bluuebunny/crossref_metadata_2025 huggingface dataset:
Analyses of Single B Cells by Polymerase Chain Reaction Reveal Rearranged VH with Germline Sequences in Spleens of Immunized Adult Rabbits: Implications for B Cell Repertoire Maintenance and Renewal
Authors: Sehgal Devinder , Schiaffella Enrico , Anderson Arthur O. , Mage Rose G.
Published: December 2022

We used PCR to amplify rearranged VHDJHgenes in single cells collected by micromanipulation from splenic germinal centers of immunized adult rabbits. In the course of the study, the objective of which was to analyze diversification of rearranged VHDJH sequences, we were surprised to find cells 7 and 10 days after immunization with rearranged VH1a2 as well as a-negative (y33 and x32) sequences that were identical or close to germline (10 or fewer changes). About 58% (82/140) of the sequences had unique CDR3 regions and were unrelated. In seven different germinal centers, we found one to four different clones with two to seven members. Clonally related cells underwent diversification by hypermutation and gene conversion. We found that contrary to published reports, adult rabbits indeed have newly diversifying B cell receptors in splenic germinal centers. The attractive idea that the rabbit, like the chicken, develops its B cell repertoire early in life and depends upon self-renewing cells in the periphery to maintain its B lymphocyte pool throughout life, is challenged by the current finding. Although a major population of B lymphocytes may be generated early in life, diversified extensively, and maintained by self-renewal in the periphery, some sources of cells with sequences close to germline do exist in adult rabbits and appear in the developing germinal centers. Although considerable repertoire diversity is generated in young rabbits, mechanisms for continued generation of B cell receptor diversity are retained in adult life, where they may confer survival advantage.
The Same Natural Ligand Is Involved in Allorecognition of Multiple HLA-B27 Subtypes by a Single T Cell Clone: Role of Peptide and the MHC Molecule in Alloreactivity
Authors: Paradela Alberto , García-Peydró Marina , Vázquez Jesús , Rognan Didier , López de Castro José A.
Published: December 2022

The human alloreactive CTL clone 27S69, raised against B*2705, cross-reacts with B*2702 and B*2703, but not with B*2701, B*2704, B*2706, or B*2710. Its natural epitope was identified by electrospray/ion trap mass spectrometry, as the proteasome-derived RRFFPYYV octamer. This is the first HLA-B27 ligand shown to be immunogenic in alloreactivity. The RRFFPYYVY nonamer, also found in the B*2705-bound peptide pool, was recognized much less efficiently, demonstrating that an alloreactive CTL distinguishes between very similar natural ligands. Molecular modeling suggested that this was due to the different conformation of each peptide in complex with B*2705. B*2702- and B*2703-RMA-S cells were lysed by CTL 27S69 when sensitized with the octamer, demonstrating that cross-reaction with these subtypes is through recognition of the same peptide as in B*2705. B*2704-, B*2706-, and B*2710-RMA-S cells were not sensitized for lysis, in spite of efficient binding of the octamer, indicating that polymorphism in these subtypes directly impairs allorecognition. B*2701-RMA-S and -C1R cells were sensitized for lysis by the octamer, suggesting lack of the endogenous peptide epitope on this subtype. Absence of the octamer in the B*2701-bound peptide pool further suggested that B*2701 polymorphism impairs the generation of this peptide.
Eliciting T Cell Immunity Against Poorly Immunogenic Tumors by Immunization with Dendritic Cell-Tumor Fusion Vaccines
Authors: Wang Jianli , Saffold Scott , Cao Xuetao , Krauss John , Chen Wei
Published: December 2022

Dendritic cells (DCs) are the most effective APCs and are being studied as natural adjuvants or Ag delivery vehicles to elicit T cell-mediated antitumor immunity. This study examined whether inoculation of DCs fused with poorly immunogenic tumor cells elicited tumor-reactive T cells for adoptive immunotherapy. DCs derived from bone marrow of C57BL/6 (B6) mice were fused with syngeneic B16 melanoma or RMA-S lymphoma cells by polyethylene glycol. The B16/DC and RMA-S/DC fusion hybrids expressed MHC class I, class II Ags, costimulatory molecules, as well as DC-specific and tumor-derived surface markers. The tumor/DC hybrids were capable of processing and presenting tumor-derived Ags, and immunization of B6 mice with irradiated B16/DC or RMA-S/DC vaccine elicited tumor-specific CTL activities. Vaccination of B6 mice with irradiated B16/DC fusion preparations induced partial host protective immunity against B16 tumor challenge. Reduced tumor incidence and prolonged survival time were observed. Adoptive transfer of T cells derived from B16/DC vaccine-primed lymph nodes into B16 tumor-bearing mice greatly reduced the number of established pulmonary metastases with or without in vivo administration of IL-2. Moreover, adoptive transfer of RMA-S/DC vaccine-primed, cultured lymph node T cells eradicated disseminated FBL-3 tumor. The results demonstrate that tumor/DC fusion products are effective cellular vaccines for eliciting T cell-mediated antitumor immunity.
Early IFN-γ Production and Innate Immunity DuringListeria monocytogenesInfection in the Absence of NK Cells
Authors: Andersson Åsa , Dai Wen Juan , Di Santo James P. , Brombacher Frank
Published: December 2022

NK cells are believed to play a mandatory role during the early phases of Listeria monocytogenes infection by producing IFN-γ, which is required for the activation of macrophage effector functions. Mice deficient in the common cytokine receptor γ-chain (γc−/−), which completely lack NK cells, were used to examine whether NK cells were essential for resistance to Listeria infection in vivo. Surprisingly, infected γc−/− mice showed normal innate immunity and macrophage responses against sublethal Listeria infection 2 days postinfection. At this time point, γc−/− mice showed increased blood IFN-γ levels compared with those in noninfected controls, demonstrating an NK-independent source of IFN-γ, which explains early resistance. Listeria-infected γc−/− × recombinase-activating gene-2−/− double-deficient mice were unable to produce IFN-γ and were highly susceptible to L. monocytogenes. Since T cells, but not B cells, are major IFN-γ producers, and γc−/− T cells were found to be efficient IFN-γ producers in vitro, we conclude from these results that T cells functionally replace NK cells for the early IFN-γ production that is necessary for activating the innate immune system following infection with L. monocytogenes. This novel observation in listeriosis underscores how the adaptive immune response can maintain and influence innate immunity.
IL-1 Receptor Accessory Protein Is an Essential Component of the IL-1 Receptor
Authors: Cullinan Emily B. , Kwee Lia , Nunes Perla , Shuster David J. , Ju Grace , McIntyre Kim W. , Chizzonite Richard A. , Labow Mark A.
Published: December 2022

The recently described IL-1R accessory protein (IL-1R AcP) interacts with IL-1β and the IL-1 type-IR (IL-1RI), but an essential requirement for IL-1R AcP in IL-1 signaling in vitro has not been established and its role in vivo has not been examined. In this study, IL-1R AcP-deficient mice and fibroblasts were produced and characterized. All IL-1 agonists bound to IL-1R AcP-deficient cells through the type I IL-1R, but failed to activate gene expression through either the nuclear factor-κB or AP-1-dependent signaling pathways. Absence of IL-1R AcP differentially affected the affinity for IL-1 ligands. IL-1R AcP-deficient fibroblasts bound murine IL-1α and human IL-1R antagonist protein (IL-1Ra) with only moderately reduced affinity when compared with wild-type cells, whereas murine IL-1β affinity was reduced by 70-fold. IL-1 also failed to produce a biologic response in vivo in IL-1R AcP-deficient mice. These data demonstrate that a type I IL-1R/IL-1R AcP complex is required for signaling by all IL-1 agonists and for high affinity binding by IL-1β. Finally, IL-1R AcP is an essential signal transducing component of the functional IL-1R and should represent a novel target for blocking IL-1 function in human disease.
The Role of Leukocyte Emigration and IL-8 on the Development of Lipopolysaccharide-Induced Lung Injury in Rabbits
Authors: Yamamoto Toshihiro , Kajikawa Osamu , Martin Thomas R. , Sharar Sam R. , Harlan John M. , Winn2 Robert K.
Published: December 2022

Leukocyte emigration and alveolar macrophage-derived cytokines may contribute to lung microvascular injury associated with adult respiratory distress syndrome. We have used mAbs against cell adhesion molecules on leukocytes (anti-CD18 and anti-CD49d) or against IL-8 to investigate these contributions. Intratracheal (i.t.) instillation of LPS (50 μg/kg) caused a significant increase in bronchoalveolar lavage polymorphonuclear leukocytes (PMNs) without an increase in mononuclear cells (MNCs) or an increase in lung permeability. Injection of LPS (10 μ/kg) i.v. at 24 h after i.t. LPS caused significant increases in bronchoalveolar lavage PMNs, MNCs, IL-8, and monocyte chemotactic protein-1, as well as increases in lung permeability. Rabbits that were administered i.t. LPS followed by i.v. LPS and treated with anti-CD18 mAb had a significantly lower lung permeability index and emigration of fewer PMNs but no change in MNC emigration compared with saline treatment. Anti-IL-8 mAb treatment resulted in a significantly lower lung permeability index with no change in PMN emigration compared with no treatment. These results suggest that PMN emigration is necessary but not sufficient for the development of LPS-induced lung injury, and that IL-8 plays a significant role in PMN-dependent lung injury, independent of PMN emigration.
Regulation of the High Affinity Receptor for IgE on Human Epidermal Langerhans Cells
Authors: Kraft Stefan , Weßendorf Jörg H. M. , Hanau Daniel , Bieber Thomas
Published: December 2022

Human epidermal Langerhans cells (LC) express variable amounts of the high affinity receptor for IgE (FcεRI); the strongest expression is characteristic of atopic dermatitis. The receptor is suggested to take part in the pathophysiology of this disease by acting as a link between aeroallergens and Ag-specific T cells in an IgE-mediated, delayed-type hypersensitivity reaction. In the present study we show that even in the absence of surface expression, normal LC maintain an intracellular pool of the α-chain of FcεRI (FcεRIα) of the same m.w. as the surface-bound FcεRIα that is able to bind significant amounts of IgE. The lack of surface expression is linked to the absence or very low expression of the γ-chain (FcεRIγ). Moreover, the amount of FcεRIα expressed at the cell surface significantly correlates with the amount of FcεRIγ. LC differentiation toward lymphoid dendritic cells is accompanied by the disappearance of transcripts for FcεRIα, but not for FcεRIγ. This leads to a rapid decrease in the intracellular and surface levels of FcεRIα, which cannot be influenced by IL-4, IgE, or other agents. Overall, our findings suggest that these mechanisms enable LC to be highly versatile APCs by rapidly adapting the surface level of FcεRI to distinct inflammatory environments.
Norepinephrine Accelerates HIV Replication Via Protein Kinase A-Dependent Effects on Cytokine Production
Authors: Cole Steve W. , Korin Yael D. , Fahey John L. , Zack Jerome A.
Published: December 2022

To explore the role of sympathetic nervous system activation in HIV pathogenesis, we examined the effect of the neuroeffector molecule norepinephrine (NE) on HIV-1 replication in quiescently infected PBMCs that were subsequently activated with Abs to CD3 and CD28. NE accelerated HIV-1 replication at concentrations ranging from 10−8 to 10−5 M. This effect could be mimicked by protein kinase A (PKA) activators (forskolin or dibutyryl-cAMP) and abrogated by β-adrenoreceptor antagonists or the PKA inhibitor rp-cAMP, indicating transduction via the adrenoreceptor signaling pathway. NE reduced cellular activation and altered the production of several HIV-modulating cytokines: IL-10 and IFN-γ were markedly suppressed; TNF-α, IL-1β, IL-2, IL-4, and IL-6 were mildly suppressed; and levels of IL-12 were not significantly altered. The addition of either exogenous IFN-γ or IL-10 abrogated the effect of NE on virus production. Thus PKA-dependent suppression of cytokine production appears to mediate the enhancement of HIV-1 replication by NE.
Growth Factor Receptor-Bound Protein 2 (Grb2) Association with Hemopoietic Specific Protein 1: Linkage Between Lck and Grb2
Authors: Takemoto Yoshihiro , Furuta Masaaki , Sato Mitsuru , Findell Paul R. , Ramble Wendy , Hashimoto Yasuhiro
Published: December 2022

To analyze the growth factor receptor-bound protein 2 (Grb2) signaling pathway in lymphoid cells, we used expression cloning to isolate the genes encoding proteins that associate with Grb2. We find that the Src homology 3 domains of Grb2 directly associate, in vitro and in vivo, with murine hemopoietic specific protein 1 (HS1), a protein identical to Lck-binding protein 1. Because HS1 associates with the p56lck and p59lyn tyrosine kinases in vitro and in vivo, and becomes tyrosine phosphorylated upon various receptor stimulations, our present data suggest that HS1 mediates linkage between Lck or Lyn and Grb2 in lymphoid lineage cells.
Qualitative Differences Between Naive and Memory T Cells Make a Major Contribution to the More Rapid and Efficient Memory CD8+ T Cell Response
Authors: Kedl Ross M. , Mescher Matthew F.
Published: December 2022

CD8+ T cells are present at a higher frequency following a primary response, and these memory cells exhibit qualitative differences from naive cells. The importance of these differences vs increased precursor frequency in making a memory response more rapid and efficient has been unclear. Adoptive transfer of 2C TCR transgenic CD8+ T cells into normal recipients, followed by i.p. challenge with allogeneic P815 tumor, results in a long-lived memory population that includes both endogenous host CD8+ T cells and 2C cells. The 2C cells can be identified, using 1B2 mAb specific for the TCR, and thus used as an indicator of the properties of the memory cells. The memory cells have a heterogeneous surface phenotype, and their distribution in lymphoid organs, blood, and peripheral sites is distinct from that of naive cells. Upon rechallenge with Ag, memory cells access the peritoneal cavity much more rapidly than do naive cells (12 h vs 5 days). This appears to result from a requirement for naive cells to interact with Ag before they can efficiently migrate to inflammatory sites, while this is not required for memory cells. In addition, memory cells exhibit some cytolytic activity before rechallenge with Ag, and potent cytolytic activity is present in the peritoneal cavity within 12 h of rechallenge. Comparison of primary and memory responses in mice having similar frequencies of Ag-specific precursors demonstrated that the more rapid migration and the immediate effector function of at least some memory cells contribute very substantially to making a memory response at a peripheral site more rapid and efficient.
Anti-CD40L Accelerates Renal Disease and Adenopathy in MRL-lprMice in Parallel with Decreased Thymocyte Apoptosis
Authors: Russell Jennifer Q. , Mooney Thomas , Cohen Philip L. , MacPherson Bruce , Noelle Randolph J. , Budd Ralph C.
Published: December 2022

The CD40/CD40L (CD40 ligand) axis regulates several interactions between T cells and B cells. Blocking of CD40 engagement by CD40L inhibits Ig class switch by B cells as well as diminishes T cell response to an immunizing Ag. For these reasons, disruption of CD40/CD40L interactions by anti-CD40L administration or by genetic disruption of CD40L has ameliorated a variety of autoimmune conditions. More recent findings suggest that a direct signal can be transmitted to T cells via their expressed CD40L, which can costimulate proliferation with CD3 or promote germinal center formation. It is therefore possible that treatment with anti-CD40L Ab might produce a different outcome than observed in genetically CD40L-deficient mice. In this regard, we observe that in contrast to the genetic deletion of CD40L in MRL-lpr mice, which diminishes autoimmune disease but has little effect on adenopathy, administration of anti-CD40L to MRL-lpr mice accelerates both of these parameters. This difference appears to result from anti-CD40L actively delivering a signal that inhibits T cell apoptosis in lpr mice. This was confirmed by in vitro studies demonstrating that CD40L cross-linking on lpr thymocytes inhibited apoptosis and surface TCR down-modulation induced by CD3 ligation.
The Chemokine Monocyte Chemotactic Protein 1 Triggers Janus Kinase 2 Activation and Tyrosine Phosphorylation of the CCR2B Receptor
Authors: Mellado M. , Rodríguez-Frade J. M. , Aragay A. , del Real G. , Martín A. M. , Vila-Coro A. J. , Serrano A. , Mayor F. , Martínez-A. C.
Published: December 2022

The chemokines are a growing family of low m.w., 70- to 80-residue proinflammatory cytokines that operate by interacting with G protein-coupled receptors. Chemokines are involved in cell migration and in the activation of specific leukocyte subsets. Using the Mono Mac 1 monocytic cell line, we show that monocyte chemotactic protein 1 (MCP-1) triggers activation of the Janus kinase 2 (JAK2)/STAT3 pathway and CCR2 receptor tyrosine phosphorylation. Both Ca2+ mobilization and cell migration are blocked in Mono Mac 1 cells by tyrphostin B42, a specific JAK2 kinase inhibitor. Within seconds of MCP-1 activation, JAK2 phosphorylates CCR2 at the Tyr139 position and promotes JAK2/STAT3 complex association to the receptor. This MCP-1-initiated phosphorylation and association to JAK2 is also observed in CCR2B-transfected HEK293 cells. In contrast, when a CCR2B Tyr139Phe mutant is expressed in HEK293 cells, it is not phosphorylated in tyrosine and triggers neither JAK2/STAT3 activation nor Ca2+ mobilization in response to MCP-1. These results implicate the tyrosine kinase pathway in early chemokine signaling, suggesting a key role for this kinase in later events.
IL-2 and IL-4 Double Knockout Mice Reject Islet Allografts: A Role for Novel T Cell Growth Factors in Allograft Rejection
Authors: Li Xian Chang , Roy-Chaudhury Prabir , Hancock Wayne W. , Manfro Roberto , Zand Martin S. , Li Yongsheng , Zheng Xin Xiao , Nickerson Peter W. , Steiger Jurg , Malek Thomas R. , Strom Terry B.
Published: December 2022

T cell growth factors (TCGFs) play a critical role in allograft rejection by promoting the activation and proliferation of alloreactive T cells. To determine whether IL-2 and IL-4 are of quintessential importance in allograft rejection and to identify possible alternative TCGFs, we have bred IL-2−/− and IL-4−/− double knockout (DKO) mice and studied islet allograft rejection using the DKO mice as allograft recipients. Although mononuclear leukocytes from DKO mice did not mount a proliferative response in vitro in response to anti-CD3 stimulation, crude islet allografts were vigorously rejected by DKO mice (mean survival time 17 ± 7, n = 8) as compared with wild-type controls (mean survival time 13 ± 4, n = 7). Treatment of DKO mice with anti-CD3 or rapamycin markedly prolonged the islet allograft survival. An analysis of intragraft cytokine gene transcripts showed robust expression of IL-7 and IL-15. In contrast, intragraft IL-9 gene transcripts were not detected in either wild-type or DKO mice. Provision of exogenous IL-2, IL-4, IL-7, or IL-15, but not IL-9, supports the proliferation of anti-CD3 activated DKO splenic leukocytes in vitro. Blocking the common γc of IL-2 receptor, a shared essential signaling component by receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, prolonged the survival of islet allografts in DKO mice. Hence, a T cell dependent allograft rejection enabled by rapamycin-sensitive signals or signals mediated by binding of the γc chain occurs in the absence of both IL-2 and IL-4. Non-T cell-derived TCGFs, especially IL-7 and IL-15, may play an active role in supporting allograft rejection.
Resistance to Antigen-Induced Airway Hyperresponsiveness Requires Endogenous Production of IL-12
Authors: Keane-Myers Andrea , Wysocka Maria , Trinchieri Giorgio , Wills-Karp Marsha
Published: December 2022

We have demonstrated previously that susceptibility of murine strains to the development of allergic airway responses is associated with a type 2 cytokine pattern. In the present study, we examine the in vivo role of IL-12 in the immune response to allergen exposure in susceptible (A/J) and resistant (C3H/HeJ, C3H) strains of mice. OVA sensitization and challenge induced significant increases in airway reactivity in A/J mice as compared with their PBS-challenged controls, while no increases in airway reactivity were observed in OVA-challenged C3H mice. OVA exposure of A/J mice resulted in marked increases in the Th2 cytokines, IL-4 and IL-10, in the bronchoalveolar lavage fluid, whereas increases in IFN-γ were observed in C3H mice. Strikingly, anti-IL-12 mAb (1 mg/mouse) treatment resulted in threefold increases in airway reactivity in OVA-challenged resistant C3H mice, concomitant with significant increases in bronchoalveolar lavage levels of Th2 cytokines and decreases in IFN-γ. IL-12 depletion of C3H mice also suppressed OVA-specific serum IgG2a levels and increased both serum OVA-specific IgG1 and IgE levels. Blockade of endogenous IL-12 levels in susceptible A/J mice resulted in further augmentation of type 2 immune responses. These results demonstrate that endogenous production of IL-12 is essential for resistance to Ag-induced airway hyperresponsiveness, and furthermore, that dysregulation of IL-12 production may lead to the development of deleterious type 2 immune responses to inhaled allergens.
The Covalent Binding Reaction of Complement Component C3
Authors: Gadjeva Mihaela , Dodds Alister W. , Taniguchi-Sidle Aiko , Willis Antony C. , Isenman David E. , Law S. K. Alex
Published: December 2022

The covalent binding of C3 to target molecules on the surfaces of pathogens is crucial in most complement-mediated activities. When C3 is activated, the acyl group is transferred from the sulfhydryl of the internal thioester to the hydroxyl group of the acceptor molecule; consequently, C3 is bound to the acceptor surface by an ester bond. It has been determined that the binding reaction of the B isotype of human C4 uses a two-step mechanism. Upon activation, a His residue first attacks the internal thioester to form an acyl-imidazole bond. The freed thiolate anion of the Cys residue of the thioester then acts as a base to catalyze the transfer of the acyl group from the imidazole to the hydroxyl group of the acceptor molecule. In this article, we present results which indicate that this two-step reaction mechanism also occurs in C3.
IL-12 Modulates Expression of Hypersensitivity Pneumonitis
Authors: Gudmundsson Gunnar , Monick Martha M. , Hunninghake Gary W.
Published: December 2022

Hypersensitivity pneumonitis (HP) is a granulomatous, inflammatory lung disease caused by inhalation of organic Ags, most commonly thermophilic actinomycetes. Only a minority of individuals exposed to these Ags develops disease, suggesting that host factors are important for the expression of HP. We compared the expression of HP in a sensitive strain of mice, C57BL/6, and in a resistant strain of mice, DBA/2. They were exposed to the thermophilic bacteria Saccharopolyspora rectivirgula (SR) or to saline alone for 3 consecutive days/week for 3 wk. After exposure to Ag, C57BL/6 mice, but not DBA/2 mice, developed granulomatous inflammation with an increase in lung index (lung weight). Both strains had similar amounts of Ag delivered to the lungs after intranasal installation, as determined with 14C-labeled Ag. Both also had similar increases in total bronchoalveolar cells after Ag exposure, but the C57BL/6 mice had more lymphocytes. Compared with the resistant strain, the sensitive strain had a significantly greater Ag-induced increase in IL-12 and IFN-γ gene expression. DBA/2 mice resembled sensitive, C57BL/6 mice if they received IL-12 augmentation therapy at the time of Ag exposure. These findings were not limited to lung, since both unstimulated and SR-stimulated spleen cells from C57BL/6 mice released significantly more IL-12 than cells from DBA/2 mice. However, spleen cells from DBA/2 mice made more IFN-γ when exposed to IL-12, than cells from C57BL/6 mice. These results suggest that the IL-12 response to Ag may modulate in part the expression of HP.
T Cell Vaccination in Experimental Autoimmune Encephalomyelitis: A Mathematical Model
Authors: Borghans José A. M. , De Boer Rob J. , Sercarz Eli , Kumar Vipin
Published: December 2022

T cell vaccination (TCV) is a method to induce resistance to autoimmune diseases by priming the immune system with autoreactive T cells. This priming evokes an anti-idiotypic regulatory T cell response to the receptors on the autoreactive T cells. Hence resistance is induced. To prevent the inoculated autoreactive cells from inducing autoimmunity, cells are given in a subpathogenic dose or in an attenuated form. We developed a mathematical model to study how the interactions between autoreactive T cells, self epitopes, and regulatory cells can explain TCV. The model is based on detailed data on experimental autoimmune encephalomyelitis, but can be generalized to other autoimmune diseases. We show that all of the phenomena collectively described as TCV occur quite naturally in systems where autoreactive T cells can be controlled by anti-idiotypic regulatory T cells. The essential assumption that we make is that TCV generally involves self epitopes for which T cell tolerance is incomplete. The model predicts a qualitative difference between the two vaccination methods: vaccination with normal autoreactive cells should give rise to a steady state of long lasting protection, whereas vaccination with attenuated cells should only confer transient resistance. Moreover, the model shows how autoimmune relapses can occur naturally without the involvement of T cells arising due to determinant spreading.
TNF Receptor-Associated Factor-3 Signaling Mediates Activation of p38 and Jun N-Terminal Kinase, Cytokine Secretion, and Ig Production Following Ligation of CD40 on Human B Cells
Authors: Grammer Amrie C. , Swantek Jennifer L. , McFarland Richard D. , Miura Yasushi , Geppert Thomas , Lipsky Peter E.
Published: December 2022

CD40 engagement induces a variety of functional outcomes following association with adaptor molecules of the TNF receptor-associated factor (TRAF) family. Whereas TRAF2, -5, and -6 initiate NF-κB activation, the outcomes of TRAF3-initiated signaling are less characterized. To delineate CD40-induced TRAF3-dependent events, Ramos B cells stably transfected with a dominant negative TRAF3 were stimulated with membranes expressing recombinant CD154/CD40 ligand. In the absence of TRAF3 signaling, activation of p38 and control of Ig production were abrogated, whereas Jun N-terminal kinase activation and secretion of IL-10, lymphotoxin-α, and TNF-α were partially blocked. By contrast, induction of apoptosis, activation of NF-κB, generation of granulocyte-macrophage CSF, and up-regulation of CD54, MHC class II, and CD95 were unaffected by the TRAF3 dominant negative. Together, these results indicate that TRAF3 initiates independent signaling pathways via p38 and JNK that are associated with specific functional outcomes.
L-Selectin-Specific Autoantibodies in Murine Lupus: Possible Involvement in Abnormal Homing and Polarization of CD4+ T Cell Subsets
Authors: Hattori Susumu , Nishimura Hiroyuki , Tsurui Hiromichi , Kato Masayuki , Endo Naoki , Abe Masaaki , Akakura Shin , Mitsui Kenichi , Ishikawa Sho , Hirose Sachiko , Shirai Toshikazu
Published: December 2022

One notable functional abnormality in murine and human systemic lupus erythematosus (SLE) is the defect in the production of IL-2 in association with the deficit in naive CD4+ T cells. The mechanism is unknown, but one idea is that naturally occurring autoantibodies with specificities to the naive CD4+ T cell subpopulation are related to this event. We selected hybridoma monoclonal autoantibodies from SLE-prone (New Zealand Black (NZB) × New Zealand White (NZW))F1 mice that reacted with restricted populations of CD4+ T cells. One of these, H32, was specific for L-selectin, as determined by 1) distribution of Ag H32 on lymphoid cells similar to Mel-14, an epitope of L-selectin; 2) shedding of 80-kDa molecules with epitope H32 from the surface of lymph node cells coincidentally with Mel-14, when stimulated with phorbol ester; 3) cross-inhibitory activities on Ag binding between H32 and Mel-14; and 4) reactivity of H32 with recombinant mouse L-selectin. Pretreatment of 51Cr-labeled lymphocytes from BALB/c mice with H32 significantly inhibited their homing to lymph nodes in vivo. The BALB/c splenic H32+ CD4+ T cell subset produced few cytokines except IL-2, thus corresponding to naive ThP-type cells. This subset was markedly selectively depleted in aged (NZB × NZW)F1 mice. There was an age-associated increase in frequencies and titers of anti-L-selectin autoantibodies in sera from (NZB × NZW)F1 mice. Thus, abnormalities of naive CD4+ T cell subset, including IL-2 production in subjects with SLE, are at least partly attributed to the generation of autoantibodies to L-selectin.
Indirect T Cell Allorecognition and Alloantibody-Mediated Rejection of MHC Class I-Disparate Heart Grafts
Authors: Pettigrew Gavin J. , Lovegrove Emma , Bradley J. Andrew , Maclean John , Bolton Eleanor M.
Published: December 2022

Recent studies in the rat have identified a role for T cell-dependent alloantibody in rejection of MHC class I-disparate allografts. RT1Aa-disparate PVG.R8 heart grafts are rejected acutely in naive, and hyperacutely in sensitized, PVG.RT1u recipients by CD4 T cell-dependent alloantibody. Here, we examined the T cell Ag recognition pathways responsible and show that direct injection into skeletal muscle of plasmid DNA, encoding a water-soluble form of the RT1Aa MHC class I heavy chain (pcmu-tAa), stimulates IgG2b cytotoxic alloantibody and markedly accelerates rejection of PVG.R8 heart grafts (median survival time 2 days). pcmu-tAa injection did not induce CTL to Aa, arguing against direct allorecognition of soluble Aa. Treatment with mAbs confirmed that the alloimmune response to pcmu-tAa injection depended on CD4, not CD8, T cells. Priming T cells for indirect allorecognition by injection of 15-mer peptides spanning the α1 and α2 domains of Aa failed to stimulate anti-Aa Ab but caused an accelerated Ab response to a PVG.R8 heart and a modest acceleration in graft rejection (median survival time 4 days). These results suggest that both soluble MHC class I and allopeptides prime CD4 T cells by the indirect pathway, but that soluble class I is a more effective immunogen for humoral alloimmunity because its tertiary protein structure provides B cell epitopes. We propose that priming humoral alloimmunity, like CTL priming, requires recognition of intact MHC on donor cells, but essential T cell help can be provided by CD4 T cells recognizing allogeneic class I exclusively by the indirect pathway.
Th2-Specific Protein/DNA Interactions at the Proximal Nuclear Factor-AT Site Contribute to the Functional Activity of the Human IL-4 Promoter
Authors: Li-Weber Min , Salgame Padmini , Hu Chenggang , Davydov Ilia V. , Laur Oskar , Klevenz Sibylle , Krammer Peter H.
Published: December 2022

IL-4 is a pleiotropic immunoregulatory cytokine secreted by activated Th2, but not Th1, cells. The proximal IL-4 promoter contains MARE, C/EBP, P0, octamer-like, P1, and activating protein-1 elements. The half c-Maf binding site (MARE), P0, and P1 sites were previously shown to be involved in Th2-specific transcriptional activity. Except the MARE and P1 site, the molecular basis for Th2 specificity of the P0 site has not been analyzed. Here, we provide the first detailed analysis of the P0 binding factors and show that in Th2, but not in Th1, cells, NF-AT and proteins of the activating protein-1 family are involved in cooperative binding to the P0 and the adjacent octamer-like site. In the mouse Th2 D10 cells, Oct-1/Oct-2 are also found to participate in formation of the P0-binding complexes. Mutation, deletion, and methylation interference analysis demonstrate that both the P0 and the octamer-like sequence are required for inducible binding. Furthermore, we provide the first report of the functional relevance of each site in the human IL-4 promoter by mutagenesis/transfection analysis and demonstrate that the octamer-like, P0 and P1 sites are important for the biologic function of the IL-4 promoter. The MARE site, although it was shown to be critical for the function of the murine IL-4 promoter, does not appear essential for human IL-4 promoter activity in Jurkat T cells. These findings suggest that besides c-Maf, another Th2-specific factor(s) may be involved in tissue-specific expression of the IL-4 gene.
Control of IL-12 and IFN-γ Production in Response to Live or Dead Bacteria by TNF and Other Factors
Authors: Zhan Yifan , Cheers Christina
Published: December 2022

When mice were infected i.v. with either Listeria monocytogenes or Brucella abortus, bioactive IL-12 was briefly detected in serum and supernatants of spleen homogenates immediately ex vivo. Although the time scale was more prolonged for the more slowly growing B. abortus, in both instances IL-12 production ceased while bacteria still persisted in high numbers. Production of IL-12, detected in serum and spleen, was neither increased nor prolonged by injecting Abs to IL-10 or IL-4. In contrast with live organisms, heat-killed bacteria did not induce detectable IL-12 in vivo and were less efficient when added in vitro to resident peritoneal cells or spleen cells. Mice lacking the receptors for TNF (TNFR−/− mice) were severely deficient in IL-12 production, suggesting a controlling role for TNF, which we have previously shown to be triggered by live, rather than dead, bacteria. Infection in the TNFR−/− mice was exacerbated, although in the Brucella-infected mice splenomegaly, the main indicator of immunopathology, was reduced. Production of NO by macrophages was deficient, but the TNFR−/− mice were not deficient in IFN-γ production. In addition to being poor inducers of IL-12, killed bacteria actively suppressed IL-12 production in response to live bacteria, by mechanism(s) unknown. The implications of these findings are discussed in light of the fact that only live bacteria satisfactorily induce cell-mediated immunity to infection.
IL-10 Regulates Thrombus-Induced Vein Wall Inflammation and Thrombosis
Authors: Downing L. Joseph , Strieter Robert M. , Kadell Amy M. , Wilke Carol A. , Austin John C. , Hare Bradley D. , Burdick Marie D. , Greenfield Lazar J. , Wakefield Thomas W.
Published: December 2022

Vein wall inflammation associated with venous thrombosis is mediated by an imbalance in proinflammatory as compared with antiinflammatory molecules. We hypothesize that IL-10 is an important antiinflammatory cytokine that influences vein wall inflammation and thrombus propagation during venous thrombosis. To test this hypothesis a model of inferior vena caval thrombosis was used. Studies were performed at sacrifice 2 days after thrombus induction and included leukocyte morphometrics, myeloperoxidase activity, vein wall permeability, thrombus weight, and IL-10 ELISA analysis from the vein wall. IL-10 was elevated in the vein wall during venous thrombosis. Neutralization of IL-10 increased inflammation, while supplementation with rIL-10 demonstrated a dose- and time-dependent decrease in inflammation. Interestingly, a low 2.5-μg rIL-10 dose given at time of initiation of thrombosis most significantly decreased inflammation. Thrombus weight was importantly diminished by reconstitution of IL-10. These studies support an important role for IL-10 in the regulation of thrombus-associated inflammation and thrombosis and suggest that IL-10 could be used as a therapeutic agent in the treatment of venous thrombosis.
Inhibition of Collagen-Induced Arthritis in Mice by Viral IL-10 Gene Transfer
Authors: Ma Yuhe , Thornton Sherry , Duwel Laura E. , Boivin Gregory P. , Giannini Edward H. , Leiden Jeffrey M. , Bluestone Jeffrey A. , Hirsch Raphael
Published: December 2022

Autoimmune arthritides are characterized by an imbalance between pro- and anti-inflammatory cytokines. Viral IL-10 (vIL-10) shares many of the anti-inflammatory properties of mouse and human IL-10, but lacks their immunostimulatory properties and may therefore offer superior immunosuppression. Viral IL-10 has a short half-life; however, genetic modification of cells in vivo offers a potential means of achieving prolonged therapeutic titers. To determine the effects on collagen-induced arthritis of vIL-10 gene transfer, DBA/1 mice were administered i.v. or intra-articular injections of Av(vIL-10), a replication-deficient adenovirus encoding vIL-10. The i.v. injection of Av(vIL-10) before disease onset delayed the onset and reduced the severity of collagen-induced arthritis, but treatment of established disease was ineffective. The preventative effects were not due to decreased anti-type II collagen Ab production. Rather, T cells from mice treated with Av(vIL-10) demonstrated a decreased in vitro proliferative response to type II collagen, and a delay was observed in up-regulation of synovial mRNA for the proinflammatory cytokines IL-2 and IL-1β. Intra-articular injection of Av(vIL-10) into knee joints did not reduce arthritis in the knees, but inhibited the development of arthritis in the paws. Humoral and cellular immune responses against Av(vIL-10) were observed. These results demonstrate that vIL-10 can significantly alter the course of autoimmune arthritis and emphasize the complexities of using gene transfer as a method of drug delivery for arthritis.
Reduced Susceptibility to Collagen-Induced Arthritis in Mice Deficient in IFN-γ Receptor
Authors: Kageyama Yasunori , Koide1 Yukio , Yoshida Atsushi , Uchijima Masato , Arai Tomio , Miyamoto Shigehito , Ozeki Takao , Hiyoshi Mitsuru , Kushida Kazuhiro , Inoue Tetsuo
Published: December 2022

Collagen-induced arthritis (CIA) is an arthritic model that was developed after immunization with type II collagen (CII). Apparently, contradictory results have been reported regarding the role of IFN-γ in the development of CIA. Therefore, we employed IFN-γR-deficient mice to study the role of IFN-γ. To introduce the CIA susceptibility gene (H-2q), IFN-γR-deficient (H-2b/b/IFN-γR−/−) mice were mated with DBA/1 (H-2q/q/IFN-γR+/+) mice; next, the F1 mice were interbred to yield F2 offspring bearing different combinations of H-2 (H-2q/q, H-2q/b, and H-2b/b) and IFN-γR (IFN-γR+/+, IFN-γR+/−, and IFN-γR−/−) genes. Although the H-2q allele appeared to confer susceptibility to CIA, mice that were homozygous for the IFN-γR mutation showed a substantially decreased incidence and severity of CIA. The CII-specific IgG levels of serum samples, which are known to be involved in the development of CIA, were remarkably reduced in IFN-γR−/− mice. Furthermore, the anti-CII IgG2a levels controlled by IFN-γR were significantly reduced in IFN-γR−/− F2 mice compared with those seen in IFN-γR+/+ and IFN-γR+/− mice, although the levels of all IgG subclass Abs examined were lower in IFN-γR−/− mice than in IFN-γR+/+ mice. No clear evidence of the imbalance of Th1/Th2 cytokines was observed in CII-immunized, IFN-γR-deficient mice. Taken together, these results suggest that IFN-γ exacerbates CIA by affecting, at least, levels of CII-specific IgG Ab rather than the imbalance of Th1/Th2 cells.
Antagonistic Action of IFN-β and IFN-γ on High Affinity Fcγ Receptor Expression in Healthy Controls and Multiple Sclerosis Patients
Authors: Van Weyenbergh Johan , Lipinski Pawel , Abadie Annie , Chabas Dorothée , Blank Ulrich , Liblau Roland , Wietzerbin Juana
Published: December 2022

Monocyte-macrophage activation by IFN-γ is characterized by a pronounced increase of high affinity Fc receptors for IgG (FcγRI), capable of triggering respiratory burst, phagocytosis, Ab-dependent cytotoxicity, and release of proinflammatory cytokines. In view of the antagonism of IFN-β on IFN-γ action, of interest in the chronic inflammatory disorder multiple sclerosis, we examined the possible effect of IFN-β on IFN-γ induction of FcγRI gene expression. We found that IFN-β significantly down-regulated IFN-γ-induced FcγRI surface expression in peripheral blood monocytes from healthy donors, in a dose- and time-dependent manner. This down-regulation of FcγRI surface levels did not correspond to a decrease in FcγRI mRNA, suggesting a posttranscriptional effect of IFN-β. Down-regulation of FcγRI surface expression correlated with diminished cellular signaling through FcγRI, since the IFN-γ-induced increase in Fcγ receptor-triggered respiratory burst was nearly completely abrogated by simultaneous addition of IFN-β. Finally, the same antagonism between both IFNs on FcγRI surface expression was observed in peripheral blood monocytes derived from multiple sclerosis patients; inhibition by IFN-β was even increased (82 ± 11%), as compared with healthy controls (67 ± 4%). These results may partially help explain the beneficial effect of IFN-β in multiple sclerosis.
Cutting Edge: Class II Transactivator-Independent Endothelial Cell MHC Class II Gene Activation Induced by Lymphocyte Adhesion
Authors: Collinge Mark , Pardi Ruggero , Bender Jeffrey R.
Published: December 2022

NK cells induce MHC class II molecules on the surface of allogeneic endothelial cells in an adhesion-dependent, IFN-γ-independent manner. Here, we demonstrate that NK cells induce HLA-DR on the surface of a mutant cell line that is defective in IFN-γ-induced MHC class II expression. RNA analysis in these cells and in a cell line that is defective in class II transactivator (CIITA) demonstrates that NK cell-induced HLA-DRα mRNA expression is also CIITA-independent. The Janus kinase-1-deficient cell line U4A expresses HLA-DRα mRNA in response to NK cell activation, and HLA-DRα promoter constructs transfected into these cells are induced by NK cells but not IFN-γ. These data indicate that the IFN-γ-independent component of the target cell HLA-DR expression induced by lymphocyte adhesion uses a signaling pathway that is distinct from the IFN-γ-dependent mechanism and also suggest that CIITA is not required.
Impaired Alloantigen-Mediated T Cell Apoptosis and Failure To Induce Long-Term Allograft Survival in IL-2-Deficient Mice
Authors: Dai Zhenhua , Konieczny Bogumila T. , Baddoura Fady K. , Lakkis Fadi G.
Published: December 2022

We examined whether IL-2 regulates alloimmune responses by studying allograft survival in wild-type (IL-2+/+) and IL-2 gene-knockout (IL-2−/−) mice. The acute rejection of vascularized, cardiac allografts and the generation of allospecific CTLs were not impaired in the absence of IL-2. In contrast, blocking the B7-CD28 T cell costimulation pathway with CTLA4Ig induced long-term allograft survival (>100 days) in IL-2+/+ recipients but failed to do so in IL-2−/− mice or in wild-type mice that had been treated with IL-2-neutralizing Ab around the time of transplantation. Allografts rejected by IL-2−/− recipients exhibited extensive mononuclear cell infiltrates despite CTLA4Ig administration. In vivo allostimulation in the absence of IL-2 led to exaggerated T lymphocyte proliferation and impaired apoptosis of activated T cells in untreated and CTLA4Ig-treated mice. These findings indicate that endogenous IL-2 is required for the induction of long-term allograft survival, and that IL-2 regulates alloimmune responses by preparing activated T lymphocytes for alloantigen-induced apoptosis.
CD1 Expression Defines Subsets of Follicular and Marginal Zone B Cells in the Spleen: β2-Microglobulin-Dependent and Independent Forms
Authors: Amano Masahiko , Baumgarth Nicole , Dick Michael D. , Brossay Laurent , Kronenberg Mitchell , Herzenberg Lee A. , Strober Samuel
Published: December 2022

We have used multicolor FACS analysis, immunohistology, and functional assays to study the expression of CD1 on B cell subsets from normal and β2m−/− mice. Two B cell subpopulations were identified that express high levels of CD1 in normal mice: splenic marginal zone B cells (IgMhigh IgDlow CD21high CD24intermediate CD23− CD43−) and a newly identified subpopulation of follicular B cells. The latter cells are unusual, because they are IgDhigh CD23+, like follicular B cells, but express high levels of CD21 and IgM, an expression pattern that is associated with marginal zone B cells. Therefore, the high-level expression of CD1 and CD21 was found to be closely associated on splenic B cells. Immunohistology confirmed the expression of CD1 on marginal zone B cells and on clusters of B cells in splenic follicles. Both the high-level CD1 expression by these cells and the low-level CD1 expression by subpopulations of B cells in the spleen, lymph node, peritoneal cavity, and bone marrow were markedly reduced in β2m−/− mice. Despite this, a CD1-restricted T cell clone proliferated vigorously in response to LPS-activated spleen cells that had been obtained from both β2m−/− and wild-type mice. This response was inhibited by the 3C11 anti-CD1 mAb. These results show the heterogeneity of B cell subsets in their expression of the β2m-dependent form of CD1. They further suggest that a β2m-independent form of CD1 is expressed on B cells that can stimulate T cells; however, this form is not easily visualized with the anti-CD1 mAb used here.
Interaction of p59fyn Kinase with the Dynein Light Chain, Tctex-1, and Colocalization During Cytokinesis
Authors: Campbell Kerry S. , Cooper Suzanne , Dessing Mark , Yates Sol , Buder Annie
Published: December 2022

The protein tyrosine kinase p59fyn (Fyn) plays important roles in both lymphocyte Ag receptor signaling and cytokinesis of proB cells. We utilized yeast two-hybrid cloning to identify the product of the tctex-1 gene as a protein that specifically interacts with Fyn, but not with other Src family kinases. Tctex-1 was recently identified as a component of the dynein cytoskeletal motor complex. The capacity of a Tctex-1-glutathione S-transferase fusion protein to effectively bind Fyn from cell lysates confirmed the authenticity of this interaction. Tctex-1 binding required the first 19 amino acids of Fyn and integrity of two lysine residues within this sequence that were previously shown to be important for Fyn interactions with the immunoreceptor tyrosine-based activation motifs (ITAMs) of lymphocyte Ag receptors. Expression of tctex-1 mRNA and protein was observed in all lymphoma lines analyzed, and immunofluorescence confocal microscopy localized the protein to the perinuclear region. Analysis of a T cell hybridoma revealed prominent colocalization of Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in cells undergoing cytokinesis. Our results provide a unique insight into a mechanism by which Tctex-1 might mediate specific recruitment of Fyn to the dynein complex in lymphocytes, which may be a critical event in mediating the previously defined role of Fyn in cytokinesis.