bluuebunny/crossref_metadata_2025 huggingface dataset:
Transendothelial migration of lymphocytes from HIV-1-infected donors: a mechanism for extravascular dissemination of HIV-1.
Authors: Birdsall H H , Trial J , Lin H J , Green D M , Sorrentino G W , Siwak E B , de Jong A L , Rossen R D
Published: December 2022

To identify factors that cause HIV-1 to establish perivascular foci of infected cells, we studied the transendothelial migration of blood mononuclear leukocytes (MNL) from 76 HIV+ patients and 41 controls. The fraction of patients' lymphocytes that migrated across endothelial cell monolayers in vitro was significantly increased (p < or = 0.03) compared with that of control donors. Migration of patients' CD4+ T cells was particularly enhanced, whereas the migration of monocytes did not differ between patients and controls. Lymphocyte migration correlated with expression of CD11a/CD18 and CD49d/CD29 and with the quantity of TNF-alpha produced as MNLs migrated through the endothelium. Measurement of HIV-1 proviral DNA copies in the patients' MNLs (n = 26) suggested that in half the cases virus-infected cells accumulated preferentially amidst the migratory leukocytes. We observed the same behavior with normal donor MNLs infected, in vitro, with each of 4 strains of HIV-1. The number of HIV-1 proviral DNA copies per million MNLs was 40 to 178 times higher in the migratory population than in the original population added to the endothelium. To test whether only certain strains of HIV-1 stimulate transendothelial migration of infected cells, we used single strand conformation polymorphism analysis to identify quasispecies of HIV-1 in the MNLs. If all strains of HIV-1 were equal in their ability to stimulate transendothelial migration, we expected to find no differences in the quasispecies present in the original and migratory cell populations. In fact the quasispecies differed in 14 of 19 paired samples, suggesting that only certain HIV-1 quasispecies promote transendothelial migration of infected cells.
Experimental autoimmune myasthenia gravis in B10.BV8S2 transgenic mice: preferential usage of TCRAV1 gene by lymphocytes responding to acetylcholine receptor.
Authors: Kaul R , Wu B , Goluszko E , Deng C , Dedhia V , Nabozny G H , David C S , Rimm I J , Shenoy M , Haqqi T M , Christadoss P
Published: December 2022

Multiple TCRBV genes have been implicated in experimental autoimmune myasthenia gravis (EAMG) pathogenesis in susceptible H-2(b) strains of mice. We studied the contribution of specific TCRBV and AV genes in EAMG pathogenesis using B10.BV8S2 transgenic mice (H-2[b]). The TCR transgenic mice predominantly have TCRBV8S2 transgene, but can use any of the endogenous AV gene repertoire. The transgenic mice were immunized with acetylcholine receptor (AChR) in CFA and evaluated for EAMG pathogenesis. Although the lymphocyte responses to AChR in B10.BV8S2 transgenic and nontransgenic TCR wild-type mice were equivalent, a marked reduction in lymphocyte response to the dominant AChR alpha chain peptide 146-162 was observed in the TCR transgenic mice. After boosting with AChR in CFA, anti-AChR Abs were detected in the serum, and 14 of 42 (33%) of the TCR transgenic mice developed clinical EAMG. Furthermore, EAMG in TCR transgenic mice was prevented by treatment with mAb to TCRBV8, which depleted BV8-expressing T cells. Cloning and sequencing of TCRAV genes from AChR-reactive T cells from B10.BV8S2 transgenic mice revealed a pattern of restricted TCRAV gene usage. The majority (60%) of the clones sequenced showed a sequence identical with that of the TCRAV1S8 gene. In the normal spleen cells of TCR transgenic mice, AV gene usage was more random. Thus, despite the presence of a complete endogenous TCRAV repertoire in B10.BV8S2 transgenic mice, T cells responding to AChR preferentially used a single endogenous TCRAV gene, thus implicating the involvement of the TCRAV1S8 gene in EAMG pathogenesis.
Functional dissection of systemic lupus erythematosus using congenic mouse strains.
Authors: Morel L , Mohan C , Yu Y , Croker B P , Tian N , Deng A , Wakeland E K
Published: December 2022

We describe the in vivo phenotypes associated with three genomic intervals containing systemic lupus erythematosus (SLE)-susceptibility genes derived from the SLE-prone NZM2410 strain on a C57BL/6 genome. These intervals were identified previously via a genome-wide analysis of SLE susceptibility in a (NZM2410 x C57BL/6)F1 x NZM2410 backcross, and transferred independently on a C57BL/6 background to produce three congenic strains: B6.NZMc1 carrying Sle1, B6.NZMc4 carrying Sle2, and B6.NZMc7 carrying Sle3. B6.NZMc1 develops high titers of IgG anti-nuclear autoantibodies in the absence of any severe nephritis. B6.NZMc4 spontaneously develops elevated levels of IgM, but not IgG Abs against several Ags, indicative of polyclonal activation or polyreactivity affecting the B cell lineage. B6.NZMc7 causes the production of IgM and IgG Abs against both nuclear and non-nuclear Ags and the development of severe lupus nephritis. Therefore, our results show that three defined genomic intervals from the NZM2410 SLE-prone strain each contribute specific component phenotypes that have been associated with SLE, which in combination can mediate severe disease.
Plasmin-induced proteolysis of tenascin-C: modulation by T lymphocyte-derived urokinase-type plasminogen activator and effect on T lymphocyte adhesion, activation, and cell clustering.
Authors: Gundersen D , Trân-Thang C , Sordat B , Mourali F , Rüegg C
Published: December 2022

Proteolysis and remodeling of the extracellular matrix occur physiologically in processes such as tissue morphogenesis and repair and may participate in the regulation of complex cell functions, including proliferation and differentiation. While matrix degradation appears to be relevant to T lymphocyte migration through tissues, little is known about whether degraded matrix affects T lymphocyte function. We have studied the interaction between T lymphocytes and tenascin-C (TN-C), a matrix protein we have previously reported to inhibit T lymphocyte activation, in the context of plasmin-induced degradation. Here we report that plasmin efficiently cleaves TN-C. Peripheral blood T lymphocytes stimulated with phorbol ester, anti-CD28, or anti-CD3 Ab, induce, within 24 to 48 h, a strong plasminogen-dependent proteolysis of TN-C. We demonstrate that stimulated T lymphocytes activate plasminogen by secreting the urokinase-type plasminogen activator (u-PA). Plasminogen activation by T lymphocyte-derived u-PA occurs efficiently in fluid phase in the absence of cells. We investigate the consequences of plasmin-induced proteolysis on three of the effects of TN-C in relation to lymphocyte functions. Plasmin proteolysis converts TN-C from a nonadhesive into an adhesive substrate for T lymphocytes and abolishes its aggregating activity on PBMC. In contrast, the inhibitory effect of TN-C on T lymphocyte activation remains unaffected. These observations demonstrate that stimulated T lymphocytes induce plasminogen-dependent proteolysis of TN-C by secreting u-PA and suggest that proteolysis of TN-C may represent a mechanism by which to regulate some of its effects on T lymphocyte functions.
Polarization of IL-4- and IFN-gamma-producing CD4+ T cells following activation of naive CD4+ T cells.
Authors: Nakamura T , Kamogawa Y , Bottomly K , Flavell R A
Published: December 2022

Naive CD4+ T cells initially transcribe both IL-4 and IFN-gamma when stimulated with either the mitogen Con A or Ag in the presence of IL-4 or IL-12 and, therefore, appear uncommitted as to the pathway of differentiation they will follow. However, when stimulated either with Con A or with Ag in the presence of IL-4, CD4+ T cells become primed to follow the Th2 differentiation pathway, and we show now that by 48 h of culture in this environment these cells extinguish IFN-gamma gene transcription. Likewise, priming in the presence of IL-12 leads to the development of Th1 cells, which switch off the expression of the IL-4 gene. To clarify the Th1 differentiation pathway, we performed ablation studies using IL-4 thymidine kinase transgenic mice. When the antiviral drug ganciclovir was added 1 day after primary stimulation in the presence of IL-12, IFN-gamma- and IL-4-producing cells were ablated. In contrast, when ganciclovir was added 2 days after primary stimulation, IL-4-producing cells, but not IFN-gamma-producing cells, were ablated. Thus, our studies show that by 48 h after activation, Th1 or Th2 cells have already become polarized to the differentiation pathway that they will follow. As the differentiation toward Th1 and Th2 effector cells proceeds, substantial amounts of IFN-gamma and IL-4 mRNA accumulate, while the mRNAs of the corresponding lineage (i.e., IFN-gamma in the case of Th2 cells, and IL-4 in the case of Th1 cells) diminish to undetectable levels. IL-4R is up-regulated during T cell differentiation by a mechanism mediated mainly by IL-4. The fact that IL-12 priming does not suppress IL-4-dependent IL-4R up-regulation shows that both IL-4 mRNA and cytokine are produced by IL-12-primed naive CD4+ T cells during differentiation into Th1 cells. Naive CD4+ T cells, therefore, begin as uncommitted cells which express both Th1 and Th2 cytokines that rapidly extinguish the expression of the inappropriate cytokine as the commitment toward the effector lineages is made.
Characterization of lidocaine-specific T cells.
Authors: Zanni M P , Mauri-Hellweg D , Brander C , Wendland T , Schnyder B , Frei E , von Greyerz S , Bircher A , Pichler W J
Published: December 2022

To investigate the cellular immune response to the drug lidocaine, we generated T cell lines and clones from the peripheral blood of four patients with proven allergy to lidocaine. The patients had contact dermatitis after topical application of lidocaine, and local swelling or generalized erythema exudativum multiforme after submucosal/subcutaneous injection of lidocaine. Two of three lidocaine-specific T cell lines were oligoclonal and one even became monoclonal, while the simultaneously analyzed immune response to tetanus toxoid was polyclonal. The lidocaine-specific T cell lines cross-reacted to mepivacaine, but not to other local anesthetics (bupivacaine, procaine, oxybuprocaine, and tetracaine). The majority of reactive T cells belonged to the CD4 cell lineage and were MHC class II restricted, but cloning also revealed some MHC class I-restricted CD8+ clones. A total of 2 of 56 lidocaine-specific T cell clones were CD4-CD8- and expressed TCR-gammadelta. The majority of 13 analyzed CD4 clones produced a rather polarized cytokine pattern, with a dominance of Th2-like cytokines showing a high IL-5 production. In addition, three CD4+ and all CD8+ (n = 7) clones secreted high IFN-gamma and low levels of IL-5/IL-4 (Th1-like). The data illustrate that a drug that sensitizes via the skin elicits a heterogeneous T cell response. The high IL-5 production and the participation of specific CD4+CD8+ and even gammadelta+ T cells appear to be distinguishing features of this hapten-specific immune response.
Structure and chromosomal location of the human CD6 gene: detection of five human CD6 isoforms.
Authors: Bowen M A , Whitney G S , Neubauer M , Starling G C , Palmer D , Zhang J , Nowak N J , Shows T B , Aruffo A
Published: December 2022

The CD6 protein has been shown to play important roles in T cell costimulation and adhesion. Recently, variably spliced isoforms of CD6 mRNA have been identified in both human and murine T cells. Here we report on the genomic organization of the human CD6 gene, its chromosomal localization, and the characterization of novel isoforms. Human CD6 is encoded by at least 13 exons. The amino terminal signal sequence, extracellular region, and transmembrane domain are encoded by seven exons, while the cytoplasmic domain of CD6 is encoded by six exons. Each of the three extracellular scavenger receptor cysteine-rich domains is encoded by a separate exon. Fluorescence in situ hybridization studies and screening of a chromosome-specific YAC (yeast artificial chromosome) library revealed that the gene encoding CD6 is located on chromosome 11 at 11q13 in close proximity to the gene encoding the related molecule CD5 and within 600 kb of CD20. Analysis of mRNA transcripts encoding CD6 isolated from mitogen-activated PBMC and from B cells obtained from patients with chronic lymphocytic leukemia revealed the presence of at least five different CD6 transcripts. These transcripts arise via variable splicing of exons encoding the cytoplasmic domain of CD6. The existence of these isoforms suggests that signaling through CD6 could be regulated via alternative splicing of cytoplasmic encoding exons.
An unbiased analysis of V(H)-D-J(H) sequences from B-1a, B-1b, and conventional B cells.
Authors: Kantor A B , Merrill C E , Herzenberg L A , Hillson J L
Published: December 2022

Previous studies conclude that the repertoire of B-1a (CD5+ B) cells is highly restricted. Studies here, which use FACS sorting and single-cell PCR methodology to develop an unbiased representation of the IgH repertoires of B-1a, B-1b, and conventional B cells from the peritoneal cavity, demonstrate that the B-1a cell repertoire is more diverse than previously thought. Furthermore, adult B-1a cells have significantly fewer noncoded nucleotide (N) insertions than conventional B cells. However, B-1a cells are not defined by the absence of these regions, since such insertions are present in two-thirds of B-1a cell transcripts. All three B cell populations use a wide spectrum of V(H), D, and J(H) elements and display considerable diversity in complementarity-determining region 3 (CDR3). However, characteristic differences in the repertoires of all three B cell populations also exist, suggesting different selective and/or developmental forces act to shape each repertoire.
The 3' untranslated region of IL-1beta regulates protein production.
Authors: Kern J A , Warnock L J , McCafferty J D
Published: December 2022

IL-1beta, a pro-inflammatory cytokine, has sequences in its 3' untranslated region (UTR) that may play a role in the post-transcriptional regulation of IL-1beta production. To test this hypothesis, a series of chimeric reporter genes were developed consisting of a chloramphenicol acetyltransferase (CAT) gene the native 3'-UTR of which was replaced by the full-length IL-1beta 3'-UTR or various 3'-UTR deletion mutants. Expression of these constructs under the SV40 late promoter in THP-1 cells showed that the full-length 3'-UTR repressed constitutive CAT activity to 28% of control CAT activity. Further analysis of the 3'-UTR localized the repressor signal to an adenosine-thymidine (AdT)-rich region. Upon exposure to LPS, the full-length IL-1beta 3'-UTR mediated almost a fivefold increase in CAT activity. The LPS response was not simply loss of AdT-mediated repression; when this sequence was tested alone, it did not respond to LPS. The LPS response effect was localized to the terminal 177 base pairs of the IL-1beta 3'-UTR. The increase in CAT activity following LPS stimulation was associated with an increased CAT.IL-1beta 3'-UTR mRNA half-life, indicating at least one effect of the 3'-UTR on a post-transcriptional process. These studies demonstrate that the IL-1beta 3'-UTR is involved in the regulation of IL-1beta protein production, and a LPS response element may be in the IL-1beta 3'-UTR.
Inhibitory effect of growth hormone on TNF-alpha secretion and nuclear factor-kappaB translocation in lipopolysaccharide-stimulated human monocytes.
Authors: Haeffner A , Thieblemont N , Déas O , Marelli O , Charpentier B , Senik A , Wright S D , Haeffner-Cavaillon N , Hirsch F
Published: December 2022

Several studies have pointed to a link between immune and endocrine systems, including a regulatory function of GH on monocyte activation. The present study demonstrates that human THP-1 promonocytic cells, engineered by gene transfer to constitutively produce human growth hormone (hGH), secreted depressed amounts of TNF-alpha in response to challenge by LPS. The effect of GH appears to occur in an autocrine fashion, since the inhibitory effect on TNF-alpha secretion by constitutive GH production could be abolished in the presence of anti-hGH mAb. The GH-induced inhibitory effect was also observed using normal human monocytes and monocyte-derived macrophages. Inhibition of TNF-alpha production by THP-1-hGH-transfected cells cultured in the presence of LPS is dependent on a selective pathway, since no inhibition of TNF-alpha production was observed when cells were cultured in the presence of PMA. Inhibition of TNF-alpha secretion by LPS-stimulated THP-1-hGH cells was associated with a decrease in nuclear translocation of nuclear factor-kappaB. The capacity of GH to inhibit LPS-induced TNF-alpha production by monocytes without altering other pathways leading to TNF-alpha production may be of potential relevance in septic shock, since GH is available for clinical use.
Molecular cloning of rat C4b binding protein alpha- and beta-chains: structural and functional relationships among human, bovine, rabbit, mouse, and rat proteins.
Authors: Hillarp A , Wiklund H , Thern A , Dahlbäck B
Published: December 2022

The C4b binding protein (C4BP) functions as a regulator of the complement system by interacting with the activated form of the fourth complement component, C4b. Human C4BP also interacts with the anticoagulant protein S and the serum amyloid P component (SAP). It is composed of seven identical 70-kDa alpha-chains and one 45-kDa beta-chain. The alpha-chain contains a binding site for C4b, whereas the beta-chain contains the protein S binding site. Recent studies have shown rabbit and bovine plasma to lack a C4BP-protein S complex, and the mouse beta-chain gene to have evolved into a pseudogene. Using a gel filtration chromatography system in combination with Western blotting, we detected a complex between C4BP and protein S in rat plasma, similar to the complex known in human plasma. Using purified rat C4BP and SAP we were unable to detect any complex between the two proteins, but rat C4BP was able to form a complex with human SAP. Rat cDNA clones encoding the C4BP alpha- and beta-chains were isolated from a rat liver cDNA library. The rat alpha-chain cDNA predicted a mature polypeptide chain of 545 amino acid residues, whereas the beta-chain cDNA predicted a mature polypeptide of 243 amino acid residues. The overall amino acid sequence identities between the rat alpha-chain and the mouse, human, rabbit, and bovine alpha-chains were 64, 60, 59, and 52%, respectively. The identities between the rat beta-chain and the human and bovine beta-chains were 68 and 57%, respectively. The rat represents the first non-primate species in which the C4BP-protein S interaction has been found to be conserved.
Expression of IL-5 in thymocytes/T cells leads to the development of a massive eosinophilia, extramedullary eosinophilopoiesis, and unique histopathologies.
Authors: Lee N A , McGarry M P , Larson K A , Horton M A , Kristensen A B , Lee J J
Published: December 2022

Transgenic mice were generated using regulatory elements from the CD3delta gene to drive T cell expression of IL-5. Expression of this cytokine resulted in white blood cell counts that expand virtually unabated (approximately 400,000 cells/mm3). This expansion is characterized by a profound eosinophilia (>60%) and commensurate increases in the absolute numbers of all other white blood cell types. In particular, circulating B220+ B lymphocyte populations increased >30-fold over wild-type (+/+) levels. Cell differentials and expression studies using a marker for eosinophil precursor cells (major basic protein gene expression) suggest that the peripheral eosinophilia is induced primarily through the establishment of extramedullary sites of eosinophilopoiesis. These mice display a massive peritoneal cavity cell exudate (1-2 x 10(8) cells) dominated by eosinophils (approximately 50%) and the infiltration of eosinophils in nearly all organ systems. Sudden unexplained death occurs in 70% of all transgenic animals by 12 mo of age. Surviving transgenic animals display severe inflammatory pathologies that include ulcerating skin lesions as well as lower bowel inflammation. These pathologies parallel clinical observations of patients with a profound eosinophilia and imply that IL-5 effector functions during some inflammatory responses may be contingent upon peripheral lymphohemopoietic expression.
Regulation of lung fibroblast alpha-smooth muscle actin expression, contractile phenotype, and apoptosis by IL-1beta.
Authors: Zhang H Y , Gharaee-Kermani M , Phan S H
Published: December 2022

IL-1 is important in regulating lung inflammation and potentially fibrosis as well. To clarify the role of this cytokine vis-à-vis changes in lung fibroblast phenotype in pulmonary fibrosis, the effects of IL-1beta on isolated lung fibroblasts were examined. Rat lung fibroblasts were treated with increasing doses of IL-1beta and examined for effects on cell number, alpha-smooth muscle actin expression, apoptosis, nitric oxide (NO) production, and contractility in collagen gels. The results show that IL-1beta caused dose-dependent down-regulation of alpha-smooth muscle actin protein and mRNA expression. The kinetics of mRNA inhibition was rapid and preceded the effects on protein expression. This IL-1beta-induced decrease in actin expression was associated with inhibition of contractility evaluated using fibroblast-populated collagen gels. Since IL-1beta inhibition of actin expression was accompanied by reduction in cell number, the effect on apoptosis was examined. Significant increase in the number of apoptotic nuclei and DNA fragmentation was observed upon IL-1beta treatment, with a dose-response curve that mirrored that for the decline in actin-positive cells. More than one-half of the apoptotic cells were actin positive at high IL-1beta doses, suggesting that the actin-expressing cells may be more susceptible to IL-1beta-induced apoptosis. IL-1beta also induced NO production in these cells, which was inhibited by NG-monomethyl-L-arginine. Similarly, IL-1beta-induced apoptosis and inhibition of actin expression were inhibited by this arginine analogue. Hence, induction of apoptosis by IL-1beta via NO production may be an important mechanism for regulating lung fibroblast alpha-smooth muscle actin expression, and consequently its contractile phenotype as well.
Linomide administration to mice attenuates the induction of nitric oxide synthase elicited by lipopolysaccharide-activated macrophages and prevents nephritis in MRL/Mp-lpr/lpr mice.
Authors: Hortelano S , Díaz-Guerra M J , Gonzalez-García A , Leonardo E , Gamallo C , Boscá L , Martínez-A C
Published: December 2022

Nitric oxide is involved as a messenger molecule in a large number of physiologic and pathologic responses. Local generation of high nitric oxide output through the expression of the calcium-independent, cytokine-inducible form of nitric oxide synthase (iNOS) can result in either protective or damaging effects. The development of drugs that specifically suppress iNOS expression or inhibit its activity may therefore provide an excellent therapeutic tool for treatment of a diverse set of dysfunctions, including asthma, inflammatory processes, and autoimmune disease. We show compelling evidence that linomide, an immunomodulator known to ameliorate autoimmune diseases, prevents accumulation in the macrophages of mRNA encoding iNOS in mice injected with LPS. This effect is partially mediated by the blocking of TNF-alpha and IL-beta production by activated macrophages. Here, we also present evidence that kidneys from MRL/Mp-lpr/lpr mice express high iNOS levels when the mice develop glomerulonephritis. The administration of linomide to MRL/Mp-lpr/lpr mice significantly decreases iNOS mRNA levels and prevents the development of glomerulonephritis, extending the half-life of mice of this strain. This linomide effect is compatible with its role in preventing the development of autoimmune disease and extends its possible use to other pathologic manifestations associated with iNOS expression, such as the systemic lupus erythematosus-associated glomerulonephritis present in MRL/Mp-lpr/lpr mice.
Targeted expression of the neuropeptide calcitonin gene-related peptide to beta cells prevents diabetes in NOD mice.
Authors: Khachatryan A , Guerder S , Palluault F , Cote G , Solimena M , Valentijn K , Millet I , Flavell R A , Vignery A
Published: December 2022

To investigate whether the immunosuppressive neuropeptide calcitonin gene-related peptide (CGRP) was a potential candidate for tissue-specific gene therapy, we engineered nonobese diabetic (NOD) mice to produce CGRP in beta cells by placing the modified calcitonin gene under the control of the rat insulin promoter. CGRP inhibits CD4 T cell production of the cytokines that have been implicated in the pathogeny of type I diabetes. Three transgene-positive mouse lines were obtained, two of which expressed immunoreactive CGRP in beta cells (NOD-CGRP mice). Isolated islets from one of these two transgene-positive founders produced active CGRP, whereas islets from transgene-negative littermates did not. The production of CGRP by beta cells prevented insulin-dependent diabetes mellitus in male and reduced its incidence by 63% in female mice. This prevention was due to a local immunosuppressive effect of CGRP as no difference was detected between NOD-CGRP and NOD littermate lymph node, spleen, and thymus cells by either FACS analysis or proliferative response to stimulation by Ag, alloantigen or anti-CD3. These data suggest that CGRP is a potential therapeutic molecule to prevent or treat diabetes and possibly other diseases and conditions in which immune cells are involved. These data also suggest that endogenous CGRP concentrated in sensory nerve endings may regulate locally the immune response, further strengthening the importance of the functional neuroimmune link.
Cutting edge: CD4+ T cells kill CD8+ T cells via Fas/Fas ligand-mediated apoptosis.
Authors: Piazza C , Gilardini Montani M S , Moretti S , Cundari E , Piccolella E
Published: December 2022

To explore the possibility that CD4+ T cells, described to mediate the elimination of themselves or B lymphocytes, could also mediate the elimination of CD8+ T cells, we analyzed apoptotic phenomena in cocultures of CD4+ and CD8+ autologous T cell lines. The data show that CD8+ T cells were lysed by activated CD4+ helper T cells by a Fas/FasL-mediated mechanism. CD4+ T cells were not lysed by activated CD8+ T cells, although Fas and FasL were equally expressed and anti-Fas Abs induced apoptosis in both CD4+ and CD8+ T cell populations. The results allowed us to speculate that CD4+ T cells not only help CD8+ T lymphocytes to mature into effector killer cells and to sustain this function but can also limit their growth.
MHC affinity, peptide liberation, T cell repertoire, and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL.
Authors: Deng Y , Yewdell J W , Eisenlohr L C , Bennink J R
Published: December 2022

MHC class I-restricted T cell responses to viral proteins focus on a limited set of peptides. To better understand this phenomenon, we examined all of the 26 nonameric peptides encoded by the influenza virus A/Puerto Rico/8/34 (PR8) conforming to the canonical Kd binding motif. Ten peptides bound strongly to Kd as assessed by a cell surface stabilization assay. Five of these 10 induced in vitro secondary CD8+ T cell responses from splenocytes derived from PR8-immunized mice. The strongest responses were induced by the two previously defined antigenic peptides, which ranked only second and fifth in relative binding affinity. To examine the limiting factors in the immunogenicity of Kd-binding peptides, we produced recombinant vaccinia viruses (rVVs) expressing cytosolic or endoplasmic reticulum (ER)-targeted peptides. rVVs expressing ER-targeted versions of the 7 peptides with the highest relative affinities for Kd rescued Kd cell surface expression in T2 cells, while those expressing the 3 lowest affinity peptides did not. The immunogenicity of several, but not all, of the highest affinity peptides was greatly enhanced when expressed as VV-encoded cytosolic or ER-targeted peptides as compared with full length proteins. We conclude that limitations in the immunogenicity of class I binding peptides reflects, in order of decreasing importance, peptide liberation by cellular proteases, T cell repertoire, and TAP-mediated peptide transport. We also observed an additional important contributing factor: suppression of T cell responses to nondominant peptides by an immunodominant peptide located in the same protein.
A major role for matrix metalloproteinases in T cell injury in the gut.
Authors: Pender S L , Tickle S P , Docherty A J , Howie D , Wathen N C , MacDonald T T
Published: December 2022

Activated lamina propria T cells responding to luminal Ags are thought to be important in celiac disease and Crohn's disease, and T cells responding to foreign MHC products are also important in intestinal graft-vs-host disease and intestinal transplant rejection. However, the mechanism(s) by which T cells mediate damage in the gut is not known. We have previously shown that activation of lamina propria T cells by PWM in explant cultures of second trimester human small intestine produces severe tissue injury, with epithelial cell shedding and loss of villi. In this study, we have investigated the role of matrix metalloproteinases in this system. Organ culture supernatants of explants stimulated with PWM showed a 3-fold increase in the concentration of interstitial collagenase and a 10-fold increase in stromelysin-1 compared with control explant culture supernatants. Tissue inhibitors of metalloproteinase-1 and -2 concentrations were unchanged. Increased metalloproteinase enzymatic activity was detected by gelatin and casein zymography. Western blotting revealed the active forms of interstitial collagenase and stromelysin-1 in PWM-stimulated culture supernatants. Up-regulation of mRNA for interstitial collagenase, stromelysin-1, and gelatinase-B was also seen. Nanomolar amounts of recombinant stromelysin-1 added directly to explants produced rapid severe tissue injury. PWM-induced mucosal injury was inhibited by a synthetic peptidomimetic inhibitor of matrix metalloproteinases. Mesenchymal cells isolated from the mucosa of human fetal small intestine produced increased amounts of interstitial collagenase, gelatinase A, and stromelysin-1 when stimulated with IL-1beta or TNF-alpha. These results suggest that T cell activation in the lamina propria results in increased production of matrix metalloproteinases, which by degrading the lamina propria matrix represent a major pathway by which T cells cause injury in the gut.
Class I-deficient hemopoietic cells and nonhemopoietic cells dominantly induce unresponsiveness of natural killer cells to class I-deficient bone marrow cell grafts.
Authors: Wu M F , Raulet D H
Published: December 2022

NK cells in normal mice reject bone marrow transplants from class I-deficient mice. In contrast, class I-deficient mice do not reject autologous cells, suggesting that NK cell tolerance is acquired. We employed fetal liver irradiation chimeras to assess two potential mechanisms that could account for the tolerance of NK cells in class I-deficient mice to class I-deficient cells: 1) a positive model, in which recognition of class I+ cells molecules by NK cells is necessary to induce functional NK cell maturation; and 2) a negative model, in which interactions of NK cells with class I-deficient cells induce tolerance. In class I+ chimeras reconstituted with mixtures of class I+ and class I-deficient fetal liver cells, the rejection of class I-deficient bone marrow cell grafts was significantly impaired, supporting the negative model. We further addressed whether nonhemopoietic cells are also able to induce NK cell tolerance. Class I- mice reconstituted with class I+ fetal liver cells were tolerant of class I-deficient cells, favoring this idea. Furthermore, class I-deficient mice reconstituted with a mixture of class I-deficient and class I+ fetal liver cells were more tolerant to class I-deficient cells than were class I+ mice reconstituted with the same fetal liver cell mixture. These results suggest that maximal tolerance induction requires the presence of class I-deficient nonhemopoietic cells.
T cell priming against vesicular stomatitis virus analyzed in situ: red pulp macrophages, but neither marginal metallophilic nor marginal zone macrophages, are required for priming CD4+ and CD8+ T cells.
Authors: Ciavarra R P , Buhrer K , Van Rooijen N , Tedeschi B
Published: December 2022

Since extensive degradation may be required to present complex Ags, we addressed whether macrophages (M phi) might function as APC for anti-viral cell-mediated immune responses. To study this question, murine splenic M phi were depleted by i.p. administration of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP-liposomes or clodronate-liposomes) before priming mice with vesicular stomatitis virus (VSV). Cl2MDP-liposome treatment resulted in the rapid (1-day) depletion of splenic M phi that was associated with a suppression of the ability of M phi-deficient mice to generate secondary anti-VSV CTL and Th cell proliferative responses in vitro. Control studies demonstrated that splenic dendritic cells were not adversely affected by treatment with Cl2MDP-liposomes. To assess the contribution of splenic M phi subpopulations to T cell priming against this virus, priming was delayed following treatment with Cl2MDP-liposomes until specific M phi subsets had repopulated the spleen. This analysis revealed that repopulation by red pulp M phi, but not with other splenic M phi subsets, was associated with the ability to mount normal secondary CTL and Th cell responses against VSV. Depletion of splenic, but not resident, peritoneal M phi by i.v. injection of Cl2MDP-liposomes did not rescue T cell priming in VSV-infected mice. Thus, only red pulp M phi, and not other splenic or peritoneal M phi populations, are necessary for T cell priming to VSV, a biochemically complex Ag.
Activation of the lymphotoxin beta receptor by cross-linking induces chemokine production and growth arrest in A375 melanoma cells.
Authors: Degli-Esposti M A , Davis-Smith T , Din W S , Smolak P J , Goodwin R G , Smith C A
Published: December 2022

The lymphotoxin beta receptor (LT beta R) was originally described as a transcribed sequence encoded on human chromosome 12p, with homology to the TNF receptor family. Subsequently, a recombinant LT beta R was shown to bind LT alpha LT beta heteromeric complexes. In this study, we have shown that LT beta R is expressed in a variety of tissues and cell lines of monocytic lineage, as well as in fibroblast and human melanoma cell lines. Unlike other members of the TNF receptor family, LT beta R is not expressed by peripheral blood T cells. A chimeric fusion protein consisting of the extracellular domain of LT beta R fused to the Fc region of human IgG1 was used to develop mAbs against LT beta R. Cross-linking LT beta R on A375 melanoma cells with these Abs generated an antiproliferative signal. In addition, the IL-8 and RANTES chemokines, early indicators of inflammation, were secreted by the A375 melanoma line and the WI38VA13 fibroblast line in response to cross-linking of LT beta R. These same activities could be induced by membrane-bound and soluble LT beta and LT alpha LT beta oligomers.
Mechanism of complement inactivation by glycoprotein C of herpes simplex virus.
Authors: Kostavasili I , Sahu A , Friedman H M , Eisenberg R J , Cohen G H , Lambris J D
Published: December 2022

Glycoprotein C (gC) of both herpes simplex virus type 1 (HSV-1) and HSV-2 interacts with complement C3b and protects the virus from complement-mediated neutralization. To study the mechanism by which gC modulates complement activation, we expressed both gC-1 and gC-2 in a baculovirus expression system. Baculovirus recombinants containing gC genes spanning the entire gC-1 sequence (gC-1-TMR) or only the extracellular domain(s) of gC-1, gC-2, or a deletion mutant of gC-1 lacking residues 33 through 123 were expressed in sf9 insect cells. Binding of the expressed proteins to human C3 and C3 fragments was assessed by direct and competition ELISA. All four expressed proteins bound to C3, C3b, and C3c but not to C3d, suggesting 1) that the binding sites for these proteins are located in the C3c region of C3; and 2) that gC, in contrast to other C3-binding proteins, interacts with native C3. We have also examined the interaction of native C3 with gC-1 expressed on the HSV-1-infected cells. Analogous to recombinant proteins, gC-1 expressed on the infected cells also bound to native C3. The ability of baculovirus-expressed gCs to inhibit the interaction of C3b with its ligands was also analyzed. We found that gC-1, but not gC-2, inhibited the binding of C5 and properdin to C3b and also inhibited the alternative pathway-mediated lysis of rabbit erythrocytes. Inhibition of alternative pathway-mediated lysis and properdin binding to C3b, but not of C5 binding to C3b, required the transmembrane segment of the gC-1. The specificity of gC interactions was examined by studying the interaction of gC with C3 from various species. In contrast to properdin, both gCs bound to cobra C3; this finding suggests that gC-1 and properdin bind to different sites on C3b. Further analyses suggested that gC-1 sterically hindered access of C5 and properdin to C3b.
Expression of lung inducible nitric oxide synthase protein does not correlate with nitric oxide production in vivo in a pulmonary immune response against Cryptococcus neoformans.
Authors: Lovchik J , Lipscomb M , Lyons C R
Published: December 2022

Mice infected intratracheally with Cryptococcus neoformans (Cne) require CD4 and CD8 T cells, IFN-gamma, and M phi production of nitric oxide (NO) for effective resolution of the pulmonary infection. Differences exist among strains of mice in clearing the infection. C.B-17 mice reduced Cne lung burden at a significantly greater rate than C57BL/6 (B6) mice and resistance correlated with greater IFN-gamma production by C.B-17 lung-associated lymph node cells. We examined whether the differences observed in the ability of B6 vs C.B-17 mice to clear Cne was due to 1) numbers of inflammatory cells recruited to the lung, 2) the activation state of the recruited cells as measured by expression of inducible nitric oxide synthase (iNOS), and/or 3) the in vivo production of NO as measured by quantitating urine nitrates. The level of iNOS protein was identical in lungs from both strains of mice during Cne infection as determined by Western blot analysis of whole lung homogenates and immunocytochemistry of isolated lung macrophages. Surprisingly, in vivo studies of iNOS activity indicated that NO production in B6 mice was significantly less than that in C.B-17 mice. While single cell suspensions from lungs of either mouse strain produced identical amounts of NO, NO production by lung explants paralleled in vivo urinary nitrate excretion, suggesting that the maintenance of pulmonary architecture and cell-cell interaction was necessary for suppression of iNOS activity in B6 mice. These data strongly implicate the existence of mechanisms that regulate NO production at the level of enzyme activity during infections and have important implications for analyzing the role of iNOS during an immune response in in vivo models.
Protective monoclonal antibody defines a circumsporozoite-like glycoprotein exoantigen of Cryptosporidium parvum sporozoites and merozoites.
Authors: Riggs M W , Stone A L , Yount P A , Langer R C , Arrowood M J , Bentley D L
Published: December 2022

The apicomplexan protozoan parasite Cryptosporidium parvum causes a diarrheal disease in humans and other mammals for which specific therapy and immunoprophylaxis are unavailable. Passive immunization with Abs against whole C. parvum organisms has variable efficacy in immunocompromised or neonatal hosts. Because apical and surface-exposed zoite Ags of the Apicomplexa are critical to infectivity and targets of protective immunity, we examined the ability of mAbs generated against such Ags in C. parvum sporozoites to passively protect against infection and identify biologically relevant parasite molecules. A panel of mAbs was produced against affinity-purified native Ags using sporozoite apical- and surface-reactive mAb C4A1 as binding ligand. One resulting mAb, designated 3E2, elicited prominent morphologic changes in sporozoites and merozoites characterized by rapid and progressive formation, posterior movement, and release of membranous Ag-mAb precipitates. These changes had a striking resemblance to the malarial circumsporozoite precipitate (CSP) reaction. Sporozoite infectivity was completely neutralized after in vitro exposure to 3E2 and the CSP-like reaction. Furthermore, orally administered 3E2 completely prevented or markedly reduced infection in neonatal BALB/c mice. 3E2 bound to apical complex and surface molecules of zoites and was demonstrated in membranous precipitates by immunoelectron microscopy. In Western blots, 3E2 recognized multiple 46 to approximately 770 kDa sporozoite Ags and an approximately 1300-kDa Ag designated CSL, also expressed by merozoites. CSL was characterized as a soluble glycoprotein exoantigen released by infectious sporozoites. Further, CSL was determined to be the molecular species mechanistically involved in the CSP-like reaction by its identification in SDS-PAGE gels and Western blots of purified membranous precipitates. These findings indicate that CSL has a functional role in sporozoite infectivity and is a candidate molecular target for passive or active immunization against cryptosporidiosis.
Identification of subdominant CTL epitopes of the GP100 melanoma-associated tumor antigen by primary in vitro immunization with peptide-pulsed dendritic cells.
Authors: Tsai V , Southwood S , Sidney J , Sakaguchi K , Kawakami Y , Appella E , Sette A , Celis E
Published: December 2022

The gp100 melanoma-associated tumor Ag was selected as a model system to study the diversity of human antitumor cytotoxic T cell responses. First, peptides corresponding to dominant gp100 HLA-A2.1-restricted CTL epitopes were tested using lymphocytes from normal volunteers and an in vitro priming protocol that uses peptide-pulsed dendritic cells as APCs and IL-7 and IL-10 as immune-enhancing cytokines. High CTL activity toward both peptide-pulsed target cells and gp100+ melanoma cells was obtained with four out of five peptides tested. Second, HLA-A2.1-binding peptides from gp100 that do not appear to represent CTL epitopes in melanoma patients were also tested for their capacity to induce CTL using the in vitro priming protocol. Three of six peptides tested induced CTL in lymphocytes from normal volunteers. One of these peptides was also immunogenic for lymphocytes derived from a melanoma patient in remission. Because these three CTL epitopes were not recognized in the natural immune response in melanoma patients but do appear as immunogens when peptides are used to induce the T cell response, they may be considered as typical "subdominant" epitopes. The results are discussed in the context of the usefulness of this approach to detail the immunologic potential of a given tumor-associated Ag and its relevance for the design of effective immune-based therapies.
Phosphatidyl serine is involved in the reduced rate of transcription of the inducible nitric oxide synthase gene in macrophages from tumor-bearing mice.
Authors: DiNapoli M R , Calderon C L , Lopez D M
Published: December 2022

Upon stimulation with LPS, peritoneal-elicited macrophages (PEM) from mammary tumor-bearing mice display a diminished ability to produce nitric oxide (NO) and lyse tumor targets. In contrast, when these cells are stimulated with LPS in combination with IFN-gamma, they perform these functions at normal levels. Kinetic studies revealed that these defects became more pronounced with tumor progression and were accompanied by similar changes in inducible nitric oxide synthase (iNOS) mRNA levels. Since this tumor is known to produce PGE2, granulocyte-macrophage CSF (GM-CSF), and phosphatidyl serine, we evaluated the effects of these products on NO production and cytolytic activity. Pretreatment of normal PEM with PGE2 or recombinant GM-CSF had negligible effects on NO production and cytolytic capacity. In contrast, phosphatidyl serine caused a concentration-dependent inhibition of these functions in response to LPS, which could be partially overcome by the addition of IFN-gamma. Moreover, iNOS mRNA levels paralleled these changes and were analogous to the alterations observed in the tumor-bearers' PEM. iNOS mRNA stability was not reduced in these cells; however, the rate of transcription was diminished relative to normal levels, suggesting that the defects causing these alterations are occurring at or before the level of iNOS transcription. These data implicate tumor-derived phosphatidyl serine in the alterations observed in tumor-bearers' macrophages and suggest that reduced iNOS transcription is responsible for the diminished capacity of these macrophages to produce NO and lyse tumor targets.
Stimulation of macrophages by lipopolysaccharide alters the phosphorylation state, conformation, and function of PU.1 via activation of casein kinase II.
Authors: Lodie T A , Savedra R , Golenbock D T , Van Beveren C P , Maki R A , Fenton M J
Published: December 2022

We previously reported that LPS stimulation of the RAW264.7 murine macrophage cell line rapidly induced a structural change within the N terminus of the transcriptional regulatory factor PU.1. PU.1 is required for the expression of a variety of cytokine, cytokine receptor, and integrin genes. Western blot analysis of nuclear extracts prepared from LPS-stimulated macrophages revealed increased phosphorylation of PU.1 at serine residues relative to that in unstimulated controls. PU.1-DNA complexes prepared using nuclear extracts from LPS-stimulated macrophages were less sensitive to protease digestion compared with PU.1-DNA complexes generated using nuclear extracts prepared from unstimulated cells. This altered protease sensitivity probably reflects a conformational change within PU.1 resulting from LPS-induced phosphorylation. This possibility was supported by the finding that in vitro-phosphorylated PU.1 was similarly resistant to protease digestion. Transient transfection studies suggest that LPS-induced phosphorylation of PU.1 at serine 148, located within a casein kinase II (CKII) consensus motif, increases the transactivation function of PU.1. Other serine/CKII sites located at positions 41, 45, 132, and 133 do not appear to be required for LPS-induced PU.1 function. Lastly, we found that LPS also increased the enzymatic activity of CKII in these cells. To our knowledge, these are the first studies to demonstrate that LPS can stimulate CKII activity, induce PU.1 phosphorylation, and enhance the capacity of PU.1 to activate transcription in macrophages.