bluuebunny/crossref_metadata_2025 huggingface dataset:
The CD56 adhesion molecule is the major determinant for detecting non‐major histocompatibility complex‐restricted cytotoxic mononuclear cells from the intestinal lamina propria
Authors: Van Tol Eric A. F. , Verspaget Hein W. , Pena A. Salvador , Kraemer Carlos V. Elzo , Lamers Cornelis B. H. W.
Published: March 2007

In this study, specific antibodies against natural killer (NK) cell surface markers identify these cells to be commonly present in normal intestinal mucosa of inflammatory bowel disease (IBD) and carcinoma patients. Cells expressing the CD56 adhesion molecule were found to be far more abundant than CD16+ cells. Functional studies revealed that cells mediating non‐major histocompatibility complex‐restricted cytotoxicity (NK activity) in the lamina propria express the CD56 surface antigen, whereas only a minority of this activity resides in the population with CD16 expression. This is in contrast with peripheral blood NK cells, which were found to be almost exclusively both CD16+ and CD56+. Moreover, in the lamina propria of the intestine we found CD3+ T lymphocytes not to be involved in spontaneous cell‐mediated killing of tumor cells.

Considerably higher numbers of cells with the CD16 or CD56 surface markers were found to be present in normal mucosa of IBD patients compared with normal mucosa of carcinoma patients, which was also reflected in higher levels of cytotoxicity detected in lamina propria mononuclear cell preparations from normal IBD mucosa. Because of the disease‐related localization of the mucosa studied from both patient groups, i.e. ileum vs. colon, the observed differences may be related to tissue characteristics. Within the IBD group, relatively high levels of cytotoxicity were found in cell preparations from normal mucosa of Crohn's disease patients compared with ulcerative colitis patients, which might support the current concept that Crohn's disease affects the whole of the gastrointestinal tract.
Differences between T helper cell type I (Th1) and Th2 cell lines in signalling pathways for induction of contact‐dependent T cell help
Authors: Kawakami Kazuyoshi , Parker David C.
Published: March 2007

B cells get help in the antibody response by presenting antigen to helper T (Th) cells. Upon antigen recognition, T cells produce lymphokines that act as growth and differentiation factors for B cells, but resting B cells require additional helper signals that depend on cell contact with an activated Th cell. Like lymphokine secretion, contact help must be induced by antigen recognition or antigen receptor cross‐linking in continuous Th cell lines. In the mouse, most CD4+ T cell lines can be classified into one of two stable differentiation states. Th1 or Th2, which produce different lymphokines and have different effector functions, activation requirements and cytoplasmic signalling mechanisms. This report demonstrates additional differences between Th1 and Th2 cell lines in the signalling pathways leading from the T cell antigen receptor to the induction of Th functions. In a system dependent on antigen presentation by B cells, B cell proliferation driven by Th2 cells but not Th1 cells was blocked by acute treatment with phorbol esters. Further experiments showed that phorbol esters blocked the induction of both contact help and lymphokine production in Th2 cells but not in Th1 cells. However, depletion of protein kinase C (PKC) activity by prolonged treatment of T cells with high concentrations of phorbol esters blocked induction of contact help and lymphokine production in Th1 cells but not in Th2 cells. These findings support the hypothesis that Th2 cells use a signalling pathway that is independent of PKC and that PKC activation can block this pathway. Since contact help and lymphokine secretion are affected in parallel, this difference between Th1 and Th2 cells probably reflects early events in the signalling pathway. Contact help and lymphokine production could be dissociated with cholera toxin and other cAMP agonists, but this dissociation could be explained by non‐cAMP‐related effects of cholera toxin on induction of contact help in Th2 cells, and by the direct effect of cAMP agonists on interleukin 2 gene transcription in Th1 cells reported by other laboratories.
Binding of a major T cell epitope of mycobacteria to a specific pocket within HLA‐DRw17(DR3) molecules
Authors: Geluk Annemieke , Bloemhoff Wim , De Vries René R. P. , Ottenhoff Tom H. M.
Published: March 2007

CD4+ T cells recognize antigenic peptides bound to the polymorphic peptide‐binding site of major histocompatibility complex (MHC) class II molecules. The polymorphism of this site is thought to dictate which peptides can be bound and thus presented to the T cell receptor. The mycobacterial 65‐kDa heat‐shock protein (hsp65) peptide 3‐13 is an important T cell epitope: it is immunodominant in the mycobacterium‐specific T cell response of HLA‐DR3+ individuals but, interestingly cannot be recognized in the context of any other HLA‐DR molecules. We, therefore, have tested whether the hsp65 epitope p3‐13 is selected for T cell recognition in the context of only HLA‐DR3 molecules by an unique binding specificity for HLA‐DR3. Using biotinylated peptides and EBV‐transformed BLCL comprising all known HLA class II specificities, we find that p3‐13 binds to HLA‐DRw17(DR3) but not to any other HLA‐DR molecule. Conversely, a control peptide p307‐319 influenza hemagglutinin binds to all known HLA‐DR molecules but only weakly to HLA‐DRw17 and HLA‐DR9. Peptide binding could be inhibited by excess unbiotinylated competitor analogue as well as by anti‐DR monoclonal antibodies but not by anti‐class I‐, anti‐DP‐ or anti‐DQ monoclonal antibodies.

The amino acid sequence of DRw17 molecules differs uniquely at five positions from the other DRβ1 sequences. Three of these five residues (positions 26, 71 and 74) are potential peptide contacting residues. These residues map closely together in the hypothetical three‐dimensional model of the DR molecule and, thus, most probably form a positively charged pocket, critical for the binding of p3‐13. Interestingly, p3‐13 does not bind to a DR3 variant, the DRw18 molecule. The DRw18β1 chain differs from DRw17 at two major positions, close to or within the DRw17‐specific pocket. These substitutions drastically change the structure and charge of the pocket and thus presumably abrogate its ability to bind p3‐13.
Memory in helper T cells of minor histocompatibility antigens, revealed in vivo by alloimmunizations in combination with Thy‐1 antigen
Authors: Lightstone Elizabeth , Marvel Jacqueline , Mitchison Avrion
Published: March 2007

A cooperative antibody response in which T helper (Th) cells recognize minor histocompatibility antigens (mha) and B cells recognize Thy‐1 antigen, is used to explore memory in the T cell compartment. In contrast to B cell memory, Th memory reaches a plateau rapidly, although Th memory of Thy‐1 itself (or an associated antigen) behaves exceptionally in this respect. The plateau then extends over several weeks at least. Single mha, among them H‐Y, generate detectable memory. Incompatible H‐2 antigens, including class I antigens on their own, inhibit this response through what appears to be a mechanism of intracellular antigenic competition. Antigen presentation in this system is by host cells, as judged by lack of donor‐specific restriction. Memory resides in both the CD45RA+ and CD45RA− compartments, although the majority of memory Th cells have the latter phenotype.
Sequence variation of cytotoxic T cell epitopes in different isolates of Epstein‐Barr virus
Authors: Apolloni Ann , Moss Denis , Stumm Robyn , Burrows Scott , Suhrbier Andreus , Misko Ihor , Schmidt Chris , Sculley Tom
Published: March 2007

Previous results have identified two distinct cytotoxic T lymphocyte (CTL) epitopes encoded by Epstein‐Barr virus (EBV), TETA (ORF BLRF3/BERF1 residues 329‐353) and EENL (ORF BERF3/BERF4 residues 290‐309). Measurement of the specificities of CTL clones (TETA‐specific clone 13 and EENL‐specific clone 7) directed against these epitopes indicated that the EENL epitope is conserved in all strains of EBV tested while the TETA epitope varied between individual virus strains. Sequencing of the DNA regions encoding these two CTL epitopes in different EBV isolates confirmed these interpretations and demonstrated that different TETA epitope sequences were encoded by B‐type EBV strains and by the B95‐8 isolate of EBV compared to the other A‐type EBV strains. Titration of synthetic variants of the TETA epitope revealed that the epitope encoded by B95‐8 was 15‐fold less efficient as a T cell epitope than the sequence encoded by other A‐type viral strains while the TETA variant encoded by the B‐type strains displayed essentially no activity as a T cell epitope.
In vitro biosynthesis of complement factor I by human endothelial cells
Authors: Julen Nathalie , Dauchel HÉLÈNe , Lemercier Claudie , Sim Robert B. , Fontaine Marc , Ripoche Jean
Published: March 2007

We have studied the secretion of the complement regulatory protein factor I by human umbilical vein endothelial cells (HUVEC). Northern and Western blot analysis and biosynthetic labeling experiments indicate that HUVEC secrete factor I at very low levels in basal conditions and that this secretion is significantly enhanced by interferon‐γ. Analysis of the proteolytic inactivation of C3b by HUVEC supernatants show that factor I is secreted in a functional form and can promote the specific proteolytic inactivation of C3b to iC3b. Together with previous studies establishing the secretion of complement factor H by HUVEC, this work demonstrates that the endothelial cell is able to secrete in its environment two complement regulatory proteins, factor I and factor H, which can mediate the degradation of C3b to iC3b. The secretion of factor I by HUVEC provides a useful in vitro model to analyze the modulation of this secretion and may be relevant to the local deposition of iC3b at the surface of the endothelium during the inflammatory reaction.
CD4+ T cell‐mediated killing of major histocompatibility complex class II‐positive antigen‐presenting cells (APC) III. CD4+ cytotoxic T cells induce apoptosis of APC
Authors: Grogg Daniel , Hahn Sinuhe , Erb Peter
Published: March 2007

A subset of CD4+ T cells, belonging to the T helper type 1 (Th1) cells, kills antigen‐presenting cells (APC) in an antigen‐specific and major histocompatibility complex (MHC) class II‐restricted way. Evidence is presented that CD4+ cytotoxic T lymphocytes (CTL) induce apoptosis or programmed cell death within susceptible APC as witnessed by quantitative DNA fragmentation. Apoptosis is more reliable to determine cell death than the 51Cr‐release assay, because some cells demonstrate resistance to CD4‐mediated lysis in the 51Cr‐release assay. Apoptosis becomes manifest after 2 to 4 h of incubation preceding the disintegration of the target cells which is detectable between 12 and 24 h as measured by the 51Cr‐release assay. Unstimulated B cells, which are not killed, but function as APC, do not undergo apoptosis, whereas lipopolysaccharide or anti‐μ‐activated B cell blasts show apoptosis and are efficiently lysed. Several CD4+ Th2‐type cells tested, which did not demonstrate killing of APC as measured by the 51Cr‐release assay, are unable to mediate programmed cell death of appropriate APC. Actinomycin D or cycloheximide, inhibitors of transcription and translation, respectively, fail to prevent apoptosis of APC excluding the involvement of newly synthesized soluble products as mediators of killing. Pretreatment of CD4+ CTL, but not of APC with 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonic acid, a specific inhibitor of the anion transport, efficiently prevents apoptosis of APC, although the secretion of interleukins is not affected. We propose, that upon contact of the CD4+ CTL with APC, molecules of yet undefined nature are activated and released in a polar fashion at the contact site and induce the endogenous pathway of programmed cell death.
Synergism between tumor necrosis factor‐α and interferon‐γ on macrophage activation for the killing of intracellular Trypanosoma cruzi through a nitric oxide‐dependent mechanism
Authors: Muñoz‐Fernández M. Angeles , Fernández Monica A. , Fresno Manuel
Published: March 2007

Intracellular replication of the protozoan parasite Trypanosoma cruzi inside macrophages is essential for the production of the disease and the development of the parasite. Two CD4+ T cell lines, A10 and A28, were established from T. cruzi‐infected BALB/c mice which specifically proliferated to parasite antigens. The trypanocidal activity of BALB/c macrophages was induced upon culture with the A10, but not with the A28 T cell line. The cell‐free supernatant from this A10 line, as well as from immune spleen cells stimulated with specific antigen or concanavalin A, but not from the A28 T cell line also activated the trypanocidal activity of peritoneal macrophages or of the J774 macrophage‐like cell line. When the lymphokine content of the supernatants from both cell lines was analyzed, it was found that the A10 T cell line secreted interferon (IFN)‐γ, tumor necrosis factor (TNF)‐α and interleukin 2, whereas the A28 line did not secrete IFN‐γ upon stimulation. Furthermore, the trypanocidal‐inducing ability of A10 supernatant was completely abrogated by neutralizing anti‐IFN‐γ antibodies and partially abrogated by neutralizing anti‐TNF‐α antibodies. When recombinant cytokines were added to J774 cells, IFN‐γ was able to induce significant trypanocidal activity whereas TNF‐α was almost ineffective. However, TNF‐α or lipopolysaccharide (LPS) showed a synergistic effect with IFN‐γ on macrophage activation. IFN‐γ triggered nitric oxide (NO) synthesis by J774 cells whereas TNF‐α was almost ineffective. TNF‐α and LPS were also synergistic with IFN‐γ in the NO production. Both the NO production and the trypanocidal activity in J774 cells induced by T cell supernatants or lymphokine combinations were inhibited by N‐monomethyl‐L‐arginine, a competitive inhibitor of NO synthase activity. A good correlation between the levels of NO production and trypanocidal activity induced by different lymphokine preparations was found. Those results suggest that IFN‐γ and TNF‐α, secreted by T. cruzi‐immune T cells, are involved in the activation of the trypanocidal activity of mouse macrophages through an NO‐dependent mechanism.
A single autosomal gene defect severely limits IgG but not IgM responses in B lymphocyte‐deficient A/WySnJ mice
Authors: Miller David J. , Hanson Keith D. , Carman Julie A. , Hayes Colleen E.
Published: March 2007

Antigen‐stimulated B lymphocytes either differentiate into IgM‐secreting plasma cells or into memory B cells that secrete other immunoglobulin isotypes upon antigen restimulation. The mechanisms that generate and maintain memory B cells are poorly understood. Previously, we described a severe B lymphocyte deficiency in adult strain A/WySnJ mice compared to subline A/J. Here we show that the single, autosomal co‐dominant locus responsible for the deficiency also diminishes IgG‐secrcting B cell formation without interfering with IgM‐secreting plasma cell differentiation. A/WySnJ secondary IgG1 responses to the protein antigens hemocyanin, bovine γ‐globulin, ovalbumin, lysozyme and β‐galactosidase were 6‐ to 50‐fold lower than A/J responses. The defect also decreased secondary IgG2a and IgG3 responses, and primary IgG1 and IgG2a responses. The reduced A/WySnJ secondary IgG1 response was not due to differential response kinetics or dose responsiveness, and could not be augmented to A/J levels by repeated immunizations. Serum IgG1, IgG2a and IgG3 levels from nonimmune A/WySnJ mice were similarly reduced. The secondary IgG1 response and splenic B cell percentage showed significant positive correlation (r = 0.72) in F2 mice, suggesting that a single locus controlled both traits. In contrast, A/WySnJ mice made good primary IgM responses to hemocyanin, β‐galactosidase, and the thymus‐independent antigen trinitrophenyl‐Ficoll. The A/WySnJ splenic adherent cells were competent in antigen‐presenting function, and A/WySnJ immune T cells proliferated in response to antigen and provided the requisite B cell stimulatory signals for an IgG1 response. Together, our results suggest that A/WySnJ mice have a genetic lesion that causes a selective IgG immune response dysfunction. The absence of IgG‐secreting cell precursors or a defect in precursor activation or differentiation are two possible mechanisms which could precipitate a selective IgG response dysfunction. We propose that the defective A/WySnJ and normal A/J strain pair offer the opportunity to use a natural genetic variation as a tool to investigate B lymphocyte development and function.
Cross‐reaction of antibodies to the nine‐amino acid repeats of Plasmodium falciparum antigen 11.1 with human serum albumin
Authors: Mercereau‐Puijalon Odile , Guillotte Micheline , Doyen Noëlle
Published: March 2007

Mice immunized with the recombinant antigen 11.1 β‐galactosidase, consisting of 22 repeats of the nine‐amino acid unit from Plasmodium falciparum antigen 11.1, produced antibodies reacting with human serum albumin. A positive reaction was observed in dot‐blot assays, in enzyme‐linked immunosorbent assay and on immunoblots of sodium dodecyl sulfate polyacrylamide gels as well as twodimensional gels. Binding was specific for human albumin, as no reaction could be detected on bovine serum albumin, hen egg ovalbumin, rat serum albumin or another abundant human serum protein, the α2‐macroglobulin. In addition, rabbit antibodies raised to human serum albumin reacted with keyhole lympet hemocyanin coupled to synthetic dimers of the nine‐amino acid repeats of the P. falciparum 11.1 antigen. These data indicate antigenic relationship between the 11.1 antigen and human albumin. The proteins have a short sequence of homology in a region where human serum albumin differs from the albumins of other species.
Preferential positive selection of Vα2+CD8+ T cells in mouse strains expressing both H‐2k and T cell receptor Vαa haplotypes: determination with a Vα2‐specific monoclonal antibody
Authors: Pircher Hanspeter , Rebai Najet , Groettrup Marcus , Grégoire Claude , Speiser Daniel E. , Happ Mary Patt , Palmer Ed , Zinkernagel Rolf M. , Hengartner Hans , Malissen Bernard
Published: March 2007

A monoclonal antibody, B20.1, was generated by fusing spleen cells from a Lou rat immunized with a soluble α/β T cell receptor (TcR; Vα2/Vβ2) to mouse myeloma cells. Analysis of a panel of Vα2 mRNA‐expressing T cell lines, hybridomas and transfectants revealed that the B20.1 antibody was specific for murine TcR Vα2 chains. The Vα2+ T cell population was examined in various inbred strains by two‐color immunofluorescence using B20.1 and CD4‐ and CD8‐specific antibodies with the following results: (a) the B20.1 antibody detected most members of the TcR Vα2 subfamily in the four TcR Vα haplotypes tested; (b) in most strains examined, TcR Vα2 expression was biased to the CD4 subset (7.4%‐17.4% Vα2+ T cells) as compared to the CD8 compartment (3.8%‐13.3%); (c) TcR Vα2 expression was not influenced by Mls gene products and (d) increased positive selection of Vα2+CD8+ T cells by H‐2k major histocompatibility complex molecules occurred in all murine strains tested of the TcR Vαa, but not in those bearing the TcR Vαb haplotype.
Engagement of major histocompatibility complex class I and class II molecules up‐regulates intercellular adhesion of human B cells via a CD11/CD18‐independent mechanism
Authors: Alcover Andrés , Juillard Véronique , Acuto Oreste
Published: March 2007

We have studied the role of major histocompatibility complex (MHC) molecules in the regulation of intercellular adhesion of human B cells. We found that molecules able to bind to MHC class II molecules, such as monoclonal antibodies or staphylococcal enterotoxins, induced rapid and sustained homotypic adhesion of Epstein‐Barr virus (EBV)‐transformed B cell lines as well as peripheral blood B lymphocytes. Moreover, anti‐MHC class I monoclonal antibodies also stimulated intercellular adherence. Adhesion induced upon MHC engagement was faster and stronger than that triggered by phorbol esters. It needed active metabolism, but divalent cations were not required. Monoclonal antibodies directed against LFA‐1 (CD11a/CD18) or its ligand ICAM‐1 (CD54) did not inhibit MHC class II‐induced homotypic adhesion of various EBV‐transformed B cell lines, nor of a variant of the B cell line Raji expressing very low LFA‐1 surface levels. Moreover, EBV‐transformed B cells from a severe lymphocyte adhesion deficiency patient, lacking surface CD11/CD18, also aggregated in response to anti‐MHC class I or class II monoclonal antibodies. Together these data indicate that engagement of MHC molecules may transduce signals to B cells resulting in up‐regulation of intercellular adhesion, via an LFA‐1‐independent mechanism. This may play a role in the stabilization of T cell / antigen‐presenting cell conjugates at the moment of antigen recognition.
A unique CD44 monoclonal antibody identifies a new T cell activation pathway
Authors: Pierres Anne , Lipcey Carol , Mawas Claude , Olive Daniel
Published: March 2007

We have identified a new T cell activation pathway mediated by the lymphocyte homing receptor/CD44 molecule. 8B2.5, a local monoclonal antibody (mAb), which recognizes two glycoproteins of 85 and 220 kDa with wide tissue distribution, is shown by sequential immunoprecipitations and competitive antibody‐binding inhibition experiments with several CD44 reference mAb to recognize the CD44 molecule. The 8B2.5 mAb, but not reference CD44 mAb, is able to induce resting peripheral blood lymphocytes to proliferate in the presence of phorbol esters. This proliferation is monocyte dependent but Fc independent and results from 8B2.5 mAb binding to CD44 molecules both expressed by both T cells and monocytes. In the absence of monocytes, proliferation can be restored by solid‐phase 8B2.5 mAb, or, to a lesser extent, by adding interleukin 2. Although CD3 and CD44 surface molecules are found physically independent, T cell activation via the CD44 pathway is inhibited by CD3 modulation. In addition to the direct role of CD44 molecules in T cell proliferation, CD44 mAb can up‐ or‐ down‐regulate the CD3 and CD28 pathways, depending on the presence of monocytes. These results suggest that T cell and monocyte binding to high endothelial venule or extracellular matrix proteins could further promote clonal expansion of resting T cells migrating in certain specific anatomic sites.
A defect in the presentation of intracellular viral antigens is restored by interferon‐γ in cell lines with impaired major histocompatibility complex class I assembly
Authors: Sibille Catherine , Gould Keith , Hämmerling Günter , Townsend Alain
Published: March 2007

Surface expression of the majority of class I major histocompatibility complex (MHC) heavy chains is known to require assembly with β2 microglobulin (β2m). To define other factors involved in class I MHC assembly, we have studied two tumor cell lines that are deficient in cell surface class I (H‐2) expression. The BC2 fibrosarcoma and the CMT lung carcinoma express only intracellular unassociated heavy chains despite the presence of β2m. As described previously, when these cell lines are treated with interferon‐γ (IFN‐γ), they are capable of assembling and transporting class I molecules to the cell surface. In this study, we have shown that in the absence of IFN‐γ these mutant cells are unable to present intracelluar viral antigens, although they can be lysed by specific cytotoxic T lymphocyte (CTL) after pre‐incubation with the corresponding synthetic peptide. Flow cytometric analysis demonstrated that extracellular peptide was capable of increasing twofold the surface expression of β2m‐heavy chain complexes. Furthermore, immunoprecipitation experiments confirmed that peptide stabilizes chain association in the BC2 cell lysates. However, infecting these mutants with vectors expressing either pre‐processed antigen or rapidly degraded antigen, failed to overcome their defect in the presentation of endogenous peptide to specific CTL or to mediate surface expression of class I MHC. Preincubation with IFN‐γ completely reversed the endogenous peptide presentation defect, even in mutant cells transfected with a vector encoding a cDNA for the H‐2 molecule restricting CTL recognition. This last result suggests that IFN‐γ corrects the defect by a mechanism separate from simple enhancement of the number of class I molecules produced by the cell. Because there is growing evidence that endogenous peptides can participate in class I MHC assembly, the defect in these mutants could be ascribed to the lack of access to class I molecules by the endogenous peptide. This would prevent stable association of the heavy and light chains and their subsequent transport. Our data suggests that IFN‐γ reestablishes class I MHC surface expression by restoring access of endogenously synthesized peptide to class I molecules.
Inhibitory activity against sporozoites induced by antibodies directed against nonrepetitive regions of the circumsporozoite protein of Plasmodium vivax
Authors: Millet Pascal , Chizzolini Carlo , Wirtz Robert A. , Bathurst Ian , Broderson J. Roger , Campbell Gary H. , Collins William E.
Published: March 2007

In previous studies, Saimiri sciureus boliviensis monkeys have been immunized with four recombinant proteins reproducing part of the circumsporozoite (CS) protein of Plasmodium vivax sporozoites (NSl81V20, rPvCS‐1. rPvCS‐2, rPv‐CS‐3), or with irradiated sporozoites of P. vivax Salvador I strain. To analyze the antibody response elicited against epitopes located outside the immunodominant repeat region of the CS protein, serum samples from these animals were tested for their ability to inhibit the in vitro development of liver stages of P. vivax VK247 strain, characterized from the other strains only by a specific repeat region on the CS protein. Results indicated that there is at least one protective B‐cell epitope outside the repeat region of the CS protein of P. vivax sporozoites, and that this epitope can be expressed by irradiated sporozoites, rPvCS‐2 and ‐3, but not by rPvCS‐1 or NS181V20. Therefore, we designed peptides from the amino acid sequences present both in rPvCS‐2 and ‐3, but not included in the recombinant proteins rPvCS‐1 and NS181V20. Anti‐peptide antibodies had no activity on the development of sporozoites of P. vivax Salvador I strain, into schizonts in primary culture of Saimiri monkey hepatocytes. In addition, antisporozoite antibodies did not react with any of the peptides. These results suggest that the in vitro inhibition observed in this study could depend upon the conformation of the CS protein. This study also demonstrates that antibody response to unnatural linear epitopes can be induced by immunization with recombinant proteins.
Major histocompatibility complex (MHC) control of CD4 T cell subset activation. II. A single peptide induces either humoral or cell‐mediated responses in mice of distinct MHC genotype
Authors: Murray Joseph S. , Pfeiffer Christiane , Madri Joseph , Bottomly Kim
Published: March 2007

CD4 T cells activated in vivo in response to human collagen type IV (hCol IV) resemble either T helper type 1 (Th1) or Th2 cells depending on the major histocompatibility complex (MHC) class II genotype of the responding mice. H‐2s mice were shown to selectively activate Th1‐like cells, releasing interleukin (IL 2) and interferon‐γ in response to hCol IV, whereas H‐2b,d mice were shown to selectively activate Th2‐like cells, releasing IL 4 and IL 5 in response to hCol IV. These results suggested that MHC class II regulated the type of effector function observed during an immune response. It was of interest to determine if the functional difference observed between the CD4 T cells of the two strains was due to the presentation of different peptides of the hCol IV molecule by the two MHC class II molecules. The present results demonstrate that a single peptide of the collagen IV molecule will elicit a Th1‐like response in H‐2s strains and Th2‐like responses in H‐2b,d strains, as was observed when using the intact hCol IV molecule. Furthermore, the failure to generate Th1‐like responses in H‐2b,d could be overcome by increasing the dose of this peptide in vitro. Compared to H‐2s, the Th1‐like response in H‐2b required 100 times the amount of peptide to reelicit an equivalent response. These data suggest that a single peptide of hCol IV can control the type of effector response observed.
Protection from lethal graft‐vs.‐host disease by donor stem cell repopulation
Authors: Rozendaal Lawrence , Pals Steven T. , Melief Cornelis J. M. , Gleichmann Ernst
Published: March 2007

Graft‐vs.‐host reaction (GVHR) induced in non‐irradiated F1 mice with DBA/2J parental spleen and lymph node (LN) cells usually does not lead to acute GVH disease (GVHD). This contrasts with the GVHR induced in other parent‐F1 combinations involving both major histocompatibility complex (MHC) class I and class II differences between donor and host. Most signs of acute GVHD in non‐irradiated F1 mice relate to immunodeficiency following destruction of the lymphohemopoietic system of the host, which leads to wasting and death due to infections. This sequence of events is prevented when donor lymphoid cells, originating from grafted stem cells, repopulate the destroyed lymphohemopoietic system of the host. To examine whether a “silent” repopulation of the F1 host by donor stem cells might underly the absence of clinical signs of acute GVHD when GVHR is induced with DBA/2J lymphoid cells, GVHR was induced with LN cells, which do not contain stem cells. Indeed. GVHR induced in (C57BL/10 × DBA/2J)F1 (BDF1) mice with 80 × 106 DBA/2J LN cells led to acute GVHD. Signs of acute GVHD such as wasting and death did not occur when donor stem cells, from an inoculum of DBA/2J spleen and LN cells, were allowed to repopulate the lymphehemopoietic system of the host. The effect of donor stem cells on clinical signs of acute GVHD was more apparent when (B10.D2 × DBA/2J)F1, instead of DBA/2J, lymphoid cells were used to induce GVHR. The detection of alloreactive anti‐host cytotoxic T lymphocyte (CTL) activity during acute GVHD induced with DBA/2J donor lymphoid cells supports the hypothesis that such CTL contribute to the destruction of the host immune system in acute GVHD.
Two signals are involved in polyclonal B cell stimulation by T helper type 2 cells: a role for LFA‐1 molecules and interleukin 4
Authors: Lohoff Michael , Koch Andreas , Röllinghoff Martin
Published: March 2007

T helper cell type 2 (Th2) cells when triggered by antibodies to CD3 acquire the capacity to stimulate the polyclonal proliferation of syngeneic, resting B cells. Here, we tested the ability of various monoclonal antibodies (mAb) to block the B cell proliferation‐inducing potential of such activated Th2 cells. We demonstrate that anti‐interleukin 4, as well as anti‐LFA‐1 antibodies interfere with the T‐B cell interaction. In kinetic studies, anti‐LFA‐1 was found to be operative during the first half and anti‐interleukin 4 during the second half of the 48‐h culture period. This defines at least two different steps in B cell triggering by Th2 cells. In addition, the data imply that the T‐B cell interaction involves an additional structure, namely an activation molecule, on the T cell surface.
Activation of natural killer cells via the FcγRIII (CD16) requires initial tyrosine phosphorylation
Authors: Ståhls Anders , Heiskala Marja , Mustelin Tomas , Andersson Leif C.
Published: March 2007

Triggering of the FcγRIII (CD16) on natural killer (NK) cells by monoclonal antibodies or antibody‐coated target cells stimulates a rapid phospholipase C (PLC)‐mediated hydrolysis of inositol phospholipids and results in subsequent delivery of the lytic hit. The role of initial tyrosine phosphorylation in these events was investigated with a tyrosine protein kinase (TPK) inhibitor, genistein. At doses that inhibited CD16‐triggered tyrosine phosphorylation of substrates in intact cells, genistein did not influence serine/threonine phosphorylation or target cell binding but prevented PLC activation, cell‐mediated cytotoxicity and antibody‐dependent cellular cytotoxicity. These findings indicate that tyrosine phosphorylation is an early and critical event during receptor‐mediated activation of the lytic machinery.
Cationic residues in pathogenic anti‐DNA autoantibodies arise by mutations of a germ‐line gene that belongs to a large VH gene subfamily
Authors: O'Keefe Theresa L. , Datta Syamal K. , Imanishi‐Kari Thereza
Published: March 2007

The F1 progeny of autoimmune NZB and normal SWR mice uniformly develop severe and accelerated lupus nephritis. The (SWR × NZB)F1 mice produce an oligoclonally expanded population of nephritogenic anti‐DNA autoantibodies that share a recurrent cross‐reactive idiotype (Id564), use highly homologous VH genes and surprisingly have the CH region allotype of the normal SWR parent. From extensive library analyses, we isolated 15 SWR germ‐line genes which are most closely related to the pathogenic autoantibody VH564 gene and which also belong to the NPb subfamily of J558 VH genes. We found that the pathogenic VH genes are probably somatic variants of a SWR germ‐line VH gene, SW6—3, and they have several basic amino acid substitutions, in addition to those already present in the SW6—3 germ‐line gene. Since these pathogenic autoantibodies are not detectable in the sera of the normal SWR mice despite the presence of the SW6—3 gene, strong selection and expansion of B cells expressing the SW6—3 VH gene is probably occurring in (SWR × NZB)F1 lupus‐prone mice. We also isolated eight germ‐line genes from the NZB mouse which are homologous to SW6—3. The autoimmune NZB parent that rarely develops nephritis lacks the SW6—3 gene, but has several highly homologous germ‐line VH genes that would encode less cationic antibodies. These results establish a correlation between structure and pathogenic potential of VH genes.
Natural mouse IgG reacts with self antigens including molecules involved in the immune response
Authors: Berneman Armand , Ternynck Thérèse , Avrameas Stratis
Published: March 2007

IgG isolated on protein A‐Sepharose from pools of normal sera from various mouse strains were examined by immunoblotting for reaction with self antigens. Homogenates of the major mouse organs, i.e. brain, skin, spleen, kidney, adrenals, thymus, heart, muscle and liver were used as the source of autoantigens. IgG stained at least 220 bands on the immunoblots. The antigens corresponding to these bands were tentatively identified by molecular mass estimation and referenced to computerized mouse protein data banks. IgG mainly recognized enzymes but it also stained intracellular structural constituents and surface molecules implicated in the functioning of the immune system. The validity of this identification was confirmed by analyzing purified antigens from mouse or other animal species by immunoblotting and enzyme immunoassays. Furthermore, extracts of 125I‐surface‐labeled cells were immunoprecipitated with IgG in the liquid phase or immobilized on beads. The proteins precipitated migrated to the same positions as those precipitated by specific monoclonal antibodies (mAb), such as class I α chain and β2‐microglobulin, class II α and β chains, CD3, CD4 and CD8 antigens. The results obtained with several enzyme immunoassay procedures using cell membrane extracts, specific mAb and normal IgG further supported the specific interaction of IgG with Ia, CD4 and CD8 molecules. Affinity chromatography indicated that at least 20% of normal mouse IgG possess polyreactive autoantibody function. Dissociation constants of these IgG were calculated for some autoantigens and found to be in the range of 2 × 10−6−7 × 10−6 M. It is concluded that normal mouse IgG exhibit autoreactivities similar to those previously described for IgM.
T cell subset‐specific expression of antigen receptor β chains in α chain‐transgenic mice
Authors: Ivars Fredrick
Published: March 2007

A large number of Vβ8 gene‐encoded cDNA were analyzed from peripheral CD4+ and CD8+ T cell subsets of T cell receptor (TcR) α chain‐transgenic mice. This analysis demonstrates that a limited repertoire of TcR β chains are co‐expressed with the transgenic α chain. Most importantly, certain Vβ8‐Jβ combinatons were found exclusively in one of the subsets and, in some cases, subset‐specific differences were localized to the VDJ junctional region of the β chain genes. In contrast, CD4−CD8−transgenic T cells, as well as CD4+ and CD8+ T cells from normal littermate controls, were found to express diverse β chain repertoires. The present study suggests that β chains with distinct structural characteristics are expressed in the CD4+ and CD8+ subsets, respectively. Moreover, the data suggest that the same structural constraints do not apply to the population of CD4−CD8− transgenic T cells.
Allorecognition of isolated, denatured chains of class I and class II major histocompatibility complex molecules. Evidence for an important role for indirect allorecognition in transplantation
Authors: Dalchau Rosemarie , Fangman Josef , Fabre John W.
Published: March 2007

Classical RT1‐A class I and RT1‐B class II major histocompatibility complex (MHC) molecules were purified from DA (RT1av1) spleens, and the individual chains separated and purified by preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. LEW (RT11) rats were immunized with the pure class I heavy chain, the RT1‐B α chain and the RT1‐B β chain with the aim of priming to indirect allorecognition (i.e. after processing and presentation of DA MHC chains on LEW antigen‐presenting cells) in the absence of any priming to direct allorecognition (i.e. to whole, undenatured, dimeric DA MHC molecules). LEW rats immunized with each of the three DA MHC chains produced alloantibodies to these chains, suggesting that indirect allorecognition did occur, because of the requirement for cognate recognition of B cells by T helper cells. This also demonstrated polymorphism of all three chains between the DA and LEW strains. The antibodies to the isolated, denatured MHC chains did not react to the whole MHC molecules on DA cells, with the possible exception of very weak reactions in some class I heavy chain‐immunized rats. DA skin grafts placed on LEW recipients immunized with each of the DA MHC chains were rejected in an accelerated fashion. Following DA skin grafting, there was an accelerated production of antibodies to whole, undenatured class I MHC molecules, even in the LEW rats preimmunized with RT1‐B α and RT1‐B β chains. These data suggest that indirect allorecognition can play an important role in the effector mechanisms of allograft rejection, and demonstrate T helper priming as one possible mechanism whereby this might be effective.
γ/δ T cell antigen receptors expressed on tumor‐infiltrating lymphocytes from patients with solid tumors
Authors: Nanno Masanobu , Seki Hidetoshi , Mathioudaki George , Suzuki Ryuzi , Itoh Kyogo , Ioannides Constantin G. , Suzuki Satsuki , Chen Pei‐Feng , Platsoucas Chris D.
Published: March 2007

The expression of γ/δ T cell antigen receptors (TcR) in T cell lines or clones derived from tumor‐infiltrating lymphocytes (TIL) from patients with solid tumors was investigated. γ/δ TcR T cell lines were derived from TIL from patients with Wilms tumor, sarcoma or metastatic melanoma by stimulation with autologous tumor cells alone and recombinant interleukin 2 and they exhibited nonspecific cytotoxicity against autologous and allogeneic tumor cells, or cells of the K562 or the MEL21 tumor cell lines. Two T cell lines were derived from a patient with Wilms tumor. One of them expressed a non‐disulfide‐linked γ/δ TcR using the 60‐kDa γ chain, whereas, the other expressed a disulfide‐linked γ/δ TcR. A T cell line was derived from a patient with sarcoma and expressed a disulfide‐linked γ/δ TcR, whereas, a T cell line derived from a patient with melanoma expressed a non‐disulfide‐linked γ chain of 62 kDa. Several T cell clones were developed from patients with metastatic melanoma or Wilms tumor and expressed either disulfide‐ or non‐disulfide‐linked γ/δ TcR. Northern analysis of RNA from certain of these clones revealed a full‐length γ chain transcript, whereas, the α or β chain transcripts were either absent or truncated. These T cell clones exhibited nonspecific cytotoxicity. Both disulfide‐ and non‐disulfide‐linked TIL T cell lines and clones expressed the δ TCS1 determinant. γ/δ TcR+ cells in freshly prepared TIL from these patients were present in low proportions (<5%) and their δTCS1/δ1 ratios were within the range observed in the peripheral blood of normal donors. These results demonstrate that both disulfide‐and non‐disulfide‐linked γ/δ TcR are expressed on T cell lines and clones derived from TIL from solid tumors. Non‐disulfide‐linked γ/δ TcR using the 56–66‐kDa γ chain are frequently found on TIL‐derived T cell lines and clones. These 56–66‐kDa γ chains are rarely expressed on T cell lines or clones derived from peripheral blood lymphocytes of normal donors.
Interleukin 2 receptor targeted fusion toxin (DAB486‐IL‐2) treatment blocks diabetogenic autoimmunity in non‐obese diabetic mice
Authors: Pacheco‐Silva Alvaro , Bastos Marcus G. , Muggia Robert A. , Pankewycz Oleh , Nichols Jean , Murphy John R. , Strom Terry B. , Rubin‐Kelley Vicki E.
Published: March 2007

Insulin‐dependent diabetes mellitus (IDDM) is strikingly similar in the non‐obese diabetic (NOD) mouse and humans. In IDDM, the systematic autoimmune destruction of insulin‐producing β cells within the pancreas is dependent on autoreactive T cells. This autoimmune process can be accelerated by transferring spleen cells from diabetic donors into irradiated syngeneic NOD mice. In a previous study we established that interleukin 2 receptor (IL 2R)‐bearing cells propagated from pre‐diabetic NOD mice promote IDDM. Therefore, we reasoned that specific elimination of IL 2R+ T cells should abort the diabetogenic process. T cell expressing IL 2R can be selectively destroyed with a diphtheria toxin‐related IL 2 fusion protein (DAB486‐IL‐2). We set DAB486‐IL‐2 the challenging task of preventing fulminant IDDM accelerated by the adoptive transfer of diabetic spleen cells. Eight weeks after the adoptive transfer only 10% and 20% of NOD mice treated with 10 and 5 μg/day of DAB486‐IL‐2, respectively, became diabetic while 100% control mice (vehicle buffer) became diabetic within 5 weeks. A dose of 1 μg/day of DAB486‐IL‐2 had no protective effect. Although the protection conferred by DAB486‐IL‐2 is not permanent, it is maintained for at least 4 weeks following cessation of treatment. Furthermore, even though these NOD mice do eventually become diabetic, the tempo of expression and severity of diabetes, as assessed by the level of hyperglycemia, is dramatically reduced. Although histologic examination of pancreas revealed minimal degree of mononuclear infiltrate within the islets in both groups, the vehicle control mice had fewer islets per section indicating many islets had already been destroyed. In addition, spleen cells from diabetic NOD mice which were pre‐treated with DAB486‐IL‐2 (10μ/day) for 1 week lost their ability to transfer disease. Taken together, these studies strongly support the concept that IL 2R‐bearing T cells are essential for the induction of IDDM and suggest that DAB486‐IL‐2 would be a promising therapeutic approach in the treatment of human IDDM.
The effect of an immunoglobulin μ transgene on B cell maturation
Authors: Iacomini John , Imanishi‐Kari Thereza
Published: March 2007

In μ 17.2.25‐transgenic (M54) mice the absolute number of surface IgM (slgM) B cells in lymphoid organs is drastically reduced compared to normal C57BL/6 mice and a high frequency of B cells express the immunoglobulin (Ig) encoded by the transgene rather than endogenous Ig on the surface. To determine the effect of a μ trangene on B cell development, adoptive cell transfers were performed using μ transgenic (M54) bone marrow and fetal liver cells. The data presented support the following conclusions: (a) adult transgenic bone marrow contains functional B cell precursors able to mature and repopulate the spleen and peritoneum of recipient mice. The relative frequency of transgene (sIgMa) and endogenous (sIgMb) surface sIgM‐positive B cells reconstituted by transgenic bone marrow in allotype‐matched C57BL/6 recipients is the same as in the M54 donors; (b) serum analysis indicates that transgenic bone marrow donor cells can reconstitute B cells in congenic and severe combined immunodeficient (SCID) recipient mice; (c) transgenic fetal liver cells are not a richer source of precursors for B cells expressing endogeneous Ig; (d) in transgenic mice sIgM+ B cells are not restricted to the CD5+ phenotype, however, the relative frequency of sIgMb B cells that are CD5+ is higher in transgenic than normal mice; and (e) bone marrow cells from adult normal and transgenic mice are able to generate CD5+ B lymphocytes in the spleen and peritoneum of allotype‐congenic and neonatal SCID recipient mice.

The results indicate that the presence of a complete μ heavy chain transgene does not result in a selective developmental block of “conventional” bone marrow‐derived pre‐B and B cells.
Diversity of T cell receptor α and β chain genes expressed by human T cells specific for similar myelin basic protein peptide/major histocompatibility complexes
Authors: Giegerich Gerhard , Pette Martin , Meinl Edgar , Epplen Jörg T. , Wekerle Hartmut , Hinkkanen Ari
Published: March 2007

T cell receptor (TcR) α and β nucleotide sequences involved in the human autoreactivity to myelin basic protein (MBP) were studied by screening cDNA libraries derived from 11 independent T lymphocyte clones (TCC) established from multiple sclerosis patients and healthy donors. The TCC with defined MBP peptide specificity and HLA‐DR restriction expressed multiple TcR. Even TCC recognizing the same human MBP peptide [amino acids (aa) 139–153] in identical or very similar HLA‐DR context expressed diverse TcR. Two TCC which recognized peptide aa 139–153 equally well in the context of both HLA‐DR2a and ‐DR1 molecules used distinct TcR α but identical β chains.

The knowledge of TcR β and TcR α chain sequences of human MBP‐specific T cells will allow studies correlating structure and function of TcR and their targets in MBP autoreactivity. This may have an impact on the development of immunotherapies in multiple sclerosis.
Constraints in antigen processing result in unresponsiveness to a T cell epitope of hen egg lysozyme in C57BL/6 mice
Authors: Kim Byung S. , Jang Yong‐Suk
Published: March 2007

T cell hybridoma clones were derived after fusion of BW‐5147 parent cells with lymphocytes from C57BL/6 mice injected with phosphorylcholine (PC)‐hen egg lysozyme (HEL) conjugates. Several T cell hybridomas were preferentially reactive with PC‐HEL over unconjugated HEL, and a particular clone (PC‐H4.1) was further analyzed. This T cell hybridoma clone could respond to its maximal level toward unconjugated HEL only when the dose of HEL was increased to 5–10‐fold of the PC‐HEL concentration. Interestingly, this clone was not stimulated by unfolded HEL (by S‐carboxymethylation) to the level of PC‐HEL. A synthetic peptide representing the amino acid position 47‐61 of HEL, which is known to be non‐immunogenic upon HEL injection in C57BL/6 mice, was able to stimulate the hybridoma only to a level comparable to that induced by unconjugated HEL. The T cell response to this synthetic peptide required an additional antigen‐processing step, based on its inability to stimulate T cells after treatment of antigen‐presenting cells with leupeptin, chloroquine or paraformaldehyde. Deletion of a single C‐terminal amino acid residue of HEL 47–61 (arginine) significantly enhanced (10–100‐fold of HEL47–61) the T cell reactivity and abrogated the necessity of further antigen processing. These results suggest that the lack of a T cell response to a certain epitope may not be due to the lack of a T cell repertoire reactive to the epitope. In some cases, the unresponsiveness may be due to the difficulty in generating the particular epitopes. Taken together, modification of the lysozyme molecule with PC conjugation may facilitate further antigen processing of HEL to generate an optimal epitope for the nonresponder mice.