bluuebunny/crossref_metadata_2025 huggingface dataset:
Fate of allogeneic or syngeneic cells in intravenous or portal vein injection: Possible explanation for the mechanism of tolerance induction by portal vein injection
Authors: Zhang Yongfa , Yasumizu Ryoji , Sugiura Kikuya , Hashimoto Futoshi , Amoh Yasuo , Lian Zhexiong , Cherry  , Nishio Nohuhiro , Ikehara Susumu
Published: March 2007

In this report we examine the fate of donor cells injected via different routes. When PKH‐26‐labeled C57BL/6 (B6) spleen cells were intravenously (i.v.) injected into BALB/c mice, the donor cells were rejected within 3 days. In contrast, when the same B6 spleen cells were portal venously (p.v.) injected, they were trapped in the recipient liver. When allogeneic or syngeneic whole bone marrow cells (BMC) or cells in a hemopoietic stem cell (HSC)‐enriched fraction were either i.v. or p.v. injected, the cells accumulated in the liver. The cells trapped in the liver were found to be wheat germ agglutinin (WGA)‐positive HSC. When B6 thymocytes were p.v. or i.v. injected into BALB/c mice, they were rapidly rejected. When BALB/c mice were i.v. preimmunized with unlabeled B6 spleen cells, BMC or thymocytes, the p.v. or i.v. injected PKH‐26‐labeled B6 spleen cells were rejected rapidly (within 2 days). In contrast, when BALB/c mice were p.v. preimmunized with B6 spleen cells or BMC, the p.v. or i.v. injected PKH‐26‐labeled B6 spleen cells were not rejected. The cells responsible for the tolerance induction were found to be HSC trapped in the liver. Delayed‐type hypersensitivity assays revealed that the tolerance could be maintained for more than 49 days by p.v. injection plus i.v. injection (at intervals of 2 weeks) of HSC. These findings indicate that HSC trapped in the liver play a crucial role in the induction and maintenance of p.v. tolerance.
The degree of CD8 dependence of cytolytic T cell precursors is determined by the nature of the T cell receptor (TCR) and influences negative selection in TCR‐transgenic mice
Authors: Auphan Nathalie , Curnow John , Guimezanes Annick , Langlet Claire , Malissen Bernard , Mellor Andrew , Schmitt‐Verhulst Anne‐Marie
Published: March 2007

Although much has been learned about CD8 structure‐function properties, it has so far not been tested whether the nature of the TCR is sufficient to transfer the property of CD8 dependence versus non‐dependence to CD8+ cytotoxic T lymphocytes (CTL) and their precursors differentiating in T cell receptor (TCR)‐transgenic (Tg) mice. In the present study, we compared the characteristics of dependence on CD8 for stimulation of CTL precursors and antigen‐specific cytolysis by CD8+ T cells from two TCR‐Tg mice expressing respectively the TCR (Tg) from a “CD8‐dependent” and from a “CD8‐independent” CTL clone, which were both reactive against the H‐2Kb alloantigen and originated from H‐2k mice. The results indicate that the property of the Tg+CD8+ cells from H‐2k TCR‐Tg mice corresponds to that of the CTL clone of origin, demonstrating that it is linked to the nature of the TCR. Consistent with this property, Tg+CD4+ cells could also differentiate into H‐2Kb‐specific CTL when originating from the “CD8‐independent”, but not from the “CD8‐dependent” Tg‐TCR. The influence of the property of “CD8 dependence” on negative selection occurring in TCR‐Tg H‐2klb mice was apparent at two levels: (i) in the thymus, the extent of deletion was much more pronounced for the “CD8‐independent” TCR‐Tg mice; (ii) in the periphery, Tg+(hi) cells with low to negative CD8 expression were present for the “CD8‐dependent” Tg‐TCR, whereas only Tg+CD4−CD8− cells with low surface Tg‐TCR and CD3 expression were found for the “CD8‐independent” Tg‐TCR, indicating that Tg+CD4−CD8− cells are susceptible to tolerance induction involving TCR/CD3 surface down‐modulation. Furthermore, different in vitro conditions led to H‐2Kb‐induced stimulation of Tg+CD4−CD8− cells to differentiate into CTL detected in an anti‐TCR clonotypic monoclonal antibody redirected cytolysis assay. Culture in interleukin‐2 of H‐2klb Tg+CD4−CD8− cells was sufficient to induce CTL activity in the “CD8‐independent” model, whereas stimulation with cells which overexpressed H‐2Kb was required in addition to interleukin‐2 to induce CTL differentiation in the “CD8‐dependent” model. These data suggest that peripheral Tg+CD4−CD8− cells present in a situation of in vivo tolerance to H‐2Kb can still be triggered by H‐2Kb with a sensitivity correlated with the degree of CD8 dependence.
Multiple self epitopes on the Rhesus polypeptides stimulate immunologically ignorant human T cells in vitro
Authors: Barker Robert N. , Elson Christopher J.
Published: March 2007

The extent of autoreactive T cell repertoire in the normal individual has previously been unclear. Here we demonstrate that T cells from healthy humans can be stimulated by multiple epitopes on a self protein to give primary proliferative responses in vitro. Synthetic 15‐mer peptides, corresponding to the sequence of a human red blood cell Rhesus polypeptide, were tested for the ability to stimulate normal T cells. Multiple peptides were found to provoke responses reproducibly, and the proliferation could be blocked consistently by antibodies to HLA‐DR, but not ‐DP or ‐DQ. T cells from each donor proliferated in response to different patterns of peptides, but this variation in pattern was less marked in individuals with the same HLA‐DR type. The responses were comparable in kinetics to those elicited by the non‐recall foreign antigen keyhole limpet hemocyanin, and the responding cells are most commonly derived from the CD45RA+ subpopulation, indicating that they had not been activated in vivo. It is considered that T cells are “immunologically ignorant” of many self peptides, presumably because they correspond to cryptic epitopes that are not normally presented in vivo.
Expression and function of CD59 on colonic adenocarcinoma cells
Authors: Bjøge Line , Vedeler Christian A. , Ulvestad Elling , Matre Roald
Published: March 2007

The expression and function of CD59, a 19–25 kDa membrane glycoprotein that inhibits formation of the membrane attack complex of complement, was analyzed on normal and malignant human colonic epithelial cells. Analysis by immuno‐fluorescence demonstrated a weak apical expression of CD59 on normal intestinal epithelium, with an increased expression on adenocarcinoma cells. The expression of CD59 was greatest on tumor cells with poor differentiation. The functional activity of CD59 on human adenocarcinoma cells was investigated using the colonic adenocarcinoma cell line HT29, CD59 on HT29 cells was glycosyl‐phosphatidylinositol‐linked, and had a molecular mass of 19–25 kDa. HT29 cells expressed approximately four times more CD59 than leukocytes, and showed a high resistance to antibody‐dependent complement‐mediated lysis. Blocking of CD59 with divalent antigen‐binding F(ab′)2 fragments of the anti‐CD59 monoclonal antibody 1F5 resulted in a dose‐dependent increase in complement‐mediated lysis, suggesting that CD59 may be of importance in protecting colonic adenocarcinoma cells against complement‐mediated cytolysis.
Flexibility of the T cell receptor repertoire
Authors: Liang Hong‐Erh , Chen Ching‐Chen , Chou Ding‐Li , Lai Ming‐Zong
Published: March 2007

Alternative T cell receptor (TcR) gene usage between mice of different Mls alleles has been demonstrated in a number of T cell responses. A clear illustration of a flexible TcR Vβ usage in the same strain of mice remains to be established. Using a model system in which I‐Ek‐restricted T cells recognizing λ repressor cI protein (cI) 12–26 and pigeon cytochrome c (pcc) 81–104 predominantly use Vβ3 in B10.A and B10.BR mice, and Vβ1 in Mls‐2a‐bearing A/J and C3H mice, we have first demonstrated that the hierarchy of TcR Vβ usage can not be inferred from one strain of mice to the other. The presumed flexibility of Vβ3 to Vβ1 did not exist in B10.BR mice in the given responses. Instead, a switch of dominant TcR from Vβ1/Vβ3 to Vβ8 was identified in C3H and B10.BR mice. In contrast, there was an absolute rigidity in TcR repertoire usage in some mouse strains such as A/J. The lack of flexibility was not due to slow generating kinetics of replacing T cells, since A/J mice treated with staphylococcal enterotoxin A from birth on still responded poorly to cI 12–26 and pcc 81–104. Therefore, whether TcR Vβ usage in a T cell response would be flexible or rigid is highly dependent on each strain of mice. However, even the plasticity seen in B10.BR mice is very limited and further tolerance of the Vβ8+ population results in non‐responsiveness toward the given antigens.
A Vβ.2‐specific superantigen from exogenous mouse mammary tumor virus carried by FM mice
Authors: Yoshimoto Takayuki , Nagase Hisashi , Nakano Hideki , Matsuzawa Akio , Nariuchi Hideo
Published: March 2007

A number of endogenous mouse mammary tumor virus (MMTV) proviruses encode superantigen that have the ability to stimulate T cells with a certain T cell receptor (TCR) β‐chain variable region (Vβ) and to mediate the Vβ‐specific clonal deletion. The tumorigenic milk‐borne MMTV carried by C3H and GR mice also have superantigenic properties in vivo. In the present study we identified and characterized a novel Vβ8.2‐specific superantigen of exogenous MMTV carried by FM mice. The open reading frame (ORF) in the 3′ long terminal repeat of the MMTV was cloned by polymerase chain reaction with primers corresponding to conserved regions spanning the ORF coding region. Sequence analysis of the ORF revealed that there is no sequence identical to those in other known MMTV in the carboxy terminus implicated in TCR Vβ recognition. Subcutaneous injection of the virus into adult BALB/c mice induced an approximately three‐ to fourfold enlargement of draining lymph nodes and a substantial increase of Vβ8.2+ CD4+ T cells in the lymph nodes within 6 days. The exposure of newborn BALB/c mice to the virus by foster nursing resulted in a marked deletion of Vβ8.2+ cells both in CD4+ and CD8+ T cells. Thus, a novel milk‐borne MMTV in FM mice expresses strong superantigenic properties capable of stimulating Vβ8.2+ T cells. Vβ8.2+ T cells have been demonstrated to be frequently involved in recognition of conventional antigens and responsible for autoimmune diseases such as experimental allergic encephalomyelitis. Therefore, the MMTV (FM) may provide a new mouse model system for inducing immunodeficiency or autoimmune disease by retroviral infection.
The invariant chain inhibits presentation of endogenous antigens by a human fibroblast cell line
Authors: Dodi Anthony I. , Brett Sarah , Nordeng Tommy , Sidhu Sasvinder , Batchelor Richard J. , Lombardi Giovanna , Bakke Oddmund , Lechler Robert I.
Published: March 2007

The human fibroblast cell line, M1, expressing the products of transfected DRA and DRB1*0101 genes (M1‐DR1) was unable to present intact influenza antigens to a series of DR1‐restricted human T cell lines and clones, but was fully able to present synthetic peptides for T cell recognition. In contrast, M1‐DR1 cells infected with live influenza virus were recognized by two polyclonal hemagglutinin‐ or whole virus‐specific T cell lines and one of four T cell clones. This difference could not be accounted for simply by the ability of infectious virus to overcome a defect in antigen uptake by the M1‐DR1 cells, in that direct studies of endocytosis showed that the M1 cells were more efficient than human B cells in the internalization of exogenous protein. These data suggested that the M1 cells were unable to present exogenous antigens but were capable of loading major histocompatibility complex (MHC) class II molecules with peptides derived from endogenous antigens. To investigate this further, the M1‐DR1 cells were super‐transfected with a cDNA encoding the p33 and p35 forms of the human invariant chain (Ii). Expression of the Ii chain was detected by intracytoplasmic staining of transfectants, and by metabolic labeling. Equimolar amounts of the p33 and p35 forms were detected, and the high level of p35 Ii was reflected by extensive retention of Ii protein in the endoplasmic reticulum. Addition of the Ii chain led to no recovery of presentation of intact antigens with DR1, but inhibited the presentation of live virus. These data indicate that MHC class II molecules in the M1‐DR1 cells can be loaded with peptides derived from endogenous proteins, possibly in the biosynthetic pathway, and that the Ii chain has a role in limiting this route of class II antigen presentation.
A novel anti‐rat CD18 monoclonal antibody triggers lymphocyte homotypic aggregation and granulocyte adhesion to plastic: Different intracellular signaling pathways in resting versus activated thymocytes
Authors: Pavlovi Miloš D. , Čolić Miodrag , Pejnović Nada , Tamatani Takuya , Miyasaka Masayuki , Dujić Aleksandar
Published: March 2007

We have raised a monoclonal antibody (mAb), NG2B12, directed against rat CD18, capable of inducing lymphocyte homotypic adhesion and granulocyte adherence to plastic. NG2B12‐induced aggregation is temperature sensitive and requires metabolic energy, an intact cytoskeleton and the presence of Mg2+, but is independent of protein synthesis. Ca2+ is not only dispensable but exerts a suppressive effect on the NG2B12‐induced adhesion. The adhesion is readily observed in thymocytes and concanavalin A blasts of thymocytes and splenocytes but is very weak in resting spleen and lymph node cells. NG2B12 also enhances phorbol 12‐myristate 13‐acetate (PMA)‐induced aggregation in an additive fashion. The NG2B12‐induced homotypic adhesion is mediated by LFA‐1. mAb against ICAM‐1 completely inhibited the induced adhesion of activated cells but inhibited only partially and in a time‐dependent manner the adhesion of resting thymocytes. The activation of protein phosphatases 1 and 2A (as assessed by the use of okadaic acid) is necessary for the NG2B12‐induced adhesion of both resting and activated thymocytes. In contrast, H‐7 (an inhibitor of protein kinase C and A), substantially suppressed the adhesion of resting thymocytes, whereas W‐7 (an inhibitor of calmodulin‐dependent protein kinase) inhibited the adhesion of activated thymocytes. NG2B12 induces both adherence to plastic and homotypic aggregation of granulocytes; the events being blocked by anti‐CD18 (WT.3) and anti‐CD11b/CD11c (OX‐42) mAb, augmented by okadaic acid and not modified by H‐7 and W‐7. Additionally, we have demonstrated that NG2B12 and PMA employ distinct intracellular signaling pathways in inducing adhesion of both thymocytes and granulocytes.
A λ1 transgene under the control of a heavy chain promoter and enhancer does not undergo somatic hypermutation
Authors: Hengstschläger Markus , Williams Marna , Maizels Nancy
Published: March 2007

To identify cis‐acting elements responsible for targeting somatic hypermutation to immunoglobulin variable regions, we generated transgenic mice which carry a rearranged λ1 gene regulated by the heavy chain intron enhancer, Eμ, and the heavy chain promoter PH186.2 from the VH186.2 variable region. C57BL/6 × SJL founders were bred with C57BL/6 mice to establish a line carrying a single copy of the transgene. Somatic hypermutation was studied by generating hybridoma cell lines from mice immunized with the hapten (4‐hydroxy‐3‐nitrophenyl)acetyl (NP) coupled to chicken gamma globulin. The immune response in this transgenic line was dominated by the endogenous VH186.2 heavy chain variable region and the transgenic λ1 light chain, and the transgene was actively expressed in all hybridomas analyzed. In this work we show that the transgenic Vλ1 regions do not undergo hypermutation, despite high levels of expression, while the expressed heavy chain V regions accumulate mutations at a rate typical of the NP response in C57BL/6 mice. Thus, within the same B cell, the PH186.2 promoter in connection with Eμ drives efficient expression of both a VH and a Vλ region, but only the VH is a target for somatic hypermutation. Our observations show that cis‐acting sequences that activate immunoglobulin gene transcription are not sufficient to target somatic hypermutation.
Entry of B lymphocytes into the persistent cell pool in non‐immunized mice is not accompanied by somatic mutation of VH genes
Authors: Chies José A. B. , Lembezat M. P. , Freitas A. A.
Published: March 2007

In this study we compare VH‐gene repertoires of short‐lived and persistent B lymphocytes in normal nonimmunized mice. Enriched populations of persistent peripheral B cells were obtained in vivo either by (i) repeated injections with hydroxyurea or (ii) maintained ganciclovir administration to herpes simplex virus‐1 thymidine kinase transgenic mice. Both approaches have previously been shown to deplete newly formed, short‐lived B cells. VH genes expressed by persistent or unselected B cell populations were amplified by polymerase chain reaction, cloned using the λ‐ImmunoZAP system (Stratagene) and sequenced. The results presented here concern a total of 116 complete VH sequences from two VH gene families of established germ‐line composition: VH7183 and VHX24. No differences were found between the two cell populations as to usage of D or JH segments and to the presence of N sequence additions at D/JH or VH/DJH junctions and CDR3 length. Over 90% of the sequenced VH genes were of germ‐line arrangement with no evidence of somatic mutation. These results show that persistent B cells in normal mice are not of embryonic origin and that somatic hypermutation is not necessary for B cell survival. They also suggest that a significant fraction of persistent IgM+ B cells in normal mice are not generated by conventional antigenic stimulation and could represent a novel class of “memory” cells expressing germ‐line repertoires.
Lipopolysaccharide‐dependent transactivation of the temporally regulated immunoglobulin heavy chain 3′ enhancer
Authors: Arulampalam Velmurugesan , Grant Patrick A. , Samuelsson Annika , Lendahl Urban , Pettersson Sven
Published: March 2007

To execute different biological functions, the expression pattern of immunoglobulin heavy chain genes (IgH) is altered during B lymphocyte differentiation. Early in B cell differentiation, it is assumed that the heavy chain promoter and the intragenic enhancer (Eμ) ensure VDJ recombination. This leads to the expression of the immunoglobulin receptor on the cell surface. An additional strong enhancer in the far 3′ end of the IgH locus has, however, prompted a re‐evaluation of the regulation of immunoglobulin gene expression. To define the temporal and spatial regulation of the IgH 3′ enhancer, transgenic mice harboring an enhancerdependent reporter gene construct were generated. Here we demonstrate that IgH 3′ enhancer activity is largely restricted to activated immunocompetent B cells. Furthermore, the enhancer can be transactivated following mitogen stimulation with lipopolysaccharide and 12‐O‐tetradecanylphorbol 13‐acetate. We propose a model whereby 3′ enhancer activation is linked to the activation of resting immunocompetant B cells. The implications of the enhancer being active in late B lymphocyte differentiation, when heavy chain class switching occurs, are discussed.
Lymphocyte populations and immune responses in CD5‐deficient mice
Authors: Tarakhovsky Alexander , Müller Werner , Rajewsky Klaus
Published: March 2007

The CD5 antigen is expressed at a high level on T cells from early on in thymocyte ontogeny and continues to be expressed on the surface of all mature T cells. In addition, it marks a population of B lymphocytes (B‐1a) with distinct physiological properties. To study the in vivo function of CD5, the murine gene was inactivated using the technique of homologous recombination in embryonic stem cells. In homozygous mutant mice the CD5 antigen is not expressed on the surface of either T or B lineage cells, indicating that in both cell populations this antigen is encoded by the same gene. CD5‐deficient (CD5T) mice are healthy and populations of T and B lymphocytes in these mice look unchanged when compared to control mice. The mutant mice are able to mount effective immune responses to T cell‐dependent and ‐independent antigens.
Expression of a transgenic class II Ab gene confers susceptibility to collagen‐induced arthritis
Authors: Brunsberg Ulrica , Gustafsson Kenth , Jansson Liselotte , Michaëlsson Erik , Ährlund‐Richter Lars , Pettersson Sven , Mattsson Ragnar , Holmdahl Rikard
Published: March 2007

The major histocompatibility complex (MHC) class II region is assumed to influence autoimmune diseases such as rheumatoid arthritis. In the mouse, the H‐2q haplotype is associated with susceptibility to collagen‐induced arthritis, while the H‐2p haplotype is not. The class II A molecules of these haplotypes differ by only four amino acids in the first domain of the β chain. To test if this difference accounts for the MHC influence on susceptibility to collagen‐induced arthritis, H‐2p mice were made transgenic with an Abp gene altered to resemble the Abq gene. The transgenic Aβ chain hybridized with the Aαp chain and was shown to be physiologically expressed by testing antigen‐presentation capacity to Aq‐restricted T cell hybridomas and with FACS analyses. These transgenic mice developed an autoimmune response to type II collagen and also collagen‐induced arthritis. The data unequivocally suggest the Ab gene as a major genetic susceptibility locus for autoimmune collagen‐induced arthritis.
High frequency of CD8αα homodimer‐bearing T cells in human fetal intestine
Authors: Latthe Manish , Terry Linda , Macdonald Thomas T.
Published: March 2007

Immunohistochemistry on frozen sections was used to identify CD8αα cells and CD8αβ cells in human intestine. As observed previously, CD8αβ cells predominate (>95%) in tonsil and post‐natal intestine. However in human fetal intestine (16–24 weeks gestation), almost half the CD8+ cells in the lamina propria are CD8αα, and many CD8αα cells can be identified in the epithelium. In contrast, in the T cell zones of the Peyer's patches, CD8αβ cells are dominant. The CD8αα cells are virtually all αβ T cell receptor positive. By analogy with the murine system, these CD8αα cells in the fetal gut may be directly derived from the marrow, undergoing thymus‐independent differentiation in the gut mucosa.
Delayed allograft rejection by T cell receptor Vβ8.1 transgenic mice peripherally tolerized to Mls‐1
Authors: Bhandoola Avinash , Bassiri Hamid , Markmann James F. , Yui Katsuyuki , Hashimoto Yasuhiro , Greene Mark I.
Published: March 2007

One commonly studied model system for peripheral tolerance is the antigen‐specific unresponsiveness of T cells from mice previously inoculated with superantigens such as Mls‐1a. In this study, we used a TcR Vβ8.1 transgenic mouse model to investigate whether mice peripherally tolerized to Mls‐1a exhibit delayed skin allograft rejection. We report dramatic prolongation of skin allograft survival in Vβ8.1 transgenic but not in non‐transgenic mice tolerized to Mls‐1a. Peripherally induced unresponsiveness to Mls‐1a can, therefore, be considered true tolerance.
Lack of association of Vβ8+ T cells with lupus‐like syndrome in MRL‐lpr/lpr mice
Authors: Fossati Liliane , Merino Ramón , Iwamoto Masahiro , Lemoine Robert , Izui Shozo
Published: March 2007

To evaluate the role of Vβ8+ T cells in the development of lupus‐like autoimmune syndrome in MRL‐lpr/lpr mice, we treated them with the F23.1 anti‐Vβ8 monoclonal antibody (mAb) from birth to 4 months of age. Here we report that almost complete depletion of Vβ8+ T cells by the F23.1 mAb treatment neither inhibited nor delayed the development of hypergammaglobulinemia, autoantibody production and autoimmune glomerulonephritis in MRL‐lpr/lpr mice. In addition, the F23.1 mAb treatment did not prevent the development of lymphadenopathy and the generation of a CD4−CD8− double‐negative T cell subset, characteristically accumulating in lpr lymph nodes. Our results strongly argue against the idea that the Vβ8+ T cells play a critical role in the development of lupus‐like autoimmune syndrome in MRL‐lpr/lpr mice.
T cells which do not express membrane tumor necrosis factor‐α activate macrophage effector function by cell contact‐dependent signaling of macrophage tumor necrosis factor‐α production
Authors: Suttles Jill , Milleru Robert W. , Taou Xiang , Stoutf Robert D.
Published: March 2007

Previous studies have suggested that T cell contact‐dependent signaling of macrophages (MΦ) is mediated by membrane tumor necrosis factor‐α (memTNF‐α), based on the observation that anti‐TNF‐α could inhibit T cell‐mediated MΦ activation. The current report confirms that anti‐TNF‐α does inhibit activation of interferon‐γ (IFN‐γ)‐primed MΦ by paraformaldehyde‐fixed activated T cells. However, the involvement of membrane molecules other than memTNF‐α in the contact‐dependent signaling is suggested by two lines of evidence. First, the TH2 clone, AK8, displayed neither secreted TNF‐α/β nor memTNF‐α/β detectable by bioassay or immunofluorescence. Nonetheless, AK8 cells were equally effective, on a per cell basis, in contact‐dependent signaling of MΦ activation as TH2 and TH1 cells which do express memTNF‐α. Second, the expression of memTNF‐α by the TH clone, D10.G4, is maximal 24 h after activation, whereas the ability of this clone to activate MΦ is maximal at 6–8 h of activation and declines thereafter. Since TNF‐α is known to play a critical role in activation of MΦ effector function, it was hypothesized that T cell membrane components other than memTNF‐α might signal MΦ production of TNF‐α, thus allowing autocrine TNF‐α stimulation of MΦ effector function. In support of this, it is demonstrated that paraformaldehyde‐fixed activated TH2 cells can induce de novo production and release of TNF‐α by MΦ. This effect was not an artifactual result of paraformaldehyde fixation since paraformaldehyde‐fixed resting T cells did not induce TNF‐α gene expression. Previous studies have demonstrated a role for autocrine TNF‐α stimulation in LPS induction of effector function in recombinant IFN‐γ‐primed MΦ. The current study confirms that TNF‐α plays a critical role in T cell contact‐dependent signaling of MΦ but indicates that memTNF on the T cells may not be a sine qua non factor for contact‐dependent signaling. The data suggest that other T cell membrane molecules contribute to activation of MΦ effector function by stimulation of MΦ TNF‐α production.
Dose‐dependent T cell tolerance to an immunodominant self peptide
Authors: Cabaniolsu Jean‐Pierre , Cibottiu Ricardo , Kourilskyu Philippe , Kosmatopoulosu Konstandinos , Kanellopoulos Jean M.
Published: March 2007

We have previously described a model of tolerance to self peptides in a mouse transgenic (Tg) line producing secreted hen egg‐white lysozyme (HEL). The HEL cDNA was placed under the control of a ubiquitous promoter expressed early in embryogenesis, so that HEL should be present in Tg mice throughout the development of the immune system. Since individual HEL Tg mice express different amounts of serum HEL, we were previously able to show that H‐2d mice with HEL blood level > 10 ng/ml are tolerant to HEL and to the immunodominant (ID) peptide 108–116. However, autoreactive T lymphocytes recognizing the HEL subdominant (SD) peptides 74–96 and 1–18 still persist and the SD‐specific responses disappear at higher blood HEL concentrations. In the present work, we have studied HEL Tg H‐2d mice with HEL serum levels < 10 ng/ml (HEL‐low Tg animals). We find that 50% of Tg animals with HEL blood concentration < 2 ng/ml are responsive to HEL in T cell proliferation assays, although these responses are lower than those seen in non‐Tg control mice. The HEL‐specific T lymphocytes react only with 15‐mer overlapping peptides encompassing the single H‐2d ID region of HEL (residues 102–122); whereas the 9‐mer minimal ID peptide 108–116, which strongly triggers non‐Tg T cells, is unable to stimulate auto‐reactive T cells in vitro from HEL‐low Tg mice. Altogether, our results suggest that T lymphocytes specific for the minimal ID peptide are deleted or inactivated, while T cell clones of lower affinity and reacting with epitopes on longer peptides persist. Thus, the high affinity ID peptide‐specific T cell clones can be negatively selected even in the presence of low amounts of HEL.
Progenies of fetal thymocytes are the major source of CD4−CD8+ αα intestinal intraepithelial lymphocytes early in ontogeny
Authors: Lin Tesu , Matsuzaki Goro , Kenai Hiroyuki , Nomoto Kikuo
Published: March 2007

Present literature supports the view of an extrathymic origin for the subset of intestinal intraepithelial lymphocytes (IEL) that express the CD4−CD8+ αα phenotype. This subset would include virtually all T cell receptor (TCR) γδ IEL and a portion of TCR αβ IEL. However, these reports do not exclude the possibility that some CD4−CD8+ αα IEL are actually thymically derived. To clarify this issue, we examined the IEL day 3 neonatally thymectomized (NTX) mice. NTX resulted in as much as 80 % reduction in total TCR γδ IEL and in a nearly complete elimination of TCR αβ CD4−CD8+ αα IEL early in ontogeny (3‐to 5‐week‐old mice). The thymus dependency of TCR γδ IEL and TCR αβ CD4−CD8+ IEL was less prominent in older mice (7‐ to 10‐week‐old mice), as the total number of these IEL increased in NTX mice, but still remained severalfold less than that in euthymic mice. Furthermore, we demonstrate, by grafting the fetal thymus of CBF1 (H‐2b/d) mice under the kidney capsule of congenitally nude athymic mice of BALB/c background (H‐2d), that a substantial number of TCR γδ IEL and TCR αβ CD4−CD8+ αα IEL can be thymically derived (H‐2b+). In contrast, but consistent with our NTX data, grafting of adult thymi into nude mice generated virtually no TCR γδ IEL and relatively less TCR αβ CD4−CD8+ αα IEL than did the grafting of fetal thymi. These results suggest that the thymus is the major source of TCR γδ and TCR αβ CD4−CD8+ αα IEL early in ontogeny, but that the extrathymic pathway is probably the major source of these IEL later in ontogeny. A reassessment of the theory that most CD4−CD8+ IEL are extrathymically derived is needed.
Cytokine synthesis analyzed at the single‐cell level before and after revaccination with tetanus toxoid
Authors: Fernandez Victor , Anderssonu Jan , Anderssonu Ulf , Troye‐Blombergf Marita
Published: March 2007

Tetanus toxoid (TT) is a potent immunogen which evokes strong antibody responses after immunization. Here, TT was used as a model antigen to study the production of cytokines at the single‐cell level during the in vitro immune response to a specific recall antigen. Peripheral blood mononuclear cells were obtained from healthy volunteers before and 9 weeks after TT vaccination and were cultured with antigen in vitro. The kinetics of cytokine synthesis as well as frequencies of cytokine‐producing cells were determined at the single‐cell level by immunofluorescent intracellular staining of the cytokine protein. The phenotype of the producer cells was revealed by concomitant staining of surface markers.

Two patterns of cytokine synthesis were induced by TT: (i) T lymphocytes expressed a number of lymphokines (interleukin (IL)‐2, IL‐3, IL‐4, IL‐10, interferon (IFN)‐γ and tumor necrosis factor (TNF)‐β), each with distinct kinetics of synthesis. This cytokine expression was strictly dependent on the previous exposure of the donor to TT and positively correlated with the level of tetanus immunity, as judged by TT‐specific Ab levels in plasma as well as lymphoproliferation. Cells producing IL‐2, IFN‐γ and particularly TNF‐β dominated this in vitro response. After 96–120 h in culture, 1.0–1.3% of the cells produced TNF‐β, i.e. frequencies at least tenfold higher than for any of the other lymphokines assayed. The addition of IL‐2 to the cultures caused a fourfold increase and a kinetics shift in the production of TNF‐β, which peaked already at 24 h. Exogenously added IL‐2 also caused a five‐ to tenfold increase in the number of IL‐2 and IFN‐γ producers but no apparent change in the kinetics of intracellular lymphokine appearance. (ii) The cytokines IL‐1α, IL‐1β, IL‐6 and TNF‐α were produced by monocytes. This inflammatory monokine response was independent of the TT‐specific immune status of the donors, characterized by a rapid onset and was transient.
Human RANTES induces the migration of human T lymphocytes into the peripheral tissues of mice with severe combined immune deficiency
Authors: Murphy William J. , Taubu Dennis D. , Anveru Miriam , Conlonu Kevin , Oppenheimu Joost J. , Kelvinu David J. , Longof Dan L.
Published: March 2007

The human cytokine, Recombinant Human Regulated on Activation, Normal T Expressed and Secreted (rhRANTES), is a small glycoprotein secreted by activated T cells and platelets and is structurally related to a family of chemotactic cytokines called chemokines. Evaluation of the effects of chemokines on human cells has largely been limited to in vitro binding assays. In an effort to study the in vivo effects of chemokines on human leukocyte migration, we examined the ability of rhRANTES to induce human T cell infiltration using a human/severe combined immune deficient (SCID) mouse model. SCID mice received human peripheral blood lymphocytes, followed by sequential subcutaneous injections of rhRANTES in the hind flank for 3 days. The skin and underlying tissue from the rhRANTES injection site were then biopsied and examined for the extent of human mononuclear cell infiltration. rhRANTES induced significant mononuclear cell accumulation 72 h after injection. Immunohistological evaluation determined that the majority of the cells recruited in response to rhRANTES injections were human CD33+ T cells with equal numbers of CD4+ and CD8+ cells. In contrast, subcutaneous injections of recombinant human macrophage colony‐stimulating factors resulted in little human cellular infiltration. Murine mononuclear cell infiltration in response to rhRANTES was also present suggesting that the in vivo effects of rhRANTES may be both direct and indirect. These results demonstrate that rhRANTES induces significant human T cell migration in vivo and suggests that the human/SCID mouse model may provide an important tool in studying the in vivo effects of chemokines on human leukocytes and leukocyte subsets.
Soluble CD23 potentiates interleukin‐1‐induced secretion of interleukin‐6 and interleukin‐1 receptor antagonist by human monocytes
Authors: Herbelin André , Elhadadu Sonia , Ouaazu Fateh , de Grooteu Donat , Descamps‐Latschaf Béatrice
Published: March 2007

The low‐affinity receptor for IgE (CD23) is cleaved into biologically active soluble fragments (sCD23), some of which have been reported to exhibit pleiotropic activities. However, it is not known whether the sCD23 fragments contribute to the induction and/or regulation of pro‐inflammatory cytokine production. In this study, this possibility was tested using interleukin (IL)‐1‐stimulated human whole blood as an ex vivo model of cytokine cascade production. We show that human recombinant 25‐kDa sCD23 significantly enhanced the production of IL‐6 in whole blood stimulated by IL‐1, but had only little or no effect in the absence of IL‐1. The potentiating effect of sCD23 was concentration dependent within the range of plasma levels occurring during various inflammatory processes in man. These results prompted us to study whether sCD23 and IL‐1 together also enhance the production of regulating factors exhibiting anti‐cytokine activities. Our data indicate that sCD23 augments the release of IL‐1 receptor antagonist induced by IL‐1. Finally, examining the effect of sCD23 on human peripheral monocytes stimulated by IL‐1, we confirmed the capacity of sCD23 to potentiate cytokine production. We suggest that sCD23 can modulate monocyte functions, thereby contributing to the amplification and regulation of immune and inflammatory processes.
Clonal expansions of Vδ1+ and Vδ2+ cells increase with age and limit the repertoire of human γδ T cells
Authors: Giachino Claudia , Granzierou Luisa , Modenau Vittorio , Maioccou Vincenzo , Lomateru Claudia , Fantiniu Flavio , Lanzavecchia Antonio , Migonef Nicola
Published: March 2007

We have investigated the complexity of the human γδ T cell repertoire by means of a VJ heteroduplex analysis method. cDNA obtained from peripheral blood mononuclear cells was amplified with Vδ1‐Cδ or Vδ2‐Cδ primers. The product was denatured and renatured to allow random reannealing of the strands and the heteroduplexes carrying mismatched junctional sequences were separated from the homoduplexes on polyacrylamide gels. Whenever one or more T cell clones were expanded to over 10% of the polyclonal background, discrete bands of homo‐ and heteroduplex appeared.

This method was applied to the analysis of the peripheral γδ compartment from healthy donors and rheumatoid arthritis patients of different ages. While samples from young individuals showed a polyclonal pattern, a clear tendency towards oligoclonality appeared with increasing age, both in normal individuals and rheumatoid arthritis patients. We also show that the VJ junctional sequence derived from the heteroduplex fragments can be successfully used to isolate and characterize the corresponding T cell clones in vitro, even after a period of 1 year. In conclusion, our findings indicate that the complexity of the γδ T cell repertoire decreases with age as a consequence of the expansion of a few T cell clones.
Decreased expression of the ligand for CD40 in newborn lymphocytes
Authors: Fuleihan Ramsay , Ahern Deborah , Geha Raif S.
Published: March 2007

Immune responses in newborn lymphocytes show a defect in isotype switching from IgM to IgG and IgA. Immunoglobulin isotype switching in B lymphocytes requires a contact‐dependent signal from T lymphocytes which is delivered by the ligand for the B cell surface antigen CD40. We investigated the capacity of newborn lymphocytes to express the CD40 ligand and to undergo CD40 ligand‐dependent immunoglobulin isotype switching. After stimulation by phorbol ester and ionomycin, newborn lymphocytes expressed markedly decreased amounts of CD40 ligand on their surface compared to normal adult lymphocytes. Northern blot analysis of mRNA derived from activated cord blood lymphocytes also revealed markedly decreased amounts of CD40 ligand mRNA. Decreased expression of CD40 ligand in newborn lymphocytes was associated with a severely decreased ability to undergo T cell‐dependent immunoglobulin isotype switching. Newborn lymphocytes synthesized little or no detectable IgE in response to T cell‐dependent stimulation by interleukin‐4 but synthesized IgE in response to T cell‐independent stimulation by CD40 monoclonal antibody and interleukin‐4. These results indicate that decreased expression of CD40 ligand in newborn lymphocytes may be the underlying cause of deficient immunoglobulin isotype switching in newborns.
Nitric oxide synthase: MRNA expression of different isoforms in human monocytes/macrophages
Authors: Reiling Norbert , Ulmer Artur J. , Duchrow Michael , Ernst Martin , Flad Hans‐Dieter , Hauschildt Sunna
Published: March 2007

To detect mRNA expression of nitric oxide synthase (NOS) isoforms in human monocytes/macrophages reverse transcription polymerase chain reaction (RT‐PCR) was used. mRNA was isolated from stimulated or unstimulated monocytes/macrophages and RT‐PCR was performed using oligonucleotide primers derived from mRNA sequences of either human endothelial constitutive (c) or human hepatocyte inducible (i) NOS. RT‐PCR of mRNA isolated from resting monocytes and macrophages resulted in the amplification of a cNOS specific mRNA fragment. When the cells were stimulated with lipopolysaccharide (LPS)/interferon‐γ (IFN‐γ) prior to mRNA extraction, RT‐PCR yielded an iNOS‐specific amplification product. Whereas the activation of both cell types was accompanied by expression of iNOS mRNA, the cNOS signal seemed to be diminished upon immunostimulation. Not only in purified human monocytes but also in the human monocytoid cell lines MonoMac 6, THP‐1, and U937 cNOS mRNA was detected. The data clearly demonstrate the presence of iNOS and cNOS mRNA in human monocytes/macrophages and provide the necessary tools to investigate the regulation of NO synthesis in these cell populations.
Soluble interleukin‐6 receptors released from T cell or granulocyte/macrophage cell lines and human peripheral blood mononuclear cells are generated through an alternative splicing mechanism
Authors: Horiuchi Sankichi , Koyanagiu Yoshio , Zhouu Yongwei , Miyamotou Hirokuni , Tanakau Yuetsu , Waki Michinori , Matsumoto Akiyoshi , Yamamotou Mikio , Yamamotof Naoki
Published: March 2007

To detect transcripts encoding the interleukin‐6 receptor (IL‐6R) molecule lacking the transmembrane (TM) domain, in various cell lines and peripheral blood mononuclear cells (PBMC), we used the polymerase chain reaction (PCR) with primer pairs that flank the TM domain and which were selected to generate a 398‐bp fragment. We detected 398‐bp and 304‐bp DNA molecules in the PCR products of the U1, J22HL60, MT‐2, MT‐4, U937 and HL60 cell lines and of PBMC isolated from several individuals. The sequencing analysis of both DNA molecules showed that a 94‐bp region consisting of the TM domain of IL‐6R was deleted in the 304‐bp molecule. Moreover, we detected a soluble (s) IL‐6R protein of 45 kDa in culture supernatants of the MT‐2, MT‐4 and U937 cell lines by radioimmunoprecipitation using specific antibodies against sIL‐6R. Our results indicate that active deletion of the TM domain by alternative splicing of mRNA represents one mechanism for release of sIL‐6R into the culture supernatants of cells, or into serum or urine.
Endocytosis of the β chain of interleukin‐2 receptor requires neither interleukin‐2 nor the γ chain
Authors: Hémar Agnès , Lieb Michéle , Subtil Agathe , Disanto James P. , Dautry‐Varsat Alice
Published: March 2007

Interleukin‐2 (IL‐2) and IL‐2 receptors (IL‐2R) critically regulate the magnitude and duration of T cell expansion required in an immune response. Modulation occurs at the level of receptor number and affinity. IL‐2R is a multisubunit receptor which contains at least three chains, IL‐2Rα (p55), IL‐2Rβ (p70) and IL‐2Rγ (p64). Some components of high‐affinity receptors (αβγ) are continuously internalized in the absence as well as in the presence of IL‐2. From studies on other receptors, it is known that endocytosis of ligand‐receptor complexes is due to an intrinsic property of the receptor. However, the specific chains responsible for endocytosis of high‐affinity IL‐2 receptors have not been fully elucidated. IL‐2Rγ has been reported to be necessary for IL‐2 internalization, based on the fact that fibroblasts transfected with IL‐2Rα and ‐β do not internalize IL‐2. However, IL‐2 dissociates too rapidly from IL‐2Rαβ receptors to allow for its internalization. From the reported results on IL‐2 internalization in transfected fibroblasts, it cannot be concluded as to the respective roles of IL‐2Rβ and/or IL‐2Rγ in endocytosis. As modulation of receptor number is important for biological activity, we have attempted to define the chains responsible for receptor internalization.

In this work, we have studied the endocytic properties of IL‐2Rβ. We demonstrate that IL‐2Rβ is constitutively endocytosed in a B cell line, derived from a X‐linked severe combined immunodeficiency patient, which lacks expression of IL‐2Rγ. IL‐2Rβ was also constitutively internalized in T and natural killer cell lines independently of IL‐2Rγ. These results suggest that IL‐2Rβ is endowed with endocytic capacity and carries internalization signals.
Differences in responsiveness to CD3 stimulation between naive and memory CD4+ T cells cannot be overcome by CD28 costimulation
Authors: Kuiper Heleen , Brouwer Miranda , de Boer Mark , Parren Paul , Van Lier René A. W.
Published: March 2007

Activation of naive CD4+ T cells is essential for the induction of primary immune responses. However, this subset is less responsive to signaling via T cell receptor/CD3 (TcR/CD3) complex than memory CD4+ cells. For mitogenic activation of T cells, in addition to triggering of the TcR/CD3 complex, costimulatory signals are required that can be generated by surface structures present on the antigen‐presenting cells. We investigated here whether differences in responsiveness to TcR/CD3 stimulation of naive and memory cells can be overcome by the costimulatory pathway B7/CD28. Using a B7‐dependent system we show that even in the presence of optimal CD28 costimulation, CD4+ naive cells still have more stringent TcR/CD3 activation requirements than memory cells. Furthermore, titration of the B7 signal revealed that for activation of naive CD4+ cells a higher level of cross‐linking of CD28 molecules is required than for memory cells. Thus, our results show that at least two signals are required for activation of both CD4+ memory and naive cells, but that for activation of naive cells higher cross‐linking of both CD3 and CD28 molecules is necessary.
The gold anti‐rheumatic drug auranofin governs T cell activation by enhancing oxygen free radical production
Authors: Vint Irene A. M. , Foreman John C. , Chain Benjamin M.
Published: March 2007

Gold‐containing drugs continue to be used in the treatment of rheumatoid arthritis, but their mode of action remains unknown. One model to explain gold action is that gold‐containing compounds can alter free radical production in cells of the immune system, but direct evidence for this hypothesis has been lacking. In this study we show that auranofin can enhance the rapid flux of reactive oxygen species (ROS), which accompanies phytohemagglutinin activation of peripheral blood T cells. Blocking this enhancement by the addition of antioxidants can reverse the functional effects of the drug on T cell responses, which we have previously demonstrated. These results provide strong experimental support for a model in which gold anti‐rheumatics act by modulating ROS production. Furthermore, our experiments suggest that auranofin may be a useful tool to investigate the postulated role of ROS in the intra cellular T cell signaling pathway.