bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential contact of disparate class I/peptide complexes as the basis for epitope cross-recognition by a single T cell receptor.
Authors: Loftus D J , Chen Y , Covell D G , Engelhard V H , Appella E
Published: December 2022

In an effort to better understand functional recognition of structurally dissimilar ligands by a single TCR, a model system for studying cross-recognition of disparate peptide/class I complexes was developed using the murine (H-2b) CTL clone AHIII12.2, which is reactive to a human self-peptide (p1049) bound to an HLA-A2.1 molecule. We identified a second complex comprised of a synthetic peptide, designated p1058, bound to H-2Db that is recognized by clone AHIII12.2. In cytolysis assays, dose-response profiles for peptides p1049 and p1058 pulsed onto the appropriate target cells were comparable, suggesting that p1049/A2.1 and p1058/Db form functionally equivalent epitopes. To probe the interaction between each complex and the TCR of AHIII12.2, singly substituted analogues of each peptide were tested for their activity in lysis assays. Differences were observed between the two epitopes with respect to permissible residue substitutions at each peptide position from P3 to P8; marked differences were evident at P3 and at P8. The results obtained suggest that this TCR forms critical contacts with atoms at peptide positions P3 and P5 of p1049/A2.1 and at P5 and P8 of p1058/Db, and that TCR cross-recognition of these ligands is a function of both shared and complex-specific contacts made with each epitope. These findings further highlight the versatile reactivity that may be shown by a single TCR and suggest a basis for the recognition of peptide ligands sharing only a limited set of structural features.
IL-2-induced IL-5 synthesis, but not proliferation, of human CD4+ T cells is suppressed by FK506.
Authors: Mori A , Suko M , Kaminuma O , Inoue S , Ohmura T , Hoshino A , Asakura Y , Terada E , Miyazawa K , Nosaka C , Okumura Y , Ito K , Okudaira H
Published: December 2022

Regulation of T cell IL-5 synthesis was investigated using human Th cell clones. Immunosuppressant FK506 suppressed IL-5 synthesis of T cells activated through TCR in a dose-dependent manner. IL-5 gene transcription and protein synthesis were also induced in the same T cell clones upon stimulation with IL-2 and were suppressed by FK506 in a dose response similar to that induced by TCR stimulation. In contrast to TCR stimulation, neither activating protein-1, nuclear factor-AT (NF-AT), nor NF-kappaB binding activity was significantly up-regulated by IL-2 stimulation. Human IL-5 promoter/enhancer-luciferase gene construct transfected to T cell clones was transcribed upon either TCR or IL-2 stimulation and was clearly down-regulated by FK506, indicating that the approximately 500-bp human IL-5 gene segment located 5' upstream of the coding region contained FK506-sensitive enhancer elements. Our present findings clearly indicate that FK506-sensitive signaling molecules are involved in T cell IL-5 production induced by both TCR and IL-2 stimulation and suggest that IL-2 receptor signal leading to IL-5 gene transcription is transduced by a unique FK506-sensitive pathway other than the Ca2+-dependent signal transduction pathway, such as the calcineurin-NF-AT system.
Platelet-activating factor abrogates apoptosis induced by cross-linking of the surface IgM receptor in a human B lymphoblastoid cell line.
Authors: Toledano B J , Bastien Y , Noya F , Baruchel S , Mazer B
Published: December 2022

B lymphocyte development is characterized by deletion, via apoptosis, of immature cells that are stimulated via the B cell receptor in the absence of a second signal. We have investigated whether platelet-activating factor (PAF), a potent B lymphocyte activator, can provide a complementary signal with B cell receptor ligation to abrogate apoptosis. Cross-linking of the surface IgM on Ramos B lymphoblastoid cells using anti-IgM Abs (2 microg/ml) caused programmed cell death in 34 +/- 5.4% of the cells. Coincubation of PAF (10(-7)M) with alphaIgM led to a significant decrease in apoptotic cells as measured by DNA laddering and TUNEL assay (13.8 +/- 3%). The effect of PAF was dose dependent (10(-7)-10(-9) M) and was inhibited by the specific PAF receptor antagonist, WEB 2170. PAF protected cells from the effect of alphaIgM for up to 1 h after it was added. alphaIgM-induced programmed cell death in Ramos cells was blocked by catalase and, therefore, is caused in part by the production of toxic hydroxyl radicals from hydrogen peroxide. We investigated the action of PAF on markers of intracellular oxidation. H2O2 in low doses induced apoptosis, via production of OH. radicals. PAF inhibited H2O2-induced apoptosis in Ramos cells; it also attenuated H2O2- and alphaIgM-mediated increases in hydroxyl radical (OH.) as measured by the oxidation of 2',7'-dichlorofluorescein diacetate to 2',7'-dichlorofluorescein and blocked the depletion of reduced glutathione induced by alphaIgM. PAF maintained IgM secretion, which was greatly inhibited by incubation with alphaIgM alone. These data indicate that PAF potentially provides an important cosignal to surface IgM-stimulated Ramos cells by inhibiting apoptosis. This is in part due to the activity of PAF in the oxidant/ antioxidant pathway.
Bone marrow can function as a lymphoid organ during a primary immune response under conditions of disrupted lymphocyte trafficking.
Authors: Tripp R A , Topham D J , Watson S R , Doherty P C
Published: December 2022

In this study we sought to better understand lymphocyte trafficking patterns and the function of secondary lymphoid organs, such as the spleen, during the generation of virus-specific T cell precursors. Treatment of mice with the Mel-14 mAb to CD62L, the lymph node homing receptor, limits trafficking of naive T cells into lymph nodes through high endothelial venules. Administering Mel-14 following respiratory infection with influenza virus forced the generation of primary virus-specific CD4+ and CD8+ T cell precursors from the mediastinal lymph nodes to the spleen. However, splenectomy did not seriously impede virus clearance from the lung and, despite a substantial reduction of the total lymphocyte pool, the acute T cell responses in the regional lymph nodes were largely normal. Mel-14 treatment of splenectomized mice did not affect clonal expansion of the virus-specific T cells in the MLN, while the response in the cervical lymph nodes was still greatly inhibited. More surprisingly, virus-specific T cell precursors were now detected from days 5 to 6 after infection in the bone marrow (BM) of the splenectomized, Mel-14-treated mice. This was not due to contamination with circulating T cells or infection of BM cells because the distribution profiles of precursor T cells for PBL and BM diverged and PCR analysis showed no evidence of virus replication in the BM. It appears that, under these conditions of disrupted lymphocyte trafficking, the BM can supplant the secondary lymphoid tissue either as a site of primary immune response or as a cache for excess T cell precursors.
Modifications of peptide ligands enhancing T cell responsiveness imply large numbers of stimulatory ligands for autoreactive T cells.
Authors: Vergelli M , Hemmer B , Kalbus M , Vogt A B , Ling N , Conlon P , Coligan J E , McFarland H , Martin R
Published: December 2022

In this report, we demonstrate for autoreactive T cell clones that single amino acid modifications of the antigenic ligand can result in not only abrogated, decreased, or unmodified, but also increased, T cell responsiveness (superagonist ligands). We further studied the effects of combinations of multiple substitutions with different effects in single peptides. Experiments with peptides carrying multiple amino acid exchanges revealed that the final outcome of TCR ligation by a given ligand is the integration of negative, neutral, and positive effects of each single residue. In addition, the introduction of superagonist substitutions together with nonconservative modifications of primary and secondary TCR contacts resulted in stimulatory ligands. These findings indicate that: 1) the specificity of a single TCR is highly degenerate; 2) ligands exist for autoreactive T cells that have higher agonist activity than the autoantigen itself; 3) the rules to search for cross-reactive epitopes in autoimmunity should take into account that amino acids at certain positions within an antigenic peptide may exert superagonist activity and compensate for the negative effects of residues at other positions that would otherwise not be tolerated.
Carbohydrate and peptide specificity of MHC class II-restricted T cell hybridomas raised against an O-glycosylated self peptide.
Authors: Jensen T , Hansen P , Galli-Stampino L , Mouritsen S , Frische K , Meinjohanns E , Meldal M , Werdelin O
Published: December 2022

MHC class II E(k)-restricted, IL-2 secreting T cell hybridomas were raised against the synthetic glycopeptide Hb(67-76)-alpha-GalNAc, (T72(Tn)), in CBA/J mice (H-2(k)). The fine specificity of the hybridomas against the glycan moiety was investigated by testing their response against a panel of Hb(67-76)-derived glycopeptides, all with a glycan attached to serine or threonine at the position 72 in the peptide, but with different glycans. The hybridomas showed a high degree of specificity for the alpha-GalNAc moiety with few and faint cross-responses to the glycopeptides having other glycans attached even though some of these were structurally very similar to alpha-GalNAc. The fine specificity of the hybridomas for the peptide moiety was investigated by testing their responses to a panel of Hb(67-76)-alpha-GalNAc glycopeptides with alanine substitutions at all positions except at the two MHC binding anchor positions, I68 and K76, and the T72 to which the alpha-GalNAc was attached. Glycopeptides substituted with alanine at positions where the amino acid side chain pointed toward the TCR did not stimulate the hybridomas, whereas glycopeptides substituted with alanine at positions orientated down into the MHC binding groove stimulated many of the hybridomas. These results indicate that the glycan attached to the peptide as well as solvent-accessible parts of the peptide are recognized with a high degree of specificity by the T cells, whereas the parts of the peptide buried in the MHC binding site are less important or totally ignored by the T cells.
Diversity of the cytotoxic melanoma-specific immune response: some CTL clones recognize autologous fresh tumor cells and not tumor cell lines.
Authors: Dufour E , Carcelain G , Gaudin C , Flament C , Avril M F , Faure F
Published: December 2022

In the present work, to analyze the heterogeneity of the tumor-specific cytotoxic immune response, a large number of T cell clones were generated from the infiltrate of a tumor-proximal invaded lymph node, and two kinds of melanoma-specific CD8+ CTL clones were derived. The majority of T cell clones (about a hundred) are characterized by a specific lysis of the autologous tumor cell lines. Among 34 of the latter clones, HLA-A2 molecule and MART-1(27-35) peptide have been shown to play a predominant role in tumor recognition. However, no significant amplification at the tumor site was observed for 3 of these CTL. The other kind of tumor-specific CTL (1 oligoclonal and 2 clonal cell lines) did not lyse the autologous melanoma cell lines but lysed the "fresh" autologous tumor cells in a MHC class I-dependent manner. Functional analysis of the two different CTL clones have shown that they did not lyse NK targets, autologous peripheral monocytes, activated T cells, and transformed B cells or any of the few allogeneic cultured and uncultured melanoma cells we tested. TCR repertoire analysis has shown that one of these CTL clones was significantly detectable "in situ" among tumor-infiltrating lymphocytes, while not detectable among PBMC. Such melanoma-specific lymphocytes, which could not have been picked out through conventional screening procedures using tumor cell lines, could potentially play a role in tumor rejection. These results suggest that the immune response analyzed toward melanoma cell lines does not totally reflect the in situ immune status.
Intracellular localization of the human receptor for the globular domains of C1q.
Authors: van den Berg R H , Prins F , Faber-Krol M C , Lynch N J , Schwaeble W , van Es L A , Daha M R
Published: December 2022

This study was performed to determine the localization of the recently described receptor for the globular domain of C1q, gC1qR. In contrast to previous reports, we were not able to detect significant surface expression of gC1qR on Raji cells, monocytes, neutrophils, human or rat mesangial cells, the endothelial cell line EA.hy 926, or HUVEC using FACS analysis. Only by using digoxigenin-conjugated Abs could some surface staining of gC1qR be observed on rat mesangial cells and neutrophils. However, after permeabilizing these cells with saponin, a strong positive intracellular staining for gC1qR was observed by FACS, fluorescence microscopy on coverslips, and confocal laser scanning microscopic analysis. By reflection contrast microscopy and electron microscopy on ultrathin sections of permeabilized Raji cells, it was shown that gC1qR is present in double membranous cytoplasmic vesicles located in the proximity of the plasma membrane. To determine whether certain conditions could induce surface expression of gC1qR, Raji cells were either stimulated with T cell growth factor, LPS, or driven to apoptosis by incubation with fenretinide or by serum depletion. None of the conditions resulted in significant surface expression of gC1qR. Our hypothesis that gC1qR is not a surface molecule but a soluble molecule that is secreted by cells is supported by the observation that gC1qR is found in significant concentrations in supernatants of several cultured cells and in normal human and rat sera. Our results suggest that the recently described gC1qR is not a cell surface receptor, but a soluble binding protein with affinity for the globular heads of C1q. Excreted gC1qR might act as a potential fluid phase regulator of complement activation.
Initiation of autoimmune diabetes in NOD/Lt mice is MHC class I-dependent.
Authors: Serreze D V , Chapman H D , Varnum D S , Gerling I , Leiter E H , Shultz L D
Published: December 2022

MHC class II alleles clearly contribute a primary genetic component of susceptibility to autoimmune insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. However, IDDM does not occur in NOD mice made MHC class I-deficient by a functionally inactivated beta2-microglobulin allele (beta2m(null)). In the present study the beta2m(null) mutation was used to examine the relative contributions of MHC class I and class II-dependent T cell responses for initiating autoimmune pancreatic beta cell destruction in NOD mice. Splenocytes from diabetic NOD donors transferred IDDM to both lymphocyte-deficient NOD-scid (class I+) and NOD-scid.beta2m(null) mice (class I-). In contrast, splenocytes from young prediabetic NOD donors only transferred IDDM to class I+, but not class I- NOD-scid recipients. However, splenocytes from prediabetic NOD donors did transfer IDDM to NOD-scid.beta2m(null) recipients previously engrafted with class I+, but not class I-, pancreatic islets. CD4+ T cell lines reactive against some syngeneic class I+ targets could be isolated from NOD.beta2m(null) mice. However, NOD.beta2m(null) T cells underwent activation-driven deletion when transferred into class I+ NOD-scid recipients. Hence, the class I autoreactive T cells present in NOD.beta2m(null) donors did not elicit IDDM when transferred into class I+ NOD-scid recipients. Collectively, these results show that autoimmune IDDM in NOD mice is initiated by MHC class I-dependent T cell responses, but this leads to the subsequent activation of additional T cell populations that can mediate pancreatic beta cell destruction in a MHC class I-independent manner.
Differential regulation of T cell activation for primary versus secondary proliferative responses.
Authors: Gupta M , Satyaraj E , Durdik J M , Rath S , Bal V
Published: December 2022

T cell activation in vivo results in proliferation and generation of effector cytokine-secreting cells, as also in development of memory cells that mount enhanced responses upon restimulation. However, differences in the signals promoting generation of effector vs memory T cells are not yet characterized. In this study, using various strategies to modulate an allorecognition system for priming human T cells in vitro, we show that there are indeed differences between the signaling requirements for a first proliferative response and those for priming T cells for enhanced recall proliferative responses. Using APCs fixed with varying concentrations of paraformaldehyde, we show that the loss of ability of these APCs to generate a first response is not matched by a similar loss in their ability to prime responder T cells for recall responses. Prevention of DNA replication during T cell priming with aphidicolin, a DNA polymerase inhibitor, is not inimical to successful T cell priming. Thus, clonal expansion during priming is less crucial than the primed activation status of T cells for the enhanced recall response. We also show that pentoxifylline, a phosphodiesterase inhibitor, inhibits the primary proliferative response, but its presence during priming enhances the recall response capabilities of T cells. On the other hand, the presence of the calcineurin inhibitor cyclosporin A during priming reduces the efficiency of priming, but at low concentrations it induces, like pentoxifylline, enhancement in recall response capability. These findings have significant implications in designing immunosuppressive therapy and in the analysis of signals for T cell memory commitment.
Role of CD3gamma and CD3delta cytoplasmic domains in cytolytic T lymphocyte functions and TCR/CD3 down-modulation.
Authors: Luton F , Buferne M , Legendre V , Chauvet E , Boyer C , Schmitt-Verhulst A M
Published: December 2022

TCR engagement leads to down-modulation of TCR/CD3 complexes from the T cell surface. The importance of this effect in T cell physiology is unknown. Here, we characterized a CTL clone deficient in TCR/CD3 surface expression that had lost both CD3delta and CD3gamma mRNA, allowing us to address the role of these chains in the assembly, signaling, and dynamics of the TCR/CD3 complex. Expression of either CD3delta or CD3gamma alone failed to reconstitute surface expression of the TCR/CD3 complex, but reconstitution with a cytoplasmically truncated CD3delta (delta t) and a native (gamma) or cytoplasmically truncated (gamma t) human CD3gamma led to reexpression of TCR/CD3 complexes in both cases. This indicated that CD3delta and CD3gamma assume specific functions in TCR/CD3 assembly independently of their cytoplasmic domains. The delta t gamma t variant specifically killed target cells, expressed the IFN-gamma gene in response to Ag, and produced TNF-alpha in response to anti-CD3 mAb, but it was affected in CD3 ligand-induced TCR/CD3 down-modulation. Both PMA- and CD3 ligand-induced TCR/CD3 down-modulation were defective in the delta t gamma t variant, whereas the delta t gamma variants were unaffected, and previously described delta gamma t variants were affected only in PMA-induced down-modulation. Specific protein kinase C (PKC) inhibitors indicated that PMA- but not CD3 ligand-induced down-modulation was dependent on PKC activity. Thus, amino acid sequences present in either the CD3delta or CD3gamma cytoplasmic domain control ligand-induced TCR/CD3 down-modulation, and neither these sequences nor this property are required for cytolysis and IFN-gamma gene expression in response to Ag.
Regulation of human T lymphocyte coactivation with an alpha4 integrin antagonist peptide.
Authors: McIntyre B W , Woodside D G , Caruso D A , Wooten D K , Simon S I , Neelamegham S , Revelle J K , Vanderslice P
Published: December 2022

The cyclic hexapeptide CWLDVC (TBC 772) is an antagonist of alpha4 integrins and a potent inhibitor of lymphocyte interactions with fibronectin, vascular cell adhesion molecule-1, and muscosal vascular addressin cell adhesion molecule-1 (MAdCAM-1). As such, peptide TBC 772 effectively inhibits the activation of freshly isolated human T lymphocytes stimulated with purified vascular cell adhesion molecule-1 coimmobilized with anti-CD3 mAb. The influence of peptide binding on distinct sites of the alpha4beta1 complex was determined by flow cytometry and cellular adhesion assays employing a panel of mAbs. Binding of the alpha4-specific mAb L25 and the beta1-specific mAb 33B6 was not altered by the peptide; however, binding of mAb 19H8, which is specific for a combinatorial epitope of alpha4beta1, was dramatically inhibited. Treatment of lymphocytes with the peptide caused an increase in a ligand-induced epitope on beta1 integrin defined by mAb 15/7. In T cell activation studies using coimmobilized anti-CD3 mAb and the anti-integrin mAbs, the peptide had broader inhibitory activity, suppressing costimulation induced by all the integrin mAbs. The peptide was not generally toxic and was integrin selective in its suppressive activity, as coactivation by ligation of CD3 in conjunction with CD28 or CD26 was not affected. These results suggest that the antagonist peptide CWLDVC can effectively neutralize integrin coactivation systems by a mechanism independent of competitive binding.
Differential effects of Flk-2/Flt-3 ligand and stem cell factor on murine thymic progenitor cells.
Authors: Moore T A , Zlotnik A
Published: December 2022

Most primitive thymic progenitors, termed CD4(low) cells (CD25- CD44+ CD117+), retain the ability to generate multiple lymphoid lineages. T cell lineage commitment occurs as CD4(low) cells differentiate into pro-T cells (CD25+ CD44+ CD117+). We previously reported that the in vitro cytokine responses of CD4(low) and pro-T cells differ. While Flt-3 ligand (Flt-3L) has been shown to be involved in early bone marrow hemopoiesis, its role in thymopoiesis has not been thoroughly examined. Here, we report that Flt-3L has no significant effect on pro-T cells, either by in vitro proliferation or in fetal thymic organ culture repopulation. In contrast, CD4(low) cells cultured in vitro for 3 days in IL-3 + IL-6 + IL-7 + Flt-3L generated a twofold increase in cell number 21 days after transfer into fetal thymic organ culture that increased to sixfold by day 35 when compared with the corresponding CD4(low) cells cultured in IL-3 + IL-6 + IL-7 + stem cell factor. Additionally, the Flt-3L-cultured CD4(low) cells displayed fetal thymic organ culture repopulation kinetics that more closely approximated those seen with freshly isolated CD4(low) cells. These data suggest that Flt-3L serves as a self-renewal or proliferation/expansion signal for CD4(low) cells, while the effect of stem cell factor is more likely to transduce a differentiation signal, resulting in more rapid repopulation at the expense of cell expansion.
Induction of IL-4-producing CD4+ T cells by antigenic peptides altered for TCR binding.
Authors: Tao X , Grant C , Constant S , Bottomly K
Published: December 2022

The adaptive immune responses to foreign Ags are primarily regulated by the cytokines produced by CD4 T cells. The generation of distinct cytokine-producing T cell subsets has been shown to be influenced by a number of factors, including cytokines, different types of APCs, and the amounts of priming Ag. We have previously reported that the affinity of an antigenic peptide for its presenting MHC class II molecules and that different doses of Ag peptide affect the outcome of the functional CD4 T cell response. In the current study, we further examined the impact of the affinity of an antigenic peptide for its TCR on CD4 T cell priming. We generated a panel of Ag peptide variants mutated at positions known to be critical for binding to a well-characterized TCR (known as altered peptide ligands, or APLs). Compared with the WT peptide, these APLs are defective in stimulating the proliferative responses of T cells. However, they can effectively prime in vitro naive CD4 T cells for differentiation into both Th1-like and Th2-like cells. In contrast, the WT peptide primes only for IFN-gamma-producing Th1-like cells. Using highly purified dendritic cells as APCs to present the APL or WT peptide leads to the same pattern of priming as using total splenic APCs. These results indicate that priming by APLs for both IL-4 production and IFN-gamma production does not require two different types of APCs. In summary, our data indicate that APL can directly stimulate naive CD4 T cells to become Th2 effector cells.
Functional characterization of the interaction between the superantigen staphylococcal enterotoxin A and the TCR.
Authors: Antonsson P , Wingren A G , Hansson J , Kalland T , Varga M , Dohlsten M
Published: December 2022

In this report, we show that despite an overall amino acid residue identity of more than 80% between the staphylococcal enterotoxins (SE) A and E, these proteins markedly differ in their absolute requirement for the MHC class II during T cell activation. The superantigens were produced as C215Fab-SE fusion proteins and analyzed for their ability to activate T cells in a MHC class II-independent manner, using C215 Ag expressing cell lines as pseudo super-APCs. C215Fab-SEA, but not C215Fab-SEE, induced T cell cytotoxicity and proliferation in these MHC class II-independent systems. Introduction of a region from SEA, comprising amino acids 20-27, to SEE transferred the ability to engage T cells in the absence of MHC class II. Analysis of the Vbeta specificity of the chimeric SEA/SEE molecules and a panel of SEA mutants demonstrated that the site for TCR interaction covers the edge surrounding the shallow cavity on top of the SEA molecule.
Two-hybrid cloning of a gene encoding TNF receptor-associated protein 2, a protein that interacts with the intracellular domain of the type 1 TNF receptor: identity with subunit 2 of the 26S protease.
Authors: Dunbar J D , Song H Y , Guo D , Wu L W , Donner D B
Published: December 2022

A protein that binds the intracellular domain of the type 1 TNFR (TNFR-1IC) has been identified by two-hybrid cloning. The 97-kDa TNFR-associated protein, TRAP2, shows sequence identity with internal amino acid sequences from subunit 2 of the 26S protease. TRAP2 antiserum recognizes subunit 2 of the 26S protease, which is consistent with the identity of these proteins. TRAP2 antiserum interacted with the 97-kDa protein in HeLa cell lysates and cytosol, the latter observation showing that TRAP2 resides in the same cellular compartment as TNFR-1IC. A fusion of glutathione-S-transferase and TNFR-1IC (GST-TNFR-1IC) precipitated TRAP2 from a HeLa cell lysate; conversely, GST-TRAP2 precipitated TNFR-1 from such a lysate. These observations show that the proteins interact in the cellular milieu. After in vitro transcription/translation and 35S labeling, TRAP2 was precipitated from a cellfree system by GST-TNFR-1IC, showing that TNFR-1IC and TRAP2 interact directly. TRAP2 was also precipitated from the cellfree translation system by a GST fusion containing the N-terminal half of TNFR-1IC, but not by a GST fusion containing the C-terminal half of TNFR-1IC that contains a "death domain" that plays an obligatory role in signaling cytotoxicity. The ability of deletion mutants of TNFR-1IC to interact with TRAP2 was tested using the two-hybrid system. This also showed that the amino acid sequences that mediate binding reside outside of the death domain in TNFR-1IC. The demonstration that a subunit of the 26S protease binds TNFR-1 may identify a novel TNF-signaling pathway.
Restricted V gene repertoire in the secondary response to insulin in young BALB/c mice.
Authors: OYu Tikhomirov , Thomas J W
Published: December 2022

Previous data on the genetic origin of insulin Abs in BALB/c mice are derived from immune responses following a single or primary immunization. This primary response, however, may not represent the repertoire established by chronic insulin administration, which is used in the treatment of diabetes mellitus and in protocols to alter beta cell destruction in pre-insulin-dependent (type 1) diabetes mellitus. In this study, the genetic composition and structure of 16 anti-insulin mAbs from the secondary response of a single BALB/c mouse were determined. In contrast to studies on other protein Ags, the V genes in the secondary response to insulin show only a low level of somatic mutation and the majority of the response is represented by restricted sets of V(H) and Vkappa genes. The restriction of V genes is not chiefly due to oligoclonal expansion but reflects recurrent usage of structurally similar V regions of independent origin. Secondary anti-insulin V(H) genes in 12 of 16 mAbs are derived from related J558 subsets, and 8 of 16 mAbs used the Vkappa19.34 gene without mutations in CDRLs. The CDHR3s of half of the mAbs are generated from an uncommon second reading frame of DSP2, suggesting that the amino acid motif STMIT may contact insulin and contribute to selection of these B cells. A total of 14 of 15 mAbs bind autologous rodent insulin with 40 to 100% of the activity on human insulin, and all mAbs bind proinsulin. These data show that a preferred set of V(H), D, and Vkappa gene segments dominate secondary responses to insulin and suggest that specific structural interactions with epitopes that are shared with autologous insulin shape this secondary response.
Identification of a natural T cell epitope presented by Salmonella-infected macrophages and recognized by T cells from orally immunized mice.
Authors: Cookson B T , Bevan M J
Published: December 2022

Murine infection with Salmonella typhimurium provides models for typhoid fever and long-lasting protective immunity conferred by oral vaccination with viable attenuated bacteria. To further understand the role of T cells in these systems, we identified a bacterial Ag recognized by murine T cells responding to a Salmonella infection. From orally infected mice, we derived a CD4+ Ak-restricted T cell clone (7.4.8) the stimulatory Ag of which was provided by S. typhimurium or its flagella, but not by other salmonellae or S. typhimurium mutants unable to synthesize the flagellar filament protein FliC. We mapped antigenic activity to FliC hypervariable region VI using a generally applicable method of sequential C-terminal truncation of recombinant MalE-FliC fusion proteins. Residues 339-350 are the minimal FliC structure capable of stimulating 7.4.8 and represent the first reported Salmonella-specific epitope recognized by T cells from infected mice. T cells with this specificity are generated by oral immunization, reactivity can be recovered for at least 5 mo afterwards, and FliC is the dominant recall Ag for CD4+ T cells from protectively immunized C3H/HeJ mice. FliC 339-350 is presented by macrophages infected with viable S. typhimurium, and presentation, but not bacterial uptake, is greatly enhanced by pretreatment of macrophages with IFN-gamma. These data point to the importance of IFN-gamma-activated macrophages in the stimulation of T cells responding to facultative intracellular pathogens like S. typhimurium and provide a model system for studying Ag-specific T cell responses in murine salmonellosis.
B cell-independent selection of memory T cells after mucosal immunization with Candida albicans.
Authors: Jones-Carson J , Vazquez-Torres F A , Balish E
Published: December 2022

B cell-deficient mice have normal T cell responses to Ags inoculated systemically; however, it is not known whether they can mount systemic and mucosal T cell responses to Ags through normally B cell-enriched gastrointestinal mucosae. Mucosal colonization of germfree, B cell-deficient J(H)D mice with the pathogenic gastrointestinal fungus, Candida albicans selected splenic CD4+ and CD8+ TCR alphabeta memory T cells, as indicated by 1) increased numbers of splenic CD4+ and CD8+ TCR alphabeta expressing T cells of the CD45RB(low) CD44(high) phenotype, 2) early expansion followed by progressive decrease in the number of splenic CD4+ and CD8+ TCR alphabeta T cells, and 3) concomitant increases in the percentage of apoptosis and proliferation in the latter subsets. Although i.v. challenge of germfree or conventional J(H)D mice with C. albicans did not increase apoptosis or induce changes in the number of splenic memory T cells, i.v. challenge of mucosally immunized germfree J(H)D mice led to further proliferation and expansion of activated splenic CD4+ and CD8+ TCR alphabeta thymic-educated memory T cells, which were first evoked by mucosal immunization. Oral colonization with C. albicans also increased the number of gammadelta and thymic and extrathymic alphabeta T cells in gastrointestinal mucosae. In conclusion, our results are the first strong evidence that thymic and extrathymic T cells participate in mucosal immunity to C. albicans in the absence of B cells; however, CD4+ and thymic-educated CD8+ TCR alphabeta memory subsets evoked by mucosal, but not parenteral (i.v.), challenge contribute to protective immunity to systemic candidiasis.
L-selectin shedding does not regulate human neutrophil attachment, rolling, or transmigration across human vascular endothelium in vitro.
Authors: Allport J R , Ding H T , Ager A , Steeber D A , Tedder T F , Luscinskas F W
Published: December 2022

Current models of the multistep adhesion cascade for leukocyte-endothelial interactions predict loss of L-selectin from the leukocyte surface before transendothelial migration. We have tested this hypothesis using in vitro adhesion and transendothelial migration assays and a zinc-dependent metalloproteinase inhibitor, Ro 31-9790 (N-2-((2s)-[(hydroxycarbamoyl)methyl)-4-methylvaleryl]-N-1,3 -dimethyl-L-valinamide), which prevents chemoattractant-induced (e.g., IL-8, FMLP, C5a, platelet-activating factor) L-selectin endoproteolytic cleavage from isolated human neutrophils. Inhibitor and vehicle-treated neutrophils exhibited identical behavior during both adhesive interactions with 4- and 24-h TNF-alpha-activated HUVEC monolayers under flow, (including rate of initial attachment, rolling velocities, stable adhesion, and transmigration) and in static adhesion assays. Flow cytometric analysis of transmigrated neutrophils with mAb to L-selectin revealed that vehicle treated neutrophils had minimal detectable surface L-selectin, whereas inhibitor-treated neutrophils retained comparable levels of L-selectin on their surface as neutrophils maintained at 37 degrees C. In addition, mAb to L-selectin that induce rapid shape change and homotypic adhesion (LAM1-116) did not enhance the rate or extent of neutrophil transmigration under flow or static conditions. Neutrophils preincubated with LAM 1-116 displayed similar behavior to neutrophils preincubated with the control anti-L-selectin mAb, LAM1-101. In summary, these results demonstrate that there is no requirement for L-selectin to be shed from the surface of neutrophils before, or during, their migration across endothelial monolayers, and that prevention of surface L-selectin proteolytic cleavage does not enhance or inhibit neutrophil-endothelial cell adhesive interactions.
Glucocorticoids inhibit the cytokine-induced proliferation of mast cells, the high affinity IgE receptor-mediated expression of TNF-alpha, and the IL-10-induced expression of chymases.
Authors: Eklund K K , Humphries D E , Xia Z , Ghildyal N , Friend D S , Gross V , Stevens R L
Published: December 2022

Mast cells are a heterogeneous family of immune cells that, when activated through their high affinity IgE receptors (Fc epsilonRI), release various granule mediators (e.g., neutral proteases and serglycin proteoglycans) and proinflammatory cytokines (e.g., IL-6 and TNF-alpha). We and others have shown that the growth and differentiation of immature, nontransformed mouse bone marrow-derived mast cells (mBMMC) can be regulated in vitro by IL-3, IL-10, and c-kit ligand. We now report that glucocorticoids inhibit the c-kit ligand- and IL-3-induced proliferation of mBMMC, the Fc epsilonRI-mediated expression of TNF-alpha, and the IL-10-mediated expression of the two chymases designated mouse mast cell protease (mMCP)-1 and mMCP-2. In contrast, glucocorticoids induce mBMMC to increase their expression of serglycin proteoglycan and carboxypeptidase A. As assessed by nuclear run-on and RNA blot analyses, dexamethasone inhibited the IL-10-mediated expression of mMCP-1 and mMCP-2, primarily by inducing rapid degradation of their transcripts. The stimulative effect on serglycin proteoglycan expression and the inhibitory effect on chymase expression were dose and time dependent and glucocorticoid specific. These findings indicate that glucocorticoids exert profound and diverse effects on the growth, cytokine expression, and granule differentiation of mouse mast cells, and that at least some of this regulation occurs through a post-transcriptional mechanism.
Activation of Ras and mitogen-activated protein kinase pathway by terminal complement complexes is G protein dependent.
Authors: Niculescu F , Rus H , van Biesen T , Shin M L
Published: December 2022

Assembly of terminal complement complexes (TCC) C5b-7, C5b-8, and C5b-9 on target cells during acute and chronic inflammation induces hydrolysis of plasma membrane phospholipids and heterotrimeric G protein activation. TCC also stimulate a variety of cellular activities, which include cytokine synthesis, proto-oncogene activation, and mitotic signaling. Now we report that sublytic TCC induced Ras, Raf-1, and extracellular signal-regulated kinase (ERK) 1 activation in JY25 B cell line. When cells were exposed to C5b-9, GTP-bound Ras in anti-C5b-9 immunoprecipitates was increased 3.2-fold at 2 min, while GTP-bound Ras in anti-Ras immunoprecipitates was increased 2-fold at 10 min. Both C5b-9 and C5b-7, but not C5b6, increased Raf-1 kinase activity maximum 3.3-fold at 2 min and 2.8-fold at 5 min, respectively. ERK1 activity was 2-fold increased by C5b-9 at 2 min and by C5b-7 at 10 min, over the C5b6 level. The role of mitogen-activated protein kinase (MAPK) pathway on TCC-inducible mitotic signaling was evaluated by assessing DNA synthesis and activator protein 1 (AP-1) DNA-binding activity. The MAPK/ERK-specific inhibitor PD 098,059 abolished the C5b-9-induced DNA synthesis. Involvement of G protein in the activation of MAPK pathway by TCC was indicated by inhibition of Raf-1 and ERK1 kinase activity, as well as the DNA synthesis by pretreatment of cells with pertussis toxin. Overexpression of beta-adrenergic receptor kinase 1 carboxyl-terminal peptide in JY25 cells also inhibited Raf-1 and ERK1 activity, indicating a direct involvement of G betagamma subunits in the signal transduction generated through activation of MAPK pathway by TCC assembly in the plasma membrane.
Expanded CD4+ and CD8+ T cell clones in elderly humans.
Authors: Schwab R , Szabo P , Manavalan J S , Weksler M E , Posnett D N , Pannetier C , Kourilsky P , Even J
Published: December 2022

The diversity of the human TCR repertoire in aging has been studied by examining the profiles of complementarity-determining region 3 (CDR3) sizes expressed by the BV families. The TCRBV CDR3 profile, which shows size heterogeneity in young adult humans, is significantly restricted in aged humans. Clonal T cell expansions were identified using a PCR-based approach, in one or more BV families from all 14 healthy persons over the age of 65 that we studied. CD4+ T cell expansions were identified in 8 of 11 donors and CD8+ T cell expansions in 7 of 10 donors. These clonal expansions were stable during a 2-year period. Interestingly, more than half of the aged persons had clonal expansions within the BV3, -14, -16, and -23 families. Although there was no homology among the eight CDR3 sequences identified in clonal T cells from 8 aged persons, selective pressure on the expanded T cell clones was suggested by the fact that the BV families used by the T cell clones were not proportional to the number of genes in the different BV families.
Molecular properties of anti-DNA induced in preautoimmune NZB/W mice by immunization with bacterial DNA.
Authors: Wloch M K , Alexander A L , Pippen A M , Pisetsky D S , Gilkeson G S
Published: December 2022

To elucidate the mechanism of Ag drive in the anti-DNA response, the Ab response to bacterial DNA has been analyzed in normal and autoimmune mice. Preautoimmune NZB/W mice immunized with Escherichia coli dsDNA produce Abs that resemble spontaneous autoantibodies and bind mammalian dsDNA. In contrast, normal mice, when immunized similarly, produce Abs that bind only bacterial dsDNA. To characterize further the responsiveness of NZB/W mice to bacterial DNA, we determined the molecular properties of mAbs from preautoimmune NZB/W mice immunized with E. coli DNA. Of nine Abs studied, all were IgM and all bound mammalian ssDNA, while four had appreciable reactivity with mammalian dsDNA. The induced anti-dsDNA resembled spontaneous anti-DNA from autoimmune mice in V gene utilization and V(H) CDR3 arginine content. These Abs lacked evidence of somatic mutation, however, indicating that affinity maturation via somatic mutation is not essential for dsDNA reactivity. The findings suggest that preautoimmune NZB/W mice have immunoregulatory defects that allow activation of mammalian dsDNA reactive B cells by bacterial DNA.
TGF-beta down-regulates stromal IL-7 secretion and inhibits proliferation of human B cell precursors.
Authors: Tang J , Nuccie B L , Ritterman I , Liesveld J L , Abboud C N , Ryan D H
Published: December 2022

Development of lymphoid progenitors in vivo requires interaction with a bone marrow stromal microenvironment containing multiple cytokines involved in the development of nonlymphoid hemopoietic lineages. We tested the effect of one such cytokine, TGF-beta, on the proliferation of early human clonogenic lymphoid progenitors using a stroma-dependent in vitro culture system. TGF-beta caused a dose-dependent inhibition of lymphoid progenitor colonies that was reversible at low TGF-beta doses by addition of exogenous IL-7 to the cultures. IL-7 was unable to reverse the inhibitory effect of higher TGF-beta concentrations or inhibition caused by IL-1alpha, IL-4, or TNF-alpha. Stromal IL-7 mRNA expression and protein secretion were markedly down-regulated by TGF-beta, suggesting that inhibition of stromal IL-7 secretion partially accounts for the inhibitory effect of TGF-beta on lymphopoiesis in this culture system. It is likely that higher TGF-beta concentrations do not inhibit lymphopoiesis by down-regulating IL-7 receptor expression, since this cytokine did not reduce IL-7R alpha or gamma c mRNA levels in normal B cell precursors. Since direct stromal contact is required for in vitro lymphopoiesis, the potential regulation of the IL-7 pathway by cell adhesion was examined. Adhesion of human B cell precursors to stroma did not alter stromal IL-7 expression or expression of IL-7R alpha or gamma c-chains by B cell precursors. These results indicate that TGF-beta is a significant negative regulator of stroma-dependent proliferation of early human lymphoid progenitors and acts in part by down-regulating stromal IL-7 secretion.
Requirement of CD4 T cells for skin graft rejection against thymus leukemia (TL) antigen and multiple epitopes on the TL molecule recognized by CD4 T cells.
Authors: Tsuji K , Obata Y , Takahashi T , Arata J , Nakayama E
Published: December 2022

By selective depletion of CD4 and CD8 T cells in vivo using the respective mAbs, we demonstrate that CD4 T cells are necessary for skin graft rejection against thymus leukemia (TL) Ag. The skin expressing T3b-TL Ag from transgenic C3H Tg.Con.3-1 mice given chimeric H-2Kb/T3b-TL gene was rejected when grafted onto C3H/He recipient mice. Depletion of CD4, but not of CD8, T cells blocked rejection. CD8 CTL were generated in MEM (control)-treated C3H/He recipient mice, while Thy-1+ CD4- CD8- CTL were generated in CD8-depleted recipient mice after rejection. However, no CTL were generated in CD4-depleted or both CD4- and CD8-depleted recipient mice. Thus, the generation of both CD8 and Thy-1+ CD4- CD8- CTL was dependent on CD4 T cells. Ab blocking indicated that both CD8 and Thy-1+ CD4- CD8- CTL were TCR alphabeta and recognized TL Ag. We furthermore demonstrated that CD4 T cells in spleen cells from C3H/He mice that had rejected C3H Tg.Con.3-1 skin showed a weak, but significant, proliferative response to in vitro stimulation with mitomycin C-treated C3H Tg.Con.3-1 spleen cells. Analysis of the reactivity of bulk CD4 T cell lines to 73 synthetic overlapping peptides encompassing the entire T3b-TL molecule showed that CD4 T cells recognized multiple epitopes on the T3b-TL molecule in an APC-dependent manner.
TGF-beta inhibits IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 in T lymphocytes.
Authors: Bright J J , Kerr L D , Sriram S
Published: December 2022

Signaling through IL-2R, IL-2 induces tyrosine phosphorylation and activation of Jak-1 and Jak-3 kinases and Stat 3 and Stat 5 transcription factors leading to cell cycle progression of activated T cells from G1 to S phase. TGF-beta is an immunosuppressive cytokine, which inhibits T cell proliferation at G1 to S phase transition. We examined the effect of TGF-beta on IL-2R signal transduction pathway in activated T cells. We show here that treatment of activated T cells with TGF-beta inhibited IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 but not Jak-3 and Stat 3. TGF-beta also inhibited IL-2-induced expression of alpha- and beta-chains of IL-2R and induced apoptotic cell death in T cells. These results suggest that TGF-beta-induced growth arrest and apoptosis are associated with the modulation of IL-2-induced activation of Jak-Stat pathway in T cells.
CD38 ligation results in activation of the Raf-1/mitogen-activated protein kinase and the CD3-zeta/zeta-associated protein-70 signaling pathways in Jurkat T lymphocytes.
Authors: Zubiaur M , Izquierdo M , Terhorst C , Malavasi F , Sancho J
Published: December 2022

CD38 ligation with the specific mAb IB4 induced early and late signaling events in Jurkat T cells, as judged by the transient induction of tyrosine phosphorylation of phospholipase C-gamma1, c-Cbl, zeta-associated protein (ZAP)-70, Shc, extracellular signal-regulated protein kinase-2 (Erk-2) as mitogen-activated protein (MAP) kinase, and increased expression of the activation Ag CD69. In addition, CD38 ligation induced Ras-dependent events such as Erk-2 mobility shift and increased Erk-2 kinase activity. Further evidence that Erk-2 activation is regulated by CD38 ligation was obtained indirectly with the observed induction of Raf-1, Lck, and Sos-1 mobility shifts, processes that are believed to be dependent, at least in part, on MAP kinase activation. Using a protein tyrosine kinase inhibitor, herbimycin A, or a protein kinase C inhibitor, Ro-31-8220, we found that the anti-CD38-induced Erk-2 activation is both protein tyrosine kinase and protein kinase C dependent. CD38 ligation also resulted in increased CD3-zeta tyrosine phosphorylation and its association with ZAP-70. CD38 ligation in a Jurkat Lck-deficient mutant, JCam1, failed to induce substrate tyrosine phosphorylation and activation of Erk-2. These data indicated that in Jurkat T cells, CD38 receptor triggering results in Lck-regulated activation of both Raf-1/MAP kinase and CD3-zeta/ZAP-70/phospholipase C-gamma1 signaling pathways.