bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of D‐3 phosphoinositides during T cell activation via the T cell antigen receptor/CD3 complex and CD2 antigens
Authors: Ward Stephen G. , Ley Steven C. , Macphee Colin , Cantrell Doreen A.
Published: March 2007

An immediate consequence of T cell activation via the T cell receptor (TcR)/CD3 complex and CD2 antigen is the hydrolysis of phosphatidylinositol‐(4,5)‐bisphosphate and the generation of inositol‐(1,4,5)‐trisphosphate and diacylglycerol which then regulate intracellular calcium and protein kinase C. Changes in cellular levels of phosphoinositides phosphorylated on the D‐4 and D‐5 position during T cell activation have been well documented. Recently it has been proposed that phosphoinositides phosphorylated on the D‐3 position of the inositol ring by a novel phosphoinositide (PI) 3 kinase may also be important in cell activation. In the present study we have examined the levels and regulation of D‐3 phosphoinositides in T cells activated by the TcR/CD3 complex and CD2 antigens. The data show the existence of phosphatidylinositol‐(3)‐monophosphate [PtdIns(3)P], phosphatidylinositol‐(3,4)‐bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol‐(3,4,5)‐trisphosphate [PtdIns(3,4,5)P3] in T cells. Activation of the TcR/CD3 complex or CD2 antigen results in modulation of PtdIns(3,4)P2 and a putative PtdIns(3,4,5)P3 in T cells but does not change levels of PtdIns(3)P. These data provide the first evidence that lipid products of a PI 3 kinase exist in T cells.
Treatment of rats with monoclonal anti‐CD4 induces long‐term resistance to streptococcal cell wall‐induced arthritis
Authors: Van Den Broek Maries F. , Van De Langerijt Lex G. M. , Van Bruggen Mieke C. J. , Billingham Mike E. J. , Van Den Berg Wim B.
Published: March 2007

To investigate the role of CD4+ cells in the induction and maintenance of streptococcal cell wall (SCW)‐induced arthritis, Lewis rats were treated with a monoclonal antibody against rat CD4 (W3/25). Injection before onset of the arthritis resulted in resistance to SCW arthritis. Treatment with anti‐CD4 during ongoing arthritis induced an amelioration of the arthritis, demonstrating that CD4+ cells are involved in both the induction and effector phases of the chronic arthritis.

After return of CD4+ cells to normal levels in the circulation, no arthritis occurred in protected rats, despite the continued presence of SCW in the body. Even reinjection of SCW could not induce arthritis in these rats, suggesting that tolerance to SCW had occurred. In addition, these tolerized rats were refractory to actively induced adjuvant arthritis (AA), but were susceptible to passively transferred AA.

Our data imply, that (a) treatment with anti‐CD4 plus SCW induces a long‐term resistance to SCW‐induced arthritis and adjuvant arthritis, (b) SCW and M. tuberculosis may use similar mechanisms of regulation of arthritis and (c) active peripheral suppression is not the mechanism of this nonresponsiveness.
Developmentally controlled selection of antibody genes: Characterization of individual VH7183 genes and evidence for stage‐specific somatic diversification
Authors: Carlsson Leif , Övermo Charlotta , Holmberg Dan
Published: March 2007

Nucleotide sequence analysis of a large number of rearranged immunoglobulin heavy chain V region genes allowed the identification of six new members of the VH7183 gene family. These six new genes plus the eight previously defined genes agrees with the previously estimated complexity of this gene family. Twelve of these genes were represented among the isolated clones. A comparison of the clones, derived from 1‐day‐ and 14‐week‐old BALB/c mice, suggested a biased and developmentally controlled VH7183 gene utilization.

Furthermore, a developmentally controlled, non‐random distribution of the functional vs. non‐functional VHDJH rearrangements was observed among clones utilizing genes of this family, suggesting unsuspected regulatory aspects of Ig rearrangements in the process of B cell differentiation. Finally, a limited junctional diversity was revealed among the neonatal clones as the result of a low frequency of N‐sequence addition. A similar discrepancy was also observed between neonatal and adult VH1558 clones. In conclusion, these data suggest a programmed generation of B cell diversity similar to what has been observed for the establishment of γ/δ T cell repertoires.
Differential effect of interleukin 1 on naive and memory CD4+ T cells
Authors: Luqman Mohammad , Greenbaum Laurence , Lu Dandan , Bottomly Kim
Published: March 2007

Freshly derived murine CD4+ T cells are divided into naive and memory cells based on the expression of CD45 isoforms. Cross‐linking the T cell receptor CD3 complex either by plastic‐bound anti‐CD3 antibodies or the antibody presented on non‐lymphoid Fcγ receptor type II‐positive Chinese hamster ovary cells in absence of competent antigen‐presenting cells fails to activate naive cells to either secrete cytokines or to proliferate. In contrast, memory cells secrete their characteristic cytokines [interleukin (IL) 2, IL4, and interferon‐γ] and show significant proliferation to this stimulus. IL 1 however, is required for their optimal clonal expansion. Differential expression of IL 1 receptor mRNA in memory cells also correlate with their responsiveness to IL 1. Thus, these data reveal a basic difference in the requirements for activation of naive and memory CD4+ T cells.
Latent help to and from H‐2 antigens
Authors: Mitchison Avrion
Published: March 2007

An optimized system for probing allo‐immunity to major histocompatibility complex (MHC) antigens by means of adoptive transfer is used to confirm and extend previous work showing that naturally occurring class I MHC antigens, while capable of inducing Th activity when presented in combination with other allo‐antigens, fail to do so on their own. The Th activity which they do induce develops slowly, after repeated immunizations, and can properly be described as “latent”. Latency, or “cripticity” as it is also termed, may help explain how autoimmune disease is initiated.
Restricted Vα2.3 gene usage by CD4+ T lymphocytes in bronchoalveolar lavage fluid from sarcoidosis patients correlates with HLA‐DR3
Authors: Grunewald Johan , Janson Carl Harald , Eklund Anders , Öhrn Mary , Olerup Olle , Persson Ulla , Wigzell Hans
Published: March 2007

The α/β T cell receptor (TcR) V gene usage of bronchoalveolar lavage (BAL) lymphocytes and peripheral blood lymphocytes (PBL) from 11 sarcoidosis patients and 4 healthy controls was investigated, using eight α/β TcR V gene product‐specific monoclonal antibodies (mAb). Twenty‐seven percent (3/11) of the sarcoidosis patients had a highly significant increase in Vα2.3+CD4+ T lymphocytes in the bronchoalveolar space, while displaying normal frequencies of these T cells in peripheral blood. The reactivities with the remaining seven TcR mAb were normal. In the control group, no compartmentalization of any T cells was seen. Four of the patients expressed the HLA‐DR3 (w17), DQw2 haplotype. Interestingly, the three patients with distinct signs of compartmentalized Vα2.3+CD4+ T cells all expressed this HLA haplotype. Additionally, a fourth patient with pronounced, although less significant, accumulation of Vα2.3+CD4+ T cells in the lung, was also HLA‐DR3(w17), DQw2+. Expression of Vα2.3+CD4+ T cells in BAL of these patients correlated with clinical disease, as revealed on re‐analyzing the four patients after 6 months or longer. Predominant TcR Vα2.3 gene usage in compartmentalized CD4+ BAL T lymphocytes, linked to HLA‐DR3(w17), DQw2 haplotype, may thus indicate presence of a specific antigen localized to the lungs of sarcoidosis patients.
CD4 binding to major histocompatibility complex class II antigens induces LFA‐1‐dependent and ‐independent homotypic adhesion of B lymphocytes
Authors: Kansas Geoffrey S. , Cambier John C. , Tedder Thomas F.
Published: March 2007

T helper cells recognize processed antigen (Ag) in the context of major histocompatibility complex (MHC) class II antigens present on the surface of B cells and other Ag‐presenting cells. This interaction is mediated through the T cell receptor complex with associate recognition of class II molecules by the CD4 molecule. In this study, the binding of a soluble recombinant CD4/Ig heavy chain fusion protein (CD4‐γ3) or monoclonal antibody (mAb) to class II antigens on human B cells was shown to induce rapid and specific homotypic adhesion of B cells and most B lymphoblastoid cell lines. mAb reactive with CD4 inhibited CD4‐γ3‐induced adhesion and a mutant B lymphoblastoid cell line deficient in class II antigens failed to respond. Induction of homotypic adhesion was dependent on energy metabolism and a functional cytoskeleton, and class II+ pre‐B cells did not exhibit adhesion in response to these stimuli, suggesting that cross‐linking of class II molecules generated a transmembrane signal and did not simply aggregate cells. In addition, MHC class II‐induced adhesion was Fc receptor independent, as 15 mAb of different Ig isotypes reactive with HLA‐D or HLA‐DQ gene products induced adhesion. Anti‐class II mAb and CD4‐γ3 were able to induce adhesion at concentrations as low as 10 ng/ml and 100 ng/ml, respectively. Suboptimal stimulation of B cell lines through HLA‐D antigens induced homotypic adhesion that was dependent on the activation of LFA‐1 (CD11a/CD18), and which could be blocked by specific mAb. However, at greater signal strengths, adhesion was not blocked by mAb against the known adhesion receptors, suggesting the induction of a novel adhesion pathway. Consistent with this, homotypic adhesion induced by engagement of MHC class II antigens was observed with LFA‐1‐deficient B cell lines, and was independent of CD49d or CD18 expression. Thus, the direct engagement of B cell class II antigens by CD4 is likely to generate transmembrane signals which trigger both LFA‐1‐dependent and LFA‐1‐independent adhesion pathways.
Interleukin 4 (IL) 4 up‐regulates gene and surface IL 1 receptor type I in murine T helper type 2 cells
Authors: Koch Karl‐Christian , Ye Ke , Clark Burton D. , Dinarello Charles A.
Published: March 2007

The T cell‐derived cytokine interleukin (IL)4 is known to increase the proliferative response of T cells stimulated with IL1. IL4 is also an autocrine growth factor for type II T helper cells (Th2) cells. In the present studies, we examined the effect of murine recombinant IL 4 on the expression of the IL 1 receptor type I (IL 1RtI) in murine Th2 cell lines at the mRNA and surface level. Using a specific anti‐murine IL 1RtI monoclonal antibody and flow microfluorometry, we found that IL 4 increased the surface expression of IL 1RtI in a dose‐dependent manner. In D10S cells, a subline of the Th2 cell line D10.G4.1, 50‐500 pg/ml IL 4 up‐regulated the receptor 1.8‐ to 3.2‐fold (p < 0.05). This up‐regulation was also seen at the mRNA level. The effect was not due to increased stability of the mRNA, since IL4 did not modify the half‐life of IL 1RtI mRNA. IL4 also up‐regulated IL 1RtI on CDC25 cells, another Th2 cell line. However, we did not observe an effect of IL 4 on gene expression of IL 1RtI in BALB/c 3T3 fibroblasts. IL2 and IL4 showed an additive effect in up‐regulating IL 1RtI and D10S cells. These studies indicate that IL4 up‐regulates IL 1RtI in murine Th2 cells by increasing gene expression for IL 1RtI without affecting mRNA stability. Thus, IL 4 production by Th2 cells may amplify the immune response via up‐regulation of IL 1RtI.
Age‐associated increase in number of CD4+CD8+ intestinal intraepithelial lymphocytes in rats
Authors: Takimoto Hiroaki , Nakamura Takao , Takeuchi Makoto , Sumi Yukiko , Tanaka Toshiyuki , Nomoto Kikuo , Yoshikai Yasunobu
Published: March 2007

A significant number of CD4+CD8+ T cells were detected in intestinal intraepithelial lymphocytes (IEL) of various strains of rats including Wistar, WKA, BN, LEW and F344. The site of the CD4+CD8+ population in IEL increased with age in all strains we examined. Most IEL bearing CD8 expressed no CD5 antigen in young rats, while all CD4+CD8+ IEL and some of CD8+ IEL in aged rats were of CD5+CD45RB− phenotype. In germ‐free Wistar rats, age‐associated increase in the number of CD4+CD8+CD5+ IEL was not evident, indicating that stimulation by the intestinal microflora was important for expansion of the CD4+CD8+CD5+CD45RB− IEL. Aged athymic F344 nude rats contained appreciable numbers of CD4+ IEL and CD8+ IEL but few CD4+CD8+ IEL, suggesting that the CD4+CD8+ IEL may be derived from thymus‐dependent populations. Unlike a majority of CD4+CD8+ thymocytes bearing a low intensity of CD3/T cell receptor (TcR) α/β, the CD4+CD8+ T cells in IEL expressed a high intensity of CD3/TcR α/β on their surface. The CD4+CD8+ IEL appear to contribute to the spontaneous proliferation of the IEL in aged rats as assessed by tritiated thymidine incorporation after in vitro culture with medium only. These results suggest that with aging a unique CD4+CD8+ IEL may expand at a local site of the intestine under the influence of intestinal microflora and may contribute to the first line of defense against various pathogens in the epithelium.
The specificity of recognition of a cytotoxic T lymphocyte epitope
Authors: Burrows Scott R. , Rodda Stuart J. , Suhrbier Andreas , Geysen H. Mario , Moss Denis J.
Published: March 2007

An Epstein‐Barr virus (EBV)‐specific CD8+ cytotoxic T lymphocyte (CTL) clone (LC13) was shown to recognize the minimal peptide determinant FLRGRAYGL from the EBNA 3 antigen of the BL74 strain of EBV. The equivalent epitope from the B95‐8 strain (FLRGRAYGI) is not recognized when endogenously presented and the peptide is 15‐fold less active than FLRGRAYGL. A replacement set of peptides was synthesized in which each residue within FLRGRAYGL was sequentially replaced with all other genetically coded amino acids. These peptides were tested for their ability to sensitize target cells to lysis by LC13. Of the 171 single‐amino acid replacement peptides only 15 were more active than the peptide FLRGRAYGI. Five peptides had significantly greater activity than FLRGRAYGL and a peptide incorporating the most active of these single‐amino acid substitutions (HIRGRAYSL) induced lysis at concentrations approximately 30‐fold less than FLRGRAYGL. Simplified theoretical calculations based on this study suggest that CTL LC13 has a specificity for its target epitope of 1 in 4.7 × 1010. This represents the first complete analysis of the role of single amino acids within a minimum epitope on the specificity of CTL recognition.
The genetics of IgG4 deficiency: Role of the immunoglobulin heavy chain constant region and HLA loci
Authors: Gallina Roberto , Bottaro Andrea , Boccazzi Cleide , Delange Gerda , Danese Paola , Mazzola Gina , Amoroso Antonio , Demarchi Mario , Carbonara Angelo Oscar
Published: March 2007

IgG4 deficiency is very common (1/400 in the Italian population) and provides a good model for analyzing the genetic factors involved in Ig subclass deficiencies. We have previously reported an association between some immunoglobulin heavy chain constant region (IGHC) polymorphisms and the IgG4 deficiency. The associated polymorphisms spanned the region between the GP and the G4 genes. A larger sample composed of 50 healthy blood donors with IgG4 deficiency (< 0.001 g/l IgG4), not carrying homozygous gene deletions, together with 82 first‐degree relatives is now examined. The results confirmed the association of the deficiency with IGHC polymorphisms, and detected a new association with the HLA‐D locus with a strong additive effect between the two systems. However, despite these associations and a highly significant risk for IgG4 deficiency within families, close linkage with either IGHC or HLA loci was not apparent by the affected sib pair method. These findings suggest that several concomitant, possibly cooperating, genetic factors may be involved in IgG4 deficiency.
Cloning and expression of an inducible lymphoid‐specific, protein tyrosine phosphatase (HePTPase)
Authors: Zanke Brent , Suzuki Haruhiko , Kishihara Kenji , Mizzen Lee , Minden Mark , Pawson Anthony , Mak Tak W.
Published: March 2007

Tyrosine phosphorylation and dephosphorylating events have been shown to be central to the process of growth regulation and signal transduction. We report here, the identification of a new gene with a tyrosine phosphatase domain (EC 3.1.3.48) which is expressed exclusively in thymus and spleen. A cDNA of 2760 bp encodes a 339‐amino acid, intracellular, single‐domain tyrosine phosphatase. When expressed as a glutathionine‐S‐transferase fusion protein, efficient lysis of p‐nitrophenyl phosphate is noted, indicating in vitro enzymatic activity of the cloned gene product. Normal mouse lymphocytes increase mRNA expression 10‐15‐fold upon stimulation with phytohemagglutinin, concanavalin A, lipo‐polysaccharide or anti‐CD3 monoclonal antibody. This new hematopoietic tyrosine phosphatase, (HePTP), may play a role in the regulation of T and B lymphocyte development and signal transduction.
Restricted utilization of germ‐line VH3 genes and short diverse third complementarity‐determining regions (CDR3) in human fetal B lymphocyte immunoglobulin heavy chain rearrangements
Authors: Raaphorst Frank M. , Timmers Erik , Kenter Marcel J. H. , Van Tol Maarten J. D. , Vossen Jaak M. , Schuurman Ruud K. B.
Published: March 2007

Twenty‐one independent immunoglobulin heavy chain VH3DJH rearrangements were cloned and sequenced from livers of human fetuses at 7, 13 and 18 weeks of gestation. The VH elements expressed were not somatically mutated. Eight out of the estimated 30 VH3 elements were utilized with a preference for five of them. One of these VH3 sequences, designated FL13‐28, represented a thus‐far unknown VH3 gene segment. From the six functional JH elements the JH3 and JH4 segments were utilized preferentially and from the estimated 30 D segments the DQ52 element and the Dxp family were found to rearrange frequently. D elements were utilized both in normal and inverted orientation, as single copies or in D to D fusions. Addition of N nucleotides, removal of nucleotides from the coding sequences and utilization of DIR elements (D genes with irregular recombination signals) further expanded the third complementarity‐determining region (CDR3) diversity. One fourth of the fetal CDR3 regions lacked N regions. Due to utilization of DQ52, the relative absence of N regions and extensive exonuclease activity operating on the D elements, the fetal CDR3 regions were significantly shorter than those found in adult B lymphocytes.
Age‐associated changes in mitogen‐induced protein phosphorylation in murine T lymphocytes
Authors: Patel Hiren R. , Miller Richard A.
Published: March 2007

Since T lymphocyte proliferation declines with age, and since activation of a still only partially defined set of protein kinases is thought to play a critical role in T cell activation, we have carried out a systematic survey of age‐related changes in protein phosphorylation in T lymphocytes. We used two‐dimensional electro‐phoretic analysis to examine lysates prepared from T cells after 10 min of exposure to mitogens [anti‐CD3, concanavalin A (Con A) and phorbol 12‐myristate 13‐acetate (PMA) plus ionomycin] and nonmitogenic activators (PMA or ionomycin used separately). Our results show a progressive, life‐long decline in the levels of anti‐CD3‐induced phosphorylation of all 16 phosphoproteins that respond vigorously in T cells of young mice. Mice 10‐18 months of age showed levels of response that were clearly below those induced in young mice, while responses of mice 22‐24 months of age were even more severely diminished. Responses to Con A, PMA, ionomycin and a combination of PMA plus ionomycin were equally blunted in old mice, except for 2 (of 12) phosphoproteins that continued to respond to PMA even in the old animals. None of the phosphoproteins which were ionomycin responsive in young mice continued to respond in old mice, although 1 (of 3) ionomycin‐inhibitable phosphoproteins remained inhibitable in old mice. We also found three phosphoproteins which became phosphorylated in response to anti‐CD3, PMA and Con A (but not ionomycin) only in old mice, and a pair of phosphoproteins which were unresponsive to all mitogens but showed a higher “baseline” phosphorylation in T cells from old mice.

Comparing phosphoprotein patterns between CD8− and CD8−CD45RB− T cells from young mice allowed us to identify nine phosphoproteins that were strongly responsive to anti‐CD3 only when when CD45RB+ (i.e. virgin) cells were present. Although the immune system of old mice consists largely of memory T cells, the change in phosphoprotein phosphorylation patterns cannot simply be explained by the accumulation of this cell type. We conclude that aging leads to a global impairment of several distinct protein kinase pathways in both virgin and memory T cell sets.
Cell kinetics of the germinal center reaction ‐ a stathmokinetic study
Authors: Hollowood Kevin , Macartney James
Published: March 2007

The changes in splenic germinal center (GC) cell proliferation were measured during primary and secondary responses to a T‐dependent antigen in vivo to examine the regulation of the GC reaction. Adult C3H/HeN mice were immunized with sheep red blood cells and boosted 7 or 21 days later. GC cell proliferation was assessed by measurement of GC cell birth rates using a stathmokinetic technique. Actual GC growth and regression were assessed in terms of total splenic volume and number. Pre‐existing GC had a mean cell birth rate of 33 cells/1000 cells/h. The GC reactions following each immunization showed a biphasic pattern of changes in cell birth rate, comprising an initial fall immediately succeeded by a transient, but significant, increase. These fluctuations occurred earlier in secondary compared to primary responses. Significant increases in total GC volumes succeeded the peaks of cell birth rate following both primary and early secondary immunization. However, there was a substantially smaller increase following later secondary immunization. We propose that the initial cell birth rate reduction is due to inhibition of pre‐existing GC clones and represents one component of the phenomenon of GC dissociation. The succeeding peak birth rate represents early, massive proliferation of newly activated antigen‐specific clones. The different patterns of GC expansion, despite similar proliferative responses, may reflect different pathways of differentiation dependent on the timing of antigenic stimulation.
The human intraepithelial lymphocyte marker HML‐1 is an integrin consisting of a β7 subunit associated with a distinctive α chain
Authors: Cerf‐Bensussan Nadine , Bègue Bernadette , Gagnon Jean , Meo Tommaso
Published: March 2007

The membrane antigen defined by the monoclonal antibody (mAb) HML‐1 is abundantly expressed on, and largely restricted to, the T cells which populate the intestinal epithelium. We show that the mature form of the antigen is a heterodimer comprising a 150‐kDa α chain and a 120‐kDa β chain. Direct sequencing of tryptic peptides cleaved from the purified β chain identified this polypeptide with the integrin β7 isotype. cDNA clones coding for the β7 chain have recently been isolated from T cell cDNA libraries, but the β7 chain had not been identified at the protein level. No information is available concerning the primary structure of the HML‐1 α chain. We show that this subunit is synthesized as a precursor form that undergoes, like several other integrin α subunits, a post‐translational cleavage of a peptide bond. Among the 11 human integrin α chains previously identified, 10 have biochemical features and/or a distribution different from those of HML‐1 α. One, VLAα4 (CD49d), has a molecular mass of 150 kDa and is expressed on HML‐1+ cells but is not recognized by HML‐1 mAb. We conclude that HML‐1 is a novel member of the integrin family made of the β7 chain and of an as‐yet‐undescribed human α chain characterized by the post‐translational cleavage of a 10‐kDa peptide. HML‐1 is, thus, probably the human counterpart of the mouse antigen M290.
Protein tyrosine kinase p59fyn is associated with the T cell receptor‐CD3 complex in functional human lymphocytes
Authors: Gassmann Martin , Guttinger Maria , Amrein Kurt E. , Burn Paul
Published: March 2007

The binding of antigen to the multicomponent T cell antigen receptor (TcR)‐CD3 complex leads to the activation of several signal transduction pathways which result in T lymphocyte proliferation and lymphokine secretion by molecular mechanisms and catalytic molecules as yet poorly defined. One of the earliest events that follows the triggering of the antigen‐specific TcR‐CD3 complex is a rapid tyrosine phosphorylation of several intracellular substrates, suggesting stimulation of at least one protein tyrosine kinase (PTK). Since none of the seven TcR‐CD3 subunits exhibits a recognizable kinase domain, it seems likely that the receptor complex is associated with an intracellular PTK. p59fyn and the T lymphocyte‐specific p56lck are two intracellular, non‐receptor, cell membrane‐associated PTK of the src family expressed in T lymphocytes. Here, we show by double immunofluorescence microscopy a specific co‐distribution of p59fyn, but not p56lck, with antibody‐induced TcR or CD3 caps in intact human T lymphocytes. These findings provide direct evidence for a significant association of p59fyn with the TcR‐CD3 complex under physiologically relevant conditions in functional T lymphocytes. They suggest that p59fyn is a crucial component of the TcR signal transduction machinery and that one of the earliest consequences of antigen recognition by the TcR is p59fyn‐mediated phosphorylation of intracellular substrates on tyrosine residues.
Liposome‐mediated delivery stimulates a class I‐restricted cytotoxic T cell response to soluble antigen
Authors: Lopes Luciene M. , Chain Benjamin M.
Published: March 2007

Most soluble protein antigens are poor at priming class I‐restricted cytotoxic cell activity. In this study we show that incorporation of one protein, ovalbumin, into liposomes converts this antigen into an effective stimulator of antigen‐specific cytotoxicity. The mechanisms responsible for this phenomenon are discussed.
H‐2‐linked control of the Yaa gene‐induced acceleration of lupus‐like autoimmune disease in BXSB mice
Authors: Merino Ramón , Fossati Liliane , Lacour Marc , Lemoine Robert , Higaki Megumu , Izui Shozo
Published: March 2007

The accelerated development of lupus‐like autoimmune disease in male BXSB mice (H‐2b, I‐E−) is associated to the presence of a mutant gene, designated Yaa, located on their Ychromosome. To investigate whether the H‐2b haplotype and/or the lack of expression of I‐E molecules play any role in the Yaa‐linked acceleration of autoimmune disease, an I‐E+ BXSB.H‐2d congenic strain was created by backcross procedures. We compared the development of autoimmune disease in the novel BXSB.H‐2d (I‐E+) strain to that of BXSB.H‐2b (I‐E−) and BXSB.H‐2b/d (I‐E+) heterozygous mice. Male BXSB.H‐2d (I‐E+) mice exhibited only a limited production of autoantibodies and a lower incidence of glomerulonephritis with a markedly prolonged survival rate, which were essentially identical to those of female BXSB mice of both‐H‐2b and H‐2d haplotypes. However. BXSB.H‐2b/d (I‐E+) heterozygous males developed an accelerated disease comparable to that of conventional BXSB.H‐2b (I‐E−) male mice. These results indicate that the expression of I‐E molecules and consequent clonal deletion or anergy of I‐E reactive T cells does not appear to be responsible for the prevention of accelerated autoimmune disease in BXSB.H‐2d (I‐E+) male mice. The finding that the Yaa gene‐induced acceleration of lupus‐like autoimmune disease is modulated by gene(s) within or closely linked to the H‐2 complex underlines the crucial role of the major histocompatibility complex and the polygenetic nature of autoimmune disease in BXSB mice.
Anti‐idiotypic immune responses against adjuvant‐free isologous IgM monoclonal antibodies and their augmentation by complex formation between IgM and albumin in bovine serum
Authors: Hannestad Kristian , Andreassen Kjetil , Kristoffersen Gunn
Published: March 2007

In previous studies we demonstrated that the hypermutated isologous myeloma protein MOPC 315 (isotype IgA; λ2) is recognized by T helper cells like an ordinary foreign protein antigen. To what extent can an immune system recognize and respond to V domains from the primary (pre‐immune) repertoire? To study this question we made 21 BALB/c hybridoma anti‐2, 4, 6 trinitrophenyl monoclonal antibodies (mAb) of IgM; λ isotype. All mAb purified from supernatants containing fetal bovine serum had formed spontaneous complexes with bovine serum albumin possibly by way of disulfide interchange. Twenty of twenty‐one mAb from this source elicited IgG1 anti‐idiotypic (Id) Ab when given as a single adjuvant‐free dose of 200 μg. For 12 of them even 10 μg was sufficient. This indicated that BSA augmented the anti‐Id responses by a carrier effect. Three of the mAb were therefore purified from ascites fluid and from serum‐free medium. Only one of them then induced humoral anti‐Id responses in BALB/c mice when given as a single adjuvant‐free dose of 100 μg. The other two became immunogenic when emulsified in Freund's complete adjuvant. The results indicate that some IgM mAb exist whose Id determinants alone can elicit substantial anti‐Id responses because they are recognized like ordinary foreign protein antigens. Since albumin in fetal bovine serum forms complexes with IgM and greatly augments its immunogenicity, serum‐free medium should be used for production of human or humanized therapeutic IgM mAb. A possible role for Id of IgM Ab as cardinal autoantigens is discussed.
Seeding of neonatal lymph nodes by T cells and identification of a novel population of CD3−CD4+ cells
Authors: Kelly Katherine A. , Scollay Roland
Published: March 2007

Mature T cells first appear in the thymus of the mouse a few days before birth, about 7–8 days after the thymus was colonized by stem cells. These mature cells are exported to the peripheral lymphoid organs beginning at about the time of birth, but because the number is very small at this stage, little is known about the phenotype or function of these early emigrants. We have examined the cells that accumulate in the peripheral lymph nodes (LN) during the first week of life to understand better the initial seeding of the periphery by T cells. Our studies showed that a high proportion of neonatal LN cells were CD4+, but that the majority of these were CD3− during the first few days of life. The CD3− population did not increase greatly in number after birth and rapidly diminished in proportion as the number of CD3+ cells increased. These CD3−CD4+CD8− cells were found to be Thy‐1loCD44+ and to lack surface expression of heat‐stable antigen, B220 and Mac‐1. They had lymphoid morphology, did not phagocytose latex, and did not exhibit any precursor activity for cells of hemopoietic lineages. Their origin (intra‐ or extrathymic) as well as their function and physiological role, therefore, remains unknown. CD3+ T cells, both CD4+CD8− and CD4−CD8+, were present in low numbers during the first 1–2 days of life, but at post‐natal day 3, a sharp increase in the accumulation of these cells occurred in both LN and spleen. By day 3 the CD4: CD8 ratio in LN was about 2:1, as in the adult. Crude estimates of the rate of export from the thymus from day 3 onwards gave values around 1% of thymocytes per day, i.e. close to our previous estimates for young adult thymus. We found no evidence of particularly high levels of emigration from the thymus during the first week after birth. Both CD4+CD8− and CD4−CD8+ T cell subsets were present in the LN as early as 1 day post‐natally with CD4−CD8+ predominating among LN T cells, even though CD3+CD4+CD8− cells predominated over CD3+CD4−CD8+ cells in the thymus. By day 3 the ratio had changed to 2:1 (as in the adult). T cells, therefore, appear to emigrate from the thymus from about the time of birth with a dramatic increase around day 3 after birth.
Altered course of visceral leishmaniasis in mice expressing transgenic I‐E molecules
Authors: Kaye Paul M. , Cooke Anne , Lund Torben , Wattie Moira , Blackwell Jenefer M.
Published: March 2007

Previous studies had shown that the outcome of infection with Leishmania donovani was exquisitely sensitive to the influence of the major histocompatibility complex. In this study, we have examined the course of infection in non‐obese diabetic (NOD) and NOD‐E‐3 mice, the latter expressing an I‐E molecule as a result of transgenic introduction of the wild‐type Edα gene. Introduction of this transgene significantly altered the course of infection allowing for enhanced parasite multiplication in the viscera from day 14 to day 28. This was associated with both a delayed and reduced tissue granulomatous response in NOD‐E‐3 mice. In vitro, spleen cells from these mice produced equivalent levels of interferon (IFN)‐γ during the early phase of infection but this originated from populations having a different balance of T cells subsets. In NOD mice CD8+ T cells contribute substantially to the total levels of IFN‐γ produced, but in transgenic mice the contribution from this subset is significantly decreased. This is reflected in a reduction in the proportion of Leishmania‐specific CD8+ T cells, which could only partially be accounted for by deletion of Vβ5‐ and Vβ3‐expressing CD8+ T cells in NOD‐E‐3 mice. This study highlights the impact of the introduction of a class II gene product on disease outcome and unexpectedly on the functional potential of CD8+ T cells.
Alloantigen‐specific regulation of cytotoxic T cell responses is mediated through the induction of clonal anergy of CD8+ T cells
Authors: Schutze Marie‐Paule , Langlade‐Demoyen Pierre , Leclerc Claude
Published: March 2007

Priming mice with an alloantigen before immunization with this same alloantigen presented in association with a second one on an F1 stimulator cell inhibits the induction of cytotoxic response directed against the second alloantigen. This inhibition is associated with the induction of a strong cytotoxic T lymphocyte (CTL) response against the first priming alloantigen. For example, a specific suppression of anti‐H‐2b CTL responses could be induced in C3H/He mice (H‐2k) by priming them with H‐2d spleen cells before immunization with F1 (H‐2dxb) spleen cells. In the present study, we have analyzed the mechanisms underlying this specific suppression of CTL responses. We have demonstrated that the reduction of H‐2b‐specific CTL responses is reflected by a decrease in the frequency of effector cells specific for H‐2b antigen. However, there was no difference in the frequencies of precursor CTL in control and suppressed mice excluding clonal deletion as the mechanism maintaining low responsiveness. Co‐culture experiments have shown that the suppression of anti‐H‐2b CTL responses was not due to suppressor cells but to the failure of CD8+ T cells of suppressed mice to collaborate with normal helper CD4+ T cells. The suppression was therefore ascribed to a functional impairment (clonal anergy) of the CD8+ T cell subset.
Human endothelial cells transfected by SV40 T antigens: characterization and potential use as a source of normal endothelial factors
Authors: Lassalle Philippe , Lagrou Christian , Delneste Yves , Sanceau Josiane , Coll Jean , Torpier Gérard , Wietzerbin Jeanne , Stehelin Dominique , Tonnel André‐Bernard , Capron André
Published: March 2007

A putative role for the vascular endothelium as target for autoantibodies has been suggested in several autoimmune disorders and connective‐tissue diseases. However, there are some difficulties linked to the use of cultured endothelial cells (EC) that limit considerably the extensive studies on the nature of endothelial target antigens involved. To overcome this problem, human EC, derived from umbilical veins, were transfected with recombinant plasmid pSV1 which contained the early genes of simian virus SV40. These transfected cells, called EC‐pSV1, are able to grow without EC growth supplement and demonstrate a population doubling time of about 50 h. Among the EC properties, EC‐pSV1 retain intracellular content of angiotensin‐converting enzyme activity, exhibit constitutive production of interleukin 6 and of a growth‐promoting activity on early passage EV, express intercellular adhesion molecule 1 (ICAM‐1) and its up‐regulation by tumor necrosis factor α, but have lost the expression of factor VIII‐related antigen. Moreover, EC‐pSV1 express a 55‐kDa antigen found on EC and human platelets, and presumably acting as an antibody target in some cases of non‐allergic asthma. However, at the 50–55th generation, morphological changes and altered growth behavior were visible. This work demonstrates that transfection of EC with SV40 T antigens may be of interest, particularly in areas of research including the study of EC targets involved in different human diseases.
Catalase inhibits nitric oxide synthesis and the killing of intracellular Leishmania major in murine macrophages
Authors: Li Yun , Severn Alison , Rogers Mark V. , Palmer Richard M. J. , Moncada Salvador , Liew Foo Y.
Published: March 2007

Mouse peritoneal macrophages activated with interferon‐γ (IFN‐γ) and lipopolysaccharide produce substantial amounts of nitric oxide (NO), which correlates with the elimination of the intracellular protozoan parasite Leishmania major. Both the production of NO and the leishmanicidal function of the activated macrophages can be significantly inhibited by catalase in a dose‐ and time‐dependent manner. These results could not be interpreted by the reduction of H2O2 by catalase since the removal of H2O2 by the addition of glutathione peroxidase had no effect on the NO synthesis or the leishmanicidal function of activated macrophages. Furthermore, catalase did not affect the induction of NO synthase in IFN‐γ‐activated macrophages. In contrast, the inhibition of NO synthesis and leishmanicidal activity by catalase was reversed in a dose‐dependent manner by the addition of tetrahydrobiopterin, a cofactor of NO synthase. Taken together, these results not only further support the central role of NO as the cytotoxic moiety, but also suggest that hydrogen peroxide may interfere with NO production by affecting the levels of cofactor needed for its synthesis.
T cell receptor activation of a ribosomal S6 kinase activity
Authors: Calvo Victor , Bierer Barbara E. , Vik Terry A.
Published: March 2007

Stimulation of the T cell receptor‐CD3 complex activates multiple signal transduction pathways, including serine/threonine and tyrosine protein kinases. Stimulation of the human T cell line Jurkat via the T cell receptor‐CD3 complex with anti‐CD3 monoclonal antibody or incubation with the tumor promoter phorbol 12‐myristate 13‐acetate caused increases in S6 kinase and microtubule‐associated protein 2 (MAP) kinase activities. An S6 kinase activity that was able to phosphorylate exogenous 40S ribosomal S6 protein was recovered in immunoprecipitates obtained using a 90‐kDa ribosomal S6 kinase‐specific antiserum and thus represents activation of a member of the 90‐kDa ribosomal S6 kinase family. Stimulation of the S6 kinase activity correlated with an increase in a kinase activity able to phosphorylate exogenous 90‐kDa ribosomal S6 kinase (rsk) attributed to a MAP kinase activity. These increases in S6 and MAP kinase activities further correlated with the appearance of a 42‐kDa phosphoprotein detected by anti‐phosphotyrosine immunoblotting. However, while the tyrosine phosphorylation of the 42‐kDa protein and the MAP kinase activity are dependent on protein kinase C activity, residual S6 kinase activity can be detected following protein kinase C depletion and subsequent anti‐CD3 stimulation. Thus, T cell activation through the T cell receptor‐CD3 complex results in activation of a member of the 90‐kDa S6 kinase family which correlates with, but can be independent of, MAP kinase activation.
Mouse autoreactive γ/δ T cells I. Functional properties of autoreactive T cell hybridomas
Authors: Wilde David B. , Roberts Kewan , Sturmhöfel Knut , Kikuchi Gary , Coligan John E. , Shevach Ethan M.
Published: March 2007

A minor population of dendritic epidermal T cells (DETC) express the Vγ1. 1Cγ4Vδ6 T cell receptor and T cell clones and hybridomas derived from this subset constitutively secrete cytokines in culture secondary to recognition of an autoantigen. Activation of these autoreactive cells requires the use of the vitronection receptor (VNR) as an accessory molecule which interacts with the Arg‐Gly‐Asp‐Ser (RGDS) sequence of extracellular matrix (ECM) proteins. We have compared the functional properties of Cγ4+ hybridomas derived from newborn thymocytes and from adult spleen with the DETC hybridomas/lines in terms of their ability to secrete cytokines spontaneously and for the use of the VNR as an accessory molecule. Almost all of the Cγ4+ thymocyte hybridomas secreted cytokines spontaneously and in the majority of lines the most prominent cytokine secreted was granulocyte‐monocyte colony‐stimulating factor. In contrast, none of the four splenic Cγ4+ hybridomas secreted cytokines spontaneously although all were capable of cytokine production following activation via the T cell receptor. Although the thymocyte hybridomas did not grow as adherent cell lines in culture, constitutive cytokine production required engagement of the VNR by its ligand in ECM proteins. In all cases, cytokine production could be inhibited by an anti‐VNR monoclonal antibody as well as by soluble RGDS. The strong correlation of functional and molecular properties (see accompanying report) between thymocyte Cγ4+ hybridomas and Cγ4+ DETC suggests that the Cγ4+ DETC may be of thymic origin and that cells with potential for autoreactivity residing in the thymus at birth may populate other peripheral locations in the mouse. The data also support the concept that the VNR, and possibly other integrins, play a role as accessory elements for autoreactive cells and may be essential for the regulation of such activity.
Transgenic T cell receptor interactions in the lymphoproliferative and autoimmune syndromes of lpr and gld mutant mice
Authors: Sidman Charles L. , Marshall Jan D. , von Boehmer Harald
Published: March 2007

To investigate the role of T cell receptor (TcR) expression and interactions in development of lymphoproliferation and autoimmunity in lpr and gld mutant mice, and to determine whether these autoimmune mutations affect T cell selection and repertoire formation, we generated mice homozygous for either the gld or the lpr mutation and containing TcR α/β transgenes (Von Boehmer, H., Annu. Rev. Immunol. 1990. 8: 531) specific for the male (H‐Y) antigen in the context of H‐2Db. Four main results emerged from analysis of these mice. First, expression of transgenic TcR had no effect on disease incidence and progression. Second, the accumulating T cells reflected normal processes of positive and negative selection. Third, cells expressing the transgenic TcR participated equally in lymphoproliferation regardless of whether their antigenic peptide and/or presenting major histocompatibility complex molecules were present or not. Fourth, expression of the TcR transgenes markedly altered the phenotype of the major accumulating lymphocyte subset. Thus, in these models of lymphoproliferation and autoimmunity, T cell repertoire formation proceeds normally, specific T cell recognition of antigen has no effect on the participation of individual clones, and the phenotype of the cells accumulating is sensitive to either the timing or the amount of TcR expression. These results are discussed in the context of the primary cause vs. secondary manifestations of autoimmunity in these models.
Different expression of the recombination activity gene RAG‐1 in various populations of thymocytes, peripheral T cells and gut thymus‐independent intraepithelial lymphocytes suggests two pathways of T cell receptor rearrangement
Authors: Guy‐Grand Delphine , Broecke Catherine Vanden , Briottet Chantal , Malassis‐Seris Michèle , Selz Françoise , Vassalli Pierre
Published: March 2007

The presence of transcripts of the recombination activating gene RAG‐1 was studied by in situ hybridization on selected populations of murine thymocytes, peripheral lymphocytes and gut intraepithelial lymphocytes (IEL), obtained by cell sorting. RAG‐1 mRNA was found in a majority of “double‐positive” (DP) thymocytes, but was absent in “single‐positive” thymocytes and peripheral T lymphocytes. The only other T lineages in which about 10%–20% of the cells contained RAG‐1 mRNA, and in smaller amounts, were “double‐negative” (DN), T cell receptor (TcR) γδ‐ cortical thymocytes and gut CD3− IEL. These observations suggest that (a) the high expression of RAG‐1 transcripts in DP thymocytes is related to the process of expansion‐selection of these cells, probably accompanied by repeated TcR rearrangements, and that (b) in contrast. CD3− IEL from the gut (which are thymus independent) as well as some DN thymocytes undergo limited TcR rearrangement giving rise locally to TcR+ T cells without prior extensive process of local expansion‐selection. A small percentage of peripheral B cells also contained RAG‐1 mRNA, raising the possibility that this protein may also be involved in immunoglobulin class switching.
Accumulation of somatic mutants in the B cell compartment after primary immunization with a T cell‐dependent antigen
Authors: Weiss Ursula , Zoebelein Rele , Rajewsky Klaus
Published: March 2007

The accumulation of somatic mutants in splenic B lymphocytes early after primary immunization with the hapten (4‐hydroxy‐3‐nitro‐phenyl)acetyl (NP) coupled to chicken gamma globulin (CG) was determined. Rearranged V186.2 heavy chain genes were amplified by the polymerase chain reaction from genomic DNA and subjected to nucleotide sequence analysis. Somatic antibody mutants become detectable on day 6 after immunization, and most of the somatic mutations accumulating in the memory compartment are introduced until day 14. At this time strong selection for mutants expressing high binding affinity for NP is apparent. Extrapolation from the mutation frequency increases between day 6 and day 14 to the previously determined mutation frequency at week 6 (Weiss, U. and Rajewsky, K., J. Exp. Med. 1990. 172: 1681) leads to the prediction that the process of mutant generation ceases to operate around day 22 after primary immunization.
An explanation for the defect in secretion of IgM Mott cells and their predominant occurrence in the Ly‐1 B cell compartment
Authors: Tarlinton David , Förster Irmgard , Rajewsky Klaus
Published: March 2007

Mott cells are a variant form of plasma cell in which the immunoglobulin (Ig), rather than being secreted, accumulates in rough endoplasmic reticulum‐derived vesicles called Russell bodies. We have examined the molecular cause of this defect and the in vivo origin of IgM Mott cells. Our examination of the Ig variable region gene sequences of two IgM Mott hybridomas derived from C. B‐20 Ly‐1 B cells showed all to be germ line. In a series of mix and match transfection experiments, the Mott phenotype was only reconstituted when the original Mott specificity was expressed as an IgM, suggesting that both the specificity and the isotype were critical to the formation of Russell bodies.

Based on our finding that Russell body formation was dependent on the Ig isotype being IgM, we suggest that the Mott phenotype is apparent only after differentiation of B cells into plasma cells and that probably the major cause of the IgM Mott phenotype is low‐affinity interaction of the Mott Ig with some as yet unknown intracellular component(s) being stabilized by the intrinsic high avidity of the pentameric secreted form of IgM. Consistent with this proposal was the finding that after in vitro lipopolysaccharide (LPS) stimulation of sorted Ly‐1 B cells derived from C.B‐20 mice, Mott cells represented up to 5% of the IgM plasma cells in the culture. LPS stimulation of conventional B cells also induced the appearance of IgM Mott cells, but at the much reduced level of 0.1%, suggesting that the major, if not the only, source of Mott cells in vivo is the Ly‐1 B cell population. A possible causal relationship between the elevated frequency of Mott cells in the Ly‐1 B cell‐derived LPS blasts and the repertoire selection inherent in the development of these B cells is discussed.