bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and Contribution of B7-1 (CD80) and B7-2 (CD86) in the Early Immune Response to Leishmania major Infection
Authors: Elloso M. Merle , Scott Phillip
Published: January 2023

CD28 interactions promote T cell responses, and whether B7-1 or B7-2 is utilized may influence Th cell subset development. CD28 blockade by CTLA-4Ig treatment or by targeted gene disruption has yielded different conclusions regarding the role of CD28 in the development of Th1 and Th2 cells following Leishmania major infection. In this study, we demonstrate that B7-mediated costimulation is required for the development of the early immune response following infection of resistant or susceptible mice. In contrast, CD28−/− BALB/c mice infected with L. major produce cytokines comparable to those of infected wild-type mice. Treatment of CD28−/− mice with CTLA-4Ig did not diminish this response, suggesting that a B7-independent pathway(s) contributes to the early immune response in these mice. In conventional BALB/c or C3H mice, B7-2 functions as the dominant costimulatory molecule in the initiation of early T cell activation following L. major infection, leading to IL-4 or IFN-γ production, respectively. The preferential interaction of B7-2 with its ligand(s) in the induction of these responses correlates with its constitutive expression relative to that of B7-1. However, B7-1 can equally mediate costimulation for the production of either IL-4 or IFN-γ when expressed at high levels. Thus, in leishmaniasis, costimulation involving B7-1 or B7-2 can result in the production of either Th1 or Th2 cytokines, rather than a preferential induction of one type of response.
Induction of Genital Immunity by DNA Priming and Intranasal Booster Immunization with a Replication-Defective Adenoviral Recombinant
Authors: Xiang Zhi Quan , Pasquini Susanna , Ertl Hildegund C. J.
Published: January 2023

Mice immunized through different routes such as i.m., intradermally, or intratracheally with a DNA vaccine to rabies virus developed high titers of serum Ab but only borderline levels of mucosal Abs determined from vaginal secretions. DNA vaccines given by either route enhanced vaginal IgA and IgG2a secretion upon a subsequent intranasal booster immunization with an E1-deleted adenoviral recombinant expressing the same Ag of rabies virus. DNA vaccine priming reduced the Ab response to the adenoviral Ags and counterbalanced the impaired B cell response to the rabies virus Ag expressed by the adenoviral recombinant in mice preimmune to adenovirus. The vaginal B cell response could further be enhanced by using the Th2-type cytokines IL-4 or IL-5 as genetic adjuvants concomitantly with the DNA vaccine before intranasal booster immunization with the recombinant vaccine.
De Novo Autoimmunity to Cardiac Myosin After Heart Transplantation and Its Contribution to the Rejection Process
Authors: Fedoseyeva Eugenia V. , Zhang Feng , Orr Patricia L. , Levin David , Buncke Harry J. , Benichou Gilles
Published: January 2023

Allograft rejection is initiated by an immune response to donor MHC proteins. We recently reported that this response can result in breakdown of immune tolerance to a recipient self Ag. However, the contribution of this autoimmune response to graft rejection has yet to be determined. Here, we found that after mouse allogeneic heart transplantation, de novo CD4+ T cell and B cell autoimmune response to cardiac myosin (CM), a major contractile protein of cardiac muscle, is elicited in recipients. Importantly, CM is the autoantigen that causes autoimmune myocarditis, a heart autoimmune disease whose histopathological features resemble those observed in rejected cardiac transplants. Furthermore, T cell responses directed to CM peptide myhcα 334–352, a known myocarditogenic determinant, were detected in heart-transplanted mice. No responses to CM were observed in mice that had received an allogeneic skin graft or a syngeneic heart transplant, demonstrating that this response is tissue specific and that allogeneic response is necessary to break tolerance to CM. Next, we showed that sensitization of recipient mice with CM markedly accelerates the rejection of allogeneic heart. Therefore, posttransplant autoimmune response to CM is relevant to the rejection process. We conclude that transplantation-induced autoimmune response to CM represents a new mechanism that may play a significant role in cardiac transplant rejection.
Helminth- and Bacillus Calmette-Guérin-Induced Immunity in Children Sensitized In Utero to Filariasis and Schistosomiasis
Authors: Malhotra Indu , Mungai Peter , Wamachi Alex , Kioko John , Ouma John H. , Kazura James W. , King Christopher L.
Published: January 2023

Infants and children are routinely vaccinated with bacillus Calmette-Guérin (BCG) in areas of the world where worm infections are common. Because maternal helminth infection during pregnancy can sensitize the developing fetus, we studied whether this prenatal immunity persists in childhood and modifies the immune response to BCG. Children and newborns living in rural Kenya, where BCG is administered at birth and filariasis and schistosomiasis are endemic, were examined. T cells from 2- to 10-year-old children of mothers without filariasis or schistosomiasis produced 10-fold more IFN-γ in response to mycobacterial purified protein derivative than children of helminth-infected mothers (p < 0.01). This relationship was restricted to purified protein derivative because maternal infection status did not correlate with filarial Ag-driven IL-2, IFN-γ, IL-4, or IL-5 responses by children. Prospective studies initiated at birth showed that helminth-specific T cell immunity acquired in utero is maintained until at least 10–14 mo of age in the absence of infection with either Wuchereria bancrofti or Schistosoma haematobium. Purified protein derivative-driven T cell IFN-γ production evaluated 10–14 mo after BCG vaccination was 26-fold higher for infants who were not sensitized to filariae or schistosomes in utero relative to subjects who experienced prenatal sensitization (p < 0.01). These data indicate that helminth-specific immune responses acquired during gestation persist into childhood and that this prenatal sensitization biases T cell immunity induced by BCG vaccination away from type 1 IFN-γ responses associated with protection against mycobacterial infection.
Cytolytic T Lymphocytes Recognize an Antigen Encoded by MAGE-A10 on a Human Melanoma
Authors: Huang Lan-Qing , Brasseur Francis , Serrano Alfonso , De Plaen Etienne , van der Bruggen Pierre , Boon Thierry , Van Pel Aline
Published: January 2023

From melanoma patient LB1751, cytolytic T lymphocytes (CTL) were generated that lysed specifically autologous tumor cells. To establish whether these CTL recognized one of the Ags that had previously been defined, a CTL clone was stimulated with cells expressing various MAGE genes. It produced TNF upon stimulation with target cells expressing MAGE-A10. The Ag was found to be nonapeptide GLYDGMEHL (codons 254–262), which is presented by HLA-A2.1. This is the first report on the generation of anti-MAGE CTL by autologous mixed lymphocyte-tumor cell culture (MLTC) from a melanoma patient other than patient MZ2, from whom the first MAGE gene was identified. MAGE genes are expressed in many tumors but not by normal tissues except male germline cells and placenta, which do not express HLA molecules. Therefore, the identification of an antigenic peptide derived from MAGE-A10 adds to the repertoire of tumor-specific shared Ags available for anti-tumoral vaccination trials.
Human Breast Carcinoma Patients Develop Clonable Oncofetal Antigen-Specific Effector and Regulatory T Lymphocytes
Authors: Rohrer James W. , Barsoum Adel L. , Dyess Donna L. , Tucker J. Alann , Coggin Joseph H.
Published: January 2023

Oncofetal Ag (OFA) is a 44-kDa glycoprotein expressed during early to mid-gestation fetal development and re-expressed as a surface Ag by tumor cells soon after transformation. The Ag is detectable on all types of human and rodent tumors tested, but is undetectable on normal cells. In experimental animals it is autoimmunogenic and induces potentially protective T cell responses both after experimental immunization and during tumor development subsequent to carcinogenic insult. To determine whether this tumor-associated Ag is also immunogenic for human T lymphocytes, breast carcinoma patients’ peripheral blood mononuclear leucocytes were stimulated in vitro with autologous tumor cells in the presence of IL-2, γ-IFN, and IL-6 for 2 wk. The tumor-reactive cells were then restimulated and cloned by limiting dilution, and the clones were analyzed. We established 24, 19, 11, and 16 tumor-reactive clones from the four respective patients. Of those, 4, 6, 4, and 7, respectively, proliferated specifically to purified OFA. Both CD4 and CD8 OFA-specific clones were established, which responded equally well to purified OFA or 32- to 44-kDa immature laminin receptor protein. All were CD3+, TCR-αβ+. All CD4 clones secreted γ-IFN, but neither secreted IL-4 nor IL-10. Both IFN-γ-secreting cytotoxic CD8 clones and IL-10-secreting inhibitory CD8 clones were established. Thus, during human cancer development, the same types of OFA-specific effector and regulatory T cells are induced as during murine T lymphomagenesis.
Cutting Edge: Chronic Intestinal Inflammation in STAT-4 Transgenic Mice: Characterization of Disease and Adoptive Transfer by TNF- Plus IFN-γ-Producing CD4+ T Cells That Respond to Bacterial Antigens
Authors: Wirtz Stefan , Finotto Susetta , Kanzler Stephan , Lohse Ansgar W. , Blessing Manfred , Lehr Hans A. , Galle Peter R. , Neurath Markus F.
Published: January 2023

We generated transgenic mice for STAT-4, a regulatory protein specifically associated with IL-12 signaling, under the control of a CMV promoter. These mice expressed strikingly increased nuclear STAT-4 levels in lamina propria CD4+ T lymphocytes upon systemic administration of dinitrophenyl-keyhole limpet hemocyanin and developed chronic transmural colitis characterized by infiltrates of mainly CD4+ T lymphocytes. The latter cells produced predominantly TNF and IFN-γ but not IL-4 upon activation with αCD3/CD28 or autologous bacterial Ags, consistent with a Th1-type cell response. Furthermore, chronic colitis in STAT-4 transgenic mice could be adoptively transferred to SCID mice by colonic and splenic CD4+ T cells that were activated with Ags from autologous bacterial flora. These data establish a critical molecular signaling pathway involving STAT-4 for the pathogenesis of chronic intestinal inflammation, and targeting of this pathway may be relevant for the treatment of colitis in humans.
Differential Effects of IL-2 and IL-15 on the Death and Survival of Activated TCRγδ+ Intestinal Intraepithelial Lymphocytes
Authors: Chu Ching-Liang , Chen Shih-Shun , Wu Tzong-Shoon , Kuo Szu-Cheng , Liao Nan-Shih
Published: January 2023

TCRγδ+ cells are enriched in the intestine mucosa and constitute approximately half of the intestinal intraepithelial lymphocytes (iIEL) in mice. They are likely activated by self and foreign Ags in situ, but little is known about how the activated γδ iIEL are regulated. In the iIEL compartment, IL-2 is produced by activated TCRαβ+ iIEL, and IL-15 message is detected in iIEL and in the epithelial cells. We found surface expression of IL-2 as well as IL-15Rs on activated γδ iIEL, and examined the effects of IL-2 and IL-15 on the survival and death of γδ iIEL during secondary stimulation through TCR. We found that both cytokines supported growth of the restimulated γδ iIEL, but exerted different effects on their survival. A significant higher number of live cells were recovered from the γδ iIEL cultures restimulated in IL-15 than in IL-2. Quantitation of apoptotic cells showed more cell death in the IL-2 group than in the IL-15 group. The cell death was associated with restimulation through TCR and was not caused by insufficient growth factor, thus representing activation-induced cell death. Western blot analyses found no difference in the levels of Bcl-2 and Bax proteins between the two groups. However, the level of Bcl-xL protein diminished with time in the IL-2 group whereas the level was sustained in the IL-15 group, which may contribute to the pro-survival effect of IL-15. These results demonstrated that the survival of activated γδ iIEL is differentially regulated by IL-2 and IL-15.
Regulation of Cell Survival During B Lymphopoiesis: Apoptosis and Bcl-2/Bax Content of Precursor B Cells in Bone Marrow of Mice with Altered Expression of IL-7 and Recombinase-Activating Gene-2
Authors: Lu Liwei , Chaudhury Prosanto , Osmond Dennis G.
Published: January 2023

B cell development in mouse bone marrow depends critically upon IL-7. To examine the possible in vivo trophic role of IL-7, we have quantitated apoptosis and Bcl-2 family proteins in populations of phenotypically defined B lineage cells in IL-7-deficient and IL-7-overexpressing mice. Using immunofluorescence labeling, multiparameter flow cytometry, and a short-term culture assay, we show that the apoptotic rates of precursor B cells, but not of more mature B cells, are enhanced by IL-7 gene deletion, associated with increased intracellular content of Bax and decreased Bcl-2, while, conversely, an IL-7 transgene suppresses precursor B cell apoptosis and produces low Bax and high Bcl-2 levels. During normal B cell development, high Bax/Bcl-2 ratios characterize cells undergoing greatest apoptotic cell death. Pro-B cells in RAG-2−/− mice, all destined to abort, show elevated Bax levels and Bax/Bcl-2 ratios. By comparison with the elevated rate of pro-B cell apoptosis in RAG-2−/− mice, provisional estimates have been made for the fraction of pro-B cells undergoing apoptosis in normal mice (70%), IL-7−/− mice (85%), and IL-7 transgenic mice (35%). The results demonstrate that IL-7 strongly promotes in vivo cell survival and maintains antiapoptotic Bcl-2/Bax ratios during the development of precursor B cells in mouse bone marrow.
Use of Antagonist Peptides to Inhibit In Vitro T Cell Responses to Par j1, the Major Allergen ofParietaria judaicaPollen
Authors: De Palma Raffaele , Wu Shenhong , Sallusto Federica , Di Felice Gabriella , Martucci Paola , Geraci Domenico , Colombo Paolo , Troise Costantino , Sacerdoti Guido , Nocera Arcangelo , Gorski Jack
Published: January 2023

Antigenic peptides with substituted side chains inhibit immune responses to a number of recall Ags from infectious agents in vitro. Here we show that the same strategy can be applied to peptides derived from a pollen protein, the major allergen of Parietaria judaica(Par j1), a plant responsible for most allergenic sensitization in the southern Mediterranean area. Three T cell lines responding to Par j1 protein were used to identify a stimulatory peptide. Two different monosubstituted altered peptide ligands (APL) were identified that bound to the HLA-DR of the responders, did not stimulate the T cell lines on their own, and decreased the response to subsaturating amounts of the unmodified stimulatory peptide. Most important, these APL were able to inhibit the response of these cell lines to intact Par j1 protein. A third monosubstituted peptide bound to the HLA-DR but did not show inhibitory activity. The two APL had a lower affinity than the unsubstituted peptide for the HLA-DR. The last two observations make MHC blockade an unlikely explanation for the observed effect. These results indicate the action of a specific peptide-mediated antagonism that may be useful in controlling the T cell component of an allergic response.
Calcium-Dependent Activation of TNF Family Gene Expression by Ca2+/Calmodulin Kinase Type IV/Gr and Calcineurin
Authors: Lobo Francis M. , Zanjani Reza , Ho Nga , Chatila Talal A. , Fuleihan Ramsay L.
Published: January 2023

CD40 ligand (L), FasL, and TNF-α are members of the TNF family of cytokines. All are expressed by T lymphocytes shortly after activation but have distinct effector functions. Transcription of these genes can be induced by stimulation of T cells by calcium ionophore alone and requires the calcineurin-dependent transcription factor NF of activated T cells. We have examined a second calcium-dependent signaling pathway, mediated by calcium/calmodulin-dependent kinase IV (CaMKIV) in transcriptional activation of TNF family genes. In reporter gene assays using constructs driven by the promoters of human CD40L, FasL, or TNF-α along with vectors expressing constitutively active CaMKIV and calcineurin, we have demonstrated that each promoter is activated by calcineurin and CaMKIV in a synergistic fashion. Furthermore, specific inhibition of CaMKIV by chemical means and by a dominant negative mutant of CaMKIV impairs the ionomycin-induced activity of all three promoters as well as protein expression of CD40L and TNF-α. Our results indicate that activation of gene expression by calcineurin and CaMKIV is common to members of the TNF cytokine family.
The Orientation and Nature of the Interaction Between Beef Insulin-Specific TCRs and the Insulin/Class II MHC Complex
Authors: Wither Joan E. , Vukusic Brian
Published: January 2023

Recent crystallographic studies suggest that TCR interact with peptide/class I MHC complexes in a single preferred orientation. Although similar studies have not been performed for class II-restricted TCR, it has been proposed that T cell recognition of peptide/class II complexes has similar orientational restrictions. This study represents a functional approach to systematic analysis of this question. Twenty-one mutant Aβd molecules were produced by alanine scanning mutagenesis and assessed for their ability to present species variants of insulin to a panel of beef insulin-specific T cell hybridomas with limited TCR α- and/or β-chain sequence differences. We demonstrate that all beef insulin-specific TCR have the same orientation on the insulin/Ad complex, such that the α-chain interacts with the carboxyl-terminal region of the Aβd α-helix, and the β-chain complementarity-determining region 3 interacts with the carboxyl-terminal portion of the peptide, consistent with that observed for crystallized TCR-peptide/class I complexes. Despite this structural constraint, even TCR that share structural similarity show remarkable heterogeneity in their responses to the panel of MHC mutants. This variability appears to result from conformational changes induced by binding of the TCR to the complex and the exquisite sensitivity of the threshold for T cell activation.
Extensive Junctional Diversity in Ig Light Chain Genes from Early B Cell Progenitors of μMT Mice
Authors: Bentolila Laurent A. , Olson Stacy , Marshall Aaron , Rougeon François , Paige Christopher J. , Doyen Noëlle , Wu Gillian E.
Published: January 2023

Nontemplated (N) nucleotide additions contribute significantly to the junctional diversity of all Ag receptor chains in adult mice except Ig light (L) chains, primarily because terminal deoxynucleotidyl transferase (TdT) expression is turned off at the time of their rearrangement in pre-B cells. However, because some Ig L chain gene rearrangements are detectable earlier during B cell ontogeny when TdT expression is thought to be maximal, we have examined the junctional processing of κ- and λ-chain genes of CD45(B220)+CD43+ pro-B cells from μMT mice. We found that both κ and λ coding junctions formed in these B cell precursors were extensively diversified with N-region additions. Together, these findings demonstrate that Ig L chain genes are equally accessible to TdT in pro-B cells as Ig heavy chain genes. Surprisingly, however, the two L chain isotypes differed in the pattern of N addition, which was more prevalent at the λ-chain locus. We observed the same diversity pattern in pre-B cells from TdT-transgenic mice. These results suggest that some aspects of TdT processing could be influenced by factors intrinsic to the sequence of Ig genes and/or the process of V(D)J recombination itself.
Functional Characterization of the Somatic Hypermutation Process Leading to Antibody D1.3, a High Affinity Antibody Directed Against Lysozyme
Authors: England Patrick , Nageotte Roland , Renard Martial , Page Anne-Laure , Bedouelle Hugues
Published: January 2023

The impact of somatic hypermutation on the affinity of Abs directed against protein Ags remains poorly understood. We chose as a model the secondary response Ab D1.3 directed against hen egg lysozyme. During the maturation process leading to this Ab, five replacement somatic mutations occurred. After reconstituting the germline Ab from which D1.3 originated, we assessed the energetic and kinetic importance of each of the somatic mutations, individually or combined, using the BIAcore apparatus. We found that the mutations induced an overall 60-fold improvement of affinity, principally due to a decrease in the kinetic rate of dissociation. We showed that their effects were additive and context independent; therefore, in the case of D1.3, the order in which somatic mutations were introduced and selected is unimportant. Interestingly, most of the affinity improvement was due to a single somatic mutation (Asn50→Tyr in VL), involving a residue that belongs to the functional interface between Ab D1.3 and lysozyme. This replacement could either establish new Van der Waals contacts between the Ab and the Ag or help stabilize the conformation of a closely situated crucial residue of the Ab paratope. The four other mutations played only a marginal part in affinity maturation; potential reasons for which these mutations were nevertheless selected are discussed.
IL-12 Administered During Chlamydia psittaci Lung Infection in Mice Confers Immediate and Long-Term Protection and Reduces Macrophage Inflammatory Protein-2 Level and Neutrophil Infiltration in Lung Tissue
Authors: Huang Jin , Wang Ming-Dong , Lenz Stephen , Gao Dongya , Kaltenboeck Bernhard
Published: January 2023

Protection against infections with the intracellular bacterium Chlamydia spp. requires Th1-polarized CD4+ T cell immunity. In BALB/c mouse lung infections, immediate innate and nascent Chlamydia-specific immune responses following intranasal inoculation of Chlamydia psittaci strain B577 were modulated by 7-day i.p. administration of murine rIL-12, the initiation cytokine for Th1 immunity. Treatment with IL-12 reduced the severity of chlamydial pneumonia, abolished mortality (37.5% in untreated mice), and significantly reduced numbers of chlamydial organisms in lungs. On day 4 after inoculation, the neutrophil:macrophage ratio in bronchointerstitial pneumonias was 1.96 in untreated mice and 0.51 in IL-12-treated mice. This immediate, IL-12-mediated shift in innate inflammatory phenotype was correlated with a significant reduction of lung concentrations of the neutrophil chemoattractant macrophage inflammatory protein (MIP)-2 (putative murine homologue of human IL-8), monocyte chemotactic protein-1, and TNF-α; and a reduction in MIP-1α and IFN-γ, at high-dose infection only, and IL-12-independent IL-10 levels. Chlamydia-specific Ab titers and Ig isotype ratios indicated an IL-12-dependent Th1 shift. Recall responses of IL-12-primed mice to secondary chlamydial lung infection eliminated chlamydiae more effectively and generated a lung cytokine profile conducive to perpetuation of the Th1 memory population. These data support the hypothesis that genetic differences in endogenous IL-12 production and response pathways could determine disease outcomes characterized by poor chlamydial clearance and a purulent inflammatory infiltrate vs effective elimination of chlamydiae in a macrophage-dominated response.
Fcγ Receptor-Mediated Activation of Phospholipase D Regulates Macrophage Phagocytosis of IgG-Opsonized Particles
Authors: Kusner David J. , Hall Clifton F. , Jackson Stephen
Published: January 2023

Receptors for the Fc portion of IgG (FcγRs) integrate the innate and acquired components of immunity by coupling the specific recognition of IgG Abs to the activation of phagocytic leukocytes. Knowledge of the molecular mechanisms that regulate phagocyte stimulation by FcγRs may permit therapeutic modulation to augment immunoprotective aspects and minimize damage to host tissues in diverse inflammatory diseases. Since phospholipase D (PLD) has been linked to the stimulation of cytotoxic leukocyte responses, we characterized FcγR-dependent activation of PLD in human macrophages. IgG-coated SRBCs (EIgG) stimulated a 9.4-fold increase in PLD activity compared with SRBCs treated with control Ab (p < 0.001), determined by formation of the PLD-specific product phosphatidylethanol in the presence of 0.5% ethanol. Levels of phosphatidic acid, the physiologic product of PLD-mediated catalyzis, were significantly increased in the absence of ethanol (6.4-fold, p < 0.001). PLD activity was also stimulated by immune complex-coated latex beads or cross-linking of Abs specific for FcγRI, FcγRII, or FcγRIII. Phagocytosis of EIgG was reduced by two inhibitors of PLD-mediated signaling, 2,3-diphosphoglycerate or 1-butanol. Addition of purified PLD restored control levels of phagocytosis in cells in which endogenous PLD was inhibited. The tyrosine kinase inhibitors genistein and herbimycin A caused concordant reductions in FcγR-stimulated PLD activity and phagocytosis. These studies demonstrate that FcγR-mediated phagocytosis is accompanied by tyrosine kinase-dependent activation of PLD and support the hypothesis that stimulation of PLD functions to regulate the ingestion of IgG-opsonized particles.
In Vitro and In Vivo Dependency of Chemokine Generation on C5a and TNF-α
Authors: Czermak Boris J. , Sarma Vidya , Bless Nicolas M. , Schmal Hagen , Friedl Hans Peter , Ward Peter A.
Published: January 2023

Under a variety of conditions, alveolar macrophages can generate early response cytokines (TNF-α, IL-1), complement components, and chemotactic cytokines (chemokines). In the current studies, we determined the requirements for TNF-α and the complement activation product C5a in chemokine production in vitro and in vivo. Two rat CXC chemokines (macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC)) as well as three rat CC chemokines (MIP-1α, MIP-1β, and monocyte chemoattractant protein (MCP)-1) were investigated. Chemokine generation in vitro was studied in rat alveolar macrophages stimulated with IgG immune complexes in the absence or presence of Abs to TNF-α or C5a. The rat lung injury model induced by IgG immune complex deposition was employed for in vivo studies. Abs to TNF-α or C5a were administered intratracheally or i.v., and effects on chemokine levels in bronchoalveolar lavage fluids were quantitated by ELISA. Both in vitro and in vivo studies demonstrated the requirements for TNF-α and C5a for full generation of CXC and CC chemokines. In vitro and in vivo blockade of TNF-α or C5a resulted in significantly reduced production of chemokines. Supernatant fluids from in vitro-stimulated macrophages revealed by Western blot analysis the presence of C5a/C5adesArg, indicating intrinsic generation of C5a/C5adesArg by alveolar macrophages and explaining the higher efficiency of intratracheal vs i.v. blockade of C5a in reducing chemokine production. These results underscore the central role of both TNF-α and C5a, which appear to function as autocrine activators to promote CXC and CC chemokine generation by alveolar macrophages.
CD95 (Fas/Apo-1) as a Receptor Governing Astrocyte Apoptotic or Inflammatory Responses: A Key Role in Brain Inflammation?
Authors: Saas Philippe , Boucraut José , Quiquerez Anne-Lise , Schnuriger Valérie , Perrin Gaelle , Desplat-Jego Sophie , Bernard Dominique , Walker Paul R. , Dietrich Pierre-Yves
Published: January 2023

Astrocytes are a major cellular component of the brain that are capable of intense proliferation and metabolic activity during diverse inflammatory brain diseases (such as multiple sclerosis, Alzheimer’s dementia, tumor, HIV encephalitis, or prion disease). In this biological process, called reactive gliosis, astrocyte apoptosis is frequently observed and could be an important mechanism of regulation. However, the factors responsible for apoptosis in human astrocytes are poorly defined. Here, we report that short term cultured astrocytes derived from different brain regions express significant levels of CD95 at their surface. Only late passage astrocytes are sensitive to CD95 ligation using either CD95 mAb or recombinant CD95 ligand. Blocking experiments using caspase inhibitors with different specificities (DEVD-CHO, z-VAD-fmk, and YVAD-cmk), an enzymatic activity assay, and immunoblotting show that CPP32/caspase-3 play a prominent role in CD95-induced astrocyte death. In contrast, early passage astrocytes are totally resistant to death, but a significant increase in astrocytic IL-8 secretion (p < 0.001, by Wilcoxon’s test for paired samples) is observed after CD95 triggering. Production of IL-8 contributes to the resistance of astrocytes to CD95 ligation. Furthermore, in the presence of IFN-γ, resistant astrocytes became sensitive to CD95-mediated death. These data suggest that microenvironmental factors can influence the consequences of CD95 ligation on astrocytes. Therefore, we propose that CD95 expressed by human astrocytes plays a pivotal role in the regulation of astrocyte life and death and may be a key factor in inflammatory processes in the brain, such as reactive gliosis.
Phosphorylation of Cytosolic Phospholipase A2 and the Release of Arachidonic Acid in Human Neutrophils
Authors: Syrbu Sergei I. , Waterman Waltraut H. , Molski Thaddeus F. P. , Nagarkatti Deepa , Hajjar Jean-Jacques , Sha’afi Ramadan I.
Published: January 2023

Kinases mediating phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in intact cells remain to be fully characterized. Platelet-activating factor stimulation of human neutrophils increases cPLA2 phosphorylation. This increase is inhibited by PD 98059, a mitogen-activated protein (MAP)/extracellular signal-regulating kinase (erk) 1 inhibitor, but not by SB 203580, a p38 MAP kinase inhibitor, indicating that this action is mediated through activation of the p42 MAP kinase (erk2). However, platelet-activating factor-induced arachidonic acid release is inhibited by both PD 98059 and SB 203580. Stimulation by TNF-α increases cPLA2 phosphorylation, which is inhibited by SB 203580, but not PD 98059, suggesting a role for p38 MAP kinase. LPS increases cPLA2 phosphorylation and arachidonic acid release. However, neither of these actions is inhibited by either PD 98059 or SB 203580. PMA increases cPLA2 phosphorylation. This action is inhibited by PD 98059 but not SB 203580. Finally, FMLP increases cPLA2 phosphorylation and arachidonic acid release. Interestingly, while the FMLP-induced phosphorylation of cPLA2 is not affected by the inhibitors of the p38 MAP kinase or erk cascades, both inhibitors significantly decrease arachidonic acid release stimulated by FMLP. SB 203580 or PD 98059 has no inhibitory effects on the activity of coenzyme A-independent transacylase.
Modulation of the Humoral Immune Response by Antibody-Mediated Antigen Targeting to Complement Receptors and Fc Receptors
Authors: Baiu Dana C. , Prechl Jozsef , Tchorbanov Andrey , Molina Hector D. , Erdei Anna , Sulica Andrei , Capel Peter J. A. , Hazenbos Wouter L. W.
Published: January 2023

During an ongoing immune response, immune complexes, composed of Ag, complement factors, and Igs, are formed that can interact with complement receptors (CRs) and IgG Fc receptors (FcγR). The role of CR1/2 and FcγR in the regulation of the immune response was investigated using OVA that was chemically conjugated to whole IgG of the rat anti-mouse CR1/2 mAb 7G6. FACS analysis using the murine B cell lymphoma IIA1.6 confirmed that the 7G6-OVA conjugate recognized CR1/2. Incubating IIA1.6 cells with 7G6-OVA triggered tyrosine phosphorylation and Ag presentation to OVA-specific T cells in vitro. Immunizing mice with 7G6-OVA at a minimal dose of 1 μg i.p. per mouse markedly enhanced the anti-OVA Ig response, which was primarily of the IgG1 isotype subclass. The 7G6-OVA did not enhance the anti-OVA response in CR1/2-deficient mice. OVA coupled to an isotype control Ab induced a considerably lower anti-OVA response compared with that induced by OVA alone, suggesting inhibition by interaction between the Fc part of the Ab and the inhibitory FcγRIIb on B cells. This finding was supported by the observation that IIA1.6 cells which were incubated with 7G6-OVA lost the ability to present Ag upon transfection with FcγRIIb. In sum, 7G6-conjugated OVA, resembling a natural immune complex, induces an enhanced anti-OVA immune response that involves at least CR1/2-mediated stimulation and that may be partially suppressed by FcγRIIb.
Differential Responses to CD40 Ligation Among Burkitt Lymphoma Lines That Are Uniformly Responsive to Epstein-Barr Virus Latent Membrane Protein 1
Authors: Henriquez Nico V. , Floettmann Eike , Salmon Mike , Rowe Martin , Rickinson Alan B.
Published: January 2023

Ligation of CD40 on the surface of B cells induces multiple phenotypic effects, many of which are mimicked by the EBV latent membrane protein 1 (LMP1) through its interaction with downstream components of the CD40 signaling pathway. Because the effects of LMP1 have been most closely studied in human Burkitt Lymphoma (BL) cell lines retaining a tumor biopsy-like phenotype in vitro, we have examined the response of a panel of such lines to CD40 ligation. Two distinct patterns of response were observed that were unrelated to the surface level of CD40 or to the EBV genome status of the lines. Following exposure to either CD40-specific mAbs or the soluble trimeric ligand (sCD40L), high responder (HR) lines showed rapid aggregation, activation of NF-κB, up-regulation of cell surface markers ICAM-1/CD54 and Fas/CD95, and growth inhibition. Aggregation was seen at lower doses than those required to elicit the other effects. By contrast, low responder (LR) lines showed no detectable response to CD40 mAbs, while their responses to sCD40L were limited to activation of NF-κB and up-regulation of CD95 only. However, in transfection experiments, LMP1 uniformly induced the full spectrum of phenotypic effects in both HR and LR lines. We conclude that some BL cell lines show a highly restricted response to CD40 ligation but remain fully susceptible to LMP1.
Postthymic Development of CD28−CD8+ T Cell Subset: Age-Associated Expansion and Shift from Memory to Naive Phenotype
Authors: Nociari Marcelo M. , Telford William , Russo Carlo
Published: January 2023

During human aging, one of the major changes in the T cell repertoire is a dramatic expansion of T cells with the atypical CD28−CD8+ phenotype. In this study, we show that this increase is a consequence not only of an expansion in the CD28−CD8+ population but also of a decrease in the number of CD28+CD8+ T cells. The decrease in circulating CD28+CD8+ T cells is dramatically accelerated after the age of 50 and is not accompanied by an equivalent reduction in the CD28+CD4+ subset. Our findings confirm that aging leads to an accumulation of CD45RO+ T cells within the CD28+CD8+ subset as previously observed. Surprisingly, we found an increase in CD45RA+ expression with age in the CD28−CD8+ subset. Immune-phenotyping for activation markers, measurement of telomere DNA content, and cytokine production analysis indicate that the large majority of CD28−CD8+ T cells are Ag-experienced, despite their CD45RA+ phenotype. Our study further demonstrates that the poor proliferative response displayed by CD28−CD8+ T cells is not a consequence of telomere shortening. Also, analysis of cytokine production at the single cell level revealed that the proportions of IFN-γ+, IL-4+, and IL-10+ T cells are considerably higher among the CD28−CD8+ than the CD28+CD8+ subset. In summary, these data explain the presence of CD45RA+ T cells in the elderly, shed light on the phylogenetic origin of CD28−CD8+ T cells, and suggest a role for these cells in the immune senescence process.
Presentation of Antigens Internalized Through the B Cell Receptor Requires Newly Synthesized MHC Class II Molecules
Authors: Forquet Frédérique , Barois Nicolas , Machy Patrick , Trucy Jeannine , Zimmermann Valérie S. , Leserman Lee , Davoust Jean
Published: January 2023

Exogenous Ags taken up from the fluid phase can be presented by both newly synthesized and recycling MHC class II molecules. However, the presentation of Ags internalized through the B cell receptor (BCR) has not been characterized with respect to whether the class II molecules with which they become associated are newly synthesized or recycling. We show that the presentation of Ag taken up by the BCR requires protein synthesis in splenic B cells and in B lymphoma cells. Using B cells transfected with full-length I-Ak molecules or molecules truncated in cytoplasmic domains of their α- or β-chains, we further show that when an Ag is internalized by the BCR, the cytoplasmic tails of class II molecules differentially control the presentation of antigenic peptides to specific T cells depending upon the importance of proteolytic processing in the production of that peptide. Integrity of the cytoplasmic tail of the I-Ak β-chain is required for the presentation of the hen egg lysozyme determinant (46–61) following BCR internalization, but that dependence is not seen for the (34–45) determinant derived from the same protein. The tail of the β-chain is also of importance for the dissociation of invariant chain fragments from class II molecules. Our results demonstrate that Ags internalized through the BCR are targeted to compartments containing newly synthesized class II molecules and that the tails of class II β-chains control the loading of determinants produced after extensive Ag processing.
Identification of a Second Major Tumor-Specific Antigen Recognized by CTLs on Mouse Mastocytoma P815
Authors: Bilsborough Janine , Van Pel Aline , Uyttenhove Catherine , Boon Thierry , Van den Eynde Benoı̂t J.
Published: January 2023

Murine mastocytoma P815 induces CTL responses against at least four distinct Ags (AB, C, D, and E). Recent studies have shown that the main component of the CTL response against the P815 tumor is targeted against Ags P815AB and P815E. The gene P1A has been well characterized. It encodes the P815AB Ag in the form of a nonameric peptide containing two epitopes, P815A and P815B, which are recognized by different CTLs. Here, we report the identification of the P815E Ag. Using a cDNA library derived from tumor P815, we identified the gene coding for P815E. We also characterized the antigenic peptide that anti-P815E CTLs recognize on the MHC class I molecule H-2Kd. The P815E Ag results from a mutation within an ubiquitously expressed gene encoding methionine sulfoxide reductase, an enzyme that is believed to be important in the protection of proteins against the by-products of aerobic metabolism. Surprisingly, immunizing mice i.p. with syngeneic tumor cells (L1210) that were constructed to express B7-1 and P815E did not induce resistance against live P815, even though a strong anti-P815E CTL response was observed with splenocytes from immunized animals.
Paradoxical Preservation of a Lipopolysaccharide Response in C3H/HeJ Macrophages: Induction of Matrix Metalloproteinase-9
Authors: Jin Fen-yu , Nathan Carl F. , Ding Aihao
Published: January 2023

C3H/HeJ mice carry a mutant allele (Lpsd) of a recently identified gene whose normal allele (Lpsn) confers responsiveness to bacterial LPS in C3H/HeN and most other mouse strains. Recently we reported a differential display analysis of matched macrophage-derived cell lines from C3H/HeJ and C3H/HeN mice under LPS-free conditions. Of the ∼12,000 transcripts evaluated, 4 were differentially expressed. One transcript represented secretory leukocyte protease inhibitor. In this study, we report another differentially expressed transcript, mouse matrix metalloprotease-9 (MMP-9). Like secretory leukocyte protease inhibitor, MMP-9 was expressed constitutively in the Lpsd macrophage cell line and not in the Lpsn cell line. Similarly, two additional macrophage cell lines that respond readily to LPS by producing nitric oxide and TNF expressed no MMP-9 under LPS-free conditions. However, in all four cell lines, LPS induced MMP-9 or augmented its expression. In primary macrophages, concentrations of LPS in the ng/ml range augmented the expression of MMP-9 mRNA. Paradoxically, macrophages from Lpsd mice expressed more MMP-9 transcripts than macrophages from Lpsn mice. In contrast, the induction of TNF in response to LPS was much more pronounced in Lpsn macrophages. The present findings with MMP-9 suggest that homozygosity at Lpsd does not so much prevent a response to LPS as dysregulate it, resulting in the suppression of some LPS signaling pathways and the preservation of others.
Clonal Dominance Patterns of CD8 T Cells in Relation to Disease Progression in HIV-Infected Children
Authors: Than Soe , Kharbanda Monica , Chitnis Vivek , Bakshi Saroj , Gregersen Peter K. , Pahwa Savita
Published: January 2023

CD8 T cells are important mediators of cellular immune responses as evidenced by clonal expansions in the CD8 TCR Vβ repertoire during primary HIV infection in adults. This study investigated the CD8 TCR Vβ repertoire by complementarity-determining region 3 length analysis using multiplex PCR in purified peripheral blood CD8 T cells of 22 HIV-infected children (age range was 0.75–15 yr, mean was 8.2 ± 4.1 yr). Evidence of clonal dominance in one or more Vβ families was obtained in 15 of 22 children. The patterns of clonal dominance were designated as major, minor, single, and none to indicate the involvement of three or more, two, one, or no Vβ families, respectively. A pattern of major or minor clonal dominance was observed in 12 children (group 1), whereas 10 children had single or no clonal dominance (group 2). In comparison with group 2, the children in group 1 had a higher percentage of CD4 cells (28.3 ± 11.6 vs 8.6 ± 4.8, p < 0.001); a higher stimulation index in lymphoproliferative responses to Candida (92.0 ± 59.5 vs 12.3 ± 14.4, p = 0.002), tetanus (76.3 ± 51.2 vs 11.2 ± 12.7, p = 0.002), and alloantigens (178.3 ± 298.9 vs 32.9 ± 35.2, p < 0.001); and a lower percentage of CD8+HLA-DR+CD38+ cells (37.4 ± 13.1 vs 54.6 ± 14.2, p < 0.01). The number of dominant CD8 T cell clones was significantly correlated with the percentage of CD4 T cells (r = 0.669, p < 0.001) but not with plasma HIV RNA. Compared with group 1, patients in group 2 had a 4.8 times greater probability of having <15% CD4 cells. These findings indicate that CD8 clonal dominance in HIV-infected children reflects robustness of immune responses, regardless of time since infection and virus load.
Cutting Edge: LFA-1 Is Required for Liver NK1.1+TCRαβ+ Cell Development: Evidence That Liver NK1.1+TCRαβ+ Cells Originate from Multiple Pathways
Authors: Ohteki Toshiaki , Maki Chikako , Koyasu Shigeo , Mak Tak W. , Ohashi Pamela S.
Published: January 2023

Using mice deficient for LFA-1, CD44, and ICAM-1, we examined the role of these adhesion molecules in NK1.1+TCRαβ+ (NKT) cell development. Although no defect in NKT cell development was observed in CD44−/− and ICAM-1−/− mice, a dramatic reduction of liver NKT cells was observed in LFA-1−/− mice. Normal numbers of NKT cells were present in other lymphoid organs in LFA-1−/− mice. When LFA-1−/− splenocytes were injected i.v. into wild-type mice, the frequency of NKT cells among donor-derived cells in the recipient liver was normal. In contrast, when LFA-1−/− bone marrow (BM) cells were injected i.v. into irradiated wild-type mice, the frequency of liver NKT cells was significantly lower than that of mice injected with wild-type BM cells. Collectively, these data indicate that LFA-1 is required for the development of liver NKT cells, rather than the migration to and/or subsequent establishment of mature NKT cells in the liver.
Activation of CD4 T Cells by Somatic Transgenesis Induces Generalized Immunity of Uncommitted T Cells and Immunologic Memory
Authors: Gerloni Mara , Miner Kent T. , Xiong Sidong , Croft Michael , Zanetti Maurizio
Published: January 2023

Cellular immune responses were analyzed in vivo after a single intraspleen inoculation of DNA coding for a 12-residue Th cell determinant associated with a 12-residue B cell epitope, a process termed somatic transgene immunization. We show that CD4 T cells are readily activated and produce IL-2, IFN-γ and IL-4, characteristics of an uncommitted phenotype. Linked recognition of the two epitopes coded in the same transgene promoted IgM-IgG1 switch and enhanced the total Ab response but had no effect on IgG2a Abs. Although originating in the spleen, T cell responsiveness was found to spread immediately and with similar characteristics to all lymph nodes in the body. A single inoculation was also effective in establishing long term immunologic memory as determined by limiting dilution analysis, with memory T cells displaying a cytokine profile different from that of primary effector T cells. These studies provide evidence that by initiating immunity directly in secondary lymphoid organs, an immune response is generated with characteristics that differ from those using vaccines of conventional DNA or protein in adjuvant administered in peripheral sites. Somatic transgene immunization can therefore be used to probe T cell responsiveness in vivo and represents a tool to further understanding of the nature of the adaptive immune response.
Naive Human CD4+ T Cells Are a Major Source of Lymphotoxin α
Authors: Ohshima Y. , Yang L-P. , Avice M-N. , Kurimoto M. , Nakajima T. , Sergerie M. , Demeure C. E. , Sarfati M. , Delespesse G.
Published: January 2023

It is generally accepted that immunologically naive T cells display a very restricted cytokine production profile consisting mainly of IL-2, which is used as an autocrine growth factor. Here we report that activated naive CD4+ T cells, of neonatal or adult origin, express very high levels of soluble lymphotoxin (LT) α (LTα3), as determined by ELISA, RNase protection assay, and intracytoplasmic staining. Besides LTα3 and IL-2, these cells also produce high levels of TNF-α together with significant amounts of IFN-γ and IL-13. Naive cells also express LTβ mRNA and the membrane form of LTα (LTαβ). On average, naive CD4+ T cells secrete four times more LTα3 than Th1-like cells, twice more than naive CD8+ T cells, and ten times more than B cells. Thus, naive T cells express a large spectrum of cytokines, mainly of the Th1 type, and the very high levels of LTα3/TNF-α that they release may play an hitherto unsuspected role in the early stage of T cell-dependent immune responses.
p38 Mitogen-Activated Protein Kinase Mediates Signal Integration of TCR/CD28 Costimulation in Primary Murine T Cells
Authors: Zhang Jian , Salojin Konstantin V. , Gao Jian-Xin , Cameron Mark J. , Bergerot Isabelle , Delovitch Terry L.
Published: January 2023

Optimal T cell activation requires two signals, one generated by TCR and another by the CD28 costimulatory receptor. In this study, we investigated the regulation of costimulation-induced mitogen-activated protein kinase (MAPK) activation in primary mouse T cells. In contrast to that reported for human Jurkat T cells, we found that p38 MAPK, but not Jun NH2-terminal kinase (JNK), is weakly activated upon stimulation with either anti-CD3 or anti-CD28 in murine thymocytes and splenic T cells. However, p38 MAPK is activated strongly and synergistically by either CD3/CD28 coligation or PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3/CD28-mediated signaling. Activation of p38 MAPK correlates closely with the stimulation of T cell proliferation. In contrast, PMA-induced JNK activation is inhibited by Ca2+ ionophore. T cell proliferation and production of IL-2, IL-4, and IFN-γ induced by both CD3 and CD3/CD28 ligation and the nuclear expression of the c-Jun and ATF-2 proteins are each blocked by the p38 MAPK inhibitor SB203580. Our findings demonstrate that p38 MAPK 1) plays an important role in signal integration during costimulation of primary mouse T cells, 2) may be involved in the induction of c-Jun activation and augmentation of AP-1 transcriptional activity, and 3) regulates whether T cells enter a state of functional unresponsiveness.