bluuebunny/crossref_metadata_2025 huggingface dataset:
HLA-Cw3 expression on porcine endothelial cells protects against xenogeneic cytotoxicity mediated by a subset of human NK cells.
Authors: Seebach J D , Comrack C , Germana S , LeGuern C , Sachs D H , DerSimonian H
Published: December 2022

There is increasing evidence that NK cells make an important contribution to human anti-porcine xenogeneic cytotoxicity. Most allogeneic as well as autologous normal cells are not susceptible to NK cell-mediated cytotoxicity because they express inhibitory molecules encoded within the MHC class I loci. The protective signal is delivered to NK cells through killer cell-inhibitory receptors expressing different MHC class I specificities. It has been proposed that xenogeneic target cells may be susceptible to NK cell-mediated lysis because their MHC class I molecules fail to be recognized by human killer cell-inhibitory receptors. To explore this hypothesis, we examined the effect of human MHC class I expression on porcine target cell lysis by human NK cells. An immortalized porcine bone marrow-derived endothelial cell line (2A2) was transfected with three different human MHC class I allelic genes (HLA-A2, -B27, or -Cw3). The cytotoxic activity of several GL183+ NK clones, which lysed untransfected porcine cells effectively, was substantially blocked by the presence of HLA-Cw3. In contrast, HLA-Cw3-positive cells were not protected against lysis by GL183- EB6+ NK clones. The expression of HLA-B27 or HLA-A2 molecules on pig target cells did not provide substantial protection from lysis by any of the NK clones tested. In addition to confirming the hypothetical basis of NK cell-mediated killing of xenogeneic targets, these results have practical implications as an approach to overcoming NK cell-mediated cytotoxicity, which may be an obstacle to pig-to-human xenotransplantation.
Nuclear localization of RelB is associated with effective antigen-presenting cell function.
Authors: Pettit A R , Quinn C , MacDonald K P , Cavanagh L L , Thomas G , Townsend W , Handel M , Thomas R
Published: December 2022

Dendritic cells (DC) are potent APCs that enter resting tissues as precursors and, after Ag exposure, differentiate and migrate to draining lymph nodes. The phenotype of RelB knockout mice implicates this member of the NF kappa B/Rel family in DC differentiation. To further elucidate the role of RelB in DC differentiation, mRNA, intracellular protein expression, and DNA binding activity of RelB were examined in immature and differentiated human DC, as well as other PB mononuclear cell populations. RelB protein and mRNA were detected constitutively in lymphocytes and in activated monocytes, differentiated DC, and monocyte-derived DC. Immunohistochemical staining demonstrated RelB within the differentiated lymph node interdigitating DC and follicular DC, but not undifferentiated DC in normal skin. Active nuclear RelB was detected by supershift assay only in differentiated DC derived from either PB precursors or monocytes and in activated B cells. These RelB+ APC were potent stimulators of the MLR. The data indicate that RelB expression is regulated both transcriptionally and post-translationally in myeloid cells. Within the nucleus, RelB may specifically transactivate genes that are critical for APC function.
IL-4 and IL-13 production in differentiated T helper type 2 cells is not IL-4 dependent.
Authors: Huang H , Hu-Li J , Chen H , Ben-Sasson S Z , Paul W E
Published: December 2022

CD4+ T cell differentiation into cells capable of producing IL-4 and IL-13 (Th2 cells) requires the presence of IL-4 and is STAT-6 dependent. Here we show that IL-4 is not required for IL-4 or IL-13 production by Th2 cells. Anti-IL-4 or anti-IL-4R Ab did not diminish IL-4 production by Th2 cells in response to TCR-mediated stimulation, nor did IL-4 enhance IL-4 production in response to stimulation of Th2 cells with limiting amounts of Ag. Th2 cells prepared from IL-4 knockout mice were capable of producing IL-13 mRNA in response to stimulation with immobilized anti-CD3. IL-4 did not increase IL-13 mRNA expression. Despite the failure of IL-4 to effect IL-4 production by primed Th2 cells, a STAT-6 binding element was demonstrated in the IL-4 promoter. The authenticity of this element was demonstrated by oligonucleotide competition, by supershifting with anti-STAT-6 Ab, and by IL-4-inducible effects on transcription of a reporter gene under the control of a multimerized element fused to an IL-4 minimal promoter. Nonetheless, an IL-4 promoter construct lacking the STAT-6 binding element was as effective as a construct containing this element in anti-CD3-induced reporter transcription. Thus, this element, if biologically active, must function at a step in T cell responsiveness distinct from the acute production of IL-4 by Th2 cells in response to Ag or anti-CD3.
Selective binding of shc-SH2 domain to tyrosine-phosphorylated zeta but not gamma-chain upon CD16 ligation on human NK cells.
Authors: Galandrini R , Palmieri G , Paolini R , Piccoli M , Frati L , Santoni A
Published: December 2022

Fc gamma RIII (CD16) is a hetero-oligomeric receptor composed of a ligand-binding alpha subunit associated with homo- or heterodimers of the TCR zeta- and Fc epsilon RI gamma-chains. We have previously demonstrated that CD16 ligation promotes complex formation between tyrosine-phosphorylated shc and Grb2, leading to activation of ras signaling pathway in human NK cells. Here we report that CD16 engagement induces rapid shc association with the tyrosine-phosphorylated receptor complex in human NK cells. In vitro binding studies demonstrate that this interaction is mediated by the shc-SH2 domain, and immunodepletion experiments indicate that the zeta- but not the gamma-chain has the capability to mediate this association. Jurkat cell clones expressing CD16-zeta or -gamma homodimers have been used to gain more information about the mechanism of shc/CD16 association. Our data show that, while engagement of both receptors induces tyrosine phosphorylation of shc and Grb2 recruitment, shc-SH2/receptor complex association is evident only in CD16-zeta but not in CD16-gamma transfectants. Overall, our data demonstrate that the adaptor protein shc can be recruited to the activated CD16 complex by interaction with tyrosine-phosphorylated zeta-chain in a SH2-dependent manner. These results also provide further support to the notion that zeta- and gamma-chains might couple to different biochemical pathways.
Signal transduction for melatonin in human lymphocytes: involvement of a pertussis toxin-sensitive G protein.
Authors: García-Pergañeda A , Pozo D , Guerrero J M , Calvo J R
Published: December 2022

We analyzed the melatonin signal transduction in human blood lymphocytes. High affinity melatonin receptors were identified by specific binding of 2-[125I]melatonin ([125I]MEL) to human lymphocyte membranes. Scatchard analysis of [125I]MEL binding revealed high affinity receptors, with a dissociation constant (Kd) of 0.45 nM and a binding capacity (Bmax) of 7.8 fmol/mg protein. Specific [125I]MEL binding was reduced markedly by GTP and its nonhydrolyzable analogues guanosine 5'-beta, gamma-imidotriphosphate (Gpp(NH)p) and guanosine 5'-O-(3-triphosphate) (GTP-gamma-S). Gpp(NH)p inhibited in a dose-dependent manner the [125I]MEL specifically bound to human lymphocyte membranes with a half-maximal inhibition (IC50) of 3.5 +/- 0.6 microM Gpp(NH)p. Treatment of human lymphocyte membranes with Gpp(NH)p increased the Kd value (Kd = 1.20 nM). Melatonin inhibited significantly and in a dose-dependent manner forskolin-stimulated cAMP production in intact human lymphocytes. Melatonin was able to stimulate diacylglycerol production in a dose-dependent manner in human lymphocyte membranes. Pertussis toxin treatment inhibited the specific [125I]MEL binding and blocked the ability of melatonin to both inhibit forskolin-stimulated cAMP production and stimulate diacylglycerol production. Pertussis toxin ADP-ribosylation and Western blot experiments demonstrated the protein expression of alpha i1/2, alpha i3/0, and beta gamma complexes of G proteins in human lymphocyte membranes. The results strongly suggest a pertussis toxin-sensitive melatonin signal transduction pathway in human lymphocytes that involves the inhibition of adenylyl cyclase and the stimulation of phospholipase C.
Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells.
Authors: Czerniecki B J , Carter C , Rivoltini L , Koski G K , Kim H I , Weng D E , Roros J G , Hijazi Y M , Xu S , Rosenberg S A , Cohen P A
Published: December 2022

Human peripheral blood contains a small subpopulation of immature dendritic cells (iDC) distinguished from circulating monocytes by their low expression of CD14. We utilized leukapheresis and countercurrent centrifugal elutriation to obtain myeloid origin mononuclear cell (MOMC) fractions of monocytes and iDC for study. These subpopulations were ultrastructurally and immunophenotypically similar before culture. After a 20- to 96-h culture either alone, with recombinant human granulocyte-monocyte CSF, or with endotoxin, greater up-regulation of costimulatory molecule expression was observed among iDC than among monocytes, and only iDC expressed the activation molecule CD83. Treatment with rhIL-4 caused many MOMC to develop morphologic properties of dendritic cells within 96 h, but costimulatory molecule up-regulation and CD14 down-regulation were heterogeneous, and CD83 expression was infrequent. In contrast, calcium ionophore (CI) treatment induced rapid and consistent effects in MOMC from both healthy volunteers and cancer patients, including down-regulated CD14 expression, acquisition of dendritic cell morphologic properties, up-regulated MHC and costimulatory molecule expression, and de novo CD83 expression. Many such effects occurred within 20 h of treatment. CI treatment activated purified CD14+ monocytes and also enhanced the spontaneous activation of purified CD14-/dim iDC in culture. Unfractionated MOMC, purified monocytes, and purified iDC displayed equivalently enhanced T cell-sensitizing efficiency following CI treatment. CD4+ T cell sensitization to keyhole limpet hemocyanin and CD8+ T cell sensitization to MART-1 melanoma-associated peptide were achieved in a single culture stimulation. Therefore, circulating monocytes and iDC can be induced by CI to manifest properties of activated DC, providing large numbers of efficient, nontransformed autologous APC for T cell sensitization strategies.
Molecular basis of HLA-C recognition by p58 natural killer cell inhibitory receptors.
Authors: Kim J , Chwae Y J , Kim M Y , Choi I H , Park J H , Kim S J
Published: December 2022

NK cells express several inhibitory receptors that recognize class I MHC molecules expressed on target cells. The NK cell inhibitory receptors (KIRs) provide a key regulatory function for NK cells via specific interaction with MHC/peptide complexes, but the molecular details for recognition of class I MHC molecules by KIRs still remain unclear. Here we report cDNA cloning and expression of p58 KIRs and a p50 killer cell activatory receptor (KAR) from a Korean blood donor and demonstrate direct binding between recombinant soluble p58 KIRs and recombinant soluble HLA-C molecules. We identified three p58/p50 killer cell receptors (KAR-K1, KIR-K6, and KIR-K7), which are homologous to p50 cl-39, p58 47.11, and p58 cl-6, respectively. Native gel shift assay revealed that p58 KIR-K6 and KIR-K7 bind both HLA-Cw3 and HLA-Cw6 molecules, but p50 KAR-K1 binds neither of the HLA-C molecules. However, binding of HLA-C molecule by p58 KIR is affected by the antigenic peptide bound on the MHC molecule, suggesting that the p58 KIR binding to the HLA-C molecule may be dependent on the peptide. In addition, the binding interaction requires the presence of both p58 Ig domains, suggesting that the binding mode of HLA-C and p58 KIR may have some similarity to that of the neonatal Fc receptor and the Fc fragment of Ab and may be distinct from that of TCR and MHC.
Bovine IgG repertoire is dominated by a single diversified VH gene family.
Authors: Sinclair M C , Gilchrist J , Aitken R
Published: December 2022

Using methods based upon the PCR, we have analyzed sequences that encode the V domain of bovine Ig heavy chain. Pairwise comparison indicates that the heavy chain repertoire in cattle is dominated by a single VH gene family that is homologous to human VHII. The third complementarity-determining region (CDR3) is particularly variable and 60% of the sequences encode a CDR3 in excess of 21 amino acids. Southern blots, single-stranded conformation polymorphism analysis, and sequencing of segments from the germline indicate that the dominant VHII gene family is of small size, comprising as few as 10 unique members, and carries a very low level of diversity in CDRs 1 and 2. Comparison of germline VH sequences with those gathered from adult bovine splenic cDNA demonstrates a pattern of nucleotide substitution that is consistent with diversification through somatic hypermutation.
Attenuation of IL-5-mediated signal transduction, eosinophil survival, and inflammatory mediator release by a soluble human IL-5 receptor.
Authors: Monahan J , Siegel N , Keith R , Caparon M , Christine L , Compton R , Cusik S , Hirsch J , Huynh M , Devine C , Polazzi J , Rangwala S , Tsai B , Portanova J
Published: December 2022

A soluble form of the human IL-5R alpha-chain (IL-5Ra) that contains the extracellular IL-5 binding domain has been evaluated for its effect on IL-5 binding to and activation of human eosinophils and basophils. The truncated receptor was expressed in Escherichia coli and recovered in biologically active form following renaturation and anion exchange chromatography. The soluble receptor formed a 1/1 complex with IL-5 in solution and bound IL-5 with affinity comparable to that of cell-associated IL-5Ra. Soluble IL-5Ra also competed with IL-5 for binding to the native alpha beta IL-5R on human cells and inhibited IL-5-mediated receptor activation and inflammatory mediator production. In this regard, the soluble receptor prevented IL-5-induced tyrosine phosphorylation of JAK2 kinase and IL-5R beta-chain and inhibited IL-5 priming of leukotriene C4 release by human basophils. However, the E. coli-derived receptor failed to inhibit IL-5 in longer term assays, including eosinophil survival and TF-1 cell proliferation, possibly due to its propensity to aggregate in a time- and temperature-dependent manner. In contrast, we observed that a soluble IL-5Ra derived from baculovirus-infected cells was less prone to aggregate and effectively antagonized IL-5-induced cell proliferation and survival. These findings indicate that the extracellular portion of the human IL-5Ra chain can prevent association of IL-5 with cell surface receptors and can attenuate signal transduction, mediator release, and survival of inflammatory cells. As such, soluble IL-5R may be useful in treating diseases such as human asthma, in which pulmonary injury is associated with the activity of IL-5R-bearing cells.
Depletion of CD8+ T lymphocytes by murine monoclonal CD8 antibodies and restored specific T cell proliferation in vivo in a patient with chronic hepatitis C.
Authors: Kiefersauer S , Reiter C , Eisenburg J , Diepolder H M , Rieber E P , Riethmüller G , Gruber R
Published: December 2022

Cellular immune mechanisms, especially those mediated by CD8+ T cells, are important in the pathogenesis and control of viral infections. On the other hand, as shown for chronic lymphocytic choriomeningitis virus infection in the mouse, CD8+ T cells may not only hinder the elimination of a virus, but make the host unresponsive to a second viral infection. In hepatitis C virus (HCV) infections, at least 50% of the patients become chronically infected, despite the detection of HCV-specific CTL and a specific proliferative response to HCV Ags in PBL and in lymphocytes isolated from the liver. To better understand the immunopathologic mechanisms of CD8+ cells in vivo and to search for a potential treatment, we applied murine CD8 mAbs to a patient with therapy-resistant chronic HCV. A drastic reduction of CD8+ circulating lymphocytes, a reduction of CD8 molecule density, and complement fixation on CD8+ cells were observed. The reduction of CD8+ cells was compensated partially by an elevation of CD4+ cells. High concentrations of neutralizing human anti-mouse Abs were induced. After the Ab infusions, the CD4/CD8 ratio in peripheral blood increased from 1.6 to values of about 3 during therapy, and gradually decreased to 2.3 1 yr after the last mAb infusion. A continuing decrease of serum aminotransferases and clinical improvement was observed. Interestingly, after initiation of treatment, a significant proliferative response to HCV-specific Ags became measurable.
Activation of monocytes by HIV-Tat treatment is mediated by cytokine expression.
Authors: Lafrenie R M , Wahl L M , Epstein J S , Yamada K M , Dhawan S
Published: December 2022

Treatment of primary monocytes with soluble HIV-Tat protein is associated with increased monocyte metalloproteinase-9 (MMP-9) expression and enhanced beta 2 integrin expression that increases monocyte/endothelial adhesion. These alterations require greater than 12 h of HIV-Tat treatment, suggesting the involvement of intermediate factors. Thus, we have examined the role of cytokines in the HIV-Tat-induced alteration of monocyte function. Treatment of monocytes with HIV-Tat rapidly up-regulated the production of IL-1 beta, IL-6, IL-8, and TNF-alpha, but not IL-3, granulocyte-macrophage CSF, basic fibroblast growth factor, or macrophage-inflammatory protein-1 alpha, and was associated with up-regulation of the corresponding cytokine mRNA. Inclusion of neutralizing anti-cytokine Abs to IL-1 beta or TNF-alpha during the HIV-Tat pretreatment period significantly inhibited the HIV-Tat-induced increase in MMP-9 production, monocyte/endothelial adhesion, and monocyte-dependent endothelial damage. In contrast, neutralizing Abs against IL-6 and IL-8 had no effect. The effects of HIV-Tat treatment, namely MMP-9 production, enhanced monocyte/endothelial cell adhesion, and monocyte-dependent endothelial damage, were mimicked by treating the monocytes with IL-1 beta or TNF-alpha, but not with IL-6 or IL-8. Therefore, the mechanism by which HIV-Tat activates monocyte function is dependent on HIV-Tat-induced production of cytokines (IL-1 beta and TNF-alpha).
Experimental Inoculation of Chickens with Hodgkin's Nodes
Authors: L'Esperance Elise S.
Published: December 2022

Summary and Conclusions

Five healthy chickens were inoculated intravenously with emulsions of lymph nodes from two cases of clinically and histologically characteristic Hodgkin's disease.

All of these chickens developed either a typical, or an atypical tuberculosis, and in the tissue smears, stained by Ziehl-Neelsen method, acid-fast granules and rods, extra- and intra-cellular, were demonstrated in three of them, and non-acid-fast granules in one of them.

Reinoculation of material from the lesions of one of these chickens into another chicken produced an identical, though more extensive manifestation of the disease.

An atypical tuberculosis developed in guinea pigs by inoculation of the tissue from the fourth chicken. A growth of bacteria with the staining and cultural characteristics of the avian tubercle bacillus was produced on egg media from material from this guinea pig.

Cultures from these birds on egg media were contaminated in all instances. In one, the chicken culture gave a symbiotic growth of a blastomyces like organism with an acid fast bacillus. These cultures were reinoculated into normal chickens and guinea pigs, but sufficient time has not elapsed to determine the results of this reinoculation.

While it is recognized that the two short series of experiments here reported cannot quite exclude the apparently unlikely alternative explanation, that we were dealing with an accidental avian infection, the findings seem to justify the conclusion that in these chickens, a lesion with the histological features of Hodgkin's granuloma, and comparable to avian tuberculosis has been produced after the inoculation of emulsified Hodgkin's nodes. This may indicate that the etiological agent in certain forms of Hodgkin's disease is pathogenic for birds, or that the avian tubercle bacilli are a factor in producing some of the lesions which are interpreted as Hodgkin's disease.
Heterogeneity of Streptococci Isolated from Sputum
Authors: Hooker Sanford Burton , Anderson Lillian M. , Follensby Edna M. , Force Helen V. , Tayian Elizabeth H.
Published: December 2022

Non-hemolytic streptococci of the alpha type are the dominant aërobic flora in sputa from minor respiratory infections. The common fermentative types in order of decreasing frequency are salivarius, mitis, and ignavus. Many sub-groups exist. Hemolytic streptococci of the true beta type are uncommonly found in these sputa. Their presence is but temporary and seems dependent upon the epidemic prevalence of certain respiratory infections. Fermentative groups are less numerous; the pyogenes type is outstanding. Alpha prime varieties have a similarly fluctuating and infrequent incidence. Gamma types are often present but rarely predominate. There is considerable fermentative diversity among strains of these two types. When many colonies of the dominant type and of indistinguishable appearance are selected from a primary plate, they can usually be separated, on the basis of other physiological characteristics, into several distinct groups. This peculiar heterogeneity has been studied in connection with 2057 colony strains from 101 specimens. Of the collections of strains isolated from virtually open surfaces about 85 per cent were diverse assemblages; those derived from closed foci or from entitative disease were homogeneous in 19 of 20 instances. The literature has been reviewed for evidence as to the homogeneity or heterogeneity of an individual's strains of streptococci and other genera. The more recent literature on the antigenic properties of alpha streptococci is reviewed and certain technologic aspects of serological classifications are critically discussed. Cross-agglutination and fixation tests have demonstrated a bewildering antigenic heterogeneity among alpha streptococci; strong cross-agglutination was observed in less than 3 per cent of 616 tests. No identical strains, from different sources, were discovered. Group reactions occurred among fermentative heterologs as well as among strains of the same fermentative type. Antigenic unlikeness also features the identical-colony strains derived from individuals. Consideration is given to the pertinent bearing which this heterogeneity has upon autogenous vaccine treatment and upon the classification of non-hemolytic streptococci.
Experimental Studies with Pyocyaneus Filtrates
Authors: Oesterlin Ernst J.
Published: December 2022

Percutaneous or intraperitoneal previous treatment with pyocyaneus filtrates protect neither guinea pigs nor rabbits from a respectively subcutaneous or intraperitoneal infection with Bacillus pyocyaneus. It is possible to protect both kinds of animals by repeated intrapleural injections of previously heated pyocyaneus filtrate against intrapleural infection with Bacillus pyocyaneus. The resistance acquired in this way is restricted to the pleural cavity. Simultaneous metastatic infections of the liver and of the kidney are common findings. In other cases the resistance is only a relative one; in spite of the previous treatment an infection of the pleura occurs, but the course of the disease is much slower than in the control animals. The question of specificity of the action of the filtrates will be studied on a larger scale. Although an occasional increase of resistance caused by the unspecific filtrates has been found, the protection produced by specific filtrates seems to be more intense and more dependable. Intrapleural injections with sterile broth have no prophylactic value against an intrapleural infection with Bacillus pyocyaneus: it is impossible to protect the animals by broth injections against such infections.
Follicular Dendritic Cell (FDC) Precursors in Primary Lymphoid Tissues
Authors: Kapasi Zoher F. , Qin Dahui , Kerr William G. , Kosco-Vilbois Marie H. , Shultz Leonard D. , Tew John G. , Szakal Andras K.
Published: December 2022

The origin of follicular dendritic cells (FDC) is unresolved, and as such, remains controversial. Based on the migration of Ag-transporting cells (ATC) into lymphoid follicles and the phenotypic similarity between FDC and ATC, one hypothesis is that ATC may represent emigrating FDC precursors. This contrasts with the view that FDC originate from local stromal cells in the secondary lymphoid tissues. Mice homozygous for the severe combined immunodeficiency (prkdcscid) mutation (scid) lack FDC. Thus, they provide a powerful tool for assessing de novo generation of FDC. To test whether FDC precursors could be found in bone marrow or fetal liver, scid/scid mice were reconstituted with either: 1) bone marrow cells from (BALB/c × C57BL/6)F1 donors, 2) bone marrow cells from ROSA BL/6 F1 (lacZ-transfected) mice, 3) rat bone marrow cells, or 4) rat fetal liver cells. Six to eight weeks after reconstitution with F1 bone marrow, cells reactive with the FDC-labeling mAb, FDC-M1, also expressed donor class I molecules on their surfaces. Similarly in mice reconstituted with lacZ-transfected bone marrow cells, these cells were also positive for the lacZ gene product. Furthermore, in spleens of animals reconstituted with either rat bone marrow or rat fetal liver, rat FDC were identified using the specifically labeling mAb, ED5. In all cases, host FDC were also present, indicating that scid/scid mice have FDC precursors that will mature in the presence of allogeneic or xenogeneic lymphoid cells. In summary, FDC can be derived from progenitor cells present in primary lymphoid tissues.
IL-12 Up-Regulates CD40 Ligand (CD154) Expression on Human T Cells
Authors: Peng Xiaohui , Remacle Jacques E. , Kasran Ahmad , Huylebroeck Danny , Ceuppens Jan L.
Published: December 2022

IL-12 is a heterodimeric cytokine produced by APC that promotes the development of CD4+ Th1 cells and their IFN-γ production after TCR/CD3 triggering. We here investigated the capacity of IL-12 to modify the expression on T cells of CD40 ligand (CD40L or CD154), a molecule transiently expressed on activated T cells and known to be of utmost importance for cognate interaction with B cells and for activation of dendritic cells and macrophages. Our data demonstrate that IL-12 up-regulates CD40L expression on anti-CD3-activated human peripheral blood T cells. For optimal induction of CD40L, IL-12 synergizes with IL-2 as well as with other costimulatory interactions, such as B7/CD28. The effect of IL-12 was observed at both the protein and the mRNA level. T cells costimulated by IL-12 provided more efficient help for IL-4-dependent B cell proliferation and for IgG production than when activated in the absence of IL-12. This helper activity was blocked by an mAb against CD40L, indicating that the effect of IL-12 on B cells is mediated indirectly through CD40L. The data thus suggest that the effects of IL-12 on cellular and humoral immune responses are partly mediated through CD40L induction.
An Intronic Silencer Regulates B Lymphocyte Cell- and Stage-Specific Expression of the Human Complement Receptor Type 2 (CR2, CD21) Gene
Authors: Makar Karen W. , Pham Christine T. N. , Dehoff Marlin H. , O’Connor Siobhan M. , Jacobi Susan M. , Holers V. Michael
Published: December 2022

Human CR2 (CD21) is a B lymphocyte protein whose surface expression is restricted primarily to the mature cell stage during development. To study the transcriptional mechanisms that govern cell- and stage-restricted CR2 expression, we first performed transient transfection analysis using constructs extending from −5 kb to +75 bp (−5 kb/+75) in the CR2 promoter. The promoter was found to be broadly active, with no evidence of cell- or stage-specific reporter gene expression. However, the addition of a 2.5-kb intronic gene segment (containing a DNase I hypersensitive site) to the (−5-kb/+75) construct resulted in appropriate reporter gene expression, defined as the silencing of the (−5-kb/+75) promoter activity only in non-CR2-expressing cells. Interestingly, appropriate reporter gene expression required stable transfection of the constructs in cell lines, suggesting nuclear matrix or chromatin interactions may be important for appropriate CR2 gene expression. Importantly, transgenic mice also required the intronic silencer to generate lymphoid tissue-specific reporter gene expression. Some transgenic founder lines did not demonstrate reporter gene expression, however, indicating that additional transcriptional regulatory elements are present in other regions of the CR2 gene. In summary, these data support the hypothesis that human CR2 expression is regulated primarily by an intronic silencer with lineage- and B cell stage-specific activity.
Internalization of Chlamydia by Dendritic Cells and Stimulation of Chlamydia-Specific T Cells
Authors: Ojcius David M. , Bravo de Alba Yolanda , Kanellopoulos Jean M. , Hawkins Ray A. , Kelly Kathy A. , Rank Roger G. , Dautry-Varsat Alice
Published: December 2022

Chlamydia species are the causative agents of trachoma, various forms of pneumonia, and the most common sexually transmitted diseases. Although the infection cycle has been extensively characterized in epithelial cells, where the Chlamydia entry-vacuoles avoid fusion with host-cell lysosomes, the cellular immune response has received less attention. Moreover, despite the abundant presence of dendritic cells (DC) in the sites of infection, the interaction between Chlamydia and DC has never been studied. We observe that DC kill Chlamydia trachomatis and Chlamydia psittaci. The chlamydiae are internalized by the DC in a nonspecific manner through macropinocytosis, and the macropinosomes fuse subsequently with DC lysosomes expressing MHC class II molecules. The interaction induces maturation of the DC, since presentation of an exogenous Ag is severely inhibited after a 1-day incubation, although chlamydial Ags are still presented and recognized by Chlamydia-specific CD4+ T cells. Thus, DC most likely play a role in initiating the T cell response in vivo and could potentially be used in adoptive transfer therapies to vaccinate against Chlamydia.
Induction of Pulmonary Allergic Responses by Antigen-Specific Th2 Cells
Authors: Li Xiu-Min , Schofield Brian H. , Wang Qian-Fei , Kim Kawn-Hyoung , Huang Shau-Ku
Published: December 2022

The development of pulmonary allergic responses was examined in mice following pulmonary transfer of Ag (conalbumin)-specific Th2 cells. The levels of serum-specific IgE, cellular infiltrates, airway mucus goblet cells, and airway responsiveness were analyzed and compared with those in Ag-sensitized and -challenged mice. Pulmonary transfer of the conalbumin-specific Th2 clone (D10) induced, in an Ag-specific manner, high levels of the Th2 cytokines IL-4 and IL-5 in the bronchoalveolar lavage fluids and mucosal eosinophils, concomitant with an increase in airway responsiveness. The D10 cell-induced responses were seen in the absence of serum specific IgE. In the presence of Ag, the transferred D10 cells not only remained in the lungs, but also increased in number 72 h post-cell transfer. Although significantly higher levels of IL-4 and IL-5 in the bronchoalveolar lavage fluids were found in D10-transferred mice, the levels of pulmonary eosinophilia, mucus goblet cells, and airway responsiveness were significantly lower than those in Ag-sensitized and -challenged mice. These results demonstrate that although Ag-specific activation of Th2 cells at mucosal sites is able to mediate the recruitment of eosinophils and the subsequent induction of airway hyper-responsiveness, the more severe pulmonary allergic responses were observed only in mice sensitized and challenged with Ag.
CC Chemokine Receptors 1 and 3 Are Differentially Regulated by IL-5 During Maturation of Eosinophilic HL-60 Cells
Authors: Tiffany H. Lee , Alkhatib Ghalib , Combadiere Christophe , Berger Edward A. , Murphy Philip M.
Published: December 2022

CC chemokine receptors 1 and 3 (CCR1 and CCR3) are expressed by eosinophils; however, factors regulating their expression and function have not previously been defined. Here we analyze chemokine receptor expression and function during eosinophil differentiation, using the eosinophilic cell line HL-60 clone 15 as a model system. RNA for CCR1, -3, -4, and -5 was not detectable in the parental cells, and the cells did not specifically bind CC chemokines. Cells treated with butyric acid acquired eosinophil characteristics; expressed mRNA for CCR1 and CCR3, but not for CCR4 or CCR5; acquired specific binding sites for macrophage-inflammatory protein-1α and eotaxin (the selective ligands for CCR1 and CCR3, respectively); and exhibited specific calcium flux and chemotaxis responses to macrophage-inflammatory protein-1α, eotaxin, and other known CCR1 and CCR3 agonists. CCR3 was expressed later and at lower levels than CCR1 and could be further induced by IL-5, whereas IL-5 had little or no effect on CCR1 expression. Consistent with the HIV-1 coreceptor activity of CCR3, HL-60 clone 15 cells induced with butyric acid and IL-5 fused with HeLa cells expressing CCR3-tropic HIV-1 envelope glycoproteins, and fusion was blocked specifically by eotaxin or an anti-CCR3 mAb. These data suggest that CCR1 and CCR3 are markers of late eosinophil differentiation that are differentially regulated by IL-5 in this model.
Subcongenic Analysis of theIdd13Locus in NOD/Lt Mice: Evidence for Several Susceptibility Genes Including a Possible Diabetogenic Role for β2-Microglobulin
Authors: Serreze David V. , Bridgett Margot , Chapman Harold D. , Chen Emmie , Richard Scott D. , Leiter Edward H.
Published: December 2022

Although they share ∼88% of their genome with NOD mice including the H2g7 haplotype, NOR mice remain free of T cell-mediated autoimmune diabetes (IDDM), due to non-MHC genes of C57BLKS/J (BKS) origin. NOR IDDM resistance was previously found to be largely controlled by the Idd13 locus within an ∼24 cM segment on Chromosome 2 encompassing BKS-derived alleles for H3a, B2m, Il1, and Pcna. NOD stocks carrying subcongenic intervals of NOR Chromosome 2 were utilized to more finely map and determine possible functions of Idd13. NOR- derived H3a-Il1 (∼6.0 cM) and Il1-Pcna (∼1.2 cM) intervals both contribute components of IDDM resistance. Hence, the Idd13 locus is more complex than originally thought, since it consists of at least two genes. B2m variants within the H3a-Il1 interval may represent one of these. Monoclonal Ab binding demonstrated that dimerizing with the β2ma (NOD type) vs β2mb isoform (NOR type) alters the structural conformation, but not total expression levels of H2g7 class I molecules (e.g. Kd, Db). β2m-induced alterations in H2g7 class I conformation may partially explain findings from bone marrow chimera analyses that Idd13 modulates IDDM development at the level of non-hematopoietically derived cell types controlling selection of diabetogenic T cells and/or pancreatic β cells targeted by these effectors. Since trans-interactions between relatively common and functionally normal allelic variants may contribute to IDDM in NOD mice, the search for Idd genes in humans should not be limited to functionally defective variants.
Cells Transfected with a Non-Neutralizing Antibody Gene Are Resistant to HIV Infection: Targeting the Endoplasmic Reticulum and Trans-Golgi Network
Authors: Zhou Paul , Goldstein Simoy , Devadas Krishnakumar , Tewari Deepanker , Notkins Abner Louis
Published: December 2022

Plasmids containing single chain Fv (scFv) non-neutralizing human anti-HIV-1 gp41 Ab cDNA, with or without endoplasmic reticulum (ER) or trans-Golgi network (TGN) retention signals, were constructed. Stable transfectants expressing these scFvs then were generated from COS-7 cells and HIV-1-susceptible CD4+ human T cells (Jurkat). scFv without a retention signal was secreted from cells, whereas scFv with an ER or TGN retention signal remained primarily within targeted intracellular compartments. The expression of scFv, scFv-ER, and scFv-TGN did not adversely affect the appearance of uninfected cells, as measured by growth rate or CD4 expression. Pulse-chase experiments revealed that the t1/2 of scFv-ER and scFv-TGN within cells was greater than 24 h and less than 9 h, respectively. The scFv-ER and scFv-TGN bound HIV gp160, and the scFv-ER-gp160 and the scFv-TGN-gp160 complexes were stable within HIV-infected transfectants. Further studies revealed that the maturation processing of gp160 into gp120 and gp41 was blocked in the scFv-ER transfectants, but not in the scFv-TGN transfectants. Moreover, HIV replication, as measured by p24, was inhibited by up to 99% in cells transfected with scFv-ER or scFv-TGN, but was not inhibited in cells transfected with the secretory form of scFv. It is concluded that the targeting of non-neutralizing anti-HIV-1 Abs to specific intracellular compartments blocks HIV replication and represents a potential therapeutic strategy for protecting uninfected lymphopoietic stem cells from HIV-1-infected patients.
A Non-Sense Mutation at Arg95 Is Predominant in Complement 9 Deficiency in Japanese
Authors: Horiuchi Takahiko , Nishizaka Hiroaki , Kojima Takeshi , Sawabe Takuya , Niho Yoshiyuki , Schneider Peter M. , Inaba Shoichi , Sakai Kouko , Hayashi Kenshi , Hashimura Chinami , Fukumori Yasuo
Published: December 2022

Deficiency of the ninth component of complement (C9D) is one of the most common genetic abnormalities in Japan, with an incidence of one homozygote in 1000. Although C9D individuals are usually healthy, it has been shown that they have an significantly increased risk of developing meningococcal meningitis. In the present study we report the molecular bases for C9D in 10 unrelated Japanese subjects. As a screening step for mutations, exons 2 to 11 of the C9 gene were analyzed using exon-specific PCR/single-strand conformation polymorphism analysis, which demonstrated aberrantly migrating DNA bands in exon 4 in all the C9D subjects. Subsequent direct sequencing of exon 4 of the C9D subjects revealed that eight of the 10 C9D subjects were homozygous for a C to T transition at nucleotide 343, the first nucleotide of the codon CGA for Arg95, leading to a TGA stop codon (R95X). R95X is a novel mutation different from those recently identified in a Swiss family with C9D. Cases 6 and 7 were heterozygous for the R95X mutation. Family study in case 10 confirmed the genetic nature of the defect. In case 6, the second mutation for C9D of the C9 gene was identified to be the substitution of Cys to Tyr at amino acid residue 507 (C507Y), while the genetic defect(s) in the other allele in case 7 remains unknown. Our results indicate that a novel mutation, R95X, is present in most cases of C9D in Japan.
Expansion of Autoreactive T Cells in Multiple Sclerosis Is Independent of Exogenous B7 Costimulation
Authors: Scholz Christian , Patton Kurt T. , Anderson David E. , Freeman Gordon J. , Hafler David A.
Published: December 2022

Multiple sclerosis (MS) is an inflammatory disease of the myelinated central nervous system that is postulated to be induced by myelin-reactive CD4 T cells. T cell activation requires an antigen-specific signal through the TCR and a costimulatory signal, which can be mediated by B7–1 or B7–2 engagement of CD28. To directly examine the activation state of myelin-reactive T cells in MS, the costimulation requirements necessary to activate myelin basic protein (MBP) or tetanus toxoid (TT)-reactive CD4 T cells were compared between normal controls and MS patients. Peripheral blood T cells were stimulated with Chinese hamster ovary (CHO) cells transfected either with DRB1*1501/DRA0101 chains (t-DR2) alone, or in combination with, B7–1 or B7–2. In the absence of costimulation, T cells from normal subjects stimulated with the recall antigen TT p830–843 were induced to expand and proliferate, but stimulation with MBP p85–99 did not have this effect. In marked contrast, T cells from patients with MS stimulated with MBP p85–99 in the absence of B7–1 or B7–2 signals expanded and proliferated. Thus, MBP-reactive CD4 T cells in patients with MS are costimulation independent and have been previously activated in vivo. These experiments provide further direct evidence for a role of activated MBP-specific CD4 T cells in the pathogenesis of MS.
Cutting Edge: Modulation of Airway Inflammation by CpG Oligodeoxynucleotides in a Murine Model of Asthma
Authors: Kline Joel N. , Waldschmidt Thomas J. , Businga Thomas R. , Lemish Jennifer E. , Weinstock Joel V. , Thorne Peter S. , Krieg Arthur M.
Published: December 2022

Asthma has been increasing in industrialized countries. Evidence suggests that asthma is caused by a Th2 immune response to inhaled environmental Ags and that childhood infections protect against this. We have shown that bacterial DNA contains motifs, centered on unmethylated CpG dinucleotides, which induce Th1-type responses. We hypothesized that the Th1 effect of these CpG motifs may oppose the Th2 type allergic response and suggest that this may account for the protective effect of childhood infection against asthma. We examined the effects of CpG-motif oligodeoxynucleotides (CpG ODN) in a murine model of asthma. Airway eosinophilia, Th2 cytokine induction, IgE production, and bronchial hyperreactivity were prevented by coadministration of CpG ODN with the Ag. Significantly, in a previously sensitized mouse, CpG ODN can prevent allergen-induced airway inflammation. These studies suggest that exposure to CpG DNA may protect against asthma.
Inhibition of T Cell Activation by Pharmacologic Disruption of the MEK1/ERK MAP Kinase or Calcineurin Signaling Pathways Results in Differential Modulation of Cytokine Production
Authors: Dumont Francis J. , Staruch Mary Jo , Fischer Paul , DaSilva Carolyn , Camacho Ramon
Published: December 2022

Productive T cell activation leading to cytokine secretion requires the cooperation of multiple signaling pathways coupled to the TCR and to costimulatory molecules such as CD28. Here, we utilized two pharmacophores, PD98059 and FK506, that inhibit, respectively, mitogen-activated protein (MAP) kinase kinase 1 (MEK 1) and calcineurin, to determine the relative role of the signaling pathways controlled by these enzymes in T cell activation. Although the two compounds had distinctive effects on CD69 induction, they both suppressed T cell proliferation induced by anti-CD3 mAb, in a manner reversible by exogenous IL-2, suggesting that PD98059, like FK506, affects the production of, rather than the responsiveness to growth-promoting cytokines. Accordingly, IL-2 production by T cells stimulated with anti-CD3 mAb in conjunction with PMA or with anti-CD28 mAb was inhibited by both compounds. However, these compounds differentially affected the production of other cytokines, depending on the mode of activation. PD98059 inhibited TNF-α, IL-3, granulocyte-macrophage (GM)-CSF, IFN-γ, and to a lesser extent IL-6 and IL-10 production but enhanced IL-4, IL-5, and IL-13 production induced by CD3/PMA or CD3/CD28. FK506 suppressed CD3/PMA-induced production of all cytokines examined here but to a lesser extent IL-13. FK506 also reduced CD3/CD28-induced production of IL-3, IL-4, IL-10, TNF-α, and IL-6 but augmented that of GM-CSF, IL-5, IFN-γ, and IL-13. Therefore, the biochemical targets of PD98059 and FK506 contribute differently to the production of various cytokines by T cells, which may have implications for the therapeutic manipulation of this production.
Generation of Intestinal Mucosal Lymphocytes in SCID Mice Reconstituted with Mature, Thymus-Derived T Cells
Authors: Camerini Victoria , Sydora23 Beate C. , Aranda Richard , Nguyen Chris , MacLean Colin , McBride William H. , Kronenberg Mitchell
Published: December 2022

Transfer of peripheral lymph node lymphocytes to SCID mice leads to the long term establishment of mucosal T lymphocytes within the epithelium and lamina propria of the small and large intestines. Analysis of engrafted intraepithelial lymphocytes (IEL) showed that they had acquired a surface phenotype that in several respects is typical of IEL. In addition, the functional profile of engrafted IEL derived from lymph node T cells was similar to that of normal IEL; as the donor-derived T cells exhibited a strong cytolytic activity, a poor proliferative response to mitogenic stimuli, and a tendency to home and expand specifically in the intestine upon transfer to secondary SCID recipients. Optimal engraftment of intestinal T cells required bacterial flora, as the number of lymphocytes was greatly reduced in SCID recipients with a reduced flora. These results demonstrate that mature, thymus-derived T cells can migrate to the intestine and become functionally specialized to the intestinal milieu. The acquisition of phenotypic markers characteristic of the intestinal microenvironment by engrafted cells suggests that T cell migration of lymphocytes to the SCID intestine is not aberrant, but it may reflect processes that are ongoing in immunocompetent mice. Furthermore, these data suggest that the homing and/or expansion of typical, thymus-derived T cells in the intestine may be driven by luminal Ags such as those derived from bacterial flora.
Potential Role of Resident Islet Macrophage Activation in the Initiation of Autoimmune Diabetes
Authors: Arnush Marc , Scarim Anna L. , Heitmeier Monique R. , Kelly Colleen B. , Corbett John A.
Published: December 2022

The purpose of this study was to evaluate the effects of resident islet macrophage activation on β cell function. Treatment of freshly isolated rat islets with TNF-α and LPS results in a potent inhibition of glucose-stimulated insulin secretion. The inhibitory actions of TNF + LPS are mediated by the intraislet production and release of IL-1 followed by IL-1-induced inducible nitric oxide synthase (iNOS) expression by β cells. The IL-1R antagonist protein completely prevents TNF + LPS-induced nitrite production, iNOS expression and the inhibitory effects on glucose-stimulated insulin secretion by rat islets. Resident macrophages appear to be the source of IL-1, as a 7-day culture of rat islets at 24°C (conditions known to deplete islets of lymphoid cells) prevents TNF + LPS-induced iNOS expression, nitrite production, and the inhibitory effects on insulin secretion. In addition, macrophage depletion also inhibits TNF + LPS-induced IL-1α and IL-1β mRNA expression in rat islets. Immunocytochemical colocalization of IL-1β with the macrophage-specific marker ED1 was used to provide direct support for resident macrophages as the islet cellular source of IL-1. IL-1β appears to mediate the inhibitory actions of TNF + LPS on β cell function as TNF + LPS-induced expression of IL-1β is fourfold higher than IL-1α, and Ab neutralization of IL-1β prevents TNF + LPS-induced nitrite production by rat islets. These findings support a mechanism by which the activation of resident islet macrophages and the intraislet release of IL-1 may mediate the initial dysfunction and destruction of β cells during the development of autoimmune diabetes.