bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in thymic export of L‐selectin+ γδ and αβ T cells during fetal and postnatal development
Authors: Witherden Deborah A. , Abernethy Nevin J. , Kimpton Wayne G. , Cahill Ross N. P.
Published: March 2007

We have used the technique of in situ intrathymic injection of fluorescein isothiocyanate to examine L‐selectin expression on γδ and αβ T cells immediately after emigrating from the thymus of fetal and postnatal animals. We found that the percentage of L‐selectin+ thymocytes exported per day decreased by half after birth and that the export of T cells from the thymus does not rely on expression of the peripheral lymph node homing receptor, L‐selectin. Analysis of L‐selectin on emigrant and mature T cell subsets revealed a remarkable heterogeneity of expression, both in terms of the numbers of cells expressing this molecule as well as the level of expression. γδ T cells, reportedly not having a propensity for homing to lymph nodes, not only contained the highest proportion of L‐selectin+ cells, but also expressed far more of this molecule than either CD4+CD8− or CD4−CD8+ αβ T cells. Furthermore, those emigrant T cells expressing L‐selectin are somewhat immature in their expression of this molecule. Subsequent maturation resulted in up‐regulation of L‐selectin on mature peripheral blood T cells, maturation that was clearly independent of extrinsic antigen. This antigen‐independent post‐thymic maturation appeared to occur as part of the normal progression from immature thymocyte to mature peripheral T cell in both fetal and postnatal animals.
CD45RB expression defines two interconvertible subsets of human CD4+ T cells with memory function
Authors: Horgan Kevin J , Tanaka Yoshiya , Luce Gale E. Ginther , Van Seventer Gijs A. , Nutman Thomas B. , Shaw Stephen
Published: March 2007

Reciprocal expression of CD45RA and CD45RO in human CD4+ T cells defines populations understood to be naive cells (CD45RA+CD45RO−) and memory cells (CD45RA−CD45RO+). We investigate two subsets of CD45RA−CD45RO+ CD4+ human T cells which differ by fourfold in their expression of the CD45RB isoform; one is CD45RBbright and the other is CD45RBintermediate. In contrast, CD45RA+ naive cells are all CD45RBbright. Both subsets of CD45RA− cells proliferate in response to recall antigens so we designate them MEM 1 (CD45RO+RBbright) and MEM 2 (CD45RO+RBintermediate). CD45RA and CD45RB expression are regulated independently during in vitro activation of naive cells. When MEM 1 cells are activated they tend to down‐regulate CD45RB expression, whereas activated MEM 2 cells tend to up‐regulate CD45RB expression. Thus, in contrast to the stability of the CD45RA−CD45RO+ phenotype, the MEM 1 and MEM 2 phenotypes are labile and may interconvert. MEM 1 and MEM 2 cells produced comparable amounts of interleukin(IL)−2, IL−4, and IL‐5 though MEM 1 cells produced slightly more interferon(IFN)‐γ (mean 1.7‐fold more). MEM 1 cells consistently proliferated more (mean 2.3‐fold more) than MEM 2 cells early during in vitro activation. Thus, differential expression of CD45RB within CD45RA− cells defines two subsets that have similar properties except for somewhast greater IFN‐γ production and proliferative responses by MEM 1 cells. Variability in CD45RB expression may represent a mechanism for fine‐tuning the responsiveness of memory cells in vivo.
Activation and internalization of p56lck upon CD45 triggering of Jurkat cells
Authors: Marie‐Cardine Anne , Maridonneau‐Parini Isabelle , Fischer Siegmund
Published: March 2007

Relationships between CD45 and p56Ick have been suggested by co‐immunoprecipitation of both proteins and by dephosphorylation of the p56lck regulatory site, Tyr 505, by CD45 in vitro. We investigated whether the kinase activity of p56lck is modulated in T cells triggered via CD45. We showed that incubation of Jurkat cells with a combination of two anti‐CD45 monoclonal antibodies (mAb) (MC5/2 + D3/9) induced an increase in p56lck kinase activity, while a single mAb did not. Under these conditions, p56lck underwent two consecutive waves of activation. This was accompanied by internalization of the kinase and by a time‐dependent increased accessibility of CD45 phosphatase at the plasma membrane. Similarly, activation and internalization of p56lck were observed using a combination of anti‐CD45 (MC5/2) and anti‐CD2(T112) mAb, suggesting that a functional complex consisting of CD45, CD2 and p56lck was formed upon cell triggering. Taken together, these results suggests that: (i) CD45 participates in the regulation of p56lck kinase activity in vivo and that (ii) CD45 could play a mediator role in the stimulation and endocytosis of p56lck through the CD2 pathway.
Differential expression of lymphocyte homing receptors by human memory/effector T cells in pulmonary versus cutaneous immune effector sites
Authors: Picker Louis J. , Martin Richard J. , Trumble Anne , Newman Lee S. , Collins Patricia A. , Bergstresser Paul R. , Leung Donald Y. M.
Published: March 2007

The heterogeneous expression of lymphocyte homing receptors (HR) by the (CD45RAlow/ROhigh) memory/effector T cell population in the human is thought to define subsets with tissue‐selective recirculatory potential. To investigate further the localization characteristics of these T cells, we used multiparameter flow cytometry to quantitate T cell subsets defined by expression of the skin‐selective HR called the cutaneous lymphocyte‐associated antigen (CLA), the peripheral lymph node (PLN) HR L‐selectin, the mucosal‐associated HR α4β7‐integrin, and the mucosal‐associated adhesion molecule αeβ7‐integrin in either cutaneous or pulmonary immune effector sites and corresponding peripheral blood. Compared to peripheral blood, skin T cells were highly enriched for the CLA+/L‐selectin+/αeβ7‐integrin− memory/effector subset, whereas lung memory/effector T cells were predominantly CLA−to low L‐selectin−, and almost half were αeβ7‐integrin+. α4β7‐integrin expressing memory/effector T cells were diminished in both skin and lung, suggesting that this HR is not a major participant in determining localization specificity in either of these sites. The characteristic pulmonary T cell HR phenotype did not significantly differ between the normal subjects and those with pulmonary inflammatory disease, and did not correlate with markers of T cell activation. Induction of a rapid up‐regulation of pulmonary inflammation via intrabronchial allergen challenge in asthmatic patients tended to decrease localization specificity, resulting in a more general importation of memory/effector subsets. Taken together, these results suggest that tissue microenvironments play a major role in determining the character of local T cell infiltrates via their ability to import and retain memory/effector subsets selectively or, more generally, depending on the intensity of local inflammatory stimuli.
Exogenous peptide ligand influences the expression and half‐life of free HLA class I heavy chains ubiquitously detected at the cell surface
Authors: Carreno Beatriz M. , Hansen Ted H.
Published: March 2007

A pool of free HLA class I chains has been detected at the plasma membrane of all cells concomitantly expressing folded and assembled class I molecules. To determine the origin of these free HLA heavy chains, we have examined the biosynthesis of a single HLA class I molecule, HLA‐B27, expressed by a murine cell line (L‐B27). In L‐B27 cells, as previously shown in Epstein‐Barr virus‐transformed lymphoblastoid cell lines, a precursor/product relationship exists, early in biosynthesis, between free (HC10‐reactive) and beta‐2‐microglobulin (β2m)‐associated (W6/32‐reactive) class I heavy chains as demonstrated by pulse/chase experiments. At later stages in class I biosynthesis, both HC10‐ and W6/32‐reactive heavy chains display complex oligosaccharides and accumulate at the cell surface. HC10‐ and W6/32‐reactive molecules are both very stable at the cell surface, with half‐lifes (t1/2) of > 7 h and ∼ 4 h, respectively. Interestingly, cell surface expression and turnover of HC10‐ and W6/32‐reactive molecules were affected by the addition of peptide ligands to the culture media. Culturing cells in the presence of HLA‐B27 ligands resulted in the increased expression of W6/32‐reactive molecules and the decreased expression of HC10‐reactive molecules. Moreover, addition of exogenous peptide extended the t1/2 of W6/32‐reactive molecules to > 7 h and reduced that of HC10‐reactive molecules to 4 h. These results indicate that surface HC10‐reactive molecules result largely from W6/32‐reactive molecules following peptide and β2m dissociation. Therefore, HC10‐reactive species are not only the precursors but also the end products in class I biosynthesis.
Assessment of alloreactive T cell subpopulations of aged mice in vivo. CD4+ but not CD8+ T cell‐mediated rejection response declines with advanced age
Authors: Rosenberg Amy S. , Sechler Joan M. G. , Horvath Judith A. , Maniero Tonnie G. , Bloom Eda T.
Published: March 2007

The present investigation explored age‐related alterations in T cell populations mediating allospecific responses in vivo. Healthy aged and young H‐2b and H‐2bxH‐2k mice were engrafted with major histocompatibility complex (MHC) class II‐disparate bm12 skin, rejection of which requires CD4+ T cells, and MHC class I‐disparate bm1 skin, rejection of which requires CD8+ T cells. Aged mice of both genders exhibited prolonged survival of bm12 skin grafts relative to their young counterparts but rejected bm1 skin grafts at a rate equivalent to that of young mice. Consistent with prolonged survival of bm12 skin grafts, markedly diminished levels of Iabm12 CTL activity were elicited from T cells of aged mice in vitro. However, no such decline was observed in the level of Kbm1 CTL from T cells of aged mice. The alterations in Iabm12 allospecific responses were not attributable to quantitative changes in CD4+ T cells of aged mice, and addition of soluble T cell helper factors to response cultures of aged mice did not augment Iabm12 cytotoxic T lymphocytes activity. These data demonstrate that aging fundamentally affects CD4+ T cell‐mediated allospecific responses particularly in vivo, and that deficient generation of soluble T cell helper factors alone cannot explain this deficit.
CD5− dendritic epidermal T cells are derived from CD5+ precursor cells
Authors: Payer Elisabeth , Kutil Raphaela , Stingl Georg
Published: March 2007

Murine Thy‐1+, TcR Vγ3/Vδ+ dendritic epidermal T cells (DETC) differ from most other T cell subsets by the absence of CD4 and CD8 antigens as well as the lack of CD5 expression. To see whether negativity for those antigens is an intrinsic feature of a given T cell population or if such triple‐negative T cells go through a maturational stage where they express these antigens, we determined the phenotype of TcR Vγ3+ fetal thymocytes which are the precursor cells of DETC. We found that TcR Vγ3+ fetal thymocytes phenotypically differ from mature DETC in that they are CD5+, mostly CD8+ and partly CD4+. The injection of fetal thymic suspensions containing TcR Vγ3+/CD5+ (but not TcR Vγ3+/CD5−) thymocytes into Thy‐1‐disparate athymic nude mice resulted in the appearance of donor‐type TcR Vγ3+/CD5− dendritic cells in the recipients' epidermis, indicating that TcR Vγ3+ thymocytes are indeed the precursors of CD5− DETC. Tracing CD5 expression on DETC precursors during their intrathymic maturation and their migration to the fetal skin, we found that (i) the earliest DETC precursor cells as defined by TcR Vγ3 expression express high levels of CD5 antigen (day 15 of gestation), (ii) after day 16 of gestation 70% of TcR Vγ3+ thymocytes express high and 30% express intermediate levels of CD5, (iii) TcR Vγ3+ cells in the fetal blood express low levels of CD5, (iv) the first TcR Vγ3+ cells entering the epidermis express very low levels of this antigen and (v) TcR Vγ3+ epidermal cells later than day 19 of gestation are CD5−. A similar down‐regulation of CD5 expression on DETC precursors was also noted when TcR Vγ3+ cells were cultured in vitro. Even the addition of PMA and ionomycin, which up‐regulates CD5 expression on TcR α/β‐bearing thymocytes and lymph node T cells, could not prevent down‐regulation on DETC precursors. The described cell system may serve as a useful tool in further experiments aimed to clarify the function of the CD5 glycoprotein as well as the mechanism(s) regulating its expression.
Negative selection of multireactive B cell clones in normal adult mice
Authors: Grandien Alf , Fucs Rita , Nobrega Alberto , Andersson Jan , Coutinho Antonio
Published: March 2007

In the absence of intentional immunizations, normal mice produce natural antibodies that react with a variety of self and foreign antigens. We have now addressed the putative physiological selection of such reactivities and some of their clonal characteristics, by analyzing antibodies produced by B cells at different stages of differentiation. Using an antigen‐specific spot‐enzyme‐linked immunosorbent assay (ELISA) with a panel of self and foreign antigens, we found that newly formed B cells, either from adult bone marrow or from newborn spleen, contain the highest frequencies of IgM antibodies with reactivities towards the panel. Resting peripheral B cells show lower frequencies of such antibodies, that are lowest among naturally activated splenic plasma cells. Analyses of monoclonal IgM antibodies derived from lipopolysaccharide‐stimulated bone marrow and spleen cell hybridomas in normal mice show that the majority of reactivities scored in spot‐ELISA originate from multireactive IgM clones. In Western blots against a large number of self antigens, each multireactive IgM antibody studied shows a unique and specific pattern of reactivity. We conclude that multireactive B cell clones are very frequent in the emergent repertoires of newborns and adults, but are subsequently negatively selected from bone marrow to periphery, and from the available repertoire to that of natural plasma cells. It, thus, seems that multireactivity of natural antibodies is not a positively selected property, but represents the sum of unique multireactive clones that have escaped inactivation or deletion.
Regulation of VH‐gene expression is a lineage‐specific developmental marker
Authors: Viale Anne‐Claire , Freitas Antonio A. , Coutinho Antonio
Published: March 2007

We have previously shown that in IgH congenic mice VH‐gene family usage in neonatal spleen B cells and adult Ig‐secreting cells is entirely determined by the IgH locus, while in adult resting B cells it is regulated by genetic element(s) located outside the IgH locus. Two observations reported here demonstrate that the differential expression of VH genes is an intrinsic property of the respective cell populations, determined by both the IgH locus and by a cis element(s) operating independently in the same animal. First, the study of F1 hybrids between the IgH congenic B6a and CB.20 strains demonstrates that cis elements control VH‐gene family expression. Second, studies in irradiation chimeras showed that the environment in which cell differentiation proceeds is unable to overcome those controls. In chimeras of IgH congenic donors, VH‐gene expression in fetal liver‐derived splenic B cells and Ig‐secreting cells is dictated by the IgH haplotype, while in bone marrow‐derived B cells is entirely determined by the cis element(s). These results show a developmental and cell lineage‐related restriction in VH‐gene expression, and suggest that most adult splenic Ig‐secreting cells may originate from precursors originally present in fetal liver, but which are rare among adult bone marrow precursors and CD5+ B cells.
B lymphocyte and macrophage expression of carcinoembryonic antigen‐related adhesion molecules that serve as receptors for murine coronavirus
Authors: Coutelier Jean‐Paul , Godfraind Catherine , Dveksler Gabriela S. , Wysocka Maria , Cardellichio Christine B. , Noël Henri , Holmes Kathryn V.
Published: March 2007

The expression of carcinoembryonic antigen (CEA)‐related glycoproteins that have been associated with intercellular adhesion and that serve as receptors for mouse hepatitis virus (MHV) was analyzed in cells from the immune system of BALB/c mice using immunolabeling and RNA polymerase chain reaction amplification of receptor transcripts. These glycoproteins, which are called biliary glycoproteins, were highly expressed in B lymphocytes, including cells of the B‐la (CD5+) lineage, and in macrophages, but were not detectable in resting T lymphocytes. Similarly, murine cell lines of B cell and macrophage origin expressed messenger RNA encoding CEA‐related molecules, while the corresponding mRNA was only slightly detectable in a T cell line. These CEA‐related cell adhesion glycoproteins were also expressed in endothelial cells. Therefore, their specific interaction with their so far unknown ligand may be of functional importance in cellular interactions in the immune response.

Monoclonal antibody directed against these glycoproteins blocked MHV‐A59 infection of the B cell‐derived SP20 cell line. Thus, the functional receptors for MHV on B lymphocytes, like those on murine fibroblasts, are isoforms of CEA‐related glycoproteins. Treatment of B cells with anti‐receptor antibody also blocked B cell‐mediated cytotoxicity against MHV‐A59‐infected fibroblasts, indicating that this phenomenon is mediated by interaction of viral attachment protein on the infected target cells with specific CEA‐related receptor glycoproteins on the effector B cells.
Preformed purified peptide/major histocompatibility class I complexes are potent stimulators of class I‐restricted T cell hybridomas
Authors: Stryhn Anette , Pedersen Lars østergaard , Ortiz‐Navarrete Vianney , Buus Søren
Published: March 2007

A panel of antigen‐specific, major histocompatibility complex class I‐restricted T cell hybridomas has been generated to examine the capacity of peptide/class I complexes to stimulate T cells at the molecular level. Peptide/class I complexes were generated in detergent solution, purified and quantitated. Latex particles were subsequently coated with known amounts of preformed complexes and used to stimulate the T cell hybridomas. Stimulation was specific, i.e. only the appropriate peptide/class I combination were stimulatory, and quite sensitive, i.e. as little as 300 complexes per bead could be detected by the T cells. Preformed complexes were about 500000 times more potent than free peptide in terms of T cell stimulation, demonstrating the physiological relevancy of the biochemically generated complexes. Surprinsingly, the majority (including the most sensitive of the hybridomas) had lost CD8 expression, suggesting that antigen‐specific stimulation of class I‐restricted T cell hybridomas, as assessed by IL‐2 release, does not depend on CD8.
Peptide transporter‐independent, stress protein‐mediated endosomal processing of endogenous protein antigens for major histocompatibility complex class I presentation
Authors: Schirmbeck Reinhold , Reimann Jörg
Published: March 2007

The peptide transporter‐defective cell line RMA‐S expressing the wild‐type simian virus 40 large T antigen (wtT‐Ag) from a transfected gene did not present two well‐defined, H‐2 class I (Db)‐restricted epitopes of T‐Ag to cytotoxic T lymphocytes (CTL). Hence, “endogenous” processing and presentation of the wtT‐Ag depended on a functional peptide transporter heterodimer. In contrast, both T‐Ag epitopes were efficiently presented to CTL by transfected RMA‐S cells expressing a truncated, cytoplasmic T‐Ag variant (cT‐Ag) or a karyophilic, amino‐terminal 272‐amino acid T‐Ag fragment. Transporter‐independent “endogenous” processing of mutant T‐Ag molecules correlated with their association with the constitutively expressed heat shock protein 73 (hsp 73). Class I‐restricted presentation of both epitopes processed from these hsp73‐associated protein antigens was sensitive to NH4Cl and chloroquine. These data indicate that selected intracellular proteins access an alternative, hsp73‐mediated pathway for class I‐restricted presentation that operates independent of peptide transporters in an endosomal compartment.
Human immunodeficiency virus‐1 reverse transcriptase immunodominant CD4+ T cell epitopes: A peptide‐based multiparametric assessment in the mouse
Authors: Borg Jean‐Paul , Ihlenfeldt Hans‐Georg , Jung Günther , Haas Gaby , Pierres Michel
Published: March 2007

We previously identified an immunodominant CD4+ T cell determinant in the carboxy‐terminal region of HIV‐1 reverse transcriptase (RT528–543). The present study aimed at enumerating all the potential sites of HIV‐1 RT recognized by Th cells in the BALB/c (H‐2d) mouse model. To achieve this we used a panel of 62 overlapping 15‐mer synthetic peptides covering the whole RT sequence to assay the following parameters: (i) immunogenicity in naive BALB/c mice injected either with peptides pools or individual peptides; (ii) antigenicity, as detected by their ability to restimulate in vitro T cells from BALB/c mice primed with native RT: (iii) MHC class II (Ad)‐binding capacity as measured by the inhibition of the antigen‐specific, Ad‐restricted presentation of unfolded apamin (4‐Acm) by fixed antigen‐presenting cells to Ad/4‐Acm‐specific, interleukin‐2‐producing T hybridoma cells; and (iv) the presence of typical or degenerate consensus Ad‐binding motifs. The results in this study permitted identification of three novel immunodominant RT mouse CD4+ T cell sites (RT276–290), RT375–389 and RT411–425) located in regions of limited polymorphism among RT from several HIV isolates. Some of these RT segments were found to be in the vicinity of B cell or H‐2Kk‐ or HLA‐A2‐restricted cytotoxic T lymphocyte epitopes. Finally, the approach used in this study was found to be very efficient for enumerating most T cell recognition sites in a complex protein, a result that would have not been achieved by a single parameter‐based analysis.
Naive T cells can mediate delayed‐type hypersensitivity response in T cell receptor transgenic mice
Authors: Sato Takehito , Sasahara Takeshi , Nakamura Yukitsugu , Osaki Takako , Hasegawa Takanori , Tadakuma Takushi , Arata Yoji , Kumagai Yoshihiro , Katsuki Motoya , Habu Sonoko
Published: March 2007

We produced transgenic mice expressing Tcell receptor‐αβ chain genes, derived from the chicken ovalbumin (OVA)‐specific I‐Ad‐restricted CD4+CD8− T helper cell clone 7–3–7. In transgenic mice with H‐2d genetic background (Tg‐d mice), delayed‐type hypersensitivity (DTH) was induced in the hind footpad by one inoculation with OVA without any previous sensitization, suggesting that naive T cells have the potential to be involved in DTH response. Spleen cells from nonimmunized Tg‐d mice showed a strong T cell proliferative response to in vitro stimulation with OVA. Furthermore, these spleen cells produce cytokines including interleukin(IL)‐2, IL‐3, interferon‐γ, granulocyte/macrophage colony‐stimulating factor, macrophage inflammatory protein (MIP)‐1α and MIP‐1β, which may play an important role in the attraction of mononuclear cells to an antigen‐challenging site.
Identification and characterization of membrane cofactor protein (CD46) in the human kidneys
Authors: Nakanishi Isao , Moutabarrik Abdelhakim , Hara Tomoko , Hatanaka Michiyo , Hayashi Terumasa , Syouji Tatsuya , Okada Noriyuki , Kitamura Eisaku , Tsubakihara Yoshiharu , Matsumoto Misako , Seya Tsukasa
Published: March 2007

Membrane cofactor protein (MCP, CD46) is an integral protein that serves as a cofactor for factor I in inactivating C3b/C4b deposited on the same cell‐membrane as C3bi/C4c+C4d. This C3b/C4b inactivation is closely associated with self‐protection of host cells from autologous complement attack. We have studied the distribution and properties of MCP in the normal human kidney by immunohistochemical and immunoblotting methods using monoclonal antibodies against MCP. MCP was predominantly expressed on the juxtaglomerular apparatus. Glomerular capillary walls, mesangial areas, and tubulus were also MCP positive. Glomerulus MCP was composed of two major bands of 45–65 kDa, which were similar to those of lymphocyte MCP. The proportion of the high and low molecular weight components in glomerulus MCP, however, was considerably different from that of lymphocyte MCP among the individual samples tested. Glomerular epithelial cells and mesangial cells from an individual having equal amounts of high and low molecular weight components in the lymphocytes were cultured seperately and the properties of their MCP investigated. MCP in the mesangial cells and glomerular epithelial cells showed profiles in which the upper band was predominant. The results may explain the unique distribution of the high and low molecular weight forms in the glomerulus. These forms of MCP together with factor I were all capable of inactivating C3b to C3bi. Message analysis suggested that glomerular epithelial cells and mesangial cells synthesized a single species of mRNA of 4.2 kb from which the polymorphic MCP species were generated. Flow cytometric analysis suggested that MCP was minimal in mesangial cells. These results, taken together with the previous reports on the distribution of other complement regulatory proteins, infer that the distribution profile of MCP is rather similar to that of DAF but differs from those of CD59 and CR1 in the normal human kidney; this may reflect the differences between their roles or functional properties in renal tissue.
Fine antigen specificity of human γδ T cell lines (Vγ9+) established by repetitive stimulation with a serotype (KTH‐1) of a gram‐positive bacterium, Streptococcus sanguis
Authors: Mochizuki Mitsuru , Suzuki Noboru , Takeno Mitsuhiro , Nagafuchi Hiroko , Harada Tatsuhiro , Kaneoka Hidetoshi , Yamashita Naomi , Hirayama Kenji , Nakajima Toshiharu , Mizushima Yutaka , Yamamoto Shoso , Sakane Tsuyoshi
Published: March 2007

We have established human γδ T cell lines specific for Streptococcus sanguis (S. sanguis) KTH‐1 present in normal oral cavity flora. The CD4−CD8− CD3+Vγ9+Vδ1−CD45RO+ CD25+ T cell lines showed a proliferative response to the streptococcal antigen (Ag) in the presence of autologous antigen‐presenting cells without apparent evidence of HLA restriction. The proliferative response of the γδ T cell lines was completely blocked by anti‐TcRγδ monoclonal antibody (mAb) and anti‐HLA class I mAb (W6/32), whereas anti‐HLA classical class Ia mAb (B‐H9; anti‐HLA‐A,B,C), anti‐HLA class II mAb (anti‐DR, anti‐DQ, and anti‐DP) and anti‐CD4 mAb did not have any inhibitory effects. Surprisingly, the γδ T cell lines showed the proliferative response against the original bacterial Ag KTH‐1 exclusively, and exhibited no cross‐reactivity with nominal Ag such as purified protein derivative of tuberculin, tetanus toxoid and Mycobacterium tuberculosis, or the same species but different strain of S. sanguis, American Type Culture Collection (ATCC) standard strain (10556), or even with the same strain but different serotype of S. sanguis, KTH‐3. Moreover, cytokine production of the γδ T cell lines was similar to the Th1 pattern [interferon‐γ, tumor necrosis factor (TNF)‐α and TNF‐β]. They also produced interleukin‐8 that functions as one of chemoattractants for polymorphonuclear cells. Using direct sequencing technique of the polymerase chain reaction products, we found that junctional diversity of the T cell receptor (TcR) used by the parental KTH‐1 specific γδ T cell line and its subclones is rather limited. It is suggested that γδ T cells with canonical TcR could preferentially respond to KTH‐1 Ag. Thus, in addition to a broad or cross‐reactivity of γδ T cells against phylogenetically conserved stress/heat‐shock protein, which is well characterized by others, some peripheral blood γδ T cells could recognize and kill exogenous agents with fine antigenic specificity to protect the body against them.
Characterization of the B cell‐macrophage lineage transition in 70Z/3 cells
Authors: Tanaka Tetsuji , Wu Gillian E. , Paige Christopher J.
Published: March 2007

The 70Z/3 cell line is able to undergo lineage conversion from a pre‐B to macrophage phenotype. Data presented here show that the transition from pre‐B to macrophage follows a reproducible pathway via a stable intermediate stage. The cells in the intermediate stage are adherent and have lost the ability to respond to lipopolysaccharide or interferon‐γ by induction of immunoglobulin χ light chains. However, these cells do not yet display the full range of macrophage‐specific properties such as receptors for macrophage‐colony stimulating factor or the β2 integrin CD11b/CD18. Subcloning experiments with the intermediate cells revealed that they retain the options of either persisting along the macrophage line of differentiation, acquiring additional macrophage traits, or reverting to the pre‐B phenotype. Further differentiation to the macrophage stage is accompanied by the apparent loss of the ability to revert. Thus, these studies define relationships among lineage‐specific traits, and begin to reveal critical stages in lineage commitment.
CD4 engagement induces Fas antigen‐dependent apoptosis of T cells in vivo
Authors: Wang Zhi‐Qin , Dudhane Anita , Orlikowsky Thorsten , Clarke Karen , Li Xun , Darzynkiewicz Zbigniew , Hoffmann Michael K.
Published: March 2007

CD4 is a T lymphocyte receptor for major histocompatibility complex class II antigens. It is referred to as coreceptor because it synergizes with the T cell receptor for antigen when both receptors become engaged simultaneously. We show here in mice that when engaged by antibody independently of the T cell antigen receptor, CD4 induces T cells to undergo apoptosis. Several features of this process were identified. The expression of an intact Fas protein is a requirement for CD4‐mediated T cell death. Mice homozygous for the lpr mutation which are defective in the expression of Fas and in their ability to delete lymphocytes apoptotically fail to delete anti‐CD4‐reactive T cells. Sessile anti‐CD4‐reactive T cells leave their homing environment in lymphoid organs and modulate their cell surface molecules, e.g. CD2, CD3, CD4. A massive influx of lymphoid cells with null‐cell phenotype occurs in the blood where they begin to reexpress cell surface markers. With their arrival in the circulation, anti‐CD4‐reactive T cells develop features of DNA degradation typical of apoptosis. More than one third of the circulating lymphoid cells show apoptotic features 7–8 h after anti‐CD4 injection. Their frequency declines subsequently presumably due to their physical disintegration via shedding of apoptotic bodies and phagocytosis. Our data show that when not obliged to the activation process by the antigen receptor, CD4 can mediate deletion signals. Thus, besides functioning as coreceptor with the antigen receptor, CD4 has a function of its own in facilitating the induction of apoptosis.
The degradation product of the C5a anaphylatoxin C5adesarg retains basophil‐activating properties
Authors: Bürgi Béatrice , Brunner Thomas , Dahinden Clemens A.
Published: March 2007

The complement cleavage product C5a is a potent agonist of different leukocyte types and also has anaphylatoxic properties through the release of mediators by basophils and tissue mast cells. C5a is very rapidly degraded by serum carboxypeptidase N which cleaves the functionally important carboxy‐terminal arginine, generating C5adesarg, a chemotactic agonist with little mast cell‐activating ability. Here we show that natural human C5adesarg is still a trigger for basophil mediator release superior to other endogenous IgE‐independent agonists such as monocyte chemotactic protein (MCP)‐1, interleukin (IL)‐8, C3a and platelet‐activating factor. On a molar basis C5adesarg is only one order of magnitude less potent and about half as efficacious as C5a at inducing basophil degranulation. Priming of basophils with either IL‐3, IL‐5, granulocyte‐macrophage‐colony‐stimulating factor (GM‐CSF) or nerve growth factor (NGF) (with comparable efficacies, but different potencies: IL‐3>NGF>IL‐5>GM‐CSF) enhanced histamine release and conditioned the cells to produce large amounts of leukotriene C4 (LTC4), which is not generated by basophils exposed to C5adesarg alone. The efficacy of C5a and C5adesarg at inducing histamine and LTC4 release by primed basophils was similar. Thus, C5adesarg is a stable inducer of release of inflammatory mediators by human basophils, particularly in primed cells, and complement may, therefore, play a role in immediate‐type hypersensitivity diseases in allergic late‐phase reactions.
Presentation of an exogenous antigen by major histocompatibility complex class I molecules
Authors: Ulmer Jeffrey B. , Donnelly John J. , Liu Margaret A.
Published: March 2007

Cytotoxic T lymphocytes (CTL) generally recognize peptides derived from endogenously expressed proteins in association with nascent major histocompatibility complex (MHC) class I molecules. In contrast, peptides derived from exogenous proteins associate with MHC class II following endocytosis to an endosomal compartment. However, we have recently demonstrated that exogenous fusion proteins consisting of the binding and translocating domains of Pseudomonas exotoxin (PE) fused with CTL epitopes derived from either influenza matrix protein (PEMa) or nucleoprotein are internalized, processed, targeted to and presented by MHC class I (Donnelly et al. 1993, Proc. Natl. Acad. Sci. USA 1993. 90: 3530). PE is known to be internalized, processed in endosomes, and translocated to the cytosol during intoxication of cells. However, our present studies demonstrate that, unlike PE, PEMa does not require translocation to the cytosol to exert its effect. First, two inhibitors of PE toxicity that exert their effects at steps subsequent to endosomal processing had no effect on the sensitization of target cells for CTL‐mediated lysis by PEMa. NH4Cl, which inhibits PE by raising endosomal pH, and brefeldin A, which inhibits PE by disrupting the Golgi complex, did not inhibit sensitization of targets cells by PEMa. Second, PEMa was capable of sensitizing for lysis T2 mutant cells, which are defective in transport of peptides from the cytosol to the lumen of the endoplasmic reticulum for presentation by MHC class I. These results suggest that PEMa is proteolytically processed in endosomes, and association with MHC class I does not require nascent MHC molecules. Such a process may involve internalized MHC class I, and subsequent expression of the peptide‐MHC complexes on the cell surface would then lead to recognition by CTL.
Characterization of avian natural killer cells and their intracellular CD3 protein complex
Authors: Göbel Thomas W. F. , Chen Chen‐Lo H. , Shrimpf Jane , Grossi Carlo E. , Bernot Alain , Bucy R. Pat , Auffray Charles , Cooper Max D.
Published: March 2007

Natural killer (NK) cell activity appears to be conserved throughout vertebrate development but NK cells have only been well characterized in mammals. Candidate NK cells have been identified in the chicken as cytoplasmic CD3+ and surface T cell receptor (TCR)/CD3– (TCRO) lymphocytes that often express CD8. The fact that the TCRO cells are abundant in the embryonic spleen before T cells enter this organ allowed us to cultivate the embryonic TCRO cells using growth factors derived from activated adult lymphocytes. These TCRO cells were cytotoxic for an NK target cell line. They expressed cell surface CD8, a putative interleukin‐2 receptor, CD45 and a receptor for IgG, but did not express CD4, major histocompatibility complex class II or immunoglobulin. Biochemical analysis of the cytoplasmic CD3 antigen revealed two of the three CD3 γ, δ and ϵ homologues, and RNA transcripts for the third. The CD3 monoclonal antibody also precipitated a 32‐kDa dimer that may represent a heterodimer of different CD3 constituents. TCR α and β gene transcripts were not detected in the TCRO cells. These results indicate that the avian TCRO cell is the mammalian NK cell homologue. The shared evolutionary features of T cells and NK cells in birds and mammals support the idea that they derive from a common progenitor.
CD4‐positive/heat‐stable antigen‐positive thymocytes cause graft‐versus‐host disease across non‐major histocompatibility complex incompatibilities
Authors: Charlton Brett , Meltzer Joseph , Fathman C. Garrison
Published: March 2007

Single‐positive thymocytes are the immediate precursors of peripheral recent thymic emigrants (RTE) which develop into mature peripheral T cells. The functional ability of RTE is unclear but their state of differentiation may be relevant to the development of tolerance to peripheral “self” antigens. Since RTE are difficult to analyze, precursor CD4+/8− thymocytes were assessed in a model in vivo to determine their functional capability and their susceptibility to tolerance induction. The ability of both heat‐stable antigen‐positive (HSA+) (immature) and HSA− (mature) single‐positive thymocytes to cause graft‐versus‐host disease (GVHD) across non‐major histocompatibility complex differences was examined. Both HSA− and HSA+ CD4+/8− thymocytes from C3H mice caused lethal GVHD in AKR recipients as did CD4+ peripheral T cells in controls. Further, neonatal C3H thymocytes also caused lethal GVHD in AKR recipients. Since CD4+/8− thymocytes are the precursors of RTE, these results suggest that RTE are not susceptible to tolerance induction to “minor” antigens and may have a normal immune function in vivo. This would suggest that peripheral tolerance may be dependent upon the manner of antigen presentation rather than T cell maturity.
Anchored polymerase chain reaction based analysis of the Vβ repertoire in the non‐obese diabetic (NOD) mouse
Authors: Sarukhanu Adelaida , Gombert Jean‐Marc , Olivi Martine , Bach Jean‐François , Carnaud Claude , Garchon Henri‐Jean
Published: March 2007

We have performed extensive analyses of T cell receptor Vβ usage in the thymus, the spleen and the infiltrated islets of preclinical non‐obese diabetic (NOD) mice. A semiquantitative anchored polymerase chain reaction (An‐PCR) protocol has been developed for this purpose. The validity of the method has been first assessed by antibody staining with a panel of anti‐Vβ monoclonal antibodies (mAb). The results obtained by An‐PCR are accurate, reproducible, and in good agreement with cell surface protein staining. A strict comparison between thymus and spleen repertoires reveals no major Vβ‐specific deletion except the already reported Vβ3 deletion due to Mtv‐3. Certain Vβ such as Vβ15, 18, 20 are found with a low frequency in the spleen, but the fact that they are also scarce in the thymus probably reflects a poor availability of these genetic elements during β chain rearrangement rather than negative selection. Other Vβ, such as Vβ2, Vβ12 and Vβ14 are significantly more abundant in the spleen than in the thymus. This finding was confirmed by mAb staining for Vβ2 and Vβ14. The expansion asymetrically affects the CD4+ subset and can be traced back to the mature, single‐positive thymocyte subset, suggesting an intrathymic positive selection event. Vβ repertoires in infiltrated islets of 13‐ and 18‐week‐old, non‐diabetic mice are polymorphic. Practically all the Vβ found in the peripheral lymphoid tissues are present in the islets, in similar proportions. The major exception is Vβ12, one of the Vβ which is subject to expansion during intrathymic differentiation and which is further augmented in the islets, both at 13 and 18 weeks. This increase probably reflects further peripheral amplification of the Vβ12‐bearing subset due to encounter with the same ligand as in the thymus or with a cross‐reactive motif. Finally, the nucleotide sequencing of all the Vβ segments in usage in the NOD strain confirms the absence of allelic polymorphism of Vβ‐coding regions.
Soluble CD14 from urine copurifies with a potent inducer of cytokines
Authors: Sundan Anders , Gullstein‐Jahr Tove , Otterlei Marit , Ryan Liv , Bazilu Vladimir , Wrightu Samuel D. , Espevik Terje
Published: March 2007

Here we report that soluble CD14 isolated from the urine of nephrotic patients (uCD14) contains a potent cytokine inducing activity. CD14 derived from urine appeared to consist of two major polypeptides of about 54 and 48 kDa. In uCD14 isolated from three different nephrotic patients the cytokine‐inducing activity appeared to co‐migrate with the 48‐kDa polypeptide which upon sequencing had the same N‐terminal sequence as native CD14. Treatment of human monocytes and the human astrocytoma cell line U373 with uCD14 resulted in a strong secretion of tumor necrosis factor (TNF) and interleukin‐6, respectively. The cytokine‐inducing activity of the uCD14 preparations was unaffected by the absence of serum. This is in contrast to the activation of human monocytes and U373 cells by lipopolysaccharide (LPS) which is highly dependent on the presence of serum. The cytokine‐inducing activity was not affected by LPS‐binding protein (LBP) or polyclonal rabbit antibodies against LBP The TNF‐inducing activity of uCD14 was also heat labile in contrast to the cytokine‐inducing activity of LPS, which was relatively heat resistant. The results suggest that CD14 may exist in at least two forms of which one is involved in cytokine induction.
Characteristics of fetal thymus‐derived T cell receptor γδ intestinal intraepithelial lymphocytes
Authors: Lin Tesu , Matsuzakiu Goro , Kenaiu Hiroyuki , Kishiharau Kenji , Nabeshimau Shigeki , Fung‐Leungu Wai Ping , Maku Tak W. , Nomotof Kikuo
Published: March 2007

We have previously demonstrated that grafting of CBF1(H‐2b/d) fetal thymus (FTG) under the kidney capsule of congenitally athymic nude mice of BALB/c background (H‐2d) generates a substantial number of T cell receptor (TCR) γδ intestinal intraepithelial lymphocytes (IEL) that were of FTG origin (H‐2b+) (see accompanying report). Here we investigated the characteristics of these FTG‐derived TCR γδ IEL and compared them to the extrathymically derived TCR γδ IEL found in nude mice. Phenotypically, FTG‐derived TCR γδ IEL were similar to their extrathymically derived counterparts in that most were Thy‐1 −, CD5− and CD8αα (homodimer). Vγ and Vδ gene usage in thymus‐derived and extrathymically derived TCR γδ IEL were found to be virtually the same. Functionally, FTG‐derived TCR γδ IEL were similar to the TCR γδ IEL found in euthymic mice as both were relatively anergic to TCR cross‐linking in vitro. However, FTG‐derived TCR γδ IEL differed slightly from extrathymically derived TCR γδ IEL, which were completely nonresponsive to the same in vitro stimulation. Overall, these findings support the view that FTG‐derived and extrathymically derived TCR γδ IEL are almost indistinguishable. Lastly, we demonstrate that despite their thymic origin, development of FTG‐derived TCR γδ IEL partially takes place extrathymically; that is positive selection of FTG‐derived Vδ4 IEL occurs extrathymically. In addition, we demonstrate that the CD8 molecule is not necessary for development and homing of FTG‐derived TCR γδ IEL. This later finding suggests that the CD8αα molecule develops extrathymically for FTG‐derived CD8αα TCR γδ IEL.
In vitro characterization of major ligands for Src homology 2 domains derived from protein tyrosine kinases, from the adaptor protein SHC and from GTPase‐activating protein in Ramos B cells
Authors: Baumann Götz , Maier Daniel , Freuler Felix , Tschopp Claude , Baudisch Kristin , Wienands Jürgen
Published: March 2007

Antigen receptors of B lymphocytes transmit their activation signal to the cell interior by associating with and activation of specific non‐receptor tyrosine kinases. Most of these kinases as well as other cytoplasmic effectors contain at least one Src homology 2 (SH2) domain, known to bind tyrosine‐phosphorylated proteins. We examined the binding specificity of SH2 domains from different signaling molecules in B cells and found that each of the SH2 domains tested bound distinct subsets of stimulation‐dependent phosphoproteins in vitro. SH2 domains from Src‐like tyrosine kinases bound predominantly to the HS1 phosphoprotein. The tandem SH2 domains of the ZAP‐70 tyrosine kinase bound to phosphorylated Ig‐β but only weakly to Ig‐α. Also the SHC‐derived SH2 domain formed complexes with the tyrosine‐phosphorylated Ig‐α/β heterodimer, while the C‐ and N‐terminal SH2 domains of GTPase‐activating protein displayed completely different binding preferences. These results suggest that cytoplasmic effector molecules can be recruited to the activated B cell receptor in an SH2‐phosphotyrosine‐mediated manner. The data also provide a possible explanation for the notion that Ig‐α and Ig‐β might couple to different biochemical pathways.
Analysis of individual immunoglobulin λ light chain genes amplified from single cells is inconsistent with variable region gene conversion in germinal‐center B cell somatic mutation
Authors: Fordu John E. , McHeyzer‐Williamsu Michael G. , Lieber Michael R.
Published: March 2007

Responding B cells in specific immune responses diversify their immunoglobulin genes and are selected on their variant antigen receptors in the microenviroment of the germinal center. The patterns of mutations previously reported for immunglobulin (Ig) genes have supported mechanistic hypotheses of either error‐prone DNA synthesis or templated variable region gene conversion as the underlying mechanism in the generation of these mutations. To assess the role of gene conversion in germinal‐center somatic mutation, we chose to examine nucleotide changes in mouse λ light chain genes which arose in response to a specific antigen. Laboratory mice possess three Vλ subexons, two of which differ from one another by only seven nucleotides, making these two subexons ideal for gene conversion. In the current study, we used six‐parameter flow cytometry to isolate single λ light chain‐expressing germinal‐center B cells from two different time points in a primary immune response. We then individually amplified and sequenced individual Vλ1 genes from these single cells for mutational analysis. None of the 32 Vλ1 genes, containing a total of 40 mutations, showed evidence of gene conversion from either of the other Vλ subexons. Features such as the replacement to silent ratio of the mutations documented at the earlier time point indicate an absence of antigen‐driven selection. These data indicate that V region gene conversion does not contribute to germinal‐center somatic mutation and that gene conversion is not responsible for targeting mutation specifically to rearranged Ig genes. The biological implications are discussed.
Monoclonal antibodies to murine CD40 define two distinct functional epitopes
Authors: Heath Andrew W. , Wu Wei Wei , Howard Maureen C.
Published: March 2007

Two rat IgG2a antibodies which define distinct epitopes on murine CD40 have been generated. These antibodies specifically bind recombinant murine CD40 expressed on L cells, and the soluble extracellular domain of murine CD40 coated onto microtiter plates. Both antibodies bind B220+ but not B220 murine spleen cells, and immunoprecipitate a 45‐kDa protein from the surface of purified murine splenic B cells. These antibodies exhibit separate functional properties, consistent with the notion that they define two distinct CD40 epitopes. One of the monoclonal antibodies (designated 1C10) directly induces a specific proliferative response from mature murine B cells, up‐regulates several B cell surface antigens, and rescues immature B lymphoma cells from anti‐IgM‐induced growth arrest. The other monoclonal antibody (designated 4F11) exhibits none of these properties, but is capable of synergizing with suboptimal amounts of either anti‐IgM antibodies or the 1C10 agonistic anti‐CD40 antibody to produce an optimal proliferative response of purified small dense B cells. Furthermore, 4F11 antibody synergizes with suboptimal amounts of 1C10 antibody to rescue B lymphoma cells from anti‐IgM‐induced growth arrest. The 1C10 and 4F11 antibodies were unable to cross‐block each other's binding to recombinant CD40 expressed in L cells, providing strong support for the notion that the antibodies recognize distinct epitopes on CD40. The potential implications of two functionally distinct CD40 epitopes are discussed.