bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo tolerization of Th1 lymphocytes following a single feeding with ovalbumin: Anergy in the absence of suppression
Authors: Melamed Doron , Friedman Aharon
Published: March 2007

Oral tolerance is a biologically relevant pathway for inducing peripheral tolerance to foreign antigens. The mechanisms responsible for the tolerant state following feeding with antigen have been shown to involve both anergy and suppression. The demonstration of anergic T lymphocytes following oral tolerance has so far been limited in in vitro systems, and a primary objective of the present study was to provide evidence, in vivo, for the existence of a state of anergy in mice orally fed with ovalbumin (OVA). In addition, it has been shown that peripheral anergy following the intravenous administration of antigen is selectively induced in Th1 lymphocytes. Thus, a second objective of this study was to investigate whether tolerance induced by a feeding regimen known to cause anergy could be selectively limited to Th1 lymphocytes, and whether tolerance induction could be explained by antigen absorption from the gut into the circulation. Oral tolerance was induced by a single feeding with OVA, and was demonstrated by diminished antibody production in vivo, and by reduced cytokine secretion or proliferation in vitro. Anergy, as a mechanism for tolerance, was demonstrated by the ability to reverse the tolerant state after culturing tolerant cells in recombinant interleukin‐2 (rIL‐2). Reversal of the tolerant state in vivo was established by antibody production in irradiated mice adoptively transferred with cells cultured in the presence of rIL‐2. The possibility that suppression was also an in vivo mechanism for tolerance was studied by adoptive transfer experiments. Our results show: 1) that a single dose of orally administered OVA leads to the selective tolerization of Th1 responses (diminished IgG2a, IL‐2 and interferon‐γ production) with intact Th2 responses (IgG1, and IL‐4), 2) that tolerance in vivo is explained by anergy in the absence of active suppression, 3) that exposure of tolerant cells to rIL‐2 in vitro abrogates the anergic state both in vitro (proliferation and cytokine secretion) and in vivo (IgG2a production), and 4) that the induction of oral tolerance is inhibited by the presence of antibodies specific for the tolerizing antigen. These findings indicate that the induction of anergy via the oral route might depend on the dissemination of antigen absorbed from the gut. It is suggested that tolerance is guaranteed by the fact that this absorbed antigen is presented to Th1 lymphocytes in the absence of inflammatory and co‐stimulatory molecules; these foreign antigens are thus not different from self antigens.
Unconventional cytotoxic T lymphocyte recognition of synthetic peptides corresponding to residues 1—23 of Ras protein
Authors: Yin Li , Thomas Claire , Hsuan J. Justin , Stauss Hans J.
Published: March 2007

A peptide corresponding to amino acids 1 through 23 of Ras protein containing a mutation at position 12 was used to induce cytotoxic T lymphocytes (CTL) in mice. Although the CTL were CD8+ and expressed α, β T cell antigen receptors (TCR), their major histocompatibility complex (MHC)‐restriction was unconventional. They recognized peptide‐treated murine cells of different H‐2 haplotypes, but not MHC class I‐negative cells. Human HLA class I molecules did not present Ras peptides and hybrid human/mouse MHC molecules revealed that all three extracellular domains α1, α2 and α3 were required for recognition by peptide‐specific CTL. Shortening the 23‐mer peptide by 5 residues at either the amino or carboxy terminus resulted in loss of CTL recognition. This demonstrates an unusual form of antigen recognition by mouse CTL in which peptide presentation requires murine H‐2 class I molecules but is not class I allele restricted, and the peptides recognized are much larger than peptides in conventional class I‐restricted responses.
Identification of collagen and laminin receptor integrins on murine T lymphocytes
Authors: Miyake Sachiko , Sakurai Tomoko , Okumura Ko , Yagita Hideo
Published: March 2007

In this study we investigated the receptors by which murine lymphocytes bind to collagen and laminin. To identify the collagen and laminin receptors, we generated three monoclonal antibodies, two of which (HMα1 and HMα2) could inhibit adhesion of activated T cells to collagen and laminin and one of which (HMα6) could inhibit that to laminin. Biochemical studies showed that the antigens recognized by HMα1, HMα2, and HMα6 are the mouse homologues of human VLA‐1, VLA‐2, and VLA‐6, respectively. Finally, we demonstrated that both VLA‐1 and VLA‐2 contribute to the functional interaction between collagen and activated T cells, since HMα1 and HMα2 specifically inhibited collagen‐induced TNF secretion from activated T cells. These results indicate that VLA‐1 and VLA‐2 play an important role in regulating adhesion and cytokine production of activated T cells.
The two soluble forms of the lipopolysaccharide receptor, CD14: Characterization and release by normal human monocytes
Authors: Durieux Jean‐Jacques , Vita Natalio , Popescu Octavian , Guette Frederique , Calzada‐Wack Julia , Munker Reinhold , Schmidt Reinhold E. , Lupker Jan , Ferrara Pascual , Ziegler‐Heitbrock H. W. Löms , Labeta Mario O.
Published: March 2007

CD14, a glycolipid‐anchored membrane glycoprotein, acts as a high affinity lipopolysaccharide receptor on leukocytes. We previously reported that the Mono‐Mac‐6 cell line releases two different soluble forms of CD14 (sCD14) (Labeta et al. Eur. J. Immunol. 1993. 23: 2144). Here we show that the two sCD 14, which we now refer to as sCD 14α (low Mr), and sCD 14β (high Mr), are also synthesized and released by normal human monocytes and present in normal plasma. Their mechanism of release was examined by using the Mono‐Mac‐6 cell line, chinese hamster ovary cell (CHO)/CD14+ transfectants and plasma from paroxysmal nocturnal hemoglobinuria (PNH) patients. It was found that: (1) sCD14β is released faster than sCD 14α and that the release of the latter is a lengthy process. (2) Monensin blocked the biosynthesis of membrane‐bound CD14 (mCD 14) and sCD 14, additionally, a 50‐kDa CD14 polypeptide accumulated in the cell lysate, suggesting that the different forms of CD14 may have a common precursor. (3) Monensin also blocked the release of mCD14α from surface‐labeled cells, suggesting that conversion of mCD14 to sCD14α involves a mechanism of endocytosis followed by exocytosis. Interestingly, (4) sCD 14α and sCD 14β were detected in PNH plasma, indicating that sCD14α may also derive from an endogenous pathway. (5) Phospholipase C‐released CD 14 was identical in size to mCD 14, thus differed from sCD 14β by ˜ 2000, indicating that release of sCD 14β involves further processing. (6) CHO cells transfected with a CD14 cDNA coding for an eight C‐terminal amino acids shorter product released an sCD 14β‐like form; thus absence of the eight C‐terminal amino acids prevented mCD 14 expression but not the secretion of sCD 14β. The characterization of sCD 14α and sCD 14β reported here may be useful for better understanding of variations in sCD 14 levels in pathological conditions and the contribution of each sCD 14 in scpsis and other, as yet unknown functions.
Conserved distribution of λ subtypes from rearranged gene segments to immunoglobulin synthesis in the mouse B cell repertoire
Authors: Boudinot Pierre , Drapier Anne‐Marie , Cazenave Pierre‐André , Sanchez Pierre
Published: March 2007

The immunoglobulin λ light chain system displays a limited diversity in inbred mice. Indeed, the λ locus is organized in two recombination units: Vλ2‐Vλx‐Jλ2‐Cλ2‐ΨJλ4‐ΨCλ4, which can produce either λ2(V2) or λ2(Vx) chains; and Vλ1‐Jλ3‐Cλ3‐Jλ1‐Cλ1, which can produce either λ1 or λ3 chains. Each of these units is associated with an enhancer, Eλ2—4 or Eλ1—3, at the 3′ side. The expression of each λ chain is, therefore, controlled by distinct promoter and/or enhancer regions. To clarify the basis of these controls, we measured, by quantitative polymerase chain reaction, the proportions of each λ subtype in BALB/c spleen mRNA and among genomic rearrangements. It appears that these distributions are similar to and consistent with the relative cellular frequencies in the spleen, as evaluated by flow cytometry. These results suggest that, in resting cells, the transcription rates are identical, regardless of the λ subtype. After lipopolysaccharide (LPS) stimulation, the transcription rates per cell remain similar for all λ subtypes despite different regulatory sequences. To detect eventual post‐transcriptional regulations, we estimated the λ light chain distribution in IgM secreted by LPS‐stimulated B cells and in serum IgG. These distributions are still similar to those of λ‐expressing cells, λ mRNA or genomic rearrangements. We conclude that the λ subtype distribution is conserved from productive V‐J rearranged genes to secreted λ immunoglobulins, despite different regulatory sequences.
Cross‐linked bispecific monoclonal antibody heteropolymers facilitate the clearance of human IgM from the circulation of squirrel monkeys
Authors: Reist Craig J. , Liang Hong‐Yu , Denny Douglas , Martin Edward N. , Scheld W. Michael , Taylor Ronald P.
Published: March 2007

We have previously demonstrated that cross‐linked bispecific monoclonal antibodies (mAb) heteropolymers (HP), specific for primate erythrocyte (E) complement receptor type 1 (CR1) and target antigen (Ag), facilitate the binding of these target Ag to human and non‐human primate E. Once bound in vitro to rhesus monkey E, upon re‐infusion these HP/Ag complexes are recognized in vivo by cells of the reticuloendothelial system (RES) and removed from the circulation without loss of the E. We now show, in squirrel monkeys, that an HP specific for E CR1 and human IgM (anti‐CR1 × anti‐IgM) can be used to facilitate in vivo E binding and clearance from the circulation of a previously injected and circulating model protein pathogen, human IgM. Approximately 70—80% of 125I‐labeled human IgM is cleared from the circulation of each of five squirrel monkeys via the HP system. We observe, in experiments analogous to previous studies on immune complex (IC) clearance, that subsequent to HP/Ag clearance there is a decrease in the number of CR1 epitopes per E which is manifested when we use both monoclonal and polyclonal anti‐CR1 probes. Our results indicate that the primary organs responsible for uptake of the complexes are the liver and spleen. This work strongly suggests that the HP/Ag complexes, bound to E, function as IC prototypes and are recognized and processed as such in vivo This, the HP‐E system may eventually serve as a viable immunotherapy for the clearance of blood‐borne pathogens from the circulation.
Expression of the mouse interleukin‐2 receptor γ chain in various cell populations of the thymus and spleen
Authors: Kondo Motonari , Ohashi Yoichi , Tada Kohtaro , Nakamura Masataka , Sugamura Kazuo
Published: March 2007

Expression of the γ chain, which is shared among functional receptor complexes for interleukin (IL)‐2, IL‐4 and IL‐7, was examined with hematopoietic cells in mouse thymus and spleen by flow cytometry. The γ chain was expressed in cell populations from the spleen. Stimulation with concanavalin A and lipopolysaccharide caused fluctuation in expression of the γ chain in T and B cells, respectively. T lineage cells developing in the adult thymus expressed the γ chain. Fetal thymus at day 15 contained mostly immature thymocytes, which also expressed the γ chain. These results demonstrate that the γ chains is widely expressed in T lineage cells, probably indicating that the γ chain plays a role not only in the proliferation of mature hematopoietic cells but also in the development of immature cells through signal transduction as a common receptor subunit for multiple cytokines.
Control of established experimental allergic encephalomyelitis by inhibition of tumor necrosis factor (TNF) activity within the central nervous system using monoclonal antibodies and TNF receptor‐immunoglobulin fusion proteins
Authors: Baker David , Butler Debra , Scallon Bernard J. , O'Neill Janet K. , Turk John L. , Feldmann Marc
Published: March 2007

Tumor necrosis factor (TNF) activity was inhibited during the development of actively‐induced, chronic relapsing experimental allergic encephalomyelitis (CREAE) in Biozzi AB/H mice, using a mouse TNF‐specific (TN3.19.12) antibody and bivalent human p55 and p75 TNF receptor‐immunoglobulin (TNFR‐Ig) fusion proteins. The development of disease could be inhibited when repeated doses of antibody were administered prior to the anticipated onset. It has now also been shown that a therapeutic effect is evident even when antibody is administered after the onset of clinical signs, further indicating an important role for TNF in pathogenic effector mechanisms in CREAE. Although biologically‐active TNF was not detected in the circulation, TNF‐α was detected in lesions within the central nervous system (CNS). This suggested that the CNS may be the main site for TNF‐specific immunomodulation and was supported by the observation that intracranial injection was significantly more potent than that administered systemically, for both antibody and TNFR‐Ig fusion proteins. The fusion proteins were as effective as antibody at doses 10—100‐fold lower than that used for antibody, reflecting their higher neutralizing capacity in vitro. Although treatment was not curative and relapse inevitably occurred in this model if treatment was not sustained, the data indicate that anti‐TNF immunotherapy, especially within the CNS, can inhibit CREAE and may, therefore, be useful in the control of human neuroimmunological diseases.
Gene transfer of the interleukin (IL)‐2 receptor β chain into an IL‐7‐dependent pre‐B cell line permits IL‐2‐driven proliferation: Tyrosine phosphorylation of Shc is induced by IL‐2 but not IL‐7
Authors: Dorsch Marion , Hock Hanno , Diamantstein Tibor
Published: March 2007

Expression of the interleukin (IL)‐2 receptor β chain in the IL‐7‐dependent pre‐B cell line I × N/2B permitted growth in presence of either IL‐2 or IL‐7, allowing for a direct comparison of intracellular signaling events. Protein tyrosine phosphorylation was essential for IL‐2‐ and IL‐7‐induced signal transduction since the tyrosine kinase inhibitor herbimycin A blocked proliferation in response to both factors. Western blot analysis of tyrosine‐phosphorylated proteins revealed that both IL‐2 and IL‐7 stimulation led to enhanced phosphorylation of proteins of 170‐, 145, 115‐ and 99‐kDa, as well as induction of phosphorylation of a 96‐kDa protein. However, a 55‐ and a 155‐kDa protein were only phosphorylated after IL‐2 stimulation. The 55‐kDa protein specifically phosphorylated by IL‐2 could be identified as p52shc which has recently been shown to be critically involved in Ras activation. Shc tyrosine phosphorylation as a result of IL‐2 stimulation was consistently found in CTLL‐2 cells and human T lymphoblasts. Taken together our results indicate that the IL‐2‐ and IL‐7‐stimulated intracellular pathways are partially different and that Shc is a target of IL2‐, but not IL‐7‐, stimulated tyrosine phosphorylation.
Induction of soluble tumor necrosis factor receptor (sTNF‐R75) release by HIV adsorption on cultured human monocytes
Authors: Rimaniol Anne‐Cécile , Boussin François , Herbelin André , De Groote Donat , Dormont Dominique , Bach Jean‐François , Descamps‐Latscha Béatrice , Zavala Flora
Published: March 2007

Soluble tumor necrosis factor (TNF) receptors were recently detected in the circulation of patients with early HIV‐induced disease, at significantly higher levels than in control subjects. They were proposed as markers of disease progression and of the degree of immunodeficiency. We report that adsorption of heat‐inactivated HIV‐1 LAI to isolated human monocytes triggers the release of both TNF‐α and its natural specific inhibitor, the soluble TNF receptor (sTNF‐R)75, but not that of sTNF‐R55. Only limited inhibition of sTNF‐R release was obtained in the presence of a fully neutralizing anti‐TNF‐α monoclonal antibody, suggesting that stimulation by TNF‐α was only partially responsible for sTNF‐R release. HIV‐1 LAI induced a higher sTNF‐R/TNF ratio than lipopolysaccharide, a well‐known monocyte activator. Monocytes thus represent a cellular source of sTNF‐R that can be detected in the circulation of HIV‐infected patients from seroconversion onwards. The release of sTNF‐R could be of great significance in the control of HIV infection via the cytokine network and especially TNF‐α.
Induction of clonal anergy by oral administration of staphylococcal enterotoxin B
Authors: Migita Kiyoshi , Ochi Atsuo
Published: March 2007

The staphylococcal enterotoxin is a major cause of food poisoning. The bacterial substance stimulates T cells expressing specific Vβ T cell receptors (TcR) and is termed “the superantigen”. We have previously demonstrated that intravenous injection of staphylococcal enterotoxin B (SEB) induces functional unresponsiveness (anergy) of reactive T cells as well as a partial deletion by activationinduced programmed cell death. In the present study, we examined the effect of oral administration of SEB in mice. Our results indicate that spleen T cells from SEB‐primed mice are hyporesponsive to SEB stimulation in vitro, but the response to SEA was normal. Vβ8+ T cells purified from SEB‐primed mice did not respond to stimulation of TcR. This SEB‐specific unresponsiveness could not be reversed by exogenous interleukin‐2, but was partially reversed by phorbol 12‐myristate 13‐acetate. Activation of mitogen‐activated protein kinase during TcR‐mediated stimulation was significantly inhibited in anergic T cells. Although the mechanisms of oral tolerance are not well understood, these results show that oral administration of SEB induce clonal anergy in peripheral T cells.
Mapping of T cell epitopes of the major fraction of rye grass using peripheral blood mononuclear cells from atopics and non‐atopics. II. Isoallergen clone 5A of Loliuum perenne group I (Lol p I)
Authors: Bungy Gholam Ali , Rodda Stuart , Roitt Ivan , Brostoff Jonathan
Published: March 2007

Rye grass is the major cause of hay fever which currently affects 20 % of the population. Lolium perenne group I (Lol p I) is a glycoprotein of 240 amino acid residues, representing the main allergen of rye grass. We have used peripheral blood mononuclear cells (PBMC) from controls and subjects allergic to rye grass and cultured them with L. perenne extract (LPE) and Lol p I and measured lymphocyte activation using thymidine incorporation. Patients were further studied against the 115 overlapping peptides of the iso‐allergen clone 5A of Lol p I to see whether the 4 amino acid residue differences between clone 1A and clone 5A affect the T cell epitope and thus, lymphocyte activation. There are 24 peptide differences between isoallergen clone 1A and clone 5A occurring in pools 4, 13, 16 and 19 each one of which could be an immunodominant epitope. The PBMC from all allergic patients studied showed a strong proliferative response to LPE and Lol p I. Five immunogenic peptide pools, pool 6, 15, 16, 17 and 19 of the isoallergen clone 5A were also identified. Most of these pools are in the C‐terminal region of Lol p I. Out of 20 pools tested in vitro 1 pool (pool‐17) induced PBMC proliferation in five out of six patients who were not restricted to an HLA class II DR gene product. However, three out of the six subjects responded to various other peptide pools in addition to the immunodominant pool. In spite of the amino acid differences between the two clones, pool 17 still remains the immunodominant T cell epitope. Control subjects showed only weak responses to LPE and no detectable response to either Lol p I or peptide pools. From within the most active pool we have defined two peptides of the isoallergen clone 5A (identical in sequence with clone 1A) which stimulate lymphocytes from rye grass‐sensitive patients in vitro. Previous studies with the two continuous sequences (193WGAVWRIDTPDK204 and 195AVWRIDTPDKLT206) tested in vivo by intradermal skin testing have shown typical delayed‐type hypersensitivity reactions after 24—48 h in one patient. Comparison of amino acid sequences of Lol p I, Lol p II and Lol p III proteins revealed a significant level of structural similarity among them. Interestingly, 50% of the residues of the second peptide sequence are also present in Lol p II and Lol p III. Clinically it would be potentially beneficial if a common immunodominant T cell epitope can be used to induce anergy or specific T cell unresponsiveness to several different rye grass allergens locally or systemically.
Lateral organization of the ICAM‐1 molecule at the surface of human lymphoblasts: A possible model for its co‐distribution with the IL‐2 receptor, class I and class II HLA molecules
Authors: Bene László , Balázs Margit , Matkó János , Möst Johannes , Dierich Manfred P. , Szöllsi János , Damjanovich Sándor
Published: March 2007

Lateral distribution of the ICAM‐1 molecule and its topological relationship (mutual proximity) to the heavy and light chains of class I HLA molecules, HLA‐DR and interleukin‐2 receptor α‐chain (IL‐2Rα) were studied in the plasma membrane of HUT‐102B2 T and JY B lymphoblastoid cell lines by the technique of flow cytometric energy transfer (FCET). Effects of adherency and treatments with recombinant interferon‐γ or tumor necrosis factor‐α on the relative expression level of ICAM‐1 to the above cell surface proteins were also investigated. While the cytokines did not significantly affect the ICAM‐1 level of either cell line, an increased ICAM‐1 expression was found on adherent JY cells. The ICAM‐1 expression varied significantly with the cell cycle and culture conditions, as well. The statistical analysis of the differences observed in the energy transfer efficiency histograms resulted in a possible model of lateral co‐distribution of these proteins in the plasma membrane. These two‐dimensional patterns proved to be different for T and B lymphoma lines. ICAM‐1 molecules showed a high degree of self‐ssociation on HUT‐102B2 (T) cells, while they were mainly expressed as monomers on the surface of JY (B) cells. Both cells showed a significant (ca. 30 %) difference between densities of the heavy and light chains of class I HLA antigen, suggesting a substantial amount of β2‐microglobulin free heavy chains on these cell lines. The class I HLA molecules also showed partial self‐association, but on both cell lines. The β2‐microglobulin and the heavy chain of the class I HLA showed strongly different proximities to the IL‐2Rα, HLA‐DR and ICAM‐1 molecules, indicating that their orientations relative to the other proteins are dissimilar. IL‐2Rα molecules of the HUT‐102B2 (T) cells are located mostly in the vicinity of the β2‐microglobulin. In contrast, the local density of HLA‐DR antigens is higher in the proximity of the heavy chain than in the vicinity of the β2‐microglobulin. The possible functional significance of these protein patterns is also discussed herein.
Autologous cytolytic T lymphocytes recognize a MAGE‐1 nonapeptide on melanomas expressing HLA‐Cw* 1601
Authors: Bruggen Pierre Van Der , Szikora Jean‐Pierre , Boël Pascale , Wildmann Claude , Somville Michel , Sensi Marialuisa , Boon Thierry
Published: March 2007

Human melanoma cell line MZ2‐MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE‐1, which codes for antigen MZ2‐E which is presented by HLA‐A1. Gene MAGE‐1 is expressed in many tumors of several types but not in normal tissues except for testis. We show here that gene MAGE‐1 directs the expression of another antigen recognized by CTL on the MZ2‐MEL cells. This antigen, which was named MZ2‐Bb, consists of MAGE‐1‐encoded peptide SAYGEPRKL bound to major histocompatibility molecule HLA‐Cw* 1601. The HLA‐Cw* 1601 allele was found to be expressed by 7 out of 99 individuals from a Caucasian population. Our results extend the range of tumor patients who could be eligible for immunization against MAGE antigens.
Presentation of a horse cytochrome c peptide by multiple H‐2b class I major histocompatibility complex (MHC) molecules to C57BL/6‐ and bm1‐derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide‐specific CTL recognition
Authors: Sheil James M. , Schell Todd D. , Shepherd Sara E. , Klimo Gerald F. , Kioschos John M. , Paterson Yvonne
Published: March 2007

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C‐H‐2bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I‐restricted recognition of this peptide in that they respond to it in the context of H‐2Kb, H‐2Db, and H‐2Kbm1 class I molecules, although the sequence lacks the usual structural Kb and Db peptide‐binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I‐presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H‐2b) targets as well as the L cell transfectants, L + Kb, L + Db, and L + Kbm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full‐length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro44 and Thr47, which occupy polymorphic positions with respect to other species‐variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H‐2Kb, H‐2Db, and mutant H‐2Kbm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain‐specific binding pockets in the MHC class I peptide‐binding site.
Differential effects of cyclic adenosine 3′, 5′‐monophosphate on T cell cytotoxicity
Authors: Østenstad Bjørn , Harboe Morten , Lea Tor
Published: March 2007

We have investigated natural killer cell and T cell cytotoxicity using different assays and report a dual effect of cyclic adenosine 3′, 5′‐monophosphate (cAMP) on T cell cytotoxicity depending on the activation status of the effector cell and the test system in question. cAMP enhanced the capacity of pre‐activated T cells to induce DNA fragmentation in the target cell, while it inhibited spontaneous T cell cytotoxicity and natural killer cell cytotoxicity in conventional assays based on 51Cr release. The enhancement was most likely mediated by the cAMP‐dependent protein kinase type II (cAKII), which is the particular isoform in T cells associated with the centrosome and the microtubule organizing center (MTOC). We show the complete co‐localization of the cAKII with the centrosome after conjugate formation. Furthermore, the reorganization of the MTOC following conjugate formation brings the type II kinase into close proximity with the T lymphocyte membrane area engaged in the effector‐target interaction. Functional studies utilizing different cAMP‐analog combinations further substantiate the involvement of the type II kinase.
Distinct binding of T lymphocytes to ICAM‐1, ‐2 or ‐3 upon activation of LFA‐1
Authors: Binnerts Minke E. , van Kooyk Yvette , Simmons David L. , Figdor Carl G.
Published: March 2007

LFA‐1 (CD11a/CD18) mediates leukocyte adhesion by binding to one of its ligands: ICAM‐1, ICAM‐2 or ICAM‐3. Here, we investigated whether stimuli known to induce adhesion to ICAM‐1 were also capable of inducing LFA‐1‐mediated adhesion of T lymphocytes to ICAM‐2 and ‐3 transfectants. We observed that phorbol 12‐myristate 13‐acetate, Mn2+, cross‐linking of CD3 or activating antibodies against LFA‐1 enhanced LFA‐1‐mediated T cell adhesion to ICAM‐2 and ‐3, although to a lesser extent than to ICAM‐1. These results indicate that, similar to what has been reported for adhesion to ICAM‐1, activation of LFA‐1 is also required for adhesion to ICAM‐2 and ‐3. Furthermore, the results suggest that ICAM‐1 is the major ligand for LFA‐1 on activated T lymphocytes. Interestingly, we observed that in contrast to activating antibodies against CD18, activating antibodies against CD11a were incapable of inducing adhesion of LFA‐1 to all three ligands. The antibody MEM‐83 stimulated binding to ICAM‐1, while at the same time inhibiting the interaction of LFA‐1 with ICAM‐2 and ‐3. The antibody NKI‐L16 selectively induced adhesion to ICAM‐1 and ‐2, but not to ICAM‐3. Our results suggest that different conformations of LFA‐1 are required to support adhesion to ICAM‐1, ‐2 or ‐3, and that ligands may bind on different sites of the LFA‐1 molecule.
Inhibition of human IgE synthesis by anti‐IgE antibodies requires divalent recognition
Authors: Stämpfli Martin R. , Miescher Sylvia , Aebischer Iwan , Zürcher Adrian W. , Stadler Beda M.
Published: March 2007

We used a selection of well‐characterized murine monoclonal anti‐IgE antibodies to investigate their effect on human in vitro IgE synthesis. We found anti‐IgE antibodies that either inhibited or enhanced interleukin‐4 plus anti‐CD40‐induced in vitro IgE synthesis in peripheral blood mononuclear cells (PBMC). This differential activity was isotype specific as neither IgM nor IgG synthesis were affected. Interestingly, only coding IgE mRNA was down‐regulated, whereas germ‐line ε RNA expression was not influenced by anti‐IgE monoclonal antibody (mAb). On purified B cells all anti‐IgE mAb inhibited interleukin‐4 plus anti‐CD40‐induced IgE synthesis, implying a role of non‐B cells for the enhancing activity observed in PBMC. Using Fab and F(ab')2 of an inhibitory anti‐IgE mAb we could show that divalent recognition was required for inhibition of IgE synthesis.
Cathepsin D, but not cathepsin B, releases T cell stimulatory fragments from lysozyme that are functional in the context of multiple murine class II MHC molecules
Authors: Van Noort Johannes M. , Jacobs Marike J. M.
Published: March 2007

In this study, the major endosomal/lysosomal proteases cathepsin D and cathepsin B were tested on their ability to release T cell stimulatory peptides from hen egg white lysozyme (HEL) in vitro. Whereas neither enzyme could cleave unreduced HEL under mild conditions, reduced HEL was readily cleaved by cathepsin D but not by cathepsin B. Instead, cathepsin B was found to be very active in the trimming of HEL peptides after their release by cathepsin D.

Following high‐performance liquid chromatography (HPLC) fractionation, cathepsin D‐released HEL fragments were screened for recognition by HEL‐specific T cells from three strains of mice, i.e. B10. A (H‐2a), C57BL/6 (H‐2b) and BALB/c (H‐2d). Peptides in a large number of different HPLC fractions triggered significant T cell responses in all three strains. Interestingly, the response profiles of T cells from the three different strains showed marked similarities. Also, several individual synthetic HEL sequences corresponding to selected cathepsin D‐released fragments were recognized by murine T cells in the context of all three major histocompatibility complex (MHC) haplotypes tested.

Our data suggest that cathepsin D rather than cathepsin B may play a central role in the initial release of HEL fragments during endosomal/lysosomal processing. The relatively long HEL fragments released by cathepsin D, containing about 20—30 amino acid residues, are significantly more promiscuous in murine class II MHC binding than the shorter synthetic HEL sequences previously employed by others for the delineation of HEL epitopes. Extensive documentation of HEL epitopes in previous investigations indicate that this promiscuity cannot be explained by simply assuming that longer peptides contain additional epitopes. Rather, an increased peptide length by itself appears to promote promiscuous MHC binding.
Detergent‐enhanced dissociation of endogenous peptides from PI‐DRB1*0401
Authors: Buelow Roland , Kuo Stephanie , Paborsky Lisa , Wilson K. Jeff , Rothbard Jonathan B.
Published: March 2007

A variety of detergents have been shown to catalyze the dissociation of bound peptides from a soluble from of DRB1*0401. By using a class II molecule lacking the hydrophobic transmembrane region, the need for solubilizing the transmembrane protein was removed and enabled the specific interaction between the class II protein and the amphiphile to be identified. The presence of detergent increased the rate of association of added peptide and the percent occupancy of the receptor, presumably because the dissociation of endogenous peptide was the rate‐limiting step in binding. The data help explain the differences reported between peptide binding to class II proteins on the surface of cells and binding to class II proteins solubilized in detergent. The interaction did not correlate with the critical micellar concentration of the detergent nor were all amphiphilic structures equally effective, consistent with a specific interaction between the amphiphile and the MHC class II protein. Of the eight detergents examined, octyl glucoside was the most efficient. These experiments did not distinguish between an allosteric mechanism or direct competition with the peptide for binding.
Intercellular adhesion molecules (ICAM)‐1 ICAM‐2 and ICAM‐3 function as counter‐receptors for lymphocyte function‐associated molecule 1 in human immunodeficiency virus‐mediated syncytia formation
Authors: Butini Luca , De Fougerolles Antonin R. , Vaccarezza Mauro , Graziosi Cecilia , Cohen David I. , Montroni Maria , Springer Timothy A. , Pantaleo Giuseppe , Fauci Anthony S.
Published: March 2007

It has been previously demonstrated that lymphocyte function‐associated molecule 1 (LFA‐1) plays a major role in human immunodeficiency virus (HIV)‐mediated syncytia formation. In the present study we investigated the involvement of intercellular adhesion molecule‐1 (ICAM‐1), ICAM‐2 and ICAM‐3 in the process. The ability of monoclonal antibodies (mAb) directed against ICAM‐1, ICAM‐2 and ICAM‐3 to block syncytia was analyzed either in phytohemagglutinin (PHA)‐activated lymphocytes infected in vitro with primary or laboratory strains of HIV or by coculturing a T cell line stably expressing HIV envelope with PHA‐activated lymphocytes. Complete inhibition of syncytia formation was observed only by the simultaneous addition to the cell cultures of all (i.e. anti‐ICAM‐1, anti‐ICAM‐2 and anti‐ICAM‐3) mAb. These results indicate that the interaction between LFA‐1 and ICAM is a critical step in HIV‐mediated syncytia formation, and that ICAM‐1, ICAM‐2 and ICAM‐3 are the receptor molecules for the LFA‐1‐dependent syncytia formation.
Class II major histocompatibility complex‐restricted T cell function in CD4‐deficient mice
Authors: Rahemtulla Amin , Kündig Thomas M. , Narendran Arumugabadiv , Bachmann Martin F. , Julius Michael , Paige Christopher J. , Ohashi Pamela S. , Zinkernagel Rolf M. , Mak Tak W.
Published: March 2007

Previously, we and others have demonstrated that CD4‐deficient mice have a normal number of T cells and B cells with a significant population of CD4‐8‐TcRαβ+ T cells. Surprisingly, however, these mice lacking CD4 show in vivo immunoglobulin isotype class switching from IgM to IgG in response to sheep erythrocytes and vesicular stomatitis virus. In this study we have depleted various subpopulations of T cells in vivo and shown that the population of CD4‐8‐TcRαβ+ T cells is responsible for providing “help” in the antibody response of CD4‐deficient mice to vesicular stomatitis virus infection. We have used antigen‐specific proliferation assays and blocking studies with class I and II major histocompatibility complex (MHC)‐specific purified antibodies to show that these cells are class II MHC‐restricted in responses against the T cell‐dependent antigen keyhole limpet hemocyanin (KLH). Finally, phenotypic analysis of the CD4‐ CD8‐thymocytes in CD4‐deficient mice shows that these cells have a more mature phenotype than the CD4‐8‐ thymocytes in wild type mice. These results indicate that CD4 is not absolutely necessary for positive selection or effector function of class II MHC‐restricted helper T cells.
Experimental allergic encephalomyelitis (EAE) in mice lacking CD4+ T cells
Authors: Koh Dow‐Rhoon , Ho Alexandra , Rahemtulla Amin , Penninger Josef , Mak Tak‐Wah
Published: March 2007

Like most experimental autoimmune disease experimental allergic encephalomyelitis (EAE) has been shown to be mediated by CD4+ helper T cells. In vivo antibody blocking studies with anti‐CD4 and adoptive transfer of activated CD4+ T cells indicate the importance of CD4+ cells in disease induction. Fourth backcross generation mutant CD4—/— PL/J mice were immunized with myelin basic protein. Despite the lack CD4+ T cells some of these mice developed EAE, albeit, at a considerably reduced frequency and with variable severity. Furthermore, antigen‐specific T cell proliferation can be demonstrated, indicating some residual helper activity that is major histocompatibility complex class II restricted. This demonstrates that, although the CD4+ T cell is the prime effector cell in EAE, in mice developmentally lacking in CD4, the expanded double‐negative T cells may subserve helper and effector functions.
Development of motility of Langerhans cell through extracellular matrix by in vitro hapten contact
Authors: Kobayashi Yasunobu , Staquet Marie‐Jeanne , Dezutter‐Dambuyant Colette , Schmitt Daniel
Published: March 2007

To study the migration capacity of Langerhans cells in vitro, a reconstituted basement membrane invasion assay (Matrigel assay) was performed. Partially enriched human epidermal Langerhans cell suspensions (6—20%) were incubated with or without haptens [2, 4, 6‐/trinitrobenzenesulfonic acid (TNBS) and fluorescein isothiocyanate (FITC)] or an irritant sodium lauryl sulfate (SLS) in Hanks' balanced salt solution for 10 min at 37°C, and were then used for the invasion assay. Fibroblast‐conditioned medium prepared from human dermal fibroblasts was used as a source of chemoattractants. Non‐treated cells showed a gradual increase in migration of HLA‐DR‐positive cells through the Matrigel‐coated filters in a time‐dependent manner. In vitro TNBS and FITC treatment resulted in a significant stimulation of this migration. By contrast, SLS treatment did not increase the number of migrating cells. The migration capacity of hapten‐treated cells decreased both in the absence of human fibroblast‐conditioned medium and in the presence of this medium in the upper compartment of the chamber, indicating that this medium actually functioned as chemoattractant. Our findings suggest that the contact between hapten and Langerhans cells could be one of the triggers to initiate and/or stimulate Langerhans cell migration from the epidermis to the dermis. In addition, some factors derived from the dermal fibroblasts may promote and regulate the directional migration of Langerhans cells.
Expression of a wide T cell receptor Vβ repertoire in human T lymphocytes derived in vitro from embryonic liver cell precursors
Authors: Poggi Alessandro , Demarest James F. , Costa Paola , Biassoni Roberto , Pella Nicoletta , Pantaleo Giuseppe , Mingari M. Cristina , Moretta Lorenzo
Published: March 2007

As shown recently, CD3+/TcR+ functional T lymphocytes can be derived in culture from embryonic liver cell precursors at a gestational age (6—8 weeks) preceeding the colonization of the epithelial thymus. In this report, we analyzed the Vβ repertoire of T lymphocytes derived from embryonic liver by applying a quantitative reverse transcriptase‐polymerase chain reaction technique. To this end, oligonucleotide primers for Cα or the various human Vβ have been used to study both freshly derived embryonic liver cell suspensions and CD3+/TcR+ populations derived after approximately 6 weeks upon stimulation with 1% phytohemagglutinin and culture in 100 units/ml recombinant interleukin‐2. In order to exclude possible contaminations with mother‐derived T lymphocytes, only T cells displaying both X and Y chromosomal sequences (i.e. derived from male embryos) were further analyzed. While neither Cα nor the various Vβ could be detected in fresh liver cells, Cα and the large majority of Vβ were detected in in vitro cultured populations. The levels of the various Vβ expressed by embryo‐derived T cells was similar to that detected in adult peripheral blood‐derived T lymphocytes. These experiments indicate that the immature liver precursors can potentially give rise in vitro to T cells which express a wide Vβ repertoire and may provide a suitable in vitro system for the analysis of the selection processes mediated by either major histocompatibility complex antigen or superantigens.
Interleukin‐12 regulates the proliferation of Th1, but not Th2 or Th0, clones
Authors: Kennedy Mary K. , Picha Kathleen S. , Shanebeck Kurt D. , Anderson Dirk M. , Grabstein Kenneth H.
Published: March 2007

Our results indicate that interleukin (IL)‐12 is an important costimulator of antigen‐dependent proliferation of murine Th1 clones. In addition, we demonstrate that IL‐10 inhibits splenic antigen‐presenting cell (APC)‐dependent proliferation of Th1 clones, at least in part, via down‐regulation of APC‐derived IL‐12. Moreover, the failure of activated B cells to provide costimulation via IL‐12 accounts for their inability to support optimal proliferative responses of Th1 clones. We also show that IL‐12 regulates the ability of Th1 clones to respond to IL‐4 and enhances their proliferation in response to IL‐2, IL‐7, or IL‐15. In contrast, Th2 and Th0 clones appear refractory to the effects of IL‐12 on antigen‐dependent or growth factor‐induced proliferation.
Distinct mouse bone marrow macrophage precursors identified by differential expression of ER‐MP12 and ER‐MP20 antigens
Authors: de Bruijn Marella F. T. R. , Slieker Walentina A. T. , van der Loo Johannes C. M. , Voerman Jane S. A. , van Ewijk Willem , Leenen Pieter J. M.
Published: March 2007

The characterization of early branch points in the differentiation of leukocytes requires identification of precursor cells in the bone marrow. Recently, we produced two monoclonal antibodies, ER‐MP12 and ER‐MP20, which in two‐color flow‐cytometric analysis divide the murine bone marrow into six defined subsets. Here we show, using fluorescence‐activated cell sorting followed by macrophage colony‐stimulating factor‐stimulated culture in soft agar, that precursors of the mononuclear phagocyte system reside only within the ER‐MP12hi20−, ER‐MP12+20+ and ER‐MP12−20hi bone marrow subsets. Together, these subsets comprise 15% of nucleated bone marrow cells. Furthermore, we provide evidence that the macrophage precursors present in these subsets represent successive stages in a maturation sequence where the most immature ER‐MP12hi20− cells develop via the ER‐MP12+20+ stage into ER‐MP12−20hi monocytes.
The immune response to Plasmodium chabaudi malaria in interleukin‐4‐deficient mice
Authors: von der Weid Thierry , Kopf Manfred , Köhler Georges , Langhorne Jean
Published: March 2007

Interleukin(IL)‐4 promotes the development of T helper (TH)2 cells, induces immunoglobulin class switching to IgG1 and is thought to be essential for switching to IgE. During a primary infection with the erythrocytic stages of Plasmodium chabaudi chabaudi, TH1 and TH2 cells specific for the parasite appear sequentially as infection progresses. To dissect the possible role of TH2 responses at the later stages of infection, mice with a targetted disruption of the IL‐4 gene were infected with P. chabaudi. IL‐4‐deficient mice were able to control and clear a primary infection, although recrudescent parasitemias were significantly higher in these mice compared with wild‐type littermates, demonstrating that IL‐4 per se is not required for parasite elimination. To evaluate the actual impairment of TH2 functions in the absence of IL‐4 in vivo during an infection with P. chabaudi, the cellular and humoral responses to the parasite generated in vitro and in vivo were compared in the two types of mice. Our data indicate that in vitro TH1 responses and ex vivo IL‐12 mRNA levels were sustained in the IL‐4‐deficient mice compared with wild‐type littermates. Correspondingly, TH2‐associated cytokine mRNA such as IL‐5 and IL‐6, but not IL‐10, were reduced early in infection in the deficient animals. However, these cytokines were expressed at comparable levels at the later stages of infection in both types of mice. Reflecting these differences in TH function, IgG1 responses were decreased in vitro and delayed in vivo, whereas IgG2a and IgG2b responses appeared earlier in vivo in the deficient mice. Strikingly, IgE secretion was not blocked in vivo in the deficient mice; the onset of the synthesis of IgE mRNA was delayed during infection and the amount of circulating IgE was five times lower than in the wild‐type littermates after 5 weeks of infection. All these impairments of TH‐related activities were insufficient to affect parasite clearance in the deficient mice, probably due to the fact that such activities were only delayed and could take place normally at the later stages of infection.
Immunological approach to inhibit formation of anti‐antibodies to allo‐ and xenogeneic anti‐T cell immunoglobulin
Authors: Mysliwietz Josef , Thierfelder Stefan , Mocikat Ralph , Kremmer Elisabeth
Published: March 2007

Inhibitory anti‐antibodies induced in patients by xenogeneic or even by humanized anti‐T cell antibodies remain an unresolved problem. Mice also produce anti‐antibodies following injection of xeno‐ or allogeneic anti‐T cell antibodies. Here we report a principle based on sequentially applied anti‐T cell antibodies generated in different species, which results in suppressed anti‐antibody formation and prolonged immunosuppression. Thus, a single priming injection in mice of mouse (MmT1 or MmT5 differing by idiotype only) or of rat (RmT1) anti‐mouse Thy‐1 monoclonal antibodies (mAb) or of rat anti‐mouse L3T4 + Ly‐2 (RmCD4 + CD8) mAb suppressed anti‐antibody formation against subsequent booster injections of one of the above antibodies, provided that they differed in species origin from the priming antibody. Correspondingly, a sixfold and longer prolongation of 50% survival of fully mismatched skin grafts was observed. Less or no anti‐antibody suppression and little prolongation of graft surival was obtained if the ‘first’ and the ‘second’ (and following) antibody injections were of the same species, differing by iso‐ or idiotype only. Finally, the suppressive principle did not manifest itself at all if the initial antibody injection included both the first and second antibody. These findings are discussed with reference to earlier studies on hapten/carrier effects as well as on immunosuppression attributed to ‘non‐depleting’ rat anti‐CD4/CD8 T cell antibodies.
Identification of T cell receptor recognition residues for a viral peptide presented by HLA B27
Authors: Bowness Paul , Allen Rachel L. , McMichael Andrew J.
Published: March 2007

The fine specificity of T cell recognition of peptide analogues of the influenza nucleoprotein epitope, NP 383–391 SRYWAIRTR, was studied using HLA B27‐restricted influenza‐specific cytotoxic T cell (CTL) clones, of defined T cell receptor (TcR) usage, derived from unrelated individuals following natural infection. Even conservative amino acid substitutions of the peptide residues P4, P7 and P8 influenced CTL recognition. These side chains are probably directly contacted by the TcR. CTL clones which used the TcR Vα14 gene segment (but not those using TcR Vα12) were also sensitive to P1 substitutions, suggesting that the TcR α chain of these clones lies over the N terminus of bound peptide, and that the “footprint” of certain TcR can span all exposed residues of a peptide bound to a major histocompatibility complex class I molecule.

These results, taken together with previous structural and functional data, suggest that, for nonamer peptides bound to HLA B27, P1, P4 and P8 are “flag” residues with TcR‐accessible side chains.