bluuebunny/crossref_metadata_2025 huggingface dataset:
Complement Opsonization Is Required for Presentation of Immune Complexes by Resting Peripheral Blood B Cells
Authors: Boackle Susan A. , Morris Margaret A. , Holers V. Michael , Karp David R.
Published: December 2022

Complement receptor 2 (CD21, CR2) is a B cell receptor for complement degradation products bound to Ag or immune complexes. The role of CD21 in mediating Ag presentation of soluble immune complexes by resting B cells was studied. Complement-coated immune complexes were formed by the incubation of influenza virus with serum from immune donors. These complexes bound to peripheral blood B cells in a complement-dependent manner. The binding required CD21 or, to a lesser extent, complement receptor 1 (CR1, CD35). B cells pulsed with immune complexes containing complement elicited a response from a panel of influenza-specific T cell clones, while those pulsed with immune complexes formed in the absence of complement did not. The expression of the early activation marker CD69 and the costimulatory molecule CD86 were not induced by CD21 ligation alone, suggesting that CD21-mediated Ag presentation occurs independently of B cell activation. Up-regulation of these markers required exposure to T cell factors elicited by the recognition of Ag derived from complement-containing immune complexes. These findings suggest that binding of Ag to CD21 enables Ag-nonspecific B cells to participate in the activation of Ag-specific T cells in a process that occurs independently of well-characterized B cell activation events.
Coreceptor-Independent T Cell Activation in Mice Expressing MHC Class II Molecules Mutated in the CD4 Binding Domain
Authors: Mostaghel Elahe A. , Riberdy Janice M. , Steeber Douglas A. , Doyle Carolyn
Published: December 2022

We have previously reported that efficient selection of the mature CD4+ T cell repertoire requires a functional interaction between the CD4 coreceptor on the developing thymocyte and the MHC class II molecule on the thymic epithelium. Mice expressing a class II protein carrying the EA137/VA142 double mutation in the CD4 binding domain develop fewer than one-third the number of CD4+ T cells found in wild-type mice. In this report we describe the functional characteristics of this population of CD4+ T cells. CD4+ T cells that develop under these conditions are predicted to be a CD4-independent subset of T cells, bearing TCRs of sufficient affinity for the class II ligand to undergo selection despite the absence of accessory class II-CD4 interactions. We show that CD4+ T cells from the class II mutant mice are indeed CD4 independent in their peripheral activation requirements. Surprisingly, we find that CD4+ T cells from the class II mutant mice, having been selected in the absence of a productive class II-CD4 interaction, fail to functionally engage CD4 even when subsequently provided with a wild-type class II ligand. Nevertheless, CD4+ T cells from EA137/VA142 class II mutant mice can respond to T-dependent Ags and support Ig isotype switching.
CBFα3 (AML2) Is Induced by TGF-β1 to Bind and Activate the Mouse Germline Ig α Promoter
Authors: Shi Meng-Jiao , Stavnezer Janet
Published: December 2022

TGF-β1 directs class switching to IgA by splenic B cells and by the surface IgM+ B cell line, I.29μ, by inducing germline (GL) Ig α transcripts. The promoter segment between −130 and +46, relative to the first initiation site for mouse GL α transcripts, is sufficient for expression and TGF-β1 inducibility of a reporter gene in B cell lines. Within this segment resides a TGF-β1-responsive element (TβRE) that is required for induction of the promoter by TGF-β1 and, when multimerized, is sufficient to transfer TGF-β1 inducibility to another promoter. In this report we show that a TGF-β1-inducible complex binds the TβRE and contains the transcription factor core-binding factor (CBF; also known as acute myeloid leukemia, AML). Although all three CBFα family members activate the GL α promoter, only CBFα3 (AML-2) is induced by TGF-β1 in splenic B and I.29μ cells. The TβRE contains two CBF binding sites. Mutation of both sites reduces but does not eliminate induction of the GL α promoter by TGF-β1 or by overexpression of CBF, possibly due to the presence of an additional CBF site in the promoter. In addition, the TβRE contains two copies of another sequence motif. Mutation of these motifs eliminates TGF-β1 induction of the GL α promoter. Together the data indicate that TGF-β1 induction of the α promoter involves induction of CBFα3, which binds to the TβRE of the promoter along with one or more proteins.
MRL/lprand MRL+/+ Macrophage DNA Synthesis in the Absence and the Presence of Colony-Stimulating Factor-1 and Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Hamilton John A. , Chan James , Byrne Robert J. , Bischof Robert J. , Jaworowski Anthony , Kanagasundaram Varuni
Published: December 2022

Macrophage accumulation and proliferation as well as altered macrophage properties have been observed in autoimmune MRL mice. To determine whether there might be innate differences in the proliferative responses, we examined the DNA synthesis responses of peritoneal macrophages and macrophages derived in vitro from bone marrow precursors (bone marrow-derived macrophages (BMM)). Murine peritoneal exudate macrophages normally require the addition of macrophage CSF (CSF-1) to enter cell cycle in vitro. In contrast, we have found that many thioglycollate-induced adherent peritoneal macrophages, but not resident peritoneal macrophages, from both MRL/lpr and MRL+/+ mice atypically underwent DNA synthesis even in the absence of added CSF-1. They also responded very well to granulocyte-macrophage CSF. These findings may help to explain the appearance of increased macrophage numbers in MRL lesions. In contrast to a previous report, it was found that MRL/lpr and MRL+/+ BMM did not have an enhanced response to CSF-1 and that modulation of CSF-1 receptor expression was not more rapid in MRL BMM. We also found no evidence for abnormal CSF-1 internalization and degradation or for the lpr mutation to have any enhanced effect on BMM survival in the absence of CSF-1. TNF-α lowered the DNA synthesis response to CSF-1 of MRL/lpr BMM rather than enhanced it, as has been reported. Our data suggest that the enhanced accumulation of macrophages in the MRL/lpr kidney cannot be explained by a proposed model of enhanced responsiveness of MRL/lpr BMM to CSF-1, including a contribution by TNF-α.
Interaction of Very Late Antigen-4 with VCAM-1 Supports Transendothelial Chemotaxis of Monocytes by Facilitating Lateral Migration
Authors: Weber Christian , Springer Timothy A.
Published: December 2022

The transient regulation of very late antigen (VLA)-4 avidity by CC chemokines may promote chemotaxis of monocytes across VCAM-1-bearing barriers, whereas late and prolonged activation of VLA-5 may mediate subsequent localization in the extracellular matrix. We demonstrate that interactions of VLA-4 with VCAM-1, fibronectin, or a 40-kDa fragment but not a 120-kDa fragment of fibronectin supported the lateral random migration of isolated blood monocytes induced by CC chemokines, termed chemokinesis. This effect was optimal at intermediate substrate concentrations. Moreover, coimmobilization of VCAM-1 with ICAM-1 allowed better migration than ICAM-1 alone. Chemokinesis on VCAM-1 appeared to be associated with transient regulation of VLA-4 avidity by CC chemokines, given that locking VLA-4 in a high avidity state markedly inhibited migration and the locomotion rate was inversely correlated with the adhesive strength of VLA-4 to VCAM-1 following stimulation with monocyte chemoattractant protein-1. Induction of VCAM-1 expression by endothelial activation with IL-4 improved chemokinesis and lateral migration toward a monocyte chemoattractant protein-1 or a monocyte inflammatory protein-1α gradient on endothelium and increased transendothelial chemotaxis of monocytes by a VLA-4-dependent mechanism. In contrast, endothelial activation with IL-4 did not affect the time required for diapedesis of monocytes itself. Hence, VCAM-1 may facilitate transendothelial chemotaxis by supporting lateral migration of attached monocytes along endothelium.
Lymphotoxin α3 Induces Chemokines and Adhesion Molecules: Insight into the Role of LTα in Inflammation and Lymphoid Organ Development
Authors: Cuff Carolyn A. , Schwartz Jessica , Bergman Cheryl M. , Russell Kerry S. , Bender Jeffrey R. , Ruddle Nancy H.
Published: December 2022

Lymphotoxin (LT) plays an important role in inflammation and lymphoid organ development, though the mechanisms by which it promotes these processes are poorly understood. Toward this end, the biologic activities of a recently generated recombinant murine (m) LTα preparation were evaluated. This cytokine preparation was effective at inducing cytotoxicity of WEHI target cells with 50% maximal killing observed with 1.2 ng/ml. mLTα also induced the expression of inflammatory mediators in the murine endothelial cell line bEnd.3. rmLTα induced expression of the adhesion molecules VCAM, ICAM, E-selectin, and the mucosal addressin cellular adhesion molecule, MAdCAM-1. When mLTα, human (h) LTα, and mTNF-α were compared, mLTα was the most potent inducer of MAdCAM-1. None of these cytokines induced the peripheral node addressin, PNAd. mLTα also induced expression of the chemokines RANTES, IFN-inducible protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1). mRNA levels peaked 4 h following treatment with mLTα and declined through the 24-h treatment period. LTα also induced chemokine protein within 8 h of treatment, which increased through the 24-h treatment period. These data demonstrate that the proinflammatory effects of LTα3 may be mediated in part through the induction of adhesion molecule and chemokine expression. Further, LTα3 may promote development of lymphoid tissue through induction of chemokines and the mucosal addressin MAdCAM-1. These data confirm previous observations in transgenic and knockout mice that LTα3 in the absence of LTβ carries out unique biologic activities.
Antineutrophil Cytoplasmic Antibodies Preferentially Engage FcγRIIIb on Human Neutrophils
Authors: Kocher Markus , Edberg Jeffrey C. , Fleit Howard B. , Kimberly Robert P.
Published: December 2022

Antineutrophil cytoplasmic Abs (ANCA) are found in the circulation of many patients with systemic vasculitis. ANCA bind to ANCA target, such as proteinase 3 and myeloperoxidase, and activate neutrophils in an FcγR-dependent manner. Human neutrophils constitutively express FcγRIIa (CD32) and FcγRIIIb (CD16), and there is clear in vitro experimental evidence of ANCA-mediated engagement of FcγRIIa. However, direct experimental evidence of ANCA engagement of neutrophil FcγRIIIb has been obscured by technical problems related to activation-induced receptor shedding and activation-induced expression of receptor on the surface of neutrophils. In this study, by blocking receptor shedding and using appropriate reporter anti-FcγR mAb, we show that human cANCA and pANCA, and murine mAb with corresponding reactivities, can indeed engage FcγRIIIb. Furthermore, our data suggest that FcγRIIIb is preferentially engaged by ANCA relative to FcγRIIa presumably due to the nearly 10-fold excess of FcγRIIIb expression relative to FcγRIIa expression. These results clearly demonstrate that the Fc region of ANCA bound to an ANCA target on the neutrophil surface engage FcγRIIIb and indicate that FcγRIIIb and FcγRIIa may both be active participants in ANCA-induced neutrophil activation. However, given the low levels of ANCA target expression on neutrophils from patients with systemic vasculitis, FcγRIIIb is likely to play a critical role in initiating and perpetuating ANCA-induced neutrophil activation.
The Role of Adhesion Molecules in Human Leukocyte Attachment to Porcine Vascular Endothelium: Implications for Xenotransplantation
Authors: Robinson Lisa A. , Tu LiLi , Steeber Douglas A. , Preis Ori , Platt Jeffrey L. , Tedder Thomas F.
Published: December 2022

Many obstacles still prevent successful xenotransplantation of porcine donor organs. When hyperacute rejection is averted, transplanted pig organs are subject to acute vascular and cellular rejection. In autologous systems, leukocyte recruitment into inflamed tissues involves selectins, integrins, and Ig family members. To determine whether these mechanisms allow human leukocytes to effectively enter porcine grafts, the pathways by which human leukocytes adhere to TNF-α-stimulated porcine aortic endothelium were examined under static and physiologic flow conditions. L-selectin and E-selectin had overlapping functions in neutrophil capture and rolling, whereas Ab blockade of E-selectin and the β2 integrins inhibited firm arrest of rolling neutrophils. Combined blockade of selectins and β2 integrins resulted in negligible human neutrophil attachment to pig endothelium. Lymphocyte attachment to porcine endothelium was primarily L-selectin mediated, whereas β2 integrin and VCAM-1/very late Ag-4 (VLA-4) interactions promoted static adhesion. Concurrent β2 integrin, VLA-4, VCAM-1, and L-selectin blockade completely inhibited lymphocyte attachment. Thus, interactions between leukocyte-endothelial cell adhesion receptor pairs remained remarkably intact across the human-porcine species barrier. Moreover, disrupting the adhesion cascade may impair the ability of human leukocytes to infiltrate a transplanted porcine organ during rejection.
Immunostimulatory DNA Sequences Inhibit IL-5, Eosinophilic Inflammation, and Airway Hyperresponsiveness in Mice
Authors: Broide David , Schwarze Jurgan , Tighe Helen , Gifford Tim , Nguyen Minh-Duc , Malek Siamak , Van Uden John , Martin-Orozco Elena , Gelfand Erwin W. , Raz Eyal
Published: December 2022

We have used a mouse model of allergen-induced airway hyperresponsiveness to demonstrate that immunostimulatory DNA sequences (ISS) containing a CpG DNA motif significantly inhibit airway eosinophilia and reduce responsiveness to inhaled methacholine. ISS not only inhibited eosinophilia of the airway (by 93%) and lung parenchyma (91%), but also significantly inhibited blood eosinophilia (86%), suggesting that ISS was exerting a significant effect on the bone marrow production of eosinophils. The inhibition of the bone marrow production of eosinophils by 58% was associated with a significant inhibition of T cell-derived cytokine generation (IL-5, granulocyte-macrophage CSF, and IL-3). ISS exerted this inhibitory effect on T cell cytokine production indirectly by stimulating monocytes/macrophages and NK cells to generate IL-12 and IFNs. The onset of the ISS effect on reducing the number of tissue eosinophils was both immediate (within 1 day of administration) and sustained (lasted 6 days), and was not due to ISS directly inducing eosinophil apoptosis. ISS was effective in inhibiting eosinophilic airway inflammation when administered either systemically (i.p.), or mucosally (i.e., intranasally or intratracheally). Interestingly, a single dose of ISS inhibited airway eosinophilia as effectively as daily injections of corticosteroids for 7 days. Moreover, while both ISS and corticosteroids inhibited IL-5 generation, only ISS was able to induce allergen-specific IFN-γ production and redirect the immune system toward a Th1 response. Thus, systemic or mucosal administration of ISS before allergen exposure could provide a novel form of active immunotherapy in allergic diseases.
CD148: A Receptor-Type Protein Tyrosine Phosphatase Involved in the Regulation of Human T Cell Activation
Authors: Tangye Stuart G. , Phillips Joseph H. , Lanier Lewis L. , de Vries Jan E. , Aversa Gregorio
Published: December 2022

Following ligation of the TCR and costimulatory molecules such as CD28, T cells proliferate and secrete cytokines. Several other cell surface molecules have been identified that are capable of augmenting activation mediated via the TCR. These include CD2, CD27, CD40 ligand, and signaling lymphocytic activation molecule. Here, we have characterized the expression and function of CD148, a recently identified receptor-type protein tyrosine phosphatase. CD148 is expressed at low levels on resting T cells, but is up-regulated following in vitro activation. Cross-linking CD148 with immobilized anti-CD148 mAb induced vigorous proliferation of anti-CD3 mAb-activated, highly purified peripheral blood T cells in an IL-2-dependent, cyclosporin A-sensitive manner. This effect was greatest after 8 days of in vitro culture, suggesting that this molecule is involved in the latter stages of a T cell response. CD148-induced proliferation was significantly greater for CD8+ T cells than for CD4+ T cells. Thus, CD148 is a receptor-type protein tyrosine phosphatase involved in the activation of T lymphocytes.
The Tetraspan Protein CD82 Is a Resident of MHC Class II Compartments Where It Associates with HLA-DR, -DM, and -DO Molecules
Authors: Hammond Craig , Denzin Lisa K. , Pan Mary , Griffith Janice M. , Geuze Hans J. , Cresswell Peter
Published: December 2022

In specialized APCs, MHC class II molecules are synthesized in the endoplasmic reticulum and transported through the Golgi apparatus to organelles of the endocytic pathway collectively called MHC class II compartments (MIICs). There, the class II-associated invariant chain is degraded, and peptides derived from internalized Ag bind to empty class II in a reaction that is facilitated by the class II-like molecule HLA-DM. An mAb raised to highly purified, immunoisolated MIICs from human B lymphoblastoid cells recognized CD82, a member of the tetraspan family of integral membrane proteins. Subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy showed that CD82 is highly enriched in MIICs, particularly in their internal membranes. Coprecipitation analysis showed that CD82 associates in MIICs with class II, DM, and HLA-DO (an inhibitor of peptide loading that binds DM). Similar experiments showed CD63, another tetraspan protein found in MIICs, also associates with these molecules in the compartment and that CD82 and CD63 associate with each other. Preclearing experiments demonstrated that both CD82 and CD63 form complexes with DM-associated class II and DM-associated DO. The ability of CD82 and CD63 to form complexes with class II, DM, and DO in MIICs suggests that the tetraspan proteins may play an important role in the late stages of MHC class II maturation.
IL-10 Is Critical in the Regulation of Autoimmune Encephalomyelitis as Demonstrated by Studies of IL-10- and IL-4-Deficient and Transgenic Mice
Authors: Bettelli Estelle , Prabhu Das Mercy , Howard Edward D. , Weiner Howard L. , Sobel Raymond A. , Kuchroo Vijay K.
Published: December 2022

Experimental autoimmune encephalomyelitis (EAE) and other organ-specific autoimmune diseases are induced by autoantigen-specific Th1 cells. In contrast, transfer of autoantigen-reactive Th2 cells that produce IL-4 and IL-10 can prevent and/or reverse EAE. The relative roles of these two Th2 cytokines in the regulation of EAE has not been evaluated. Utilizing IL-4 and IL-10 knockout mice deficient for these cytokines and IL-10 and IL-4 transgenic mice overexpressing these cytokines, we demonstrate that IL-10-deficient mice (IL-10−/−) are more susceptible and develop a more severe EAE when compared with IL-4-deficient mice (IL-4−/−) or wild-type mice. T cells from IL-10−/− mice exhibit a stronger Ag-specific proliferation, produce more proinflammatory cytokines (IFN-γ and TNF-α) when stimulated with an encephalitogenic peptide, and induce very severe EAE upon transfer into wild-type mice. In contrast, while IL-4 transgenic mice develop similar disease compared with their nontransgenic littermates, mice transgenic for IL-10 are completely resistant to the development of EAE. Taken together, our data suggest that IL-10 plays a more critical role in the regulation of EAE by regulating autopathogenic Th1 responses.
Neonatal Murine B Lymphocytes Respond to Polysaccharide Antigens in the Presence of IL-1 and IL-6
Authors: Chelvarajan Ralph L. , Gilbert Nikole L. , Bondada Subbarao
Published: December 2022

Unlike adults, neonates are unable to respond to polysaccharide Ags, making them especially vulnerable to pathogenic encapsulated bacteria. Since the Ab response to polysaccharides in adult mice requires certain cytokines, it was hypothesized that neonatal murine B cells may be competent to respond to such Ags, but may fail to do so due to a deficiency of cytokines. Neonatal splenocyte cultures, which were otherwise unresponsive to trinitrophenyl (TNP)-Ficoll, a haptenated polysaccharide Ag, mounted an adult-like Ab response when supplemented with IL-1. However, IL-1 failed to induce such a response to TNP-Ficoll when purified B cells were used instead. Although IL-6 alone did not induce a response in whole spleen cells or purified B cells from neonates, it synergized with IL-1 in inducing purified neonatal B cells to respond to TNP-Ficoll. The avidity of the cytokine-induced neonatal anti-TNP Abs was comparable to that of Abs made by adult splenocyte cultures. One effect of IL-1 may be at the level of clonal expansion, since it induced neonatal B cells to proliferate in response to anti-IgM, which was further enhanced by IL-6. The spontaneous secretion of IL-1 by neonatal splenocytes was below the detection limit, while adult splenocytes secreted 30.8 ± 5.2 U/ml, which is of the same order of magnitude as what was required to stimulate neonatal B cells to respond to TNP-Ficoll. Thus, the neonatal unresponsiveness to polysaccharide Ags could be due to the inability of a non-B cell population resident in the neonatal spleen to secrete sufficient quantities of IL-1.
Expression and Function of CTLA-4 in Th1 and Th2 Cells
Authors: Alegre Maria-Luisa , Shiels Helena , Thompson Craig B. , Gajewski Thomas F.
Published: December 2022

CTLA-4 is expressed on T cells after activation and shares homology with the CD28 costimulatory receptor. In contrast to CD28, CTLA-4 is thought to be a negative regulator of T cell activation. Cross-linking of CTLA-4 during activation of peripheral T cells reduces IL-2 production and arrests T cells in G1. Much less is known about the function of CTLA-4 in differentiated T cells. We have investigated the expression and function of CTLA-4 in established Th1 and Th2 clones and in bulk populations of Th1 and Th2 cells freshly derived in vitro from TCR transgenic splenocytes. We found that CTLA-4 was induced under similar conditions and with similar kinetics following activation of both Th1 and Th2 clones. However, CTLA-4 expression was much higher in Th2 than Th1 clones and lines. This was confirmed by flow cytometry, confocal microscopy, and Northern blot analysis. The ratio of surface to intracellular expression of CTLA-4 and its rate of endocytosis were similar in Th1 and Th2 clones. Inhibition of binding of CTLA-4 to its ligands using soluble anti-CTLA-4 mAb during stimulation with Ag increased the production not only of IL-2 by Th1 clones, but also that of IL-3 and IFN-γ by Th1 clones and of IL-3, IL-4, IL-5, and IL-10 by Th2 clones. In contrast, when anti-CTLA-4 was coimmobilized with anti-CD3 and anti-CD28 mAbs, a decrease in the production of multiple cytokines was observed. We conclude that CTLA-4 can function to suppress the production of cytokines produced by both Th1 and Th2 cells.
Caspase-Independent Cell Death Induced by Anti-CD2 or Staurosporine in Activated Human Peripheral T Lymphocytes
Authors: Déas Olivier , Dumont Céline , MacFarlane Marion , Rouleau Matthieu , Hebib Chafika , Harper Francis , Hirsch François , Charpentier Bernard , Cohen Gerald M. , Senik Anna
Published: December 2022

We examined the effects of the cell-permeable, broad spectrum peptide caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD.fmk), and BOC-Asp(OMe)-fluoromethyl ketone (BOC-D.fmk), on apoptosis induced by anti-CD2, anti-Fas, and the protein kinase inhibitor staurosporine in activated human peripheral T lymphocytes. We monitored ultrastructural, flow cytometric, and biochemical apoptotic changes, including externalization of phosphatidylserine, cleavage of poly(ADP-ribose) polymerase (PARP) and lamins, activation of caspase-3 and caspase-7, decrease in mitochondrial membrane potential, and DNA fragmentation. Z-VAD.fmk and BOC-D.fmk completely inhibited all the biochemical and ultrastructural changes of apoptosis in anti-Fas-treated cells. In marked contrast, neither Z-VAD.fmk nor BOC-D.fmk inhibited CD2- or staurosporine-mediated cell shrinkage, dilatation of the endoplasmic reticulum (seen in anti-CD2-treated cells), externalization of phosphatidylserine, and loss of mitochondrial membrane potential that accompanied cell death. However, these inhibitors did inhibit the cleavage of PARP and lamins and the formation of hypodiploid cells, and partially inhibited chromatin condensation. These results demonstrate that in activated T cells, anti-CD2 and staurosporine induce a caspase-independent cell death pathway that exhibits prominent cytoplasmic features of apoptosis. However, caspase activation is required for the proteolytic degradation of nuclear substrates such as PARP and lamins together with the DNA fragmentation and extreme chromatin condensation that occur in apoptotic cells.
γ3 Gene-Disrupted Mice Selectively Deficient in the Dominant IgG Subclass Made to Bacterial Polysaccharides Undergo Normal Isotype Switching After Immunization with Polysaccharide-Protein Conjugate Vaccines
Authors: Shapiro David A. , Threadgill Deborah S. , Copfer M. Janna , Corey Deborah A. , McCool Tera L. , McCormick Laura L. , Magnuson Terry R. , Greenspan Neil S. , Schreiber John R.
Published: December 2022

Bacterial polysaccharides (PS) are T-independent type 2 Ags that elicit restricted Ab responses of IgM and IgG3 in mice and IgM and predominantly IgG2 in humans. Immunodeficiency in the dominant IgG subclass made to PS is associated with chronic sinus and pulmonary infections with PS-encapsulated bacteria. To elucidate the biologic role of the dominant IgG subclass in the immune response to PS and to make an animal model of human IgG subclass deficiency, we generated mice with a targeted disruption of the exon encoding the CH1 domain of the γ3 heavy-chain constant region gene. Homozygotes had no detectable serum IgG3, and their splenocytes did not produce IgG3 after LPS stimulation. IgG3−/− mice immunized with PS from Pseudomonas aeruginosa LPS O-side chain or Streptococcus pneumoniae type 19F capsule did not produce any IgG3 anti-PS Abs, in contrast to wild-type mice in which IgG3 was the major IgG subclass. Immunizing both wild-type and IgG3−/− mice with 19F PS-protein conjugate elicited IgG1 Abs. We conclude that IgG3−/− mice have a selective deficiency in the dominant murine IgG subclass made to T-independent type 2 Ags and may be a useful animal model of IgG subclass deficiency. In addition, we show that the anti-PS Ab class switching to IgG1 that occurs when mice are immunized with a PS-protein conjugate vaccine does not require sequential Ig expression or an intact, upstream γ3 heavy-chain gene.
IL-15 Promotes IL-12 Production by Human Monocytes Via T Cell-Dependent Contact and May Contribute to IL-12-Mediated IFN-γ Secretion by CD4+ T Cells in the Absence of TCR Ligation
Authors: Avice Marie-Noëlle , Demeure Christian E. , Delespesse Guy , Rubio Manuel , Armant Myriam , Sarfati Marika
Published: December 2022

At inflammatory sites, the number of activated bystander T cells exceeds that of Ag-activated T cells. We investigated whether IL-15, a monocyte-derived cytokine that shares several biologic activities with IL-2, may contribute to bystander T cell activation in the absence of IL-2 and triggering Ag. The addition of IL-15 to cocultures of monocytes and T cells stimulates CD4+ but not CD8+ T cells to produce IFN-γ. IFN-γ production requires endogenous IL-12, the production of which in turn is dependent upon CD40/CD154 interactions between CD4+ T cells and monocytes. Indeed, non-TCR-activated CD4+ but not CD8+ T cells express significant levels of CD154. IL-15 may enhance IFN-γ in this system by up-regulating CD40 expression on monocytes and IL-12Rβ1 expression on CD4+ T cells. Conversely, using neutralizing anti-IL-15 mAb, we show that the ability of IL-12 to augment IFN-γ secretion is partly mediated by endogenous IL-15. Finally, in the absence of monocytes, a synergistic effect between exogenous IL-12 and IL-15 is necessary to induce IFN-γ production by purified CD4+ T cells, while IL-15 alone induces T cell proliferation. It is proposed that this codependence between IL-12 and IL-15 for the activation of inflammatory T cells may be involved in chronic inflammatory disorders that are dominated by a Th1 response. In such a response, a self-perpetuating cycle of inflammation is set forth, because IL-15-stimulated CD4+ T cells may activate monocytes to release IL-12 that synergizes with IL-15 to induce IL-12 response and IFN-γ production.
Role of Nuclear Factor-κB and Mitogen-Activated Protein Kinase Signaling Pathways in IL-1β-Mediated Induction of α-PDGF Receptor Expression in Rat Pulmonary Myofibroblasts
Authors: Lindroos Pamela M. , Rice Annette B. , Wang Yi-Zhe , Bonner James C.
Published: December 2022

Induction of the α-platelet-derived growth factor receptor (PDGF-Rα) by IL-1β in lung myofibroblasts enhances mitogenic and chemotactic responses to PDGF, and this could be a mechanism of myofibroblast hyperplasia during lung fibrogenesis. Since the regulation of many genes by IL-1β involves activation of NF-κB and mitogen-activated protein (MAP) kinases, we examined these signaling pathways in the control of PDGF-Rα expression by IL-1β in cultured rat lung myofibroblasts. Treatment of cells with pyrrolidine dithiocarbamate (PDTC), an antioxidant that inhibits NF-κB activation, completely blocked PDGF-Rα up-regulation by IL-1β as assayed by [125I]PDGF-AA binding and PDGF-Rα mRNA expression, suggesting a role for NF-κB. However, while IL-1β and TNF-α both induced nuclear binding of the Rel proteins p50 and p65 to an NF-κB consensus oligonucleotide in gel shift assays and caused transient degradation of inhibitor of NF-κB-α (IκB-α) in the cytoplasm of myofibroblasts, only IL-1β up-regulated PDGF-Rα. These results suggest that NF-κB activation alone is not sufficient for up-regulation of PDGF-Rα. An investigation of MAP kinase signaling pathways revealed that IL-1β or PDTC activated extracellular signal-regulated kinase-2 (ERK-2) and c-jun NH2 terminal kinase-1 (JNK-1) phosphorylation of PHAS-1 and c-Jun substrates, respectively. Pretreatment of cells with the MAP kinase kinase-1 (MEK1) inhibitor PD 98059 blocked IL-1β-induced activation of ERK-2 by more than 90% but enhanced IL-1β-stimulated induction of PDGF-Rα expression fourfold. Taken together, these data suggest that IL-1β activates both positive and negative signaling pathways that control the expression of PDGF-Rα. IL-1β appears to mediate its negative effects on PDGF-Rα expression via MAP kinase activation, while the factor(s) that mediate induction of PDGF-Rα remain to be elucidated.
Specific Antagonism of Type I IL-4 Receptor with a Mutated Form of Murine IL-4
Authors: Schnare Markus , Blum Horst , Jüttner Stefan , Röllinghoff Martin , Gessner André
Published: December 2022

IL-4 is a pleiotropic cytokine that is essential for the differentiation of Th2 cells and is critically involved in the pathogenesis of certain infectious and allergic diseases. We have produced and functionally characterized a mutant of murine IL-4 (IL-4.Y119D) as a potential antagonist of IL-4. The analysis of IL-4R binding revealed no differences between wild-type and mutated IL-4. Despite this finding, IL-4.Y119D was unable to induce proliferation of several IL-4-responsive T cell lines mediated via the type I IL-4R (IL-4Rα/common γ chain (γc chain)) and specifically inhibited the proliferative effect of wild-type IL-4. In contrast, with IL-4.Y119D we found induction of MHC class II and CD23 molecules on resting splenic B cells as well as proliferation of B9 plasmocytoma cells. In addition, IL-4.Y119D induced mRNA for soluble IL-4R, leading to the release of soluble IL-4R protein by spleen cells. In macrophages, mutated IL-4 in combination with IFN-γ induced TNF-α-dependent killing of Leishmania major parasites such as wild-type IL-4. The agonistic effects of IL-4.Y119D were observed on cells expressing the IL-13R α-chain, including an IL-13R α-chain transfected T cell line, but were absent in T cells that lack this molecule, indicating that IL-4.Y119D conveys its activity via the type II IL-4R (IL-4Rα/IL-13Rα). The described IL-4 mutant, therefore, represents a new tool to use in dissecting different IL-4 functions that are mediated by either type I or type II IL-4R complexes.
The Molecular and Functional Characterization of a Dominant Minor H Antigen, H60
Authors: Malarkannan Subramaniam , Shih Patty P. , Eden Peter A. , Horng Tiffany , Zuberi Aamir R. , Christianson Greg , Roopenian Derry , Shastri Nilabh
Published: December 2022

Minor histocompatibility (H) Ags elicit T cell responses and thereby cause chronic graft rejection and graft-vs-host disease among MHC identical individuals. Although numerous independent H loci exist in mice of a given MHC haplotype, certain H Ags dominate the immune response and are thus of considerable conceptual and therapeutic importance. To identify these H Ags and their genes, lacZ-inducible CD8+ T cell hybrids were generated by immunizing C57BL/6 (B6) mice with MHC identical BALB.B spleen cells. The cDNA clones encoding the precursor for the antigenic peptide/Kb MHC class I complex were isolated by expression cloning using the BCZ39.84 T cell as a probe. The cDNAs defined a new H locus (termed H60), located on mouse chromosome 10, and encoded a novel protein that contains the naturally processed octapeptide LTFNYRNL (LYL8) presented by the Kb MHC molecule. Southern blot analysis revealed that the H60 locus was polymorphic among the BALB and the B6 strains. However, none of the H60 transcripts expressed in the donor BALB spleen were detected in the host B6 strain. The expression and immunogenicity of the LYL8/Kb complex in BALB.B and CXB recombinant inbred strains strongly suggested that the H60 locus may account for one of the previously described antigenic activity among these strains. The results establish the source of an immunodominant autosomal minor H Ag that, by its differential transcription in the donor vs the host strains, provides a novel peptide/MHC target for host CD8+ T cells.
Therapeutic Preparations of Normal Polyspecific IgG (IVIg) Induce Apoptosis in Human Lymphocytes and Monocytes: A Novel Mechanism of Action of IVIg Involving the Fas Apoptotic Pathway
Authors: Prasad Nagendra K. A. , Papoff Giuliana , Zeuner Ann , Bonnin Emmanuelle , Kazatchkine Michel D. , Ruberti Giovina , Kaveri Srini V.
Published: December 2022

Therapeutic preparations of normal human IgG for i.v. use (IVIg) exhibit a broad spectrum of immunoregulatory activities in vitro and in vivo. IVIg has been shown to inhibit the proliferation of activated B and T lymphocytes and of several autonomously growing cell lines. In this study, we demonstrate that IVIg induces apoptosis in leukemic cells of lymphocyte and monocyte lineage and in CD40-activated normal tonsillar B cells, involving, at least in part, Fas (CD95/APO-1) and activation of caspases. IVIg-induced apoptosis was higher in Fas-sensitive HuT78 cells than in Fas-resistant HuT78.B1 mutant cells, and soluble Fas inhibited IVIg-induced apoptosis. IVIg immunoprecipitated Fas from Fas-expressing transfectants and recognized purified Fas/glutathione-S-transferase fusion proteins upon immunoblotting. Affinity-purified anti-Fas Abs from IVIg induced apoptosis of CEM T cells at a 120-fold lower concentration than unfractionated IVIg. Inhibitors of cysteine proteases of the caspase family, caspase 1 (IL-1β-converting enzyme) and caspase 3 (Yama/CPP32b), partially inhibited IVIg-induced apoptosis of CEM cells. Furthermore, cleavage of poly(A)DP-ribose polymerase into an 85-kDa signature death fragment was observed in CEM cells following IVIg treatment. Thus, normal IgG induces apoptosis in lymphocytes and monocytes. Our results provide evidence for a role of Fas, bring new insights into the mechanisms of action of IVIg in autoimmune diseases, and suggest a role of normal Ig in controlling cell death and proliferation.
Cutting Edge: Anti-CD1 Monoclonal Antibody Treatment Reverses the Production Patterns of TGF-β2 and Th1 Cytokines and Ameliorates Listeriosis in Mice
Authors: Szalay Gudrun , Ladel Christoph H. , Blum Carmen , Brossay Laurent , Kronenberg Mitchell , Kaufmann Stefan H. E.
Published: December 2022

Protection against intracellular bacteria by T cells is regulated by Ag-presenting molecules, which comprise classical MHC class I molecules, MHC class II molecules, and nonclassical MHC class Ib molecules. The role of CD1 molecules, which are structurally similar to classical MHC class I gene products, but less polymorphic, is not understood so far. We show that CD1 surface expression increased on APC in Listeria-infected mice. The in vivo treatment with anti-CD1 mAb reduced TGF-β2 levels and concomitantly increased secretion of the proinflammatory cytokine TNF, the Th1 cell promoting cytokine IL-12, and the Th1 cell cytokine IFN-γ at the onset of listerial infection. These findings point to a regulatory role of CD1-reactive cells in the immune response against listeriosis.
Cutting Edge: Silencing Virus-Specific Cytotoxic T Cell-Mediated Immune Recognition by Differential Splicing: A Novel Implication of RNA Processing for Antigen Presentation
Authors: Kienzle Norbert , Sculley Tom B. , Greco Sonia , Khanna Rajiv
Published: December 2022

Persistent viruses have developed potent strategies to overcome host immune defenses. In particular, viral interference with Ag presentation by HLA class I molecules can effectively impair the host’s CTL function. Here we provide evidence for a novel aspect of differential splicing on endogenous processing of a latent viral transcript resulting in dominant protein isoforms from which the CTL determinant has been deleted. Consequently, virus-infected cells expressing these isoforms were poorly recognized by CTLs. Molecular analysis revealed that this splicing significantly reduced expression of the viral transcript encoding the relevant epitope to levels below the threshold required for CTL recognition. The importance of splicing was further reinforced by the observation of efficient CTL recognition of target cells expressing a truncated viral transcript that abolished differential splicing. Thus, differential splicing, which is a common mechanism of gene regulation in many pathogens, may unexpectedly interfere with immune recognition.
Enhancing or Suppressive Effects of Antibodies on Processing of a Pathogenic T Cell Epitope in Thyroglobulin
Authors: Dai Yang , Carayanniotis Karen A. , Eliades Petros , Lymberi Peggy , Shepherd Philip , Kong Yi-chi M. , Carayanniotis George
Published: December 2022

Thyroglobulin (Tg)-specific Abs occur commonly in thyroid disease, but it is not clear to what extent they affect Tg processing and presentation to T cells. Here we show that generation of the nondominant pathogenic Tg epitope (2549–2560), containing thyroxine (T4) at position 2553 (T4(2553)), is augmented by Tg-specific IgG mAbs that facilitate FcR-mediated internalization of Tg. However, other mAbs of the same (IgG1) subclass enhanced Tg uptake by APC but had no effect on the generation of this peptide. Treatment of APC with chloroquine or glutaraldehyde abrogated enhanced generation of T4(2553). The boosting effect was selective, since the enhancing mAbs did not facilitate generation of the neighboring cryptic (2495–2511) peptide, which is also pathogenic in mice. When Tg was simultaneously complexed to a mAb reactive with T4(2553) and to a mixture of boosting mAbs, the presentation of this epitope was totally suppressed. These results suggest that Tg-specific Abs alter Tg processing and may boost or suppress the presentation of nondominant pathogenic determinants during the course of disease.
Protein Kinase C Activation Inhibits Tyrosine Phosphorylation of Cbl and Its Recruitment of Src Homology 2 Domain-Containing Proteins
Authors: Liu Yuhong , Liu Yun-Cai , Meller Nahum , Giampa Leslie , Elly Chris , Doyle Melissa , Altman Amnon
Published: December 2022

One of the major proteins that is rapidly tyrosine phosphorylated upon stimulation of the TCR/CD3 complex is the 120-kDa product of the c-cbl protooncogene (Cbl). Upon activation, tyrosine-phosphorylated Cbl interacts with the Src homology 2 (SH2) domains of several signaling proteins, e.g., phosphatidylinositol 3-kinase (PI3-K) and CrkL. In the present study, we report that pretreatment of Jurkat T cells with PMA reduced the anti-CD3-induced tyrosine phosphorylation of Cbl and, consequently, its activation-dependent association with PI3-K and CrkL. A specific protein kinase C (PKC) inhibitor (GF-109203X) reversed the effect of PMA on tyrosine phosphorylation of Cbl and restored the activation-dependent association of Cbl with PI3-K and CrkL. We also provide evidence that PKCα and PKCθ can physically associate with Cbl and are able to phosphorylate it in vitro and in vivo. Furthermore, a serine-rich motif at the C terminus of Cbl, which is critical for PMA-induced 14-3-3 binding, is also phosphorylated by PKCα and PKCθ in vitro. These results suggest that, by regulating tyrosine and serine phosphorylation of Cbl, PKC is able to control the association of Cbl with signaling intermediates, such as SH2 domain-containing proteins and 14-3-3 proteins, which may consequently result in the modulation of its function.
Crosstalk Between Keratinocytes and T Lymphocytes via Fas/Fas Ligand Interaction: Modulation by Cytokines
Authors: Arnold Ralf , Seifert Martina , Asadullah Khusru , Volk Hans Dieter
Published: December 2022

Apoptosis mediated by Fas/FasL interaction plays an important role during many inflammatory skin disorders. To estimate whether the expression of FasL, the ligand for Fas, might be regulated by cytokines we stimulated primary human keratinocytes with several pro- and anti-inflammatory cytokines. Keratinocytes cultured to subconfluence expressed FasL constitutively. Cells stimulated with the proinflammatory cytokines IL-1β, TNF-α, IFN-γ, and IL-15, respectively, increased significantly their intracellular as well as cell surface-bound FasL expression in a time- and dose-dependent manner. This cytokine-induced FasL expression was dependent on new protein synthesis. Despite enhanced expression of cell surface-bound FasL, no release of soluble FasL was measured in the cell supernatants determined by ELISA. Stimulation of the cells with IL-6, IL-10, IL-12, TGF-β1, and GM-CSF did not modulate the constitutive FasL expression, but IFN-γ-mediated FasL up-regulation was significantly diminished by IL-10 and TGF-β1, respectively. Up-regulation of FasL on IFN-γ-stimulated keratinocytes led to increased apoptosis within monolayers cultured for 48 h. Moreover, coculture experiments performed with Fas+ Jurkat T cells revealed that enhanced FasL expression on IFN-γ-stimulated keratinocytes induced apoptosis in cocultured T cells, demonstrating that up-regulated FasL was functionally active. In summary, our data suggest the important regulatory role of cytokine-controlled Fas/FasL interaction in the cross-talk between keratinocytes and skin-infiltrating T cells for maintenance of homeostasis in inflammatory skin processes.
Single Amino Acid Replacements in an Antigenic Peptide Are Sufficient to Alter the TCR Vβ Repertoire of the Responding CD8+ Cytotoxic Lymphocyte Population
Authors: Kalergis Alexis M. , Ono Toshiro , Wang Fuming , DiLorenzo Teresa P. , Honda Shinichiro , Nathenson Stanley G.
Published: December 2022

Cytotoxic CD8+ T lymphocytes are activated upon the engagement of their Ag-specific receptors by MHC class I molecules loaded with peptides 8–11 amino acids long. T cell responses triggered by certain antigenic peptides are restricted to a limited number of TCR Vβ elements. The precise role of the peptide in causing this restricted TCR Vβ expansion in vivo remains unclear. To address this issue, we immunized C57BL/6 mice with the immunodominant peptide of the vesicular stomatitis virus (VSV) and several peptide variants carrying single substitutions at TCR-contact residues. We observed the expansion of a limited set of TCR Vβ elements responding to each peptide variant. To focus our analysis solely on the TCR β-chain, we created a transgenic mouse expressing exclusively the TCR α-chain from a VSV peptide-specific CD8+ T cell clone. These mice showed an even more restricted TCR Vβ usage consequent to peptide immunization. However, in both C57BL/6 and TCRα transgenic mice, single amino acid replacements in TCR-contact residues of the VSV peptide could alter the TCR Vβ usage of the responding CD8+ T lymphocytes. These results provide in vivo evidence for an interaction between the antigenic peptide and the germline-encoded complementarity-determining region-β loops that can influence the selection of the responding TCR repertoire. Furthermore, only replacements at residues near the C terminus of the peptide were able to alter the TCR Vβ usage, which is consistent with the notion that the TCR β-chain interacts in vivo preferentially with this region of the MHC/peptide complex.
Tumor Cell Surface Expression of Granulocyte-Macrophage Colony-Stimulating Factor Elicits Antitumor Immunity and Protects from Tumor Challenge in the P815 Mouse Mastocytoma Tumor Model
Authors: Soo Hoo William , Lundeen Katherine A. , Kohrumel Joshua R. , Pham Nhat-Long , Brostoff Steven W. , Bartholomew Richard M. , Carlo Dennis J.
Published: December 2022

A novel membrane-bound form of GM-CSF (mbGM-CSF) was expressed on the surface of the mouse mastocytoma cell line P815 to target tumor cell-associated Ags to epidermal Langerhans cells. Transfected clones stimulated the proliferation of syngeneic bone marrow cells, indicating that mbGM-CSF is biologically active. We evaluated the in vivo effects of mbGM-CSF by comparing the growth of mbGM-CSF cells (termed 1D6.1E5) to that of wild-type P815 cells in DBA/2 mice. The growth rates of tumors initiated by P815 and 1D6.1E5 were similar until day 12, after which P815 tumors grew to large sizes while 1D6.1E5 tumors were rejected. In contrast, the growth of both tumors was unimpeded when injected into nude mice, suggesting that a T cell-dependent antitumor response was induced by 1D6.1E5 in normal mice. Lymphocytes from 1D6.1E5-vaccinated mice were able to kill 51Cr-labeled P815 cells in a dose-dependent fashion that was inhibited by anti-CD8 Abs, suggesting that the antitumor response involved CD8+ CTL. We then tested whether vaccination with these cells would elicit a protective antitumor response by injecting mice with either irradiated 1D6.1E5 or P815 cells and challenging them with nonirradiated P815 cells. 1D6.1E5-treated mice grew small tumors that soon disappeared in all animals. In contrast, the majority of animals receiving the irradiated wild-type tumor vaccine grew large tumors, and 50% died. These data demonstrate that mbGM-CSF expressed on the surface of tumor cells is biologically active and elicits protective antitumor immunity.