bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of thymic B cells by signals of CD40 molecules plus interleukin‐10
Authors: Inaba Muneo , Inaba Kayo , Fukuba Yoh , Mori Shin‐Ichiro , Haruna Hiroki , Doi Hiroshi , Adachi Yasushi , Iwai Hiroshi , Hosaka Naoki , Hisha Hiroko , Yagita Hideo , Ikehara Susumu
Published: March 2007

We have previously found that thymic B cells, particularly thymic CD5+ B cells, show low responsiveness to the usual B cell stimulants such as lipopolysaccharide or anti‐IgM plus interleukin (IL)‐4, although they proliferate and produce antibodies after direct interaction with major histocompatibility complex class II‐restricted T blasts. These findings raise the possibility that a CD40‐CD40 ligand (L) interaction is involved in the activation of thymic B cells. In the present study, we therefore examine this possibility using CD40L‐transfected Chinese hamster ovary (CHO) cells or anti‐CD40 monoclonal antibody (mAb). When B cells in the spleen and peritoneal cavity were stimulated, they proliferated and produced immunoglobulin (Ig) in the presence of CD40L‐CHO cells or anti‐CD40 mAb alone. However, another signal delivered by IL‐10 in addition to CD40L‐CHO cells or anti‐CD40 mAb was found to be necessary for thymic B cells to proliferate and secrete Ig. Other interleukins acting on B cells, such as IL‐4, IL‐5, and IL‐6, had no effect on the activation of thymic B cells, which thus have unique characteristics not found in peripheral B cells. This report discusses the physiological significance of IL‐10‐ and CD40‐driven signals in the activation of thymic B cells.
Alloreactive natural killer cells in the rat: complex genetics of major histocompabitility complex control
Authors: Naper Christian , Vaage John Torgils , Lambracht Doris , Løvik Guro , Butcher Geoffrey W. , Wonigeit Kurt , Rolstad Bent
Published: March 2007

A major role for the nonclassical major histocompatibility complex (MHC) class I region, i.e. RTI.C, in controlling rat natural killer (NK) cell alloreactivity has recently been established, and several findings suggested the existence of NK‐triggering alloantigens coded for by this region. Here, we have extended our studies on the MHC control of NK cell cytotoxicity against concanavalin A‐activated T cell blasts by comparing semi‐syngeneic and fully allogeneic combinations, and we show the following: (a) The self MHC exerted a strong influence on the NK allorecognition repertoire. (b) When anti‐F1 hybrid cytolytic activities of parental strain NK cells were measured, both recessively and non‐recessively inherited susceptibility patterns emerged. (c) In most combinations parental strain cells were lysed by F1 hybrid NK cells, thus resembling the hybrid resistance phenomenon described in mice. The cytotoxicity was lower in strain combinations where NK susceptibility was inherited non‐recessively, i.e. when parent anti‐F1 reactivity was detected, than in recessive combinations. (d) LEW.1LM1 (RT11m1) target cells, with a deletion in the RTI.C region that includes expressed class I genes, were more sensitive to lysis by MHC matched NK cells (PVG.IL(LEW), RT11) than were parental LEW (RT11) cells. The effect of the deletion was the opposite when MHC allogeneic (RT1c, RT1u) as well as semi‐syngeneic (RT11/c) NK cells were employed, i.e. sensitivity was decreased. We conclude that certain MHC‐encoded antigens, depending on the haplotype combination of effector and target cells, may either trigger or inhibit rat NK cell cytotoxicity. Furthermore, the potential role of peptides bound to MHC class I molecules recognized by NK cells is discussed.
Human eosinophil peroxidase enhances tumor necrosis factor and hydrogen peroxide release by human monocyte‐derived macrophages
Authors: Spessotto Paola , Dri Pietro , Bulla Roberta , Zabucchi Giuliano , Patriarca Pierluigi
Published: March 2007

Inhibition of growth or eradication of experimentally induced tumors has been shown to be accompanied by infiltration of eosinophils and macrophages into the tumor mass. Since macrophages are important mediators of host antitumor activity, the possibility arises that a collaboration may exist between these two cell types in the control of tumor growth.

In this study, we report the effect of eosinophil peroxidase (EPO), a basic protein contained in eosinophils that binds to several cell types including macrophages, on tumor necrosis factor (TNF) production and hydrogen peroxide release by human monocyte‐derived macrophages. After incubation with EPO, the macrophages produced large amounts of TNF and displayed an enhanced phorbol 12‐myristate 13‐acetate‐triggered hydrogen peroxide release. These effects were accompanied by an increased cell protein content and by morphologic changes leading the large, round macrophages of the control cultures to become elongated, pear‐like or spindle shaped cells after treatment with EPO. The stimulatory effect of EPO on hydrogen peroxide release was insensitive to addition of exogenous catalase, a H2O2‐degrading enzyme, suggesting that an extracellular catalytic activity of EPO was not involved. In addition, myelo‐peroxidase, the homologous peroxidase of neutrophils with a catalytic activity similar to that of EPO, was ineffective. The EPO‐induced effects differed in several aspects from the effects of lipopolysaccaride and interferon‐γ, two well‐known macrophage activators. These findings provide supportive evidence for a functional interrelationship between eosinophils and macrophages that may be physiologically relevant in the tumoricidal activity of macrophages.
T cell receptor ι an alternatively spliced product of the T cell receptor ζ gene
Authors: Nocentini Giuseppe , Ronchetti Simona , Bartoli Andrea , Testa Giuseppe , D'Adamio Francesca , Riccardi Carlo , Migliorati Graziella
Published: March 2007

It has been previously suggested that three alternative splicings of the murine T cell receptor (TCR) ζ gene are involved in the regulation of TCR/CD3 transduction signals. We here describe a new alternative splicing of this gene (TCRι), cloned by reverse transcriptase‐polymerase chain reaction, that is encoded by exons 1–7 and 10. The protein putatively encoded by TCRθ mRNA differs in its carboxy terminus from that coded by TCRθ as a consequence of the reading frame shift of exon 10. The possible role of this new splicing in TCR modulation is briefly discussed.
Fc receptor binding of anti‐CD3 monoclonal antibodies is not essential for immunosuppression, but triggers cytokine‐related side effects
Authors: Vossen Ann C. T. M. , Tibbe G. John M. , Kroos Martin J. , van de Winkel Jan G. J. , Benner Robbert , Savelkoul Huub F. J.
Published: March 2007

A major drawback to the use of OKT3, a mouse anti‐CD3 monoclonal antibody (mAb), as an immunosuppressive agent is the associated cytokine release syndrome. We used a mouse model to elucidate the properties of anti‐CD3 mAb responsible for these cytokine‐related side effects. We have previously demonstrated that the hamster anti‐CD3 mAb 145‐2C11 induced strong cytokine release and morbidity in vivo, whereas two rat anti‐CD3 mAb 17A2 and KT3 did not. In the current study, we show that the mitogenic capacity of soluble anti‐CD3 mAb in vitro correlates with their induction of side effects in vivo. Mitogenesis in vitro and tumor necrosis factor‐α (TNF‐α) release in vivo induced by anti‐CD3 mAb could be inhibited by the anti‐FcγR mAb 2.4G2, indicating that FcγR binding of anti‐CD3 mAb is responsible for their mitogenic properties and for their induction of side effects. Importantly, the two non‐mitogenic rat anti‐CD3 mAb were equally capable of suppressing skin allograft rejection as the mitogenic hamster anti‐CD3 mAb, suggesting FcγR binding of anti‐CD3 mAb is not essential for their immunosuppressive properties. This suggestion is reinforced by our demonstration that administration of 2.4G2 in vivo did not interfere with immunosuppression of skin allograft rejection by 145‐2C11. These findings suggest that clinical use of non‐mitogenic anti‐CD3 mAb will result in effective immunosuppression without cytokine‐related side effects.
Expression of CD34 in endothelial cells, hematopoietic progenitors and nervous cells in fetal and adult mouse tissues
Authors: Lin Guosheng , Finger Elizabeth , Gutierrez‐Ramos José C.
Published: March 2007

The human cell surface molecule CD34 is selectively expressed on uncommitted and committed hematopoietic progenitor cells and on vascular endothelial cells. It has been suggested that CD34 regulates early events in blood cell migration and differentiation, possibly as a cell adhesion molecule. To characterize the patterns of expression of CD34 in the mouse embryo and in the adult, as well as to dissect the function of different portions of the extracellular domain of this molecule, we have generated the first monoclonal antibodies (mAb) specific for mouse CD34. The epitope(s) recognized by these mAb are not carbohydrate moieties, and are comprised either within the immunoglobulin‐like domain or within a portion of the mucin domain, containing approximately half of the predicted O‐ and N‐linked carbohydrate attachment sites. The specificity of the antibodies was established by ELISA and Western blotting. Western analysis revealed that these mAb recognize a protein of approximately 110 kDa in PA6 stromal cell lysates, which can be specifically blocked by the recombinant CD34 protein. To establish the reactivity of these mAb on different cell lineages, a panel of cell lines was stained. This analysis showed strong reactivities with 3T3 fibroblasts, stromal cell lines from fetal liver and with the endothelial cell line D10. Bone marrow hematopoietic progenitors were also stained by these mAb. Immunostaining of frozen sections from embryonic and adult tissues revealed a strong reactivity against vascular endothelial cells at different stages of development, including sinusoidal cells in the fetal liver, yolk sac, and in the fetal bone marrow, endothelial cells from adult lung and kidney, and neural cells, including those of the neural tube of midgestation embryos and neuronal bodies in adult brain.
Prolonged inhibition of an antigen‐specific IgE response in vivo by monoclonal antibody against lymphocyte function‐associated antigen‐1
Authors: Tanaka Yoshitaka , Takahashi Akiko , Arai Iwao , Inoue Tomoyuki , Higuchi Shohei , Otomo Susumu , Watanabe Kazuhito , Habu Sonoko , Nishimura Takashi
Published: March 2007

Intraperitoneal injection of ovalbumin (OVA) into BALB/c mice caused the induction of OVA‐specific IgE production in vivo. However, administration of monoclonal antibody against lymphocyte function‐associated antigen‐1 (anti‐LFA‐1 mAb) at days 0 and 1 after OVA immunization resulted in an inhibition of OVA‐specific primary and secondary IgE production in a dose‐dependent manner. The inhibition of the antigen‐specific IgE response due to anti‐LFA‐1 mAb was seen up to 8 weeks after anti‐LFA‐1 mAb administration. The OVA‐specific IgG1 response was also blocked by anti‐LFA‐1 mAb. The spleen cells obtained from OVA‐immunized mice showed enhanced proliferation against secondary stimulation with OVA in vitro. However, the spleen cells obtained from the mice treated with both OVA and anti‐LFA‐1 mAb revealed a markedly decreased proliferative responses to OVA, while they showed no reduced responses against keyhole limpet hemocyanin stimulation, indicating that anti‐LFA‐1 mAb might induce antigen‐specific anergy in vivo. It was also demonstrated that treatment of the mice with anti‐LFA‐1 mAb significantly inhibited the interleukin‐4‐producing ability of OVA‐immunized mouse spleen cells. These results demonstrated that LFA‐1‐dependent cell‐cell interaction is essential for the production of IgE in vivo and may be important in IgE‐dependent allergic disease.
A syngeneic idiotype is immunogenic when borne by IgM but tolerogenic when joined to IgG
Authors: Reitan Solveig Klæbo , Hannestad Kristian
Published: March 2007

Some syngeneic monoclonal antibodies (mAb) elicit immune responses like conventional T‐dependent antigens. To find out whether the heavy chain class (isotype) plays a role for the immunogenicity of an idiotype (Id), we isolated rare subclones of an IgM mAb (termed Id3) in which the variable region of the heavy chain (VH) is associated with a new constant region (CH). The VH‐Id3 gene is a member of the murine 36–60 family and probably has three replacement mutations. The light chain V gene is germ‐line Vλ2. IgM, IgG1, IgG2a and IgG2b variants of Id3 were purified from protein‐free medium and injected without adjuvant into BALB/c mice. The parental 19S IgM mAb given subcutaneously (s.c.) elicited a vigorous humoral response against Id3; in comparison, monomeric 8S IgM was a much weaker immunogen. Unlike IgM, multiple challenges with the IgG switch variants failed to induce anti‐Id3 Ab. IgG variants gained immunogenicity if they were purified from medium containing fetal calf serum, mixed with complete Freund's adjuvant or injected into mice primed with IgM‐Id3. Pretreatment with 100 μg s.c. + 50 μg of the IgG2a variant extinguished the Ab response to parental IgM, but the response to adjuvant‐free bovine serum albumin was intact. Therefore, the tolerance induced by the IgG2a switch variant is antigen‐specific and not due to toxicity. Significant inhibition of the Ab response to parental IgM was observed after treatment with 4 μg of the IgG2a switch variant. Administration of the IgG1 and IgG2b switch variants also inhibited this response significantly. Thus, the outcome of an encounter with Id3 is strongly influenced by the CH isotype to which the Id is joined. This suggests novel ways to minimize unwanted Ab responses against Id of human therapeutic mAb. In the context of the theory of Id networks, we suggest that dominant B cell clones can preempt anti‐Id Ab responses against themselves by early switching from IgM to IgG secretion, before immunogenic IgM Ab have had time to activate anti‐Id B cells.
Enhanced production of biologically active interleukin‐1α and interleukin‐1β by psoriatic epidermal cells ex vivo: evidence of increased cytosolic interleukin‐1β levels and facilitated interleukin‐1 release
Authors: Debets Reno , Hegmans Joost P. J. J. , Troost Roger J. J. , Benner Robbert , Prens Errol P.
Published: March 2007

The expression of interleukin (IL)‐1 is altered in psoriatic lesions. However, little is known about the actual production of IL‐1α and IL‐1β by psoriatic epidermal cells (EC). We monitored IL‐1 in the extracellular, the membrane and the intracellular compartment of freshly isolated EC from untreated lesional psoriatic (PP) and normal healthy (NN) skin during non‐stimulated short‐term cultures, representing a psoriasis model ex vivo. Cytokines were measured using bioassays combined with neutralizing antibodies and enzyme‐linked immunosorbent assay in parallel. PP EC released significantly increased amounts of biologically active IL‐1α and IL‐1β in a ratio of 3:1, whereas NN EC only released IL‐1α. Also, the release of IL‐6, but not of TNF‐α, by PP EC was significantly increased. Membrane‐associated IL‐1 activity, analyzed using glutaraldehydefixed EC, was low and not unique to PP EC. The cytosol of PP EC contained significantly increased levels of immunoreactive IL‐1β. Furthermore, PP EC displayed loss of membrane integrity, as determined by trypan blue exclusion and release of cytosolic lactate dehydrogenase. This facilitated release of intracellular IL‐1. Depletion of CD45+ cells showed that intraepidermal leukocytes did not contribute to the production of IL‐1. Our observations show that resident PP EC express enhanced IL‐1 production ex vivo, which is due to an increased cytosolic IL‐1β content and facilitated IL‐1 release. This study provides the first evidence that PP EC can produce bioactive IL‐1β.
Characterization of self‐reactive T cells that evade tolerance in hepatitis B e antigen transgenic mice
Authors: Milich David R. , Schödel Florian , Peterson Darrell L. , Jones Joyce E. , Hughes Janice L.
Published: March 2007

Previous studies of hepatitis B e antigen (HBeAg)‐expressing transgenic (Tg31e) mice have indicated that the degree of T cell tolerance was epitope specific. For example, T cells specific for residues 120–131 of HBeAg are profoundly tolerant, whereas a proportion of T cells specific for residues 129–140 escape tolerance induction in B10. S × B10‐Tg31e mice. To understand the basis for differential tolerance towards two T cell sites on the same self antigen, we characterized T cell recognition of HBeAg by primary T cells and T cell hybridomas derived from HBeAg‐Tg and non‐Tg mice. The self‐reactive T cells surviving in B10‐Tg31e mice exhibited a unique fine specificity, albeit still focussed on HBeAg residues 129–140, which could be distinguished from the HBeAg‐specific T cell repertoire in non‐Tg B10 mice. Further, self‐reactive T cells were comprised predominantly of Th2‐type cells that preferentially evaded tolerance induction as compared to their Th1 counterparts. Because HBeAg may act as a tolerogen during the vertical transmission of chronic hepatitis B virus (HBV) infection, these results suggest that a predominance of HBeAg‐specific Th2 cells expressing a limited repertoire may influence the initiation or the maintenance of the HBV chronic carrier state.
Measurement of affinity in serum samples of antigen‐free, germ‐free and conventional mice after hyperimmunization with 2,4‐dinitrophenyl keyhole limpet hemocyanin, using surface plasmon resonance
Authors: Bakker René , Lasonder Edwin , Bos Nicolaas A.
Published: March 2007

We previously investigated the primary and secondary responses and hyperimmunization to the T cell‐dependent antigen 2,4‐dinitrophenyl keyhole limpet hemocyanin (DNP‐KLH) in antigen‐free (AF), germ‐free (GF) and conventional (CV) mice. Both the absolute and relative numbers of DNP‐specific IgG‐secreting cells in the spleen of AF mice were considerably higher compared to GF and CV mice, especially after hyperimmunization. In the present study we measured the total and DNP‐specific IgG concentration in the sera of these hyperimmunized mice using a sensitive sandwich enzyme‐linked immunosorbent assay. With respect to the total IgG concentration before and after hyperimmunization, the AF mice showed an almost 13‐fold increase after boosting with the antigen; the GF mice showed an approximately 8‐fold increase. A slight but non‐significant increase was observed in the CV mice. The total as well as the DNP‐specific IgG levels in the AF‐immunized mice were 2‐fold and 5‐fold higher compared to GF and CV mice, respectively. With the use of Surface Plasmon Resonance instrumentation (BIAcoreTM, Pharmacia, Uppsala, Sweden) we obtained mean binding affinities (KA) of the polyclonal samples of the three groups of hyperimmunized mice. IgA and IgM samples displayed low affinity for DNP‐lysine. The AF mice displayed the highest KA value among IgG antibodies, followed by GF mice, while CV mice showed a 3‐fold lower KA compared to AF mice. These differences were mainly determined by the dissociation rate constant (kdiss), since no significant changes were observed in the association rate constant (kass). Furthermore, the sera of the CV mice have a lower percentage of high‐affinity antibodies compared to GF and AF mice. These results suggest that besides a higher overall binding affinity seen in AF mice, and to a lesser extent in GF mice, the relative contribution of high‐affinity IgG is greater in AF mice compared to CV mice.
CD4+ T cells determine the ability of spleen cells from F1 hybrid mice to induce neonatal tolerance to alloantigens and autoimmunity in parental mice
Authors: González Mercedes , Schurmans Stephane , Ramos Angeles , Merino Ramón , Lambert Paul Henri , Merino Jesús
Published: March 2007

Spleen cells from F1 hybrid mice injected into newborn parental mice induce a state of cytolytic unresponsiveness to the corresponding alloantigens. However, these mice develop a transient autoimmune syndrome characterized by the production of multiple autoantibodies and glomerulonephritis. Previous reports indicated that the depletion of F1 donor T cells, shortly prior the injection into parental mice, does not interfere with any of these events. Here, we have explored whether the continuous absence of T cells in F1 mice influences the ability of their spleen cells to induce neonatal tolerance to alloantigens and the associated autoimmune manifestations. Our results revealed that spleen cells from athymic (BALB/c × C57BL/6) F1 hybrid (CB6F1) nulnu mice or from euthymic CB6F1 mice depleted from birth of CD4+ T cells, but not of CD8+ T cells, are unable to induce neonatal tolerance to alloantigens and autoimmune manifestations. By contrast, the partial reconstitution of T cells in CB6F1 nulnu mice, after the neonatal graft of a syngeneic thymus, restored the capacity of spleen cells from these mice to induce tolerance and autoimmunity when injected into newborn BALB/c mice. These results demonstrate that the functional defect of spleen cells from athymic CB6F1 nulnu mice to induce neonatal tolerance to alloantigens is directly related to the long‐term absence of mature CD4+ T cells. Interestingly, a new increase in the titers of anti‐DNA Ab was observed when spleen cells from athymic CB6F1 nulnu mice were injected into adult BALB/c mice that had been tolerized at birth with normal CB6F1 spleen cells. This finding indicates that B cells from CB6F1 nulnu mice recover their capacity to interact with alloreactive Th2 cells when they are placed into mice having functional CD4+ T cells. These data indicate that the continuous absence of CD4+ T cells causes a reversible functional defect in F1 spleen cells that determines their inability to induce neonatal tolerance and autoimmunity.
The effects of natural altered peptide ligands on the whole blood cytotoxic T lymphocyte response to human immunodeficiency virus
Authors: Klenerman Paul , Meier Ute‐Christiane , Phillips Rodney E. , McMichael Andrew J.
Published: March 2007

Cytotoxic T lymphocytes (CTL) directed against human immunodeficiency virus (HIV)‐1 are detectable in the majority of infected individuals, and their early appearance as the initial viremia is suppressed is thought to represent a potent antiviral response. Variation which arises in CTL epitopes can affect recognition by CTL, and we have observed previously that variant epitopes in HIV‐1 gag which arise in HIV‐1‐seropositive donors may act as T cell receptor (TCR) antagonists of their own CTL (Klenerman et al., Nature 1994. 369: 403). The most important question arising from these observations is the extent of these immune escape mechanisms in vivo.

Here we show that fresh, uncultured lymphocytes taken directly from HIV‐1‐infected patients are susceptible to TCR antagonism by variants present within their own virus. In contrast to HLA Class II‐restricted T cell responses, where anergy may be induced, we find that in vitro, natural variants may stimulate and sustain growth of CTL. These CTL lines retain lytic specificity exclusively for the original peptide. If this represents events in vivo, natural HIV altered peptide ligands (APL) have the capacity to inhibit the range of CTL directed against an epitope, not simply those clones selected in vitro. Partial activation of CTL by APL could also act to drive an ineffectual CTL response incapable of lysing infected cells bearing these natural antigenic variants. Distortion of lymphocyte populations and function by APL might represent a further mechanism of immune evasion by HIV.
A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H‐2b mice: Fine specificity and T cell receptor Vβ expression of encephalitogenic T cells
Authors: Mendel Itzhack , de Rosbo Nicole Kerlero , Ben‐Nun Avraham
Published: March 2007

A predominant response to myelin oligodendrocyte glycoprotein (MOG) was recently observed in patients with multiple sclerosis (MS). To study the possible pathogenic role of T cell response to MOG in MS, we have investigated the encephalitogenic potential of MOG. Synthetic MOG peptides, pMOG 1‐21, 35–55, 67–87, 104–117 and 202–218, representing predicted T cell epitopes, were injected into C57BL/6J and C3H.SW (H‐2b) mice. The mice developed significant specific T cell responses to pMOG 1–21, pMOG 35–55 and pMOG 104–117. However, pMOG 35–55 was the only MOG peptide which could induce neurological impairment. The highly reproducible disease was chronic, with ascending paralysis and neuropathology comparable with those observed in experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein or proteolipid protein, except that in H‐2b mice the disease was consistently non‐remitting. These features differ markedly from those which we recently observed in PL (H‐2u) mice with pMOG 35–55‐induced disease. In PL mice, pMOG 35–55‐induces atypical chronic relapsing EAE, the expression and progression of which are unpredictable. Hence, in different mouse strains, the same MOG peptide can induce typical EAE characterized by ascending paralysis, or atypical EAE with unpredictable clinical signs. pMOG 35–55‐specific T cells from H‐2b mice recognized an epitope within amino acids 40–55 of the MOG molecule, and pMOG 40–55‐reactive T cell lines were encephalitogenic upon transfer into syngeneic recipients. The encephalitogenic pMOG 35–55‐reactive C57BL/6J T cell lines expressed Vβ1, Vβ6, Vβ8, Vβ14 and Vβ15 gene segments, and the pMOG 35–55‐reactive C3H.SW T cell lines expressed Vβ1, Vβ2, Vβ6, Vβ8, Vβ10, Vβ14, and Vβ15 gene segments. However, in both mouse strains, the utilization of the Vβ8 gene product was predominant (40–43 %). The highly reproducible encephalitogenic activity of pMOG 35–55 strongly suggests a pathogenic role for T cell reactivity to MOG in MS and supports the possibility that MOG may also be a primary target antigen in the disease.
Restriction of self‐antigen presentation to cytolytic T lymphocytes by mouse peptide pumps
Authors: Gournier Héène , Pascolo Steve , Siegrist Claire‐Anne , Jehan Josette , Pérarnau Béatrice , Garcia Zacarias , Rose Thierry , Neefjes Jacques , Lemonnier François A.
Published: March 2007

Transport of an immunogenic self‐peptide from the second domain of the mouse major histocompatibility complex (MHC) H‐2Kd class I molecule is blocked at the TAP1‐TAP2 peptide pump level due to its amino acid sequence and is not presented to cytolytic T lymphocytes (CTL). We demonstrate that first, TAP1‐TAP2 pumps can restrict antigen presentation by selecting against internal peptide motifs which are not involved in peptide binding to MHC class I molecules. Second, some molecules targeted to the endoplasmic reticulum are processed for MHC class I presentation in the cytosol. Third, some abundantly expressed immunogenic self‐peptides are cytosolically sequestered. The advantage for the host, in terms of the peripheral T cell repertoire is that the spared CTL can be used to recognize foreign antigens. It is, however, anticipated that this advantage will be exploited by pathogens to evade immune surveillance by similar strategies.
A point mutation in the human CD45 gene associated with defective splicing of exon A
Authors: Thude Hansjörg , Hundrieser Joachim , Wonigeit Kurt , Schwinzer Reinhard
Published: March 2007

CD45 is a receptor‐type protein tyrosine phosphatase involved in the regulation of lymphocyte activation. Different CD45 isoforms are generated by alternative splicing of three variable exons (A, B and C). The pattern of CD45 splicing depends upon cell type and state of activation. CD45RA isoforms (containing exon A‐encoded sequences) can usually be found on a subset of resting T cells, but not on activated T cells. We have recently described a variant pattern of CD45RA expression which is characterized by continuous expression of CD45RA molecules on activated and memory T cells. Here, we demonstrate that this phenotype is associated with heterozygosity for a point mutation at nucleotide position 77 of exon A, leading to a C → G transition. This mutation does not change the protein sequence of the CD45RA isoform. We conclude that position 77 is part of a motif necessary for splicing of exon A, which supports the hypothesis that sequences within exons have significant effects on alternative splicing. The mutation of this motif might prevent binding of a transacting splice factor. In the heterozygous state, this mutation is not associated with impaired T cell reactivity. Functional consequences of the homozygous state remain to be elucidated.
Macrophages, but not dendritic cells, present collagen to T cells
Authors: Michaëlssony Erik , Holmdahl Meirav , Engström Åke , Burkhardt Harald , Scheynius Annika , Holmdahl Rikard
Published: March 2007

Dendritic cells, such as epidermal Langerhans cells, play a crucial role for the antigen‐specific priming of T cells. We have addressed the question whether dendritic cells present collagen, a major protein component in tissues through which dendritic cells migrate, i.e. the basement membrane, dermis, and synovial tissue. Langerhans cells, spleen cells and peritoneal macrophages were compared for antigen‐presenting capacity using a panel of mouse T cell hybridomas reactive with different determinants on type II collagen, myelin basic protein, ovalbumin and pepsin. Langerhans cells did not present any of the type II collagen determinants, unless the antigen was administered as a 15‐mer peptide, but did present myelin basic protein, ovalbumin and pepsin. Spleen cells and peritoneal macrophages, in contrast, presented all type II collagen determinants. This biased antigen presentation was also observed when Langerhans cells were pulsed with antigen in vivo. The inability to present type II collagen is related to the collagen sequence as such, since both native type II collagen, type II collagen α chains, as well as a type II collagen determinant incorporated in type I collagen, were not presented by Langerhans cells. In addition, granulocyte/macrophage colony‐stimulating factor‐expanded blood dendritic cells displayed the same biased antigen presentation, suggesting that the inability to present collagen is not restricted to dendritic cells localized in epidermis. B cell‐deficient mice could prime a type II collagen‐reactive T cell response, thus excluding B cells as obligatory antigen‐presenting cells for the priming of collagen‐reactive T cells. We suggest that neither Langerhans cells nor B cells, but macrophages are the primary antigen‐presenting cells in the immune response towards type II collagen.
Local Fas/APO‐1 (CD95) ligand‐mediated tumor cell killing in vivo
Authors: Rensing‐Ehl Anne , Frei Karl , Flury Renata , Matiba Bernd , Mariani Sara M. , Weller Michael , Aebischer Patrick , Krammer Peter H. , Fontana Adriano
Published: March 2007

Fas/APO‐1 (CD95) is a cell surface receptor which mediates apoptosis when ligated by specific antibodies or by its recently cloned natural ligand, FasL. We have studied the cytotoxic potential of FasL in vivo using Fas/APO‐1‐expressing Yac‐1 cells as targets. Supernatant harvested from Neuro‐2a cells transfected with the murine FasL cDNA contains FasL and transduces a potent apoptotic signal to Yac‐1 cells in vitro. Specificity of FasL‐mediated cytotoxicity was confirmed by competition assays using soluble Fas or anti‐Fas/APO‐1 F(ab')2 fragments which specifically interfere with FasL‐Fas/APO‐1 interactions. Intraperitoneal injection of FasL‐containing supernatant efficiently killed Yac‐1 target cells which had been implanted in capsules into the peritoneal cavity of mice. Analysis of the target cells revealed DNA fragmentation and nuclear changes typical of apoptosis. As previously shown, intraperitoneal injection of anti‐Fas/APO‐1 antibodies caused liver failure (Ogasawara, J., Watanabe, F. R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T. and Nagata, S., Nature 1993. 364: 806) and was observed at doses which did not reduce Yac‐1 cell viability. In contrast, FasL did not induce histopathology in the liver when applied intraperitoneally at doses cytotoxic for Yac‐1 cells. However, intravenous administration of FasL induced lethal liver hemorrhages and hepatocyte apoptosis. Thus, locally applied FasL kills tumor cells very efficiently without systemic toxicity and may therefore represent a candidate for local tumor treatment.
Macrophages regulate induction of delayed‐type hypersensitivity and experimental allergic encephalomyelitis in SJL mice
Authors: Cua Daniel J. , Hinton David R. , Kirkman Linda , Stohlman Stephen A.
Published: March 2007

The relationship between the delayed‐type hypersensitivity (DTH) response and susceptibility to experimental allergic encephalomyelitis (EAE) was examined using a unique age‐dependent defect in the DTH response in an EAE‐susceptible mouse strain. Young adult male SJL mice (< 10 weeks of age) are defective in DTH responses following immunization with a variety of soluble antigens. By contrast, they respond to antigens applied to the skin, demonstrating a normal contact sensitivity response. In this report, we show that the nonresponder male SJL are also unable to mount a DTH response to soluble neuroantigens or neuroantigens emulsified in complete adjuvant, and are additionally resistant to actively induced EAE. This contrasts with the DTH response in older males (> 10 weeks of age) and young adult females (6 weeks of age), which are both DTH responders and susceptible to EAE. By contrast, all three groups are susceptible to EAE mediated by the transfer of activated effector T cells, suggesting that the defect in young adult males is in the induction of effectors. Furthermore, transfer of a macrophage population from female responders to young male non‐responders mediates the induction of both DTH responsiveness and EAE susceptibility. The phenotype of this antigen‐presenting cell is (I‐A+, Mac‐1+, Mac‐2−, Mac‐3+), identical to the phenotype of the macrophage regulating DTH responsiveness in this strain of mice. These data are consistent with the hypothesis that a defect in this cell inhibits induction of both CD4+ Th1 DTH and EAE effector T cells.
Regulatory T cells in thymic epithelium‐induced tolerance. I. Suppression of mature peripheral non‐tolerant T cells
Authors: Modigliani Yves , Pereira Pablo , Thomas‐Vaslin VÉRonique , Salaün Josselyne , Burlen‐Defranoux Odile , Coutinho Antonio , Douarin Nicole Le , Bandeira Antonio
Published: March 2007

Athymic mice grafted at birth with allogeneic thymic epithelium (TE) display life‐long tolerance to tissue grafts of the TE donor strain, in spite of harboring peripheral T cells capable of rejecting those grafts. Tolerance is maintained in these chimeras by TE‐specific regulatory CD4 T cells. We presently address the quantification and the mechanisms of this dominant tolerance process. C57BL/6 mice containing variable but defined numbers of peripheral, resident T cells received cell transfers of graded numbers of peripheral T cells from B6(BALB E10) chimeras (C57BL/6 nude mice grafted with TE from 10‐day‐old BALB/c embryos), resulting in a series of animals containing a wide range of donor (tolerant) versus host (non‐tolerant) T cell chimerism. Increasing the relative representation of donor T cells results in a progressive delay in the rejection of BALB/c skin grafts, life‐long tolerance being achieved at a ratio of tolerant and non‐tolerant T cell populations of 1. In recipients displaying full tolerance, graftreactive non‐tolerant T cells were not deleted, anergized or committed to noninflammatory functions. Thus, sorted host T cells from tolerant recipients readily rejected BALB/c skin grafts upon transfer to immunodeficient animals. Finally, measurements of “helper” and inflammatory activities, as well as interleukin‐4 and interferon‐γ production, failed to discriminate between T cell populations from tolerant and non‐tolerant animals after specific in vitro stimulation. We conclude that: (a) TE‐selected regulatory T cells can suppress, in a quantitative manner, in vivo T cell responses against major and minor histocompatibility antigens expressed by the TE and, (b) this suppressive activity neither inactivates mature non‐tolerant T cells, nor does it seem to drive their differentiation along noninflammatory pathways.
Expression of a gastric autoantigen in pancreatic islets results in non‐destructive insulitis after neonatal thymectomy
Authors: Barrett Simon P. , Van Driel Ian R. , Tan Seong‐Seng , Alderuccio Frank , Toh Ban‐Hock , Gleeson Paul A.
Published: March 2007

Autoimmune gastritis, induced by day‐3 thymectomy of BALB/c mice, is a destructive CD4+T cell‐mediated disease characterized by leukocyte infiltrates in the gastric mucosa, loss of parietal and chief cells and anti‐gastric H/K ATPase autoantibodies. Our previous studies have indicated that a T cell response to the H/K ATPase β subunit is required for the onset of autoimmune gastritis (Alderuccio, F., Toh, B. H., Tan, S. S., Gleeson, P. A. and van Driel, I. R.,J. Exp. Med.1993.178:419). To determine whether a response to the β subunit autoantigen is alone sufficient to induce autoimmunity, or whether other tissue‐specific factors are required, we have generated transgenic mice expressing the gastric H/K ATPase β subunit in β islet cells of the pancreas (RIP‐H/Kβ). RIP‐H/Kβ mice developed autoimmune gastritis and insulitis after day‐3 thymectomy. Significantly, insulitis, observed as a peri‐islet infiltrate, was only detected in thymectomized mice with autoimmune gastritis. There was no apparent immune destruction of the pancreas as insulitis did not progress to invasion of the islets or diabetes. Double transgenic mice, expressing the gastric H/K ATPase β subunit in the thymus and in the pancreas, were protected from both gastritis and insulitis after day‐3 thymectomy. Therefore, insulitis in the RIP‐H/Kβ mice appears to be dependent on a T cell response to the H/K ATPase β subunit. This is the first example where an organ‐specific initiating autoantigen has been expressed in another peripheral tissue. Autoimmune destruction in the stomach, but not the pancreas, indicates that tissue‐specific factors play a fundamental role in the development of organ‐specific autoimmunity.
CD4/major histocompatibility complex class II interaction analyzed with CD4‐ and lymphocyte activation gene‐3 (LAG‐3)‐Ig fusion proteins
Authors: Huard Bertrand , Prigent Philippe , Tournier Muriel , Bruniquel Denis , Triebel Frédéric
Published: March 2007

We analyzed CD4 major histocompatibility complex (MHC) class II interactions with CD4 and lymphocyte activation gene (LAG)‐3 recombinant fusion proteins termed CD4Ig and LAG‐3Ig. CD4Ig bound MHC class II molecules expressed on the cell surface only when used in the micromolar range. This weak CD4Ig binding was specific, since it was inhibited by anti‐CD4 and anti‐MHC class II mAb. LAG‐3Ig bound MHC class II molecules with intermediate avidity (Kd = 60 nM at 37°C). Using LAG‐3Ig as a competitor in a CD4/MHC class II‐dependent cellular adhesion assay, we showed that this recombinant molecule was able to block CD4/MHC class II interaction. In contrast, no inhibition was observed in a CD4/MHC class II‐dependent T cell cytotoxicity assay. Together, these results suggest that co‐engagement of the TcR with CD4 alters the CD4/MHC class II molecular interaction to become insensitive to LAG‐3Ig competition.
The VDJ repertoire expressed in human preB cells reflects the selection of bona fide heavy chains
Authors: Milili Michèle , Schiff Claudine , Fougereau Michel , Tonnelle Cécile
Published: March 2007

In early steps of B cell differentiation, μ chains are transiently expressed in association with a surrogate light chain (ΨL) composed of the λ‐like and VpreB monomorphic polypeptides, thus forming a putative preB receptor. Using a monoclonal anti‐VpreB antibody, preB cells were isolated from two adult human bone marrow samples and their VDJ repertoire analyzed at the transcription level. All VH families were identified and further analysis focused on VH3 sequence analysis of 37 distinct VDJ cDNA clones. The VH3 genes expressed in the two bone marrow samples were also encountered in fetal liver and adult peripheral blood lymphocytes with a roughly similar contribution of 3.30, 3.23, 3.9 and 3.53. The characteristic features of the preB repertoire as compared to the activation B repertoire include the quasi absence of somatic mutations, limited N diversity and a shorter third complementarity‐determining region (CDR3). It also significantly differs from the fetal repertoire, which makes higher usage of DQ52 and has CDR3 of even shorter lengths. The almost constant presence of glycine residues in the CDR3 and predominance of JH4 with a low level of DQ52 DH usage, suggest that preB cell clones are submitted to an initial selective pressure which should be antigen independent. The bona fide heavy chains would be merely selected for their ability to interact with the surrogate light chains, thus shaping the repertoire that will be co‐expressed with immunoglobulin light chains in IgM molecules.
Nerve growth‐factor and anti‐CD40 provide opposite signals for the production of IgE in interleukin‐4‐treated lymphocytes
Authors: Brodie Chaya , Oshiba Akihiro , Renz Harald , Bradley Katherine , Gelfand Erwin W.
Published: March 2007

Nerve growth factor (NGF) is a well‐known neurotrophic factor acting on both the peripheral and the central nervous systems. In addition, it has been shown to play a role in the function of the immune system through specific receptors. Both high‐affinity and low‐affinity NGF receptors (NGFR) are expressed on human B lymphocytes. The low‐affinity NGFR has been shown to have structural homology with another specific B cell surface molecule, CD40, which plays an important role in IgE production. In view of the structural similarities of the p75 NGFR and CD40 we examined whether NGF may also be involved in the regulation of IgE production. We found that NGF and anti‐CD40 exerted opposite effects on the induction of IgE by IL‐4 in peripheral blood mononuclear cells. NGF inhibited the induction of IgE by IL‐4 and this inhibition was not mediated through blocking of the induction of CD23 nor through inhibition of IL‐4R expression. The inhibition of IL‐4‐dependent IgE production was observed on surface (s)IgE+ and sIgE−/sIgM+ B lymphocytes. Anti‐CD40 on the other hand, exerted an enhancing effect on IgE production and its addition to IL‐4 provided a signal that was resistant to the inhibitory effect of NGF. Antagonistic effects of NGF and IL‐4 were also observed for other Ig isotypes since IL‐4 prevented the increase in IgA and IgM production induced by NGF. These data indicate that although NGFR and CD40 belong to the same receptor super‐family and exert similar proliferative effects on B lymphocytes, they interact differently with IL‐4 in the regulation of IgE production.
Activation via the antigen receptor is impaired in T cells, but not in B cells from patients with common variable immunodeficiency
Authors: Fischer Michael B. , Wolf Hermann M. , Hauber Ilona , Eggenbauer Heinz , Thon Vojtech , Sasgary Maria , Eibl Martha M.
Published: March 2007

The patients included in this study belong to a subset of common variabie immunodeficiency (CVID) patients whose peripheral blood T cells have a T cell receptor (TCR)‐mediated activation defect leading to impaired expression of the interleukin (IL)‐2 gene upon stimulation with recall antigens (tetanus toxoid, Escherichia coli) or superantigens (staphylococcal enterotoxins). In the present report we demonstrate that the patients' peripheral blood T cells failed to generate the second messenger inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) following stimulation with superantigen or mAb specific for the monomorphic region of the TCR β‐chain. Patients' T cell lines were also impaired in generating Ins(1,4,5)P3 when stimulated with tetanus toxoid‐pulsed autologous monocytes. Addition of a second or third co‐stimulatory signal provided by recombinant IL‐2, CD28 or both had no effect on the Ins(1,4,5)P3 formation of the patients' antigen‐driven T cell lines. The T cell activation defect, however, was not absolute, as Ins(1,4,5)P3 formation in the patients' T cells after phytohemagglutinin or aluminium fluoride stimulation was normal. The impairment in signal transduction via the T cell antigen receptor was limited to the patients' T cells, as no activation defect after ligation of surface immunoglobulin, the antigen receptor on B cells, could be detected.
Divergent evolution in the mechanisms controlling major histocompatibility complex class II gene transcription in mouse and human
Authors: De Lerma Barbaro Andrea , Rigaud Gildas , Sartoris Silvia , Nicolis Morena , Cestari Tiziana , Accolla Roberto S.
Published: March 2007

The expression of the major histocompatibility complex (MHC) class II gene family is developmentally regulated and, in general, in a coordinate manner. In this study, we show that the expression of the entire repertoire of human class II genes, otherwise transcriptionally silent in the bare lymphocyte syndrome‐derived BLS1 cell line, can be rescued by somatic cell hybridization with normal mouse spleen cells. The analysis of the interspecies cell hybrids revealed a particularly important and unprecedented aspect. A return to the BLS1‐like, human MHC class II‐negative phenotype due to segregation of mouse chromosomes was accompanied in certain hybrids by loss of IE, but not IA cell surface antigen expression. At the molecular level, this was the result of lack of Eα‐specific mRNA in the presence of Eβ‐, Aα‐ and Aβ‐specific mRNA. Thus, the mouse trans‐acting function operating across species barriers and able to complement the defect of human BLS1 cells diverged in mice to control Ea, but not Eb, Aa and Ab gene expression. These findings suggest that evolutionary pressure has maintained the expression of the MHC class II multigene family under the control of quite distinct species‐specific transcriptional mechanisms.
Alteration of a model antigen by Au(III) leads to T cell sensitization to cryptic peptides
Authors: Griem Peter , Panthel Klaus , Kalbacher Hubert , Gleichmann Ernst
Published: March 2007

Certain metal ions are known to be potent sensitizers, but the self proteins modified by metal ions and the self peptides recognized by ‘metal‐specific’ T cells are unknown. In humans and mice treatment with gold anti‐rheumatic drugs, containing Au(I), may lead to allergic and autoimmune side effects. Human and murine T cells do not react to Au(I), however, but to the reactive metabolite Au(III). Here we show that alteration by Au(III) of a model antigen, bovine ribonuclease (RNase) A, results in T cell sensitization to cryptic peptides of this protein. Upon immunization of mice with Au(III)‐pretreated RNase [RNase/Au(III)], CD4+ T cell hybridomas specific for RNase/Au(III) were obtained in addition to those recognizing the immunodominant peptide RNase 74–88; the latter also were obtained after immunization with native RNase. RNase/Au(III)‐specific T cell hybridomas reacted against RNase/Au(III) and RNase denatured by S‐sulfonation of cysteine residues, but not against native RNase, or RNase pretreated with Au(I), Al(III), Cu(II), Fe(II), Fe(III), Ni(II), Mn(II), or Zn(II). Using a panel of overlapping, synthetic RNase peptides which were devoid of gold or gold‐induced modifications, epitope mapping revealed that RNase/Au(III)‐specific T cell hybridomas recognized the cryptic peptides 7–21 and 94–108, respectively. Comparison of the proliferative response of bulk CD4+ T cells, prepared from splenocytes after immunization with either RNase/Au(III) or native RNase, revealed that Au(III) pretreatment of RNase led to a markedly enhanced response to the two cryptic peptides while it did not influence the response to the immunodominant peptide. The cryptic peptides were also presented after preincubation of bone marrow‐derived macrophages with RNase and Au(I), but not with RNase alone, suggesting that oxidation of Au(I) to Au(III) and subsequent protein alteration by Au(III) can happen in mononuclear phagocytes. We conclude that Au(III) alteration of proteins alters antigen processing and, thus, leads to presentation of cryptic peptides. This mechanism may shed light on the development of allergic and autoimmune side effects of Au(I) anti‐rheumatic drugs. In addition, it might provide a general mechanism of how metal ions act as T cell sensitizers.