bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of heavy chain residues in a humanized anti-CD3 antibody important for efficient antigen binding and T cell activation.
Authors: Zhu Z , Carter P
Published: December 2022

A previously identified humanized anti-CD3 Ab variant, v9, binds T cells with > 100-fold higher efficiency than the original variant, v1, and almost as efficiently as a chimeric molecule containing corresponding murine variable domains. Variants v1 and v9 differ at six positions in the H chain second CDR. Here a mutational analysis was used to identify which of these six replacements are primarily responsible for the difference in binding efficiency. These anti-CD3 variants were used to probe the relationship between Ag binding efficiency and potency in stimulating T cell proliferation. The human to mouse mutations T57S and V63F increase the binding efficiency of variant v1 for T cells by 8- and 12-fold, respectively, and together in variant M18 enhance binding by 26-fold to within 4-fold of variant v9. A framework mutation, 169L, was identified that enhances the binding of variants v1 and M18 by 14- and 3-fold, respectively. The Ag binding efficiencies of anti-CD3 variants correlate directly with their potencies in stimulating the proliferative activity of both resting human PBMC and IL-2-activated human T lymphocytes. Humanized variant v9 is equipotent to the murine parent Ab in stimulating ATL activity. PBMC activated by variants v1 and v9 IgG in a short term culture are equally cytotoxic against human breast carcinoma cells. Thus, high efficiency Ag binding by anti-CD3 variants is important for stimulating efficient T cell proliferation, but not cytotoxicity, in vitro.
The Ig heavy chain switch region is a hotspot for insertion of transfected DNA.
Authors: Baar J , Shulman M J
Published: December 2022

The Ig heavy chain class switch usually occurs by breaking and rejoining DNA in the switch (S) regions, which consist of tandemly repeated sequences 5' of the constant region exons. Various studies have suggested that S DNA can also recombine with non-S sequences. To measure the frequency of such recombination events, the hybridoma cell line igm692, a deletion mutant that lacks the C mu 1 and C mu 2 exons and the 3' end of the S mu region, was transfected with a fragment bearing the C mu 1-2 exons, but no S mu DNA. Insertion of this fragment into the residual VDJ-C mu intron of igm692 can restore a functional mu gene, yielding a transformant that is detected as a plaque-forming cell (PFC). PFC comprise approximately 8 x 10(-7) of the surviving transfected cells. In 10 of 12 PFCs, the C mu 1-2 fragment inserted into the 2.5-kb residual S mu region, whereas insertion in two cases occurred in the 3.5-kb segment 5' of S mu. Using a PCR assay to measure the frequency of insertion of the transferred fragment elsewhere in the hybridoma genome, we found that approximately 9% of the surviving transfected cells had stably acquired the C mu 1-2 fragment. These results indicate that the S mu region is approximately 100-fold more recombinogenic than the average genomic site, and approximately 7-fold more recombinogenic than the non-S mu segment of the residual VDJ-C mu, i.e., the S mu region is a hotspot for insertion of transfected DNA.
Bacterial infection of the testis leading to autoaggressive immunity triggers apparently opposed responses of alpha beta and gamma delta T cells.
Authors: Mukasa A , Hiromatsu K , Matsuzaki G , O'Brien R , Born W , Nomoto K
Published: December 2022

The mechanisms that lead to the breakdown of self-tolerance and testis-specific immune reactivity in the murine orchitis model are understood only in part. We investigated the histopathologic and immunologic consequences of a unilateral bacterial (Listeria monocytogenes) infection of the testis. Both infected and contralateral sides of this bilateral organ suffered severe inflammatory responses despite a conspicuous absence of bacteria in the contralateral tissue. Also, in both testicles, T cell populations increased, involving both alpha beta and gamma delta T cell subsets. Concomitant with the bilateral orchitis, testis-specific delayed type hypersensitivity and Ab responses developed. Ab depletion experiments indicated that in this orchitis model, as in others, alpha beta T cells are initiators of the autoaggressive reactivity. In contrast, Ab depletion of gamma delta T cells accelerated the inflammatory response in both testicles, suggesting a regulatory role for this type of T cells in both infection-induced and autoimmune orchitis.
Mice with the xid B cell defect are less susceptible to developing Staphylococcus aureus-induced arthritis.
Authors: Zhao Y X , Abdelnour A , Holmdahl R , Tarkowski A
Published: December 2022

To investigate the role of B cells in the development of experimental Staphylococcus aureus-induced arthritis, we used X-linked immunodeficiency (xid) mice that carry a Bruton's tyrosine kinase mutation affecting the function of B cells. NFR/N.xid and congenic NFR/N mice were inoculated i.v. with a toxic syndrome toxin-1 producing S. aureus LS-1 strain. B cell-deficient NFR/N.xid mice developed less frequent (p < 0.01) and less severe (p < 0.01) arthritis than NFR/N mice did. These clinical findings were corroborated by histopathologic evaluation, indicating that NFR/N.xid mice had significantly lower (p < 0.01) erosivity of the disease. Interestingly, infected NFR/N.xid mice showed decreased bacterial burden in blood, joints, and other organs compared with the control mice. Serologic studies displayed poor B cell responses to staphylococcal cell walls, toxic shock syndrome toxin-1, and ssDNA, accompanied by a low level of Igs in infected NFR/N.xid mice. More importantly, xid defect affected cytokine profile. The in vitro experiments showed that the lymphocytes from NFR/N.xid mice had low IL-6, but high IFN-gamma production upon stimulation with staphylococcal cell walls compared with NFR/N mice. Furthermore, the in situ hybridization technique revealed the relative increase of IFN-gamma, but marked decrease of IL-1 beta mRNA expression in spleens of infected NFR/N.xid mice. No significant difference in IL-4, IL-10, and TNF-alpha mRNA expression was found between both strains. Our findings demonstrate that B cells may, directly or indirectly, contribute to the pathogenesis of septic arthritis. The results indicate that increased IFN-gamma production along with low IL-6 and IL-1 beta synthesis found in xid mice may provide a more favorable outcome of S. aureus arthritis.
Endotoxin receptors (CD14) are found with CD16 (Fc gamma RIII) in an intracellular compartment of neutrophils that contains alkaline phosphatase.
Authors: Detmers P A , Zhou D , Powell D , Lichenstein H , Kelley M , Pironkova R
Published: December 2022

CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein on the surfaces of monocytes and polymorphonuclear leukocytes (PMN) that binds and initiates cellular responses to bacterial LPS. PMN also contain an intracellular pool of CD14 that can be deployed rapidly to the cell surface in response to stimulation with a variety of agonists. To determine which of the well-characterized subcellular compartments of PMN contains CD14, cells were cavitated and fractionated on Percoll gradients. The gradient fractions were assayed for CD14 by ELISA and Western blot and for the marker proteins beta-glucuronidase (azurophil granules), vitamin B12 binding protein (specific granules), alkaline phosphatase (secretory vesicles and plasma membrane), and HLA (plasma membrane). Approximately one-half of the CD14 ran with plasma membrane fractions and one-half with intracellular membranes of light density. Both intracellular and cell surface CD14 were associated tightly with membrane, and both forms showed identical electrophoretic mobility. The intracellular CD14 was clearly not present in azurophil granules or specific granules, but ran precisely with alkaline phosphatase, a marker for secretory vesicles. Parallel studies showed that an additional GPI-linked protein, Fc gamma RIII (CD16), also fractionated precisely with CD14 and alkaline phosphatase. Association of CD14 with secretory vesicles were confirmed by studies on cells stimulated with the formyl peptide fNLLP for 20 min at 37 degrees C before fractionation. This treatment caused translocation of CD14 from intracellular fractions to plasma membrane fractions. No release of the specific granule marker vitamin B12 binding protein was observed under these conditions, whereas two other GPI-anchored proteins, alkaline phosphatase and CD16, moved coincidentally with CD14 to comigrate with the plasma membrane. Time course studies of CD14 and CD16 surface expression confirmed the rapid and coordinate up-regulation of these proteins. Thus, the intracellular compartment containing CD14 and CD16 had the properties of secretory vesicles. These vesicles may represent a specialized membrane domain of PMN enriched in GPI-anchored proteins.
Expression of Bcl-2, Bcl-x, and Bax after T cell activation and IL-2 withdrawal.
Authors: Broome H E , Dargan C M , Krajewski S , Reed J C
Published: December 2022

Bcl-2, bcl-x, and bax genes code for proteins that affect the susceptibility of cells to apoptosis. In general, the expression of bcl-2 or bcl-x inhibits apoptosis while bax promotes apoptosis. We examined the levels of these proteins by immunoblotting in resting and activated T cells and in thymocytes. Bcl-2 and Bax proteins vary coordinately, but Bcl-x varies independently: Bcl-2 and Bax are higher in splenic T cells than in thymocytes, and their levels increase even more after T cell activation. In contrast, Bcl-x is almost undetectable in splenic T cells but is manyfold greater in thymocytes and in activated splenic T cells. When CTLL-2 cells or activated T cells are starved of IL (IL-2), the level of Bcl-x but not Bcl-2 protein drops before the onset of apoptosis. Stable transfection of either bcl-2 or bcl-x expression plasmids promotes the survival of CTLL-2 cells in the setting of IL-2 withdrawal. Over 70 to 90% of the transfected cells remain viable at 48 h after IL-2 withdrawal when all of the control transfected cells are apoptotic. These findings suggest that a decrease in Bcl-x protein levels precedes apoptosis after IL-2 withdrawal in T cells and that transfected bcl-2 promotes survival after IL-2 withdrawal by functionally masking this drop in Bcl-x.
Allelic differences in the VHOx-1 gene explain the absence of a B cell clonal dominance in the primary response of C57BL/6 mice to phthalate.
Authors: Winter D B , Diamond M E , Abu-hadid M , Falkenberg S , Bankert R B
Published: December 2022

A highly conserved Id (CRIXmp-1) associated with the murine (BALB/c) humoral immune response to the hapten phthalate (Xmp) is conspicuously absent in C57BL/6 mice. The absence of this Id in C57BL/6 mice is shown here to be due to the absence of the appropriate VH gene (VHOx-1) usage in the Xmp response. To determine whether the failure to utilize this VH was due to an active suppression or to the lack of the requisite VH gene in the available repertoire, VHOx-1 gene-specific primers were used to amplify the germ-line VHOx-1 gene from genomic DNA from BALB/c and C57BL/6 mice. The germ-line coding sequence of the C57BL/6 allele of the VHOx-1 gene is 99% similar to the germ-line coding sequence of the BALB/c allele. Amplification of cDNA made from splenic RNA from C57BL/6 mice confirmed that this gene is expressed. There are four nucleotide differences that lead to three amino acid changes in the predicted protein sequence. Each change is either in or immediately adjacent to a complementarity-determining region (CDR). Two of these changes are unique to the C57BL/6 allele and are not shared with CRIXmp-1-expressing strains. These two changes are predicted to alter the Xmp binding capabilities of the C57BL/6 allelic form of this VH gene, thereby explaining the absence of the Xmp-1 clonotype, which is dominant in the primary Xmp immune response of most other strains of mice.
Extensive TCR junctional diversity of V gamma 9/V delta 2 clones from human female reproductive tissues.
Authors: Christmas S E , Brew R , Thornton S M , Deniz G , Flanagan B F
Published: December 2022

Panels of gamma delta T cell clones bearing the V gamma 9/V delta 2 form of TCR were derived from human first trimester decidualized endometrium and cervix. Seventy-three percent of these clones expressed the human mucosal lymphocyte Ag HML-1 compared with only 14% of PBL V gamma 9/V delta 2 clones, indicating that most clones were derived from the tissue itself rather than contaminating peripheral blood. All 13 clones isolated expressed V gamma 9JPC gamma 1- and V delta 2(D)J delta 1-encoded receptors; TCR gamma and delta junctional regions from most of these were sequenced and analyzed, together with the TCR-delta junctional region of a sequence obtained from bulk CD3+ decidual leukocytes. There was considerable junctional diversity of both gamma- and delta-chains with a similar extent of germline V and J gene trimming and N-region nucleotide addition to that found in PBL V gamma 9/V delta 2 cells. Eight of eleven TCR-delta junctional sequences contained a strongly hydrophobic amino acid in position 97, as has been found in > 90% o V gamma 9/V delta 2 clones. Thymic V gamma 9/V delta 2 cells show much less junctional diversity and less pronounced selection at residue 97 of the delta-chain. Thus, unlike the mouse, gamma delta T cells from human female reproductive tissues exhibit extensive TCR junctional as well as combinatorial diversity. This suggests that V gamma 9/V delta 2 cells in these human tissues have undergone selective but diverse peripheral expansion in response to antigenic stimuli in a similar manner to those in peripheral blood.
Organization, regulatory sequences, and alternatively spliced transcripts of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) gene.
Authors: Sampaio S O , Li X , Takeuchi M , Mei C , Francke U , Butcher E C , Briskin M J
Published: December 2022

The mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is expressed selectively at venular sites of lymphocyte extravasation into mucosal lymphoid tissues and lamina propria, where it directs local lymphocyte trafficking. MAdCAM-1 is a multifunctional type l transmembrane adhesion molecule comprising two distal lg domains involved in alpha 4 beta 7 integrin binding, a mucin-like region able to display L-selectin-binding carbohydrates, and a membrane-proximal lg domain homologous to lgA. We show in this work that the MAdCAM-1 gene is located on chromosome 10 and contains five exons. The signal peptide and each one of the three lg domains are encoded by a distinct exon, whereas the transmembrane, cytoplasmic tail, and 3'-untranslated region of MAdCAM-1 are combined on a single exon. The mucin-like region and the third lg domain are encoded together on exon 4. An alternatively spliced MAdCAM-1 mRNA is identified that lacks the mucin/lgA-homologous exon 4-encoded sequences. This short variant of MAdCAM-1 may be specialized to support alpha 4 beta 7-dependent adhesion strengthening, independent of carbohydrate-presenting function. Sequences 5' of the transcription start site include tandem nuclear factor-kappa B sites; AP-1, AP-2, and signal peptide-1 binding sites; and an estrogen response element. Our findings reinforce the correspondence between the multidomain structure and versatile functions of this vascular addressin, and suggest an additional level of regulation of carbohydrate-presenting capability, and thus of its importance in lectin-mediated vs alpha 4 beta 7-dependent adhesive events in lymphocyte trafficking.
Complement component C5: engineering of a mutant that is specifically cleaved by the C4-specific C1s protease.
Authors: Ogata R T , Low P J
Published: December 2022

Previous studies showed that simply inserting or substituting a few amino acid residues immediately downstream of the proteolytic activation site in C component C3 renders that site susceptible to the C4-specific C1s protease. This report describes the results of extending those studies to the closely related component C5. We found that small changes, similar to those that made C3 susceptible to C1s, were insufficient to render C5 C1s-sensitive; and neither more extensive substitution downstream of the cleavage site with a 14 residue long segment from C4, nor upstream substitution with an 8 residue long C4 segment gave C1s cleavage. However, substitution of both the upstream and downstream segments gave a hybrid C5 protein, designated ASC4, which was cleaved by C1s. The protease sensitivity of ASC4 was curious, however, in that C1s was more active against the secreted extracellular biosynthetic precursor, pro-ASC4(E) than mature ASC4, whereas a C5-specific convertase cleaved the mature protein but not the precursor. In contrast, both mature and precursor forms of wild-type C5 were cleaved by the C5 convertase, but neither of course is recognized by C1s. These results demonstrate that a mutant C5 molecule can be constructed that is cleaved at the activation site by both C1s and C5 convertase. This suggests that the structures necessary for specific recognition by the two proteases have little or no overlap and that recognition by C5 convertase involves residues that are distant from the activation site itself.
Immunogenic HIV variant peptides that bind to HLA-B8 can fail to stimulate cytotoxic T lymphocyte responses.
Authors: McAdam S , Klenerman P , Tussey L , Rowland-Jones S , Lalloo D , Phillips R , Edwards A , Giangrande P , Brown A L , Gotch F
Published: December 2022

Cytotoxic T lymphocyte responses in HIV infection can be impaired through variation in the epitope regions of viral proteins such as a gag. We report here an analysis of variant epitope peptides in three gag epitopes presented by HLA B8. Fifteen variant peptides were examined for their binding to HLA-B8; all but one bound at concentrations comparable to known epitopes. All except two of those that bound could be recognized by CTL from an HLA-B8 positive HIV-1-infected patient and were therefore immunogenic. However, in a hemophiliac patient studied in detail, there was a failure to respond to two immunogenic peptide epitopes representing virus present as provirus in the patient's peripheral blood. In one case, the patient's CTL had previously responded to the peptide; in the other case, there was a good response to a peptide of closely related sequence. Thus there was a selective failure of the CTL response to some proviral epitopes. This impaired reaction to new variants could contribute to the loss of immune control of the infection.
Reversal of acute experimental autoimmune encephalomyelitis and prevention of relapses by treatment with a myelin basic protein peptide analogue modified to form long-lived peptide-MHC complexes.
Authors: Samson M F , Smilek D E
Published: December 2022

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease induced by immunization with myelin basic protein (MBP), proteolipid protein, or encephalitogenic peptides from these myelin components. EAE resembles basic protein multiple sclerosis in some of its clinical and histologic features, and serves as an experimental model for this and other autoimmune diseases. In this study, we examine i.v. peptide therapy of EAE in detail, and show that repeated i.v. injections of MBP peptides effectively treat EAE in (PLJxSJL)F1 mice. In this study, administration of the immunodominant epitope (MBP Ac1-11) prevents MBP-induced disease, whereas the subdominant epitope MBP 31-47 is neither required nor sufficient. Intravenous administration of substituted MBP peptide analogues is also effective in treating EAE, provided the peptide side chains presumed to be involved in TCR contact and MHC binding are preserved. A substituted MBP peptide analogue that forms long-lived peptide-MHC complexes in vivo is more effective than the unmodified MBP peptide. Lower doses of the substituted peptide analogue are effective, and the effect is longer lasting than treatment with the unmodified peptide. Clinical signs of EAE are reversed by injection of the substituted peptide during the acute phase of disease. Moreover, treatment of mice in the remission phase of EAE results in a dramatically reduced incidence of relapse. In summary, we have shown that EAE can be reversed after onset and treated during remission with an MBP peptide analogue that has been modified for improved therapeutic potency.
The IFN pregnancy recognition hormone IFN-tau blocks both development and superantigen reactivation of experimental allergic encephalomyelitis without associated toxicity.
Authors: Soos J M , Subramaniam P S , Hobeika A C , Schiffenbauer J , Johnson H M
Published: December 2022

Multiple sclerosis (MS) is an inflammatory demyelinating autoimmune disease if the central nervous system (CNS). Recently, the type I IFN, IFN-beta-1b was demonstrated to be a useful immunotherapy for MS. During treatment with IFN-beta-1b, toxicity at higher doses has been observed. IFN-tau, discovered for its role in the reproductive cycle, possesses all of the functions normally ascribed to the type I IFNs but lacks the toxicity normally associate with IFN treatment in vitro. We have examined the effects of IFN-tau treatment on experimental allergic encephalomyelitis (EAE), an animal model useful for the study of MS. EAE is a model of Ag-induced autoimmunity that can be modulated by bacterial superantigen to resemble the relapsing-remitting pattern of autoimmune disease observed in MS. IFN-tau was able to prevent development of EAE as effectively as IFN-beta but without associated toxicity such as lymphocyte suppression and weight loss. In addition, IFN-tau was able to prevent superantigen reactivation of EAE akin to the reduction in disease exacerbations observed in IFN-beta-1b treated MS patients. Mechanisms by which IFN-tau may prevent EAE include reduced proliferation in response to the autoantigen myelin basic protein and reduced TNF-alpha production. Thus, IFN-tau may prove to be a promising new IFN therapy for MS in light of its ability to prevent EAE and the lack of toxicity exhibited by this novel IFN.
Unmasking of anti-ribosomal P autoantibodies in healthy individuals.
Authors: Stafford H A , Anderson C J , Reichlin M
Published: December 2022

Autoantibodies to the ribosomal P proteins (anti-P) are found almost exclusively in a subset of patients with systemic lupus erythematosus. To determine if anti-P autoantibodies are present in healthy individuals but in a masked state, sera from healthy adults were applied to affinity columns coated with ribosomes, and the affinity-purified fractions were analyzed for anti-P Abs by ELISA and immunoblot. Anti-P Abs were detectable in serum only after affinity chromatography, were predominantly of the IgG isotype, stained HEp-2 cells in the characteristic anti-P pattern, and demonstrated specificity for all three ribosomal P phosphoproteins. Using batch affinity chromatography, we identified anti-P autoantibodies in the affinity-purified serum fractions of 54 of 55 healthy individuals. Anti-P Abs from healthy adults generally bound less ribosomal P Ag per mg/ml IgG than anti-P from patients. Inhibition studies revealed that autologous serum contained an IgG inhibitor of anti-P Ab. We conclude that masked anti-P autoantibodies are present in the healthy population and hypothesize that detection of these autoantibodies in systemic lupus erythematosus represents disruption of these regulatory networks.
A comparative study of experimental autoimmune encephalomyelitis in Lewis and DA rats.
Authors: Stepaniak J A , Gould K E , Sun D , Swanborg R H
Published: December 2022

We compared the T cell responses of Lewis (LEW) and DA rats to guinea pig myelin basic protein (MBP), the synthetic peptides corresponding to the epitopes that are encephalitogenic in the LEW strain (MBP73-86, MBP68-86, and MBP87-99), and bovine proteolipid protein (PLP). DA and LEW rats were susceptible to experimental autoimmune encephalomyelitis (EAE) induced with MBP or MBP68-86, but the peptide was less active in DA rats than in intact MBP molecule. MBP73-86 and MBP87-99 induced EAE in LEW rats but not in the DA strain. MBP89-169 was also encephalitogenic in DA rats. Encephalitogenic CDa+ T cell lines and clones derived from MBP-sensitized DA rats secreted IFN-gamma and TNF-alpha and proliferated to MBP and MBP89-169, but not to MBP68-88. However, T cells from MBP68-86-sensitized DA or LEW rats proliferated specifically in an I-A-restricted response to MBP68-86. T cells from MBP87-99-immunized LEW rats responded to MBP87-99 in the context of I-E, whereas the peptide-specific response of MBP87-99 immunized DA rats was I-A-restricted, although FACScan analysis indicated that DA rats express both I-A and I-E. DA rats were also highly susceptible to EAE induced with PLP; 0.6 nmol was Encephalitogenic for DA rats, but did not induce clinical EAE in LEW rats. Although both DA and LEW rats are highly susceptible to EAE, we demonstrate marked differences in the array of myelin epitopes capable of inducing the disease, as well as the MHC restriction of these epitopes, between the two rat strains.
Differential activation of a calcium-dependent endonuclease in human B lymphocytes. Role in ionomycin-induced apoptosis.
Authors: Aagaard-Tillery K M , Jelinek D F
Published: December 2022

The state of B cell maturation profoundly influences the outcome, i.e., activation, growth arrest, or programmed cell death, of a variety of stimuli, including the calcium ionophore, ionomycin. Initial studies confirmed the observation that cell lines representative of immature B cells, i.e., Burkitt lymphoma cell lines, were induced to undergo apoptosis in response to ionomycin, whereas more mature B cell lines did not, and instead underwent cell cycle arrest in the G1 interval. To understand this differential outcome, we have focused on comparing the expression and activation of an endonuclease(s) in cells induced by ionomycin to undergo programmed cell death (Ramos) with cells resistant to ionomycin-induced programmed cell death (Ly1). Our results demonstrated that a low m.w. fraction of an endogenous Ca2+/Mg(2+)-dependent endonuclease was activated in Ramos cells, but not in activated Ly1 cells, following the addition of ionomycin. Of interest, however, low m.w. endogenous endonuclease(s) activity was induced when isolated Ly1 cell nuclei were treated with exogenous calcium instead. Use of field inversion gel electrophoresis further indicated that cleavage of DNA into large m.w. (> 50 kbp) DNA fragments does not precede ionomycin-induced internucleosomal cleavage in Ramos cells or in ionomycin-resistant Ly1 cells. In summary, these data support the conclusion that ionomycin-induced apoptosis involves the activation of a latent, low m.w., calcium-responsive endonuclease and suggest that control of endonuclease depression may contribute to cell-specific regulation of calcium ionophore-induced apoptosis.
Characterization of cyclic adenosine diphosphate-ribose-induced Ca2+ release in T lymphocyte cell lines.
Authors: Guse A H , da Silva C P , Emmrich F , Ashamu G A , Potter B V , Mayr G W
Published: December 2022

Ca2+ release from intracellular stores is one of the major events transducing extracellular signals into living cells. Recently, a metabolite of nicotinamide adenine dinucleotide+ (NAD+), termed "cyclic adenosine diphosphate-ribose" (cADPr), has been described to release Ca2+ from caffeine-sensitive internal stores of cells. Jurkat T cells possess intracellular Ca2+ stores sensitive to caffeine, so a potential involvement of cADPr in Ca2+ signaling was investigated. cADPr released Ca2+ in a dose-dependent manner from intracellular stores of permeabilized Jurkat T cells. Half maximal release was obtained at 2.25 microM cADPr. Prior addition of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) or thapsigargin did not influence cADPr-induced Ca2+ release, indicating the presence of different Ca2+ pools sensitive to Ins(1,4,5)P3 and cADPr. The specificity of the response was confirmed using the inhibitors ruthenium red, 8-NH2-cADPr, and 8-Br-cADPr. All three compounds blocked cADPr-induced, but not Ins(1,4,5)P3-induced, Ca2+ release in a dose-dependent manner. Cyclic GMP (cGMP)-induced Ca2+ release was also partly antagonized by ruthenium red, indicating involvement of a cGMP-dependent step in the formation of cADPr. The presence of endogenous cADPr was analyzed directly by HPLC. Sequential separation on strong anion exchange HPLC and reverse-phase, ion-pair HPLC resulted in a single symmetric peak co-eluting with standard cADPr. The identity of this endogenous material was further confirmed by its ability to release Ca2+ in saponin-permeabilized Jurkat T cells.
A soluble gradient of endogenous monocyte chemoattractant protein-1 promotes the transendothelial migration of monocytes in vitro.
Authors: Randolph G J , Furie M B
Published: December 2022

Chemokines secreted by endothelium may promote diapedesis of leukocytes by a gradient-dependent chemotactic mechanism or by stimulating random motility so that leukocytes transmigrate in a gradient-independent manner. Alternatively, chemokines may bind to endothelium and extracellular matrix to stimulate haptotactic migration. We first analyzed the role of the chemokine monocyte chemoattractant protein-1 (MCP-1) in the migration of human monocytes across untreated or IL-1-stimulated HUVEC monolayers cultured on human amnion. Then we further examined whether MCP-1-dependent transmigration occurred through a chemokinetic, chemotactic, or haptotactic mechanism. A neutralizing mAb against MCP-1 inhibited passage of monocytes across untreated or IL-1-stimulated HUVEC by 74 +/- 3% and 45 +/- 4%, respectively. Addition of MCP-1 itself to the apical compartment of unstimulated HUVEC/amnion cultures also reduced the transmigration of monocytes, in this instance by 73 +/- 9%. MCP-1 suppressed diapedesis only when present above the endothelium at a concentration equal to or greater than that endogenously deposited beneath the endothelium, and its inhibitory action could be overcome by addition of more concentrated MCP-1 below the HUVEC cultures. As much as 90% of the MCP-1 secreted into the underlying basement membrane and connective tissue could be washed out of HUVEC/amnion cultures; this procedure decreased transmigration by 69 +/- 4%. These data indicate that MCP-1 promotes transmigration of monocytes, but only when present in a gradient across endothelial monolayers. They further suggest that this gradient is predominantly soluble, rather than haptotactic.
Intercellular adhesion molecule-2 (CD102) binds to the leukocyte integrin CD11b/CD18 through the A domain.
Authors: Xie J , Li R , Kotovuori P , Vermot-Desroches C , Wijdenes J , Arnaout M A , Nortamo P , Gahmberg C G
Published: December 2022

The interactions between the leukocyte-specific beta 2-integrins cluster of differentiation (CD) Ag CD11/CD18 and their ligands, the intercellular adhesion molecules (ICAMs), play important roles in many adhesion-dependent leukocyte functions. ICAM-1 is known to be a ligand for both CD11a/CD18 and CD11b/CD18. ICAM-2, whose two extracellular Ig domains show the highest homology to the two NH2-terminal domains of ICAM-1, has been previously shown to be a ligand for CD11a/CD18. We recently found that a 22-amino acid CD11a/CD18-binding peptide, P1, derived from the first domain of ICAM-2, also binds to purified CD11b/CD18. In the present study, we demonstrate that the ICAM-2 protein interacts with CD11b/CD18, and the binding is through the CD11b A domain.
Altered target organ. A mechanism of postrecovery resistance to murine autoimmune oophoritis.
Authors: Lou Y H , McElveen F , Adams S , Tung K S
Published: December 2022

Experimental murine autoimmune oophoritis, a model of human premature ovarian failure, is induced by immunization with a peptide of the ZP3 glycoprotein from mouse zona pellucida (ZP3(330-340)) in CFA. The ovarian pathology is mediated by ZP3-specific, CD4+ T cells, and not by Abs. We now show that mice recovered from autoimmune oophoritis in 4 mo, as characterized by regression of ovarian inflammation. Recovery was associated with disease resistance upon rechallenge with ZP3(330-340) in CFA. Oophoritis resistance was not explicable by immunosuppressive effect of CFA priming, nor by suppression of pathogenic T cells. ZP3-specific, proliferative T cell response could be detected, and a ZP3-specific, IFN-gamma-producing pathogenic T cell line was derived readily from the recovered mice by in vitro stimulation with the ZP3(330-340) peptide. Moreover, recovered mice, when challenged with ZP3(330-340) in CFA, produced Abs of IgG class to the ZP3(330-340) peptide. Suppressor T cells are not readily demonstrable. Most importantly, oophoritis occurred in normal ovaries implanted under the renal capsule of the recovered mice. That oophoritis developed in the implanted ovaries, but spared the endogenous ovaries, further indicates that the latter is refractory to oophoritis. Disease resistance of the ovaries is not explicable by limitation of accessible target Ags. When mated, recovered mice were fertile and produced normal litters; and, as recipients of a ZP3-specific T cell line, their ovaries developed oophoritis. We conclude that altered local environment of the target organ following autoimmune disease recovery can contribute to the complex disease-resistant state.
Induction of bcl-x by CD40 engagement rescues sIg-induced apoptosis in murine B cells.
Authors: Wang Z , Karras J G , Howard R G , Rothstein T L
Published: December 2022

CD40L, a membrane protein of activated T cells, interacts with the B cell receptor CD40. This interaction has been implicated in the rescue of germinal center B cells from apoptosis and in the rescue of WEHI-231 B lymphoma cells from sIg-induced apoptosis. In this report, we have demonstrated that the signal mediated by CD40L acts upon bcl-x, a bcl-2 homologue. bcl-x expression is strongly enhanced by CD40 receptor engagement, while there is little or no induction by sIg cross-linking. The expression of bax and bcl-2 is not significantly affected by either CD40L or sIg cross-linking. Antisense but not sense phosphorothioate oligonucleotide for bcl-x can partially block this CD40-mediated apoptotic rescue. This result suggests that the up-regulation of bcl-x by CD40L plays an important role in CD40-mediated apoptotic rescue in murine B cells.
B lymphocytes can be competent antigen-presenting cells for priming CD4+ T cells to protein antigens in vivo.
Authors: Constant S , Schweitzer N , West J , Ranney P , Bottomly K
Published: December 2022

The potential role of different subsets of APCs to stimulate naive CD4+ T cells to peptide and protein Ags in vivo was examined. Mice lacking B cells (microMT knockout mice) were impaired in their priming to protein but not peptide Ags, suggesting a requirement for B cells in priming to protein Ags in vivo. Experiments designed to determine the ability of splenic dendritic cells (DCs) and B lymphocytes to take up peptide or protein Ags in vivo demonstrated that peptide Ags were taken up preferentially by DCs, whereas proteins were taken up by Ag-specific B cells in vivo. A further examination of the Ag-specific B cells pulsed in vivo with protein Ags revealed a marked up-regulation in surface expression of B7-2 costimulatory molecules, detectable as early as 4 h after Ag administration. Based on their potency in the uptake and processing of protein Ags as well as their ability to up-regulate costimulatory molecules through Ag internalization, we suggest that Ag-specific B cells will be an important APC in priming naive CD4+ T cells to protein Ags in vivo.
The intracellular transport of MHC class II molecules in the absence of HLA-DM.
Authors: Green J M , DeMars R , Xu X , Pierce S K
Published: December 2022

The HLA-DM alpha and HLA-DM beta genes encode a nonpolymorphic, class II-like molecule that functions by an as yet undefined mechanism in the assembly of processed antigen-HLA class II complexes. Mutant cells that fail to express HLA-DM are deficient in Ag processing. We previously isolated a subcellular compartment in mouse B cells in which functional processed Ag-class II complexes are first formed, referred to as the peptide-loading compartment. Here, evidence is provided that HLA-DM resides in a subcellular compartment with the characteristics of a peptide-loading compartment in a human B lymphoblastoid cell line, but is not required for the intracellular transport of HLA-DR3 molecules to a corresponding compartment in HLA-DM-deficient cells. Thus, the primary defect in HLA-DM-deficient cells does not appear to be a failure in the intracellular trafficking of class II molecules.
Modulation of anti-IgM-induced B cell apoptosis by Bcl-xL and CD40 in WEHI-231 cells. Dissociation from cell cycle arrest and dependence on the avidity of the antibody-IgM receptor interaction.
Authors: Merino R , Grillot D A , Simonian P L , Muthukkumar S , Fanslow W C , Bondada S , Núñez G
Published: December 2022

The demise of B cell progenitors expressing functional IgM receptors for self appears to be the main mechanism by which B cell tolerance is accomplished. The genetic mechanisms that regulate the death process during this critical step of B cell development are still poorly understood. We have studied the regulation of apoptosis in WEHI-231 lymphoma cells after treatment with a panel of anti-IgM mAbs as an in vitro model of clonal B cell deletion. We showed that a product of bcl-x, Bcl-xL, can inhibit anti-IgM-induced apoptosis but not cell cycle arrest in a dose-dependent manner. Bcl-xL was efficient in protecting B cells from low but not high avidity anti-IgM mAbs. In contrast to that observed with Bcl-xL, CD40 stimulation was efficient in inhibiting both cell cycle arrest and apoptosis after IgM cross-linking regardless of the binding avidity of the anti-IgM Ab. Moreover, activation through IgM receptors but not CD40 induced up-regulation followed by rapid down-modulation of Bcl-xL. Thus, the capacity of Bcl-xL to modulate anti-IgM-induced apoptosis in WEHI-231 cells is highly dependent on the avidity of the Ab-IgM receptor interaction.
Alloreactive gamma delta thymocytes utilize distinct costimulatory signals from peripheral T cells.
Authors: Penninger J M , Timms E , Shahinian A , Jezo-Bremond A , Nishina H , Ionescu J , Hedrick S M , Mak T W
Published: December 2022

Interactions between CD28/CTLA-4 on T cells and CD80 (B7.1) and CD86 (B7.2) counter receptors provide crucial costimulatory signals for TCR-alpha beta+ lymphocytes. To test the role of CD28 in thymic development and activation of TCR-gamma delta+ T cells, we introduced the alloreactive V gamma 2V alpha 11.3 TCR into CD28-deficient mice (CD28-/-). We show that positive and negative selection of gamma delta Tg thymocytes proceeded normally in the absence of CD28. Although mature Tg gamma delta+ thymocytes required a second costimulatory signal for proliferation, gamma delta+ thymocytes from CD28-/- and CD28+/- littermates responded equally well to the alloantigen Tlab. Alloreactivity of CD28-/- and CD28+/- Tg gamma delta+ thymocytes could not be blocked with mAbs against CD80 and CD86 ligands. Thus gamma delta thymocytes utilize a costimulatory system during development and alloresponses that is independent of CD28/CD80 and CD28/CD86 interactions. By contrast to V gamma 2V alpha 11.3+ thymocytes, alloreactivity of V gamma 2V alpha 11.3+ lymph node T cells depended on CD28 costimulation and was severely impaired in CD28-/- mice. These data provide functional evidence that maturation and selection of gamma delta cells is independent of CD28. These results also indicate that distinct costimulatory pathways are operational in mature thymocytes and peripheral T cells.
Costimulation of human CD4+ T cells by fibroblast growth factor-1 (acidic fibroblast growth factor).
Authors: Zhao X M , Byrd V M , McKeehan W L , Reich M B , Miller G G , Thomas J W
Published: December 2022

T cell infiltration is prevalent in wound healing, atherosclerosis, vascular lesions in chronic allograft rejection, and autoimmune diseases. Whether T cells play a role in the migration and proliferation of vascular smooth muscle cells and endothelial cells in these lesions is not known. We previously reported that some human T cells express FGF-1, a potent growth factor for vascular smooth muscle cells and endothelial cells. In this study, we extend this observation and examine the expression and function of FGF receptors on human T cells. Using reverse transcription-PCR, Northern analysis, and immunohistochemistry, we found that some human T cells also express high affinity FGF receptor 1 (FGFR-1) respond to FGF-1. In the presence of anti-CD3, exogenous FGF-1 functions as a costimulator for these T cells, while FGF-1 alone does not induce T cell proliferation. [3H]Thymidine incorporation is sevenfold higher in T cells costimulated with FGF-1 compared with stimulation with anti-CD3 alone. Using limiting dilution, we demonstrate that FGF-responsive T cells are present in normal peripheral blood at a mean frequency of 1:19780 (95% confidence limits, 1:15100-1:23000), and similar T cells are increased in the peripheral blood of heart transplant recipients (mean frequency, 1:4210; 95% confidence limits, 1:3420-1:6781). In addition, a subline of Jurkat, a human T cell tumor, expresses FGFR-1 receptor. The function of FGFR-1 receptor in Jurkat T cells is demonstrated by the production of IL-2 after stimulation with FGF-1 and anti-CD3. IL-2 levels are sevenfold higher in Jurkat T cells costimulated with FGF-1 compared with those stimulated with anti-CD3 alone. FGF-1 alone has no effect on Jurkat T cells. These findings thus provide evidence that a subset of human T cells expresses a receptor for vascular cell growth factors, and this receptor functions to increase IL-2 production consistent with costimulation. The potential role of FGF-responsive T cells in a variety of vascular and inflammatory lesions is discussed.
Characterization of herpes simplex virus type-1 infection and herpetic stromal keratitis development in IFN-gamma knockout mice.
Authors: Bouley D M , Kanangat S , Wire W , Rouse B T
Published: December 2022

Herpetic stromal keratitis (HSK) has an immune-mediated pathogenesis that involves T cells that have a type 1 cytokine profile. IFN-gamma is suspected to be the type 1 cytokine involved in ocular pathology, and to test this notion more directly the pathogenesis of HSK was compared in mice deficient in the IFN-gamma gene (gamma knockout or gko) and control mice (wild-type littermates or BALB/c mice). The clinical course of HSK in gko mice closely paralleled that in control mice, yet virus persisted in the corneas of gko mice for an extended period of time, severe periocular skin lesions developed, and gko mice were far more susceptible to encephalitis. Delayed-type hypersensitivity to viral Ag was present, though diminished, in knockout mice, and serum herpes simplex virus-specific IgG isotypes indicated a Th2 shift. No differences existed in proliferative responses to in vitro Ag stimulation in gko vs control mice nor in T cell or proinflammatory cytokine mRNA levels in the corneas of infected mice. However, up-regulation of Th2 cytokine mRNA did occur in in vitro Ag-stimulated gko immune splenocytes. Histopathologic lesions were not statistically different between any of the groups of mice analyzed. These observations indicate that although IFN-gamma plays an important role in the clearance of virus from the eye, the pathogenesis of HSK lesions most likely involves additional cytokines, inflammatory mechanisms, or immune responses to nonviral Ags.
Serum amyloid A induces calcium mobilization and chemotaxis of human monocytes by activating a pertussis toxin-sensitive signaling pathway.
Authors: Badolato R , Johnston J A , Wang J M , McVicar D , Xu L L , Oppenheim J J , Kelvin D J
Published: December 2022

We have previously reported that serum amyloid A (SAA) induces adhesion and chemotaxis of human monocytes and polymorphonuclear neutrophils, in vitro as well as in vivo. Since the mechanism of SAA signaling is unknown, we have investigated the possibility that SAA, like other chemoattractants such as the chemotactic peptide FMLP and chemokines, might induce migration of monocytes by G protein activation. We report here that preincubation of monocytes with pertussis toxin (PTx) inhibited SAA chemotaxis, while incubation with cholera toxin (CTx) did not. Staurosporine and H-7, both inhibitors of protein kinase C (PKC), significantly decreased rSAA-induced chemotaxis of monocytes, suggesting that PKC may be involved in the rSAA signaling pathway. Moreover, rSAA, at concentrations that were effective in chemoattracting monocytes, resulted in transient elevation of cytoplasmic calcium concentration ([Ca2+]i), and incubation of cells with PTx markedly inhibited the mobilization of Ca2+ in response to rSAA. This suggests that both chemotaxis and the rise in [Ca2+]i, are mediated by G proteins of the Gi class. The increase in [Ca2+]i, induced in monocytes by rSAA, was comparable to that elicited by FMLP, and was severalfold greater than that induced by optimal concentrations of chemokine beta-family members such as RANTES, MCAF/MCP-1, and MIP-1 alpha. The chemoattractants FMLP, RANTES, MIP-1 alpha, and MCAF/MCP-1, all failed to desensitize rSAA-induced Ca2+ influx and chemotaxis in monocytes. This suggests that SAA uses a distinct receptor that is coupled to PTx-sensitive G proteins.
Isoforms of human C4b-binding protein. I. Molecular basis for the C4BP isoform pattern and its variations in human plasma.
Authors: Sánchez-Corral P , Criado García O , Rodríguez de Córdoba S
Published: December 2022

Human C4b-binding protein (C4BP) is an important regulator of the complement system that also binds and inactivates the anticoagulant vitamin K-dependent protein S. These two activities are performed by two distinct polypeptides of 70 kDa and 45 kDa known as alpha- and beta-chains, respectively. C4BP is present in plasma in various isoforms with different alpha beta composition. Here we report multiple discrete variations of the relative levels of the C4BP isoforms among normal individuals and provide evidence that they are determined by genetic factors that segregate with the regulator of complement activation gene cluster. We also report the characterization of the C4BP molecules secreted by HepG2 and Hep3B cells, as well as transfection experiments in COS cells, to illustrate that the relative levels of expression of the C4BPA and C4BPB genes play a major role in determining the proportion in which the different C4BP isoforms are synthesized. Altogether, the data indicate that the human C4BP isoform pattern is genetically determined, but can be modified by factors with a differential effect on the expression of the C4BPA and C4BPB genes. These observations provide a new way to explore the possible association between elevated levels of C4BP and an increased risk to thromboembolic disorders.