bluuebunny/crossref_metadata_2025 huggingface dataset:
The fate of human peripheral blood lymphocytes after transplantation into SCID mice
Authors: Martino Gianvito , Anastasi John , Feng Jiajia , Shan Carol Mc , Degroot Leslie , Quintans Josè , Grimaldi Luigi M. E.
Published: March 2007

Human peripheral blood lymphocytes (hu‐PBL) can be adoptively transferred by intraperitoneal injection into mice with severe combined immunodeficiency (SCID). The transplanted lymphocytes can produce immunoglobulin (Ig), respond to antigens, and survive for months in this chimeric model (hu‐PBL SCED). However, whether the lymphocytes actually repopulate and reconstitute lymphoid structures and organs has been subject of some debate. To address this question and to characterize the hu‐PBL SCID model better, we employed a novel technique for the identification of human cells in xenogeneic mice. We used fluorescence in situ hybridization (FISH) with a biotinylated DNA probe to all human centromeres. We demonstrated that FISH could be used to detect human cells when they accounted for less than 1 % of human/mouse cell mixtures; it could also be employed for the identification and localization of individual human cells in tissue sections. By using FISH, we studied 31 SCID mice injected with 1.5 × 107 −4 × 107 hu‐PBL via intravenous (i.v.) or intraperitoneal (i.p.) routes. In the 6 i.v ‐injected mice, we found that the human cells were removed from the circulation into the lung within 1 h. In 22 of 25 i.p ‐injected animals, 90–3716 μg/ml of human IgG was found in the sera at 3 to 13 weeks after transplantation (a.t.). Human cells colonized the peritoneal cavity and persisted for up to 13 weeks a.t. and, in the 12 mice studied, accounted for 4 % to 57 % of the cells in the peritoneal fluid. However, only rare, isolated human cells were found in the spleen, blood, bone marrow, lung or Peyer's patches. In 7 of 19 mice that received hu‐PBL i.p. from Epstein‐Barr virus‐seropositive donors, we found masses of human cells usually beneath the peritoneal lining but sometimes infiltrating normal tissue. We conclude that FISH offers a simple means for accurate identification of human cells in the xenogeneic mouse. Although there is colonization of the peritoneal cavity in most mice, and development of lymphoid masses in some, there is no reconstitution of lymphoid structures and only minimal engraftment of lymphoid organs by human cells in conventionally‐prepared hu‐PBL SCID constructs.
Linkage disequilibrium between TAP2 variants and HLA class II alleles; no primary association between TAP2 variants and insulin‐dependent diabetes mellitus
Authors: Rønningen Kjersti S. , Undlien Dag E. , Ploski Rafal , Maouni Nicki , Konrad Robert J. , Jensen Elisabeth , Hornes Erik , Reijonen Helena , Colonna Marco , Monos Dimitri S. , Strominger Jack L. , Thorsby Erik
Published: March 2007

The TAP1 and TAP2 genes, located in the HLA class II region, encode subunits of a peptide transporter. Both genes display limited genetic variability; four different nucleotide substitutions have been found in theTAP2 gene. Here studies on linkage disequilibrium between TAP2 variants and HLA class II alleles are reported, in an attempt to evaluate whether TAP2 variants are associated with insulin‐dependent diabetes mellitus (IDDM). As reported previously, a significant decrease of homozygosity for TAP2 alleles encoding alanine at residue 665 (665 Ala) and glut amine at 687 (687 Gin) paralleled by an increase in homozygosity for TAP2 alleles encoding threonine at residue 665 (665 Thr) and a stop codon at 687 (687 Stop), was found in both Finnish and Norwegian IDDM patients compared to random controls. However, a strong linkage disequilibrium between these TAP2 polymorphisms and given HLA‐DR and ‐DQ genes was observed among healthy controls. The frequent 665 Thr and 687 Stop variants were in linkage disequilibrium both with the DR4‐DQ8 and the DR3‐DQ2 haplotypes, haplotypes which are strongly associated with IDDM. In contrast, the DR1‐DQ5 and DR13‐DQ6 (e.g. DQB1*0603) haplotypes, which are decreased among IDDM patients, were associated with the 665 Ala and 687 Gin variants. Thus, when DR‐ and DQ‐matched patients and controls were compared, associations of the investigated TAP2 variants and IDDM were no longer detectable. These data, therefore, indicate that the associations previously found between certain TAP2 variants and IDDM are secondary to a primary association between this disease and particular DQαβ heterodimers.
Age‐related accumulation of LFA‐1high cells in a CD8+CD45RAhigh T cell population
Authors: Okumura Meinoshin , Fujii Yoshitaka , Takeuchi Yukiyasu , Inada Keiji , Nakahara Kazuya , Matsuda Hikaru
Published: March 2007

The differential expression of CD45 isoforms has been suggested as a marker for stages of post‐thymic T cell development, that is, CD45RA+CD45R0− T cells and CD45RA−CD45R0+ T cells are supposed to be virgin and memory cells respectively. Recently, several adhesion molecules have been shown to be up‐regulated on the cell surface of memory T cells, and have been suggested to serve as a memory marker. In this study, we investigated the levels of LFA‐1 expression on T cells in various subpopulations defined by CD45 isoform expression in donors of various ages.

In CD4+ T cells, the proportion of LFA‐1high cells among CD45RAhighCD45R0‐T cells remained low in all age groups and did not show significant accumulation with age. CD4+CD45RA−CD45R0highTcells expressed LFA‐1 at a higher level than CD4+CD45RAhighCD45R0− T cells. Thus, the currently prevailing view that CD45RA and CD45R0 can be markers for virgin and primed cells was consistent with LFA‐1 expression in CD4+ T cell population.

In CD8+ T cells, however, CD45RAhighCD45R0− T cells consisted of two distinct subpopulations, LFA‐1low and LFA‐1high cells, whereas CD45RA−CD45R0high T cells were almost exclusively LFA‐1high When CD29 expression was examined in place of LFA‐1 expression, similar results were obtained; CD45RAhigh CD45R0− T cells consisted of two distinct subpopulations, CD29‐to low and CD29high cells, while CD45RA‐CD45R0high T cells were mostly CD29high.

The proportion of LFA‐1high cells in the CD8+CD45RAhigh T cell subpopulation increased significantly as a function of age (r = 0.9, p < 0.001). It ranged from 8% in a 14‐year‐old donor to 94% in a 79‐year‐old donor. Furthermore, the proportion of CD8+CD45RAhighLFA‐1high cells in the CD8+ T cell population increased significantly as a function of age (r = 0.85, p < 0.001). On the other hand, the proportion of LEA‐1high cells in CD8+CD45RA− T cell subpopulation exceeded 90% in most donors irrespective of age.

These results indicate that the CD8+CD45RAhighCD45R0− T cell subpopulation contains a considerable number of LFA‐1high cells and CD29high cells, phenotypically similar to previously activated cells. Thus, in terms of LFA‐1 and CD29 expressions, the simple scheme that CD45RA is a marker of virgin cells is not applicable to the CD8+ T cell population.
Human CD3−CD16+ natural killer cells express the hGATA‐3 T cell transcription factor and an unrearranged 2.3‐kb TcR δ transcript
Authors: Biassoni Roberto , Verdiani Simonetta , Cambiaggi Anna , Romeo Paul‐Henri , Ferrini Silvano , Moretta Lorenzo
Published: March 2007

In this study we analyzed the Tcell receptor(TcR) δ transcripts expressed by CD3−CD16+ cells and we investigated whether these cells expressed the hGATA‐3 Tcell transcription factor and the recombination‐activating gene (RAG)‐l.

Multiple TcR δ transcripts deriving from an unrearranged TcR δ gene were detected in both polyclonal and clonal CD3−CD16+ natural killer(NK) cell lines. Two unrearranged TcR δ transcripts had a size similar to that of the functional TcR δ mRNA (2.3 and 1.3 kb) found in TcR γ/δ+ T lymphocytes. Sequence analysis of nine different 2.3‐kb cDNA clones obtained from NK‐derived polyA+ RNA confirmed that they corresponded to an unrearranged TcR δ gene. These cDNA were 2343 bp long and their transcription initiation site was located 814 bp upstream from the Jδ1 segment. The sequence located upstream of the Jδ1 segment corresponded to the previously reported germ‐line sequence. The Jδ1 segment was correctly spliced to Cδ; in addition the four Cδ exons were found to be already assembled. Two polyadenylation sites were present in the fourth Cδ exon. However, only that located at the 3′ end appeared to be utilized in the 2.3‐kb cDNA. The expression of hGATA‐3, a T cell‐specific factor known to be involved in the regulation of the transcription of TcR δ locus, was analyzed by Northern blot, in cultured NK cell population and clones (but not in freshly derived cell populations). All NK clones and cell lines studied were found to express hGATA‐3‐specific mRNA, suggesting that hGATA‐3 may be involved in the regulation of the unrearranged TcR δ gene expression in NK cells. Finally, no transcription of the RAG‐1 gene could be detected in all NK cell lines or clones analyzed.
Real‐time measurement of antigenic peptide binding to empty and preloaded single‐chain major histocompatibility complex class I molecules
Authors: Ojcius David M. , Godeau François , Abastado Jean‐Pierre , Casanova Jean‐Laurent , Kourilsky Philippe
Published: March 2007

Cytotoxic T lymphocytes (CTL) recognize peptides in association with major histocompatibility complex (MHC) class I proteins, but how peptides bind to class I is not well understood. We used a fluorescence technique to measure antigenic peptide binding to a soluble, single‐chain Kd (SC‐Kd) molecule in which the Kd heavy chain was connected by a 15‐residue link to β2‐microglobulin. Peptides were covalently labeled at their N terminus with dansyl, and binding of dansylated Kd‐restricted peptides to SC‐Kd resulted in significant fluorescence enhancement, which could be inhibited by unmodified Kd‐restricted peptides. Real‐time binding of a dansylated peptide could be followed by monitoring the fluorescence at 530 nm. The dansylated Plasmodium berghei circumsporozoite (PbCS) 263–260 peptide bound to “empty” SC‐Kd with an association rate constant of 1140 M−1s−1, and the subsequent spontaneous dissociation of the SC‐Kd‐peptide complex was slow. The dissociation increased dramatically after addition of excess unlabeled PbCS 253–260 peptide, but with a slower association constant for unlabeled peptide, 77 M−1s−1. Thus, the Kd‐peptide complex on the surface of antigen‐presenting cells should be stable, but high concentrations of peptides in the endoplasmic reticulum (ER) lumen would allow for peptide exchange on Kd before export to the surface. The apparent activation energy for PbCS 253–260 peptide binding to SC‐Kd was 6.78 ± 0.64 kcal/mole, similar to values previously reported for antigen‐antibody interactions.
86Rb+ ion fluxes in resting and immunologically stimulated mucosal mast cells
Authors: Pilatus Ulrich , Pecht Israel
Published: March 2007

We studied fluxes of Rb+ ions, using its 86Rb isotope as a radioactive tracer in living rat mucosal mast cell cultures (RBL‐2H3 line) grown to high density on beads. Continuously perfused samples of these cells could be immunologically stimulated by antigen clustering of IgE bound to the cells type I Fee receptors (FcεRI) and both the cellular response, as measured by the secreted mediators, as well as the uptake of 86Rb+ of the perfused sample could be monitored.

The following results were obtained, (i) In resting cells, 86Rb+ influx is observed upon exposure to extracellular 86Rb+. It proceeds with a monoexponential time course (τ = 30.6 ± 8 min) reaching a steady‐state distribution of [86Rb+]int/[86Rb+]ext = 31.6 ± 6.4 and can be inhibited by ouabain, (ii) FcεRI clustering‐mediated stimulation of these cells causes an immediate and marked increase in both amplitude and rate of 86Rb+ uptake, which also fits a monoexponential function (τ = 26.8 ± 8.6 min). (iii) This stimulated 86Rb+ uptake can also be inhibited by ouabain. It is not caused by Ca2+ influx or by the exocytotic process as evidenced by the fact that it is also observed in buffer to which no Ca2+ ions were added. Analysis of these results by a simple model taking into account unidirectional 86Rb+ influx by the Na+/K+‐dependent ATPase and its efflux by K+ channels yields a resting cells unidirectionel K+ uptake of 3.0 ± 1.1 107 ions/cell/s, which is increased by ca. 10% upon clustering of the FcεRI by IgE and antigen. The stimulated influx is suggested to be due to enhanced activity of the Na+/K+‐dependent ATPase, reflecting increased permeability for Na+ ions.
Physical association of CD5 and the T cell receptor/CD3 antigen complex on the surface of human T lymphocytes
Authors: Osman Narin , Lazarovits Andrew I. , Crumpton Michael J.
Published: March 2007

The physical association of CD5 and the Tcell antigen receptor (TcR)/CD3 complex on the surface of intact human lymphocytes was investigated using co‐capping experiments and fluorescence resonance energy transfer (FRET) analyses. Antibody‐induced capping of CD5 or CD3 and double indirect immunofluorescence labeling revealed a specific co‐localization of a significant fraction of CD3 and CD5 molecules on the Tcell surface. By means of FRET measurements we studied further the physical proximity of CD5 and the TcR/CD3 complex at the surface of normal lymphocytes. Significant fluorescence energy transfer was measured between CD5 and CD3 molecules indicating that the associated molecules were within 10 nm of one another. No energy transfer was observed between the integrin α4β7 and CD3 or CD5. The close physical proximity measured between CD5 and CD3 correlates with our co‐capping data and taken together the results show that the association of CD5 and the TcR/CD3 complex first detected by immunoprecipitation occurs on the surface of human T cells under physiologically relevant conditions.
Lymphocytes bearing the γδ T cell receptor in acute Brucella melitensis infection
Authors: Bertotto Alberto , Gerli Roberto , Spinozzi Fabrizio , Muscat Christopher , Scalise Francesco , Castellueei Giuseppe , Sposito Maddalena , Candio Francesco , Vaeearo Renato
Published: March 2007

A phenotypical analysis carried out by indirect immunofluorescence and two‐color cytofluorometry showed that the number of lymphocytes bearing the γδ T cell receptor (TcR) heterodimer was dramatically increased in the blood of six children with Brucella melitensis infection. Most in vivo expanded γδ T cells reacted with a monoclonal antibody which identifies Vδ2 gene products and a significant proportion expressed CD25 and HLA‐DR activation antigens. In addition, whereas only a few γδ T lymphocytes were CD8+, nearly all were CD4−. Highly enriched populations of both αβ and γδ T cells were obtained by negative immunoselection from three subjects with brucellosis sampled during convalescence. Despite the different form of their TcR, the proliferation of these two major T cell subsets in response to a mitogenic anti‐CD3 monoclonal reagent (OKT3) was optimal. In contrast, αβ, but not γδ, T lymphocytes proliferated vigorously in response to the antigenic stimulus elicited by heat‐killed Brucella. Further studies are, therefore, needed to determine whether the selective expansion of the γδ T cell subpopulation observed during the clinical course of the infection is driven by antigenic determinant(s) borne by the pathogen in vivo or is due to host‐derived stimuli, such as autologous heat‐shock proteins expressed on the surface of the infected cells.
Strict peptide length is not required for the induction of cytotoxic T lymphocyte‐mediated antiviral protection by peptide vaccination
Authors: Kast W. Martin , Brandt Remco M. P. , Melief Cornelis J. M.
Published: March 2007

Sendai virus nuclear protein peptides of different lengths were titrated in peptide vaccination experiments. We observed that peptide length was not important in inducing cytotoxic T lymphocyte‐mediated protective immunity in vivo against a challenge with a lethal dose of virus. These results suggest that long peptides are trimmed in vivo to peptides that fit into the groove of major histocompatibility complex class I molecules. In addition several adjuvants were screened for their effectiveness in peptide vaccination protocols. Incomplete Freund's adjuvant and Titermax™ turned out to be useful, whereas alum was much less effective.
A monoclonal antibody reactive with a glycophosphatidylinositol‐anchored molecule on T cells defines CD4+ T cell subsets
Authors: Nagata Norikazu , Taketani Shigeru , Adachi Yasushi , Hosaka Naoki , Miyashima Shigeo , Tokunaga Rikio , Ikehara Susumu
Published: March 2007

A hybridoma, 25T3 (IgM, χ), was established from MRL/+ mice immunized with an autoreactive T cell line (1/+T1). The antigenicity of the antigen recognized by hybridoma 25T3 (25T3‐Ag) expressed on thymic and splenic cells was abolished by treatment with phosphatidylinositol‐specific phospholipase C, showing that 25T3‐Ag is a glycophosphatdidylinositol‐anchored Ag. 25T3‐Ag was expressed on approximately 90% of thymocytes. Double‐negative, double‐positive and CD8 single‐positive cells were highly positive for the expression of 25T3‐Ag, whereas CD4 single‐positive cells were weakly positive (approximately 40 %) or negative (approximately 60%). In the spleen, only CD3+ cells (and not B220+ nor Mac‐1+ cells) reacted with 25T3 monoclonal antibody (mAb), indicating that 25T3 mAb is specific for T cells. The majority of splenic CD8+ T cells were positive for the expression of 25T3‐Ag, although the intensity was weaker than that of thymocytes. In contrast, splenic CD4+ T cells were divided into negative (60‐70%) and positive (30‐40%) populations. Similar staining profiles were observed in BALB/c, C57BL/6, C3H/HeN and AKR/J mice. When BALB/c CD4+ T cell subsets were sorted and cultured with irradiated (25 Gy) antigen‐presenting cells, stimulation with immobilized anti‐CD3 mAb for 2 days resulted in CD4+25T3+ cells secreting more interleukin‐2 and less interleukin‐4 than did CD4+25T3− subsets, although the proliferative responses of the cells on day 2 of culture were similar. This suggests that CD4+ T cells can be divided into two populations and relatively defined as T helper 1 and T helper 2 cells using this 25T3 mAb. Immunoprecipitation and SDS‐PAGE revealed that 25T3‐Ag was approximately 70 kDa. These findings are discussed in relation to CD4+ T cell subsets.
Clonal deletion as direct consequence of an in vivo T cell response to bacterial superantigen
Authors: Wahl Claudia , Miethke Thomas , Heeg Klaus , Wagner Hermann
Published: March 2007

To date clonal deletion of peripheral mature T cells is restricted to in vivo model systems characterized by prolonged exposure of mice to antigens and clonal T cell expansion preceding clonal deletion. Here we describe that upon challenge of mice with the superantigen staphylococcal enterotoxin B two immediate events become imposed on ligand‐reactive Vβ8+ T cells in lymph node cells draining the local site of injection. First, and within hours Vβ selective clonal deletion is initiated via an apoptotic process. Second, the remaining Vβ8+ T cells first develop a profound state of ligand‐specific unresponsiveness and subsequently initiate clonal in vivo growth. It is suggested that the dichotomy of events observed reflects a direct consequence of T cell receptor occupancy in the context of inappropriate signalling.
Identification of peptide sequences that potentially trigger HLA‐A2.1‐restricted cytotoxic T lymphocytes
Authors: Nijman Hans W. , Houbiers Jos G. A. , Vierboom Michel P. M. , van der Burg Sjoerd H. , Drijfhout Jan W. , D'Amaro Joe , Kenemans Peter , Melief Cornelis J. M. , Kast W. Martin
Published: March 2007

We used the human processing defective cell line 174CEM.T2 (T2) to identify potential cytotoxic T lymphocyte (CTL) epitopes of human proteins. Exogenously added peptides can increase the number of properly folded HLA‐A2.1 molecules on the cell surface of T2 cells, as shown by immunofluorescence measurements using the mouse monoclonal antibody BB7.2 (anti‐HLA‐A2.1) and fluorescein isothiocyanate‐labeled goat anti‐mouse F(ab')2 antibody. The peptides were selected on the basis of a computer score derived from the recently described HLA‐A2.1 specific motif. Analysis of the influenza matrix protein showed that 15 out of 35 high‐scoring peptides up‐regulate the expression of HLA‐A2.1 molecules on theT2 cell surface. The combination of the computer scoring program and an immunofluorescence‐based peptide binding assay allows rapid detection of potential CTL target peptides.
Modulation of interferon‐γ receptor during human T lymphocyte alloactivation
Authors: Novelli Francesco , Giovarelli Mirella , Gentz Reiner , Zucca Mario , Pierro Francesco Di , Garotta Gianni , Forni Guido
Published: March 2007

Previous work has shown that neutralization of physiologically secreted interferon(IFN)‐γ or blockade of its receptor during T lymphocyte activation inhibits both proliferation and cytotoxic T lymphocyte generation, suggesting that IFN‐γ plays a crucial role in T lymphocyte induction and differentiation. In this study, the kinetics of the surface expression of the 90‐kDa IFN‐γ receptor (IFN‐γR) was followed during human mixed lymphocyte reaction (MLR) to alloantigens. IFN‐γR mRNA is constitutively expressed on resting peripheral blood lymphocytes emerging from nylon wood column (NW‐PBL) and its expression increases two‐ to threefold on alloactivated NW‐PBL. IFN‐γR protein is poorly expressed on the membrane of resting CD3+ cells, but up‐modulates after 3‐day MLR and sharply down‐modulates at day 6. Both the p55 and the p75 chains of interleukin‐2 receptor (IL‐2R) were shown to up‐modulate in parallel with IFN‐γR, whereas they were still highly expressed at day 6. After alloactivation, IFN‐γ and IL‐2 secretion starts at 24 h, peaks at day 3 and decreases just when IFN‐γR and IL‐2R begin to up‐modulate. Proliferation peaks at day 6. Lastly, stimulation with distinct cell populations showed that the intensity of lymphocyte proliferation, IFN‐γR membrane up‐modulation, and IFN‐γ and IL‐2 secretion are regulated in a parallel manner, thus suggesting that they are interrelated. Taken as whole these results demonstrate that increased expression of IFN‐γR on T lymphocytes can be a critical event during their activation, and strongly support the hypothesis that IFN‐γ/IFN‐γR interaction provides a signal for its progression.
A pattern search method for putative anchor residues in T cell epitopes
Authors: Hobohm Uwe , Meyerhans Andreas
Published: March 2007

The binding affinity between an antigenic peptide and its particular major histocompatibility complex (MHC) molecule seems to be largely determined by only a few residues. These residues have been called “anchors” because of their property of fitting into “pockets” inside the groove of the MHC molecule. To predict natural antigenic epitopes within a longer sequence, it therefore appears to be important to know the motif or pattern describing the anchors, i.e. the anchors amino acid residue preference and the distance between anchor residues.

A large set of MHC class I‐restricted peptides has been described. Peptide sequences vary in length and lack an obvious common sequence motif. For a list of peptides belonging to one type of MHC class I molecule, we describe a method to find the most prominent sequence motif with at least two anchor residues. Briefly, antigenic sequences are aligned, and two anchor positions are searched for, where all anchor residues share a high similarity. The alignments are scored according to the similarity of their anchor residues. We show that the motifs predicted for the MHC alleles A2.1, B27, Kb, Kd, Db are in substantial agreement with experimental data. We derive binding motifs for the MHC class I alleles HLA‐A1, All, B8, B14, H‐2Ld and for the MHC class II alleles I‐Ab and I‐As. In some cases, higher scores were obtained by allowing a slight variation in the number of residues between anchors. Therefore, we support the view that the length of epitopes belonging to a particular class I MHC is not uniform.

This method can be used to predict the natural short epitope inside longer antigenic peptides and to predict the epitopes anchor residues. Anchor motifs can be used to search for antigenic regions in sequences of infectious viruses, bacteria and parasites.
Systematic study of human αβ T cell receptor V segments shows allelic variations resulting in a large number of distinct T cell receptor haplotypes
Authors: Cornélis François , Pile Kevin , Loveridge Jason , Moss Paul , Harding Rosalind , Julier Cécile , Bell John
Published: March 2007

The variation of the αβ T cell receptor (TCR) results mainly from rearrangements of germ‐line V, D and J elements combined with the processes of N‐ and P‐region addition. In addition to this extensive diversity, diallelic polymorphism is also recognized in V regions of β loci. Four such polymorphisms have previously been defined, but the full extent of such variation has not yet been established. To investigate allelic polymorphism, we used a strategy based on V locus‐specific polymerase chain reaction and single‐strand conformation polymorphisms. Studying the two Vβ 2 loci and the Vα8.1 locus, we found that all exhibited a coding polymorphism. One of the Vβ 2 loci proved to be the first multiallele segment to be recognized, with three common variants. The second Vβ 2 locus, for which none of the two alleles has been identified in cDNA, appeared in fact to be a Vβ orphon, in abnormal location on the chromosome 9. A yeast artificial chromosome containing part of the TCRB locus allowed us to place the first Vβ 2 segment on the known map to define haplotypes with two other polymorphic segments: Vβ 1 and Vβ 6.7. Multiple distinct haplotypes result from combinations between these polymorphic loci, showing that Vβ regions are highly variable between individuals. Two alleles exist at the Vα8.1 segment and both are expressed. This represents the first example of a frequent coding polymorphism for TCRA gene. The distribution of allele frequencies for these segments suggest the action of balancing selection. These data add a further dimension to TCR polymorphism and suggest new candidates to explore TCR‐encoded susceptibility to autoimmune diseases.
Exon skipping without splice site mutation accounting for abnormal immunoglobulin chains in nonsecretory human myeloma
Authors: Cogné Michel , Guglielmi Paul
Published: March 2007

The proliferating plasma cells of patient COM with nonsecretory myeloma synthesized truncated 42 kDa γ1 chains made of a complete constant region but devoid of variable domain. In the absence of light chain expression, the shortened γ chains were retained intracellulary and were subsequently degraded within 12 h. COM neoplastic plasma cells contained short γ1 heavy chain transcripts in which the leader peptide exon was directly joined to the CH1 exon using the regular splice sites. However, study of the productive γ gene showed that the skipped variable exon was bounded by normal splicing signals and that the adjacent intron organization was not altered. Since this unusual splicing pattern was maintained when COM γ gene was transfected in murine plasmocytoma cells, exon skipping possibly relates to the modified structure of COM variable region. The latter showed a 2‐base pair deletion introducing a translation frameshift in the Vh region and a DNA insertion at the VH‐DJH junction consisting in a perfect duplication of the first 54 nucleotides of the recombined D JH segment. The lack of light chain production by COM cells was explained by alterations of the variable region of the rearranged χ gene leading to abnormally spliced transcripts.
The assembly of H2‐Kb class I molecules translated in vitro requires oxidized glutathione and peptide
Authors: Bijlmakers Mariejose J. E. , Neefjes Jacques J. , Wojcik‐Jacobs Eldine H. M. , Ploegh Hidde L.
Published: March 2007

Association of the mouse major histocompatibility complex (MHC) class I heavy chain H2‐Kb with mouse β2‐microglobulin (β2m) was studied in an in vitro translation system. Formation of stable class I complexes was found to be dependent on the presence of presentable peptides and oxidized glutathione, which promotes the formation of disulfide bridges. Translocation of peptides into microsomes was demonstrated by showing that a radioiodinated peptide containing an N‐glycosylation acceptor site became glycosylated. Class I complex formation was observed only when heavy chains and β2m were translated simultaneously, and thus occurs in the microsomes and not after their solubilization. However, peptide binding takes place only after solubilization of the microsomes. The class I complexes translated in vitro show the same specificity and length preference for peptides as their counterparts in RMA‐S cells. Assembly of in vitro translated class I complexes was found to occur also in the absence of peptides, resulting in the formation of unstable molecules that are stabilized by incubation with peptides.
Fetal thymocyte potential for T cell receptor Vγ3‐Jγ1 junctional modification
Authors: Larché Mark , Hurwitz Julia L.
Published: March 2007

Junctional modifications of T cell receptor (TcR) and immunoglobulin (Ig) gene joining regions provide great diversity to respective protein repertoires. The addition of non‐germ‐line‐encoded nucleotides (N‐regions) in the V‐Jγ junction is one such modification which is developmentally regulated, rarely evident in the fetal animal, but common in the adult. A question has recently arisen as to whether developmentally patterned N‐region additions in V‐Jγ joins are a reflection of T cell progenitors which are committed to particular types of rearrangement prior to the event, or of changing environmental influences on uncommitted cell populations. To address this question with regard to theVγ3‐Jγ1 join, T cells were examined in the fetal thymic organ culture (FTOC), a system with which the environment of early progenitor cells could be deliberately altered. At various times following FTOC initiation, cells were isolated for examination by the polymerase chain reaction, cloning and sequencing. Vγ3‐Jγl sequences within genomic DNA as well as cDNA were evaluated. Data from these studies revealed frequent N‐region additions within V‐Jγ joins among day 14 fetal thymocyte populations, a situation dissimilar from that in vivo. Also dissimilar from the in vivo situation was the degree of exonuclease activity evident in FTOC. The canonical Vγ3‐Jγl join (a frequent junction lacking N‐region addition) was recognized in all experiments, but was least common among DNA versus cDNA sequences. Results illustrate that early progenitor cell populations are not programmed to exclude junctional modifications from Vγ3‐Jγ1 joins.
The role of the interleukin‐2 (IL‐2)/IL‐2 receptor pathway in MRL/lpr lymphadenopathy: The expanded CD4−8− T cell subset completely lacks functional IL‐2 receptors
Authors: Tanaka Toshiyuki , Yasuhiko  , Nagasaka  , Kitamura Fujiko , Kuida Keisuke , Suwa Hiroshi , Miyasaka Masayuki
Published: March 2007

Autoimmune MRL/MP‐lpr/lpr (MRL/lpr) mice spontaneously develop a systemic lupus erythematosus‐like disease accompanied by a profound lymphadenopathy that consists of CD4−8−B220+ a P T cells. By the use of cross‐linking experiments with radiolabeled interleukin‐2 (IL‐2), these abnormal T cells have been reported to constitutively express the IL‐2 receptor β chain (IL‐2Rα), a signal transducing component of IL‐2R, in the absence of the a chain (IL‐2Rα).To critically reevaluate the role of the IL‐2/IL‐2R pathway in the pathogenesis of lymphadenophathy we examined expression of the IL‐2Rα and IL‐2Rβ in MRL/lpr mice by 125I‐IL‐2 binding analysis and also by flow cytometric analysis using monoclonal antibodies against each component of the receptor. We found that, contrary to the previous report, the CD4−8−B220+ α β T cells in lymph node (LN) of MRL/lpr mice were negative for both IL‐2Rα and IL‐2Rβ expression. The lpr liver CD4−8−B220+ a P T cells that had been implicated in the genesis of these abnormal LN T cells were also negative for IL‐2Rβ expression. Therefore, our results indicate that the IL‐2/IL‐2R system plays little role, if any, in the expansion of abnormal CD4−8− B220+ α β T cells in MRL/lpr mice.
Age‐related changes of T cell subsets in intestinal intraepithelial lymphocytes of mice
Authors: Takeuchi Makoto , Miyazaki Hiroyuki , Mirokawa Katsuku , Yokokura Tenio , Yoshikai Yasunobu
Published: March 2007

Age‐related changes in T cell subsets were examined in intestinal intraepithelial lymphocytes (i‐IEL), which contain unique T cells differentiating extrathymically. In 2‐month‐old mice bred under conventional condition, i‐IEL consisted of a large number of CD4−CD8α/α+ cells bearing either T cell receptor (TcR)α/β or TcRγ/δ and only a few CD4+CD8α− cells. In aged mice (6 months old and 24 months old), unique CD4+CD8α/α+ i‐IEL bearing TcRα/β increased in number and conversely the proportion of TcRγ/δ+ i‐IEL was decreased. Such an increase in number of CD4+CD8α/α+ cells was detected in i‐IEL from aged (14‐months old) nude mice, but not in aged (14 months old) germ‐free mice, suggesting that a significant fraction of TcRα/β T cells such as CD4+CD8α+ i‐IEL can develop along an extrathymic pathway under the influence of intestinal microflora with age.
Conservation of T cell receptor usage by HLA B27‐restricted influenza‐specific cytotoxic T lymphocytes suggests a general pattern for antigen‐specific major histocompatibility complex class I‐restricted responses
Authors: Bowness Paul , Moss Paul A. H. , Rowland‐Jones Sarah , Bell John I. , McMichael Andrew J.
Published: March 2007

Eight HLA B7‐restricted influenza A virus nucleoprotein 383–391‐specific cytotoxic T lymphocyte (CTL) clones were obtained from three unrelated donors following natural infection. T cell receptor (TcR) usage was studied using the “anchored” polymerase chain reaction. TcR α‐chain usage was restricted with three predominant Vα (Vα 12.1, 14.1, 22) and two predominant Jα segments. β‐chain variable‐region usage was also conserved, with Vβ7 being used by five clones despite contributing less than 2% of peripheral blood lymphocyte Vβ sequences of one individual studied. The TcR β‐chain junctional region was highly conserved even between CTL clones from unrelated individuals, with a negatively charged amino acid, contributed to by N‐region addition, encoded at position 97 in all but two clones. This study shows that peptide‐specific HLA B27‐restricted CTL following influenza virus infection use very similar TcR and, when considered with previous studies, suggests a pattern of TcR conservation for major histocompatibility complex class I‐restricted responses. No difference in TcR usage was detected between one healthy donor and two with HLA B27‐associated arthritis.
Fine specificity of monoclonal antibodies directed at human T cell receptor variable regions: Comparison with oligonucleotide‐driven amplification for evaluation of Vβ expression
Authors: Diu Anita , Romagné Francois , Genevée Catherine , Rocher Corinne , Bruneau Jean‐Michel , David Alain , Praz Françchise , Hercend Thierry
Published: March 2007

Seven distinct anti‐human T cell receptor (TcR) V region monoclonal antibodies (mAb) were generated by immunizing mice with either human T cell lines or transfected murine cells expressing human TcR Vβ genes. The specificity of these reagents was determined as follows: T cells recognized by each mAb were purified from the peripheral blood of healthy donors and TcR transcripts expressed in these cells were analyzed using oligonucleotide‐driven amplification and cDNA sequencing. Four mAb were found to delineate the Vβ3, Vβ8,Vβ17 and Vβ19 subfamilies, respectively. The remaining reagents recognize subsets within the Vβ2,Vβ5 and Vβ13 subfamilies. Reactivity of the mAb with circulating T cells from 18 unrelated healthy individuals was determined. Limited variability was found from an individual to another. In four donors, mAb staining was compared to oligonucleotide‐driven amplification for evaluation of Vβ3, Vβ8,Vβ17 and Vβ19 subfamily expression in the peripheral blood. Although the V gene subfamily‐specific oligonucleotides used in this study belong to a carefully controlled series, our results show that this method does not give an accurate estimate of the percentage of peripheral T cells expressing a given TcR β chain. The present data confirm the necessity to establish a complete set of well‐characterized monoclonal reagents to study human T cell responses.
Evidence for clonal anergy as a mechanism responsible for the maintenance of transplantation tolerance
Authors: Braun Michel Y. , McConnack Ann , And Gillian Webb , Batchelor J. Richard
Published: March 2007

The main stimulus triggering early acute allograft rejection is known to be delivered by the allogeneic “passenger” leukocytes present within the grafts. Once these cells have been replaced by cells of recipient origin, subsequent rejection episodes are generally less frequent and less acutely destructive. How this replacement affects the cell populations responsible for allograft rejection is not known. Here we report that rat alloreactive non‐cytotoxic AS (RT11) anti‐August (RT1C) CD4+ T cells, that were shown to be specific for RT1.BC+ August spleen stimulators, were able to cause acute rejection of normal August kidney allografts transplanted into sublethally irradiated AS recipients. These cells, however, failed to reject passenger cell‐depleted (PCD) August kidneys, despite the substantial expression of RT1.BC+ products on the graft tubular epithelium. In experiments in vitro, August kidney tubular epithelial cells expressing RT1.BC+ antigens were found to be unable to stimulate the alloreactive T cells to proliferate. Moreover, preincubation with class II‐positive August kidney epithelial cells specifically abrogated the alloreactivity of the T cells. Adding recombinant interleukin‐2, however, restored the response to alloantigens. These results are consistent with the hypothesis that T cell populations capable of mediating early acute allograft rejection are different from those mediating late rejection, when donor passenger leukocytes are no longer present. They also suggest clonal anergy as one of the mechanisms responsible for maintaining long‐term transplantation tolerance.
Occurrence of a silencer of the interleukin‐2 gene in naive but not in memory resting T helper lymphocytes
Authors: Mouzaki Athanasia , Rungger Duri , Tucci Alessandra , Doucet Arlette , Zubler Rudolf H.
Published: March 2007

In the immune system the first activation of a naive T cell by antigen is a key step in the shaping of the peripheral T cell specificity repertoire and maintenance of self‐tolerance. In the present study, analysis of the interleukin‐2 (IL‐2) gene activation shows that naive human helper T cells (cord blood CD4+ T cells, adult CD4+CD45RO− T cells) regulate IL‐2 transcription by a mechanism involving both a silencer and an activator acting on the purine‐rich IL‐2 promoter elements (NF‐AT binding sites). By contrast, memory cells, either in vitro activated helper T cells reverting to a resting state, or CD4+ T (memory) clones, or CD4+CD45RO+ T cells isolated ex vivo, no longer have a silencer. Their IL‐2 transcription seems to be controlled solely by the transition from inactive to active functional state of a positive transcription factor binding to these promoter elements as well as its cytoplasmic or nuclear location: in resting memory T cells the activator is located in the cytoplasm and is inactive, whereas in stimulated cells it is functional in promoting transcription and now resides in the nucleus. Thus, the regulation of the gene coding for the main T cell growth factor changes irreversibly after the first encounter of T cells with antigen. It is most likely that the presence of a silencer contributes to the more stringent activation requirements of naive CD4+ T cells.
Pathogenesis of the toxic shock syndrome: T cell mediated lethal shock caused by the superantigen TSST‐1
Authors: Miethke Thomas , Duschek Katja , Wahl Claudia , And Klaus Heeg , Wagner Hermann
Published: March 2007

The pathogenesis of the toxic shock syndrome (TSS) is only incompletely understood. We now present evidence that TSS toxin‐1 (TSST‐1), one of the superantigens produced by Staphylococcus aureus, induces lethal shock in D‐galactosamine sensitized mice. In this model TSS is dependent on T cells, since cyclosporin A (CsA) completely blocked development of shock, and since T cell‐deficient SCID mice did not show signs of disease upon injection with TSST‐1. However, SCID mice repopulated with T cells succumbed to lethal shock. The disease is characterized by a burst of lymphokines like interleukin‐2 (IL‐2) and tumor necrosis factor (TNF) released into the sera of TSST‐1‐treated animals. Already 1–2 h after TSST‐1 application TNF serum levels peaked and IL‐2 levels peaked around 4 h after treatment. TNF appears as key mediator of TSS, because anti‐TNF monoclonal antibodies protected TSST‐1‐challenged mice. Interestingly, the burst of TNF in serum was noted well in advance of detectable markers of T cell activation. Thus, about 5 % of all peripheral T cells started to express the IL‐2 receptors as late as 4 h after treatment. Comparing TSST‐1‐ and endotoxin‐induced shock we conclude that TNF effects shock in both diseases. However, the type of cells involved appears distinct in that T cells cause TSS triggered by the exotosin TSST‐1 while macrophages mediate the shock induced by endotoxins.
Suppression of B cell differentiation by ligation of membrane‐bound IgM
Authors: Grandien Alf , Modigliani Yves , Coutinho Antonio , Andersson Jan
Published: March 2007

Using B cells from the transgenic mouse line B6‐Sp6 and control littermates, stimulated by lipopolysaccharide (LPS) under novel culture conditions that provide for the response of all B cells, we show here that specific ligation of the surface IgM molecules always results in inhibition of terminal differentiation and immunoglobulin secretion by activated cells, regardless of the ligand. Thus, monoclonal antibodies to (a) the CH region of Ig (anti‐μ. and anti‐allotype), (b) the Cx region, (c) the V region (anti‐idiotype) of surface IgM, as well as (d) multivalent antigen (2,4,6‐trinitrophenyl‐bovine serum albumin), all show similar effects and dose‐response curves. IgD‐negative transgenic B cells are equally sensitive to IgM ligation‐dependent inhibition, as control (IgD‐positive) B cells. The allotype specificity of this inhibition, assessed by using anti‐u, allotype reagents to inhibit and assay the responses, suggests that B cells expressing transgenic or endogenous IgM in transgenic B6‐Sp6 mice are largely independent populations. These observations establish that anti‐IgM antibodies in conjunction with appropriate LPS stimulation, provide a universal model system for functional characterization of B cell responses.
Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen‐specific T cells
Authors: Moll Heidrun , Fuchs Harald , Blank Christine , Röllinghoff Martin
Published: March 2007

Murine epidermal Langerhans cells (LC) have been shown to internalize Leishmania major, a cause of human cutaneous leishmaniasis, and to stimulate a vigorous parasite‐specific T cell response. The present study emphasizes the critical role of LC in leishmaniasis by documenting directly that LC have the ability to transport L. major from the skin to the draining lymph node (LN). This was revealed by irreversible labeling of LC with a fluorescent cell linker and in vivo tracking. In contrast, no migration to the LN was seen with L. major‐infected macrophages. These findings were consistent with the results of mixed labeling immunohistology showing that early in infection the expression of parasite antigen in the LN draining the lesion was confined to dendritic cells and could not be detected in macrophages. Furthermore, dendritic cells in LN draining the site of cutaneous infection stimulated L. mayor‐primed T cells in vitro and, most notably, were able to activate unprimed T cells capable of mediating parasite‐specific delayed‐type hypersensitivity reactivity in vivo. Taken together, the results indicate that LC capture L. major in the skin and transport it to the regional LN for initiation of the specific T cell immune response.
The mucosal T cell integrin αM290β7 recognizes a ligand on mucosal epithelial cell lines
Authors: Roberts Kevan , Kilshaw Peter J.
Published: March 2007

The integrin αM290β7 is expressed at high levels on mucosal T cells, particularly on those within the epithelium of the gut. We now report that a mouse T cell hybridoma, MTC‐1, with similar surface expression of this molecule, adhered strongly to cells of the mouse rectal carcinoma line CMT93 and that adhesion was blocked completely by the monoclonal antibody (mAb) M290. Other mAb to the αM290 or β7 subunits had little or no inhibitory effect. M290 also inhibited adhesion of the hybridoma to cells of the mouse lung carcinomas CTM64/61 and KLN205 but had little or no effect on adhesion to seven other mouse epithelial cell lines or to the human colon carcinoma line, HT29. Intraepithelial lymphocytes (IEL) isolated from the small intestine of BALB/c mice displayed potent Tcell receptor‐dependent cytotoxic effector function against CMT93 in the presence of low concentrations of Phytolacca americana lectin. This cytotoxic activity also was inhibited by the M290 mAb. Treatment of CMT93 cells with tumor necrosis factor‐a and interferon‐γ induced expression de novo of ICAM‐1 and reduced the inhibitory effect of M290 in tests both for adhesion and cytotoxicity. In further experiments cytotoxic activity of IEL against the mastocytoma P815 was investigated. This target cell was considered not to possess a ligand for the integrin. In this case cytotoxic effector function was triggered by anti‐CD3 mAb and, in contrast to results with CMT93, target cell lysis was increased in the presence of M290 and other antibodies to the integrin, suggesting a co‐stimulatory effect. These results show that αM290β7 recognizes a ligand on the surface of certain epithelial cell lines. Further, they provide the first clear indication that this integrin may play an important role in functional interactions between T cells and the mucosal epithelium.
Environmental modulation of the autonomy of cytotoxic T lymphocytes
Authors: Bodmer Helen , Obert Georges , Chan Susan , Benoist Christophe , Mathis Diane
Published: March 2007

The extent to which one compartment of the immune system depends on another for efficient function is important to establish to fully comprehend disease phenotypes arising from selective immunodeficiency. Just how much the major histocompatibility complex class I‐restricted cytotoxic T cell responses depend on class II‐restricted T cell help has been controversial. Using the influenza A virus system, we show that mice unable to make class II‐restricted T cell responses due to an engineered defect in class II molecule expression are able to mount virtually normal cytotoxic responses when bred under specific‐pathogen‐free conditions. However, when exposed to the more diverse environmental challenges of a conventional breeding facility, a situation that more closely parallels immuno‐deficient states in man, they show impaired cytotoxic responses.