bluuebunny/crossref_metadata_2025 huggingface dataset:
Definition of specific peptide motifs for four major HLA-A alleles.
Authors: Kubo R T , Sette A , Grey H M , Appella E , Sakaguchi K , Zhu N Z , Arnott D , Sherman N , Shabanowitz J , Michel H
Published: December 2022

Allele-specific motifs for the human MHC class I molecules, HLA-A1, A3, A11, and A24 were characterized by three complementary approaches. First, amino acid sequence analysis of acid eluted peptide pools from affinity purified class I molecules defined putative motifs 9 or 10 amino acids in length and bearing critical anchor residues at position 2 and at the COOH-terminal. These motifs were distinct, with the exception of the HLA-A3 and A11 motifs that were very similar to each other. Second, the correctness of these putative motifs was verified by analyzing the binding capacity of polyalanine peptide analogues to purified HLA-A molecules. Several alternative anchor residues that were not obvious from the pooled peptide sequencing analysis were identified. Third, sequences of individual peptides eluted from HLA-A1, A11, and A24 were determined by tandem mass spectrometry. Nonamers were the predominant species, although peptides of 8, 10, 11, and 12 amino acids in length were also identified. These peptides displayed anchor residues predicted by the specific motifs at position 2 and at the COOH-terminal, regardless of peptide length. Synthetic versions of the naturally processed peptides were shown to bind to the appropriate HLA-A alleles with IC50 values in the 0.3- to 200-nM range. A rational approach to search Ags with known amino acid sequences for epitopes restricted by some of the most common HLA-A types and of potential clinical importance is now feasible.
Ligand-induced adhesion to activated endothelium and to vascular cell adhesion molecule-1 in lymphocytes transfected with the N-formyl peptide receptor.
Authors: Honda S , Campbell J J , Andrew D P , Engelhardt B , Butcher B A , Warnock R A , Ye R D , Butcher E C
Published: December 2022

Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.
Induction of IL-3 and granulocyte-macrophage colony-stimulating factor by substance P in bone marrow cells is partially mediated through the release of IL-1 and IL-6.
Authors: Rameshwar P , Ganea D , Gascón P
Published: December 2022

Experimental data strongly suggest that the nervous and immune systems are interrelated. One example of this interrelation is anatomical and is represented by innervation of the lymphoid organs by substance P (SP) immunoreactive fibers, among others. Neurotransmitters/neuropeptides can exert functional receptor-mediated immunologic responses. SP binding to its receptor induces cytokine production in macrophages and T cells and stimulates IgG secretion from B cells. SP has also been associated with inflammation and other immune-mediated diseases such as arthritis. We have previously reported an in vitro stimulatory effect of SP on hematopoiesis that was mediated mostly by the induction of two relevant hematopoietic growth factors, IL-3 and granulocyte-macrophage-CSF (GM-CSF). In this study, we have shown that SP, through the carboxyl terminus, induces the production of IL-3 and GM-CSF in bone marrow mononuclear cells. This production requires de novo synthesis and is blocked by two different SP-R antagonists, spantide and CP-96,345-1. The induction of IL-3 and GM-CSF is partially mediated by IL-1 and IL-6, which are also produced by bone marrow mononuclear cells. Furthermore, the production of IL-3 and GM-CSF correlated with an accumulation of their respective steady state mRNAs. T cells found within the bone marrow are responsible for most of the induced IL-3. Because SP mediates the release of IL-1, IL-3, IL-6, and GM-CSF, all important hematopoietic regulators, by bone marrow cells, this study further suggests the possibility of a regulatory role of the nervous system in hematopoiesis mediated by neuropeptides such as SP.
Influence of H-2 class II antigens on the development of murine AIDS.
Authors: Makino M , Tang Y , Murphy D B , Fredrickson T N , Okada Y , Fujiwara M , Chattopadhyay S K , Mizuochi T , Komuro K , Morse H C
Published: December 2022

Inbred strains of mice differ markedly in their relative susceptibility to the development of lymphoproliferation and immunodeficiency, a syndrome termed mouse AIDS (MAIDS), after infection with the LP-BM5 mixture of murine leukemia viruses (MuLV). The etiologic virus in this mixture is replication defective (BM5def) and encodes only a variant gag protein. Genetic determinants of resistance and susceptibility to induction of MAIDS reside both within and outside the MHC. In strains with C57BL background genes, the MHC haplotypes associated with resistance to disease include d and a, whereas haplotypes b, s, and q are associated with sensitivity. Previous studies showed that MHC class I genes (H-2Dd, H-2Ld) mapping in the D end of H-2 and other genes mapping proximal to the D end determine resistance to MAIDS. This paper examines the nature of these non-D end MHC genes using assays of MHC recombinant and transgenic mice. We demonstrate that expression of E alpha d confers significant resistance to MAIDS, even in mice that do not express H-2Dd/H-2Ld. Unexpectedly, we found that E alpha polymorphisms can significantly influence resistance, with H-2b mice bearing E alpha d as a transgene having greater resistance to MAIDS than mice bearing an E alpha k transgene. E alpha d-mediated resistance to MAIDS was associated with decreased levels of the BM5def genome in splenic DNA, suggesting that E alpha genes exert their effect by enhancing antiviral activity.
Human acetylcholine receptor presentation in myasthenia gravis. DR restriction of autoimmune T epitopes and binding of synthetic receptor sequences to DR molecules.
Authors: Manfredi A A , Yuen M H , Moiola L , Protti M P , Conti-Tronconi B M
Published: December 2022

Autoimmune Th cells in myasthenia gravis recognize several sequence regions of the human muscle acetylcholine receptor (AChR). Most AChR Th epitopes are presented by HLA class II DR molecules (DR). Four sequence regions of the AChR alpha-subunit form Th epitopes recognized by most myasthenic patients, irrespective of their DR haplotype. In this study we first identified in five myasthenic patients the DR molecule(s) likely to be involved in presentation of T immunodominant AChR sequences. We then investigated the binding to the affinity purified DR molecules thus identified (DR2/w51, DR4/w53, and DR7/w53) and to the DR1 molecule, of a panel of overlapping synthetic peptides screening the human alpha-subunit sequence, previously used to identify AChR Th epitopes in myasthenic patients. The AChR peptides that stimulated anti-AChR autoimmune Th cells all bound the relevant DR molecules. Some AChR peptides never recognized by Th cells of myasthenic patients also bound well to one or more DR molecules. The relative ability to bind to DR molecules of different sequence regions of the AChR, i.e., an autoantigen, agrees well with the results of previous studies on the DR binding of synthetic sequences of exogenous antigens. Some peptide sequences uniquely bound one DR molecule, others bound several DR molecules, and others did not bind any of the DR molecules tested.
How many specific B cells are needed to protect against a virus?
Authors: Bachmann M F , Kündig T M , Kalberer C P , Hengartner H , Zinkernagel R M
Published: December 2022

The size of the Ab repertoire has been estimated to comprise theoretically somewhere between > 10(10) and approximately 10(4) specificities, dependent on the criteria used. In an attempt to estimate the anti-viral protective Ab repertoire of the mouse the B cell and Ab-forming cell (AFC) frequencies and protective neutralizing Ab levels during the course of an infection with vesicular stomatitis virus (VSV) were analyzed. Determination of AFC frequencies, limiting dilution assays, and adoptive transfer experiments to SCID mice revealed that during the acute phase (day 8) of the immune response, more than 50% of all IgG2a-producing AFCs were specific for VSV, most of them recognizing the neutralizing determinant. In a later phase (days 21 or 50), 10 to 20 times fewer VSV-specific AFCs were present, corresponding to a frequency of approximately 1:10(4) spleen cells. Finally, in a protection assay in SCID mice, adoptively transferred protective Ab concentrations were found to be approximately 1 to 10 micrograms Ab/ml mouse serum. Because during the memory phase of the anti-VSV response usually 10(4) AFC/mouse are engaged to maintain a high level of memory IgG against the neutralizing determinant on VSV and if one assumes a total number of about 10(6) AFCs/mouse, these data suggest a rather limited neutralizing anti-viral protective memory-AFC repertoire of 10(2) to 10(4) different specificities.
Control of the rat T cell response to retroviral and bacterial superantigens by class II MHC products and Tcrb-V8.2 alleles.
Authors: Herrmann T , Hochgrebe T , Torres-Nagel N E , Huber B T , Hünig T
Published: December 2022

The in vitro response of unprimed rat T cells to retroviral and bacterial superantigens (SAg) was analyzed with TCR V beta 8.2-, 8.5-, 10-, and 16-specific mAbs. Specific stimulation of V beta 8.2 and 8.5 CD4 cells was observed in the response to Mls1a, the retroviral SAg encoded by integrated provirus Mtv-7 (Mtv-7 SAg), which was presented by mouse B cells or mouse fibroblasts transfected with DR1 genes and the Mtv-7 SAg. Additionally, a strong response of V beta 16 CD4 cells to an as yet unidentified mouse SAg was found. Only some of the bacterial SAg known to stimulate mouse and human T cells also activated rat lymph node cells. SEA, SEE, and TSST-1 stimulated rat T cells well; SEB, SEC1, and SED did not. This defect was apparently a result of weak binding to rat MHC class II molecules because presentation by human MHC class II molecules restored T cell activation. Under these conditions, SEB stimulated V beta 8.2+ and 8.5+ CD4 and CD8 cells from Lewis rats. A comparison of several rat strains revealed an unresponsiveness to SEB or Mtv-7 SAg for V beta 8.2 cells from F344 and DA rats. Determination of the nucleotide sequences of the Tcrb-V8.2 of these strains revealed differences between SAg-responsive and SAg-unresponsive Tcrb-V8.2 in seven amino acids, four of them located in the putative SAg contact site. The significance of these findings for the evolution of TCR-SAg interactions is discussed.
B7/CD28-dependent IL-5 production by human resting T cells is inhibited by IL-10.
Authors: Schandené L , Alonso-Vega C , Willems F , Gérard C , Delvaux A , Velu T , Devos R , de Boer M , Goldman M
Published: December 2022

We analyzed the effects of rIL-10 on IL-5 production by human resting T cells isolated from peripheral blood. Resting T cells of healthy individuals required activation for 48 h with either anti-CD3 mAb cross-linked on B7/CD32-transfected mouse fibroblasts or PMA in conjunction with anti-CD28 mAb for optimal IL-5 secretion. In each condition, IL-5 secretion measured by ELISA was inhibited in a dose-dependent manner by rIL-10, whereas IFN-gamma production was not suppressed. The inhibitory effect of rIL-10 on IL-5 synthesis induced by PMA and anti-CD28 mAb was also observed at the mRNA level. In contrast with its action on T cells costimulated by B7/CD28 signaling, rIL-10 did not block IL-5 secretion in response to PMA and A23187 calcium ionophore. The inhibition of IL-5 production by rIL-10 was not due to IL-2 deprivation because it was not modified by the addition of exogenous rIL-2. Moreover, cyclosporin A, which inhibited IL-2 more efficiently than rIL-10 in response to anti-CD3 mAb and B7/CD32 transfected fibroblasts, did not reduce and even enhanced IL-5 production. Finally, we analyzed the influence of endogenously produced IL-10 on IL-5 secretion by T cells stimulated by PMA and anti-CD28 mAb. Addition of a neutralizing anti-IL-10 mAb increased IL-5 release in this system, indicating that endogenous IL-10 controls IL-5 production. We conclude that both rIL-10 and endogenous IL-10 inhibit IL-5 production by T cells costimulated by B7/CD28 signaling.
Antigen presentation is enhanced by targeting antigen to the Fc epsilon RII by antigen-anti-Fc epsilon RII conjugates.
Authors: Squire C M , Studer E J , Lees A , Finkelman F D , Conrad D H
Published: December 2022

Targeting Ag to the Fc epsilon RII by Ag-specific IgE has been shown to be an efficient means of enhancing Ag presentation by B cells to Ag-specific T cells. To take advantage of the Fc epsilon RII as a targeting molecule and to investigate whether IgE was required for mediation of the enhanced stimulation, Ag was covalently coupled to anti-Fc epsilon RII by using heterobifunctional crosslinking reagents. These Ag-Ab conjugates were used with T cell lines specific for the Ags, OVA (BB6.5) or rabbit gamma-globulin (CDC35 and D1.6), and splenic B cells to examine both B cell and T cell proliferation in vitro. Significant presentation of Ag-anti-Fc epsilon RII conjugates was apparent at doses of Ag 1,000- to 10,000-fold lower than seen with unconjugated Ag alone. Ag presentation with the use of anti-Fc epsilon RII-Ag conjugates was as good as or better than conjugates with Ab to the adhesion molecule Pgp-1 or control Ab in T cell proliferation and better than those conjugates in B cell proliferation assays (10- to 100-fold). Anti-Fc epsilon RII-Ag conjugates were clearly more effectively presented than Ag-anti-Fc gamma RII conjugates (> 100-fold). Mouse Fc epsilon RII is presently known to be expressed on B cells and follicular dendritic cells and these in vitro results suggest that the conjugates would be useful tools for investigating the role of IgE-mediated B cell Ag presentation in vivo. BALB/c mice immunized with OVA-anti-Fc epsilon RII conjugates made a quite significant OVA-specific IgG1 response and a detectable IgE response. No detectable Ab was produced in response to OVA alone and a minimal response was seen when an isotype-matched control conjugate was used. Thus, the results indicate that Fc epsilon RII targeting is operative both in vivo and in vitro.
Acute rejection of marrow grafts in mice. Dependence on and independence of functional TCR in the rejection process.
Authors: Takeda K , Moore M W , Dennert G
Published: December 2022

The question of how irradiated mice acutely reject marrow grafts has remained controversial, and evidence in support of T cell- and natural killer cell-mediated rejection mechanisms has been provided. Here we show in support of previous data that CB17 severe combined immunodeficiency mice acutely reject allogeneic marrow but the specificity of rejection cannot be mapped within the MHC. It is shown that a similar rejection specificity is also expressed in normal CB17 mice and that it is caused by CD3- or CD3+ effector cells that do not utilize TCR. In search of TCR-independent rejection mechanisms in other mouse strains, use is made of TCR transgenic mice expressing a defect in recognizing H-2Dd. It is shown that, although marrow graft rejection is impaired in these mice, pointing to participation of TCR in the rejection process, residual resistance does exist. This resistance maps to the MHC, cannot be shown to involve TCR, and appears to be expressed by NK1+ CD3+ cells. It is concluded that acute marrow graft rejection in normal mice can be mediated by both TCR-mediated and NK cell receptor-dependent effector mechanisms, depending on the particular mouse strains.
Phosphatidylinositol synthesis is a proximal event in intracellular signaling coupled to T cell receptor ligation. Differential induction by conventional antigen and retroviral superantigen.
Authors: Weber G F , Cantor H
Published: December 2022

There is increasing evidence that at least two functionally distinct signal transduction pathways may be coupled to the TCR complex. A conventional signal transduction pathway coupled to TCR ligation by peptide/MHC complexes includes increased [Ca2+]i, phosphatidylinositol (PI) hydrolysis, and cytokine expression. TCR ligation by MIs-1a/MTV superantigens may be coupled to an alternative signal transduction pathway without increased [Ca2+]i or detectable PI hydrolysis. We asked whether early events in the PI hydrolytic pathway might account for differential levels of PI breakdown after TCR ligation by conventional Ag and superantigen. We show that TCR ligation by conventional peptide Ag is coupled to a burst of PI synthesis in nontransformed T cells, which may represent a rate-limiting step for downstream PI hydrolysis. Incorporation of radiolabeled glucose by newly synthesized PI as well as inositol-1,4,5-trisphosphate provided direct evidence that this intracellular pool of PI represents an important substrate for this signaling pathway. By contrast, TCR ligation of the same T cell clone by retroviral superantigen leads to similar levels of T cell proliferation without detectable PI synthesis. Increased levels of radiolabeled intracellular PI reflected de novo synthesis of PI from glucose-6-phosphate rather than recycling of inositol phosphates because labeled phosphate and glucose were incorporated into PI but extracellular [3H]inositol was not. These findings suggest that differential PI synthesis represents an early biochemical event that distinguishes two functionally distinct signaling pathways coupled to TCR ligation by peptide Ag and retroviral superantigen.
Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells.
Authors: Marquardt D L , Walker L L , Heinemann S
Published: December 2022

Adenosine potentiates the stimulated release of mast cell mediators. Pharmacologic studies suggest the presence of two adenosine receptors, one positively coupled to adenylate cyclase and the other coupled to phospholipase C activation. To identify mast cell adenosine receptor subtypes, cDNAs for the A1 and A2a adenosine receptors were obtained by screening a mouse brain cDNA library with the use of PCR-derived probes. Mouse bone marrow-derived mast cell cDNA libraries were constructed and screened with the use of A1 and A2a cDNA probes, which revealed the presence of A2a, but not A1, receptor clones. A putative A2b receptor was identified by using low stringency mast cell library screening. Northern blotting of mast cell poly(A)+ RNA with the use of receptor subtype probes labeled single mRNA bands of 2.4 kb and 1.8 kb for the A2a and A2b receptors, respectively. In situ cells. An A2a receptor-specific agonist failed to enhance mast cell mediator release, which suggests that the secretory process is modulated through the A2b and/or another receptor subtype. By using RNase protection assays, we found that mast cells that had been cultured in the presence of N-ethylcarboxamidoadenosine for 24 h exhibited a decrease in both A2a and A2b receptor RNA levels. Cells that had been cultured for 1 to 2 days in the presence of dexamethasone demonstrated increased amounts of A2a receptor mRNA, but no identifiable change in A2b receptor mRNA. Mast cells possess at least two adenosine receptor subtypes that may be differentially regulated.
The major surface glycoprotein (gp63) from Leishmania major and Leishmania donovani cleaves CD4 molecules on human T cells.
Authors: Hey A S , Theander T G , Hviid L , Hazrati S M , Kemp M , Kharazmi A
Published: December 2022

The effect of Leishmania major and L. donovani surface protease gp63 on surface markers on human T cells was studied using fluorescence-activated flow cytometry. Purified gp63 (63,000 m.w. glycoprotein) at concentrations above 10 micrograms/ml completely inhibited binding of six different anti-CD4 Abs to human T cells, whereas the binding of one Ab, OKT4, was not inhibited. Heat inactivation of the protease before the incubation with cells abolished the effect on binding of anti-CD4 Abs. Cells incubated for 2 h with the protease and subsequently washed free of the protease showed a gradual re-expression of CD4, reaching 50% of the initial level after 72 h of incubation in medium. Preincubation of cells with live promastigotes showed an inhibitory effect on CD4 comparable to that seen with purified gp63. The binding of Abs directed against other surface markers present on human T-cells--CD2, CD3, CD5, CD8, CD11A, CD25, CD45RO, CD45RA, CD58, TCR-alpha, TCR-gamma, and HLA DQ--was not inhibited by gp63. These data suggest that gp63, both in its purified form and in the form anchored to the parasite membrane, cleaves CD4 on human T cells. The cleavage of CD4 by the protease might play a role in interfering with the induction of the immune response and thus disease progression in Leishmania infections.
The resolution of acute malaria in a definitive model of B cell deficiency, the JHD mouse.
Authors: van der Heyde H C , Huszar D , Woodhouse C , Manning D D , Weidanz W P
Published: December 2022

Because the role of cell-mediated immunity (CMI) in the resolution of blood-stage malaria remains unclear, we examined the question of whether mice completely lacking Ab-mediated immunity (AMI) but possessing some CMI can resolve experimental malaria previously reported not to require AMI for resolution. Severe combined immunodeficient mice reconstituted with enriched immune T cells (< 0.5% B220+ cells) suppressed acute Plasmodium chabaudi adami parasitemia, suggesting that T, but not B, cells are required to clear this form of malaria. In addition, JHD mice, which are a definitive model of B cell deficiency, were also shown to resolve P. chabaudi adami, Plasmodium vinckei petteri and Plasmodium chabaudi chabaudi malaria. These observations collectively establish that CMI alone can mediate the clearance of acute malaria caused by these subspecies of Plasmodium. Moreover, the protective cell-mediated immune response involved depends upon CD4+ T cells because JHD mice treated with anti-CD4 mAb do not resolve their infections. These results suggest that evaluation of immunization regimens to activate CD4+ T cell dependent cell mediated immunity against Plasmodium falciparum may be appropriate.
Mast cells augment lesion size and persistence during experimental Leishmania major infection in the mouse.
Authors: Wershil B K , Theodos C M , Galli S J , Titus R G
Published: December 2022

Mast cells are a source of a variety of cytokines that may influence the host response to Leishmania major. To investigate the role of mast cells during L. major infection, we performed a morphometric analysis of mast cells at cutaneous sites in resistant C57BL/6 mice and susceptible BALB/c mice injected with L. major. Extensive dermal mast cell degranulation was found at sites of L. major infection in both strains of mice. We also examined the course of L. major infection in genetically mast cell-deficient WBB6F1-W/Wv or WCB6F1-Sl/Sld mice, their respective congenic normal (WBB6F1-(+/+) or WCB6F1-(+/+)) littermates, and WBB6F1-W/Wv mice that had been selectively and locally repaired of their cutaneous mast cell deficiency. We found that mast cells significantly augmented the intensity and maximal size of the cutaneous lesions at sites of L. major infection, and in some cases substantially prolonged the persistence of the reactions. However, the lesions ultimately resolved in both the mast cell-deficient and the congenic normal mice. In addition, the presence or absence of mast cells had little or no effect on the numbers of viable parasites recovered from the cutaneous lesions. Moreover, mast cell-deficient W/Wv mice and the congenic normal (+/+) mice produced similar levels of IFN-gamma mRNA in lymph nodes draining the cutaneous lesions whereas no IL-4 mRNA was detectable. Taken together, these data suggest that mast cells significantly augment the size of cutaneous lesions during L. major infection in mice. However, mast cells do not appear to influence significantly either the parasite burden or the ultimate resolution of the infection.
Induction of vascular addressins and adhesion molecules in the pancreas of IFN-gamma transgenic mice.
Authors: Lee M S , Sarvetnick N
Published: December 2022

"Vascular addressins" play an important role in lymphocyte homing to chronic inflammatory areas. However, it is not yet known which cytokines induce vascular addressins and other adhesion molecules in vivo. Because IFN-gamma induces adhesion molecules in vitro, we tested the ability of IFN-gamma to induce vascular addressins and other adhesion molecules in vivo employing a transgenic mice model that expresses IFN-gamma in pancreatic beta-cells and develops insulitis (Ins-IFN-gamma mice). Two vascular addressins were induced in the transgenic pancreata. The first was MadCAM-1, a mucosal vascular addressin recognized by MECA-367 and required for lymphocyte homing to Peyer's patches. The second was a peripheral lymph node vascular addressin recognized by MECA-79 and required for homing to peripheral nodes. ICAM-1 was expressed on ductal epithelial cells, endothelial cells, and the majority of infiltrating lymphocytes as well as its ligand LFA-1. Lymphocyte-deficient transgenic mice were also induced to express vascular addressins and ICAM-1, suggesting that probably IFN-gamma itself stimulated the expression of those molecules, not cytokines secreted from infiltrating lymphocytes. LPAM and L-selectin, potential counter-receptors for vascular addressins, were expressed on infiltrating lymphocytes. These results underscore the importance of IFN-gamma as an inducer of vascular addressins in vivo.
Acquired C1 inhibitor deficiency as a result of an autoantibody to the reactive center region of C1 inhibitor.
Authors: Mandle R , Baron C , Roux E , Sundel R , Gelfand J , Aulak K , Davis A E , Rosen F S , Bing D H
Published: December 2022

An autoantibody that we hypothesize to react with the reactive center of the plasma serine proteinase inhibitor, C1 inhibitor (C1INH), has been found in a patient with acquired C1INH deficiency. The Ab blocks the ability of C1INH to inhibit the hydrolysis of N-carbobenzyloxy-L-lysine thiobenzylester by purified C1s. A cryoprecipitate from the patient's plasma as well as the Ig fraction were able to block C1INH inhibition of C1s. The immunoaffinity purified Ab to C1INH from the patient's plasma Ig fraction neutralizes the inhibitory activity of C1INH in a dose-dependent manner and blocks the ability of normal C1INH to form a complex with C1s. The neutralizing activity of the purified Ab is reversed by a synthetic peptide that corresponds to the amino acid sequence in the P1 to P15 positions of the reactive center of C1INH but not by a 34-amino-acid trypsin peptide or 37-amino-acid elastase peptide derived from the C-terminus of C1INH. Western blot analysis indicated that the Ab is an oligoclonal Ig with kappa light chains.
A single amino acid substitution in core residues of S-antigen prevents experimental autoimmune uveitis.
Authors: Singh D P , Kikuchi T , Singh V K , Shinohara T
Published: December 2022

We have previously reported that microbial Ags, having a three- to six-amino-acid-sequence homology with a uveitopathogenic epitope (peptide M) of retinal soluble protein (S-Ag), induce experimental autoimmune uveitis (EAU). Another uveitopathogenic epitope (peptide G) of S-Ag also was characterized. In addition, we have characterized the core sequences by truncating peptides G and M from amino acid and carboxyl termini. In this study, we have further defined the core sequences using synthetic octapeptides with a single amino acid substitution. In addition, the analogues of peptides Gm5 or Mm4 are capable of inhibiting the proliferative response of T-lymphocytes from rats immunized with peptides G-8 or M-8, respectively. Co-injection of a pathogenic peptide with nonpathogenic substitution analogues blocked the induction of EAU. These results suggest that specific nonpathogenic analogues with single amino acid substitution derived from pathogenic peptides have potential for prevention and therapy of autoimmune diseases.
Transcriptional regulation of CD6 expression on human T lymphocytes by phorbol ester.
Authors: Bott C M , Doshi J B , Li L L , McMurtry S A , Sanders J L , Fox D A
Published: December 2022

Activation of protein kinase C (PKC) has emerged as a major common signal-transducing mechanism for T cell activation and regulation of expression of T cell surface glycoproteins. Surface expression of the CD6 Ag is known to increase with T cell activation and CD6 itself may be involved in regulation of T cell activation. Therefore, we performed experiments to determine whether activation of PKC by phorbol ester induced an increase in CD6 expression and to investigate the mechanisms of such an effect. CD6 surface expression was up-regulated substantially in response to PMA on both mature and immature T cells, but only negligibly on B cell lines. This increase was blocked by PKC inhibitors. The PMA-induced increase in CD6 surface expression was accompanied by an increase in total CD6 protein, as detected by using Western blot analysis of whole cell lysates. After PMA stimulation, Northern blot analysis showed that steady state levels of CD6 mRNA increased in response to PMA treatment. This increase was blocked by cycloheximide, demonstrating that it was dependent on new protein synthesis. Nuclear run-on analysis showed that the rate of CD6 mRNA transcription increased by approximately two to threefold after PMA stimulation of Jurkat cells. Experiments in which we used actinomycin D showed that PMA had no significant effect on the t1/2 of CD6 mRNA. The data suggest that the effect of PMA on CD6 expression is mediated primarily by an increase in CD6 mRNA transcription after PKC activation. mAbs were used to determine whether augmented CD6 expression could be induced by perturbation of specific T cell surface molecules. Up-regulation of CD6 expression occurred when thymocytes were cultured with anti-CD2 Abs, but not with Abs to other functional T cell surface structures, and not when mature T cells were cultured with the anti-CD2 mAbs. Up-regulation of CD6 expression by activation of PKC, triggered in thymocytes by ligation of CD2, could allow CD6 to provide additional regulatory signals required for events in later stages of T cell activation and differentiation.
Human T cell-dependent B cell differentiation induced by staphylococcal superantigens.
Authors: Stohl W , Elliott J E , Linsley P S
Published: December 2022

Microbial superantigens (SAgs), by virtue of their binding to TCR V beta elements on T cells and to class II MHC molecules on accessory cells (AC), trigger T cell activation. Although anti-CD3 mAb (which also trigger T cell activation via surface CD3/TCR) can readily induce T cell-dependent B cell differentiation in unmanipulated PBMC cultures, induction of Ig production in SAg-stimulated cultures has usually required special manipulation of the T cells, such as irradiating them or treating them with mitomycin C. We now demonstrate that eight different staphylococcal SAgs, typically at concentrations 10- to 100-fold lower than those required for proliferation, can each trigger unmanipulated peripheral blood and tonsil T cells to drive polyclonal B cell differentiation. Such SAg-induced T cell-dependent generation of Ig-secreting cells (IgSC) requires T cells and B cells only and occurs in the absence of monocytes as long as there are adequate numbers of B cells to serve as (DR+) AC. Physical contact among T cells, responder B cells, and AC (when different from the responder B cells) is required. The fusion protein CTLA4Ig inhibits SAg-induced IgSC generation in a dose-dependent fashion, whereas a control fusion protein has no such effect. In contrast, CTLA4Ig has, at best, only modest effects on SAg-induced T cell proliferation, indicating that CD28 (CTLA4)/B7 (B7-like) interactions play a more prominent role in SAg-induced IgSC generation than in SAg-induced T cell proliferation. These results establish SAg-induced T cell-dependent B cell differentiation as a useful model for T cell/B cell interactions, inasmuch as no other cell types are necessary for successful B cell differentiation; these results also demonstrate the importance of CD28 (CTLA4)/B7 (B7-like)-dependent mechanisms in this process.
IL-4- and IL-5-dependent protective immunity to Onchocerca volvulus infective larvae in BALB/cBYJ mice.
Authors: Lange A M , Yutanawiboonchai W , Scott P , Abraham D
Published: December 2022

A significant reduction in challenge worm survival occurred when BALB/cBYJ mice were vaccinated against Onchocerca volvulus infective third stage larvae (L3) by using irradiated O. volvulus L3. Challenge infections consisted of L3 implanted in diffusion chambers, which were used as a means to contain, and thus efficiently recover, the larvae from the host. The goal of the present study was to describe the mechanism of immune-mediated killing of O. volvulus L3 in diffusion chambers in mice. Direct contact between host cells and parasites was required for killing of larvae in immunized hosts. To define the mechanism of immune-mediated killing in this system, the time of influx of cells and cytokines into the infection site was compared with the time challenge infections were killed. The only cell type that was found to increase in diffusion chambers in immunized mice was eosinophils; maximal levels of eosinophils were coincident with the time of parasite killing. IL-5 was found in diffusion chambers of immunized mice coincident with the time of parasite killing; IL-5 was not found in diffusion chambers recovered from control mice. Significant levels of IFN-gamma were absent in the diffusion chambers of both groups. Immunized mice were treated with mAb to eliminate IL-5 or IL-4 to assess the role these cytokines or their by-products play in larval killing. Elimination of either IL-5 or IL-4 significantly reduced the protective effects of vaccination against larval O. volvulus.
Treatment of mice with IL-1 before infection increases resistance to a lethal challenge with Salmonella typhimurium. The effect correlates with the resistance allele at the Ity locus.
Authors: Morrissey P J , Charrier K
Published: December 2022

Administration of a single dose of IL-1 alpha to various strains of mice approximately 16 h before a lethal infection with Salmonella typhimurium resulted in a significant augmentation of survival in most Ityr, but in no Itys strains of mice. Lower numbers of bacteria were observed in the liver and spleen in response to IL-1 pretreatment shortly after infection in all Ityr strains of mice tested, including the congenic C.D2-Ityr mice. Treatment with IL-1 alpha after infection had no effect on survival in either Ityr or Itys mice. A combination of IL-1 alpha pretreatment with IL-1 alpha post-treatment did not increase survival over the effect of IL-1 alpha pretreatment alone in Ityr mice and did not increase the survival of the Itys mice. The combination of IL-1 alpha pretreatment with GM-CSF post-treatment was effective in Ityr but not in Itys strains of mice. Thus, IL-1 alpha pretreatment enhances the resistance of Ityr, but not Itys strains of mice to a lethal challenge with S. typhimurium by up-regulating antibacterial mechanisms shortly after infection.
IL-1 down-regulates platelet-derived growth factor-alpha receptor gene expression at the transcriptional level in human osteoblastic cells.
Authors: Xie J F , Stroumza J , Graves D T
Published: December 2022

Regulation of the platelet-derived growth factor (PDGF)-alpha receptor is thought to play an important role in pathophysiologic processes. Previously, we have reported that IL-1 has the potential to regulate PDGF-induced biologic activity in both normal human osteoblastic cells and the human osteoblastic cell line, MG-63, by decreasing the expression of PDGF-alpha receptor mRNA. In the present studies, we analyzed the effects of IL-1 on transcription rates and the stability of PDGF-alpha receptor mRNA in MG-63 cells. The data indicate that the t1/2 of PDGF-alpha receptor mRNA is approximately 3.3 h after incubation with the RNA II polymerase transcription inhibitor 5,6-dichloro-1 beta-D-ribofuranosylbenzimidazole (DRB). Approximately the same t1/2 (3.1 h) was obtained when osteoblastic cells were incubated with IL-1. The t1/2 for PDGF-alpha receptor mRNA for cells incubated with both IL-1 and DRB was 3 h. This finding suggests that the levels of PDGF-alpha receptor mRNA transcripts are not regulated by post-transcriptional mechanisms. Results of nuclear run-on analysis were consistent with this conclusion, demonstrating that IL-1 modulates PDGF-alpha receptor gene expression at the transcriptional level. Surprisingly, incubation of cells with cycloheximide also caused down-regulation of PDGF-alpha receptor mRNA, which suggests that synthesis of a labile factor is necessary for constitutive expression. The functional consequence of down-regulation of PDGF-alpha receptors by IL-1 was also assessed. By using chemotaxis assays, we demonstrated that IL-1 significantly inhibited PDGF-AA-mediated migration in human MG-63 osteoblastic sarcoma cells.
Costimulation of tumor-reactive CD4+ and CD8+ T lymphocytes by B7, a natural ligand for CD28, can be used to treat established mouse melanoma.
Authors: Li Y , McGowan P , Hellström I , Hellström K E , Chen L
Published: December 2022

Interactions between the costimulatory molecule B7 on APC and its counter-receptor CD28 on T lymphocytes play a key role in the induction of cell-mediated immune responses. We studied the role of costimulation of tumor-reactive T cells by B7 in the immune destruction of the K1735-M2 mouse melanoma into which the gene encoding the human melanoma-associated Ag, p97, had been transfected. Previous work has demonstrated that the p97 transfectant cl62 is immunogenic but still grows progressively in immunocompetent C3H/HeN mice and that adoptive transfer of p97-specific CD4+ T cells can induce the regression of small established cl62 tumors metastatic to the lungs. We have now shown that expression of B7 in cl62 after retroviral-mediated gene transfer eliminated its ability to grow in immunocompetent mice but not in T cell-deficient nude mice. Mice immunized with B7-transduced p97+ cells had an increased activity of both CD4+ T cells, which could proliferate in response to the p97 Ag, and CD8+ CTL, which could lyse a broad spectrum of cultured syngeneic p97+ and p97- tumor lines but not allogeneic tumor lines or syngeneic lymphoblasts. Both CD4+ and CD8+ T cell subsets were required for tumor rejection, and the depletion of CD4+ T cells in vivo decreased the tumoricidal activity of CD8+ CTL. Treatment of mice bearing an 8-day established s.c. cl62 melanoma by i.p. injection of B7+ cells from 2A, a highly immunogenic p97 transfectant, resulted in complete tumor regression and cure, injection of B7- 2A cells did not. The therapeutic effect was specific for the cl62 tumor. Our results demonstrate that costimulation by B7 can amplify both CD4+ and CD8+ T cell responses against small tumors toward therapeutic benefit.
CD45-mediated regulation of extracellular calcium influx in a CD4-transfected human T cell line.
Authors: Mittler R S , Schieven G L , Dubois P M , Klussman K , O'Connell M P , Kiener P A , Herndon V
Published: December 2022

Transfection of a CD4- Jurkat leukemic T cell line with the human wild-type CD4 gene resulted in the constitutive mobilization of calcium by these cells. The altered calcium phenotype was dependent on expression of a completely functional CD4 molecule on the cell surface. Transfectants receiving the vector alone or those in which a mutated CD4 gene lacking a functional Lck binding region failed to generate a constitutive calcium response. In addition, CD4 wild-type transfectants over time lost CD4 expression. These CD4- revertants failed to constitutively mobilize calcium. Treatment of CD4 wild-type transfected cells with either anti-CD45 mAb, EGTA, or PMA rapidly restored the cells to basal levels of intracellular calcium. Analysis of CD45 cross-linking on CD4+ and CD4- normal Jurkat lines demonstrated that CD4 expression was required for CD45-mediated inhibition of TCR induced calcium responses. CD45-mediated inhibition affected the duration of the response rather than its magnitude. These results, taken together with the observations obtained with CD4 transfected Jurkats suggested that influx of extracellular calcium was the predominant reason for the elevated levels of calcium within the cell. In support of this hypothesis, we could find no evidence of phosphorylated phospholipase C gamma 1 or constitutive inositol 1,4,5 trisphosphate generation in the CD4 wild-type transfected cells, yet both were detectable after anti-CD3 mAb-induced activation. Immunokinase assays of Lck and Fyn precipitated from untreated or anti-CD45-treated wild-type transfectants demonstrated that CD45 cross-linking dephosphorylated Lck and reduced its capacity to phosphorylate enolase. In contrast, neither Fyn autophosphorylation nor its activity was affected by CD45 cross-linking.
Leishmania major-parasitized macrophages augment Th2-type T cell activation.
Authors: Chakkalath H R , Titus R G
Published: December 2022

We studied the ability of resident peritoneal M phi, parasitized in vitro with Leishmania major, to present Ag to three Th2-type T cell clones that are specific for non-leishmanial Ags. Results indicated that L. major-infected M phi enhanced the proliferation and IL-4 secretion of Th2 T cells in response to stimulation with their cognate Ag. Augmentation of Th2 T cell proliferation was seen in Ag-driven responses but not in mitogenic Con A stimulation. Furthermore, live L. major promastigotes and amastigotes, but not killed parasites, augmented the Th2 T cell response. To delineate the augmentative effect of L. major-infected M phi for Th2 T cell activation, we analyzed the cytokines produced by infected M phi in the presence or absence of Th2 T cell clones. The supernatants contained IL-1 but not IL-6 or IL-10. Interestingly, addition of a neutralizing anti-IL-1 alpha mAb to the cultures reduced the augmentative effect for Th2 proliferation. Moreover, additional experiments showed that IL-1 alpha could substitute for L. major and enhance T cell activation in cultures consisting of Th2 cells, normal M phi, and Ag. Thus, our study shows that L. major-infected M phi present Ags in a manner that augments Th2 T cell proliferation and IL-4 synthesis, and that the phenomenon is at least in part mediated by IL-1 secreted by the infected M phi. For purposes of comparison, two non-leishmanial Ag specific Th1-type T cell clones were stimulated with L. major-infected M phi. Our data, as already reported by others, showed that infected M phi inhibited the response of Th1 T cells to their cognate Ag. Taken together, this report shows that leishmanial-infected M phi favor the activation of Th2 T cells over Th1 cells and that Th1 and Th2 cells require distinct activation signals from M phi.
Enhancement of intracellular replication of Toxoplasma gondii by IL-6. Interactions with IFN-gamma and TNF-alpha.
Authors: Beaman M H , Hunter C A , Remington J S
Published: December 2022

Toxoplasma gondii is a major pathogen of immunocompromised hosts, and one defense mechanism against the parasite is activation of macrophages (M phi) for toxoplasmacidal activity by IFN-gamma after triggering by TNF-alpha. IL-6, IFN-gamma, and TNF-alpha are cytokines involved in inflammatory responses and are induced by T. gondii infection. We studied the interaction of these three cytokines using an in vitro model of T. gondii infection. Pretreatment (but not post-treatment) of unelicited murine peritoneal M phi with IL-6 enhanced T. gondii replication in a dose-dependent manner. Pretreatment with IFN-gamma resulted in active killing of parasites whereas the addition of IL-6 to IFN-gamma pretreatment resulted in a reversal of IFN-gamma-mediated toxoplasmacidal activity. Combining TNF-alpha with IL-6 and IFN-gamma pretreatment resulted in restoration of toxoplasmacidal activity. Addition of a polyclonal anti-TNF-alpha Ab to IL-6 and IFN-gamma pretreatment resulted in enhancement in the IL-6-mediated impairment of IFN-gamma function. These data taken together suggest that IL-6 enhances intracellular replication of T. gondii and reverses IFN-gamma mediated activation of murine peritoneal M phi, and that certain of the interactions between these two cytokines may be at the level of TNF-alpha triggering.
Biologic activities of the murine beta-chemokine TCA3.
Authors: Luo Y , Laning J , Devi S , Mak J , Schall T J , Dorf M E
Published: December 2022

The murine beta-chemokine TCA3 was purified to homogeneity. The biologic activities of the purified glycoprotein were evaluated in vivo and in vitro. Mice injected i.p. with 1- to 100-ng purified rTCA3 exhibited a rapid influx of neutrophils and macrophages. Increased numbers of neutrophils and monocytes were observed in peripheral blood within 15 min and peak at 45 min. After 45 min neutrophil and macrophage levels were increased in the peritoneal exudate with peak levels occurring at 2 h, followed by a subsequent decline by 24 h. Inflammatory responses were induced in a dose-dependent fashion. The in vivo inflammatory responses were mirrored by the pattern of TCA3-induced chemotaxis in vitro. Neutrophils and macrophages responded to similar concentrations of TCA3 (3 x 10(-9) to 10(-8) M). Lymph node cells responded to other chemokines but did not migrate to TCA3. We also demonstrated that rTCA3 stimulates a transient increase in cytoplasmic free calcium in monocytic cells through a PTX-sensitive pathway. Cross-desensitization studies indicate that TCA3 acts independently of other beta-chemokines (MIP-1 alpha and RANTES) and the alpha-chemokine IL-8. Furthermore, TCA3 does not induce a Ca2 lux in cells transfected with cDNA for the C-C CKR-1 chemokine receptor, supporting the conclusion that there are distinct receptors for TCA3.