bluuebunny/crossref_metadata_2025 huggingface dataset:
Il-7 supports D-J but not V-DJ rearrangement of TCR-beta gene in fetal liver progenitor cells.
Authors: Tsuda S , Rieke S , Hashimoto Y , Nakauchi H , Takahama Y
Published: December 2022

The rearrangement of TCR-beta gene, one of the earliest events in T cell development, consists of two consecutive steps: D-J rearrangement and V-DJ rearrangement. The present study examined the signals supporting D-J beta and V-DJ beta rearrangements during early T cell development from progenitor cells that reside in fetal liver. We have found that there is an interval of 1 to 2 days between D-J beta and V-DJ beta rearrangements during the early T cell development from fetal liver progenitor cells in deoxyguanosine-treated thymus lobes. We have also found that IL-7, a cytokine expressed in the subcapsular area of the thymus, can promote D-J beta rearrangement of fetal liver progenitor cells, and that anti-IL-7 and anti-IL-7R Abs inhibit the D-J beta rearrangement and further T cell development of fetal liver progenitor cells in the thymus environment. Interestingly, unlike the thymus environment, IL-7 alone was not capable of supporting V-DJ beta rearrangement in the fetal liver cell cultures. These results indicate that D-J beta rearrangement during fetal liver-derived early T cell development is supported in the thymus by IL-7. Furthermore, the present results demonstrate that IL-7, supporting D-J beta rearrangement, does not promote V-DJ beta rearrangement of fetal liver progenitor cells, suggesting that intrathymic molecules promoting V-DJ beta rearrangement are distinct from IL-7 that supports the D-J beta rearrangement.
Identification and characterization of a C(K/R)TC motif as a common epitope present in all subtypes of hepatitis B surface antigen.
Authors: Qiu X , Schroeder P , Bridon D
Published: December 2022

The a determinant of hepatitis B surface antigen (HBsAg) is the most critical determinant for both diagnosis and immunoprophylaxis of the hepatitis B virus. We have used synthetic peptides and an anti-a mAb to identify a peptide sequence corresponding to amino acid residues 117 to 128 of HBsAg as an antigenic epitope contributing to the a determinant. Compared to the native protein HBsAg, the cyclic form of the peptide (aa 117-128) is only 20-fold less effective, whereas the linear form of the peptide is 160-fold less effective in the inhibition of mAb binding to HBsAg. Based on these results, we have postulated a previously unidentified disulfide bond between residue Cysl21 and Cysl24. Individual substitution of amino acids in the peptide (aa 117-128) with alanine identified three residues Cys121, Thr123, and Cys124 as the most critical residues for mAb recognition. Substitution of alanine for any one of the three residues caused a substantial loss in binding free energy (greater than 4.5 kcal/mol). Sequence analysis indicated that the C(K/R)TC motif is highly conserved among 100 subtypes and mutants of HBsAg isolates. Collectively, these results show that the cyclic C(K/R)TC motif is an essential part of the a determinant of HBsAg. Synthetic peptides containing the C(K/R)TC motif are potentially useful as alternative hepatitis B vaccines and as diagnostic reagents for the detection of the hepatitis B virus.
Modulation of endogenous IL-1 beta and IL-1 receptor antagonist results in opposing effects on HIV expression in chronically infected monocytic cells.
Authors: Goletti D , Kinter A L , Hardy E C , Poli G , Fauci A S
Published: December 2022

A proportion of HIV-infected individuals experience episodes of localized or systemic bacterial infections caused by Gram-negative bacteria. Many of the clinical side effects of these infections are associated with the production of proinflammatory cytokines, which are induced primarily by LPS, a constituent of the bacterial cell wall of Gram-negative bacteria. The present study examines the mechanisms involved in LPS-mediated induction of HIV expression in U1 cells, a promonocytic cell line chronically infected with HIV. Stimulation of U1 cells by LPS alone induced minimal levels of HIV expression, which was significantly enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF). Costimulation of U1 cells with LPS plus GM-CSF resulted in the accumulation of steady-state levels of HIV RNA; however, only a weak induction of HIV long terminal repeat-driven transcription, which was not associated with the activation of the cellular transcription factor nuclear factor-kappa B, was noted. Costimulation of cells with LPS plus GM-CSF induced the production of proinflammatory cytokines, IL-8, IL-1 beta and IL-6, but not TNF-alpha. IL-1 receptor antagonist (ra) inhibited LPS enhancement of HIV expression in GM-CSF-stimulated cells, suggesting that endogenous IL-1 was involved in LPS-mediated viral production. In this regard, anti-inflammatory cytokines inhibited LPS plus GM-CSF-stimulated HIV expression, and this effect closely correlated with inhibition of IL-1 beta release and, in particular, with up-regulation of endogenous IL-1ra production. Thus, the balance between an endogenously produced viral inducer (IL-1 beta ) and an inhibitor (IL-1ra) may represent an important pathway leading to modulation of HIV expression from monocytic cells.
IL-10 production is enhanced in human T cells by IL-12 and IL-6 and in monocytes by tumor necrosis factor-alpha.
Authors: Daftarian P M , Kumar A , Kryworuchko M , Diaz-Mitoma F
Published: December 2022

IL-10, an immunoregulatory cytokine produced by T cells and monocytes, inhibits the expression of inflammatory and hemopoietic cytokines as well as its own expression. To evaluate the regulation of IL-10 production by T cells and monocytes, we measured IL-10 levels by ELISA in supernatants of PHA-stimulated PBMC following depletion of either T cells or monocytes. IL-10 production was significantly down-regulated in both T cell- and monocyte-depleted PBMC compared with undepleted PBMC, and IL-10 production could be restored by the addition of monocyte-conditioned medium (supernatant of PHA-stimulated, T cell-depleted PBMC), suggesting that IL-10 production by T cells is regulated by a monokine(s) produced by activated monocytes. To further clarify the monokine(s) responsible for IL-10 induction, we stimulated monocyte-depleted PBMC, purified CD4+, and CD8+ T cells with PHA and measured IL-10 production by ELISA and semiquantitative reverse transcriptase-PCR following monokine(s) addition. Addition of IL-6 and IL-12 enhanced IL-10 production in monocyte-depleted PBMC in a dose-dependent and additive manner. Furthermore, anti-IL-6 and anti-IL-12 Abs neutralized the IL-10-inductive effect of monocyte-conditioned medium. Similarly, IL-12 and IL-6 induced IL-10 production by purified CD4+ and CD8+ T cells. With respect to regulation of IL-10 produced by monocytes, TNF-alpha was found to induce IL-10 production by resting as well as by LPS-stimulated purified monocytes/macrophages. Taken together, these findings suggest that IL-10 production by human T cells and monocytes is differentially regulated. IL-12 and/or IL-6 can induce the expression of IL-10 by PHA-stimulated T cells, whereas TNF-alpha induces IL-10 production by monocytes. Since IL-10 inhibits the production of IL-6, IL-12, and TNF-alpha, these results may indicate a potential mechanism of negative feedback regulation of the immune response.
Multiple interactive residues of recognition: elucidation of discontinuous epitopes with linear peptides.
Authors: Gao B , Esnouf M P
Published: December 2022

The discontinuous epitopes of beta-factor XIIa for three mAbs were mapped by a linear peptide-based immunoblotting technique, referred to as multiple interactive residues of recognition. The Abs were incubated with a set of overlapping synthetic peptides, deduced from the cDNA sequences of beta-factor XIIa, on a polymer membrane, and the signal was amplified by an ECL assay. Several discrete sequences of the protein were recognized by each Ab. The recognized peptides were further characterized using alanine substitution analogues and peptides of different lengths. The discontinuous epitopes found for each Ab were formed by several peptides and were composed of 20 to 31 residues. The sequence FLQEA was recognized by all three Abs and was the immunodominant peptide. Abs 201/9 and 2/15 bound to very similar discontinuous sequences, but with subtle differences. The sequences 76RLHEAFSP83, 88HDLALLRLQE97, 178GFLEG182, 146FLQEA150, and 237IRE239 formed the epitope for mAb 201/9, whereas 76RLHEAFSP83, 90LALLRLQE97, 146FLQEA150, and 235AWIREHT241 formed the epitope for Ab 2/15. The third Ab 202/7 recognized the sequences 79EAFSP83, 93LRLQE97, 133WGHQF137, and 146FLQEA150. We suggest that these sequences represent the sites to which the Abs bind. This procedure provides a sensitive and convenient tool to elucidate discontinuous epitopes for the binding of Abs, receptor ligand-binding sites, or enzyme inhibitor binding sites.
Identification, molecular cloning, biologic properties, and tissue distribution of a novel isoform of murine low-affinity IgG receptor homologous to human Fc gamma RIIB1.
Authors: Latour S , Fridman W H , Daëron M
Published: December 2022

A cryptic splice donor site in the first intracytoplasmic (IC) exon of the murine Fc gamma RIIB gene generates a previously unknown Fc gamma RIIB isoform. Three membrane Fc gamma RIIB polypeptides of 37, 32, and 30 kDa were immunoprecipitated by Fc gamma RIIB-specific Abs, and three Fc gamma RIIB cDNAs of 1071, 987, and 930 bp were amplified by reverse transcriptase-PCR with Fc gamma RIIB-specific oligonucleotides from the mastocytoma cells P815. The 1071-bp cDNA contains all sequences of IC exons and encodes the 37-kDa Fc gamma RIIB1 isoform. The 930-bp cDNA lacks sequences of the first IC exon and encodes the 30-kDa Fc gamma RIIB2 isoform. The 987-bp cDNA has an 84-nucleotide deletion of the first IC exon 3' sequences. When stably transfected in the lymphoma B cells IIA1.6, or in the mast cells RBL-2H3, this cDNA encoded 32-kDa Fc gamma RIIB whose biologic properties were undistinguishable from those of Fc gamma RIIB1: they inhibited B cell activation when coaggregated to B cell receptors and capped when aggregated at 37 degrees C, but failed to mediate endocytosis or phagocytosis. Sequences responsible for capping and inhibition of internalization, previously assigned to sequences encoded by the first IC exon, can thus be mapped in the 19 N-terminal residues. These residues are highly conserved in human Fc gamma RIIB1. The 87-bp first IC exon of the human gene ends by a single splice site in the downstream intron and encodes a 19-amino acid insertion. The 32-kDa Fc gamma RIIB is the murine homologue of human Fc gamma RIIB1. We propose to name it Fc gamma RIIB1'. Fc gamma RIIB1' was expressed in myeloid and lymphoid cell lines, in normal spleen cells, and in resting or LPS-activated B cells.
Increased susceptibility of mice to Salmonella infection following in vivo treatment with the substance P antagonist, spantide II.
Authors: Kincy-Cain T , Bost K L
Published: December 2022

Successful resolution of salmonellosis in naive mice depends in large part upon IL-12-induced IFN-gamma production to eliminate this intracellular pathogen of macrophages. In the present study we questioned the contribution that expression of substance P receptors makes to the protective response following oral inoculation with a lethal dose of Salmonella. Such a relationship was suggested when oral inoculation with Salmonella induced rapid and dramatic increases in substance P receptor mRNA expression within Peyer's patches and mesenteric lymph nodes and subsequently in the spleen. The importance of substance P receptor expression in vivo was further suggested by pretreatment of mice with the substance P antagonist, spantide II, before oral inoculation with Salmonella. Mice pretreated with spantide II and then orally inoculated developed advanced salmonellosis and had significantly reduced survival rates compared with mice pretreated with a control peptide. Treatment with spantide II significantly reduced early Salmonella-induced IL-12p4O and IFN-gamma mRNA expression at mucosal sites, suggesting a mechanism for the reduced ability of spantide II-treated mice to resist this pathogen. Increased susceptibility to salmonellosis was not due to 1) spantide II-induced alterations in the uptake of this pathogen from the gut, 2) global spantide II-mediated immune suppression, or 3) nonsubstance P receptor-mediated effects of spantide II on macrophages. The ability of Salmonella to induce substance P receptor expression on cultured macrophages suggested that one mechanism for resistance against this intracellular pathogen might be a direct effect of substance P on this cell population.
DNA double strand breaks in epidermal cells cause immune suppression in vivo and cytokine production in vitro.
Authors: O'Connor A , Nishigori C , Yarosh D , Alas L , Kibitel J , Burley L , Cox P , Bucana C , Ullrich S , Kripke M
Published: December 2022

UV irradiation of the skin causes immune suppression by a mechanism involving epidermal cytokines. To determine the role of epidermal DNA damage in immune suppression, we used HindIII restriction endonuclease encapsulated in liposomes to cause DNA strand breaks in epidermal cells in vivo and in vitro. Topical application of HindIII in liposomes to murine skin in vivo impaired the induction of contact hypersensitivity responses initiated either locally or at distant sites and impaired the function of APCs. Unlike UV-B radiation, however, treatment of mice with HindIII in liposomes before contact sensitization did not induce tolerance or transferable suppression. The liposome-encapsulated HindIII caused double strand breaks in DNA and induced IL-10 and TNF-alpha production when added to cells of a murine keratinocyte line in vitro. Topical application of liposomal HindIII also induced TNF-alpha in the epidermis of mice. Liposomes containing heat-inactivated HindIII or an endonuclease specific for pyrimidine dimers in DNA did not exhibit these effects. These results support the hypothesis that DNA damage is a trigger for the production of cytokines that modulate immune responses. They also suggest that immune suppression and suppressor cell induction are separate consequences of cutaneous injury that require different stimuli.
Effect of endotoxin in IL-1 beta-deficient mice.
Authors: Fantuzzi G , Zheng H , Faggioni R , Benigni F , Ghezzi P , Sipe J D , Shaw A R , Dinarello C A
Published: December 2022

IL-1 plays an important role in the pathophysiologic responses to infection and inflammation, in part by mediating its own production and that of other proinflammatory cytokines. However, the relative contribution of IL-1 alpha and IL-1 beta to the inflammatory response has not been well clarified. Using IL-1 beta-deficient (IL-1 beta -/-) mice, we investigated the specific role of IL-1 beta in the in vivo and in vitro response to LPS. No differences between IL-1 beta +/+ and IL-1 beta -/- mice were observed in circulating levels for IL-1 alpha, IL-6, or TNF-alpha after the systemic administration of either a low (5 micrograms/kg) or high (5 mg/kg) dose of LPS. IL-1 beta -/- mice also had a normal response to LPS in terms of activation of the hypothalamus-pituitary-adrenal axis, hypoglycemia, serum amyloid A production, and anorexia. IL-1 beta -/- mice were normally sensitive to the lethal effect of LPS and were protected against LPS toxicity when pretreated with low-dose LPS. However, in vitro, peritoneal macrophages from IL-1 beta -/- mice stimulated with LPS produced significantly less IL-1 alpha than macrophages from IL-1 beta +/+ mice (p < 0.05). No differences in IL-6 or TNF-alpha synthesis were observed between macrophages from IL-1 beta +/+ and IL-1 beta -/- mice. In summary, our results suggest that either IL-1 beta is not essential for the in vivo systemic response to LPS or that its role can be fulfilled by other cytokines with overlapping activities.
Acidic sphingomyelinase-generated ceramide is needed but not sufficient for TNF-induced apoptosis and nuclear factor-kappa B activation.
Authors: Higuchi M , Singh S , Jaffrezou J P , Aggarwal B B
Published: December 2022

Previously we have shown that treatment of ML-1a cells with TNF in the presence of cycloheximide triggers apoptosis within 90 min. In the present report we used this system to investigate the role of ceramide in TNF action. We found that while maximum DNA fragmentation response was induced by 1 nM TNF, the synthetic membrane-permeable C-2, C-6, and C-8 ceramides had no effect even up to 40 microM concentration. To investigate the roles of ceramide in TNF action, we used ((2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxy-phenethyl)amino] propyloxy)benzenesulfonyl))indolizine (SR33557), a potent inhibitor of acidic but not neutral sphingomyelinase (SMase). Even though ceramides by themselves did not mimic TNF, we found that SR33557 inhibited TNF-induced apoptosis and the addition of ceramide reversed this effect, indicating that ceramide generated by acidic SMase is involved in TNF action. The addition of SR33557 to cells at 0 or at 30 min after TNF treatment showed inhibition, but the addition 60 min later had no effect, indicating that an SR33557-sensitive step is located between 30 and 60 min of signal transduction. Since ceramide has been shown to play a role in TNF-mediated activation of nuclear factor-kappa B (NF-kappa B), we examined the effect of SR33557 on this early cellular response of TNF. Surprisingly, this inhibitor of ceramide production was found to have no effect on TNF-mediated NF-kappa B activation, thus suggesting that SR33557-sensitive acidic SMase is not involved in this process. From these results, we concluded that ceramide is needed for certain TNF-mediated cellular responses, but it alone may not be sufficient.
TNF-alpha enhances colony-stimulating factor-1-induced macrophage accumulation in autoimmune renal disease.
Authors: Moore K J , Yeh K , Naito T , Kelley V R
Published: December 2022

The lpr mutation on the MRL background accelerates autoimmune nephritis in which macrophage (M phi) accumulation is prominent. Renal disease is absent in other strains with lpr. TNF-alpha and CSF-1 are increased in the kidney of MRL-lpr mice with loss of renal function. We have established that CSF-1 can incite renal injury in mice with the lpr mutation, and M phi from the MRL strain hyper-respond to this growth factor. We hypothesized that TNF-alpha enhanced the M phi response to CSF-1 in MRL-lpr mice. We now report that TNF-alpha enhanced CSF-1-induced bone marrow M phi proliferation in MRL-lpr mice, and not in congenic MRL +/+, normal C3H +/+, and BALB/c, or another strain with lpr (C3H-lpr). Using a gene transfer approach to deliver CSF-1 together with TNF-alpha into the kidney, we evaluated the impact on renal injury. Tubular epithelial cells genetically modified to produce CSF-1 (CSF-1-TEC) and TNF-alpha (TNF-TEC) placed under the renal capsule caused a greater accumulation of M phi in the implant site than CSF-1-TECs alone in MRL-lpr, but not MRL +/+ mice. We noted in tissues adjacent but not distal to the implanted TECs, an increase in M phi in the interstitium and surrounding glomeruli of MRL-lpr but not MRL +/+ mice. This indicated that CSF-1 and TNF-alpha released by TECs were responsible for promoting renal pathology. Taken together, these data suggest that the simultaneous expression of TNF-alpha and CSF-1 in the MRL-lpr kidney fosters M phi accumulation. We speculate that the increase in M phi in the kidney in response to CSF-1 and TNF-alpha is responsible for the rapid tempo of autoimmune renal injury in MRL-lpr mice.
The signaling activity of murine CD19 is regulated during cell development.
Authors: Krop I , Shaffer A L , Fearon D T , Schlissel M S
Published: December 2022

CD19, a B cell-specific transmembrane protein, is essential for murine B-1 cell development and T cell-dependent B cell immune responses. Whereas signaling by the human B cell Ag receptor can be modulated by CD19, less is known about the biochemical properties of murine CD19. We have used a novel rat mAb specific for murine CD19 to study the biochemical properties of the murine protein. We demonstrate that murine CD19 shares with human CD19 an association with complement receptor CD21 and CD81, tyrosine phosphorylation, binding of phosphatidylinositol-3 kinase, and synergistic signaling with membrane IgM. Murine CD19 is shown also to enhance signaling through the micro-surrogate light chain complex of primary pre-B cells. We found that although expressed in the earliest B cell precursors, CD19 ligation does not activate Ca2+ mobilization until the pre-B cell stage of development. In mature B cells, CD19 cross-linking activates Ca2+ flux in B-2 cells but not in B-1 cells, although it can synergize with surface IgM in both B-1 and B-2 cells. These biochemical properties of CD19 will be important for understanding its function in B cell development and the humoral immune response.
Invariant chain association with MHC class I: preference for HLA class I/beta 2-microglobulin heterodimers, specificity, and influence of the MHC peptide-binding groove.
Authors: Vigna J L , Smith K D , Lutz C T
Published: December 2022

MHC class I molecules require the assembly of heavy chain with beta2-microglobulin (beta 2m) and peptide in order to present Ag on the cell surface. Endoplasmic reticulum resident proteins associate with class I molecules and aid assembly. Free class I heavy chains associate with calnexin, which may facilitate association with beta 2m. Invariant chain (Ii) also associates with MHC class I molecules, but its role in class I assembly is not clear. We report here that Ii strongly associates with HLA class I/beta 2m heterodimers, but weakly with free class I heavy chains in HLA-B7-transfected T2 cells. Ii/HLA class I complexes persist stably within the endoplasmic reticulum/cis-Golgi compartment in peptide-processing deficient cells, but are much less prominent in normally processing cells. Furthermore, Ii differentially associates with variant HLA-B7 molecules that have peptide-binding groove mutations, and the degree of association correlates with HLA-B7 variant cell surface expression. Ii also shows HLA class I molecule specificity, associating to a greater degree with HLA-B7 than HLA-A2. Together these observations suggest that Ii stabilizes particular HLA class I/beta 2m heterodimers until peptide is loaded, and that this association may enhance class I cell surface expression.
Mechanisms of stimulation of the respiratory burst by TNF in nonadherent neutrophils: its independence of lipidic transmembrane signaling and dependence on protein tyrosine phosphorylation and cytoskeleton.
Authors: Dusi S , Della Bianca V , Donini M , Nadalini K A , Rossi F
Published: December 2022

This study concerns the controversial problem of whether the TNF-alpha (TNF) induces a respiratory burst in human neutrophils in suspension. The results have shown that in these cells TNF induces a classical respiratory burst. In fact, the production of oxygen free radicals 1) is linked to the translocation of NADPH oxidase components from cytosol to the plasma membrane, 2) does not take place in neutrophils from a patient lacking the cytochrome b558, and 3) does not involve other sources such as mitochondrial respiratory chain or xanthine oxidase. Signal transduction studies have demonstrated that this respiratory burst 1) is not accompanied by calcium transients, stimulation of phosphoinositide turnover, and phospholipase D activity (moreover, this burst is associated with the stimulation of the activity of phospholipase A2, but not of sphingomyelinase); 2) is strictly dependent on activation of tyrosine kinases, which is functional to the translocation to the plasma membrane of the cytosolic NADPH oxidase component rac; and 3) is dependent on the integrity of the cytoskeleton because it is completely suppressed by cytochalasin B. The integrity of the cytoskeleton is required for a full translocation of all the NADPH oxidase components and for an optimal activation of tyrosine kinases, but not for phospholipase A2 activation. Taken together, these findings demonstrate that TNF activates the NADPH oxidase through stimulation of tyrosine kinases, whose function is cytoskeleton-dependent, and raise the problem of whether the activation of this respiratory burst involves signals arising from TNF-activated beta2 integrins.
Human natural killer cell receptors involved in MHC class I recognition are disulfide-linked heterodimers of CD94 and NKG2 subunits.
Authors: Lazetic S , Chang C , Houchins J P , Lanier L L , Phillips J H
Published: December 2022

CD94 receptors expressed on NK cells have been implicated in the recognition of certain HLA class I allotypes. We now demonstrate that CD94 glycoproteins form disulfide-bonded heterodimers with the NKG2A/B, NKG2C, and NKG2E glycoproteins. NKG2A/B possesses two immunoreceptor tyrosine-based inhibition motif (ITIM) sequences in its cytoplasmic domain, which may be responsible for the inhibitory function of these receptors, whereas other NKG2 proteins lack ITIMs and may potentially transmit positive signals. Structural heterogeneity in the NKG2 gene family and the formation of heterodimers with CD94 provides for the creation of a diverse NK cell repertoire.
Regulation of surface and intracellular expression of CTLA4 on mouse T cells.
Authors: Alegre M L , Noel P J , Eisfelder B J , Chuang E , Clark M R , Reiner S L , Thompson C B
Published: December 2022

CTLA4 is a cell surface molecule that shares 30% homology with CD28 and binds B7 family members with high affinity. Analysis of surface expression on murine T cells revealed up-regulation after stimulation with anti-CD3 mAb in vitro and further augmentation after the addition of exogenous IL-2 or anti-CD28 mAb. The effects of IL-2 and anti-CD28 mAb were additive and in part independent, as anti-CD28 mAb increased anti-CD3 mAb-induced T cell CTLA4 expression in IL-2-deficient mice. In contrast, CTLA4 expression was only minimally augmented by the addition of IL-4, IL-6, IL-7, or IL-12. Expression of CTLA4 induced by anti-CD3 mAb was inhibited by anti-IL-2 plus anti-IL-2R mAbs. Inasmuch as these agents prevented T cell proliferation, the effects of cell cycle inhibitors also were examined. Drugs blocking at G1 (cyclosporin A, mimosine) or S (hydroxyurea) phase inhibited the up-regulation of CTLA4 induced by anti-CD3 mAb, suggesting that entry into the cell cycle was necessary to increase the expression of CTLA4. The kinetics of intracellular expression of CTLA4 after stimulation with anti-CD3 mAb paralleled those of surface expression, but surprisingly, much more CTLA4 was localized in the cytoplasm of T lymphocytes than on the cell surface at each time point. Importantly, surface CTLA4 was rapidly internalized intracellularly, which may explain the low levels of expression generally detected on the cell surface. We conclude that both CD28 and IL-2 play important roles in the up-regulation of CTLA4 expression. In addition, the cell surface accumulation of CTL4 appears to be primarily regulated by its rapid endocytosis.
Allospecific CD8+ Tc1 and Tc2 populations in graft-versus-leukemia effect and graft-versus-host disease.
Authors: Fowler D H , Breglio J , Nagel G , Eckhaus M A , Gress R E
Published: December 2022

Allogeneic CD8+ T cells mediate both a graft-vs-leukemia (GVL) effect and graft-vs-host disease (GVHD). To evaluate whether CD8 cells of defined cytokine phenotype differentially mediate these processes, alloreactive donor CD8+ T cells preferentially secreting type I or type II cytokines were generated by alloantigenic priming in vitro in the presence of IL-12 or IL-4, respectively. Both cytokine-secreting subsets lysed allogeneic tumor targets in vitro ("Tc1" and "Tc2" subsets). A transplantation model was established (B6 into B6C3F1, 1050 cGy host irradiation) using the 32Dp210 myeloid line (bcr/abl transfected, H-2k; 1 x 10(4) tumor cells/recipient). Compared with leukemia controls (death at 12.9 days post-bone marrow transplantation), both Tc1 and Tc2 recipients were conferred a survival advantage. At cell doses of 2 to 2.5 x 10(7), the Tc1-mediated GVL effect (mean survival of 34.2 days) was more potent than the Tc2-mediated GVL effect (mean survival of 20.5 days; Tc1 > Tc2, p = 0.009). On day 15, histologic examination showed that Tc1 recipients had undetectable tumor burdens, whereas Tc2 recipients had extensive leukemic infiltrates. However, Tc2 recipients had essentially no histologic evidence of GVHD, whereas Tc1 recipients had mild to moderate GVHD (average GVHD scores of 1/40 and 9.3/40, respectively). In contrast, recipients of uncultured CD8+ donor T cells developed severe GVHD (average GVHD score of 26.7/40). Because in vitro-generated, alloreactive Tc1 and Tc2 populations mediated GVL with reduced GVHD, we conclude that both subsets may improve the therapeutic outcome of allogeneic T cell transfers in patients with leukemia.
CpG DNA rescue of murine B lymphoma cells from anti-IgM-induced growth arrest and programmed cell death is associated with increased expression of c-myc and bcl-xL.
Authors: Yi A K , Hornbeck P , Lafrenz D E , Krieg A M
Published: December 2022

One of the principal mechanisms thought to maintain B cell tolerance to self Ags is deletion of cells bearing functional IgM receptors for self Ag via apoptosis in the bone marrow. Because of its characteristic growth arrest and apoptosis in response to surface IgM cross-linking, the B cell line WEHI-231 has been a useful model system for studies of Ag receptor-mediated apoptosis. Unmethylated CpG dinucleotides in oligonucleotides (CpG DNA) can be strong B cell mitogens. In the present study we evaluated whether CpG DNA can rescue WEHI-231 cells from anti-IgM-induced cell cycle arrest and apoptosis. The addition of CpG DNA protected WEHI-231 cells from anti-IgM-mediated apoptosis as well as growth arrest. The protective effect of CpG DNA was dependent on the presence of unmethylated CpG dinucleotides. Kinetic analyses showed that the addition of CpG DNA can be delayed for up to 3 h after anti-IgM treatment with no decrease in the protection. CpG DNA reversed anti-IgM-induced down-regulation of c-myc expression in WEHI-231 and up-regulated myn, bcl2, and bcl-xL mRNA expression. Our results suggest that CpG DNA protection of WEHI-231 cells from anti-IgM-induced apoptosis may be mediated by specific and/or cooperative interactions of multiple genes and that CpG DNA could be a useful tool for studies of B cell tolerance.
Eosinophils from schistosome-induced hepatic granulomas produce superoxide and hydroxyl radical.
Authors: McCormick M L , Metwali A , Railsback M A , Weinstock J V , Britigan B E
Published: December 2022

Human peripheral blood eosinophils generate superoxide (O2.-) in response to PMA stimulation. These cells are also capable of forming the highly reactive hydroxyl radical (HO.) by a process that is dependent on the presence of active eosinophil peroxidase. To extend this work to tissue-resident cells, we chose to study a murine model of Schistosoma mansoni infection in which parasite ova induce granulomas whose cellular content is 50% eosinophils. In contrast to peritoneal lavage eosinophils, dispersed granuloma cells were unable to reduce ferricytochrome c (as an indicator of O2.-) in response to PMA stimulation. Furthermore, when human neutrophils were pretreated with conditioned medium from the granuloma cells, they also failed to reduce ferricytochrome c following PMA stimulation, implying the existence of an inhibitory factor. However, using a 5,5-dimethyl-1-pyrroline-N-oxide spin-trapping system, we were able to demonstrate significant generation of O2.- in response to PMA stimulation, not only in the granuloma cells, but also in the conditioned medium-treated neutrophils, demonstrating that the inhibitory factor was not affecting O2.- generation, but rather was interfering with ferricytochrome c reduction. In addition, using an alpha-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone/ethanol spin-trapping system, we were able to detect HO. formation by these same cells following PMA stimulation. This HO. formation was inhibited by superoxide dismutase, azide, and thiocyanide, and NaSCN, consistent with a mechanism requiring O2.- and enzymatic peroxidase activity. These results demonstrate that tissue eosinophils associated with the schistosome-induced granuloma have the ability to form both O2.- and HO., and point out potential problems associated with the measurement of O2.- in whole tissue preparations.
Autoantibodies to murine type II collagen in collagen-induced arthritis: a comparison of susceptible and nonsusceptible strains.
Authors: Brand D D , Marion T N , Myers L K , Rosloniec E F , Watson W C , Stuart J M , Kang A H
Published: December 2022

Collagen-induced arthritis (CIA) is an experimental autoimmune disease elicited in genetically susceptible strains of mice by immunization with heterologous type II collagen. This experimental disease is mediated by the immune response of both T cells and B cells, and susceptibility is restricted by the class II molecules of the MHC. In this study we identify specific epitopes bound by autoantibodies elicited through immunization of several haplotypes of C57BL/10 mice with chick alpha1 (II)-CB11. ELISA analysis using a panel of 15-mer murine type II collagen peptides revealed a pattern of autoantibody epitope specificity that was remarkably similar among CIA-susceptible and nonsusceptible congenic strains, regardless of class II haplotype. However, one epitope was identified that was bound only by autoantibodies from CIA-susceptible strains bearing I-A(q) (B10.Q and B10.QbetaBR). In addition, this epitope was also present within affinity-purified Ab obtained from the CIA-susceptible strain DBA/1 (I-A(q)). Analyses of immune serum from B10.Q and B10.QbetaBR mice revealed that a subset of the antibodies binding this epitope were of the IgG2 subclass, and therefore efficient at fixing complement, a requirement for pathogenicity of the Abs in CIA.
Impaired survival and proliferation in IL-7 receptor-deficient peripheral T cells.
Authors: Maraskovsky E , Teepe M , Morrissey P J , Braddy S , Miller R E , Lynch D H , Peschon J J
Published: December 2022

Mice genetically deficient in IL-7R(alpha) are highly lymphopenic in the peripheral lymphoid organs. The functional competence of T cells that have developed in the absence of an IL-7R signal was investigated. Three important observations were made using several in vitro activation regimens. First, stimulation of T cells from IL-7R -/- mice at limiting dilution with immobilized Abs to CD3, CD4 or CD8, and CD18 revealed a six- to sevenfold reduction in the frequency of clonogenic T cells compared with T cells from IL-7R +/+ mice. IL-7R -/- T cells were also significantly less responsive to alloantigen as well as to receptor-independent stimuli such as PMA and ionomycin. Furthermore, the average clone size of single IL-7R -/- T cells was 50% smaller than that of IL-7R +/+ T cells. These data suggest that the reduced clonogenicity was predominantly due to intrinsic deficiencies in the ability of IL-7R -/- T cells to proliferate upon stimulation. Second, analysis of the kinetics of cell growth of IL-7R -/- T cells revealed that a significant proportion of T cells failed to proliferate within the first 72 h of in vitro stimulation, with the majority undergoing programmed cell death. Third, both clonogenic IL-7 -/- T cells and IL-7R +/+ T cells showed a similar proliferative response in the presence of IL-2 and similar survival kinetics, indicating that a subpopulation of IL-7R -/- T cells is functionally mature. We propose that an absence of IL-7R signaling not only affects T cell development in the thymus, but also results in the accumulation of functionally inactive T cells in the periphery.
Cloning of the mouse fusin gene, homologue to a human HIV-1 co-factor.
Authors: Heesen M , Berman M A , Benson J D , Gerard C , Dorf M E
Published: December 2022

Previous studies have demonstrated that mouse cells do not become infected with HIV-1 despite transfection with human CD4. Recently, a human protein termed "fusin" with characteristics of a seven-transmembrane-spanning receptor was found to be a co-factor required for the entry and fusion of HIV-1 with human CD4-bearing lymphocytes. Thus, cloning of the murine homologue of the human fusin (also termed CXCR-4) gene could provide an important comparative tool for identification of the structures crucial for fusin function. Using degenerate PCR, the mouse homologue of human fusin was cloned from a peritoneal exudate cell cDNA library. The predicted amino acid sequence is 91% identical to human fusin. Twenty-eight of the 37 amino acid differences between mouse and human fusin are located in the ectodomains, suggesting that the intracytoplasmic components that mediate G protein binding and signaling are highly conserved. Northern blot analysis showed a message of 2.2 kb in thymus, spleen, neutrophils, and primary astrocyte cultures. Lymphoid and monocyte cell lines also expressed message for fusin. The coding regions of most chemokine receptors lack introns. In contrast, cloning of genomic DNA for mouse fusin revealed the presence of a 2.3-Kb intron separating the first seven amino acids from the remaining 352 residues. Therefore, the mouse fusin gene has a unique genomic organization compared with other chemokine receptors.
A nine-amino acid peptide from IL-1beta augments antitumor immune responses induced by protein and DNA vaccines.
Authors: Hakim I , Levy S , Levy R
Published: December 2022

The idiotypic determinants of B cell lymphoma provide a tumor-specific Ag and a target for immunotherapy. We have developed several generations of idiotype vaccines that were tested in an animal model, the 38C13 mouse B cell lymphoma. Initially we showed that effective tumor immunity was elicited by the syngeneic Id when it was conjugated to a carrier protein and mixed with an adjuvant. A subsequent generation of Id vaccines eliminated the need for a carrier protein and for an adjuvant by incorporating cytokines into fusion proteins containing the Id. A third generation of vaccines consisting of naked DNA encoding the Id-granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion proteins was equally effective in inducing tumor immunity. To determine whether Ig variable regions, in the absence of constant regions, could be immunotherapeutic in this model, we tested the use of single-chain Fv (scFv). scFv proteins, produced in bacteria, and naked DNA encoding scFv were used in this study. scFv was tested alone or fused to GM-CSF or an immunoenhancing peptide derived from IL-1beta. Here we demonstrate that scFv-GM-CSF was effective only when injected as a protein, not as a DNA vaccine. In contrast, both scFv-IL-1beta peptide fusion protein and naked DNA encoding it induced tumor immunity that protected mice from tumor challenge.
Immunodominance analysis of CTL responses to influenza PR8 virus reveals two new dominant and subdominant Kb-restricted epitopes.
Authors: Vitiello A , Yuan L , Chesnut R W , Sidney J , Southwood S , Farness P , Jackson M R , Peterson P A , Sette A
Published: December 2022

In the present study, a systematic analysis of the influenza (Flu) PR8 determinants recognized by H-2b mice was undertaken. A single Db-restricted immunodominant epitope (NP(366)) was previously known in this system. Twenty-three different Flu PR8-derived peptides that bound either Kb or Db molecules in vitro were identified. Sixteen were immunogenic following peptide immunization of C57BL/6 mice, yet CTL induced by peptide immunization recognized PR8-infected target cells only in the case of the NP(366) and NS2(114) epitopes. Similarly, CTL responses following whole-PR8 virus immunization were detected only for the same two determinants. CTL recognizing these dominant epitopes had high avidity for peptide-pulsed target cells, with 5 to 200 pM of peptide required for 30% specific lysis. In contrast, most (80%) of the remaining epitopes were recognized with lower avidity (30% effective concentration in the range of 0.4-50 nM). Repeated in vitro stimulation of primary CTL cultures revealed one additional Kb-restricted epitope (M1(128)). This peptide bound Kb with high affinity (4.6 nM) and induced CTL that effectively recognized PR8-infected cells. These results suggest that 1) this epitope is produced by natural processing in relatively high amounts and 2) low precursor frequency might be related to the subdominant status of the M1(128) epitope. Taken together, these results illustrate the crucial contributions of MHC-binding capacity, and T cell repertoire availability, to the shaping of the repertoire of CTL specificities for Flu Ag virus.
Peripheral blood mononuclear cells of insulin-dependent diabetic patients respond to multiple islet cell proteins.
Authors: Brooks-Worrell B M , Starkebaum G A , Greenbaum C , Palmer J P
Published: December 2022

Insulin-dependent diabetes (IDDM) results from autoimmune destruction of pancreatic beta cells mediated predominantly by cellular effector mechanisms. To date, investigators have studied a limited number of islet cell proteins stimulatory to T cells. However, before development of clinical IDDM, the majority of the beta cells are impaired or destroyed. Thus, numerous proteins from lysed beta cells would be accessible to the immune system of the patient. Our goal was to investigate the PBMC reactivity of IDDM patients to the full spectrum of fractionated human pancreatic islet cell proteins to determine whether numerous islet cell proteins or a select few would be recognized. We observed that PBMCs from IDDM patients responded reproducibly (mean stimulation index, >2.0) to the proteins in all m.w. regions, whereas the mean stimulation index for controls from all m.w. regions was <2.0. Using three different islet protein preparations, PBMC responses of IDDM patients (n = 30) and controls (n = 39) to the islet cell proteins were significantly different. Dose responses were also demonstrated for the lymphocyte reactivity of the IDDM patients (n = 29) vs controls (n = 56) to the islet cell preparations. Proteins, presumably irrelevant to the IDDM disease process, from a human osteosarcoma cell line and normal human spleen cells did not stimulate PBMCs from IDDM patients or controls. Moreover, IDDM patients and controls responded similarly to mitogens and tetanus toxoid. These studies show that at the time of diagnosis of IDDM, PBMCs from IDDM patients are stimulated by a wide array of islet cell proteins.
Specific clonal T cell accumulation in intestinal lesions of Crohn's disease.
Authors: Nakajima A , Kodama T , Yazaki Y , Takazoe M , Saito N , Suzuki R , Nishino H , Yamamoto K , Silver J , Matsuhashi N
Published: December 2022

The initial cellular events that take place at the outset of autoimmune diseases and that may be the most important in terms of understanding their pathogenesis are poorly understood, especially in humans. This is mainly due to the difficulties in the identification of primary lesions and the accessibility to such material. In this respect, it is noteworthy that the appearance of small reddish erosions, known as aphthoid lesions, is known to be an early event in Crohn's disease. In the present study, accumulation of lesion-specific clonal TCR bands in the intestinal lesions of Crohn's disease patients was demonstrated by means of a highly sensitive method based on single strand conformational polymorphism. Such clonal accumulation was demonstrated in both aphthoid and discrete ulcer lesions. There were several TCR BV bands that were present in both aphthoid lesions and discrete ulcer lesions, but some were specific to the aphthoid lesions. In addition, the same aphthoid lesion-specific/dominant T cell clones were found to be expanded in separate aphthoid lesions of a single patient, and were absent from intervening, non-inflamed mucosa. Some of these bands may represent T cell clones activated primarily at the onset of disease.
Restricted TCR repertoire and long-term persistence of donor-derived antigen-experienced CD4+ T cells in allogeneic bone marrow transplantation recipients.
Authors: Vavassori M , Maccario R , Moretta A , Comoli P , Wack A , Locatelli F , Lanzavecchia A , Maserati E , Dellabona P , Casorati G , Montagna D
Published: December 2022

We investigated the contribution of transfer of Ag-experienced donor T cells to the immune reconstitution of allogeneic bone marrow transplantation (BMT) recipients. To this purpose, we used a combination of cell culture methods to isolate tetanus toxoid (TT)-specific T cell clones, and a sensitive and specific heteroduplex analysis to monitor the presence of a particular clonotype using TCR N region sequences. We document that patients after BMT display a small response to TT, entirely accounted for by few donor-derived clones. These patients show a strong polyclonal response to TT vaccination; however, the T cell clones transferred with the transplant can still be detected within the polyclonal T cell lines for up to at least 5 yr after BMT. We also demonstrate that vaccination of donors with TT before BMT results in a more relevant transfer of Ag-experienced T cells, allowing the recipients to mount a strong polyclonal response without need of vaccination. These findings provide a rationale for vaccinating donors to optimize adoptive transfer of protective T cell immunity into recipients, and suggest the possibility of using preventive T cell adoptive therapy in conjunction with marrow infusion.
Tyrosine phosphorylation of Shc is not required for proliferation or viability signaling by granulocyte-macrophage colony-stimulating factor in hematopoietic cell lines.
Authors: Durstin M , Inhorn R C , Griffin J D
Published: December 2022

The receptor for human granulocyte-macrophage (GM)-CSF (GMR) is a heterodimer, consisting of an alpha-chain (GMR alpha) and a beta-chain (GMR beta). While GMR alpha is capable of binding GM-CSF, GMR beta is necessary for signal transduction. Phosphorylation of one or more tyrosine residues in GMR beta is an early event in signaling. We have recently demonstrated that tyrosine 750 (Y750) in GMR beta is a site of GM-CSF-induced phosphorylation and this site may contribute to the maintenance of cellular viability in response to GM-CSF. To investigate possible contributions made by additional GMR beta cytoplasmic tyrosine residues to receptor function, we mutated other selected tyrosine residues to phenylalanine and tested for any defects in signaling. in the present study, we show that Y577 is required for phosphorylation of Shc and an Shc-associated p140 in response to GM-CSF. Y577 is also required for association of Shc with GRB2. Y577 does not appear to be necessary for GM-CSF-induced proliferation and survival. GMR beta with a mutated Y577 is able to transduce signals leading to the activation of the Raf-1 pathway and the Jak-Stat pathway. Interestingly, mutation of Y750 reduced detectable GM-CSF-induced tyrosine phosphorylation of GMR beta, suggesting that the reduction of Shc phosphorylation associated with that mutant might be actually due to a failure to phosphorylate Y577. These data indicate that the phosphorylation of Shc in response to GM-CSF is not required for proliferation or viability signaling in these cells.