bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of thymic subpopulations expressing the activation antigen GL7. Expression, genetics, and function.
Authors: Hathcock K S , Pucillo C E , Laszlo G , Lai L , Hodes R J
Published: December 2022

Previously, we reported an mAb, GL7, that defines an activation Ag expressed by in vitro-stimulated B and T cells as well as by a subpopulation of thymocytes. The current study analyzes the GL7-expressing populations of adult and fetal thymus and demonstrates that: 1) The majority of GL7+ adult thymocytes are CD4+CD8- cells that are CD3 epsilon high, TCR-alpha beta high, HSAlow, and bimodal for CD69 expression. The 3G11-6C10- subset of CD4+CD8- thymocytes is enriched in GL7-expressing cells. 2) Strain differences exist in the expression of GL7 on adult CD4+CD8- thymocytes; 21.9 +/- 5.9% of BALB/c CD4+CD8- thymocytes are GL7+, whereas 4.4 +/- 1.7% of C57BL/6 CD4+CD8- thymocytes are GL7+. The low GL7 expression phenotype is dominant in CB6F1 thymocytes (7.0 +/- 2.0%), and analysis of BALB/c x CB6F1 mice suggests that low GL7 expression is determined by multiple genes. 3) CD4+CD8- GL7+ thymocytes from BALB/c mice, but not C57BL/6 mice, are skewed toward a high proportion of V beta 8+ cells. 4) Adult GL7+ CD4+CD8- thymocytes can be activated by TCR-specific stimuli to proliferate and to secrete high amounts of IL-4. 5) Fetal thymocytes contain GL7+ cells, which are predominantly CD4-CD8-, HSAlow, CD69-, and bimodal for TCR-gamma delta. Thus, GL7 expression defines a subpopulation of functionally competent TCR-alpha beta+ CD4+CD8- thymocytes as well as TCR-gamma delta+ and TCR- subpopulations of fetal CD4-CD8- thymocytes.
CD40 function in nonhematopoietic cells. Nuclear factor kappa B mobilization and induction of IL-6 production.
Authors: Hess S , Rensing-Ehl A , Schwabe R , Bufler P , Engelmann H
Published: December 2022

CD40 is a member of the TNF receptor family that was first characterized as an important T-B cell interaction molecule. This receptor is also expressed on many other cell types, including normal basal epithelium, carcinomas, and transformed cell lines. The functions of CD40 in non-B cells are largely unknown. Our studies demonstrate that CD40 mediates nuclear factor kappa B (NF-kappa B) mobilization and IL-6 production in nonhematopoietic cells. Stimulation of the transformed fibroblast cell line SV80 with CD40 ligand (CD40L) or anti-CD40 Ab resulted in the production of IL-6; this could be increased by IFN-gamma pretreatment, which is known to up-regulate CD40 expression. Studies with transfectants overexpressing CD40 demonstrated that activation of CD40 alone is sufficient to induce IL-6 production. The transcription factor NF-kappa B appears to play a central role in CD40-mediated activation of the IL-6 gene; NF-kappa B mobilization directly preceded CD40-mediated IL-6 production, and suppression of NF-kappa B mobilization with the metabolic inhibitor D609 also suppressed the IL-6 response. A striking similarity to the requirements for TNF-induced IL-6 production, which is mediated by the p55TNF receptor in SV80 cells, was observed. In view of the intracellular homologies between CD40 and the p55TNF receptor, it should be considered that the two receptors share common components in their signaling pathways that lead to IL-6 production.
Expression of ecto-adenosine deaminase and CD26 in human T cells triggered by the TCR-CD3 complex. Possible role of adenosine deaminase as costimulatory molecule.
Authors: Martín M , Huguet J , Centelles J J , Franco R
Published: December 2022

The expression of surface adenosine deaminase (ADA) and CD26 in activated human T cells was studied by flow cytometry. PBLs and CD3+ or CD4+ cells, when subjected to a variety of stimuli (anti-CD3 Abs plus IL-2 or phorbol esters), presented two structurally different cell populations, which differed in size and cellular complexity (populations B1 and B2). In PBLs triggered by an anti-CD3 mAb there was no significant increase of expression of either surface ADA or CD26 in cells of population B1, whose structure is similar to that of nonstimulated cells. In contrast, there was a significant increase in the percentage of expression of ADA and CD26 in the population B2, which corresponds to structurally more complex and larger cells. In the case of activation via TCR-CD3 but in the presence of IL-2 or via phorbol esters, the increase was found in cells from both populations, but B2 cells always showed a higher percentage of expression than B1 cells. The results of increased expression of surface ADA and CD26 were similar in whole T cells or in purer preparations such as CD3+ or CD4+ lymphocytes. Polyclonal Abs against ADA were not able to induce an activation response in T cells even when cross-linked by a secondary Ab. Interestingly, these Abs produced anergy in CD4+ cells subjected to an anti-CD3 stimulus. In contrast, addition of ADA produced an enzyme-independent synergism in the response through the TCR-CD3 complex. In T cells, ADA and CD26 colocalized on the surface of T cells; thus, the effect of exogenous ADA seems to be mediated by CD26 molecules that are not interacting with endogenous ADA (spare CD26 molecules). The presence of spare CD26 molecules on the surface of CD4+ cells was demonstrated by flow cytometry in the presence of exogenous ADA and also by confocal microscopy. The set of results strongly indicates that ADA binding to CD26 produces a costimulatory response in T cell activation events.
TAP associates with a unique class I conformation, whereas calnexin associates with multiple class I forms in mouse and man.
Authors: Carreno B M , Solheim J C , Harris M , Stroynowski I , Connolly J M , Hansen T H
Published: December 2022

To define the rules governing de novo assembly of the trimeric class I complex, we have identified the class I folding/assembly intermediates associated with calnexin or TAP, using both human and mouse cell lines. To better characterize the class I H chain structure associated with TAP, mouse mAb that distinguish open (64-3-7+) vs folded (30-5-7+) Ld heavy (H) chains were used. We report here that open forms of Ld are uniquely and specifically associated with TAP and that the conformational change in the class I H chain coincident with peptide binding induces TAP release. Chimeric Ld/Q10 displayed TAP association, demonstrating that soluble class I molecules can bind TAP. As previously reported, beta 2m was found to be required for H chain association with TAP. Interestingly, beta 2m was associated with TAP in the human class I-negative cell line LCL 721.221, suggesting that beta 2m can bind to TAP before class I H chain. In contrast to TAP, which binds a specific class I conformation, calnexin was detected in association with multiple forms of both mouse and human class I. Most significantly, we show for the first time that beta 2m-assembled forms of human as well as mouse class I molecules interact with calnexin. Based on these findings, we propose a model for the sequential assembly of class I heterotrimers and their respective interactions with TAP and calnexin.
Expression of activated H-rasval12 in nontumorigenic and non-cross-reactive syngeneic cells induces tumor antigens cross-reactive with rat mammary adenocarcinoma 13762.
Authors: Frey A B , Cestari S
Published: December 2022

Adenocarcinoma 13762 expresses tumor Ags that can induce protective immunity to tumorigenic challenge. Syngeneic fibroblast Rat1 cells transformed by expression of H-rasval12 (Rat1/ras) but not parental Rat1 cells, and p53-transformed Rat1 cells, or other syngeneic cells or tumors, can immunize rats against tumorigenic challenge of 13762 tumor. Coincident with induced resistance to 13762 tumors, immunization of rats with Rat1/ras tumor induces CD4+ T cells that cross-react with adenocarcinoma 13762 in vitro and transfer protective immunity to tumorigenic 13762 challenge in vivo. Cross-reactive tumor Ags expressed in Rat1/ras tumor are not derived from ras protein, because immunization with purified H-rasval12 protein induces protective immunity to challenge by Rat1/ras tumor but not to adenocarcinoma 13762. In addition, immunization with H-rasval12 protein induces anti-ras CD4+ T cells that are uniquely reactive with Rat1/ras tumor: anti-ras T cells are not reactive with 13762 tumor in vitro and do not confer protective immunity to challenge with 13762 tumor in vivo. Tumor Ags expressed in Ras-transformed Rat1 cells that elicit cross-protective immunity likely arise as a consequence of transformation mediated by activated ras oncogene but are not derived from the Ras protein sequence.
Nonviable bacterial antigens administered with IL-12 generate antigen-specific T cell responses and protective immunity against Listeria monocytogenes.
Authors: Miller M A , Skeen M J , Ziegler H K
Published: December 2022

The development of effective vaccine strategies for intracellular pathogens, including bacteria, viruses, and parasites, is one of the major frontiers of scientific research. For the studies described here, the murine model of Listeria infection was used to evaluate the adjuvant effects of IL-12 when used as an immunization component. These studies revealed that typically nonimmunogenic doses of heat-killed Listeria monocytogenes, or soluble listerial Ag preparations, elicit intense Th1-type Listeria-specific T cell responses when administered i.p. along with recombinant murine IL-12. In addition to the Ag-specific production of IL-2 by CD4+ peritoneal cells that was elicited, several other correlates of protective responses were noted, including dramatic induction of CD3+ and alpha beta TCR+ cell populations in the peritoneal cavity and increased expression of class II MHC and production of IL-12 (upon in vitro restimulation) by peritoneal macrophages. Protection studies demonstrated that the T cell responses elicited by a IL-12-potentiated, heat-killed L. monocytogenes vaccine were sufficient to effectively protect mice against challenge with a large dose of virulent Listeria.
Lipopolysaccharide-induced change of phosphorylation of two cytosolic proteins in human monocytes is prevented by inhibitors of ADP-ribosylation.
Authors: Heine H , Ulmer A J , Flad H D , Hauschildt S
Published: December 2022

Interaction of LPS with human monocytes causes altered phosphate labeling of cytosolic proteins of 36 kDa and 38 kDa (p36/38). This property, determined by in vitro studies, is shared by other monocyte activators. Phosphorylated p36/38 are distinct from p38, 42-kDa, and 44-kDa isoforms of mitogen-activated protein kinases expressed in monocytes. Occupation of LPS binding sites by a LPS antagonist, the synthetic tetraacylated bisphosphate precursor of Escherichia coli lipid A (also known as compound 406, lipid IVa, or precursor Ia), prevents LPS-induced changes in the phosphate labeling of the two proteins. Abs against CD14 inhibit protein phosphorylation induced by low concentrations of LPS (10 ng/ml), whereas at high concentrations (1 microgram/ml), the Abs fail to prevent phosphorylation. In addition to phosphorylation, ADP-ribosylation of proteins has been implicated in a number of biologic processes. Here we show that inhibitors of ADP-ribosylation, namely meta-iodobenzylguanidine and nicotinamide, inhibit LPS-initiated altered phosphorylation of p36/38. This loss of phosphate labeling of p36/38 is accompanied by an inhibition of TNF-alpha and Il-6 mRNA and protein production. The synthesis of IL-1 is not affected. This suggests that the inhibitors interfere with specific steps in IL-6 and TNF-alpha production, which are not required for IL-1 synthesis. Taken together, the data indicate that ADP-ribosylation may be involved in LPS-induced alteration of the phosphorylation state of two cytosolic proteins (p36/38) and that these proteins modulate cellular processes leading to TNF-alpha and IL-6 release.
Differential effects of granulocyte-macrophage colony-stimulating factor on eosinophil and neutrophil superoxide anion generation.
Authors: Nagata M , Sedgwick J B , Busse W W
Published: December 2022

Eosinophils (EOS) are specifically recruited to sites of allergic inflammation and parasitic infection, while neutrophil (NEUT) influx predominates in bacterial infections. This biologic selectivity suggests that granulocytes may respond differently to inflammatory mediators such as granulocyte-macrophage-CSF (GM-CSF). To establish the mechanisms of the response of granulocytes to GM-CSF, isolated human peripheral blood EOS and NEUT were obtained from allergic rhinitis patients and incubated in vitro with this cytokine. Incubation with GM-CSF (10 or 100 pM) significantly enhanced FMLP-stimulated EOS superoxide anion (O2-) generation, LTC4 release, and adhesion to tissue culture plates. Both GM-CSF-enhanced EOS adhesion and O2- generation were inhibited by an anti-beta 2 (CD18) Ab suggesting that this beta 2 integrin was associated with increased cell function. In contrast, FMLP + cytochalasin B were required to demonstrate GM-CSF enhancement of NEUT O2- and LTB4 generation; GM-CSF had no effect on NEUT adhesion. Furthermore, GM-CSF augmentation of FMLP + cytochalasin B-activated NEUT O2- generation was not affected by anti-beta 2 Ab but was blocked by a 5-lipoxygenase-activating protein antagonist, BAY x 1005. Finally, 0.1 to 10 nM LTB4 mimicked the GM-CSF-priming effect on NEUT O2- generation, thus suggesting that the augmented NEUT respiratory burst was the result of LTB4 production and its effect on the NEUT. These data demonstrate that GM-CSF promotes EOS and NEUT O2- generation to FMLP via distinct mechanisms: enhanced adhesion of EOS vs autocrine-priming by enhanced LTB4 generation of NEUT.
Delineation of additional genetic bases for C8 beta deficiency. Prevalence of null alleles and predominance of C-->T transition in their genesis.
Authors: Saucedo L , Ackermann L , Platonov A E , Gewurz A , Rakita R M , Densen P
Published: December 2022

We studied the molecular bases for C8 beta deficiency in 34 unrelated families from the United States and the former Soviet Union. These families represented 69 unrelated null alleles of which 59 (86%) were found to be due to a previously described C-->T transition in exon 9. Six additional null alleles were also caused by C-->T transitions, of which four (6%) were located at base 388 in exon 3, one (2%) at base 298 in exon 3, and one (2%) involved cytosine 847 in exon 6. All of the null alleles affecting cytosine 388 were linked to the sequence polymorphism at base 376, which determines the uncommon C8 beta acidic allotype. Two null alleles were caused by single base pair deletions of cytosines at positions 430 and 632 in exons 3 and 5, respectively. Of the characterized null alleles, 97% were due to C-->T transitions in which an arginine (64 alleles) or a glutamine (one allele) was replaced by a stop codon. The basis for this apparent high frequency of C-->T transitions occurring in a relatively short stretch of DNA is uncertain.
T cell-dependent loss of proliferative responsiveness to colony-stimulating factor-1 by a murine epidermal-derived dendritic cell line, XS52.
Authors: Kitajima T , Ariizumi K , Bergstresser P R , Takashima A
Published: December 2022

We have reported previously that XS52 cells, a long-term dendritic cell (DC) line established from mouse epidermis, proliferate maximally in response to CSF-1, and that XS52 cells expanded in this manner induce brisk proliferation of HDK-1 T cells (KLH-specific Th1 clone) and 5S8 T cells (DNBS-specific Th0 clone) in the presence of Ag. Our purpose was to determine whether CSF-1-dependent mitotic potential of XS52 cells might be affected upon Ag-dependent interaction with these T cell clones. Both surface CSF-1R expression and mitotic responsiveness to CSF-1 became undetectable within 24 h after incubation with each T cell clone in the presence of relevant Ag. By contrast, incubation with T cells alone or Ag alone had minimal effect, indicating a requirement for both T cells and Ag. Exposure of fresh XS52 cells to the supernatant collected from complete XS52/HDK-1/KLH or XS52/5S8/DNBS coculture was sufficient to abrogate both CSF-1R expression and CSF-1 responsiveness. Importantly, both were restored by mAb against IFN-gamma, and both were diminished by rIFN-gamma in the absence of T cells or Ag. Thus, IFN-gamma, which was detected in relatively large amounts in the above supernatants, serves as a major mediator. rIFN-gamma reduced the number of CSF-1 binding sites on XS52 cell surface, without affecting CSF-1R mRNA expression. Thus, it appears that IFN-gamma down-regulates CSF-1R by a post-transcriptional mechanism. We interpret these results to document a novel, bi-directional signaling event in which Ag-dependent DC-T cell interaction promotes the growth of T cells, but inhibits the growth of DC.
IL-4 expression in human T cells is selectively inhibited by IL-1 alpha and IL-1 beta.
Authors: Sandborg C I , Imfeld K L , Zaldivar F , Wang Z , Buckingham B A , Berman M A
Published: December 2022

Imbalances in anti-inflammatory and proinflammatory cytokines may be responsible for initiation or progression of diverse pathologic states including autoimmune and infectious diseases. IL-4 production of proinflammatory cytokines and IL-12 promotes differentiation and activation of IFN-gamma-producing T cells, but does a counter-regulatory effect of proinflammatory cytokines on IL-4 production exist? This study evaluates the effect of proinflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-12, and TNF-alpha) on IL-4 production in primary human T cell cultures. PBMCs from healthy individuals were tested for IL-4 production in response to PHA and various cytokines. IL-4 was measured by proliferation of the IL-4-sensitive T cell line (CT.h4S) or ELISA. IL-1 alpha and IL-1 beta inhibited IL-4 production by 20 to 80% in > 92% of healthy individuals (p = 0.0001, paired t-test). IL-12 had an inhibitory effect on PBMC IL-4 production as previously described, but neither IL-6 nor TNF-alpha inhibited IL-4 production. IL-1 had no effect on PHA-induced PBMC or purified T cell proliferation or IL-2 production. IL-4 production by purified T cells stimulated by PHA or the combination of PMA with calcium ionophore (A23187) was inhibited by IL-1, and reconstitution with peripheral blood-derived adherent macrophages had no effect. IL-12 did not inhibit IL-4 production in stimulated purified T cells. Steady state IL-4 mRNA levels were determined by semiquantitative competitive reverse transcribed PCR (RT-PCR). Marked inhibition of IL-4 mRNA levels were seen at 5 h after exposure to IL-1. This interaction between IL-1 and IL-4 may be an important physiologic regulator of the balance between proinflammatory cytokines from activated macrophages and anti-inflammatory cytokines from T cells.
Extracellular Nef protein regulates productive HIV-1 infection from latency.
Authors: Fujinaga K , Zhong Q , Nakaya T , Kameoka M , Meguro T , Yamada K , Ikuta K
Published: December 2022

In HIV-1-infected asymptomatic carriers, the vast majority of infected cells in PBMCs are believed to be latently or nonproductively infected. We have isolated a subclone (MOLT-20-2) from an infected T cell line that expressed HIV-1 Ags at a very low level. However, viral Ag expression was markedly up-regulated by stimulation with either TNF-alpha, A23187, or PMA, indicating that the subclone might provide a suitable model of HIV-1 latency. Our previous studies have shown that the carboxyl-terminal region of the extracellular form of HIV-1 Nef played an important role in the interaction of infected cells with uninfected T cells, and could induce the cytostatic state. This suggested that Nef might contribute to intracellular signal transduction through an interaction with latently infected cells. We show in this study that stimulation of MOLT-20-2 with soluble Nef leads to HIV-1 activation from latency in a dose-dependent manner. Moreover, using a total of 14 overlapping Nef-related synthetic peptides, stimulatory activity was mapped to a discrete peptide (amino acid residues 132-147) that had the potential to activate latent HIV-1. This novel Nef function was confirmed by activation of virus production from the PBMCs of asymptomatic carriers. In addition, Nef-dependent HIV-1 activation from latency was also observed in another independently derived, latently infected cell line, U1, though not in cell line ACH-2. These results extend the significance of the Nef activity in vivo to the regulation of productive HIV-1 infection from latency, and define the regions of the protein involved.
Nitric oxide expression in the spleen, but not in the liver, correlates with resistance to blood-stage malaria in mice.
Authors: Jacobs P , Radzioch D , Stevenson M M
Published: December 2022

The production and function of nitric oxide during the early phase of blood-stage infection with Plasmodium chabaudi AS was analyzed using two inbred strains of mice that differ in the level of resistance to this parasite. Northern blot analysis of in vivo expression of inducible nitric oxide synthase (iNOS) revealed that early during infection resistant C57BL/6 mice, which clear the infection by 4 wk, have higher levels of iNOS mRNA in the spleen than susceptible A/J mice. In contrast, susceptible A/J mice have significantly increased levels of iNOS mRNA in the liver later in the course of infection just before death occurs. Splenic macrophages recovered from resistant C57BL/6 mice on day 7 postinfection express iNOS mRNA which is up-regulated following overnight stimulation of the cells with LPS. Furthermore, during the first week postinfection, splenic macrophages recovered from resistant hosts produce significantly higher levels of nitrite (NO2-) in vitro in response to LPS than similarly stimulated macrophages from susceptible A/J mice. Increased levels of nitrate (NO3-) were only detected in serum of resistant C57BL/6 mice at the time of peak parasitemia. Treatment with the iNOS inhibitor, aminoguanidine, reduced NO3- levels in serum of C57BL/6 mice and eliminated resistance of these hosts to P. chabaudi AS malaria without affecting parasitemia. These results demonstrate that the ability to produce high amounts of nitric oxide (NO) early during infection with blood-stage P. chabaudi AS correlates with resistance, but that NO may not be involved in parasite killing. Moreover, the tissue site of NO production, that is, spleen vs liver, appears to be critical and correlates with resistance vs susceptibility to P. chabaudi AS malaria, respectively.
A hierarchy for integrin expression and adhesiveness among T cell subsets that is linked to TCR gene usage and emphasizes V delta 1+ gamma delta T cell adherence and tissue retention.
Authors: Nakajima S , Roswit W T , Look D C , Holtzman M J
Published: December 2022

To define the relationship between T cell phenotype and adhesiveness, we examined T cell adhesion to endothelial cell, fibroblast, and epithelial cell monolayers as well as extracellular matrix proteins (collagen and fibronectin) using a three-color flow cytometry-based adherence assay that minimizes basal adhesion levels and facilitates quantitative lymphocyte subtyping. Regardless of monolayer type, monolayer stimulation conditions, or T cell activation status, we found that the gamma delta-TCR-bearing T cells adhered more efficiently than alpha beta T cells. The difference was based predominantly on increased levels of activatable LFA-1 (and to a lesser degree VLA-4) because: 1) it correlated precisely with inhibitability by anti-LFA-1 (and VLA-4) mAbs and the levels of LFA-1 (and VLA-4) on the cell surface, and 2) it persisted after maximal LFA-1 (and VLA-4) activation with phorbol dibutyrate. In contrast to most cases of alpha beta T cell behavior, gamma delta T cell adhesion to cell monolayers was not linked to memory status, i.e., there was no difference between naive V delta 1+ and memory V delta 2+ populations in levels of LFA-1 (or VLA-4) expression or LFA-1- (or VLA-4-) dependent adhesion to cell monolayers. However, V delta 1+ cells exhibited higher levels of VLA-5 that correlated with an increased adhesiveness to fibronectin and to a 120-kDa fibronectin fragment (FN-120) that contains only the VLA-5-binding domain but not to type I collagen or to a fibronectin fragment (FN-40) that binds only VLA-4. Taken together, the results define a hierarchy for integrin (LFA-1, VLA-4, and VLA-5) expression and consequent adhesion among T cell subsets that is linked to TCR gene usage (but not necessarily linked to memory status) and may thereby help to explain the accumulation and retention of V delta 1+ gamma delta T cells in epithelial and connective tissues.
Soluble antigen can impede affinity maturation and the germinal center reaction but enhance extrafollicular immunoglobulin production.
Authors: Pulendran B , Smith K G , Nossal G J
Published: December 2022

The primary immune response to T cell-dependent Ags develops in two pathways. These comprise the extrafollicular pathway, in which foci of Ab-secreting cells develop, and the intrafollicular pathway that gives rise to germinal centers and affinity maturation. We have previously shown that de-aggregated (4-hydroxy-3-nitrophenyl) acetyl (NP) coupled to the protein carrier human serum albumin (HSA), (NP-HSA), injected 6 days after challenge immunization with aggregated NP-HSA, resulted in impaired development of NP-specific, higher-affinity cells. Studies presented here describe the cellular basis underlying this impairment of affinity maturation. Using multiparameter flow cytometry, we show that mice injected with soluble NP-HSA ("tolerant" mice) develop significantly fewer NP-binding IgG1+ B220+ cells of germinal center origin than do the control ("immune") mice. In addition, using immunohistology, we noted that the spleens of tolerant mice had a marked reduction in the number of germinal centers that contained lambda-bearing cells, these being characteristic of the NP response in C57BL/6 mice. Curiously, germinal centers in the spleens of tolerant mice had more than twice the volumes of those in the immune spleens. In contrast to its effect on the germinal center pathway, soluble Ag enhanced the extrafollicular pathway, reflected by the increased numbers of B cells secreting IgM and IgG1 Abs specific for NP, HSA, and undefined Ags. Thus, soluble NP-HSA given after challenge immunization can impede affinity maturation of NP-specific cells, but enhance the extrafollicular pathway. These results are discussed in the context of the known capacity of some persisting Ags, e.g., malaria parasites, to frustrate affinity maturation and memory cell generation.
Small amounts of exogenous IL-4 increase the severity of encephalitis induced in mice by the intranasal infection of herpes simplex virus type 1.
Authors: Ikemoto K , Pollard R B , Fukumoto T , Morimatsu M , Suzuki F
Published: December 2022

The effect of murine rIL-4 on the development of herpesvirus encephalitis (HSE) in mice infected intranasally with herpes simplex virus type 1 (HSV-1) was investigated. The mortality rates of mice infected with a 0.5 LD50 dose of HSV-1 were greatly increased after the administration of rIL-4 at doses ranging from 0.01 to 1.0 U/mouse 2 h before and 2, 4 and 6 days after the infection. In contrast, survival rates of mice exposed to a 5 LD50 dose of HSV-1 were clearly increased when these mice were treated with anti-IL-4 mAb. Cervical lymph node (CLN) cells and cerebrospinal fluid (CSF) cells from mice with HSE (HSE mice) produced IL-4 in their culture fluids when they were stimulated in vitro with HSV-1 Ag. Increased amounts of HSV-1 infection in mice resulted in the increased production of IL-4 in the culture fluids of local lymphocytes. However, significant amounts of IL-4 were not produced in serum specimens or in culture fluids of spleen cells from HSE mice. IL-4 production in culture fluids of CLN and CSF cells from HSE mice was clearly reduced after treatment of HSE mice with anti-IL-4 mAb. Furthermore, IL-4 production by CLN and CSF cells was greatly enhanced when the cells were prepared from HSE mice previously treated with rIL-4. The IL-4 was mainly produced from CD4+ T cells. These results demonstrate that small amounts of exogenous IL-4 increase the severity of HSE in HSV-1-infected mice through the increased production of IL-4 from local CD4+ T cells.
Lipoarabinomannan inhibits nonopsonic binding of Mycobacterium tuberculosis to murine macrophages.
Authors: Stokes R W , Speert D P
Published: December 2022

The initial phagocytic interaction between Mycobacterium tuberculosis and macrophages (M phi) in the lung is probably nonopsonic, which would mean that M phi receptors will bind directly to bacterial ligands without the involvement of serum opsonins. Lipoarabinomannan (LAM) is a major component of the cell wall of mycobacteria. The possibility that LAM is involved in the nonopsonic binding of M. tuberculosis to M phi was investigated by using competitive inhibition assays. LAM inhibited binding of M. tuberculosis to murine peritoneal M phi in a dose-dependent manner. LAM also inhibited the binding of Mycobacterium avium and Mycobacterium bovis BCG to M phi. Phosphatidylinositol mannoside and lipomannan have the same phosphatidylinositol (PI) moiety as LAM, but differ in their glycosylation patterns. Both molecules inhibited binding of M. tuberculosis to M phi. Deacylation of LAM abrogated its capacity to inhibit binding of M. tuberculosis to M phi. These observations indicated that it was the PI moiety of LAM that was important in mediating its inhibitory properties. In support of this hypothesis, commercial PI was shown to inhibit the binding of M. tuberculosis to M phi. Our results suggest that cellfree LAM is able to inhibit the binding of mycobacteria to M phi, but that it does not do so by competing with any interaction between M phi receptors and cell-associated LAM, because the PI end of the molecule is believed to be anchored in the bacterial plasma membrane, and therefore not available as a ligand on the cell surface. However, LAM that is released from M. tuberculosis in the course of its active replication during infection may be able to interfere with the phagocytic clearance of mycobacteria.
Self-thyroid epithelial cell (TEC)-reactive CD8+ T cell lines/clones derived from autoimmune thyroiditis lesions. They recognize self-thyroid antigens directly on TEC to exhibit T helper cell 1-type lymphokine production and cytotoxicity against TEC.
Authors: Sugihara S , Fujiwara H , Niimi H , Shearer G M
Published: December 2022

Self-thyroid epithelial cell (TEC)-reactive CD8+ and CD4+ T cell lines were established by culturing T cells that infiltrate in autoimmune thyroiditis lesions. We investigated the properties of CD8+ T cell lines and clones in comparison with previously characterized CD4+ T cell lines/clones. Although the recognition of self-Ag by anti-TEC CD4+ T cell lines/clones required the cooperation of syngeneic spleen cells as APC, a representative CD8+ line (N4C) was stimulated with syngeneic TEC in the absence of APC. Precise analysis of MHC restriction using N4C-derived clones revealed that CD8+ clones recognize self-Ag on TEC in the context of class I MHC molecules. Most CD8+ clones were also found to express TCR with V beta specificities that were different from those observed for anti-TEC CD4+ clones. N4C cells produced IL-2, IFN-gamma, and TNF-alpha beta, but not IL-4 and IL-5 after stimulation with TEC, thus exhibiting the profile of lymphokine production similar to that expressed by CD4+ Th1 on one hand, but on the other, they showed the functional property that has not been observed for anti-TEC CD4+ clones. Namely, they elicited appreciable levels of cytolytic effects on syngeneic TEC in a short-term (4-h) 51Cr release assay. Thus, these results indicate that self-TEC-reactive CD8+ T cell lines/clones recognize Ag directly on TEC in a class I MHC-restricted way so as to exhibit various functions including the Th1-like profile of lymphokine production and anti-TEC cytolysis. The results are also discussed in terms of the nature of self-Ag presented with class I MHC molecules on TEC, as well as the potential roles of anti-TEC CD8+ T cells in the pathogenesis of thyroiditis.
Induction of tolerance to heart allografts in high responder rats by combining anti-CD4 with CTLA4Ig.
Authors: Yin D , Fathman C G
Published: December 2022

It has been difficult to induce donor-specific transplantation tolerance in high responder Lewis rats. Results presented below demonstrate that amounts of pretransplant anti-CD4 sufficient to allow allograft tolerance in low responder strains (5 mg/kg x 4 days) did not prevent the acute rejection of ACI heart allografts in high responder Lewis recipients. Higher doses of pretransplant anti-CD4 (10 mg/kg, 15 mg/kg, and 20 mg/kg) given alone could delay but not prevent allograft rejection. Pretransplant anti-CD4 combined with anti-CD8, thymectomy, and total lymphoid irradiation all failed to produce tolerance to ACI heart allografts. However, a regimen of anti-CD4 combined with CTLA4Ig allowed indefinite survival of ACI heart allografts (mean survival time, > 100 day). Second-donor matched heart grafts were permanently accepted, and third-party heart grafts were permanently accepted, and third-party heart allografts were rejected by the tolerant recipients. These results suggest a new combination therapeutic strategy for clinical transplantation.
Lymphotoxin-alpha-deficient mice. Effects on secondary lymphoid organ development and humoral immune responsiveness.
Authors: Banks T A , Rouse B T , Kerley M K , Blair P J , Godfrey V L , Kuklin N A , Bouley D M , Thomas J , Kanangat S , Mucenski M L
Published: December 2022

Targeted mutagenesis in embryonic stem cells was used to generate mice deficient in lymphotoxin-alpha (LT-alpha). Mice lacking LT-alpha -/- (LT-alpha -/- mice) exhibit a phenotype dominated by defects in secondary lymphoid organ development. LT-alpha -/- mice lack lymph nodes and Peyer's patches, and possess spleens in which the usual architecture is disrupted. However, in a few of the mutants, abnormal lymph node-like structures were observed, mainly within the mesenteric fat. Abnormal clusters of lymphocytes were also found to accumulate in the periportal and perivascular regions of the liver and lung of LT-alpha -/- mice. Yet, lymphocytes from LT-alpha -/- mice appeared phenotypically normal, expressing the expected ratios of B and T cell surface markers as well as the lymphocyte homing marker, L-selectin. In addition, bone marrow cells from LT-alpha -/- mice were able to successfully reconstitute the lymphoid organs of severe combined immunodeficient mice. However, LT-alpha -/- mutant mice examined for humoral immune responsiveness were found to be impaired in their ability to respond to different Ag. These data illustrate the utility of this mouse model as a system for understanding lymphoid organ development and its effects on immune responsiveness.
Membrane and soluble Fc gamma RII/III modulate the antigen-presenting capacity of murine dendritic epidermal Langerhans cells for IgG-complexed antigens.
Authors: Esposito-Farese M E , Sautès C , de la Salle H , Latour S , Bieber T , de la Salle C , Ohlmann P , Fridman W H , Cazenave J P , Teillaud J L
Published: December 2022

Murine dendritic epidermal Langerhans cells (LC) are APC. This implies that LC take up, process, and present Ag to T cells. One way of doing so that could allow Ag internalization is provided by the low affinity receptors for the Fc region of IgG (Fc gamma R), which murine LC are known to express, although their isoform(s) and function(s) have not been defined. By using molecular biology and biochemical approaches, we demonstrated that LC expressed Fc gamma RIIb2 and Fc gamma RIII. Furthermore, LC internalized Fc gamma R by receptor-mediated endocytosis, as observed with gold-labeled anti-Fc gamma RII/III mAb or immune complexes. We demonstrated the biologic relevance of this process by observing that Fc gamma R-mediated Ag internalization improved by approximately 300-fold the Ag-presenting capacity of LC to T cells. Moreover, analysis of cell culture supernatants showed that two forms of soluble Fc gamma R (sFc gamma R) were released by LC: the first most probably was the secreted transmembrane-deleted Fc gamma RII isoform, Fc gamma RIIb3, and the second was a soluble receptor probably derived from the membrane-associated Fc gamma RII/III. The ability of two recombinant forms, corresponding to the two sFc gamma R released by LC, to inhibit Fc gamma R-mediated presentation enhancement was assayed. Preincubation of IgG-complexed Ag with either rsFc gamma R led to a dose-dependent decrease in the Ag-presenting capacity of LC. Taken together, our results suggest that, in vivo, LC express membrane Fc gamma R, which increase their Ag-presenting capacity for IgG-complexed Ag, and release sFc gamma R, which might be able to modulate this Ag presentation.
CD28-mediated signaling in vivo prevents activation-induced apoptosis in the thymus and alters peripheral lymphocyte homeostasis.
Authors: Shi Y , Radvanyi L G , Sharma A , Shaw P , Green D R , Miller R G , Mills G B
Published: December 2022

Activation of T lymphocytes can result in a functional immune response, anergy or apoptosis. Functional T cell activation requires the interaction of the TCR with Ag presented by MHC molecules on APC concurrent with appropriate interactions between cell surface accessory molecules. Interestingly, the level of CD28 expression is regulated during T cell development as well as during T cell activation and proliferation, suggesting that CD28 could play a role in determining the outcome of activation of TCR during T cell ontogeny. We identify, herein, a novel function of murine CD28 in the regulation of activation-induced apoptosis in thymocytes. In vivo, or combined in vivo and in vitro treatment with mAbs to CD28 prevents apoptosis of CD4+CD8+ thymocytes induced by Abs to the TCR complex. Prolonged administration of anti-CD28 Abs increased the number of both CD4+CD8- and CD4-CD8+ T cells in the thymus, while the number of CD4+CD8+ T cells is relatively unchanged. Furthermore, this treatment leads to a dramatic enlargement of peripheral lymphoid organs characterized primarily by the expansion of B cells. The number of CD4+CD8- T cells in the spleen of anti-CD28-treated mice is also moderately increased, while the number of CD4-CD8+ cells is relatively unchanged.
Glucocorticoids accelerate anti-T cell receptor-induced T cell growth.
Authors: Wiegers G J , Labeur M S , Stec I E , Klinkert W E , Holsboer F , Reul J M
Published: December 2022

To study steroid regulation of cell-mediated immunity, we used anti-TCR-stimulated rat splenic lymphocyte mitogenesis as our experimental paradigm. Surprisingly, we found that the principal glucocorticoid of the rat, corticosterone (CORT), potently enhanced anti-TCR-induced lymphocyte proliferation after 2 to 3 days in culture, followed by inhibited cell growth after 5 to 7 days. Thus, glucocorticoids appeared to accelerate anti-TCR-induced lymphocyte mitogenesis. This effect occurred at physiologic concentrations (50-1000 nM), which are known to be released in vivo after an immune challenge. Kinetic experiments showed that CORT had to be present within 60 min after the initiation of TCR activation to produce maximal enhancing effects; a delay of 2 h or more left CORT ineffective. The lymphocytes incubated with CORT may have an increased sensitivity to IL-2 because 1) CORT suppressed IL-2 production throughout the culture period, and 2) an anti-IL-2R mAb completely blocked both control and CORT-treated anti-TCR-induced lymphocyte proliferation. Although the IL-2R alpha- and beta-chain mRNA concentrations were not altered in CORT-treated splenocyte cultures, we observed by FACS analysis an increased expression of the IL-2R alpha-chain on CORT-treated TCR alpha beta + and CD4+ T cells after 48 to 72 h of culture, suggesting an increased sensitivity of these T cells to IL-2 during the phase of enhanced proliferation. These results demonstrate a clear distinction between the enhancing effects of glucocorticoids on anti-TCR-induced lymphocyte proliferation and their well known inhibitory actions. Thus, the present study expands the regulatory role of glucocorticoids in cellular immunity, adding a novel effective stimulatory component to their inhibitory properties.
Differential stability of HLA-DR alleles independent of endogenous peptides.
Authors: Devaux B , Wilson K J , Aguilar B , Jorgensen B , Rothbard J B
Published: December 2022

Purified HLA DRB1*0101 was shown to be inherently more stable to dissociation than DRB1*0401. The residues responsible for the differential stability were defined by constructing hybrid molecules, which contained a small number of residues from DRB1*0101 substituted into the framework of DRB1*0401. One of the hybrid molecules, containing six substituted amino acids, was as stable as DRB1*0101, but exhibited the binding specificity of DRB1*0401. This result indicated that the differential stability between the alleles arose from structural differences, and was not due solely to varying populations of endogenous peptides.
Transforming growth factor-beta stimulates arginase activity in macrophages. Implications for the regulation of macrophage cytotoxicity.
Authors: Boutard V , Havouis R , Fouqueray B , Philippe C , Moulinoux J P , Baud L
Published: December 2022

Macrophage arginine metabolism via nitric oxide (NO) synthase and arginase pathways reduces and enhances tumor cell proliferation, respectively. Transforming growth factor-beta (TGF-beta) has been shown to down-regulate the NO synthase pathway. The present study describes the effect of TGF-beta on the arginase pathway. TGF-beta up-regulated arginase activity in rat peritoneal macrophages as assessed by measuring the generation of [14C]urea from [14C]-L-arginine in the presence of NG-monomethyl-L-arginine (L-NMMA). The stimulation, which reached fivefold after a 48-h exposure of macrophages to 10 ng/ml TGF-beta, was due to reduction in Km value of arginase. TGF-beta-induced up-regulation of arginase activity led to the release of more polyamines, mainly putrescine. The role of this up-regulation on macrophage cytotoxicity toward L-929 tumor cells was analyzed in coculture experiments. Macrophages blunted DNA synthesis by L-929 cells as assessed by measuring the incorporation of [3H]TdR into the cells and the proportion of cells in the G2 phase. Addition of TGF-beta in the presence of L-NMMA permitted L-929 cells cocultured with macrophages to resume DNA synthesis. The mechanism responsible for this restoration was the up-regulation of arginase activity rather than the down-regulation of NO synthase activity since TGF-beta in the presence of L-NMMA failed to further reduce NO synthase activity whereas it still enhanced arginase activity; synthetic putrescine (1-10 microM) also blunted macrophage cytotoxicity toward L-929 cells. This is the first evidence that TGF-beta up-regulates arginase activity in macrophages and, hence, limits macrophage-dependent cytostasis.
Differential up-regulation of HLA class I molecules on neuronal and glial cell lines by virus infection correlates with differential induction of IFN-beta.
Authors: Dhib-Jalbut S S , Xia Q , Drew P D , Swoveland P T
Published: December 2022

Adult neurons normally lack the expression of MHC class I molecules, which has implications on virus clearance from the central nervous system. The author previously demonstrated that HLA class I up-regulation in measles virus (MV)-infected glial cells is primarily mediated by IFN-beta. In contrast, this study demonstrates that MV-infection of the neuronal cell lines IMR-32 and CHP-126 fails to up-regulate HLA class I expression, which was associated with an inability of MV to induce IFN-beta in the neuronal cell lines. However, treatment with IFN-beta on coculture of the IMR-32 neuronal cell line with MV-infected glioma cells resulted in the up-regulation of HLA class I on the former, which could be neutralized by anti-IFN-beta Ab. The inability of MV to up-regulate HLA class I expression on the neuronal cell line IMR-32 was not virus specific because similar findings were observed with mumps virus or stimulation with the synthetic dsRNA polyinosinic polycytidylic acid (PIPC). Induction of IFN-beta gene expression by virus requires binding of NF-kappa B to the positive regulatory domain II element of the IFN-beta promoter. Our studies indicate that MV, TNF-alpha, or PIPC induces NF-kappa B (p50 and p65 subunits) binding to positive regulatory domain II in the glioma cell line. In contrast, such activity was induced by TNF-alpha but not MV or PIPC in the neuronal cell line IMR-32. This indicated that HLA class I expression is differentially regulated in glial and neuronal cell lines in response to MV, which correlates with differential binding of NF-kappa B to the IFN-beta promoter and induction of IFN-beta gene expression.
Cell surface molecules related to factor J in human lymphoid cells and cell lines.
Authors: Jiménez-Clavero M A , González-Rubio C , Larrucea S , Gamallo C , Fontán G , López-Trascasa M
Published: December 2022

Factor J (FJ) is a cationic glycoprotein that is able to inhibit in vitro both the classical and alternative pathways of complement. FJ was purified to homogeneity from human urine by sequential chromatographic steps. To examine the expression of FJ in human cells we obtained mAbs against urine-purified FJ. Preliminary studies by immunocytochemistry revealed that one of the anti-FJ mAbs recognized cell surface components of certain cell lines, such as K562 and U937 cells, so we have focused subsequently on the detection of these homologue membrane-bound FJ Ags (FJ-h Ags) in cell lines of lymphoid (Ramos and Jurkat) and mieloyd (U937 and K562) origin, as well as in peripheral blood cells. The flow cytometry analysis of the examined cell lines revealed partial staining ranging from 10% (U937) to 29% (K562) positive cells. Flow cytometry of peripheral blood cells showed a positive staining in a small but consistent population of lymphocytes (mean = 11%, n = 17) but none at all on monocytes, granulocytes, erythrocytes, or platelets. Double Ab immunostaining of lymphocytes showed that the FJ-h positive population included mainly B lymphocytes (a mean of 63% CD19+ were FJ-h positive). When we analyzed peripheral blood lymphocytes from a patient with chronic lymphocytic leukemia B (95% CD19+/CD5+), the majority of these (55%) bore FJ-h on their surface. Acid strip of these cells did not abrogate the surface staining, which supports the finding that the Ag is tightly bound to the membrane. Immunoprecipitation from U937 cell lysates showed a single 65 kDa band under reducing conditions. FJ-h Ags purified from K562 and U937 cells displayed inhibitory activity in the functional EAC14 assay for the classical complement pathway, as did urine FJ, and they were recognized immunochemically by five different (one polyclonal and four monoclonal) anti-FJ Abs. In conclusion, FJ-homologues are present in the membranes of several human cell lines that show functional and antigenic characteristics similar to soluble urine FJ. They are also found in a small subset of peripheral blood lymphocytes, mainly B cells. The structural relationship between both soluble urine FJ and these membrane-bound FJ-h remains to be established.