE levels were found between controls and IL-4-treated mice. These data suggest that the anti-inflammatory properties of IL-4 are mediated in part by down-regulation of Th1 responses rather than up-regulation of Th2 responses.
Ligation of the V7 molecule on T cells blocks anergy induction through a CD28-independent mechanism.
Authors: Soares L R , Rivas A , Tsavaler L , Engleman E G
Published: December 2022

Previous studies have demonstrated that a mAb that recognizes the leukocyte surface Ag V7 inhibits TCR/CD3-dependent T cell activation. In the current study, we demonstrate that in addition to inhibiting T cell proliferation and IL-2 production, anti-V7 blocks tyrosine phosphorylation of TCR/CD3-associated substrates. PMA overcomes this effect, and both PMA and exogenous IL-2 overcome anti-V7-mediated inhibition of T cell proliferation and IL-2 production. T cells stimulated with anti-CD3 in the absence of CD28 or V7 ligation become unresponsive (anergic) to restimulation with anti-CD3; T cells primed in the presence of either anti-V7 or anti-CD28 retain their ability to respond to restimulation with anti-CD3. When T cells are primed in the presence of optimal concentrations of anti-V7 and anti-CD28 Abs, they proliferate normally, indicating that the costimulatory signals generated through CD28 dominate the inhibitory signals generated through V7. However, as the anti-CD28 stimulus is diluted, the V7 effect becomes dominant and proliferation is inhibited. Thus, although both anti-V7 and anti-CD28 Abs prevent anergy, they induce distinct, competing intracellular signals. Wortmannin, which blocks phosphoinositol 3-kinase-dependent signaling, has little effect on V7-mediated inhibition, while herbimycin, an inhibitor of tyrosine kinase, synergizes with anti-V7 to inhibit T cell activation. On the basis of these findings, V7-mediated signals appear to inhibit TCR-dependent tyrosine kinases that are required for IL-2 production and cellular proliferation.
High affinity rheumatoid factor transgenic B cells are eliminated in normal mice.
Authors: Wang H , Shlomchik M J
Published: December 2022

Although systemic autoimmune diseases can be accompanied by multiple autoantibodies, certain specificities are dominant. Presumably, these specificities and their cognate Ags have properties that make them particularly amenable to autoimmune induction. Rheumatoid factors (RFs) are a dominant class of autoantibodies in rheumatoid arthritis and certain other autoimmune syndromes. To study the regulation of RFs in normal and autoimmune animals, we previously created a RF Ig transgenic model based on an RF isolated from an autoimmune MRL/lpr mouse. Using this model, called AM14, we were surprised to find that normal mice do not regulate disease-related RF B cells. This raised the question of whether RFs in general are not susceptible to tolerance induction, perhaps due to the unique properties of serum IgG and its FcRs. Alternatively, RFs can be tolerized, and the disease-related RFs are below the affinity threshold for such tolerance. To distinguish these possibilities, we generated a second RF transgenic model with the same specificity but much higher affinity than AM14. We found that, in contrast to AM14, high affinity RF B cells are subject to central tolerance, showing that there is not an absolute defect in RF B cell tolerance, but, rather, that RF B cell tolerance is affinity dependent even in normal animals. This is also the first model in which a disease-related specificity has been shown clearly to delete in a system in which Ag-positive and negative mice can be produced and compared.
Neutralizing IL-12 during induction of murine acute graft-versus-host disease polarizes the cytokine profile toward a Th2-type alloimmune response and confers long term protection from disease.
Authors: Williamson E , Garside P , Bradley J A , More I A , Mowat A M
Published: December 2022

Injection of parental spleen cells into BDF1 mice results in a graft-vs-host disease (GVHD), the nature of which is critically dependent on the parental haplotype. B6-->BDF1 mice develop a Th1-mediated immunosuppressive lethal GVHD, whereas DBA/2-->BDF1 mice develop a Th2-dependent chronic GVHD, characterized by autoantibody production and glomerulonephritis. In this study we show that neutralizing endogenous IL-12 for a brief period during the initiation of acute GVHD in B6-->BDF1 mice not only confers long term protection from the acute disease, but also permits full repopulation of the recipient with donor B6 lymphocytes. Antibody-treated animals showed normal T cell proliferation in response to Con A stimulation and remained healthy throughout the study. Splenocytes from such mice showed reduced in vitro production of IFN-gamma and enhanced production of IL-5 and IL-10, suggesting a permanent switch from a Th1 to a Th2 cytokine response, comparable to that associated with chronic GVHD in DBA/2-->BDF1 mice. In contrast to DBA/2-->BDF1 mice, however, anti-IL-12-treated B6-->BDF1 mice displayed only mild B cell hyper-responsiveness, as evidenced by a modest increase in serum IgG and IgE levels and moderate levels of anti-dsDNA Abs. Importantly, however, anti-IL-12-treated B6-->BDF1 mice showed no evidence of immune complex-mediated glomerulonephritis. These results demonstrate that neutralizing IL-12 is an effective means of preventing acute GVHD and does not result in the development of chronic GVHD, which might otherwise limit its application.
Protein kinase C alpha-mediated chronic signal transduction for immunosenescence.
Authors: Ohkusu K , Du J , Isobe K I , Yi H , Akhand A A , Kato M , Suzuki H , Hidaka H , Nakashima I
Published: December 2022

In CD2/protein kinase C alpha (PKC alpha)-overexpressing human CD2/rabbit PKC alpha transgenic mice, an aging-dependent increase in PKC alpha expression and a decrease in proliferative responsiveness of splenic T cells were promoted. We found that an aging-associated accumulation of CD44(high) CD45RB(low) memory CD4+ T cells in exchange for CD44(low) CD45RB(high) naive CD4+ T cells was promoted in transgenic mice. A disequilibrium between Ag-dependent generation and subsequent elimination of memory T cells in these mice was shown to underlie this phenomenon. When stimulated with Ag, the PKC alpha transgenic mice responded poorly regarding Ab production and produced cytokines biased for high IFN-gamma/IL-12 and low IL-4/IL-10 levels. These results prove, for the first time, a causal role for chronic signal transduction through PKC alpha in aging-associated immunodysfunction and provide the first animal model for genetically promoted immunosenescence.
Perforin-dependent cytotoxic activity and lymphokine secretion by CD4+ T cells are regulated by CD8+ T cells.
Authors: Williams N S , Engelhard V H
Published: December 2022

Factors influencing the development of CD4+ T cell subpopulations with differing lymphokine profiles are well established. However, CD4+ cells can show both perforin- and Fas ligand-dependent cytotoxicity, and little is known about conditions favoring the development of these effector activities. We now report that CD8+ cells regulate the development of perforin-dependent cytotoxicity in CD4+ cells. CD4+ cells activated in either the presence or absence of CD8+ cells developed Fas ligand-dependent cytotoxic activity. However, CD4+ cells developed perforin-dependent cytotoxicity only in the absence of activated CD8+ cells. CD8+ cells also inhibited the development of IL-4-secreting CD4+ cells; however, there was no correlation between the expression of perforin-dependent cytotoxic activity and the ability to secrete IL-4, and perforin-dependent cytotoxic CD4+ cells represented only 10% of isolated clones. This suggests that the two characteristics are expressed in different CD4+ subsets and might be regulated by distinct effects of the CD8+ cells. In keeping with this, regulation of the lymphokine profile of CD4+ cells by CD8+ cells was consistent with mediation by IFN-gamma, but only when delivered at high concentrations requiring close proximity of the cells. In contrast, regulation of perforin-dependent cytotoxic activity of CD4+ cells by CD8+ cells seemed inconsistent with an IFN-gamma-dependent mechanism, suggesting either direct cell contact or close proximity to allow delivery of an unidentified soluble factor. The characteristics of perforin-dependent CD4+ CTL and their regulation by activated CD8+ cells suggest that they represent a previously unrecognized subpopulation that plays a defensive role when a CD8+ cell response is absent.