bluuebunny/crossref_metadata_2025 huggingface dataset:
The proximal promoter of the IL-4 gene is composed of multiple essential regulatory sites that bind at least two distinct factors.
Authors: Hodge M R , Rooney J W , Glimcher L H
Published: December 2022

Immune responses to pathogens often lead to the generation of polarized T helper subsets designated Th1 and Th2. Th1 cells, characterized by the production of IL-2 and IFN-gamma, stimulate cellular immune responses important for protection against intracellular pathogens. In contrast, Th2 cells, which produce IL-4, are potent stimulators of B cells and stimulate protection against extracellular pathogens. IL-4 has also emerged as a key cytokine in T cell differentiation since it has been shown to direct the development of naive T cells toward a Th2 phenotype. Recent studies have provided insights into the transcriptional regulation of IL-4, including the identification of multiple binding sites for a subunit of the IL-2 transcription factor NF-AT. In this study we describe the characterization of an essential region of the IL-4 promoter located immediately upstream of the TATA element. High-resolution mutagenesis of this 33-bp region revealed multiple sites indispensable for inducible IL-4 transcription. Included in this region are overlapping binding sites for the cyclosporin A-sensitive factor NF-ATp and a novel constitutively expressed factor designated PCC. An additional sequence adjacent to the TATA element is also shown to be critical for IL-4 transcription in Th2 cells.
Coding end processing is similar throughout ontogeny.
Authors: Nadel B , Tehranchi S , Feeney A J
Published: December 2022

During the recombination process, extensive processing of the coding ends provides tremendous potential diversity to the joint of any two gene segments. However, the diversity of the newborn B and T cell repertoires is greatly reduced compared with that of the adult. At the mechanistic level, this difference is primarily due to the absence of terminal deoxynucleotidyltransferase expression until the first week after birth. Additionally, one direct consequence of the lack of N regions early in ontogeny is the more frequent occurrence of homology-directed recombination, reducing even further the potential of diversity. Other enzymatic factors could also contribute to this ontogenic difference. However, the use of the homology-directed recombination pathway early in life obscures the analysis of the coding end processing. In this study we compared the coding end processing throughout ontogeny, in normal and terminal deoxynucleotidyltransferase -/- mice in the presence of minimal homology-directed recombination. The analysis of partial D-J joints allowed us to avoid potential bias by early selection events. Our results show that the extent of nucleotide deletion of a given end is consistent throughout ontogeny in the presence or absence of terminal deoxynucleotidyltransferase. However, a distinctive processing pattern is observed for each coding end.
Antibodies to murine complement receptor 1 and 2 can inhibit the antibody response in vivo without inhibiting T helper cell induction.
Authors: Gustavsson S , Kinoshita T , Heyman B
Published: December 2022

Administration to mice of mAb against CR1/CR2 inhibits the primary and secondary in vivo Ab response. We have addressed the question of whether induction of T helper cells, in addition to B cells, is inhibited by this treatment. A hapten-carrier system was used, where one group of mice was immunized with a monoclonal CR1/CR2-specific Ab and Ag (SRBC or keyhole limpet hemocyanin (KLH)), another with Ag alone, and a third group left nonimmunized. After 2 to 15 wk, spleen cells from these mice were transferred to irradiated syngeneic recipients, together with Ag coupled to the hapten 4-hydroxy-5-iodo-3-nitro-phenacetyl (NIP). The number of NIP-specific, IgG-producing B cells, which in this system is an indication of T cell priming, was determined in an ELISPOT assay. Although the primary SRBC- or KLH-specific IgG response was inhibited markedly (74-98%) by CR1/CR2-specific Ab, no decrease in the number of NIP-specific IgG-producing B cells was detected after secondary immunization. In sharp contrast, the SRBC- or KLH-specific secondary IgG responses were low or undetectable. These findings suggest that CR1/CR2-specific Ab can inhibit the primary Ab response without affecting T helper cells and that the induction of B cell memory is decreased markedly by this treatment.
Induction of opioid receptor-mediated macrophage chemotactic activity after neonatal brain injury.
Authors: Milligan C E , Webster L , Piros E T , Evans C J , Cunningham T J , Levitt P
Published: December 2022

Macrophages have a prominent role in the injury response of the brain, yet the molecular mechanisms that control their invasion to the site of neuronal degeneration is unknown. After removal of the posterior cortex at birth, there is massive and specific targeting of nonresident macrophages to axotomized neurons in the lateral thalamus. The present study has identified an injury-induced, brain-derived chemotactic factor (BDCF) capable of eliciting chemotactic responses from resident peritoneal macrophages and brain macrophages. Conditioned media collected from tissue slices containing the axotomized central nervous system neurons exhibit BDCF activity. Initial experiments indicated that BDCF is a small peptide and, thus, we used specific pharmacologic reagents to characterize further BDCF activity. Naloxone, a pan opioid receptor antagonist, completely blocks BDCF activity. Although both kappa and mu opioid receptor antagonists failed to modify BDCF-induced macrophage chemotaxis, two specific delta receptor antagonists blocked BDCF. Analysis of BDCF by reverse phase HPLC and RIA revealed peak chemotactic activity in fractions consistent with the presence of an opioid peptide. The results suggest that cells in the brain respond to neuronal injury by producing and releasing opioids that can initiate a specific macrophage response.
Curative and protective effects of IL-10 in experimental autoimmune thyroiditis (EAT). Evidence for IL-10-enhanced cell death in EAT.
Authors: Mignon-Godefroy K , Rott O , Brazillet M P , Charreire J
Published: December 2022

We studied the effects of in vivo administration of rhIL-10 in two models of experimental autoimmune thyroiditis (EAT): 1)-in EAT induced by injection of mTg emulsified in adjuvant, and 2) in EAT induced by adoptive transfer of mTg-specific T lymphocytes. Furthermore, we tried to assess both the protective and curative potential of IL-10 in EAT, by administering rhIL-10 either at the time of priming and challenge with mTg, or only at the time of challenge. We demonstrated that proliferative and cytotoxic responses of splenic cells to mTg were markedly reduced by in vivo rhIL-10 treatment. Cell surface marker studies revealed a 40 to 45% reduction in CD4+ and in CD8+ lymphoblastoid spleen cells from mice treated with rhIL-10 either in the early or in the late EAT. The severity of EAT was significantly reduced in mice treated with high-dose rhIL-10, whereas levels of autoantibodies to mTg were not altered. Furthermore, when analyzing purified T lymphocytes from rhIL-10-treated animals, an increase of cells undergoing apoptotic cell death became evident in the rhIL-10 treated group, as compared with controls. This IL-10-mediated enhancement of activation-induced cell death critically depended on the applied therapeutic dose of rhIL-10. Thus, IL-10 exerts beneficial effects on the development and course of EAT through a mechanism that could imply an IL-10-mediated enhancement of activation-induced cell death in T lymphocytes, findings that call for considering IL-10 in the immunotherapy of early-phase and likewise of already established autoimmune thyroiditis.
Constitutive presentation of dominant epitopes from endogenous naturally processed self-beta 2-microglobulin to class II-restricted T cells leads to self-tolerance.
Authors: Guéry J C , Sette A , Appella E , Adorini L
Published: December 2022

The mouse beta 2-microglobulin (m beta 2-m) peptide corresponding to residues 25-40 binds to the MHC class II molecules I-Ad and I-Ed and is immunogenic in BALB/c beta 2-m-deficient but not in normal BALB/c mice. The self-m beta 2-m peptide 25-40 is presented by both I-Ad and I-Ed class II molecules as demonstrated by the activation of T cell hybridomas specific for this sequence obtained from beta 2-m knock-out mice. By analyzing the effect of N- and C-terminal truncations of m beta 2-m25-40 on binding to class II molecules and on activation of T cell hybridomas, the minimum epitopes recognized by I-Ad and I-Ed-restricted T cells are included within amino acid residues 26-39 and 24-36, respectively. Both sets of T hybridomas are also activated by the corresponding naturally processed self-epitope presented by APC from BALB/c mice and from other H-2d strains, irrespective of their Mls phenotype. Therefore, the sequence 25-40 contains dominant naturally processed self-epitopes of the mouse beta 2-m. Processing of endogenous m beta 2-m is sensitive to protease inhibitors and lysosomotropic amines, and is not caused by reuptake of shed or released protein. These results indicate that self-beta 2-m-peptide-MHC class II complexes derive from constitutive processing of the endogenous intracellular pool of m beta 2-m in an acidic endosomal compartment. Antigenic complexes between m beta 2-m peptides and I-Ad or I-Ed class II molecules are constitutively expressed by APC of different tissues, including the thymus, and they are able to induce T cell tolerance, as shown by the lack of T cell response to m beta 2-m25-40 in BALB/c mice.
CD45-cross-linking stimulates IFN-gamma production in NK cells.
Authors: Shen F , Xu X L , Graf L H , Chong A S
Published: December 2022

The in vitro demonstration of the ability of NK cells to secrete cytokines prompted in vivo studies that illustrated the importance of NK cell-derived cytokines in regulating immune responses. Cross-linking of CD16 on NK cells can stimulate cytokine production. CD16-independent interactions capable of stimulating cytokine production have also been described, but molecules mediating such stimulations remain to be biochemically defined. We report here that cross-linking of CD45 specifically stimulates IFN-gamma production in human NK cells. The NK cells used were IL-2-activated adherent NK cells and from the NK3.3 cell line. The ability of CD45 mAbs to stimulate NK cells appears not to be dependent on CD16, as CD45 mAbs of both IgG1 and IgG2a isotypes were equally stimulatory, as were F(ab')2 compared with whole anti-CD45 mAbs. Resting NK cells, like T cells, express predominantly CD45RA, whereas IL-2 activated adherent NK cells acquire expression of CD45RO. Abs specific for CD45RO, but not CD45RA, were able to stimulate IFN-gamma production in NK cells. It has been reported that one ligand for CD45RO is CD22 beta. We tested the ability of CD22-expressing transfectants to bind to and stimulate NK cells. Whereas NK cells bound to CD22 alpha and CD22 beta transfectants, this interaction was not inhibited by CD45RO Abs. In addition, neither of the CD22-transfectants were able to stimulate NK3.3 cells to secrete IFN-gamma. These observations collectively suggest that binding of NK3.3 cells to CD22 may be independent of CD45RO on NK3.3 cells.
Cis-acting DNA elements of mouse granulocyte-macrophage colony-stimulating factor gene responsive to Fc epsilon receptor cross-linking stimulation in the mouse mast cell line MC/9.
Authors: Nishikomori R , Kawai M , Jung E Y , Tai G , Miyajima A , Arai N , Mayumi M , Heike T
Published: December 2022

Mouse mast cells produce many kinds of cytokines in response to cross-linking of high affinity Fc epsilon receptor (Fc epsilon RI). Among these cytokines, granulocyte-macrophage CSF (GM-CSF) gene induction in mouse mast cells has been reported to be regulated at both the transcriptional level and the post-transcriptional level. We analyzed the mechanism of the transcriptional regulation of GM-CSF gene induction through Fc epsilon RI cross-linking stimulation in the mouse mast cell line MC/9. In MC/9, the GM-CSF gene was activated transcriptionally by Fc epsilon RI cross-linking stimulation. The 5' deletion analysis of GM-CSF gene promoter indicated that the 5' boundary of the responsive promoter region lay between positions -113 and -95. When the deletion was extended to positions -72 or -60, the stimulatory effect was significantly diminished. We then examined 3' deletion of pmGMCAT -113 from position -60. This analysis indicated that the 3' boundary lay between positions -84 and -72. No subfragments of the region spanning positions -113 to -72 could cover the full induction level. A site-directed mutagenesis experiment revealed that the sequence spanning positions -108 to -72 was needed for full activation. These data indicate that GM-CSF gene in mast cells is activated mainly through the sequence spanning positions -108 to -72.
HLA-A*0201 complexes with two 10-Mer peptides differing at the P2 anchor residue have distinct refolding kinetics.
Authors: Robbins P A , Garboczi D N , Strominger J L
Published: December 2022

The immune response to viruses partially depends on the biochemical interaction between viral peptides and histocompatibility molecules. In this study, the refolding of recombinant HLA-A*0201 heavy chain and beta 2-microglobulin (beta 2-m) in the presence of peptides from influenza B nucleoprotein (BNP), influenza A matrix protein, and HIV gp120 and their analogues was examined. The plateau value for the amount of refolded complex with three peptides, a 10-mer BNP 85-94 (A86) with alanine substituted for leucine at the P2 anchor residue and two BNP 8-mers, was significantly lower than the native peptide epitope BNP 85-94 or with other peptides tested. To attempt to understand the basis for the lower yield of complex, equilibrium dissociation constants (KdS) for the two 10-mers, BNP 85-94 (A86) and BNP 85-94, were determined from association and dissociation rates and from Scatchard plots, all measured at 10 degrees C. In addition, dissociation rates were measured at 0 degrees, 26 degrees, and 37 degrees C. Although the kinetics were similar at 0 degrees and 10 degrees, at 37 degrees these two complexes had distinct rates of dissociation, resulting in relatively stable or unstable complexes. The behavior of the unstable complexes paralleled the behavior of empty complexes described in vivo; they are unstable at physiologic temperature, produced in low yield, and stabilized by low temperature. Comparison of all of the kinetic data suggests that the equilibrium amounts of the two HLA/peptide complexes (plateau values) result from distinct reaction pathways, i.e., that the molecules that form stable complexes may undergo an additional reaction to those that form unstable complexes.
A cDNA clone coding for a novel allergen, Cla h III, of Cladosporium herbarum identified as a ribosomal P2 protein.
Authors: Zhang L , Muradia G , Curran I H , Rode H , Vijay H M
Published: December 2022

Mold allergens represent a major cause of atopic disorders. Progress in the molecular characterization of allergens has been hampered by batch-to-batch variation and poor yields in mold extracts. In the present study, we established a cDNA library in lambda ZAP II by using mRNA isolated from a major allergen-producing mold, Cladosporium herbarum. From this library, a novel allergen has been cloned and sequenced. The clone encodes a full length protein of 111 amino acids with a molecular mass of 11.1 kDa and pI of 3.94. By using sequence homology analysis, the allergen was found to belong to the ribosomal P2 protein family. Approximately 60% peptide sequence homology was found between the cloned protein and other known fungal ribosomal P2 proteins. In addition to conserved C-terminal sequences and serine blocks for phosphorylation in all eukaryotic ribosomal P2 proteins, this protein also contains a potential N-glycosylation site at position 15-17. Northern blots demonstrated that mRNA molecules of this gene are present even at late stages of culture. The gene was expressed in Escherichia coli by using the pMAL-2c system, and murine antisera against the recombinant allergen (rCh2.1) were generated. The antisera revealed that the native allergen corresponding to rCh2.1 was present in extracts prepared by grinding mycelia under liquid nitrogen in the presence of protease inhibitors, but absent in extracts prepared by conventional methods. This result indicates the usefulness of recombinant allergens in characterization and standardization of mold allergenic extracts.
Activation-induced CD4+ T cell death by apoptosis in experimental Chagas' disease.
Authors: Lopes M F , da Veiga V F , Santos A R , Fonseca M E , DosReis G A
Published: December 2022

Infection of mice with Trypanosoma cruzi, the causative protozoan agent of human Chagas' disease, leads to immunosuppression of the T cell compartment and to chronic cardiac inflammation which resembles the human infection. Recently, reinduction of programmed cell death by apoptosis in mature T cells has been demonstrated. It has been suggested that mature T cell apoptosis could play a role in immunosuppression caused by virus infection. In this report, we have investigated the occurrence of mature T cell apoptosis in murine experimental Chagas' disease. Infection with T. cruzi metacyclic forms led to a relative accumulation of CD8 T cells over CD4 T cells in the spleens of infected mice. Splenic T cells from T. cruzi-infected donors, but not from control littermates, died in vitro upon stimulation with T cell mitogens Con A and anti-TCR-alpha beta mAb in a dose-dependent fashion. DNA fragmentation into nucleosome-sized bands was detected in the supernatants of CD4+ T cells from infected origin, after stimulation with the T cell mitogen Con A. Upon in vitro stimulation with either anti-TCR-alpha beta or Con A, CD4+ T cells were susceptible to elimination, whereas CD8+ T cells were not. Splenic T cells from infected donors were markedly unresponsive to anti-TCR mAb in proliferative assays and underwent apoptosis in vitro, as assessed by electron microscopy. Apoptosis also occurred in vivo in the course of acute infection, as seen by DNA fragmentation in freshly explanted splenic cells and purified T cell subsets. The data indicate that activation-induced CD4+ T cell death by apoptosis is a prominent feature of experimental infection with T. cruzi, and could play a role in immunosuppression and parasite persistence in infected hosts.
Generation of tumor-specific CTLs from melanoma patients by using peripheral blood stimulated with allogeneic melanoma tumor cell lines. Fine specificity and MART-1 melanoma antigen recognition.
Authors: Stevens E J , Jacknin L , Robbins P F , Kawakami Y , el Gamil M , Rosenberg S A , Yannelli J R
Published: December 2022

PBLs were isolated from 13 patients with metastatic melanoma. Mixed lymphocyte tumor cell cultures (ML TCs) were established (15 times) by using irradiated HLA-matched (one class I locus) allogeneic melanoma tumor cell lines (13 times) or autologous melanoma tumor cell lines (two times) in medium containing 120 IU/ml IL-2 and 100 IU/ml IL-4. PBLs grew to levels that could be assessed for functional reactivity 9 of 15 times. In seven of nine cases, CD3+CD8+ CTLs grew from MLTCs that were tumor specific; five were restricted by HLA-A2 and two were restricted by HLA-A24. Four of the tumor-specific CTL lines lysed autologous fresh tumor cells. Tumor-specific CTLs from two of three patients had cytolytic activity identical with tumor-infiltrating lymphocytes (TIL) derived from tumor biopsies removed earlier and grown in high concentrations (6000 IU/ml) of IL-2. Three of the HLA-A2-restricted tumor-specific CTLs were shown to recognize 293 cells transfected with HLA-A2.1 cDNA and the gene encoding the melanoma Ag, MART-1. In addition, these CTLs recognized the T2 cell line pulsed exogenously with the peptide MART-1(27-35), which is the nine-amino acid immunodominant epitope of the MART-1 Ag recognized on melanoma tumor cells by nearly all HLA-A2-restricted TIL. Thus, we have demonstrated the ability to generate tumor-specific CTLs from PBLs that are similar in their reactivity to TIL. This technique obviates the need for autologous tumor tissue and suggests that PBLs contain sufficient CTL precursors for use in generating antitumor CTLs for cellular immunotherapy trials.
NK cell response to viral infections in beta 2-microglobulin-deficient mice.
Authors: Tay C H , Welsh R M , Brutkiewicz R R
Published: December 2022

Because class I MHC Ags have been implicated as modulators of target cell sensitivity to NK cell-mediated lysis, the regulation of virus infections and the fate of NK cells and their natural targets was examined in beta 2-microglobulin-deficient mice, which have defective class I MHC expression. Infections with either the NK cell-sensitive murine cytomegalovirus (MCMV) or the NK cell-resistant lymphocytic choriomeningitis virus (LCMV) significantly augmented NK cell activity in either C57BL/6 (+/+) or beta 2-microglobulin knockout (-/-) mice. Depletion of NK cells in vivo with antiserum to asialo-GM1 markedly enhanced the synthesis of MCMV but had no effect on the synthesis of LCMV in either strain of mouse. Analysis of naturally NK cell-sensitive thymocyte targets from these virus-infected -/- mice revealed no cell surface expression of class I MHC detectable by conformation-dependent or -independent Abs, but the virus infections enhanced class I expression on thymocytes from +/+ mice. The sensitivity of +/+ thymocytes to NK cell-mediated lysis was markedly reduced after in vivo poly inosinic:cytidylic and treatment or viral infection; in contrast, the sensitivity of the -/- thymocytes was significantly less affected by poly inosinic:cytidylic acid treatment or viral infection. These data indicate that the normal expression of class I MHC Ags on NK cells or their targets is not required for the antiviral functions of NK cells against a NK-sensitive virus (MCMV) nor do they protect a NK-resistant virus (LCMV) from the antiviral activity of NK cells.
IL-13 selectively induces vascular cell adhesion molecule-1 expression in human endothelial cells.
Authors: Bochner B S , Klunk D A , Sterbinsky S A , Coffman R L , Schleimer R P
Published: December 2022

Previous studies with human umbilical vein endothelial cells (HUVEC) have shown that the cytokine IL-4 induces adherence of human eosinophils, but not neutrophils, because of its ability to selectively induce surface expression of vascular cell adhesion molecule-1 (VCAM-1). Because the cytokine IL-13 shares a number of biologic properties with IL-4, we examined the effect of IL-13 on the expression and function of adhesion molecules on HUVEC. Incubation of HUVEC for 4 to 48 h with IL-13 (0.1 to 15 U/ml) induced surface expression of VCAM-1, as detected by indirect immunofluorescence and flow cytometry, without significantly affecting expression of E-selectin or intercellular adhesion molecule-1. The kinetics and maximal IL-13-induced expression of VCAM-1 were similar to those seen with IL-4. Treatment of HUVEC with an optimal concentration of IL-13 (15 U/ml for 24 h) induced adhesiveness for eosinophils, but not for neutrophils, and adhesion was completely inhibited by mAb recognizing VCAM-1 or alpha 4 integrin (CD49d). These results demonstrate that IL-13, like IL-4, selectively stimulates HUVEC to express functional cell surface VCAM-1 and suggest a possible role for IL-13 in promoting VCAM-1/alpha 4 integrin-dependent accumulation of eosinophils during allergic and other inflammatory reactions in vivo.
Complement component C7 is a plasminogen-binding protein.
Authors: Reinartz J , Hänsch G M , Kramer M D
Published: December 2022

Ab deposition, whether by reaction with the specific Ag or by preformed immune complexes, is followed by activation and deposition of complement components. Tissue destruction is observed in the Ab- and complement-induced lesions. The proteolytic enzyme plasmin is thought to participate in the Ab- and complement-mediated organ pathology. Plasmin is generated from plasma-derived plasminogen by cell-derived plasminogen activators (PAs). Two types of PAs are known, urokinase-type PA (uPA) and tissue-type PA (tPA). We investigated whether the PA system and the complement system can interact to promote local plasmin generation. Among the terminal complement components C5b6, C7, C8, and C9, the nonenzymatic component C7 is a plasminogen-binding protein. Radioligand binding studies revealed that the isolated component, as well as C7 after its incorporation into the terminal complement complex C5b-9, can bind plasminogen. Binding was inhibited by the lysine analogues 6-aminohexanoic acid and tranexamic acid, implicating the lysine binding sites of plasminogen into the binding interaction. tPA-mediated plasminogen activation was enhanced in the presence of C7. Based on these findings, an interaction is proposed between the complement system and the plasminogen activator system; a mechanism that may focus plasmin activity to structures that have been tagged by Ab and complement deposition.
Heparan sulfate initiates signals in murine macrophages leading to divergent biologic outcomes.
Authors: Wrenshall L E , Cerra F B , Singh R K , Platt J L
Published: December 2022

We have previously shown that the interaction of heparan sulfate (HS), a constituent of cell surfaces and extracellular matrices, with murine macrophages causes activation of the macrophages leading to the production of cytokines and PGE2 and profound changes in the cellular immune responses triggered by the macrophages. Here we describe the molecular mechanisms that underlie these immunoregulatory changes. We demonstrate that HS delivers signals to macrophages through at least two pathways, one involving the activation of a tyrosine kinase and of nuclear factor-KB, and the other involving the activation of protein kinase C and the elevation of intracellular calcium. The former pathway is associated with the production of IL-6, and the latter pathway is associated with the production of PGE2. Our findings suggest a model in which components of the microenvironment, such as HS, may determine the functional state of an APC, thereby modifying immune responses.
Synergistic effect of rapamycin and cyclosporin A in the treatment of experimental autoimmune uveoretinitis.
Authors: Martin D F , DeBarge L R , Nussenblatt R B , Chan C C , Roberge F G
Published: December 2022

Immunosuppressive drugs currently available for the treatment of autoimmune diseases display a narrow therapeutic window between efficacy and toxic side effects. The use of combinations of drugs that have a synergistic effect may expand this window and reduce the risk of toxicity. We evaluated the combination effect of rapamycin (Rapa) and cyclosporin A (CsA) in an autoimmune disease model of the eye. The dose-effect relationship of Rapa with CsA was measured in vitro on the inhibition of proliferation of retinal S-Ag-primed lymphocytes. A median effect analysis was performed and a combination index (CI) calculated for 50% inhibition of proliferation. Rapa and CsA were markedly synergistic over a wide dose range (lowest CI = 0.31). Calculated dose reduction factors indicated that Rapa could be reduced nine-fold and CsA reduced five-fold when these drugs were used in combination. These reduced doses were tested in vivo for the treatment of experimental autoimmune uveoretinitis (EAU). Twelve of 15 rats treated with CsA, 2 mg/kg/day, developed EAU with a median severity of 2.5. Fourteen of 15 rats treated with Rapa, 0.01 mg/kg/day, developed EAU with a median severity of 3.25. Complete inhibition of EAU was achieved in all 15 animals treated with the combination of Rapa and CsA (combined vs CsA alone, p < 0.0002; combined vs Rapa alone, p < 0.00001). The demonstrated synergistic relationship between Rapa and CsA will allow the use of reduced doses of each drug to achieve a therapeutic effect. The use of lower doses may reduce the toxicity of these drugs for the treatment of autoimmune uveitis.
In vitro transformation of murine pro-B and pre-B cells by v-mpl, a truncated form of a cytokine receptor.
Authors: Fichelson S , Vigon I , Dusanter I , Charon M , Velu T , Baillou C , Gisselbrecht S , Lemoine F M
Published: December 2022

v-mpl is a constitutively activated, truncated form of a cytokine receptor that has been transduced in a murine retrovirus, the myeloproliferative leukemia virus (MPLV). Expression of this oncogene results in the factor-independent proliferation of myeloid, erythroid, megakaryocytic, and mast precursor cells, which retain the ability to differentiate. However, no lymphoid disease was ever reported. To determine whether MPLV could infect and transform very early B cells and their precursors (BCPs), lymphoid long-term bone marrow cultures were infected with a helper-free MPLV. Within 3 wk after infection, highly proliferating BCPs could be isolated. These cells were able to clone spontaneously in semi-solid cultures, grown in the absence of feeder cell layer or exogenous growth factor and rapidly produced tumors after s.c. injection into synegic irradiated mice. In addition, MPLV transformation of pre-B cells led to the induction of an autocrine activity. Immunophenotypic and molecular analysis indicated that MPLV transformed early pro-B, pro-B, and pre-B cells, according to the expression of HSA, CD43, B220, Thy1, s-IgM and BP1 Ags, and to the rearrangements of Ig genes. Interestingly, MPLV-transformed BCPs expressed Mac1 Ag without acquiring further characteristics of macrophagic differentiation. Although the v-mpl cytoplasmic domain is devoid of tyrosine kinase consensus sequence, MPLV-transformed pre-B cells contained a major approximately 105-kDa tyrosine-phosphorylated protein that was not detected in uninfected cells or in cells transformed by the Abelson viral oncogene (v-abl). These results demonstrate that, like v-abl, the truncated cytokine receptor v-mpl is able to transform BCPs in vitro and suggest that the oncogenic transformation of BCPs by either v-mpl or v-abl use different pathways.
Activation of intracellular proteases is an early event in TNF-induced apoptosis.
Authors: Voelkel-Johnson C , Entingh A J , Wold W S , Gooding L R , Laster S M
Published: December 2022

The serine protease inhibitor tosyl-argenine methyl ester inhibits TNF-induced apoptosis, suggesting that proteolysis is necessary for this response. To test this hypothesis, we asked whether protein fragmentation occurs during the death of C3HA fibroblasts, a 3T3-like cell that was rendered sensitive to TNF by cycloheximide. Our results show that the binding of fluorescamine, which binds primary amines, was increased in apoptotic cells by approximately 50%. We also found that 10-15% of the protein in apoptotic cells was no longer precipitable by TCA. Evidence for proteolysis was also revealed by SDS-PAGE analysis and from Western blots. We observed fragmentation and/or degradation of lamin B, topoisomerase I, histone H1, protein kinase C beta 1, and cPLA2, indicating that proteolysis during apoptosis is non-specific. We also found evidence of proteolysis in C3HA cells sensitized to TNF by the adenovirus dl758 (which lacks the E3 14.7-kDa resistance gene) suggesting that protease activation is common in TNF-induced apoptosis. In contrast, the adenovirus E3 14.7-kDa resistance gene prevented proteolysis suggesting that this protein acts at, or upstream of the proteases activated in this response. Finally, because tosyl-argenine methyl ester inhibits the release of [3H]arachidonic acid from apoptotic cells, we tested whether proteolysis of cPLA2 is necessary for enzyme activation. Our results failed, however, to reveal a common proteolytic fragment in different cell types, and when tested in vitro the cytosol from apoptotic cells had less cPLA2 activity. It is unlikely, therefore, that proteolysis is necessary for the activation of this enzyme during TNF-induced apoptosis.
Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif.
Authors: Carlow D A , Marth J , Clark-Lewis I , Teh H S
Published: December 2022

In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4+8+ thymocytes upon TCR cross-linking. In contrast, expression of Tgtp is peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of approximately 50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation.
Accumulation of distinct T cell clonotypes in human solid tumors.
Authors: Yamamoto K , Masuko K , Takahashi S , Ikeda Y , Kato T , Mizushima Y , Hayashi K , Nishioka K
Published: December 2022

Tumor-infiltrating lymphocytes (TIL) have been described in a variety of human solid tumors. It is unknown whether such T cells are nonspecific inflammatory cells or a subset of specific host immune responses. To examine this question, we have analyzed the clonotypes of TCR beta-chain messages expressed in TIL, draining lymph nodes, and PBL of 10 patients with uterine or ovarian tumors. We report here that TIL bears distinct T cell clonotype accumulations only in patients without obvious metastasis. In contrast, accumulations of clonally expanded T cells were also found in lymph nodes and PBL of patients with metastatic cancer. The numbers and locations of the accumulated T cell clonotypes seemed to correlate with the stage of tumor invasion and the degree of metastasis. These data support the existence of Ag-driven immune responses to solid tumors in vivo.
Estrogen modulation of JE/monocyte chemoattractant protein-1 mRNA expression in murine macrophages.
Authors: Frazier-Jessen M R , Kovacs E J
Published: December 2022

The chemotactic cytokine, monocyte chemoattractant protein-1 (MCP-1), and its murine homologue, JE, have been detected in atherosclerotic lesions but not in normal arteries, implicating that these proinflammatory cytokines may be involved in the pathogenesis of atherosclerosis. Epidemiologic studies reveal that postmenopausal women receiving estrogen replacement for treatment of osteoporosis have a greatly reduced risk of developing cardiovascular disease. Because JE/MCP-1 and estrogen play regulatory roles in the development of atherosclerotic lesions, we chose to examine the effect of estrogen treatment on JE/MCP-1 mRNA expression in macrophages. 17 beta-estradiol (E2) inhibited LPS-stimulated JE/MCP-1 mRNA expression in ANA-1 and J774A.1 murine macrophage cell lines and in thioglycolate-elicited murine peritoneal macrophages. Inhibition of JE/MCP-1 mRNA ranged from 50 to 90%, with a maximal effect occurring at a concentration of 300 pg/ml E2. Conversely, E2 had little effect on LPS-stimulated TNF-alpha mRNA production. Treatment of LPS-stimulated macrophages with moxestrol, an estrogen agonist, resulted in a similar inhibition, and the addition of the estrogen antagonist, tamoxifen, reversed E2 inhibition of JE/MCP-1 mRNA expression. Progesterone failed to inhibit LPS-induced JE/MCP-1 mRNA expression. Immunohistochemical analysis revealed the presence of estrogen receptors in ANA-1 cells, indicating that E2 inhibition of LPS-induced JE/MCP-1 mRNA expression in murine macrophages may be mediated through the estrogen receptor. Thus, another mechanism whereby estrogen exerts antiatherogenic effects may be through prevention of macrophage accumulation in the atherosclerotic lesion.
Structure and repertoire usage of rat TCR alpha-chain genes in T cells infiltrating heart allografts.
Authors: Shirwan H , Barwari L , Fuss I , Makowka L , Cramer D V
Published: December 2022

We have studied the structure and diversity of TCR alpha-chain genes used by graft-infiltrating lymphocytes (GIL) in the ACI-to-LEW rat cardiac allograft model. We previously reported the structure of 16 different V alpha and 17 different J alpha genes isolated in two different cDNA libraries established from LEW thymocytes and GIL. In this report, we obtained new sequence information for 17 additional V alpha and J alpha genes from the GIL cDNA library. This study completes our characterization of 33 different V alpha and 23 different J alpha genes in rats. The V alpha genes are classified into 14 different families. Nucleotide sequence comparison with mouse alpha genes revealed the existence of mouse homologues for all the J alpha and 32 of the 33 V alpha genes. One of the rat V alpha genes seemed to have no known mouse counterpart and is classified here as a V alpha 24 gene family. The definition of rat TCR alpha-chain genes derived from these studies should facilitate a better understanding of the T cell role in pathologic conditions including autoimmune diseases and graft rejection. As for the TCR alpha-chain repertoire usage in allograft rejection, we completed the characterization of 36 productively rearranged TCR alpha-chain cDNA clones from the GIL cDNA library and found 31 different V alpha and 23 different J alpha genes among these clones. Unlike the TCR beta-chain that uses a limited repertoire, the alpha-chain repertoire usage seems to be relatively more diverse in this allograft model. These results suggest that the interaction of beta-chain with allogeneic MHC-encoded determinants may dictate the T cell reaction to the heart allograft in this model.
Molecular analysis of the same HIV peptide functionally binding to both a class I and a class II MHC molecule.
Authors: Takeshita T , Takahashi H , Kozlowski S , Ahlers J D , Pendleton C D , Moore R L , Nakagawa Y , Yokomuro K , Fox B S , Margulies D H
Published: December 2022

Although several peptides have been found to bind to both class I and class II molecules, the basis for this binding of the same peptide to two classes of MHC molecules has not been compared previously. We have analyzed one such peptide, P18 from the V3 loop of HIV-1 gp160, which we have previously shown to be recognized by CD8+ CTL with the class I molecule H-2Dd, and by CD4+ Th cells with the class II molecule I-Ad. With the use of truncated and substituted peptides, we found that the minimal core peptides are very similar, that the residues required for class I binding precisely fit the recently identified consensus motif for peptides binding to Dd (XGPX[R/K/H]XXX(X) [L/I/F]), and that at least three of the same residues are involved in binding to class II I-Ad. In addition, several of the same residues are involved in TCR interaction when the peptide is presented by class I and class II molecules. Modeling shows results to be consistent with the crystal structure of a peptide-class II MHC complex. Thus, the recognition of this versatile peptide by CD4+ Th cells with class II MHC molecules and by CD8+ cytotoxic T cells with class I MHC molecules is remarkably similar in both the core peptide used and the role of different residues in the ternary complex.
DNA mimics a self-protein that may be a target for some anti-DNA antibodies in systemic lupus erythematosus.
Authors: Zack D J , Yamamoto K , Wong A L , Stempniak M , French C , Weisbart R H
Published: December 2022

Recent studies suggest that some anti-DNA Abs in systemic lupus erythematosus may actually be Abs to specific proteins and that binding to dsDNA is a nonspecific cross-reactive event. To identify such proteins that bind to anti-DNA Abs, a cDNA expression library from human placenta was screened with mAb 3E10, a pathogenic anti-dsDNA Ab. MAb 3E10 was shown to bind to a 44-amino-acid fragment of HP8, a newly identified protein with amino acid sequence homology to the family of SPARC extracellular matrix proteins. To determine if Ab binding to both dsDNA and HP8 protein occurs through a common binding site, and therefore represents molecular mimicry, the Ab binding domains for protein and DNA were mapped. Chain recombinations between mAb 3E10 and a non-anti-DNA mAb showed that both the heavy and the light chains of mAb 3E10 were essential for anti-dsDNA and anti-HP8 reactivity. Mutagenesis experiments demonstrated that dsDNA and HP8 shared several critical binding residues located in all three complementarity-determining regions of mAb 3E10 VH. Moreover, Abs to HP8 were demonstrated in the sera of a subset of lupus patients. These results indicate that DNA mimics the HP8 protein in binding a lupus Ab, and that this protein may be a target for a subpopulation of anti-dsDNA Abs in systemic lupus erythematosus.
Expression of a novel integrin beta 1 chain epitope and anti-beta 1 antibody-mediated enhancement of fibronectin binding are dependent on the stage of T cell differentiation.
Authors: Wadsworth S A , Chang A C , Hong M J , Halvorson M J , Otto S , Coligan J E
Published: December 2022

Beta 1 integrins are a family of alpha beta heterodimers that serve as cell surface receptors for extracellular matrix proteins. We demonstrate that the anti-mouse integrin beta 1 chain mAb KMI6 selectively recognizes a beta 1 epitope that is constitutively expressed by certain immature thymocytes and is induced only slightly on mature thymocytes and peripheral T cells by activation with Con A. Because virtually all cells examined expressed beta 1 integrins on their surface, expression of the KMI6 epitope is T cell differentiation stage specific. Most CD3-4-8- thymocytes were KMI6+, with the lowest level of staining observed on the earliest CD44+IL-2R- cells within this subset. Expression was down-regulated during the CD3-4-8- to CD3-4-8+ transition, and lost by the CD4+8+ stage. Mature single positive thymocytes and resting peripheral T cells were also KMI6-. In contrast with the loss of the epitope before TCR expression by other thymocytes, most CD3+4-8- and certain CD8+ gamma delta TCR+ thymocytes were KMI6+ Addition of KMI6 to cell adhesion assays enhanced CD4-8- thymocyte, but not activated mature thymocyte or peripheral T cell, binding to fibronectin (via alpha 4 beta 1 and alpha 5 beta 1), whereas laminin binding (via alpha 6 beta 1) was unaffected. These properties distinguish the KMI6 epitope from other epitopes involved in beta 1 integrin activation in mice and other species. The unique selectivity of KMI6 recognition of beta 1 integrins, and its selective enhancement of ligand binding suggest that beta 1 integrin structure and factors that regulate beta 1 integrin binding are correlated with the stage of T cell differentiation.
Sequence constraints and recognition by CTL of an HLA-B27-restricted HIV-1 gag epitope.
Authors: Nietfield W , Bauer M , Fevrier M , Maier R , Holzwarth B , Frank R , Maier B , Riviere Y , Meyerhans A
Published: December 2022

Previous studies on the variation of an immunodominant HLA-B27-restricted HIV-1 gag p24 epitope (KRWIIL GLNK, amino acids 263-272) have demonstrated the persistence of variants recognized by CTL. Sequence comparisons of HIV isolates showed that this region is relatively conserved and as a consequence might restrict antigenic variation. To evaluate the possibility of HIV-1 to yield infectious mutants of this epitope that lack the ability to bind to HLA-B27 or escape HLA-B27-restricted CTL recognition, single-point mutations were constructed in the infectious molecular clone of HIV-1 Lai. Changes of arginine 264, the anchor amino acid for HLA-B27, to lysine or glycine resulted in infectious HIV-1 variants. The respective synthetic peptides showed reduced ability to sensitize target cells for CTL recognition and a corresponding loss of binding affinity to HLA-B27. In contrast, mutation of glycine 269 to lysine or glutamate abrogated HIV-1 infectivity. The corresponding peptides were able to bind to HLA-B27 but were not recognized by CTL. These data show that HIV-1 tolerates some genetic variation of the HLA-B27-restricted CTL epitope in gag p24 and that single-point mutations can alter quantitatively the immunologic properties. Further, it demonstrates that the mere nonrecognition of peptides derived from quasispecies analysis of small regions might simply correspond to nonviable virus variants and cannot be taken as evidence for CTL escape mutants. Together with the previously published data on the persistence of CTL epitopes, these results suggest that CTL do not play a major role in driving HIV-1 evolution in vivo.
Domain interactions and antigen binding of recombinant anti-Z-DNA antibody variable domains. The role of heavy and light chains measured by surface plasmon resonance.
Authors: Polymenis M , Stollar B D
Published: December 2022

The heavy (H) and light (L) chain V domains of anti-Z-DNA mouse mAb Z22 were expressed separately in bacteria. When mixed in vitro, the V domains associated stoichiometrically to reconstitute the Ag binding site of Z22, as judged by specific reactivity with Z-DNA and anti-Z22 ld Abs. The apparent Kd of the Z22 VH-VL association was 5.47 x 10(-8) M, measured by surface plasmon resonance. A replacement at VL position 96, which reduced Ag binding affinity of a single chain Fv (clone LZ1-2) by two orders of magnitude, did not reduce the affinity of interaction between the VH and VL domains (apparent Kd = 1.93 x 10(-8) M for VH association with LZ1-2). Fab prepared from native Z22 bound specifically to a 30-bp Z-DNA oligonucleotide with an apparent Kd = 1.56 x 10(-8) M. The VH domain alone bound Z-DNA specifically with an affinity similar to that of the Fab or Fv's of Z22 (Kd = 1.68 x 10(-8) M), whereas Z22 VL domain alone did not interact with nucleic acids. Z22 VH binding to Ag was inhibited by association with the mutant LZ1-2 VL. These results indicate that the Z22 H chain makes important contributions to specific binding of Z-DNA. Although the L chain does not add greatly to the binding energy, an appropriate L chain is required to permit Ag binding in the Fv domain. These in vitro results resemble the in vivo modulation of H chain autoreactivity that occurs with L chain substitution in receptor editing.
Regulatory regions 3' of the immunoglobulin heavy chain intronic enhancer differentially affect expression of a heavy chain transgene in resting and activated B cells.
Authors: Sigurdardottir D , Sohn J , Kass J , Selsing E
Published: December 2022

We have compared the expression patterns of three Ig heavy chain transgenes. The three constructs differ only by deletion of J-C intron sequences located downstream of the Emu enhancer region. When stably transfected into a myeloma cell line, all three constructs are expressed at comparable levels. However, transgenic mice carrying each construct show dramatic differences in transgene expression. Our results indicate that, in addition to the Emu enhancer, at least two regions, RegA and RegS, within the J-C intron influence transgene expression. RegA, located directly downstream of the core Emu enhancer, is involved in up-regulation of transgene expression after LPS activation of splenocytes. RegS, located within or downstream of the Smu switch region, is important for normal levels of transgene expression in splenocytes of heavy chain transgenic mice.