bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic differences in the T cell receptor alleles of LEW rats and their encephalomyelitis‐resistant derivative, LER, and their impact on the inheritance of EAE resistance
Authors: Blankenhorn Elizebeth P. , Stranford Sharon A. , Smith Paul D. , Hickey William F.
Published: March 2007

Experimental allergic encephalomyelitis (EAE) is an animal model for the human disease, multiple sclerosis. The LEW rat strain is very susceptible to induction of EAE, whereas the closely related, major histocompatibility complex (MHC)‐identical, inbred strain LER is resistant. In this report, the two rat strains have been compared for differences at a number of immunologically relevant loci by restriction fragment length analysis and by nucleotide sequencing. A major difference between the two strains was discovered at the T cell receptor β chain locus (TcR β). Both variable (Vβ8) and constant (Cβ1) region elements of TcR β showed allelic variation between LEW and LER.

The known genetic influences in rat models of autoimmunity are currently limited to those encoded by the rat MHC, RT‐1. In this study we report our characterization of the allelic differences in TcR β chains between two rats which differ in their susceptiblity to induced EAE, with the goal of understanding the role played by these allelic forms of TcR in the pathogenesis of EAE. The importance of the TcR β allelic difference in resistance or susceptibility to EAE was assessed in a study of backcross rats scored for both EAE and for the novel LER TcR β allele. We found that the TcR β allele from the susceptible strain was present in three out of four susceptible rats, suggesting that it is an important, but not the only, genetic factor in EAE. Supporting this conclusion were the observations that 12 of 13 rats with homozygous LER‐derived TCR β alleles were resistant to EAE.
Hemolytic anemia in non‐obese diabetic mice
Authors: Baxter Alan G. , Mandel Thomas E.
Published: March 2007

The non‐obese diabetic mouse (NOD mouse) is widely used as a model of organ‐specific autoimmunity because it develops specific autoimmune destruction of pancreatic β cells mediated by T cells and culminating in insulin‐dependent diabetes mellitus. Here, we report that the NOD mouse also develops Coombs'‐positive hemolytic anemia, a B cell‐mediated autoimmune disease.

Aged NOD mice were found to have splenomegaly and jaundice predominantly due to raised unconjugated serum bilirubin. Their hematocrits were markedly lowered, and there was a reciprocal increase in the reticulocyte count. Red blood cells (RBC) from anemic mice showed a normal lytic response to hypotonicity. RBC from non‐anemic mice had normal half lives in non‐anemic, non‐diabetic NOD mice by 51Cr labeling but, dramatically shortened half lives in anemic mice. Similar results were obtained with RBC from anemic mice. Hemolysis could be transferred with serum from anemic mice resulting in reticulocytosis. The antibody‐mediated nature of the anemia was confirmed with the direct Coombs' test. Anemia was found only in mice aged > 200 days and was more common in diabetic (4/8) than non‐diabetic (1/16) mice at 300 days. However, by 550 days, 14/17 non‐diabetic mice were affected.
Cytotoxic T lymphocyte precursor cells specific for the major histocompatibility complex class I‐like antigen, Qa‐2, require CD4+ T cells to become primed in vivo and to differentiate into effector cells in vitro
Authors: Muraoka Shizuko
Published: March 2007

Experiments were performed to determine whether CD4+ T cells are required for the generation of cytotoxic T lymphocytes (CTL) specific for the nonpolymorphic major histocompatibility complex (MHC) class I‐like antigen, Qa‐2. Splenic T cells from BALB/cBy (Qa‐2b) mice that had been immunized with irradiated BALB/cJ (Qa‐2a) splenocytes generated CTL following in vitro stimulation with BALB/cJ splenocytes. These CTL lysed all Qa‐2+, but not Qa‐2− targets, regardless of the H‐2 haplotypes of target cells or their non‐MHC backgrounds. This apparent MHC class I‐unrestricted recognition of Qa‐2 antigen was confirmed using Qa‐2‐specific CTL clones. The Qa‐2‐primed CTL precursor cells (CTLp) and CTL were found to be CD8+ T cells. Primed splenocytes depleted of CD4+ T cells prior to culture failed to generate CTL, but addition of lymphokines to the culture restored the CTL generation. Stimulation of primed splenic T cells with irradiated Qa‐2+ T blast cells, instead of splenocytes or B blast cells, led to little to no CTL generation, suggesting that MHC class II molecules are involved in the presentation of Qa‐2 antigen to CD4+ T cells. This was also supported by the results of experiments using Qa‐2+, class II− thymoma cells of BALB/c origin. Stimulation of the thymoma‐primed splenic T cells with the mitomycin C‐treated thymoma cells resulted in no generation of anti‐Qa‐2 CTL, despite the fact that high levels of CTL specific for minor histocompatibility (H) antigens and H‐2d were generated by immunizing the corresponding allogeneic hosts with the thymoma. However, the addition of lymphokines rendered thymoma‐primed T cells capable of generating anti‐Qa‐2 CTL. Both CD4+ and CD8+ T cell populations, isolated from the BALB/cJ splenocyte‐primed responder cells, proliferated in vitro in response to the Qa‐2+ splenocytes, suggesting that Qa‐2‐reactive CD4+ T cells were present in the immunized mice. Depletion of CD4+ T cells from thymectomized BALB/cBy mice with anti‐L3T4 monoclonal antibodies markedly reduced, but did not eliminate anti‐Qa‐2 CTL generation, In contrast, depletion of CD8+ T cells led to a complete abrogation of the CTL response. Addition of lymphokines to the culture of responder cells depleted of either T cell subset did not restore their reactivity. It is concluded that anti‐Qa‐2 CTLp need “help” from CD4+ T cells to become primed in vivo. Furthermore, primed CTLp also need “help” or lymphokines provided by CD4+ T cells to differentiate into effector CTL in vitro. This dependency of anti‐Qa‐2 CTLp on CD4+ T cells is different from that of CTLp specific for Qa‐1, minor H antigens, or MHC class I antigens.
Differential activity of recombinant lymphokines on mouse B cell proliferation and cell cycle progression are revealed by 5‐bromo‐2′‐deoxyuridine/Hoechst 33258 dye flow cytometry
Authors: Seyschab Helga , Hoehn Holger , Rabinovitch Peter S. , Chen Una
Published: March 2007

Activation of resting mouse B cells with anti‐μ chain antibodies (anti‐μ) leads to cell proliferation. We have investigated the effect of recombinant T cell interleukins (IL 2 to IL 6) on such anti‐μ‐induced proliferation. No proliferative response was detected when IL 2, IL 3 and IL 6, either alone or in combination with anti‐μ, were studied. Furthermore, neither IL 4 nor IL 5 could induce proliferation when added alone to B cell cultures. However, when combined with anti‐μ, IL 4 as well as IL 5 stimulated cell growth. Analysis by 5‐bromo‐2′‐deoxyrudine/Hoechst 33258 flow cytometry revealed distinct effects of IL 4 and IL 5 on B cell growth. In the presence of anti‐μ, both IL 4 and IL 5 co‐stimulated unfractionated splenic B cells. However, when B cells were separated into subpopulations by density, IL 4 proved to be a cell cycle progression factor, stimulating the majority of resting B cells to enter the cell cycle. In contrast, IL 5 had little effect on the resting fraction of B cells. Rather, IL 5 acted as a co‐competence factor, stimulating predominantly low‐density B cells. Following exposure of anti‐μ alone, most B cells accumulated in the G1 of the second cycle. Upon addition of IL 4, the cells acquired the ability to progress into the next S phase compartment. Contrary to what is seen when B cells are stimulated by other mitogens, very few cells are in the G2 compartments after anti‐μ plus IL 4 stimulation. This phenomenon was not due to a differential cell cycle progression rate. Our findings provide an analytical basis for fractionating cell‐cycle‐compartment‐specific B cells for their molecular study.
Involvement of both T cell receptor Vα and Vβ variable region domains and α chain junctional region in viral antigen recognition
Authors: Brändle Daniel , Bürki Kurt , Wallace Valerie A. , Rohrer Urs Hoffmann , Mak Tak W. , Malissen Bernard , Hengartner Hans , Pircher Hanspeter
Published: March 2007

We have studied the lymphocytic choriomeningitis virus (LCMV)‐specific cytotoxic T cell response in transgenic mice expressing either the T cell receptor (TcR) α (Vα2/JαTA31) or the corresponding TcR β (Vβ8.1/Dβ/Jβ2.4) chain originally isolated from the LCMV glyconrotein specific (residues 32–42), H‐2Db‐restricted T cell clone P14. The expression of single transgenic TcR chains did not influence the corresponding endogenous TcR V gene usage in unstimulated T cells indicating that one particular TcR α or β chain can randomly pair with different Vβ or Vα chains without any obvious bias. However, upon infection with LCMV, reactive cytotoxic T lymphocytes (CTL) from P14 β‐transgenic mice were predominantly Vα2+ whereas CTL from P14 α‐transgenic mice preferentially expressed Vβ8.1 and unexpectedly also Vβ8.3 (but not Vβ8.2). Correspondingly the LCMV‐specific CTL response in both α and β TcR‐transgenic mice was strongly biased to the original P14 T cell epitope (LCMV glycoprotein residues 32–42). Sequence analysis of a large panel of LCMV‐reactive “half‐transgenic” TcR from P14 single receptor chain‐transgenic mice revealed a highly conserved VJα and a more diverse VDJβ junctional region. This report demonstrates that the antigen specificity of the studied TcR depends on the specific combination of both TcR α and β chains which implies that amino acids located in the TcR Vα and Vβ segments as well as in the junctional region are involved in binding of the viral antigenic fragment.
Structural requirements of the cytoplasmic domains of the human macrophage Fcγ receptor IIa and B cell Fcγ receptor IIb2 for the endocytosis of immune complexes
Authors: Engelhardt Waltraud , Gorczytza Harald , Butterweck Anette , Mönkemann Heike , Frey Jürgen
Published: March 2007

Two isotypes of the monocyte/macrophage as well as B cell Fcγ receptor type II (FcRIIa and FcRIIb2, respectively) mainly differ in the length (76 vs. 44 amino acids) and amino acid sequence of their cytoplasmic domains. Only the eight amino acids just behind the putative transmembrane region are identical. Despite this marked difference, both FcRII mediate endocytosis of immune complexes. To determine the functional significance of the cytoplasmic domains, we expressed truncated FcRIIa and FcRIIb2 in FcR− BHK‐21 cells. Mutants of both receptors containing only one amino acid (tail‐minus) of the cytoplasmic domain failed to mediate immune complex uptake. The significance of the cytoplasmic domain of the receptors could be further demonstrated using a chimeric FcRIII‐FcRIIa construct. Therefore we expressed an FcRIII lacking the hydrophobic carboxyl terminus (containing the putative phosphatidyl — inositol — glycan anchor site) fused inframe to the transmembrane and cytoplasmic domain of the FcRIIa in BHK‐21 cells. In contrast to the wild type FcRIII, this chimeric receptor mediated immune complex uptake indistinguishable from that mediated by the FcRIIa. Receptor mutants with relatively short cytoplasmic domains (FcRIIb2: 13, and FcRIIa: 16 amino acids) revealed, that these short amino acid stretches are sufficient to allow reduced receptor‐mediated endocytosis of bound ligand. Furthermore, using FcRIIa deletion mutants with a cytoplasmic domain consisting of 62, 46, and 28 amino acids, respectively, we found that the capability of these mutants to mediate immune complex uptake decreased gradually with the truncation of the cytoplasmic tails. Thus, only short amino acid sequences of the cytoplasmic domain are sufficient to enable an, albeit reduced, receptor‐mediated endocytosis.
Clonal persistence of B lymphocytes in normal mice is determined by variable region‐dependent selection
Authors: Thomas‐Vaslin Véronique , Andrade Luiz , Freitas Antonio , Coutinho Antonio
Published: March 2007

Many adult splenic B cells die within 1 week in the spleen of adoptive adult recipient mice; in contrast, the cellular environment of newborn recipients allows for their expansion and persistence for several weeks. In the present study, we show that the local environment of adult peritoneal cavity also allows adult splenic B cells to persist for over 2 weeks after intraperitoneal transfer. In order to determine whether the persistence of donor B cells in newborn hosts and in the peritoneum of adult recipients results from a selection process involving the clonal specificities expressed, the variation in time of VH gene family repertoires of donor B cells was analyzed in the hosts. At different times after the transfer of splenic cells from lipopolysaccharide (LPS)‐reactive mice into LPS‐non responder histocompatible recipients, mRNA colony blot assays were performed. The results show that among the donor adult LPS‐reactive B cells, the VH genes are differently used by the expanding or persisting B cells, in both kinds of recipients. Thus, cells expressing J558 or VH11 gene families are, in particular, positively selected, while those expressing D‐proximal or J606 and 36‐30 VH gene families are less selected. These findings demonstrate that the propensity of B cells to persist and expand is determined by their selection through their immunoglobulin variable regions, rather than by genetic properties linked to particular B cell subsets.
Coordinate secretion and functional synergism of T cell‐associated serine proteinase‐1 (MTSP‐1) and endoglycosidase(s) of activated T cells
Authors: Vettel Ulrike , Bar‐Shavit Rachel , Simon Markus M. , Brunner Georg , Vlodavsky Israel , Kramer Michael D.
Published: March 2007

Cell lysates and exocytosed soluble mediator(s) (ESM) released from CD8+ T cell lines (TCL) by receptor‐triggered secretory exocytosis were tested for degradation of proteoglycahs associated with in vitro produced subendothelial extracellular matrix (ECM). ESM was found to release low‐molecular weight (kav 0.5–0.6) fragments from the sulfated proteoglycans in ECM. In the presence of heparin, an inhibitor for endoglycosidase activity, only high‐molecular‐weight products (kav 0.2) were formed. Preincubation of ESM with HD‐prolylphenylalanyl‐arginyl‐chloromethylketone (PFR‐CK) an inhibitor for the T cell‐associated serine proteinase‐1 (MTSP‐1) totally prevented release of high‐ and low‐molecular weight proteoglycan fragments. Furthermore, it was shown that purified MTSP‐1 is able to release from ECM high‐molecular weight proteoglycans and that this process is inhibitable by PFR‐CK but not by heparin. Further treatment of these soluble high‐molecular weight sulfated proteoglycans with ESM from TCL 1.D9 led to appearance of low‐molecular weight split products (kav 0.5–0.6). This conversion was inhibitable by heparin but not by PFR‐CK. These findings indicate that activated T cells contain two enzymatic activities, i.e. MTSP‐1 and at least one endoglycosidase, which after receptor‐triggered secretion can synergize in the degradation of sulfated proteoglycans in subendothelial ECM.
Differential migration of T and B cells during an acute inflammatory response
Authors: Lee Chee‐Seong , Meeusen Els , Brandon Malcolm
Published: March 2007

Inflammation in the liver and mammary glands of sheep caused by challenge infection with Taenia hydatigena or infusion of killed Staphylococcus aureus, respectively were characterized by the recruitment of both T and B cells. The patterns of migration of these two major lymphocyte subpopulations were distinctly different. While T cells seemed to migrate out of existing, flat endothelium‐lined blood vessels resulting in a diffuse distribution at the sites of inflammation, B cells were characteristically present as clusters of tightly packed cells at restricted sites in the inflamed tissue. Within these B cell clusters distinct capillary vessels lined with plumb endothelial cells were always present suggesting that they were the sites of intense migration of B cells originating from the draining lymph nodes. These results indicate differential regulation of adhesion molecules on B and T cells and/or their ligands on endothelium during acute inflammatory reactions.
Lack of H‐2 restriction of the Plasmodium falciparum (NANP) sequence as multiple antigen peptide
Authors: Pessi Antonello , Valmori Danila , Migliorini Paola , Tougne Chantal , Bianchi Elisabetta , Lambert Paul‐Henri , Corradin Giampietro , Del Giudice Giuseppe
Published: March 2007

The major surface antigen of malaria sporozoites, the circumsporozoite protein, contains a region of tandem amino acid repeats, which in the case of the human malaria parasite Plasmodium falciparum, consist of four amino acids Asn‐Ala‐Asn‐Pro (NANP) repeated up to about 40 times. This repetitive sequence has been considered as the basis for the development of subunit vaccines against P. falciparum malaria. We and others had previously shown that synthetic and recombinant NANP peptides were immunogenic only in H‐2b mice. In the present report we show that, when mice with different H‐2 haplotypes are immunized with the repetitive NANP sequence incorporated in a synthetic branching multiple antigen peptide (MAP), all except one of the mouse strains tested mounted an anti‐peptide antibody response. Such a response does not appear to be due to the peculiar assembly of the NANP sequence. In fact, MAP containing repetitive sequences from circumsporozoite proteins of other malaria parasites did not overcome the genetic restriction of the immune response to the linear peptides. These data show that in the case of the P. falciparum NANP repeats, their immunogenicity can be dramatically changed and increased when these peptides are assembled as MAP. This unexpected finding may be of interest in the design of synthetic candidate malaria vaccines.
Exogenous β2‐microglobulin is required for antigenic peptide binding to isolated class I major histocompatibility complex molecules
Authors: Kane Kevin P. , Sherman Linda A. , Mescher Matthew F.
Published: March 2007

Binding of antigenic peptides to purified class I major histocompatibility complex (MHC) molecules, as measured by antigen‐specific cytolytic T lymphocyte (CTL) degranulation, was found to occur in the presence of serum but not in its absence. The role of soluble β2‐microglobulin (β2m), a normal component of serum, in class I‐peptide complex formation was therefore examined. Sera depleted of β2m did not support effective peptide binding to class I, but binding was restored in the presence of low concentrations of purified human β2m. Sequential incubation of immobilized class I with human β2m first, followed by peptide, resulted in antigenic complex formation, while reversing the order of pulsing could not. Similar results were obtained in experiments examining H‐2Db, Kb and Kd with appropriate peptides and CTL. These results demonstrate that mature class I proteins are not able to directly bind peptide, but that interaction with exogenous β2m results in a structure that will subsequently bind peptide. Binding of exogenous β2m appears to result in “empty” class I molecules, possibly by exchange for endogenous β2m, with a concomitant loss of endogenous peptide.
Efficiency of peptides and lipopeptides for in vivo priming of virus‐specific cytotoxic T cells
Authors: Schild Hansjörg , Deres Karl , Wiesmüller Karl‐Heinz , Jung Günther , Rammensee Hans‐Georg
Published: March 2007

Synthetic peptides and a novel type of lipopeptide vaccine, both containing T cell epitopes recognized by Kd‐restricted, influenza virus‐specific cytotoxic T cells (CTL) were compared in their efficiency to induce virus‐specific CTL in vivo. All attempts to induce virus‐specific CTL with synthetic peptide (in the absence of adjuvants) failed. However, a latent immunization was observed, resulting in an increased response to the injected peptide seen only after boosting the recipients with immunogenic virus. In contrast, priming with synthetic lipopeptide vaccine (tripalmitoyl‐S‐glycerylcysteinyl‐seryl‐serine [P3CSS] coupled to peptide) was successful under most conditions, and matched the priming efficiency seen with infectious virus. The requirements for in vivo priming of virus‐specific CTL using lipopeptide suggest that attachment of the lipopeptide to a hydrophobic entity, such as the cell membrane, is responsible for its efficiency.
The chicken D locus and its contribution to the immunoglobulin heavy chain repertoire
Authors: Reynaud Claude‐Agnès , Anquez Viviane , Weill Jean‐Claude
Published: March 2007

Sixteen D elements were characterized from the chicken genome, 15 of which are extremely homologous. Early expression of this D repertoire was studied for both DJ and VDJ alleles. No N diversification occurs at either DJ or VD junctions. Only P additions were observed, the length of which does not appear restricted to a dinucleotide. A selection for the almost exclusive usage of the first reading frame of the D elements takes place during B cell expansion in the bursa, in parallel with the selection of productive rearrangements. All three reading frames were observed for the DJ allele at each developmental stage, although some bias for the first reading frame occurs already at the junctional stage. The high incidence of D‐D junctions observed (25% among DJ sequences) might represent the major functional contribution of this multigene cluster in a system in which diversity will be generated later on by successive superimposed gene conversions. Other possible functions are discussed. The onset of D diversification through gene conversion between day 15 and day 18 of embryonic development is further documented.
Terminal differentiation of human bone marrow cells capable of spontaneous and high‐rate immunoglobulin secretion: Role of bone marrow stromal cells and interleukin 6
Authors: Roldán Ernesto , Brieva José A.
Published: March 2007

Human bone marrow (BM) is a major site for in vivo immunoglobulin (Ig) formation. A subset of BM cells has been described which is capable of high‐rate Ig secretion for 14 days in vitro without additional stimuli. Therefore, it provides a suitable model for analyzing the terminal B cell differentiation within the BM. The pleiotropic cytokine interleukin (IL) 6 was found to be essential for the further maturation of BM spontaneous Ig‐secreting cells, as can be deduced from the following findings: (a) the addition of anti‐IL‐6 antibodies inhibited most of their Ig production; (b) when endogenous IL6 synthesis in the culture was restricted by using serum‐free medium, the missing IgG secretion could be restored by the addition of exogenous IL6; and (c) active IL6 synthesis by BM cells in fetal calf serum‐containing cultures was confirmed by direct quantitation (range 0.37‐2.1 ng/ml). The presence of IL 6 during the first 3 days of culture was necessary for the induction of Ig secretion. Since neither the proliferation of these cells was elicited by IL6 nor the inhibition of the DNA synthesis in these cultures prevented the IL6‐mediated Ig secretion, IL6 must act on the BM Ig‐secreting cells as a differentiation factor. The source of the endogenous IL6 was, apparently, an adherent cell, since most of the IL 6 production was present in this cell fraction. In contrast, the nonadherent BM cell fraction contained all of the mature Ig‐secreting cells even though it produced little, if any, IL6; the combination of both populations completely restored Ig secretion. Finally, homogeneous populations of fibroblastic stromal cells derived from long‐term BM cultures were totally efficient in inducing Ig secretion by purified BM CD38+ cells; this phenomenon was also demonstrated to be IL6 mediated. Taken together, these findings appear to indicate that BM Ig‐secreting cells are not terminally differentiated, suggesting that their final maturation could be mediated by the BM microenvironment via the paracrine production of IL6.
Interferon‐γ and growth factor production by murine T cells derived from three different lymphoid tissues
Authors: Höién Ingmarie , Möller Göran
Published: March 2007

Various antigen‐presenting cells and the environment in different lymphoid tissues have been suggested to influence the type of lymphokine produced by T cells. We have investigated the mitogen‐induced proliferation, interferon‐γ (IFN‐γ) and growth factor production by cells isolated from spleen, mesenteric and peripheral (axilliary, brachial and inguinal) lymph nodes (LN). We found that stimulation with concanavalin A or staphylococcus enterotoxin B induced IFN‐γ synthesis in spleen cells but not in LN cells. Proliferation and growth factor production were comparable in the three cell populations. The addition of the phorbol ester phorbol 12‐myristate 13‐acetate (PMA), which is commonly used as a substitute for accessory cells, did not influence the IFN‐γ synthesis by LN cells. The growth factor production was, on the other hand, elevated by the addition of PMA. A high number of IFN‐γ‐producing peripheral LN cells were obtained if they were stimulated in the presence of splenic adherent cells. The growth factor synthesis was marginally affected by the presence of these cells. Thus, splenic adherent cells provide a co‐stimulatory signal to the T cell necessary for IFN‐γ synthesis.
A humanized monovalent CD3 antibody which can activate homologous complement
Authors: Routledge Edward G. , Lloyd Irene , Gorman Scott D. , Clark Mike , Waldmann Herman
Published: March 2007

The rat monoclonal antibody (mAb) YTH12.5, specific for the CD3 antigen complex on human T cells has been modified in order to improve its efficacy in human therapy. With the aim of rendering it less immunogenic, it has been humanized using the method of framework grafting. During this process sequence analysis of the YTH12.5 VL gene indicated that it was of the λ subclass, however, it was markedly dissimilar from previously published rat and mouse Vλ gene sequences and may represent a new Vλ gene family. The humanization of this light chain represents the first successful reshaping of a λ light chain V region. To improve the effector function of the antibody we have created a monovalent form (1 Fab, 1 Fc) using a novel method involving the introduction of an N‐terminally truncated human IgG1 heavy chain gene into cells producing the humanized CD3 mAb. Comparison of the mono‐ and bivalent humanized mAb in a complement‐mediated cell lysis assay revealed that the monovalent antibody mediated lysis of human T cell blasts whereas the bivalent form did not. The availability of a humanized, complement‐fixing CD3 mAb may improve opportunities for human therapy, in the management of organ rejection, autoimmunity and the treatment of T cell lymphoma.
Major histocompatibility complex controls clonal proliferation of CD5+ B cells in H‐2‐congenic New Zealand mice: a model for B cell chronic lymphocytic leukemia and autoimmune disease
Authors: Okada Takashi , Takiura Fumiaki , Tokushige Katsutoshi , Nozawa Shingo , Kiyosawa Tomoharu , Nakauchi Hiromitzu , Hirose Sachiko , Shirai Toshikazu
Published: March 2007

By employing H‐2‐congenic NZB, NZW and (NZB x NZW)F1 mice with either the homozygous H‐2d/H‐2d, H‐2z/H‐2z or heterozygous H‐2d/H‐2z haplotype, we found that in the spleen of all the congenic strains homozygous for H‐2z, there were extremely high frequencies of CD5+ B cells. These cells eventually proliferated in an oligoclonal or even monoclonal fashion, and B cell‐chronic lymphocytic leukemia (B‐CLL) developed in some cases. Because this feature was not observed in H‐2d/H‐2d homozygotes or H‐2d/H‐2z heterozygotes, the high CD5+ B cell frequencies are apparently controlled by the homozygosity of a locus or cluster of loci closely linked to H‐2z complex of NZW strain. As the CD5+ B cell frequencies in the peritoneal cavity did not differ among the H‐2‐congenic strains, the frequencies of these cells in the peritoneal cavity and in the spleen appear to be at least in part under separate control. Flow cytometry and Southern blot analyses using an immunoglobulin gene JH probe revealed that the H‐2z/H‐2z homozygotes, there was a propagation of distinct clonal populations between the spleen and the peritoneal cavity, a finding which suggested that in the major histocompatibility complex (MHC)‐related microenvironments for CD5+ B cell propagation differ between the two compartments. All our findings taken together imply that certain different but related MHC haplotypes may predispose either to B‐CLL or to autoimmune disease, in close relatives.
Correlation of lymphocyte lipid composition membrane microviscosity and mitogen response in the aged
Authors: Huber Lukas A. , Xu Qing‐Bo , Jürgens Günther , Böck Günther , Bühler Ernst , Fred Gey K. , Schönitzer Dieter , Traill Karine N. , Wick Georg
Published: March 2007

Healthy aged and young blood donors were investigated for the role of membrane lipid composition in the age‐related increase in membrane microviscosity and decline of mitogen responsiveness. Membrane microviscosity was shown to correlate positively with membrane cholesterol/phospholipid molar ratios, which were significantly elevated in the elderly. A positive correlation also was confirmed between lymphocyte membrane microviscosity, which was measured using the probe 1,6‐diphenyl 1,3,5‐hexatriene, and phytohemagglutinin responsiveness of cells from the same donor. Using stepwise regression statistical analysis, the variables age, cholesterol, cholesterol/total phospholipid and phosphatidyl ethanolamine/phosphatidyl choline molar ratios were all shown to have a significant positive influence on membrane microviscosity, whereas total phospholipids had a negative effect. No statistically significant difference was seen in content of any single saturated or unsaturated fatty acid between young and old donors. After pooling, however, the proportion of all unsaturated fatty acids was significantly higher in cells from the elderly as a consequence of an increase of n‐6 and n‐3 polyunsaturated fatty acids. Changes in lipid composition and physical properties of lymphocyte plasma membranes may, therefore, be responsible (at least partially) for the diminution of immune reactivity in old age.
Different types of allospecific CTL clones identified by their ability to recognize peptide loading‐defective target cells
Authors: Aosai Fumie , Öhlen Claes , Ljunggren Hans‐Gustaf , Höglund Petter , Franksson Lars , Ploegh Hidde , Townsend Alain , Kärre Kias , Stauss Hans J.
Published: March 2007

Allospecific immune responses against the MHC of another individual are remarkably strong, due to a high number of responding T cell clones. Although it has been demonstrated that some allospecific cytotoxic T lymphocytes (CTL) recognize peptides presented by allogeneic MHC class I molecules, it has remained unclear whether MHC molecules can be recognized directly. We used the H‐2b‐derived murine lymphoma mutant RMA‐S, which has a defect affecting peptide loading of class I molecules, to test whether recognition by allospecific CTL always requires the presence of peptides. Three types of anti‐H‐2Kb CTL clones can be distinguished by their ability to lyse RMA‐S target cells. Type A CTL clones efficiently lyse these target cells, the lysis by type B CTL clones is inefficient, and type C clones fail to lyse RMA‐S. Up‐regulation of the levels of H‐2Kb density improved lysis by type B clones, but did not lead to lysis by type C clones. Some type A and B CTL clones apparently can recognize class I molecules devoid of peptides, while others are likely to recognize peptides which are not affected by the presentation defect of RMA‐S. We suggest that type C clones are specific for peptides which are not presented by the mutant cells. The results show that the majority of alloreactive CTL recognize peptide/MHC complexes, while some CTL behave as if they can recognize class I molecules in the absence of MHC‐bound peptides.
Cloning, heterologous expression and characterization of murine interleukin 1 receptor antagonist protein
Authors: Shuck Mary E. , Eessalu Thomas E. , Tracey Daniel E. , Bienkowski Michael J.
Published: March 2007

A cDNA coding for the human interleukin 1 receptor antagonist protein (IL 1Ra) was used to clone the corresponding murine cDNA. The nucleotide sequence of the open reading frame coding for the processed form of mIL 1Ra predicted a 152‐residue protein that was 77% identical to human IL 1Ra. The cellular and tissue distribution of murine IL 1Ra (mIL 1Ra) transcripts showed high levels in macrophages and skin while lower levels were detected in tissues that contain significant numbers of resident macrophages. The portion of the mIL 1Ra cDNA that codes for the mature form of the protein was placed under the control of a Trp promoter and expressed in E. coli at a level of 37% of total cell protein. The expressed protein was secreted into the periplasm and was purified to homogeneity in a single step by cation‐exchange chromatography. Recombinant mIL 1Ra competitively inhibited 125I‐labeled IL 1α binding to murine type I IL 1R present on EL4 6.1 cells (Ki value of 0.21 nM) and antagonized IL 1‐stimulated co‐mitogenesis in murine thymocytes (0.7 x 106‐1.1 x 106 units/mg).
Evidence for multiple sites of interaction in C3 for complement receptor type 2 (C3d/EBV receptor, CD21)
Authors: Esparza Inmaculada , David Becherer J. , Alsenz Jochem , De La Hera Antonio , Lao Zhege , Tsoukas Constantine D. , Lambris John D.
Published: March 2007

Multivalent but not monovalent CR2 ligands are required to elicit Raji cell proliferation as well as other B cell responses. It has been reported (C. Servis and J. D. Lambris, J. Immunol. 1989. 142: 2207) that the tetrameric peptide T‐(C31202–1214)4, which represents the CR2‐binding site in C3d, was able to support Raji cell growth. We show here that the tetrameric peptide T‐(gp35019–30)4, which contains the CR2‐binding site in gp350 protein of EBV also induces Raji cell growth and this effect is inhibited by the monomeric peptides gp35019–30 and C31201–1214. We also investigated the nature of the interaction between C3 fragment and CR2 in order to explain the Raji cell growth‐supporting effect exerted by C3. The following findings suggest that there are multiple sites in the C3 molecule able to interact with CR2: (1) both C3c and C3d immobilized on microspheres are able to bind to Raji cells through CR2.(2) soluble C3d inhibits to a greater extent the binding of CR2 to fixed C3d than to fixed C3b, which suggests the existence of additional CR2‐binding sites within C3b not present in the C3d portion of the molecule; (3) synthetic peptides C31187–1214, C3741–757 and C3295–307 which represent regions of similarity in the C3 molecule bind specifically to CR2 on Raji cells and compete with each other for binding to the receptor and (4) preincubation of microtiter plate‐fixed C3b with monoclonal or polyclonal anti‐peptide antibodies (C3‐9, anti‐C3727–768) recognize the N terminus of the α chain of C3 (including residues 741‐757) inhibited CR2 binding. Therefore, these data suggest that the N terminus of the α chain of C3 is involved in binding to CR2.
Minor histocompatibility antigens, defined by graft‐vs.‐host disease‐derived cytotoxic T lymphocytes, show variable expression on human skin cells
Authors: De Bueger Marleen , Bakker Astrid , Van Rood Jon J. , Goulmy Els
Published: March 2007

Little is known on the effector mechanisms inducing the cutaneous lesions observed during acute graft‐vs.‐host disease (aGvHD) after allogeneic bone marrow transplantation (BMT). Histological findings have indicated that infiltrating CD8+ lymphocytes probably play a role. We addressed the question of whether host minor histocompatibility (mH) antigen‐reactive cytotoxic T lymphocytes (CTL) could account for this phenomenon via direct lysis of the epidermal cell layer. Six CTL clones, obtained from peripheral blood lymphocytes of patients suffering from aGvHD, each recognizing a well‐characterized MHC class I‐restricted mH antigen epitope, were tested on cultured keratinocytes of nine MHC and mH antigen‐typed donors. Four of six mH antigen‐specific CTL clones lysed unstimulated MHC class I‐expressing, as well as recombinant interferon‐γ (rlFN‐γ)‐activated, ICAM‐1, MHC class I‐ and II‐expressing keratinocytes. Two strongly cytolytic CTL clones showed no recognition of keratinocytes of donors whose phytohemagglutinin‐activated T cell lines were readily lysed. With respect to aGvHD, the results imply that some class I‐restricted CTL obtained from peripheral blood lymphocytes of aGvHD patients have the in vitro potential to destroy resting as well as IFN‐γ‐activated epidermal cells, whereas others do not. In other words, CTL‐defined human mH antigens vary with respect to their expression in the skin. It is intriguing that those minor H antigens which cannot be detected on human keratinocytes in vitro are those known to be associated with the occurrence of GvHD.
Signaling by vascular cell adhesion molecule‐1 (VCAM‐1) through VLA‐4 promotes CD3‐dependent T cell proliferation
Authors: Burkly Linda C. , Jakubowski Aniela , Newman Barbara M. , Rosa Margaret D. , Chi‐Rosso Gloria , Lobb Roy R.
Published: March 2007

Vascular cell adhesion molecule, VCAM‐1, is an adhesion molecule expressed on activated endothelium thought to play a role in leukocyte migration to sites of inflammation. VCAM‐1 adheres to leukocytes through the VLA‐4 integrin. Recombinant soluble VCAM‐1 (rsVCAM) and anti‐CD3 mAb OKT3 were utilized to address the role of the VCAM‐1/VLA‐4 pathway in antigen‐dependent T cell activation. Monocyte‐depleted T cells proliferated upon exposure to co‐immobilized OKT3 and rsVCAM but to neither alone. In contrast, an anti‐VLA‐4 mAb HP1/2 failed to co‐activate with OKT3, despite the fact that both rsVCAM and HP1/2 support T cell adhesion comparably. These data indicate that adhesive function is not sufficient for co‐stimulatory activity. They also reveal that VCAM‐1 may play a role in regulating T cell immune responses as well as migration in vivo.
Limiting dilution analysis of proliferative T cell responses to mycobacterial 65‐kDa heat‐shock protein fails to show significant frequency differences between synovial fluid and peripheral blood of patients with rheumatoid arthritis
Authors: Fischer Heinrich Peter , Sharrock Claire E. M. , Colston M. J. , Panayi Gabriel S.
Published: March 2007

Recent evidence has pointed to the mycobacterial 65‐kDa heat‐shock protein (hsp 65) as an antigen that may be important in the pathogenesis of rheumatoid arthritis (RA). Using limiting dilution analysis the frequency of purified protein derivative of tuberculin (PPD) and hsp 65‐responsive T cells was measured in paired peripheral blood and synovial fluid samples of patients with RA. There was no increase in the anti‐PPD or anti‐hsp 65 frequency in synovial fluid compared with peripheral blood. In addition, no difference was found between peripheral blood of RA patients and healthy controls. These results do not support the idea of an important pathogenic role of T cells responding to hsp 65, or a cross‐reacting antigen, in RA.
In vivo evaluation of the effects of interleukins 2, 4 and 7 on enhancing the immunotherapeutic efficacy of anti‐tumor cytotoxic T lymphocytes
Authors: Lynch David H. , Namen Anthony E. , Miller Robert E.
Published: March 2007

The draining lymph nodes of mice injected with viable syngeneic tumor cells contain lymphoid cells capable of generating anti‐tumor cytotoxic T lymphocytes (CTL) during a 4‐day in vitro culture period and intravenous infusion of relatively low numbers of these CTL can effectively eliminate a challenge of fibrosarcoma cells at distal skin sites that would otherwise result in the death of the host. Using this model system the effects of addition of interleukin (IL)2, IL4 or IL7 to the culture medium on the therapeutic efficacy of the anti‐tumor CTL generated was investigated. In this regard, IL7 was found to be the most potent of the cytokines tested. Addition of IL7 either alone, or in combination with low doses of IL2, resulted in the generation of CTL with significantly (6–8‐fold) enhanced therapeutic efficacy in vivo. Anti‐tumor effector cells generated in the presence of IL7 were also found to be at least 4‐fold more effective at eliminating established tumors than CTL generated in medium alone. Of the other cytokines tested, addition of IL2 resulted in elevated CTL activity in vitro, but only a modest (approximately 2–3‐fold) enhancement of the therapeutic efficacy in vivo. Addition of IL4, either alone or in combination with IL2, also resulted in the generation of effector cells with enhanced tumoricidal activity in vitro and yet the therapeutic efficacy of these cells was decreased compared to that of CTL generated in medium alone. These observations indicate that (a) inclusion of IL 7 in the medium in which tumor‐reactive T cells are cultured can markedly enhance the immunotherapeutic efficacy of the resulting effector cell populations; and (b) in vitro assays of tumoricidal activity cannot be used as a reliable predictor of therapeutic efficacy in vivo.
Functionally distinct subsets of human γ/δ T cells
Authors: Morita Craig T. , Verma Sunita , Aparicio Pedro , Martinez‐A. Carlos , Spits Hergen , Brenner Michael B.
Published: March 2007

To determine if effector subsets exist among human γ/δ T cells, we examined the cytokine production and cytotoxic activity of γ/δ T cell clones with different accessory molecule phenotypes, Vδ and Vγ gene expression, and Jγ rearrangements. T cell clones bearing γ/δ T cell receptor produce an array of cytokines like α/β T cell clones. Individual γ/δ T cell clones produced a characteristic array of cytokines without correlation with Vδ or Vγ gene expression. However, when phenotypic subsets were considered, CD4+ γ/δ clones produced significantly higher levels of interleukin 2 and granulocyte‐monocyte colony‐stimulating factor compared with CD4−CD8− and CD8+ γ/δ clones. Similarly, when cytotoxic potential was assessed, CD4+ γ/δ clones exhibited minimal activity when compared with CD4−CD8− and CD8+ adult peripheral blood γ/δ clones. We conclude that functionally distinct γ/δ T cell subsets exist and suggest that these subsets may correlate with expression of the CD4 accessory molecule.
A strong propensity toward loop formation characterizes the expressed reading frames of the D segments at the Ig H and T cell receptor loci
Authors: Abergel Chantal , Claverie Jean‐Michel
Published: March 2007

A compilation of murine and human Ig H and TcR β D segment sequences was used to estimate the relative usage of the various reading frames and to look for associated sequence patterns. We confirm a strong bias in the expression of the Ig H D segments, with more than 90% (murine) and 85% (human) expressed peptides resulting from a preferred reading frame. Remarkably, 86% (mouse) and 90% (human) of those peptides contain at least one glycine residue. All but one of the atypical preferred D peptides contain serine or proline residues and are found in the immediate vicinity of glycine residues provided by specific JH segments. The presence of tyrosine residues is also a characteristic feature of expressed reading frames in both mouse (75%) and human (90%). These results suggest that the constraints of forming a flexible loop within the third complementarity‐determining region, is a factor in the preference for a particular reading frame in Ig H D. For the TcR β D segments, glycine is specified in most reading frames, and no significant preference is observed.
T lymphocyte adhesion to the fibronectin and laminin components of the extracellular matrix is regulated by the CD4 molecule
Authors: Hershkoviz Rami , Miron Shmuel , Cohen Iran R. , Miller Ariel , Lider Ofer
Published: March 2007

The adhesion of T cells to components of the extracellular matrix (ECM) is mediated by the β1 subfamily of integrin receptors, designated VLA. It has been recently demonstrated that the binding of VLA receptors to protein components of the ECM is rapidly augmented by the activation of the T cells without, however, any actual change in the level of expression of the VLA receptors for fibronectin (FN) or laminin (LN). Thus, it is likely that activation of existing VLA receptors is required for binding. The activation must be regulated by T cell surface molecules capable of transducing signals into the cell. We studied the role of the CD4 molecule in the binding of rat CD4+ T cells to the FN and LN components of the ECM. We now report that the CD4 molecule appears to play a major role in regulating T cell interactions with ECM. This conclusion is based on the following observations: (a) monoclonal antibodies directed against the CD4 molecule inhibited T cell adhesion to both FN and LN; (b) down‐regulation of the CD4 molecule resulted in partial loss of the ability of CD4+ T cells to adhere to FN and LN; (c) a CD4+ T cell clone adhered to both FN and LN while a CD4−CD8− clone expressing an identical T cell receptor bound weakly to both proteins and (d) treatment of the CD4+ T cells with an inhibitor of the CD4‐associated tyrosine protein kinase activity inhibited T cell adhesion to both ECM proteins.
Pre‐B lymphocyte‐specific transcriptional control of the mouse VpreB gene
Authors: Okabe Taijiro , Bauer Steven R. , Kudo Akira
Published: March 2007

The VpreB genes, which encode surrogate immunoglobulin light chain molecules, are expressed as RNA almost exclusively in pre‐B cells. We have investigated the transcriptional control mechanisms which are responsible for the pre‐B cell‐specific RNA expression of the mouse VpreB1 and VpreB2 genes. Nuclear run‐on analyses demonstrate that the pre‐B cell‐specific expression of both VpreB genes is controlled primarily at the level of initiation of transcription. S1 nuclease protection‐mapping defined two or three major start sites of transcription for the VpreB genes. To find a promoter and other potential cis‐acting regulatory elements, a 700‐bp fragment 5′ of the transcription start sites of the VpreB1 gene was used in gene transfer experiments and found to act as a promoter in pre‐B lymphocytes. Deletion experiments showed that 191 bp upstream of the most 5′ transcription start site is required for the pre‐B cell promoter activity. DNA sequence analysis of the 5′ region of the mouse VpreB1, VpreB2 and human VpreB genes reveal that this region of approximately 200 bp is strongly conserved. This 200‐bp promoter region contains several conserved nucleotide sequence motifs which may act to mediate the pre‐B cell‐specific transcription of the VpreB genes.
A pre‐B‐ and B cell‐specific DNA‐binding protein, EBB‐1, which binds to the promoter of the VpreB1 gene
Authors: Okabe Taijiro , Watanabe Takeshi , Kudo Akira
Published: March 2007

The VpreB1 protein is thought to be expressed on the surface of pre‐B cells in association with λ5 and μ, heavy chain, and to play an important role on B cell differentiation. The expression of VpreB1 and λ5 is pre‐B cell specific, and regulated at the initiation of transcription. We have identified at least two sequence‐specific DNA‐binding proteins which bind to the region −191 to ‐74 of the promoter of the mouse VpreB1 gene. These DNA‐binding proteins also bind to the promoter of the mouse λ5 gene. One of the two DNA‐binding proteins, called EBB‐1, is restricted to pre‐B and B cells, but not detected in plasma cells, T cells and cells of other lineages. Transient transfection analysis of reporter constructs revealed that the binding sites of these proteins play a significant role in the activity of the promoter, especially the binding site of EBB‐1. Taken together these results suggest that EBB‐1 might be one of the crucial factors which regulates a series of intracellular events in B cell differentiation.