bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen‐presenting human T cells and antigen‐presenting B cells induce a similar cytokine profile in specific T cell clones
Authors: Wyss‐Coray Tony , Gallati Harold , Pracht Inge , Limat André , Mauri David , Frutig Karin , Pichler Werner J.
Published: March 2007

One of the factors that may influence the cytokine secretion profile of a T cell is the antigen‐presenting cell (APC). Since activated human T cells have been described to express major histocompatibility complex (MHC) class II molecules as well as costimulatory molecules for T cell activation, like e.g. ICAM‐1, LFA‐3 and B7, they might play a role as APC and be involved in the regulation of T‐T cell interactions. To define further the role of T cells as APC we tested their capacity to induce proliferation and cytokine production in peptide‐ or allospecific T cell clones and compared it with conventional APC, like B lymphoblasts (B‐LCL) or HTLV‐1 ‐ transformed T cells, or with non‐classical APC, like activated keratinocytes or eosinophils. CD4+, DP‐restricted T cell clones specific for a tetanus toxin peptide (amino acids 947‐967) and CD4+, DR‐restricted allospecific Tcell clones produced interleukin (IL)‐2, IL‐4, tumor necrosis factor‐α and interferon‐γ (IFN‐γ) after phorbol 12‐myristate 13‐acetate and ionomycin stimulation and a more restricted cytokine pattern after antigen stimulation. Dose‐response curves revealed that the antigen‐presenting capacity of activated, MHC class II+, B7+ T cells was comparable to the one of B‐LCL. Both APC induced the same cytokine profile in the T cell clones despite a weaker proliferative response with T cells as APC. Suboptimal stimulations resulted in a lower IFN‐γ/IL‐4 ratio. Cytokine‐treated, MHC class II+ keratinocytes and eosinophils differed in the expression of adhesion molecules and their capacity to restimulate T cell clones. The strongly ICAM‐1‐positive keratinocytes induced rather high cytokine levels. In contrast, eosinophils, which express only low densities of MHC class II and no or only low levels of adhesion molecules (B7, ICAM‐1 and LFA3), provided a reduced signal resulting in a diminished IFN‐γ/IL‐4 ratio. We conclude that non‐classical APC differ in their capacity to restimulate T cell clones, whereby the intensity of MHC class II and adhesion molecules (B7, ICAM‐1) expressed seems to determine the efficacy of this presentation.
The lymphocyte surface antigen CD38 acts as a nicotinamide adenine dinucleotide glycohydrolase in human T lymphocytes
Authors: Gelman Laurent , Deterre Philippe , Gouy Hélène , Boumsell Laurence , Debré Patrice , Bismuth Georges
Published: March 2007

The extracellular domain of the lymphocyte surface antigen CD38 has been recently shown to share a high sequence homology with a nicotinamide adenine dinucleotide (NAD+)‐specific hydrolyzing enzyme cloned from the ovotestis of the gastropod Aplysia (E. States, D. J., Walseth, T. F., Lee, H. C, Trends Biochem. Sci. 1992. 17: 495). In agreement with this finding, we present here evidence that CD38‐overexpressingT cells, such as human thymocytes and cells from the human HPB‐ALL T cell line, exhibit a NAD+‐hydrolyzing enzymatic activity present on the outer surface of the cell membrane. In contrast, T lymphocytes with relatively low levels of CD38 marker, such as the human Jurkat cell line, display a lower activity. This suggests a relationship between ecto‐NAD+ glycohydrolase activity and CD38 expression, as confirmed here when comparing wild‐type Jurkat cells and a Jurkat cell variant overexpressing the CD38 molecule. Moreover, CD38 immunoprecipitates from thymocytes behave as an authentic NAD+ glycohydrolase enzyme: it transforms NAD+ stoichiometrically into nicotinamide plus adenosine 5′‐diphosphoribose. Altogether these results strongly support the assumption that CD38 is actually a lymphocyte‐specific NAD+‐hydrolyzing enzyme, a finding that give new prospects to understand the in vivo function of this cell membrane protein.
Calcium ionophore‐induced apoptosis of human B cells is preceded by the induced expression of early response genes
Authors: Ning Zhi‐Qiang , Murphy John J.
Published: March 2007

We have investigated the effects of calcium ionophore on apoptosis of Ramos human B cells. Our results show that the calcium ionophore A23187 at denned concentrations leads to apoptosis of Ramos cells. The majority of cells (> 90%) undergo apoptosis in response to ionophore. The response is rapid and nuclear condensation and DNA degradation can be detected within 2 h after addition of ionophore. In attempts to define the changes in gene expression preceding apoptosis, we investigated the expression of a panel of early response genes in these cells after ionophore addition. We show that calcium ionophore‐induced apoptosis of Ramos cells is preceded by the induced expression of a number of early response genes. These results are consistent with calcium ionophore initiating changes in gene expression which may be important in signaling these cells to undergo apoptosis.
A novel 120‐kDa antigen shared by immature human thymocytes and long‐term‐activated T cells
Authors: Fabbi Marina , Tiso Micaela , Gangemi Rosaria M. R. , Favre Anna , Demartini Paolo , Bargellesi‐Severi Antonio
Published: March 2007

In this study we report the characterization of monoclonal antibody (mAb) 8B4/20, raised against immature human thymocytes, that identifies a novel leukocyte antigen. The molecular characterization of the antigen by immunoprecipitation and immunoblotting yields, under nonreducing conditions, a specific band of 120 kDa which, under reducing conditions, displays a slightly lower molecular mass (110 kDa). mAb 8B4/20 detects a molecule found on the majority of thymocytes with an inverted gradient of expression when compared to CD3. It appears at high density on the CD3−/low thymocytes, at reduced density on the CD3med and double‐positive thymocytes, and is absent on CD3hi and singlepositive thymocytes and on peripheral blood T cells. Immunohistochemistry on frozen sections demonstrates cortical staining of the thymic lobules. Flow cytometric analysis of the different subsets of peripheral blood mononuclear cells shows that mAb 8B4/20 detects an antigen expressed only on CD56+/CD16+ natural killer cells and on a fraction of CD14+ monocytes. T cells, B cells, erythrocytes, granulocytes and platelets are consistently negative. The expression of the molecule on tumor cell lines does not show lineage restriction. Analysis of phytohemagglutinin plus recombinant interleukin‐2‐activated peripheral blood lymphocytes shows that mAb 8B4/20 identifies an antigen expressed on CD3+ cells by week 3 of culture. Thus, it recognizes a very late activation antigen (VLA) on mature T cells. The cell distribution and the electrophoretic pattern of the molecule identified by mAb 8B4/20 is distinct from that of known CD and of integrin/VLA molecules. Its function on thymocytes is so far unknown; however, the binding of mAb 8B4/20 to tumor lines induces changes in the morphology and adhesive properties of the 8B4/20+ cells growing in suspension. We suggest that mAb 8B4/20 recognizes a molecule that may be involved in interactions between thymocytes and other thymic structures that may be relevant for the selection process.
Beneficial effect of polyclonal immunoglobulins from malaria‐infected BALB/c mice on the lupus‐like syndrome of (NZB × NZW)F1 mice
Authors: Hentati Basma , Sato Maria Notomi , Payelle‐Brogard Béice , Avrameas Stratis , Ternynck Thérèse
Published: March 2007

We previously reported that infection of BALB/c mice with the parasite Plasmodium chabaudi induces high production of natural autoantibodies. Here we demonstrate that such an infection of lupus‐prone (NZB × NZW)F1 (B/W) mice retards the development of their autoimmune disease. Survival and disease hallmarks (high‐grade proteinuria and IgG anti‐DNA antibodies) were delayed for 6 months when parasite inoculation was given at either 3 or 7 months of age, i.e. before or after the onset of the clinical symptoms. Similar beneficial effects, although less pronounced, were obtained when mice were treated with a total of 800 m̈g of IgG (P‐IgG) or IgM (P‐IgM) or 300 m̈g of cryoglobulin preparations isolated from P. chabaudi‐infected BALB/c mice while similarly prepared fractions from uninfected mice had little effect. Compared to these fractions, P‐IgG and P‐IgM contained higher levels of natural antibodies bearing the D23 idiotype characteristic of polyreactive natural autoantibodies with enhanced activity against Fab and Fc fragments of IgG.

In surviving mice, the level of anti‐DNA antibodies, particularly those of IgG1 isotype, were significantly decreased. Flow cytometric analysis of various T cell subsets showed that the number of cells expressing γδ T cell receptor (TcR) antigens which did not vary with age was not modified after P‐IgG or P‐IgM treatment. In contrast, the number of T cells expressing Vβ8.1,2, Vβ10 and Vβ14 TcR antigens, which increased with age, were significantly reduced. Taken together, these results indicate that parasite infection of mice induces the synthesis of populations of IgM and IgG natural autoantibodies with immunoregulatory properties and that these antibodies attempt, at least transitorily, to rescue a natural autoantibody network that is deficient in B/W mice.
Trypanosoma cruzi infection suppresses nuclear factors that bind to specific sites on the interleukin
Authors: Soong Lynn , Tarleton Rick L.
Published: March 2007

Interleukin‐2 (II‐2) gene expression, a critical early event during T lymphocyte activation, is severely suppressed in mice infected with the protozoan parasite Trypanosoma cruzi, the causative agent of human Chagas disease. Our previous observation that reduction of IL‐2 mRNA in T cells from T. cruzi‐infected mice is not due to an increased degradation of the mRNA suggests a repression of the IL‐2 gene at the transcriptional level. In this study, we have measured the level of nuclear factors that bind to specific sites on the IL‐2 enhancer. Splenocytes and splenic T cells from acutely infected mice show a marked decrease in the level of AP‐1, and a modest decrease in the level of NF‐xB and nuclear factor of activated T cells (NF‐AT). DNA‐binding activity of Oct‐1 was least affected in T cells from infected mice. Although the basal level of AP‐1 activity is comparable in cells from uninfected and infected mice, mitogen‐induced AP‐1 activation is absent in the cells from T. cruzi‐infected mice. Sodium deoxycholate treatment slightly enhances NF‐xB‐binding activity in splenocyte nuclear and whole‐cell extracts from infected mice, suggesting that a blockage of the activation of NF‐xB is only partially responsible for the decrease in the level of NF‐xB in T cells from T. cruzi‐infected mice. These data identify the molecular basis of IL‐2 gene regulation in T. cruzi infection and suggest that T cells are anergized as a result of the infection.
Regulated expression of p150,95 (CD11c/CD18; αX/β2) and VLA‐4 (CD49d/CD29; α4/β1) integrins during myeloid cell differentiation
Authors: Bellóan Teresa , López‐Rodríguez Cristina , Rubio Miguel A. , Jochems Gijs , Bernabeu Carmelo , Corbí Angel L.
Published: March 2007

Integrins are a family of cell surface heterodimers which mediate both cell‐cell and cell‐extracellular matrix interactions and affect cellular differentiation through their signal transduction capacity. Integrin expression is regulated during differentiation as well as by numerous growth factors and cytokines. We have analyzed the changes in p150,95 (CD11c/CD18 or αX/β2) and VLA‐4 (CD49d/CD29 or α4/β1) integrin subunits mRNA levels that take place during the myeloid differentiation of HL60 and U937 cells, and compared them to other integrins with similar functional activities. Northern blot analysis revealed that the monocytic differentiation of U937 and HL60 cells alters the αX and α4 mRNA steady‐state levels: αX mRNA is induced de novo whereas α4 mRNA decreases to undetectable levels. Both changes were dependent on the activity of protein kinase C and were also observed upon granulocytic differentiation of HL60 cells. Parallel analysis of other integrin subunits mRNA (β1, α5, β7) demonstrated that the mRNA levels for the α subunits of the fibronectin receptors α4/β1 (VLA‐4) and α5/β1 (VLA‐5) are differentially regulated during the monocytic differentiation of myeloid cell lines, and suggested that myeloid cells express a heterodimer formed by the association of β7 with an integrin α subunit distinct from α4. Nuclear transcription assays and functional analysis of the αX and α4 promoter regions demonstrated that the transcription rate of the αX gene is considerably elevated after phorbol 12‐myristate 13‐acetate treatment of U937 cells, while that of α4 is almost unaffected, suggesting that posttranscriptional mechanisms are causing the extremely low α4 mRNA levels observed in differentiated U937 cells.
Professional presentation of antigen by activated human T cells
Authors: Barnaba Vincenzo , Watts Colin , de Boer Mark , Lane Peter , Lanzavecchia Antonio
Published: March 2007

Activated human T cells express class II molecules, but their capacity to present soluble antigens and stimulate T cells has been repeatedly questioned. Two lines of evidence indicate that T cells may indeed function as professional antigen‐presenting cells. First, T cells that have been recently activated can efficiently capture, process and present tetanus toxoid to class II‐restricted T cell clones. This capacity correlates with the rate of class II synthesis. Second, activated T cell clones express high levels of B7, are powerful stimulators in mixed lymphocyte reactions, and their stumulatory capacity is inhibited by soluble CTLA4 or anti‐B7 antibody. Furthermore, expression of B7 can be detected in vivo on T cells from biopsies of patients with liver disease. Presentation of soluble antigen by activated T cells may play a role in the amplification of the specific response, and possibly in immunopathological states.
Determination of amino acids on agretopes of pigeon cytochrome c‐related peptides specifically bound to I‐A allelic products
Authors: Itoh Yasushi , Ogasawara Kazumasa , Takami Kimitaka , Gotohda Toshihiko , Naruse Hirohito , Good Robert A. , Onoé Kazunori
Published: March 2007

In our prior study it was demonstrated that residues 46 and 54 on a synthetic peptide, AEGFSYTVANKNKGIT (50V), work as an agretope (site contacts with major histocompatibility complex molecules) and residues 50 and 52 function as an epitope (site contacts with T cell receptor), when tri‐molecular complexes are formed among 50V, I‐Ab and the T cell receptor. 50V was composed of residues 43 to 58 of pigeon cytochrome c (p43‐‐58) except that the aspartic acid (D) at residue 50 was substituted by valine (V). Substitution of agretopic residues on 50V changed this I‐Ab‐binding peptide to an I‐Ak‐binding peptide, suggesting that positions 46 and 54 work as an agretope in I‐Ak‐restricted T cell responses. In the present study we examined whether residues 46 and 54 of 50V worked as agretopes in T cell responses restricted to other I‐A haplotypes. The 50V‐related peptides with phenylalanine (F) at position 46 and alanine (A) at position 54 bound tightly to I‐Ab, I‐Ad, I‐Aq and I‐As molecules and stimulated T cells most potently in mice bearing these I‐A haplotypes. In contrast, 50V‐related peptides carrying D at position 46 and A at position 54 bound most potently to I‐Ak molecules, and the peptides with arginine (R) at position 46 and A at position 54 bound most efficiently to I‐Av molecules. The present findings, thus, demonstrate that the agretopic positions on the p43‐‐58 related peptides are preserved in T cell responses restricted to each I‐A haplotype studied, and that the specific amino acids on the agretopic positions exist a priori for each I‐A allele‐specific structure.
Evidence for recombinatorial hot spots at the T cell receptor Jα locus
Authors: Rytkönen Marja , Hurwitz Julia L. , Tolonen Kirsti , Pelkonen Jukka
Published: March 2007

The complex genomic organization of the murine T cell receptor (TcR) δ‐α region has hindered detailed studies of α gene rearrangement and Jα gene usage in individual differentiating T cell precursors. We have isolated a novel set of Jα probes which, in combination with a few restriction enzyme digests, enable a reliable, simple and nearly complete analysis and location of any rearrangement at the Jα locus by conventional Southern blotting. The probes were used to analyze TcR α gene rearrangements in T cell hybridomas derived from an in vitro culture system that supports T cell differentiation of bone marrow cells. Our results indicate that Jα genes are unequally accessible for rearrangement and two hot spots for rearrangement could be demonstrated. In addition, only a restricted set of Jα genes was rearranged in each culture indicating that the slightly variable composition of factors can influence the recombinatorial accessibility of Jα genes. The hot spots for rearrangement were not only limited to T cells differentiating in vitro but could also be demonstrated among functional T cell clones based on the published sequence information from isolated TcR α gene rearrangements. The demonstration and the location of the hot spots for rearrangement in the T cell differentiation culture system opens up the possibility to study factors and mechanisms that regulate recombinatorial accessibility of TcR α genes.
Induction of B cell costimulatory function by recombinant murine CD40 ligand
Authors: Kennedy Mary K. , Mohler Kendall M. , Shanebeck Kurt D. , Baum Peter R. , Picha Kathleen S. , Otten‐Evans Carol A. , Janeway Charles A. , Grabstein Kenneth H.
Published: March 2007

T cell‐dependent regulation of B cell growth and differentiation involves an interaction between CD40, a B cell surface molecule, and the CD40 ligand (CD40L) which is expressed on activated CD4+ T cells. In the current study, we show that recombinant membrane‐bound murine CD40L induces B cells to express costimulatory function for the proliferation of CD4+ Tcells. CD40L‐ or lipopolysaccharide (LPS)‐activated, but not control‐cultured B cells were strong costimulators of anti‐CD3 or alloantigen‐dependent T cell responses. The molecular interactions responsible for the increased costimulatory functions were examined by analyzing the activated B cells for changes in the expression of two costimulatory molecules, B7 and heat‐stable antigen (HSA), as well as by the use of antagonists of B7 and HSA (CTLA4.Fc and 20C9, respectively). The expression of both B7 and HSA was enhanced on B cells activated with LPS. As observed in previous studies, the costimulatory activity of the LPS‐activated B cells was dependent on both B7 and HSA and was completely inhibited in the presence of a combination of CTLA4.Fc and 20C9. In contrast, activation of B cells with CD40L induced the expression of B7 but did not enhance the expression of HSA. In addition the costimulatory activity of the CD40L‐activated B cells was partially, but not completely, inhibited by the combination of CTLA4.Fc and 20C9. These results demonstrate that CD40L regulates costimulatory function of B cells in part by inducing the expression of B7 and suggest that CD40L‐activated B cells express an additional costimulatory activity that is not associated with LPS‐activated B cells.
Differential regulation of interleukin‐6 receptors by interleukin‐6 and interferons in multiple myeloma cell lines
Authors: Lasfar Ahmed , Wietzerbin Juana , Billard Christian
Published: March 2007

Interleukin‐6 (IL‐6) mediates pleiotropic functions through specific receptors (IL‐6R) composed of an 80‐kDa binding protein, associated with a non‐ligand binding protein (gp130) which transduces the signal. Because IL‐6 is the major tumor growth factor in multiple myeloma, we investigated the regulation of IL‐6R in two human multiple myeloma cell lines. Binding experiments with 125I‐labeled IL‐6 showed that IL‐6R were expressed at a high density on RPMI‐8226 cells (15 000 receptors/cell), but no specific binding was detected on XG‐1 cells, whose growth depends on the presence of exogenous IL‐6. However, when IL‐6 was removed from the culture medium, high‐affinity IL‐6R appeared on the surface of XG‐1 cells (5300 sites/cell). Treatment of RPMI‐8226 cells with IL‐6 reduced the number of IL‐6R without changing their affinity. This reduction was dose dependent and was not affected by acid treatment which dissociates ligand‐receptor complexes. Cross‐linking experiments showed that the formation of one IL‐6/receptor complex of 160 kDa markedly decreased upon IL‐6 treatment, while the other complex of 190 kDa became undetectable. These data provide evidence for ligand‐induced down‐regulation of membrane IL‐6R expression in myeloma cells. Treatment of RPMI‐8226 cells with interferon‐α (IFN‐α), which inhibits the growth of these cells, stimulated IL‐6R expression and increased the formation of the 160‐kDa IL‐6/receptor complex. This stimulation was specific for IFN‐α, since IFN‐γ reduced the number of IL‐6R. These data indicate that, in myeloma cells, IL‐6R are differentially regulated by IL‐6 and IFN‐α.
Identification of a signaling complex involving CD2, ζ chain and p59fyn in T lymphocytes
Authors: Gassmann Martin , Amrein Kurt E. , Flint Nicholas A. , Schraven Burkhart , Burn Paul
Published: March 2007

CD2 is a cell surface receptor molecule which has been implicated in cell‐cell adhesion and signaling functions in T lymphocytes and natural killer cells. The mechanism by which extracellular stimuli induce CD2‐regulated signal transduction events is largely unknown. However, there is increasing evidence that in cells of hematopoietic origin several receptor‐mediated signaling mechanisms involve transmembrane polypeptides related to the CD3 ζ chain and the activation of protein tyrosine kinases. We have therefore investigated the potential involvement of ζ chain and src family protein tyrosine kinases in signal transduction pathways initiated by CD2. Using in vitro kinase assays on CD2 immunoprecipitates from detergent lysates of T lymphocytes, we identified a complex consisting of CD2, ζ chain and the src family kinases p59fyn and p56lck. Furthermore, using double indirect immunofluorescence combined with capping techniques, we have revealed such complexes in viable T lymphocytes. These findings provide evidence for a multimolecular signaling complex consisting of at least CD2, ζ chain and p59fyn in T lymphocytes and suggest a critical role for this complex in the initiation of CD2‐mediated cellular activation by regulating the activation of intracellular signaling molecules.
Regulation of RAG‐1 and CD69 expression in the thymus during positive and negative selection
Authors: Brändle Daniel , Müller Stefan , Müller Christoph , Hengartner Hans , Pircher Hanspeter
Published: March 2007

Successful interaction of the T cell receptor (TCR) with major histocompatibility complex (MHC) molecules during thymic selection down‐regulates the expression of the recombination activating genes (RAG)‐1 and ‐2 in cortical thymocytes and thereby prevents further endogenous TCR α‐chain gene rearrangements (Borgulya, P., Kishi, H., Uematsu, Y. and von Boehmer, H., Cell. 1992. 69: 529‐‐537; Brändle, D., Müller, C., Rülicke, T., Hengartner, H. and Pircher, H., Proc. Natl. Acad. Sci. USA 1992. 89: 9529‐‐9533). To address the question whether down‐regulation of RAG‐1 activity represents an irreversible process we have blocked TCR‐MHC interactions of thymocytes with thymic stromal cells. Firstly, transgenic (Tg) mice expressing a virus‐specific MHC class I (H‐2Db)‐restricted TCR were injected with anti‐Db or anti‐CD8 monoclonal antibodies and RAG‐1 expression was examined by in situ hybridization on thymus sections. The results show that cortical thymocytes up‐regulated RAG‐1 expression within 24 h after antibody administration. Secondly, immature thymocytes from TCR Tg mice were released from the thymic microenvironment and cultured in vitro for 14 h in single‐cell suspension. The amount of RAG‐1 mRNA was increased sixfold in cultured cells when compared to freshly isolated thymocytes. In addition, we show that immature thymocytes from TCR transgenic mice bearing non‐selective MHC molecules (H‐2d) down‐regulated RAG‐1 expression after antigen‐induced TCR engagement. Cytofluorometric analysis further revealed that surface expression of CD69 on immature thymocytes inversely correlated with RAG‐1 expression during positive and negative selection processes.
B cells from CBA/N mice do not proliferate following ligation of CD40
Authors: Hasbold Jhagvaral , Klaus Gerry G. B.
Published: March 2007

The CBA/N mouse carries the X‐linked immunodeficiency xid, resulting in defective B cell development. B cells from these animals cannot mount antibody responses to type 2 T‐independent antigens, and do not synthesize DNA when stimulated with anti‐immunoglobulin (Ig) antibodies which are mitogenic for normal B cells. The primary antibody responses of CBA/N mice to T‐dependent antigens have also been reported to be abnormal. Here we describe the results of experiments which demonstrate that the B cells from these animals respond abnormally to ligation of CD40, a B cell surface molecule now known to play a key role during T cell‐B cell interactions, via its interaction with the counterligand (CD40L) expressed by activated T cells. Hence, xid B cells fail to proliferate when cultured with preactivated T helper type 2 (Th2)T cells (known to express CD40L), with a soluble CD40L‐CD8 fusion protein, or in response to monoclonal antibodies to CD40, even in the presence of IL‐4 and/or anti‐Ig reagents. However, xid B cells do receive abortive activation signals following ligation of CD40, as manifested by up‐regulation of class II major histocompatibility complex and CD23 antigens. Since the xid defect has now been identified as a point mutation in the protein tyrosine kinase Btk, our results point to an important role for this kinase in the downstream signaling cascade elicited in response to ligation of either surface Ig or CD40.
Rat interleukin‐2‐activated natural killer (A‐NK) cell‐mediated lysis is determined by the presence of CD18 on A‐NK cells and the absence of major histocompatibility complex class I on target cells
Authors: Smits Katinka M. , Kuppen Peter J. K. , Eggermont Alexander M. M. , Tamatani Takuya , Miyasaka Masayuki , Fleuren Gert Jan
Published: March 2007

The precise mechanism by which target cells are recognized and subsequently lysed by interleukin‐2‐activated natural killer (A‐NK) cells is poorly understood. In this study the role of major histocompatibility complex (MHC) class I and adhesion molecules in the recognition and lysis of tumor cells was investigated in a syngeneic Wag rat model. Preincubation of tumor cells with F(ab′)2 fragments of anti‐MHC class I monoclonal antibody (mAb) OX18 strongly enhanced the A‐NK cell‐mediated lysis. Also normal syngeneic cells such as T cells and A‐NK cells became highly sensitive for lysis by A‐NK cells after preincubation with mAb OX18. Two other mAb against MHC class I had no effect on lysis of target cells. These data indicate that masking of MHC class I on syngeneic tumor and normal cells by mAb OX18 is sufficient for A‐NK cells to recognize target cells as non‐self, resulting in lysis. In addition, we found that the presence of mAb against the β2 (CD18)‐integrins blocked the lysis of all tumor cell lines by A‐NK cells in 51Cr‐release assays, also when target cells were preincubated with mAb OX18. Because of the absence of CD18 on most tumor cells we concluded that a CD18‐associated integrin on A‐NK cells is essential for lysis of target cells. These results show that in this syngeneic rat model CD18 on A‐NK cells together with MHC class I on tumor cells determine A‐NK cell‐mediated lysis. Furthermore, we hypothesize that the anti‐MHC class I OX18 recognizes an epitope on rat MHC class I which is, or is very close to, the restriction element determining A‐NK cell‐mediated lysis.
HLA‐G: expression in human keratinocytes in vitro and in human skin in vivo
Authors: Ulbrecht Matthias , Rehberger Bernd , Strobel Isolde , Messer Gerald , Kind Peter , Degitz Klaus , Bieber Thomas , Weiss Elisabeth H.
Published: March 2007

Classical, polymorphic major histocompatibility complex class I molecules are expressed on most nucleated cells. They present peptides at the cell surface and, thus, enable the immune system to scan peptides for their antigenicity. The function of the other, nonclassical class I molecules in man is controversial. HLA‐G which has been shown by transfection experiments to be expressed at the cell surface, is only transcribed in placental tissue and in the fetal eye. Therefore, a role of HLA‐G in the control of rejection of the allogeneic fetus has been discussed. We found that HLA‐G expression is induced in keratinocytes by culture in vitro. Three different alternative splicing products of HLA‐G can be detected: a full length transcript, an mRNA lacking exon 3 and a transcript devoid of exon 3 and 4. Reverse transcription followed by polymerase chain reaction also revealed the presence of HLA‐G mRNA in vivo in biopsies of either diseased or healthy skin.
A genetic association between systemic lupus erythematosus and tumor necrosis factor alpha
Authors: Wilson Anthony G. , Gordon Caroline , di Giovine Francesco S. , de Vries Niek , van de Putte Leo B. A. , Emery Paul , Duff Gordon W.
Published: March 2007

We have investigated the significance of tumor necrosis factor alpha (TNF‐α) polymorphism in relation to systemic lupus erythematosus (SLE) and autoantibody production. Typing of HLA‐B, ‐DR and TNF was performed in 81 Caucasian SLE patients and 168 Caucasian controls. The presence of anti‐Ro and anti‐La antibodies was also determined in patients. The frequency of the TNF2 allele increased in SLE compared with controls [0.24 vs. 0.17, p = 0.04, odds ratio (OR) = 1.6], as did HLA‐DR3 (0.25 vs. 0.13, p < 0.01, OR = 2.3) and HLA‐B8 (0.23 vs. 0.15, p = 0.02, OR = 2). Although HLA‐DR3 showed the strongest disease association, we could not demonstrate association of HLA‐DR3 or TNF2 with SLE independently of each other. Within SLE a much stronger association of TNF2 was seen with autoantibody production: anti‐Ro antibody (0.39 vs. 0.16, p < 0.001, OR = 3.4) and anti‐La antibody (0.43 vs. 0.19, p < 0.001, OR = 3.2). When analyzed independently of each other, however, HLA‐DR3 remained significantly associated with autoantibodies, while TNF2 did not. These data suggest that on the B8‐DR3 haplotype, TNF‐α polymorphism may play a role in SLE susceptibility, but it is not primarily associated with autoantibody production.
Generation of mature T cell populations in the thymus: CD4 or CD8 down‐regulation occurs at different stages of thymocyte differentiation
Authors: Marodon Gilles , Rocha Benedita
Published: March 2007

We have studied the differentiation and repertoire selection during the maturation of CD4+CD8+ (DP) thymocytes into CD4+CD8‐ (CD4SP) and CD8+CD4‐ (CD8SP) T cells, in normal mice, mice transgenic for T cell receptor (TcR)‐αβ restricted by either class I or class II major histocompatibility (MHC), and in mice deficient in class I or class II MHC expression. Our data suggest that mature CD4 and CD8 T cells derive from different pathways of T cell differentiation in the thymus. Thus, interaction of DP thymocytes with MHC class II leads to the immediate down‐regulation of CD8, which occurs simultaneously with an increase in TcR expression; DPTcRloHSAhi thymocytes mature into a CD4+CD8lo TcRhiHSAhi intermediate population. This cell population generates CD4SP thymocytes, the majority of which are still HSAhi. In contrast, interaction with MHC class I induces the up‐regulation of TcR, which precedes the down‐regulation of CD4; DPTcRlo generate DPTcRhi thymocytes, the majority of which are the committed precursors of CD8SP cells. Further differentiation results in CD4 down‐regulation and the transition from DPTcRhi into CD8+CD4lo TcRhiHSAlo and CD8SPTcRhiHSA‐ T cells. Since down‐regulation of CD4 and CD8 occurs at different stages of thymocyte differentiation, our results do not support a stochastic/selective model of lineage commitment in the thymus.
Skin disease‐related T cells bind to endothelial selectins: expression of cutaneous lymphocyte antigen (CLA) predicts E‐selectin but not P‐selectin binding
Authors: Rossiter Heidemarie , Van Reijsen Frank , Mudde Geert C. , Kalthoff Frank , Carla A. F. M. , Bruijnzeel‐Koomen  , Picker Louis J. , Kupper Thomas S.
Published: March 2007

Cutaneous lymphocyte antigen (CLA), defined by the HECA‐452 antibody, is a cell surface glycoprotein found on a subset of T cells in peripheral blood that binds specifically to E‐selectin. This marker is present on the majority of T cells at sites of cutaneous inflammation and immune responses. Based upon such evidence, an association between T cell CLA expression and skin homing has been proposed. To understand better this relationship, we asked whether putative disease‐related, antigen‐specific T cells expressed CLA. In this study, we employed T helper type 2 (TH2) T cell clones specific for house dust mite (Dermatophagoides pteronyssinus) antigens. These cells were derived from challenged skin of an individual known to react positively to epicutaneous challenge with this agent. In this study, we show that these cloned T cells showed very high homogeneous expression of CLA (nearly 500‐fold higher than T cell clones derived from peripheral blood) and bound specifically to recombinant E‐selectin. The CLA molecule on these cells was identified not only by HECA‐452, but also by CSLEX‐1, indicating that it contained sialyl‐Lex (S‐Lex) determinants. T cells cloned under similar conditions from peripheral blood were CLA negative or low and bound poorly to E‐selectin. Surprisingly, both skin and blood clones bound comparably to P‐selectin. This binding was independent of S‐Lex or CLA expression. We conclude that in sensitized individuals, antigen‐specific T cells expressing high levels of CLA localize in skin promptly after epicutaneous challenge. This localization is likely to involve the interaction of S‐Lex determinants on the CLA molecule with E‐selectin on the dermal microvasculature. We further conclude that T cells can interact with P‐selectin on endothelium and that S‐Lex does not appear to be necessary for this interaction.
Granzyme B and perforin can be used as predictive markers of acute rejection in heart transplantation
Authors: Legros‐Maïda Sabine , Soulié Annie , Benvenuti Christophe , Wargnier Alain , Vallée Nathalie , Berthou Christian , Guillet Jacqueline , Sasportes Marilyne , Sigaux Nicole
Published: March 2007

We have investigated perforin and granzyme B expression in graft‐infiltrating lymphocytes of patients who underwent heart transplantation. Those proteins are commonly present in the cytoplasmic granules of cytotoxic T lymphocytes and are released upon effector‐target cell interaction. From 28 patients 103 endomyocardial biopsies were obtained and examined by histology and immunocytochemical analysis using relevant monoclonal antibodies. We found that “high” biopsy histological grades were associated with perforin and granzyme B expression in graft‐infiltrating lymphocytes of patients with acute severe rejection crisis. In contrast, these markers were not detected in patients without rejection or during graft stabilization. Interestingly, in patients with mild rejection and “low” histological grades, two groups could be distinguished with a differential expression of the two intracytoplasmic proteins. The presence of perforin and granzyme B‐expressing cells was found to be predictive of rapid progression to severe rejection, so that this situation required additional treatment; in contrast, their absence seemed to correlate with a good graft outcome without additional treatment. Moreover, perforin and granzyme B expression seemed to be down‐regulated by immunosuppressive drugs, which coincided with graft stabilization. In conclusion, our data suggest that detection of granzyme B and perforin in graft‐infiltrating lymphocytes might be helpful for routinely monitoring heart transplant patients.
Early events in the assembly of major histocompatibility complex class II heterotrimers from their free subunits
Authors: Nijenhuis Marga , Neefjes Jacques
Published: March 2007

Endogenous antigen presentation by major histocompatibility complex class II molecules can be understood if class II αβ heterodimers bind peptide in the endoplasmic reticulum (ER) before they associate with the invariant chain (Ii). We have studied the assembly of class II molecules from the free α, β and Ii subunits to examine the existence of a class II αβ heterotrimer as an intermediate in the assembly of class II αβIi heterotrimers in the ER. In human kidney cell transfectants, the free class II α and β subunits and the class II αβ heterodimer are retained in the ER by association with the chaperonin immunoglobulin binding protein (BiP) and Ii is retained through its cytoplasmic tail. Co‐expression of Ii results in release of BiP from class II αβ complexes and exit of class II αβIi heterotrimers from the ER. We show that the cytoplasmic tail and the transmembrane region of the class II α and β chain is not essential for proper assembly of the class II αβ heterodimer. We followed assembly of the class II αβIi heterotrimers in wild‐type cells. The class II subunits assemble post‐translationally. No class II αβ heterodimers could be isolated as intermediates in the formation of class II αβIi heterotrimers, suggesting that peptide binding by class II molecules in the ER is necessarily inefficient.
Discrete early pro‐B and pre‐B stages in normal human bone marrow as defined by surface pseudo‐light chain expression
Authors: Guelpa‐Fonlupt Valérie , Tonnelle Cécile , Blaise Didier , Fougereau Michel , Fumoux Francis
Published: March 2007

Vpre‐B and δ‐like genes are selectively expressed in B cell precursors and encode polypeptide chains associated in a m̈‐pseudo light chain (m̈‐PSL) complex which is thought to regulate some early steps of B cell differentiation. We have generated anti‐Vpre‐B monoclonal antibodies which allowed us to identify different steps of differentiation from the pro‐B to the immature B cells by following surface expression of Vpre‐B, m̈ and light chains in normal adult human bone marrow. Already present at the surface of a small fraction of B cell progenitors (CD34+/CD19+) the Vpre‐B molecule was consistently found coexpressed with CD19 and was also found with the sequentially occurring CD10, CD20, CD21, CD22 and CD5 markers. Three discrete cell types were identified: (i) a subpopulation expressing Vpre‐B without m̈ and which represented an early stage of differentiation, (ii) a minor subpopulation co‐expressing Vpre‐B and m̈ without the conventional light chains and (iii) a major subpopulation co‐expressing Vpre‐B, m̈ and χ or δ chains, considered an intermediate pre‐B/B stage. The presence of the PSL chain in various cell subpopulations, in possible association with discrete molecules and/or different contexts, suggests its involvement at several steps of early B cell differentiation.
Cytokines induce lymphocyte migration in vitro by direct, receptor‐specific mechanisms
Authors: Pleass Robert , Camp Richard
Published: March 2007

Several cytokines have been found to induce human peripheral blood lymphocyte (PBL) migration in vitro. The mechanisms involved are unclear, therefore experiments were carried out to determine whether PBL migration in response to selected cytokines is due to a direct effect, or to the generation of nonspecific secondary mediators, and whether migration is receptor specific. Purified human PBL were incubated with the lymphocyte chemotactic cytokines interleukin (IL)‐1α and IL‐8, followed after 30‐‐60 min by addition of specific neutralizing monoclonal antibody. Supernatants of these mixtures were shown by subsequent assay to be devoid of PBL attractant activity, whereas positive control supernatants containing no antibody induced dilution‐related migration. Addition of antibody to the high affinity IL‐2 receptor abolished the potent attractant effect of IL‐2 on PBL, but had little effect on responses to IL‐1α and IL‐8. These results demonstrate that the in vitro locomotor responses of human PBL to selected cytokines are due to direct, receptor‐specific effects and are not dependent upon the generation of secondary mediator(s).
Development and proliferation of lymphocytes in mice deficient for both interleukins‐2 and ‐4
Authors: Sadlack Benjamin , Kühn Ralf , Schorle Hubert , Rajewsky Klaus , Müller Werner , Horak Ivan
Published: March 2007

Interleukin (IL)‐2 and IL‐4 are considered as important regulators of growth and differentiation of lymphocytes. We report that in mice made deficient for both IL‐2 and IL‐4 by gene targeting all major T cell subsets and B cells were normal, indicating that IL‐2 and IL‐4 are not essential for development of the immune system. Paradoxically, proliferation of T cells was increased in both IL‐2‐ and IL‐4‐deficient homozygous mice.
Tolerance induction as a multi‐step process
Authors: Schönrich Günther , Alferink Judith , Klevenz Alexandra , Küblbeck Günter , Hämmerling Günter J. , Arnold Bernd , Auphan Nathalie , Schmitt‐Verhulst Anne‐Marie
Published: March 2007

Tolerant T cells are characterized by their partial or full resistance to activation by antigen. We investigated whether tolerant T cells were still receptive to further tolerogenic signals. T cells expressing a transgenic T cell receptor (TCR) specific for the major histocompatibility complex (MHC) class I molecule Kb were deleted in mice carrying Kb but not in mice expressing the mutant Kb‐molecule Kbm1 [TCR (H‐2bm1 × k) mice]. These T cells were tolerant in vivo but could be activated in vitro by the Kb antigen. This in vitro reactivity was abolished after the tolerant T cells encountered Kb‐positive cells that had been intravenously injected. Furthermore, in TCR (H‐2bm1 × k), mice expressing Kb only on hepatocytes, no T lymphocytes bearing the transgenic TCR could be found in the periphery, indicating that the additional contact with Kb on hepatocytes led to deletion of the tolerant T cells. These findings demonstrate that tolerance induction can be a multi‐step process.
The envelope glycoprotein of HIV‐1 gp120 and human complement protein C1q bind to the same peptides derived from three different regions of gp41, the transmembrane glycoprotein of HIV‐1, and share antigenic homology
Authors: Stoiber Heribert , Ebenbichler Christoph , Dierich Manfred P. , Thiele Nicole M. , Arlaud Gerard J.
Published: March 2007

Gp41, the transmembrane glycoprotein of HIV‐1, has been shown to be non‐covalently associated with gp120. We have shown that it also binds human C1q. To analyze the interaction site(s) of gp41 with these two molecules, we established an enzyme‐linked immunosorbent assay (ELISA) system using recombinant soluble gp41 [amino acids (aa) 539–684] and peptides thereof. In the cell‐external part of gp41 three sites (aa 526–538, aa 590–613 and aa 625–655) were found to bind both gp120 and C1q. That gp120 and C1q use the same sites was evidenced by the fact that these proteins competed with each other for the same sites in recombinant soluble gp41 and gp41 peptides. It could be demonstrated by ELISA, that rabbit antibodies against human C1q recognized gp120, and rabbit antibodies against gp120 cross‐reacted with C1q. Rabbit anti‐gp120, HIV‐1‐positive human sera and anti‐gp120 obtained from such sera agglutinated sensitized sheep erythrocytes with human C1q (EAC1q).

These data suggest that in addition to functional homology between C1q and gp120 structural homology between these two molecules exists. This molecular mimicry might become the basis for immunologically relevant autoimmune phenomena.
Neuronal interferon‐γ immunoreactive molecule: Bioactivities and purification
Authors: Olsson Tomas , Bakhiet Moiz , Höjeberg Bo , Ljungdahl Åke , Kelic Sofija , Edlund Conny , Kristensson Krister , Van Der Meide Peter H.
Published: March 2007

An interferon (IFN)‐γ immunoreactive molecule, localized to small neurons in peripheral sensory ganglia (N‐IFN‐γ), has been detected with two mouse monoclonal antibodies (DB1 and DB16) directed against different epitopes of rat IFN‐γ. To define N‐IFN‐γ with regard to its protein characteristics and bioactivities, DB1 and DB16 were used to purify N‐IFN‐γ from rat trigeminal ganglia in a two‐step sequential antibody‐affinity procedure. Sodium dodecylsulfate polyacrylamide gel electrophoresis (PAGE) and silver staining of purified N‐IFN‐γ displayed three bands with an approximate molecular mass of 66, 62 and 54 kDa. The N‐IFN‐γ bioactivity was confined to the protein stained on gel when native material was run on PAGE. Biological effects of pure N‐IFN‐γ were examined and compared with those of lymphocyte‐derived recombinant IFN‐γ. N‐IFN‐γ had antiviral effects in vitro and induced major histocompatibility complex class I and II antigens on macrophages and in cells in skeletal muscle cell cultures. N‐IFN‐γ also stimulated myoblast proliferation and affected cholinergic receptor distribution on myotubes similar to recombinant IFN‐γ. Both molecules potently stimulated Trypanosoma brucei brucei growth. These data suggest that, although N‐IFN‐γ is a protein distinct from lymphocyte‐derived IFN‐γ, the two molecules have enough structural similarities to allow for antibody recognition of at least two epitopes, and action on similar target structures on both parasite and mammalian cells.
Murine epidermal Langerhans cells and splenic dendritic cells present tumor‐associated antigens to primed T cells
Authors: Cohen Philip J. , Cohen Peter A. , Rosenberg Steven A. , Mulé James J. , Katz Stephen I.
Published: March 2007

We examined the ability of epidermal Langerhans cells and splenic dendritic cells to present tumor‐associated antigens (Ag) to immune T cells. Methylcholanthrene (MCA)‐induced subcutaneous fibrosarcomas derived from C57BL/6 mice were used as tumor models. Our data demonstrate that both murine Langerhans cells and splenic dendritic cells have the capacity to present tumor‐associated Ag to primed T cells. We found that variously treated tumor preparations (irradiated viable tumor cells, irradiated frozen‐stored tumor cells, mitomycin C‐treated viable tumor cells, and snap freeze‐thawed tumor cell lysates) can be utilized for tumor Ag‐pulsing. Primed CD4+ T cells demonstrated in vitro specificity towards their respective tumors and did not cross‐react to other syngeneic MCA‐induced or non‐MCA‐induced tumors. The T cell proliferative response critically depended on the presence of immune CD4+ T cells. We discuss the implications of these findings for the adoptive immunotherapy of cancer using immune CD4+ T cells.
Identification of an HLA‐DQ2 peptide binding motif and HLA‐DPw3‐bound self‐peptide by pool sequencing
Authors: Verreck Frank A. W. , van de Poel Anja , Termijtelen Annemarie , Drijfhout Jan‐Wouter , Koning Frits , Amons Reinout
Published: March 2007

Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele‐specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II‐bound peptides and found that HLA‐DQ2‐eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA‐DPw3/DPw4‐eluted peptide pool yielded a sequence matching an epitope from the endogeneous enzyme glyceraldehyde‐3‐phosphate dehydrogenase. This self‐peptide and a partially identical, known allo‐epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II‐eluted peptides.