bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of granulomatous experimental autoimmune thyroiditis in mice with in vitro activated effector T cells and anti-IFN-gamma antibody.
Authors: Stull S J , Sharp G C , Kyriakos M , Bickel J T , Braley-Mullen H
Published: January 2023

Experimental autoimmune thyroiditis (EAT) can be induced in mice after the transfer of mouse thyroglobulin (MTg)-sensitized donor spleen cells that have been activated in vitro with MTg. CD4+ T cells are required for the transfer of EAT in this model. Because CD4+ T cells produce various lymphokines, such as IFN-gamma, that may be involved in the activation or regulation of the immune response to MTg and the development of EAT, the present study was undertaken to determine whether a neutralizing mAb to IFN-gamma could modulate the induction or expression of EAT. The anti-IFN-gamma mAb XMG-1.2 had no effect on sensitization of donor cells. However, addition of XMG-1.2 mAb during in vitro activation of MTg-primed spleen cells resulted in more severe EAT in recipient mice. The thyroid lesions in recipients of cells cultured with MTg and XMG-1.2 mAb also exhibited granulomatous changes, which differed qualitatively from the predominantly lymphocytic cell infiltrates in recipients of cells cultured with MTg alone. Recipients of MTg-activated spleen cells also developed severe granulomatous EAT when they were given injections of XMG-1.2 mAb. The effects of XMG-1.2 could be neutralized by IFN-gamma. Recipients of cells cultured in the presence of XMG-1.2 mAb had augmented autoantibody responses, although there were no apparent differences in the IgG subclass distribution of the anti-MTg autoantibody responses. These studies suggest that neutralization of endogenous IFN-gamma results in increased activity of cells capable of inducing granulomatous EAT in mice.
A human systemic lupus erythematosus-related anti-cardiolipin/single-stranded DNA autoantibody is encoded by a somatically mutated variant of the developmentally restricted 51P1 VH gene.
Authors: Van Es J H , Aanstoot H , Gmelig-Meyling F H , Derksen R H , Logtenberg T
Published: January 2023

We report the Ig H and L chain V region sequences from the cDNAs encoding a monoclonal human IgG anti-cardiolipin/ssDNA autoantibody (R149) derived from a patient with active SLE. Comparison with the germ-line V-gene repertoire of this patient revealed that R149 likely arose as a consequence of an Ag-driven selection process. The Ag-binding portions of the V regions were characterized by a high number of arginine residues, a property that has been associated with anti-dsDNA autoantibodies from lupus-prone mice and patients with SLE. The VH gene encoding autoantibody R149 was a somatically mutated variant of the 51P1 gene segment, which is frequently associated with the restricted fetal B cell repertoire, malignant CD5 B cells, and natural autoantibodies. These data suggest that in SLE patients a common antigenic stimulus may evoke anti-DNA and anti-cardiolipin autoantibodies and provide further evidence that a small set of developmentally restricted VH genes can give rise to disease-associated autoantibodies through Ag-selected somatic mutations.
Ricinoleated Streptococcic Vaccines
Authors: Larson W. P.
Published: January 2023

This brief paper is in reply to a recent article by Kozlowski (1), in which he reports the results of studies on the immunizing properties of ricinoleated streptococcic (scarlet fever) cultures.

Kozlowski observed ulcerations in his experimental animals at the site of the injection, and concluded that the reaction was due to the sodium ricinoleate, which he used as a detoxifying agent.

Kozlowski states that he purified the ricinoleic acid, from which the soap was prepared, through the barium salts. This undoubtedly is the key to his difficulty. I have found that soap prepared from fatty acids which have been purified with barium, invariably causes abscesses, since it is very difficult, and from a practical standpoint almost impossible, to remove the last trace of barium in the final purification processes. For this reason I have long since discontinued that method of purification.
Studies in Hypersensitiveness
Authors: Brown Aaron
Published: January 2023

The nature of the protective effect of the injection of pollen extracts in hay fever subjects is still unknown.

This mode of treatment of hay fever was introduced in medical practice after hay fever was recognized as a condition of hypersensitiveness, and it was looked upon as a procedure analagous in its operation to that of desensitization in anaphylaxis. However, recent studies from this department have shown that the prophylactic effect of the treatment is not due to a neutralization of the substances which appear to be responsible for the state of hypersensitiveness, because, at the end of a successful course of treatment, these substances (the atopic reagins) are not diminished in the blood and, in fact, they are sometimes increased.

These unexpected findings removed from consideration the only explanation of the phenomenon of protection that was compatible with our knowledge of the hypersensitive state and with the assumption that the protective effect of the injections is a specific one.
HLA-A1 and HLA-A3 T cell epitopes derived from influenza virus proteins predicted from peptide binding motifs.
Authors: DiBrino M , Tsuchida T , Turner R V , Parker K C , Coligan J E , Biddison W E
Published: January 2023

The potential value of peptide binding motifs of HLA class I molecules for the prediction of viral epitopes presented to T cells has been analyzed for two common HLA alleles. CTL generated against type A influenza virus recognize peptide epitopes derived from the nucleoprotein (NP) and basic polymerase 1 presented by HLA-A1, and epitopes derived from NP presented by HLA-A3. Distinct peptide binding motifs with characteristic anchor residues were previously identified for each of these class I molecules based on the sequences of endogenous peptides: for HLA-A1, position 3 = Asp or Glu and position 9 = Tyr; for HLA-A3, position 2 = Leu and position 9 = Lys or Tyr. Six peptides containing the HLA-A1 binding motif were identified within the sequences of the NP and basic polymerase 1 proteins, and one peptide containing the HLA-A3 motif was identified in the NP molecule. Three of the six HLA-A1 peptides and the one HLA-A3 NP peptide could bind to HLA-A1 or HLA-A3, respectively, in an in vitro peptide binding assay. Two of the HLA-A1-binding peptides could sensitize target cells for lysis by influenza virus-immune CTL populations restricted by HLA-A1 (NP 44-52 CTELKLSDY and PB1 591-599 VSDGGPNLY), and the one HLA-A3 NP peptide (NP 265-273 ILRGSVAHK) could sensitize target cells for lysis by HLA-A3-restricted influenza-immune CTL. Each peptide was also shown to be able to induce peptide-specific class I-restricted CTL in vitro, and the CTL generated against two of these peptides could specifically recognize virus-infected targets. Thus, these peptide binding motifs can be used to construct immunogenic synthetic epitopes which are capable of inducing antiviral T cell-mediated immune responses.
Identification of a novel population of CD8 alpha+ beta- bone marrow-derived dendritic epidermal cells. This unique population is subsequently outnumbered by thymus-independent expansion of the CD8 alpha+ beta+ dendritic epidermal cells.
Authors: Hayakawa J , Shiohara T , Moriya N , Arahari K , Nagashima M , Yagita H , Ishikawa H
Published: January 2023

Maturation of some T cell populations has been suggested to occur in epithelial tissues. The CD8 molecule, which is expressed as heterodimers made of alpha/beta-chains on virtually all peripheral T cells, is preferentially expressed as homodimers made only of alpha-chains on a subset of CD8+ mouse gut intraepithelial lymphocytes, which arise from bone marrow (BM) precursors in a thymus-independent mechanism. This unique population of CD8 alpha+ beta- T cells, however, has not been identified in other lymphoid tissues in normal adult mice. Because our previous study demonstrated that reconstitution of thymectomized irradiated mice with T cell-depleted BM cells resulted in the appearance of CD8+, TCR-alpha beta+ dendritic epidermal cells (DEC) and suggested that a proportion of the BM-derived DEC may mature within the epidermis, we asked whether the unique phenotype of CD8 alpha+ beta- could be detected among the CD8+ BM-derived DEC. In this report for the first time we identified this unique population of CD8 alpha+ beta- cells among the DEC. Although this population comprised the predominant fraction of the BM-derived DEC in the early post-transfer period (2 to 3 mo), gradual shift from the CD8 alpha+ beta+ phenotypes occurred during the late post-transfer period (4 to 6 mo) independently of the presence of the thymus. Kinetic studies on the BM-derived DEC revealed that this phenotypic shift could be caused by the subsequent expansion of the CD8 alpha+ beta+ DEC. In contrast, the CD8 alpha+ beta- population was the major subset of the BM-derived intraepithelial lymphocytes throughout the entire observation period and the phenotypic shift with age was never observed. These results indicate that CD8 alpha+ beta- cells are preferentially detected on T cells that home to the epithelial tissues and that in the epidermis, but not in the gut epithelia, the subsequent expansion of the CD8 alpha+ beta+ DEC would dilute the high number of the CD8 alpha+ beta- cells.
Avidity for antigen can influence the helper dependence of CD8+ T lymphocytes.
Authors: Heath W R , Kjer-Nielsen L , Hoffmann M W
Published: January 2023

To examine the influence of avidity on the helper dependence of CD8+ T cells, purified CD8+ T cells from transgenic mice expressing a TCR specific for H-2Kb were tested for their responsiveness to spleen cells expressing different densities of H-2Kb. High avidity interactions resulted in the induction of strong proliferative responses by purified CD8+ T cells. These cells synthesized their own IL-2 and IL-2R. By contrast, low avidity interactions failed to induce proliferation or detectable IL-2 synthesis, although IL-2R were induced and addition of rIL-2 induced a proliferative response. These results are consistent with a model in which TCR avidity for Ag can determine the helper dependence of a CD8+ T cell.
Keratinocyte-derived IL-7 serves as a growth factor for dendritic epidermal T cells in mice.
Authors: Matsue H , Bergstresser P R , Takashima A
Published: January 2023

Dendritic epidermal T cells (DETC) are CD3+, CD45+, CD4-, CD8-, TCR-V gamma 3/V delta 1+ T lymphocytes that reside in symbiosis with keratinocytes in mouse epidermis. To address mechanisms by which these cells survive and proliferate within the epidermal environment, we have tested the hypothesis that cytokines secreted by neighboring keratinocytes play relevant roles. The present study was conducted to determine whether keratinocytes produce biologically relevant amounts of IL-7, and, if so, to study its effects on DETC. The long term cultured DETC line, 7-17, and freshly isolated DETC exhibited dose- and time-dependent proliferative responses to rIL-7. These responses were blocked completely by anti-IL-7 antibodies, whereas anti-IL-2 had no effect, indicating that DETC respond to IL-7 by an IL-2-independent mechanism. Proliferative responses depended on the state of cell activation; DETC stimulated 2 to 5 days earlier with Con A proliferated optimally to added IL-7, whereas cells stimulated 10 days earlier did not proliferate. DETC that failed to proliferate responded to IL-7 by showing prolonged survival; cells maintained for 7 days with IL-7 alone retained their capacity to proliferate in response to Con A. Mouse epidermal cells and Pam 212 keratinocyte line both expressed IL-7 mRNA constitutively, as demonstrated by reverse transcription-polymerase chain reaction analyses. The production of IL-7 by mouse keratinocytes was also confirmed; Pam 212 culture supernatants supported DETC proliferation, and this activity was diminished by 50% with added anti-IL-7 antibodies. These results indicate that keratinocytes produce IL-7 in biologically relevant amounts, which, in turn, serve to promote the survival and growth of DETC. IL-7-mediated communication between epithelial cells and gamma delta T cells may represent one mechanism to sustain the indefinite residence of gamma delta T cells in epithelial tissues of mice.
Dissociation of the peptide-MHC class I complex limits the binding rate of exogenous peptide.
Authors: Ojcius D M , Abastado J P , Casrouge A , Mottez E , Cabanie L , Kourilsky P
Published: January 2023

Soluble, single-chain molecules for two MHC class I alleles, H-2Kd and H-2Kb, were used to analyze the kinetics of antigenic peptide binding to MHC. After MHC preloading with radiolabeled or fluorescent peptides, the observed rate of MHC-peptide complex dissociation increased after addition of an excess of unlabeled competitor peptide. Although exogenous peptides conforming to the allele-specific motif were required for the enhanced complex dissociation to occur, the dissociation rate of the complex was independent of exogenous peptide concentration. Similarly, the association rate of exogenous peptides was independent of concentration, reflecting the presence of low affinity peptides in the binding sites of the recombinant MHC proteins; the sequences of these endogenous peptides conform to the consensus motif for the MHC allele studied. Finally, the association rate of exogenous peptide decreased when MHC molecules were preloaded with high affinity peptides, and the binding of labeled high affinity peptide to isolated recombinant MHC was faster than the subsequent dissociation observed in the presence of competitor peptide. Taken together, these results imply that the rate of exogenous peptide binding is limited by the dissociation rate of the previously bound peptides.
Two separate mechanisms of T cell clonal anergy to Mls-1.
Authors: Yui K , Ishida Y , Katsumata M , Komori S , Chused T M , Abe R
Published: January 2023

T cell tolerance to superantigen can be mediated by clonal anergy in which Ag-specific mature T cells are physically present but are not able to mount an immune response. We induced T cell unresponsiveness to minor lymphocyte stimulations locus antigen (Mls)-1a in mice transgenic for TCR V beta 8.1 in three different systems: 1) injection of Mls-1a spleen cells, 2) mating with Mls-1a mice, and 3) bone marrow (BM) chimeras in which Mls-1a is present only on nonhematopoietic cells. CD4+8-V beta 8.1+ cells from all these groups did not proliferate in response to irradiated spleen cells from Mls-1a mice. We compared the response of these cells by T cell/stimulator cell conjugate formation, Ca2+ mobilization, and proliferation assays. The mechanisms underlying the unresponsiveness of these T cells appear to differ. CD4+8-V beta 8.1+ cells from Mls-1a spleen cell-injected mice mobilized cytoplasmic Ca2+ but proliferated at a reduced level in response to cross-linking with anti-TCR mAb. However, these cells formed conjugates, mobilized Ca2+, and proliferated in response to Mls-1a when activated B cells were used as stimulators, although they produced reduced levels of IL-2. In Mls-1a/b V beta 8.1 transgenic mice, a subset in CD4+8-V beta 8.1+ cells did not mobilize cytoplasmic Ca2+ after TCR cross-linking. Their conjugate formation, Ca2+ mobilization, or proliferation in response to Mls-1a on activated B cells was undetectable. Finally, CD4+8-V beta 8.1+ cells from the BM chimeras proliferated to TCR cross-linking at a partially reduced level and formed conjugates, mobilized Ca2+, and proliferated in response to Mls-1a on activated B cells. These features suggest that the mechanisms underlying the maintenance of anergy in Mls-1a spleen cell-injected mice are distinct from those in Mls-1a mice.
Third component of trout complement. cDNA cloning and conservation of functional sites.
Authors: Lambris J D , Lao Z , Pang J , Alsenz J
Published: January 2023

Of the 30 distinct complement proteins recognized to date, C3 is probably the most versatile and multifunctional molecule known, interacting with at least 20 different proteins. It plays a critical role in both pathways of complement activation and participates in phagocytic and immunoregulatory processes. Structural and functional analysis of C3 from different species, in addition to phylogenetic information, provides insights into the structural elements mediating the various functions. This study describes the cDNA cloning of one of two isoforms of the third complement component, C3-1, of rainbow trout (Salmo gairdneri) and the analysis of its functional sites. By screening a trout liver lambda gt11 library with anti-trout C3 chain-specific antibodies and polymerase chain reaction we have determined the cDNA sequence of trout C3-1. The obtained sequence is in complete agreement with the protein sequence of several tryptic peptides, corresponding to different regions of trout C3-1. C3-1 consists of 1640 amino acids with a calculated molecular mass of 181,497 Da. The sequence contains two potential N-glycosylation sites, one on each chain of C3. The deduced protein sequence showed 44.1, 43.3, 44.2, 44.9, 43.1, 43.8, 45.9, 29.9, and 33.1% amino acid identities to human, mouse rat, guinea pig, rabbit, cobra, frog, hagfish, and lamprey C3, whereas the identities to human C4, C5, and alpha 2M are 30.4, 28, and 22.9%, respectively. The trout C3 amino acid sequence shows clusters of high and low similarity to C3 from other species. In the regions of high similarity belong the C3 domains that contain the thiolester site and the properdin binding sites, whereas the regions that correspond to regions of human C3 where CR1 and CR2 bind show low amino acid sequence similarity. The deduced amino acid sequence shows that the C3 convertase cleavage site (Arg-Ser) is conserved in trout C3, whereas the factor I cleavage sites are Arg-Ala and Arg-Thr instead of Arg-Ser, which is found in the C3 of other species. Protein sequencing of the trout C3 fragments fixed on zymosan during complement activation confirmed the cleavage of trout C3 by trout C3 convertase and factor I at Arg-Ser and Arg-Thr, respectively.
cAMP activates the IL-5 promoter synergistically with phorbol ester through the signaling pathway involving protein kinase A in mouse thymoma line EL-4.
Authors: Lee H J , Koyano-Nakagawa N , Naito Y , Nishida J , Arai N , Arai K , Yokota T
Published: January 2023

Expression of the IL-5 gene in T cells is induced in response to Ag stimulation; however, functional analysis of the IL-5 gene has been limited by lack of an appropriate transfection assay to facilitate measurement of the IL-5 promoter activity in response to T cell activation signals. Here, we describe a transient transfection system with which the IL-5 promoter activity can be assayed quantitatively. Using mouse thymoma line EL-4 cells, which produce several lymphokines including IL-2, IL-3, IL-4, IL-10, and GM-CSF in response to PMA, the effect of cAMP on IL-5 production was examined. These cells produce a low level of IL-5 when stimulated with PMA alone; however, N6, O2-dibutyryl cAMP (Bt2cAMP), in combination with PMA, augmented by more than tenfold the IL-5 production at the mRNA and the protein levels. Likewise, a transient transfection assay revealed that Bt2cAMP activated the IL-5 promoter more than tenfold, in a PMA-dependent manner, thereby indicating that two signals, PMA and cAMP, are required for optimal activation of the IL-5 promoter. Activation of the IL-5 promoter in response to Bt2cAMP and PMA depends on the region spanning from nucleotide position -1,200 to +33 relative to the transcription initiation site. Action of cAMP on the IL-5 promoter is mimicked by cotransfection of the expression plasmid containing cDNA encoding the catalytic subunit of protein kinase A, hence, cAMP probably exerts its action through the signaling pathway that involves protein kinase A. In contrast, Bt2cAMP almost completely inhibited the PMA-dependent activation of the endogenous IL-2 gene as well as the transfected IL-2 promoter. These results indicate that the IL-5 gene in EL-4 cells is positively regulated by cAMP in a manner opposite that for the IL-2 gene.
Met-ase: cloning and distinct chromosomal location of a serine protease preferentially expressed in human natural killer cells.
Authors: Smyth M J , Sayers T J , Wiltrout T , Powers J C , Trapani J A
Published: January 2023

A cDNA clone encoding a human NK serine protease was obtained by screening a lambda-gt10 library from the Lopez NK leukemia with the rat natural killer Met-ase (RNK-Met-1) cDNA clone. In Northern blot analysis human Met-ase (Hu-Met-1) cDNA hybridized with a 0.9-kb mRNA in two human NK leukemia cell lines, unstimulated human PBMC, and untreated purified CD3-CD56+ large granular lymphocytes. Unlike other members of the granzyme family that are highly expressed in activated peripheral T cells, the Hu-Met-1 transcript was barely detected in a population of PMA and ionomycin or IL-2-treated high density T cells. Several in vitro cultured Burkitt lymphomas, chronic- and promyeloid leukemias, acute lymphoblastic leukemias, and colon and ovarian carcinomas and colon and ovarian carcinomas did not express Hu-Met-1 mRNA. Hu-Met-1 mRNA expression in a small number of human T cell tumor lines did not correlate with any particular phenotype or stage of development. The presence of Hu-Met-1 mRNA closely correlated with the Met-ase activity of cellular lysates prepared from these various human peripheral blood subsets and in vitro cultured cell lines. Met-ase activity detected in whole cell lysates of cytotoxic lymphocytes was associated with the cytoplasmic granules of these cells. The nucleotide sequence of the Hu-Met-1 cDNA clone encodes a predicted serine protease of 257 amino acids. The predicted protein is an active enzyme of 232 amino acids with a calculated unglycosylated m.w. of 27,100. Hu-Met-1 is 66% identical to RNK-Met-1 at the amino acid level. The human and rat mature protein sequences conserve the active site His, Asp, and Ser amino acids that form the catalytic triad of serine proteases, all 8 cysteine residues, and several amino acids critical in the formation of the substrate binding pocket. The gene for the Hu-Met-1 serine protease is located on chromosome 19, which distinguishes it from any other member of the human granzyme family.
Very late antigen-5 and complement receptor type 3 cooperatively mediate the interaction between Bordetella pertussis and human monocytes.
Authors: Hazenbos W L , van den Berg B M , van Furth R
Published: January 2023

Nonopsonized Bordetella pertussis, the causative agent of whooping cough, can attach to and become ingested by human monocytes. It has been reported that complement receptor type 3 (CR3) on human monocyte-derived macrophages binds filamentous hemagglutinin expressed on B. pertussis. In the present study, the role of very late antigen-5 (VLA-5) in the attachment of B. pertussis to adherent human monocytes was investigated. It was found that soluble fibronectin and soluble mAb against VLA-5 markedly inhibited the attachment of B. pertussis to monocytes. When VLA-5 on monocytes was cross-linked by plating these cells onto surfaces precoated with fibronectin or mAb against VLA-5, the binding of both B. pertussis and C3bi-coated sheep erythrocytes to these cells was significantly enhanced, whereas the binding of a B. pertussis mutant strain deficient in filamentous hemagglutinin was not affected. The enhanced attachment of B. pertussis to monocytes plated onto fibronectin-coated surfaces was markedly inhibited by soluble mAb against CR3. Neutrophils, which express similar levels of CR3 and about 10-fold lower levels of VLA-5 as compared with monocytes, did not bind B. pertussis. Together, these results indicate that VLA-5 is involved in the attachment of B. pertussis to monocytes and that cross-linking of VLA-5 enhances the attachment of B. pertussis to monocytes by augmenting the binding activity of CR3. We propose that the attachment of B. pertussis to monocytes occurs in two steps: binding and cross-linking of VLA-5 by B. pertussis enhances the binding activity of CR3, which in turn facilitates the subsequent binding of these bacteria to the latter receptor.
Differential expression in rheumatoid synovium and synovial fluid of alpha 4 beta 7 integrin. A novel receptor for fibronectin and vascular cell adhesion molecule-1.
Authors: Lazarovits A I , Karsh J
Published: January 2023

T lymphocyte adhesion to vascular endothelium plays an important role in the immunopathogenesis of rheumatoid arthritis. The migration of T lymphocytes into the synovium is mediated by a variety of adhesion molecules, notably the integrins. We have prepared Act I, a murine mAb that identifies a novel integrin termed alpha 4 beta 7. The natural ligands for alpha 4 beta 7 are vascular cell adhesion molecule-1 and fibronectin; both molecules are up-regulated in the rheumatoid synovium. We investigated the expression of alpha 4 beta 7 in the three compartments of rheumatoid arthritis, the peripheral blood, synovial fluid, and synovial membrane, utilizing the FACS and immunoperoxidase microscopy of frozen tissues. The results of our experiments show a striking differential expression of alpha 4 beta 7 integrin in rheumatoid arthritis. Sixty-two percent of synovial membrane T cells expressed high density alpha 4 beta 7, in contrast to only 4.7% of synovial fluid and 9.1% of PBL. These data suggest that the expression of alpha 4 beta 7 integrin may provide a mechanism whereby certain T cells adhere to rheumatoid synovium while others remain in the synovial fluid. The augmented expression of alpha 4 beta 7 in the synovial membrane T cells may contribute to the development and perpetuation of rheumatoid arthritis.
Polyclonal B cell activation arises from different mechanisms in lupus-prone (NZB x NZW)F1 and MRL/MpJ-lpr/lpr mice.
Authors: Merino R , Iwamoto M , Fossati L , Izui S
Published: January 2023

The polyclonal B cell activation is the earliest and most common immunologic abnormality in lupus-prone mice. However, its cellular mechanism(s) has not been well defined. To determine the contribution of CD4+ T cells in this immunologic abnormality, we have depleted CD4+ T cells in lupus-prone (NZB x NZW)F1 and MRL/MpJ-lpr/lpr mice by treating them with anti-CD4 mAb from birth and determined the development of IgM and IgG polyclonal antibody formation. Our results indicate that first, different mechanisms control the development of IgM polyclonal B cell activation in these two autoimmune mice; in (NZB x NZW)F1 mice, IgM polyclonal B cell activation is likely to be a result of an intrinsic B cell defect, whereas CD4+ T cells seem to be responsible for this immunologic abnormality in MRL/MpJ-lpr/lpr mice. Second, the increased production of IgG antibodies, including the IgG3 subclass, was totally regulated by CD4+ T cells in both autoimmune mice. Because IgG3 antibodies can be highly nephritogenic, independent of their immunologic specificities, which is the result of the antibodies' cryoglobulin activity, the active role of CD4+ T cells in the production of IgG3 antibodies in lupus-prone autoimmune mice further strengthens the implication of CD4+ T cells in murine systemic lupus erythematosus.
Preferential use of a H chain V region in antitumor-associated glycoprotein-72 monoclonal antibodies.
Authors: Rixon M W , Gourlie B B , Kaplan D A , Schlom J , Mézes P S
Published: January 2023

The DNA sequence of the mouse H chain V regions from five hybridomas directed against the human tumor Ag tumor-associated glycoprotein-72 (TAG-72) have been determined. This includes a previously determined VH gene sequence from a first-generation anti-TAG-72 mAb, B72.3, and the VH gene sequences from four second-generation anti-TAG-72 mAb, CC49, CC83, CC46, and CC92. A sequence comparison revealed a high degree of shared sequence identity between the five productively rearranged VH genes, suggesting derivation from a common germ line V region gene. In the process of cloning the unrearranged germ line gene, two highly related VH germ line genes were identified and designated VH alpha TAG-1 and VH alpha TAG-2. A comparison of the productively rearranged anti-TAG-72 VH sequences with the two germ line VH genes demonstrated that they were all derived from VH alpha TAG-1. In contrast, the L chain V regions are all derived from separate germ line V region genes. The preferential use of VH alpha TAG-1 in these five mouse hybridomas suggests that VH alpha TAG-1 is a preferred anti-TAG-72 H V chain region germ line gene and that the H chain plays a predominant role in the recognition of this Ag.
VH4.21-encoded natural autoantibodies with anti-i specificity mirror those associated with cold hemagglutinin disease.
Authors: Schutte M E , van Es J H , Silberstein L E , Logtenberg T
Published: January 2023

The Ig VH regions of virtually all human pathogenic cold agglutinin (CA) anti-i/l autoantibodies are encoded by a single Ig VH gene segment, VH4.21, in conjunction with a highly variable CDR3 structure. The anti-I specificity is often associated with V kappa III-encoded L chains, whereas anti-i autoantibodies appear to use a broader array of kappa and lambda VL gene segments. B cells expressing VH4.21 are abundantly present in adult lymphoid tissues from healthy individuals but their relationship with B cells secreting pathogenic CA is unknown. Herein we have analyzed the distribution of VH4.21-expressing B cells in fetal, neonatal, and adult B cell populations using the monoclonal anti-VH4.21 antibody 9G4. In addition, we have analyzed the anti-i and anti-I binding capacity and V regions of 19 VH4.21-encoded mAb secreted by cord and adult blood-derived cell lines from healthy individuals. The results show that VH4.21 expressing B cells are overrepresented in all repertoires studied and are evenly distributed over cord blood CD5+ and CD5- B cell populations. VH4.21-encoded H chains strongly predispose for anti-i binding capacity, regardless of the VL regions or H chain CDR3 structure. The avidity of some of these antibodies was similar to those of pathogenic CA. In addition, we found evidence for monospecific anti-I binding in antibodies encoded by other members of the VH4 gene family. We conclude that VH4.21-encoded antibodies with anti-i specificity from the normal B cell repertoire mirror their pathogenic counterparts and that naturally occurring anti-I antibodies may be encoded by a more diverse array of VH4 genes.
TSG-6: a TNF-, IL-1-, and LPS-inducible secreted glycoprotein associated with arthritis.
Authors: Wisniewski H G , Maier R , Lotz M , Lee S , Klampfer L , Lee T H , Vilcek J
Published: January 2023

TSG-6 (TNF-stimulated gene 6) was originally discovered by differential screening of a cDNA library prepared from TNF-stimulated human diploid FS-4 fibroblasts. We show that the 35-kDa protein encoded by TSG-6 was undetectable in the medium of untreated FS-4 cultures, whereas its production reached approximately 1400 and 700 ng/10(6) cells after 24-h treatment with IL-1 or TNF, respectively. Stimulation of TSG-6 protein and mRNA levels was also demonstrated in normal human mononuclear cells by treatment with TNF and, especially, by LPS. In view of the inducibility of TSG-6 by inflammatory cytokines and its earlier demonstrated affinity for hyaluronan, we examined the presence of TSG-6 protein in the synovial fluids from patients with various forms of arthritis. TSG-6 protein was undetectable in the joint fluids of persons with no known history of arthritis, but high levels of TSG-6 oere demonstrated in the synovial fluids of a majority of arthritis patients. TSG-6 protein was also detected in the sera of some of the arthritis patients, albeit at concentrations that were less than in the joint fluids. To investigate the source of TSG-6 in the synovial fluids, we examined the production of TSG-6 protein in cultures of synovial cells. Synoviocytes from rheumatoid arthritis patients produced TSG-6 protein constitutively, and this production was increased by treatment with TNF or IL-1, but not with TGF-beta. Steady-state levels of TSG-6 mRNA were also increased in synoviocytes after treatment with TNF or IL-1. The presence of high levels of TSG-6 protein in the synovial fluids of arthritis patients and its inducibility by inflammatory cytokines in fibroblasts, mononuclear cells, synoviocytes, and chondrocytes suggest a role for TSG-6 in arthritis and inflammation.
Cryptic epitopes on the nicotinic acetylcholine receptor are recognized by autoreactive CD4+ cells.
Authors: Bellone M , Ostlie N , Karachunski P , Manfredi A A , Conti-Tronconi B M
Published: January 2023

Experimental autoimmune myasthenia gravis is induced in C57BL/6 mice by injection of Torpedo nicotinic acetylcholine receptor (TAChR). We investigated here the presence of cryptic CD4+ epitopes on the TAChR molecule, and their relationship with potentially autoreactive CD4+ cells, which survived clonal deletion. CD4+ cells from C57BL/6 mice immunized with native or denatured TAChR were challenged in vitro with overlapping synthetic peptides, 20-residue long, screening the sequences of TAChR alpha, gamma, and delta subunits. Only three epitopes on the alpha subunit were recognized consistently. Mice immunized with large doses (nanomoles) of TAChR clearly recognized only the immunodominant sequence T alpha 150-169. Anti-TAChR CD4+ cells did not cross-react with murine alpha subunit sequences, or with any synthetic sequence of human gamma and delta subunits, which are very similar to the corresponding murine subunits. To facilitate recognition of cryptic epitopes, we injected mice with pools of synthetic peptides corresponding to the sequences of TAChR alpha, gamma, and delta subunits. In addition to the three immunodominant alpha subunit epitopes, other epitopes were recognized by CD4+ cells within the sequences T alpha 304-322, T gamma 105-124, T gamma 120-139, T gamma 401-420, T gamma 357-376, T delta 16-35, T delta 61-80, T delta 121-140, and T delta 301-320. CD4+ cells thus sensitized cross-reacted with the mammalian sequences alpha 304-322, gamma 105-124, gamma 120-139, and delta 301-320. Mice were immunized with large doses (approximately 40 nmol) of individual TAChR synthetic cryptic epitopes. CD4+ cells sensitized to five cryptic epitopes (the ones listed above plus delta 121-140) cross-reacted with autologous sequences. We determined the dose dependence of the sensitization of CD4+ cells in vivo to the strongly immunodominant epitope peptide T alpha 150-169 and to the cryptic epitope peptides T gamma 120-139 and T delta 301-320 by immunizing mice with increasing doses of peptide (approximately 1.2 to approximately 20 nmol), and testing the in vitro anti-peptide response of the CD4+ cells. No difference was found for the epitopes tested. Doses of 3 to 10 micrograms induced a strong CD4+ sensitization, and the dose dependence of the in vitro response of the sensitized cells to the relevant peptide was comparable. Production of cryptic epitopes upon in vitro TAChR processing was investigated by testing peptide-sensitized CD4+ cells with native TAChR: only two cryptic epitopes were produced.
Rapamycin treatment depresses intragraft expression of KC/MIP-2, granzyme B, and IFN-gamma in rat recipients of cardiac allografts.
Authors: Wieder K J , Hancock W W , Schmidbauer G , Corpier C L , Wieder I , Kobzik L , Strom T B , Kupiec-Weglinski J W
Published: January 2023

Rapamycin (RPM) treatment prevents accelerated rejection of cardiac allografts in sensitized rats. The prominent feature of this brisk 24-h rejection, which includes a panoply of both cellular and humoral host immune responses, is a massive infiltration of rejecting grafts with neutrophils. In this study we tested the hypothesis that RPM-mediated therapeutic effects on accelerated rejection may be linked to decreased expression of protein encoded by gro/melanoma-growth stimulatory activity gene (KC) and macrophage inflammatory protein-2 (MIP-2) genes, the operational rat homologues of the human intercrine-alpha cytokines with proinflammatory IL-8-like neutrophil activation/chemotactic properties. The induction of these genes was then correlated with mRNA profiles encoding for Th1-selective IFN-gamma and CTL-specific granzyme B proteins. Northern blot analysis of RNA from cardiac allografts of sensitized untreated recipients, revealed maximal levels of KC and MIP-2 mRNA at 3 to 6 h after transplantation. In contrast, IFN-gamma mRNA, which was at most very weakly expressed at 3 h, peaked between 6 to 12 h. As with IFN-gamma, granzyme B transcripts were undetectable at 3 h, but peaked around the time of actual graft rejection at 24 h. RPM therapy abrogated accelerated rejection and prolonged cardiac allograft survival to ca. 46 days. This effect was associated with markedly reduced expression of KC and MIP-2 mRNA in the first 24 h as well as at 7 and 34 days after transplantation. Moreover, RPM completely blocked intragraft appearance of granzyme B and IFN-gamma mRNA in long term cardiac allografts. Immunohistologic analysis has revealed that accelerated rejection was associated with extensive neutrophil infiltration, which peaked at 18 to 24 h. At this time, leukocytes and endothelium were intensely stained for IL-8 and IFN-gamma antibodies. In contrast, the allografts from RPM-treated hosts showed essentially no neutrophil infiltration and minor, focal staining for IL-8 and IFN-gamma. This study demonstrates an association between the early expression of genes for proinflammatory IL-8-dependent neutrophil chemotactic activity, and later expression of genes associated with activation/effector activity of CTL and NK cells. It also documents a novel effect of RPM in vivo, which results in the suppression of intragraft IL-8-like and CTL-dependent mRNA/protein production and diminished neutrophil infiltration; these may contribute to the striking efficacy of RPM therapy in sensitized graft recipients.
Functionally anergic lpr and gld B220+ T cell receptor (TCR)-alpha/beta+ double-negative T cells express CD28 and respond to costimulation with phorbol myristate acetate and antibodies to CD28 and the TCR.
Authors: Giese T , Allison J P , Davidson W F
Published: January 2023

Mice homozygous for lpr and gld develop lymphadenopathy characterized by the progressive accumulation of an unusual population of CD4-, CD8-, CD2-, IL-2R- double-negative (DN) T cells that express reduced levels of TCR-alpha/beta, high levels of CD45 (B220) and Ly-6C and variable levels of CD69. These cells are refractory to most stimuli, including staphylococcal entertoxins and cross-linking of the TCR, Ly-6C, and CD69. For normal T cells, the binding of ligand to the TCR alone is insufficient to induce a proliferative response and can result in the induction of a state of prolonged anergy. Efficient stimulation is dependent on the delivery of a second or costimulatory signal. Recently it was reported that CD28 can provide costimulatory signals to T cells and, that these signals can prevent anergy induction in T cell clones. We investigated the possibility that lpr and gld DN T cells are unresponsive because they fail to transduce signals via CD28. These studies showed that highly purified B220+ TCR-alpha/beta+ DN T cells expressed high levels of CD28, responded weakly to stimulation with PMA and anti-CD28 mAb and quite strongly to PMA, anti-CD28 antibody and high concentrations of immobilized anti-TCR-alpha/beta antibodies. The latter stimulus also induced low levels of expression of CD2 and IL-2R and secretion of modest amounts of IL-2. Although DN T cells proliferated and secreted IL-2, these responses differed qualitatively and quantitatively from those of +/+ and lprB220- T cells. Consistent with its effects on normal T cells, cyclosporin A partially inhibited the response of DN T cells to TCR cross-linking and CD28 ligation. Studies of synergism between CD28-, Ly-6C-, and CD69-mediated signals revealed that ligation of CD28 enhanced the proliferative response induced by cross-linking of Ly-6C or CD69 on +/+, lpr and gld B220- T cells but had no effect on the unresponsiveness of DN T cells to these stimuli. Ligation of CD28 did not reverse the unresponsiveness of DN T cells to SEB and had only a weak synergistic effect on the response of B220- T cells. Together, these observations suggest that the mechanisms leading to immunosuppression of DN T cells are complex and appear to involve abnormalities in signal transduction via the TCR and CD28 and possibly via Ly-6C and CD69 as well.
Effect of bcl-2 on Fas antigen-mediated cell death.
Authors: Itoh N , Tsujimoto Y , Nagata S
Published: January 2023

Fas Ag is a cell surface protein that can mediate apoptosis and belongs to the TNF receptor family. The product of protooncogene bcl-2, a membrane-associated protein, has been shown to inhibit apoptosis in various hematopoietic cells including B cells and T cells. To examine the possible interaction of the Fas Ag and bcl-2, we coexpressed human Fas Ag and bcl-2 cDNA in murine IL-3-dependent FDC-P1 cell line and murine lymphoma WR19L. FDC-P1 transformants expressing bcl-2 showed a prolonged survival to IL-3 depletion. FDC-P1 transformants expressing the Fas Ag alone were killed by anti-Fas antibody in the presence of IL-3. Overexpression of bcl-2 in FDC-P1 resulted in a partial inhibition of Fas-induced cell death. WR19L transformants expressing bcl-2 were partially resistant to the cytolytic activities of the TNF-alpha and anti-Fas antibody treatment. These results suggest that the Fas Ag and TNF receptor may share the same signaling pathway, and that bcl-2 interferes with the apoptotic process mediated by the Fas Ag and TNF receptor.
Tyrosine phosphorylation of phospholipase C concomitant with its activation by platelet-activating factor in a human B cell line.
Authors: Kuruvilla A , Putcha G , Poulos E , Shearer W T
Published: January 2023

Platelet-activating factor (PAF) is a powerful inflammatory mediator in a variety of systems. Upon binding its receptor on B lymphocytes, it activates phospholipases C and A2, thus initiating a cascade of events culminating in changes in the program of the target cell. We have now extended our previous studies of the effects of PAF on EBV-transformed human B cell lines to examine the mechanism by which phospholipase C (PLC) is activated. PAF-induced incorporation of 32P into phosphatidylinositol (PtdIns) was markedly diminished by the tyrosine kinase inhibitors, genistein, and those of the tyrphostin family, tyrphostins 25, 47, and 51. The generation of inositol phosphates induced by PAF was also significantly inhibited by these inhibitors. Correlating with this inhibition of PtdIns turnover, the elevation of intracellular calcium concentrations stimulated by PAF was observed to be inhibited in the presence of inhibitors of tyrosine kinases. In addition, the induction of expression of the proto-oncogene, c-fos, was substantially attenuated by these inhibitors. Finally, employing anti-phosphotyrosine immunoprecipitates of lysates from PAF-stimulated cells in an in vitro PLC assay, we have provided evidence for an increase in the tyrosine phosphorylation levels of PLC upon stimulation, and the inhibition of this increase by tyrosine kinase inhibitors. In summary, we have shown that in B cells tyrosine kinase activity is essential for the PtdIns turnover, the generation of inositol phosphates, and the calcium flux induced by PAF and that platelet-activating factor-induced tyrosine phosphorylation of PLC is likely to be required for the activation of this enzyme.
T-cell presentation of antigen requires cell-to-cell contact for proliferation and anergy induction. Differential MHC requirements for superantigen and autoantigen.
Authors: LaSalle J M , Toneguzzo F , Saadeh M , Golan D E , Taber R , Hafler D A
Published: January 2023

MHC class II+ human T-cell clones are able to simultaneously present and respond to peptide Ag and superantigen resulting in both proliferation and subsequent anergy. A major question remains as to whether a single T cell can present to itself or whether T-T cell interactions are required. We have employed a novel technique for inhibiting cell-to-cell contact that encapsulates individual T cells in agarose gel microdrops. Myelin basic protein-reactive individual CD4+ T-cell clones entrapped within these microdrops neither proliferated nor became anergized to either peptide Ag or Staphylococcal enterotoxin B (SEB), suggesting that cell-to-cell contact was required for T-cell presentation of Ag leading to proliferation and anergy. PMA treatment induced T-cell migration out of gel microdrops, restoring cell-to-cell contact and resulting in proliferation and anergy after T-cell coculture with peptide or superantigen. However, analysis of [Ca+2]i release revealed differences in T-cell responses to SEB versus peptide Ag. The addition of SEB, but not peptide Ag, induced a calcium flux in solitary T cells. Additionally, alpha HLA-DR mAb blocked peptide but not SEB-induced proliferation and anergy induction. Thus, SEB generated an early signal in solitary T cells that may not be a result of self stimulation via MHC class II. However, subsequent cell-to-cell contact was required for proliferation and anergy induction by SEB. These results indicate that peptide Ag requires a MHC class II-dependent cell-to-cell interaction for calcium flux, proliferation, and anergy induction, whereas SEB requires a MHC class II independent cell-to-cell interaction for proliferation and anergy induction after a TCR-generated calcium flux.
Differential activation of CD8+ T cells by transforming growth factor-beta 1.
Authors: Lee H M , Rich S
Published: January 2023

Transforming growth factor-beta (TGF-beta) is a highly conserved multifunctional factor that broadly regulates cell growth and differentiation, and exhibits diverse regulatory roles in the immune system. In contrast to other studies describing TGF-beta as a potent inhibitor of lymphocyte growth, we have shown previously that TGF-beta 1 can also costimulate proliferation of murine splenic T cells activated by immobilized anti-CD3 antibody. In the present studies, we further investigate the subsets of T cells that are responsive to TGF-beta 1 costimulation. T cells were isolated into CD45RBhi/lo or CD4+/8+ populations, and their responses to TGF-beta 1 were examined. Sorted CD45RBhi cells were highly responsive to TGF-beta 1 costimulation, and proliferated to a level similar to that of unsorted T cells in response to TGF-beta 1. TGF-beta 1 also costimulated proliferation in the CD45RBlo population that was distinguished by low response and delayed kinetics. In contrast, sorted CD4+ and CD8+ T cells showed a striking differential response. Anti-CD3-stimulated proliferation of sorted CD8+ or CD4- T cells was substantially enhanced by TGF-beta 1, whereas sorted CD4+ or CD8- T cells were unresponsive. TGF-beta 1 also down-regulated CD45RB and increased CD44 expression on responsive CD8+ and CD45RBhi T cells, thereby leading to a population of T cells enriched in mature phenotype. Generation of anti-CD3-redirected lytic activity by these TGF-beta 1-costimulated CD8+ cells was strongly suppressed. However, these CD8+ T cells exhibited cytotoxic activity after restimulation in the absence of TGF-beta. TGF-beta 1 precultured CD8+ T cells also had heightened IL-2 and IFN-gamma secretion upon restimulation in comparison to cells activated initially without TGF-beta. CD8+ T cells precultured with anti-CD3 and TGF-beta remained responsive to growth enhancement by TGF-beta, although re-exposure to TGF-beta depressed other functions of these cells. Thus, TGF-beta 1 demonstrates important costimulatory roles in both growth and maturation of CD8+ T cells.