bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoimmune Sjögren's-like lesions in salivary glands of TGF-beta1-deficient mice are inhibited by adhesion-blocking peptides.
Authors: McCartney-Francis N L , Mizel D E , Redman R S , Frazier-Jessen M , Panek R B , Kulkarni A B , Ward J M , McCarthy J B , Wahl S M
Published: December 2022

The targeted disruption of the TGF-beta1 gene in mice (TGF-beta1 -/-) leads to extensive inflammation in vital organs, cachexia, and death within 3 to 4 wk. Significant inflammatory lesions develop initially in the periductal regions of the salivary glands and escalate as the animals become symptomatic. These inflammatory sites, characterized by lymphocytic infiltration and increased proliferation, cytokine mRNA expression, and IgG-positive cells, resemble lesions of Sjögren's syndrome. Moreover, the inflammatory pathology, enhanced MHC expression, and Ab production are consistent with an autoimmune-like etiology. Glandular atrophy and loss of acini with reduced saliva production appear to contribute to the wasting syndrome characteristic of the TGF-beta1 -/- mice. To determine whether the structural and functional defects were developmental due to the absence of TGF-beta1 or secondary to the inflammation, TGF-beta1 -/- mice were treated with synthetic fibronectin peptides, which block leukocyte infiltration. Daily systemic injections of RGD, CS-1, and/or peptides derived from the heparin-binding region of the A chain not only prevented leukocyte infiltration in the salivary glands of the TGF-beta1 -/- mice, but also reversed the acinar and ductal derangements. These data suggested that salivary gland development is not jeopardized in the absence of TGF-beta1, but that the extensive infiltration of inflammatory cells compromises glandular structure and function. The essential nature of TGF-beta1 in controlling inflammatory and immune processes is confirmed by these studies. Moreover, these TGF-beta1 -/- mice provide an important model of autoimmune disease that can be used in the design of therapeutic interventions.
HIV-1-Tat protein promotes chemotaxis and invasive behavior by monocytes.
Authors: Lafrenie R M , Wahl L M , Epstein J S , Hewlett I K , Yamada K M , Dhawan S
Published: December 2022

Monocytes are susceptible to HIV infection and to activation by a regulatory gene product of the HIV genome, HIV-Tat. Recently, we have demonstrated that treatment with HIV-Tat up-regulates monocyte adhesion to the endothelium and increases metalloproteinase production. in the present study, we have examined the ability of the HIV-Tat protein to alter the migratory and invasive behavior of monocytes. Monocytes pretreated for 24 h with 10 ng/ml HIV-Tat exhibited enhanced migratory behavior compared with untreated monocytes in chemotaxis assays, both in the absence of a chemoattractant as well as in response to FMLP. in addition, HIV-Tat itself induced the migration of both untreated and HIV-Tat pretreated monocytes. Checkerboard analysis showed that monocytes migrated in response to an HIV-Tat concentration gradient, thus confirming the chemotactic characteristics of the HIV-Tat protein. Pretreatment of monocytes with 10 ng/ml HIV-Tat for 24 h also increased their ability to invade reconstituted extracellular membrane (Matrigel)-coated filters by 5-fold in the absence of chemoattractant. The presence of FMLP or HIV-Tat further enhanced invasion by both untreated and HIV-Tat-pretreated monocytes by more than 10-fold. Monocyte invasion was partially inhibited by the inclusion of anti-beta integrin Ab or tissue inhibitor of metalloproteinase (TIMP). Thus, for the first time, we present evidence that HIV-Tat can enhance the chemotactic and invasive behaviors of monocytes and propose an active role for HIV-Tat in the recruitment of monocytes into extravascular tissues, a process which may contribute to the destruction of tissues and cellular architecture often seen in patients with acquired immunodeficiency syndrome.
Nebulized IFN-gamma inhibits the development of secondary allergic responses in mice.
Authors: Lack G , Bradley K L , Hamelmann E , Renz H , Loader J , Leung D Y , Larsen G , Gelfand E W
Published: December 2022

The effects of nebulized IFN-gamma on primary and secondary IgE production and development of airway hyper-responsiveness (AHR) were investigated. BALB/c mice received primary exposure to aerosolized OVA daily for 10 days and developed anti-OVA IgE responses, immediate cutaneous reactivity to OVA, and altered airway function when assayed on day 12. After secondary exposure to OVA challenges on days 30 and 31, these mice developed an amplified IgE response, heightened cutaneous reactivity to OVA and AHR when measured on day 37. Administration of IFN-gamma for 13 days, beginning 3 days prior to and during primary OVA sensitization, resulted in a decrease in anti-OVA IgE, increases in serum anti-OVA IgG2a levels, a decrease in cutaneous reactivity to OVA, and normal airway function when assessed on day 12 after primary sensitization. This treatment also prevented the development of secondary anti-OVA IgE responses and altered airway responsiveness but did not induce a secondary rise in anti-OVA IgG2a in the serum measured on day 37. Treatment with IFN-gamma on days 26 to 30, well after primary responses were established but just prior to secondary OVA challenge, abolished the development of secondary anti-OVA IgE responses, resulted in an increase in anti-OVA IgG2a in the serum, and prevented the development of AHR. In vitro, CD4+ T cells obtained from OVA-sensitized mice treated with either "early" or "late" IFN-gamma inhibited IgE production. Delivery of IFN-gamma to the airways can prevent secondary allergen sensitization even after primary sensitization has been achieved and this effect is mediated by CD4+ T cells.
Comparison of CD45 extracellular domain sequences from divergent vertebrate species suggests the conservation of three fibronectin type III domains.
Authors: Okumura M , Matthews R J , Robb B , Litman G W , Bork P , Thomas M L
Published: December 2022

Mammalian CD45 is a transmembrane protein tyrosine phosphatase expressed by all nucleated cells of hematopoietic origin. In lymphocytes, CD45 is required for Ag-induced signal transduction due to its ability to positively regulate Src family members. The mechanisms by which CD45 function is regulated are unknown. Indeed, the interactions of CD45 extracellular domains are largely undefined. To gain insight into potentially important regions of the extracellular domain, we sought to identify conserved features from divergent species. cDNAs encoding the putative CD45 homologue from Heterodontus francisci (horned shark) were isolated. The cDNA sequence predicts a protein of 1200 amino acids that contains a 452-amino acid extracellular domain, a 22-amino acid transmembrane region, and a 703-amino acid cytoplasmic domain. Alignment searches revealed that the Heterodontus cytoplasmic domain sequence was most identical to mammalian CD45 and a transmembrane protein tyrosine phosphatase sequence identified from chickens, ChPTP lambda. A dendrogram with other transmembrane protein tyrosine phosphatase sequences suggest that the Heterodontus and chicken sequences represents CD45 orthologues for their respective species. Analysis of vertebrate CD45 extracellular domain sequences indicates the conservation of three structural regions: a region containing potential O-linked carbohydrate sites, a cysteine-containing region, and a region containing three fibronectin type III domains. For each vertebrate species, multiple isoforms are generated by alternative splicing of three exons that encode a portion of the region containing potential O-linked glycosylation sites. These studies provide evidence for a conservation in CD45 extracellular domain structure between divergent species and provide a basis for understanding CD45 extracellular domain interactions.
IL-12 is both required and sufficient for initiating T cell reactivity to a class I-restricted tumor peptide (P815AB) following transfer of P815AB-pulsed dendritic cells.
Authors: Bianchi R , Grohmann U , Belladonna M L , Silla S , Fallarino F , Ayroldi E , Fioretti M C , Puccetti P
Published: December 2022

Delayed-type hypersensitivity responses, mediated by CD8+ cells and detected by skin test assay, occur in sensitized mice in response to challenge with a class I-restricted synthetic peptide related to a poorly immunogenic tumor rejection Ag, P815AB, of murine mastocytoma cells. Efficient priming for this response, which requires functional CD4+ cells and production of IFN-gamma in the host, is achieved by transfer of dendritic cells (DC) pulsed in vitro with a physical mixture of P815AB and T helper peptides, such as a class II-restricted synthetic peptide of tetanus toxin. We now show that the adjuvant effect of the T helper peptide was associated with the appearance of early and late IL-12 transcripts in the spleens of DC recipient mice, correlated with a late IFN-gamma response, and was negated by serologic ablation of endogenous IL-12 at the time of cell transfer. rIL-12, administered in vivo to the DC recipient mice, could substitute for the T helper peptide in initiating skin test reactivity following transfer of DC pulsed with P815AB alone, leading to Ag-specific production of IFN-gamma by CD4+ and CD8+ T cells. In vitro and in vivo cell depletion experiments suggested the following: 1) the exogenous IL-12 required both CD4+ and CD8+ cells for activity; 2) the immune response initiated by IL-12 relied on later production of IL-12 by the host; and 3) the early adjuvanticity of the exogenous IL-12 involved improved recognition of class II-restricted epitopes of this otherwise poorly immunogenic tumor peptide.
Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria.
Authors: Yañez D M , Manning D D , Cooley A J , Weidanz W P , van der Heyde H C
Published: December 2022

We determined the requirement for selected lymphocyte subsets and cytokines in the pathogenesis of experimental murine cerebral malaria (CM) by using gene-targeted knockout and mAb-suppressed mice. Plasmodium berghei ANKA infection induced CM in A 0/0 mice, which lack expression of surface MHC class II glycoproteins and consequently express a severe and chronic reduction in numbers of CD4+ T cells. However, when A 0/0 mice, which are on a C57BL/6 x 129 genetic background, or immune-intact C57BL/6 controls treated with anti-CD4 mAb were infected, none developed CM. The latter finding confirms an earlier report that CD4+ T cells are required for CM to occur and additionally indicates that the reduced numbers of CD4+ T cells present in A 0/0 mice are sufficient for CM development. Neither the recently described CD4+, NK1.1+ T cell subset shown to be present in A 0/0 mice nor traditional NK cells seem to be required for the induction of CM because A 0/0 and C57BL/6 mice severely depleted of both NK1.1+ populations with mAb developed CM as readily as did normal Ig-treated controls. Deficiency of Th1-associated cytokines (IFN-gamma or IL-2) in mice by gene-targeted disruptions completely inhibited CM development, whereas the lack of Th2-associated cytokines (IL-4 or IL-10) did not prevent this disease. Our observation that B cell-deficient JHD and microMT mice developed CM provides evidence that neither B cells, their products, nor B cell Ag presentation are a requisite for CM pathology. We further observed that neither beta 2m 0/0 knockout mice, which lack CD8+ alpha beta T cells, nor C57BL/6 mice depleted of CD8+ T cells with anti-CD8 mAb treatment developed CM, leading us to conclude that CD8+ T cells are also crucial for the development of CM.
CD88 antibodies specifically bind to C5aR on dermal CD117+ and CD14+ cells and react with a desmosomal antigen in human skin.
Authors: Werfel T , Zwirner J , Oppermann M , Sieber A , Begemann G , Drommer W , Kapp A , Götze O
Published: December 2022

The expression of the C5aR (CD88) on human epidermal and dermal cells was studied with five anti-C5aR mAb directed to the N-terminal domain of the receptor. All mAb bound to suspended dermal CD117+ mast cells and to dermal CD14+ cells. The binding to CD14+ and CD117+ cells could be blocked by rC5a and by peptide EX-1 representing amino acid residues 1-31 of the C5aR. In acetone-fixed frozen or in paraformaldehyde-fixed, paraffin-embedded tissue, we detected a binding of the Abs to dermal perivascular cells and, additionally, to keratinocytes and dermal epithelial cells that could be blocked by EX-1. Immunoelectromicroscopy revealed a binding of anti-C5aR mAb to desmosomal regions in human epidermis. However, the following results indicate that CD88 mAb cross-react with epithelium in a specific way: 1) the binding to suspended epidermal cells and to the epidermal cell line HaCat could be blocked by EX-1 but not by rC5a; 2) FITC-labeled C5a bound to CD117+ and to CD14+ cells but not to epidermal cells; 3) C5a led to transient calcium fluxes in CD14+ and CD117+ dermal but not in epidermal cells; 4) C5aR mRNA was detectable by reverse transcription PCR in granulocytes but not in keratinocytes or in HaCat. Our results show that CD88 mAb are good tools for the investigation of the C5aR on hemopoietic cells. Results with epithelial cells should be considered with caution, as the binding of CD88 mAb that were raised to a synthetic peptide sequence may be due to a cross-reactivity.
Inhibition of superantigen-induced proinflammatory cytokine production and inflammatory arthritis in MRL-lpr/lpr mice by a transcriptional inhibitor of TNF-alpha.
Authors: Edwards C K , Zhou T , Zhang J , Baker T J , De M , Long R E , Borcherding D R , Bowlin T L , Bluethmann H , Mountz J D
Published: December 2022

We have used fas-defective MRL-lpr/lpr mice to study the effects of the staphylococcal enterotoxin superantigens on the development of autoimmune, inflammatory joint disease in animals that are susceptible to the development of rheumatoid arthritis-like disease. We show that systematic administration by a single i.p. injection of staphylococcal enterotoxin B (SEB; 10 micrograms/mouse) caused a mild, inflammatory arthritis +30 days postchallenge in the knee joints of young (< 2-mo-old) MRL-lpr/lpr mice, but not aged-matched MRL +/+ mice. In aged (> 8-mo-old) MRL-lpr/lpr mice, but not in aged MRL +/+ mice, SEB caused a severe, inflammatory arthritis, as assessed histologically, and systemic autoimmune disease, including glomerulonephritis and autoantibody production. Furthermore, in aged MRL-lpr/lpr mice, SEB but not heat-denatured SEB caused acute weight loss and elevated levels of serum proinflammatory cytokines. Compared with highly purified peritoneal macrophages obtained from either aged MRL +/+, young MRL-lpr/lpr, or young MRL +/+, peritoneal macrophages obtained from aged MRL-lpr/lpr mice constitutively expressed 2- to 10-fold greater levels of TNF-alpha, IL-1 beta, IL-6, and IL-10, and produced elevated amounts of these cytokines when treated in vitro with SEB. SEB-challenged aged MRL-lpr/lpr mice treated with anti-TNF mAb (100 micrograms/mouse; every other day), anti-V beta 8 TCR mAb (250 micrograms/mouse; every other day), or orally with the novel TNF-alpha inhibitor MDL 201,449A (9-[(1R, 3R)-trans-cyclopentan-3-ol] adenine; 25 mg/kg/day) exhibited reduced inflammatory arthritis, autoantibody formation, and serum TNF-alpha levels, but not IL-10 levels, after +30 days of treatment. These data suggest that SEB is an extremely potent macrophage-activating factor in vitro and in vivo, enhancing several aspects of autoimmune disease in MRL-lpr/lpr mice, and that anti-TNF therapies may have potential use in inflammatory arthritis.
Defective ribosomal products (DRiPs): a major source of antigenic peptides for MHC class I molecules?
Authors: Yewdell J W , Antón L C , Bennink J R
Published: December 2022

MHC class I molecules predominantly bind to peptides derived from a cytosolic pool of polypeptides. Little is known about the nature of the polypeptides that serve as substrates for peptidogenic cytosolic proteases. We propose that a significant source of self and viral peptides are defective ribosomal products (DRiPs), which consist of prematurely terminated polypeptides and misfolded polypeptides produced from translation of bona fide mRNAs in the proper reading frame. DRiPs are produced entropically, due to the inevitable imperfections inherent to protein synthesis or folding. To accelerate recognition of cells harboring intracellular parasites such as viruses, DRiP formation may be enhanced by changes in the cellular physiology induced by infection or by exposure of cells to cytokines released at the site of inflammation.
Intraocular injection of class II-restricted peptide induces an unexpected population of CD8 regulatory cells.
Authors: Kosiewicz M M , Streilein J W
Published: December 2022

Intraocular injection of exogenous protein induces an Ag-specific impairment of systemic delayed hypersensitivity (DH), termed anterior chamber associated immune deviation (ACAID). The ACAID-inducing signal is carried by blood-borne cells from the eye to the spleen and can also be generated in vitro by incubating APCs with Ag plus TGF-beta. Paradoxically, class I-restricted CD8 regulatory cells are induced in the spleens of mice with ACAID, and previous studies suggest that CD8 cells are important, and even necessary, for the expression of ACAID. To explore this issue further, we asked whether ACAID could be induced with a class II-restricted peptide, and if so, what type of regulatory cells are generated. An intraocular, but not an i.v., injection of OVA (323-339) peptide resulted in impairment of native OVA-specific DH in both naive and previously sensitized mice. Furthermore, i.v. injection of APCs pretreated with TGF-beta plus OVA peptide also prevented native OVA-specific DH. Surprisingly, both CD8+ and CD4+ spleen cells capable of impairing expression of DH were induced by either intraocular injection of peptide or i.v. injection of APCs pretreated with peptide plus TGF-beta. In summary, ACAID can be induced by a class II-restricted peptide and is accompanied by the generation of two unusual populations of cells: 1) CD8+ regulatory cells unexpectedly induced by class II-restricted peptide; and 2) a novel population of CD4 regulatory cells induced by peptide, but not native protein. Potential mechanisms involved in the processing and presentation of exogenous protein in the ACAID model are discussed in light of the present data.
Fas system-mediated apoptosis suppresses lymphopoiesis.
Authors: Yasutomo K , Nagasawa H , Hisaeda H , Good R A , Kuroda Y , Himeno K
Published: December 2022

The lymphoproliferation (lpr) mutation causes the defective expression of Fas Ag, which normally transduces an apoptotic signal into cells. T cells from mice homozygous for this mutation overexpress the counter-receptor, Fas ligand. In this study, we investigated the effects and regulatory influences attributable to Fas ligand overexpression on lymphocyte development to clarify the role of Fas system-mediated apoptosis in lymphopoiesis in vivo. Nonirradiated severe combined immunodeficient (SCID) mice grafted with a fetal thymus (FT) plus fetal liver cells (FLC) from MRL-lpr/lpr mice (Fas Ag-defective mice), or with FT from C3H-gld/gld mice (Fas ligand-defective mice) plus FLC from C3H +/+ mice, developed FLC-derived T and B cells. In contrast, SCID mice grafted with FT from MRL-lpr/lpr Thy-1.1 mice plus FLC from MRL +/+ Thy-1.2 mice (chimera 1) developed few FLC-derived T and B cells in the spleen, and the thymus of the recipients also contained few FLC-derived T cells. In addition, when SCID mice grafted with FT from MRL-lpr/lpr Thy-1.2 mice (H-2k) were co-transplanted with FLC from C57BL/10 Thy-1.1 mice (H-2b) (chimera 2), FLC-derived T and B cells developed normally. Thy-1.1 + cells from chimera 1 expressed Fas ligand mRNA about threefold higher than those from chimera 2, and seven- to eightfold higher than Thy-1.2+ cells from SCID mice grafted with FT from MRL +/+ Thy-1.2 mice by Northern blot analysis. These findings indicate that overexpression of Fas ligand on T cells significantly impairs both T and B cell development. Furthermore, the Fas ligand overexpression sufficient to impair lymphopoiesis appears to require MHC-restricted T cell activation. These results suggest that the Fas system suppresses lymphopoiesis in vivo.
Transmembrane-4 superfamily proteins CD81 (TAPA-1), CD82, CD63, and CD53 specifically associated with integrin alpha 4 beta 1 (CD49d/CD29).
Authors: Mannion B A , Berditchevski F , Kraeft S K , Chen L B , Hemler M E
Published: December 2022

Anti-alpha 4 integrin mAb coprecipitated CD81 (TAPA-1), a 25-kDa cell surface protein, from various alpha 4 beta1 -positive hemopoietic cell lines, including Molt4, Jurkat, Ramos, and alpha 4-transfected K562 (KX4C4) cells. In reciprocal experiments, the integrin alpha 4 beta 1 (VLA4, CD49d/CD29) could be reprecipitated from CD81 immunoprecipitates. Anti-alpha 4 integrin mAb also coprecipitated CD81 from the alpha 4 beta 7-positive B cell line RPMI 8866. In contrast, no CD81 was identified in alpha 2 beta 1, alpha 5 beta 1, or alpha L beta 2 immunoprecipitates. Abs to other members of the transmembrane-4 superfamily, including CD53, CD63, and CD82, also coprecipitated alpha 4 beta 1. As shown by confocal microscopy, CD81 and CD82 colocalized with alpha 4 beta 1 in cell surface clusters. The cytoplasmic domain of the alpha 4 integrin was not necessary for alpha 4 beta 1/CD81 association, nor was the association influenced by divalent cations, EDTA, integrin-activating mAb, or alpha 4 subunit cleavage. Notably, two independent alpha 4 adhesion-deficient mutants (D346E and D408E) were deficient in their ability to associate with CD81. Thus, CD81 and other transmembrane-4 superfamily members may participate in functionally relevant interactions with alpha 4 beta 1 and other integrins.
Regulation of IgM and IgD heavy chain gene expression: effect of abrogation of intergenic transcriptional termination.
Authors: Yuan D , Witte P L , Tan J , Hawley J , Dang T
Published: December 2022

Early IgM+ B cells express little or no membrane IgD due to the low abundance of delta mRNA. Extensive transcriptional termination regulated by sequences in the intronic region between mu and delta heavy chain genes may be the primary reason for the lack of delta gene transcription. We have examined the effect of deletion of these sequences on the regulation of IgM and IgD heavy chain gene expression in transfectants as well as mice carrying this otherwise intact transgene. By run-on transcriptional measurement, we show that the delta exons are transcribed in bone marrow B cells from these transgenic mice. However, in spite of the induced premature synthesis of the full-length mu-delta transcript in pre-B cells, processing to delta mRNA does not occur until the lymphocytes express cell surface IgM. Therefore, during B cell development, synthesis of the full-length transcript is a necessary but not sufficient condition for initiation of delta mRNA synthesis. Furthermore, unexpectedly, the abrogation of transcriptional termination was found to also affect the processing of the primary transcript to microM mRNA. These results show that expression of IgD in primary B cells is stringently regulated and closely linked to IgM expression.
Human gamma delta T cell subset-proliferative response to malarial antigen in vitro depends on CD4+ T cells or cytokines that signal through components of the IL-2R.
Authors: Elloso M M , van der Heyde H C , Troutt A , Manning D D , Weidanz W P
Published: December 2022

We examined the cellular and molecular basis of the proliferative response of human gamma delta T cells in cultures of PBMC stimulated with blood-stage Plasmodium falciparum malarial Ag. Flow cytometry revealed that maximal gamma delta T cell proliferation occurs after maximal CD4+ alpha beta T cell proliferation. Depletion of CD4+ T cells from PBMC before stimulation with malarial Ag markedly reduces the number of proliferating gamma delta T cells, which suggests that CD4+ T cells function in providing help to gamma delta T cells to respond to this parasite Ag. Removal of gamma delta T cells, however, did not alter the expansion of the CD4+ T cell subset. The addition of exogenous IL-2, IL-4, or IL-15 restored the capacity of gamma delta T cells to proliferate in Ag-stimulated cultures of PBMC depleted of CD4+ T cells. mAbs specific for the alpha- and beta-subunits of the IL-2 receptor inhibit the gamma delta T cell subset expansion in cultures stimulated with malarial Ag. Taken together, these findings suggest that the proliferation of gamma delta T cells in response to malarial Ag is dependent on the presence of CD4+ alpha beta T cells, but the requirement for CD4+ alpha beta T cells can be met by cytokines that use the IL-2R.
Altered T cell ligands derived from a major house dust mite allergen enhance IFN-gamma but not IL-4 production by human CD4+ T cells.
Authors: Tsitoura D C , Verhoef A , Gelder C M , O'Hehir R E , Lamb J R
Published: December 2022

Changes in the affinity of the interaction between T cell Ag receptors and their ligands can modulate selected T cell effector functions. Since both allergen-specific Th2 and Th0 cells are present in the peripheral CD4+ T cell pool of atopic individuals, the potential to inhibit cytokine production by Th2 cells and promote Th1-type cytokines from Th0 cells may contribute to the down-regulation of allergic inflammation. The aim of this study was to investigate the effects of peptide analogues of a dominant T cell epitope of the group II allergen derived from house dust mite, residues 28 to 40, on proliferation and cytokine production by human Th2 and Th0 cells. From both functional competition and proliferation assays, using analogues substituted with alanine or charged amino acids, the influence of different positions in p28-40 on TCR recognition and/or MHC class II binding was determined. For the specific Th0 cells, generally those analogues that lead to a reduction in proliferation also decreased both IL-4 and IFN-gamma production. However, the p28-40 analogues with alanine residues at positions 34 and 36 altered the IFN-gamma:IL-4 ratio by selectively enhancing IFN-gamma secretion. In the case of Th2 cells, stimulation with the peptide analogues induced different patterns of effector function. Selected analogues were capable of inducing IL-4 production in the absence of proliferation, whereas in response to other peptide variants of p28-40, both IL-4 production and proliferation were inhibited.
Anti-phospholipid autoantibodies bind to apoptotic, but not viable, thymocytes in a beta 2-glycoprotein I-dependent manner.
Authors: Price B E , Rauch J , Shia M A , Walsh M T , Lieberthal W , Gilligan H M , O'Laughlin T , Koh J S , Levine J S
Published: December 2022

Anti-phospholipid autoantibodies (aPL) are associated with a clinical syndrome of hypercoagulability, thrombocytopenia, and fetal loss. Several groups have shown that the in vitro target of many aPL is not a pure phospholipid Ag, but is either a complex between anionic phospholipid and the plasma protein beta2-glycoprotein I (beta 2GPI) or the protein beta 2GPI alone. Anionic phospholipids are normally absent from the extracellular surface of cell membranes but redistribute from the inner to the outer leaflet during apoptosis. We show that aPL bind specifically to apoptotic, but not viable, thymocytes, and that binding is dependent upon the presence of beta 2GPI. Moreover, we show that beta 2GPI binds selectively to the surface of apoptotic thymocytes to generate an epitope for antiphospholipid autoantibodies. These findings suggest that apoptotic cells may be the natural immunogen and/or target for aPL. Moreover, we propose that the interaction of circulating beta 2GPI with redistributed anionic phospholipid may itself generate a novel ligand by which apoptotic cells are recognized directly for phagocytic clearance.
The mechanisms of peptide exchange and beta 2-microglobulin exchange on cell surface Ld and Kb molecules are noncooperative.
Authors: Cook J R , Myers N B , Hansen T H
Published: December 2022

It has previously been shown that the presence of exogenous beta 2-microglobulin (beta 2m) can dramatically enhance the binding of exogenous peptide to cell surface class I MHC. However, the mechanism by which this enhancement takes place is unknown. Two models have been proposed to explain this phenomenon. In the first model, the exchange of peptide and the exchange of beta 2m are cooperative processes. In the alternative model, beta 2m stabilizes free class I heavy chains to increase the total number of peptide binding sites available. In this report, we have examined the relationship between peptide exchange and beta 2m exchange. Comparisons of Ld and Kb complexes formed with peptides possessing widely disparate affinities revealed a reciprocal correlation between the peptide off-rate and the rate of beta 2m exchange. This result indicates that peptide exchange and beta 2m exchange are noncooperative processes that may, in fact, antagonize one another. These findings provide the first demonstration of peptide-specific influences on the rate of beta 2m exchange and suggest that exogenous beta 2m promotes peptide binding by maintaining class I heavy chains in a peptide-receptive state.
Peptide-specific T cell clonal expansion in vivo following immunization in the eye, an immune-privileged site.
Authors: Egan R M , Yorkey C , Black R , Loh W K , Stevens J L , Woodward J G
Published: December 2022

To visualize the primary antigen-specific T cell response to Ag introduced into the eye, we have used an adoptive transfer system in which a limiting number of OVA peptide (323-339)-specific T cells from a TCR-transgenic mouse were transferred into nonirradiated, syngeneic recipients and then tracked in vivo by staining for FACS analysis or immunohistochemistry with the clonotypic mAb KJ1-26. Following posterior chamber injection of Ag, KJ1-26+ cells accumulated primarily in the draining, submandibular lymph node (LN) within 3 days. Although reduced in number, by day 6 these cells were primarily in the paracortical regions and were able to proliferate and secrete IL-2 in response to Ag stimulation. In contrast, following i.v. injection of Ag, the KJ1-26+ cells accumulated in the paracortical regions of the LN to a comparable degree, but did not proliferate or secrete IL-2. The day 3 accumulation of KJ1-26+ cells in the submandibular LN was inhibited if the eye was removed within 5 h after injection of Ag. In the spleen, foci of KJ1-26+ cells were observed in the periarteriolar lymphoid sheaths at day 3; these were not observed to the same degree following other forms of immunization. These results demonstrate that the submandibular LN is the primary site for early clonal expansion of antigen-specific T cells following intraocular Ag administration and that these cells show changes consistent with immunity rather than tolerance.
Inhibition of gamma delta T cell development and early thymocyte maturation in IL-7 -/- mice.
Authors: Moore T A , von Freeden-Jeffry U , Murray R , Zlotnik A
Published: December 2022

While early thymic T cell precursor populations and their maturational sequence have been recently identified, the signals driving differentiation are unknown. While cytokines may play an integral role in T cell development, various mouse models rendered genetically deficient for specific cytokines do not display abnormalities in T cell development. Recently, we have generated IL-7 -/- mice and reported that IL-7 plays a unique and nonredundant role in lymphopoiesis. These mice displayed a 10- to 20-fold reduction in the total number of T and B cells. Here, we show that IL-7 -/- mice display a sharp reduction in both the frequency and absolute number of adult thymic gamma delta T cells while retaining normal frequencies of alpha beta T cells. This defect in gamma delta T cell production extends to peripheral organs as IL-7 -/- mice are essentially devoid of splenic and intestinal intraepithelial gamma delta T cells. This aberrant phenotype was traced back to impaired fetal gamma delta T cell maturation. In the absence of IL-7, differentiation of immature V gamma 3 low-CD24+ fetal T cells to mature V gamma 3 high CD24- cells is inhibited. In contrast, NK cell maturation appears to be only mildly affected in the absence of IL-7. To further clarify the role of IL-7 in thymic development, detailed analysis of CD3-4-8- thymic precursors was performed. A partial inhibition in the differentiation of CD44+25+ pro-T cells into CD44-25+ pre-T cells was observed. Unexpectedly, the lack of IL-7 resulted in decreased expression of CD117 (c-kit) on both CD4 low and pro-T cells, suggesting that IL-7 may influence the expression of other cytokine receptors involved in early hemopoietic development. Together, these data clarify the developmental abnormalities during T cell development due to the absence of IL-7.
Granzyme B induces apoptosis and cyclin A-associated cyclin-dependent kinase activity in all stages of the cell cycle.
Authors: Shi L , Chen G , He D , Bosc D G , Litchfield D W , Greenberg A H
Published: December 2022

Granzyme B rapidly induces apoptosis in the presence of the pore-forming protein perforin. We have examined the cell cycle restriction of this apoptosis by separating Jurkat cells into fractions representing different stages of the cell cycle by centrifugal elutriation. Cells were susceptible to apoptosis from G1 through to G2/M, with no significant resistance detected at any stage. Similarly, cells arrested at G1/S or G2/M with either hydroxyurea or nocodazole were slightly more sensitive than asynchronously growing cells. Granzyme B induces Cdc2 kinase activity and requires its induction for apoptosis. Cyclin-dependent kinase (CDK) activity is regulated by phosphorylation and association with cyclins that also control subcellular localization of the CDK/cyclin complexes. Cdc2 associates with both cyclin A, which is synthesized at G1 and S, and cyclin B, which is produced later during S and G2 before G2/M transition. We find that the CDK activity induced by granzyme B is associated primarily with cyclin A in both asynchronous and G1/S-arrested cells, while cyclin B-associated kinase activity is minimal. Because cyclin A is also able to associate with Cdk2, a kinase that is important for G1/S transition, we examined the activation of this CDK during granzyme B-induced apoptosis and find that Cdk2 is induced as rapidly as Cdc2. In conclusion, we have identified a lack of cell cycle restriction of granzyme B-induced apoptosis and the rapid activation of both cyclin A/Cdc2 and cyclin A/Cdk2 kinase activity.
Viral peptide specific induction of MHC class II expression by murine T cell clones.
Authors: Thomas D B , Smith C A , Graham C M
Published: December 2022

We report that I-Ab-restricted T cell clones, elicited by influenza infection of C57BL/10 mice and specific for the hemagglutinin peptide HA1 186-205, express class II. They respond to peptide stimulation by IL release (IL-3 or IFN-gamma) without a requirement for APC but do not proliferate. Moreover, surface expression of class II requires de novo synthesis in the presence of the stimulatory peptide and is inhibited by coculture with TCR-specific Ab, or brefeldin A or cycloheximide. Clonotypic specificity of peptide induction was confirmed by failure of other allele specific peptides to enhance class II expression. Addition of the viral peptide to T cells induced homotypic adhesion, which provides a physical basis for stabilization of class II-peptide complexes at the cell surface. Extinction of class II expression was evident in the corresponding T cell hybridomas, which might account for the failure to report class II expression by murine T cells. Control studies indicated that class II was not passively acquired from APC by demonstrating 1) failure of processed Ag to induce class II expression, 2) allo-class II (Ak) was not acquired by coculture with peptide and semisyngeneic (H-2 b/k) APC, 3) absence of class II expression by a NP peptide-specific Th2 clone under identical culture conditions, and most significantly, 4) reverse-transcriptase PCR amplification and surface expression of class II using highly purified preparations of FACS-selected CD4+ class II- cells cocultured with the stimulatory peptide.
Correlation between the amino acid position of arginine in VH-CDR3 and specificity for native DNA among autoimmune antibodies.
Authors: Krishnan M R , Jou N T , Marion T N
Published: December 2022

Autoimmunity to DNA in mouse models for the systemic autoimmune disease systemic lupus erythematosus (SLE) has all of the characteristics of an Ag-driven secondary immune response to DNA. Since the pathogenesis of anti-DNA Ab in SLE is correlated to Ab specificity for native DNA (dsDNA), understanding how such specificity is generated is important. As with immune A responses to most Ags, autoimmune Ab responses to DNA are dependent upon the clonal selection of B cells expressing particular H and L chain V-region structures. The VH structures of most autoimmune anti-DNA Abs include at least one arginine in VH-CDR3; moreover, previous results led us to propose that anti-DNA Ab specificity for dsDNA may be dependent upon the relative position of arginines in VH-CDR3. The present results demonstrate a strong correlation between specificity for dsDNA and arginine position in VH-CDR3, for Abs with V, encoded by VH genes from the VH7183, VHQ52, and VHS107 families but not from the VH558 family. Specificity for dsDNA was not only correlated to the presence of VH-CDR3 arginines but also to the relative position of the arginines in VH-CDR3. The majority of the VH-CDR3 arginines appeared to have been encoded by sequences generated during V-D-J recombination. These results are not only important for understanding how A specificity for dsDNA is generated but also how somatically derived structures generated during V-D-J recombination may influence clonotype selection of an immune response within an individual mouse.
HOXB cluster genes in activated natural killer lymphocytes: expression from 3'-->5' cluster side and proliferative function.
Authors: Quaranta M T , Petrini M , Tritarelli E , Samoggia P , Carè A , Bottero L , Testa U , Peschle C
Published: December 2022

The expression of HOXB cluster genes (i.e., B1 through B9) was evaluated in purified IL-2/IL-1 beta-activated NK lymphocytes from normal adult peripheral blood by RNase protection and reverse transcription-PCR. In quiescent NK cells these genes are essentially not expressed. After IL-2/IL-1 beta addition, we observed a coordinate induction wave in the 3'-5' HOXB cluster direction, i.e., from B1 through B9. As notable exceptions, B8 is silent, while B9 RNA is detected starting from 6 h through day 11. Furthermore, the 3' located B2/B3/B4 are expressed earlier and at higher level than the 5' located B5/B6/B7/B8. In IL-2/IL-1 beta-activated NK cells, treatment with antisense oligonucleotides targeting B2 mRNA causes a significant inhibition of both cell proliferation and expression of activation markers (i.e., IL-2R alpha-chain and transferrin receptor). These studies provide novel evidence of the role of HOX genes in adult NK cell proliferation. Thus, 1) a coordinate activation of HOXB genes from the 3'-->5' cluster side apparently underlies IL-2/IL-1 beta-induced NK cell activation. 2) Since NK cell activation and survival induced by IL-12 and c-kit ligand, respectively, are not associated with cell proliferation of HOXB gene expression, it is apparent that HOXB gene induction is specifically associated with IL-2-induced NK cell proliferation. 3) Studies with antisense oligomer targeting HOXB2 mRNA suggest an important role for 82 in NK cell proliferation, possibly in part via the IL-2R.
Probing HLA-B7 conformational shifts induced by peptide-binding groove mutations and bound peptide with anti-HLA monoclonal antibodies.
Authors: Smith K D , Mace B E , Valenzuela A , Vigna J L , McCutcheon J A , Barbosa J A , Huczko E , Engelhard V H , Lutz C T
Published: December 2022

To determine the influence of peptide-binding groove residues and MHC-bound peptide on HLA-B7 conformation, we investigated the binding sites of nine locus- or allele-specific mAbs using a panel of 82 HLA-B7 variants. The functional mAb epitopes encircle the HLA-B7 peptide-binding groove. Three mAbs are affected by mutations at solvent-accessible peptide-binding groove mutations. Mutations in peptide-binding groove residues 45, 63, and 150 affect multiple nonoverlapping mAb epitopes, probably by interaction with other MHC residues or bound peptide. However, 18 of 24 peptide-binding groove mutations do not affect mAb binding, indicating that the conformation of solvent-accessible HLA-B7 structures is largely dissociated from changes in the peptide-binding groove. To test whether bound peptides alter HLA-B7 conformation, we loaded HLA-B7 heavy chains on acid-stripped cells with beta2-microglobulin and 20 individual synthetic peptides. Two of eight mAbs are sensitive to HLA-B7-bound peptides. A likely interpretation of these data is that the conformational flexibility of HLA-B7 is due to peptide-induced conformational shifts in MHC side chains, rather than major shifts in the MHC main chain. These results suggest that HLA-B7 conformation is largely maintained in the context of different bound peptides and different peptide-binding grooves.
IL-10 enhances the growth of Legionella pneumophila in human mononuclear phagocytes and reverses the protective effect of IFN-gamma: differential responses of blood monocytes and alveolar macrophages.
Authors: Park D R , Skerrett S J
Published: December 2022

Legionella pneumophila is a facultative intracellular pathogen that parasitizes human alveolar macrophages and blood monocytes recruited to the lungs. The inhibitory cytokines IL-10, TGF-beta, and IL-4 generally deactivate macrophages and permit enhanced microbial growth in some models of intracellular infection, but their effects on human alveolar macrophages are unknown. We hypothesized that inhibitory cytokines could facilitate the infection of human alveolar macrophages and monocytes by virulent intracellular lung pathogens. Therefore, we tested the effects of IL-10, TGF-beta, and IL-4 in an in vitro model of human alveolar macrophage and monocyte infection with L. pneumophila. We found that unstimulated alveolar macrophages supported over 100-fold greater L. pneumophila growth than did unstimulated monocytes. IL-10 treatment significantly enhanced L. pneumophila growth in monocytes, and completely reversed the protective effect of IFN-gamma against intracellular L. pneumophila replication. IL-10 had similar but less potent effects on alveolar macrophages. In contrast, TGF-beta and IL-4 had no significant effects on L. pneumophila growth in resting or IFN-gamma-activated monocytes or alveolar macrophages. IL-10 blocked TNF-alpha production by infected cells, but exogenous TNF-alpha did not reverse the activating defect in cells cocultured with IFN-gamma and IL-10. Finally, L. pneumophila-infected monocytes produced substantially more IL-10 than did infected alveolar macrophages. In summary, IL-10 significantly enhances the growth of L. pneumophila in human monocytes, reverses the protective effect of IFN-gamma, blocks TNF-alpha secretion, and is secreted by infected monocytes and alveolar macrophages. Induction of IL-10 may be a virulence mechanism that promotes intracellular bacterial replication in human legionellosis.
Mycobacterial lipoarabinomannan recognition requires a receptor that shares components of the endotoxin signaling system.
Authors: Savedra R , Delude R L , Ingalls R R , Fenton M J , Golenbock D T
Published: December 2022

Phagocytic leukocytes respond to a variety of bacterial products including Gram-negative bacterial LPS and mycobacterial lipoarabinomannan (LAM). Anti-CD14 mAbs have been shown to block LPS and LAM activation of myeloid cells, suggesting that CD14 is required for cellular recognition of both ligands. Activation of undifferentiated promonomyelocytic THP-1 cells with LAM or LPS under serum-free conditions was enhanced in the presence of recombinant soluble CD14 (rsCD14). LPS binding protein (LBP), which is present in normal serum, further enhanced the sensitivity of undifferentiated THP-1 cells to both ligands even in the absence of rsCD14. Although CD14-transfected Chinese hamster ovary and human HT1080 fibrosarcoma cell lines can be activated by LPS, neither cell line was activated by LAM. Furthermore, U373 astrocytoma cells, which respond to LPS using sCD14 and LBP, failed to be activated by LAM in the presence of rsCD14 and rLBP. We then tested the effects of lipid IVA and Rhodobacter sphaeroides lipid A, compounds that function as endotoxin inhibitors in human cells by interacting with a molecule thought to be a CD14-dependent LPS signal transducer. Both lipid IVA and R. sphaeroides lipid A inhibited the effects of LPS and LAM in THP-1 cells. Thus, the LPS and LAM receptors share CD14, LBP, and a putative endotoxin antagonist-inhibitable signal transducing component. However, the LAM signaling system appears to require an additional receptor component whose expression is restricted to cells of hemopoietic origin.
Autoantibodies in insulin-dependent diabetes recognize distinct cytoplasmic domains of the protein tyrosine phosphatase-like IA-2 autoantigen.
Authors: Lampasona V , Bearzatto M , Genovese S , Bosi E , Ferrari M , Bonifacio E
Published: December 2022

Protein tyrosine phosphatase-like IA-2 is a major autoantigen in insulin-dependent diabetes. It has been identified as both a specificity of cytoplasmic islet cell Abs and one of the precursors of the 40- and 37-kDa tryptic fragment islet autoantigens. To characterize autoantibody binding to IA-2 and determine whether humoral autoimmunity extends to other tyrosine phosphatases, we analyzed serum reactivity in 100 patients with insulin-dependent diabetes against different in vitro translated portions of the IA-2 protein as well as the tyrosine phosphatase domains of HPTPbeta and HPTPdelta. All autoantibody reactivity was confined to the cytoplasmic portion of IA-2 (amino acids 601-979). At least four epitopes were found. These were contained within amino acids 605 to 620 and 605 to 682 of the juxtamembrane region and within amino acids 777 to 937 and 687 to 979 in the IA-2 tyrosine phosphatase-like domain. Footprinting studies confirmed the presence of multiple epitopes. Fifty-six percent of sera with IA-2 Abs bound epitopes within the juxtamembrane region, and 83% bound epitopes in the tyrosine phosphatase-like domain; 39% had Abs to both regions. No reactivity against the IA-2 ectodomain or the tyrosine phosphatase domains of HPTPbeta and HPTPdelta was found. These data suggest that the cytoplasmic region, in particular the tyrosine phosphatase-like domain, is the major target of IA-2 Abs in insulin-dependent diabetes, and that autoantibody reactivity is specific for IA-2 or IA-2-like molecules.
IL-4 induces Fas resistance in B cells.
Authors: Foote L C , Howard R G , Marshak-Rothstein A , Rothstein T L
Published: December 2022

Activated B cells express Fas (CD95) and are targets for apoptosis induced by CD4+ Th1 effector cells that kill in a Fas-dependent fashion. We report here that IL-4 reverses the susceptibility to Fas-mediated apoptosis that characterizes CD40-stimulated primary B cells. IL-4-induced Fas resistance is not associated with an alteration in the elevated level of Fas expression produced by CD40 ligand and does not depend on additional receptor-mediated signals. However, IL-4-induced resistance to Th1 cell-mediated cytotoxicity (Th1-CMC) develops more slowly than resistance mediated by surface Ig and is not affected by protein kinase C depletion, unlike anti-Ig-induced Fas resistance. By these two criteria, IL-4-and anti-Ig-induced resistance to Th1-CMC appear to be driven through distinct mechanisms; in keeping with this, suboptimal doses of IL-4 and anti-Ig act in synergy to induce marked protection against Th1-CMC. An important role for IL-4-induced Fas resistance is suggested by the observation that sera from IL-4-overexpressing transgenic mice contain autoreactive Abs.