bluuebunny/crossref_metadata_2025 huggingface dataset:
Viral superantigen‐induced hyporesponsiveness of T cells and polyclonal B cell activation in HIV‐1 infection
Authors: Scott‐Algara Daniel , Lafon Monique , Vuillier Françoise , Pialoux Gilles , Dauguet Charles , Dighiero Guillaume
Published: March 2007

The mechanisms of CD4 depletion and hyporesponsiveness during human immunodeficiency virus (HIV) infection are still unknown. Given the ability of superantigens to stimulate a higher number of lymphocytes than conventional antigens, they may play a major role in this process. Recently, a novel superantigen, the rabies virus nucleocapsid (NC), was described in humans. In the present work, we tested the responses of peripheral blood lymphocytes from asymptomatic HIV‐infected patients to this superantigen. In contrast to its effect in normal controls, NC failed to expand T cells from HIV‐infected individuals expressing the Vβ8 family, and induced a strong decrease in the response to CD3 activation. This absence of response was not the consequence of programmed cell death, and was explained by an anergic state induced by the superantigen. NC superantigen was also able to induce polyclonal activation of B cells, as measured by the secretion of anti‐HIV antibodies and autoantibodies. Moreover, Vβ8 depletion experiments showed that induction of autoantibody secretion was Vβ8 dependent, whereas secretion of HIV‐1 antibody was not. Interleukin secretion studies showed that NC was able to induce high levels of interleukin‐4 and interleukin‐10. Taken together, our results suggest a role for exogenous viral superantigens such as NC in the induction of T cell hyporesponsiveness and polyclonal B cell activation during HIV infection. The induction of a Th2 response and the role of these superantigens in the immunopathogenesis of acquired immunodeficiency syndrome are discussed.
Tyrosine kinase activity associated with the CD7 antigen: correlation with regulation of T cell integrin function
Authors: Chan Anissa S. H. , Reynolds Pamela J. , Shimizu Yoji
Published: March 2007

Rapid up‐regulation of the functional activity of integrin adhesion receptors is a hallmark of T cell activation. Monoclonal antibody engagement of the CD7 antigen on human T cells results in an increase in β1 and β2 integrin‐mediated adhesion within minutes. This suggests that CD7 is capable of transducing intracellular signals, and is consistent with other indirect studies implicating CD7 as a signaling receptor on T cells. In this report, we have explored the intracellular mechanism by which CD7 modulates integrin functional activity. First, CD7‐mediated up‐regulation of T cell adhesion was found to be unique when compared to phorbol ester stimulation and CD3/T cell receptor cross‐linking, based on differences in the kinetics of activation‐dependent integrin‐mediated adhesion and lack of increase in CD2 functional activity. Second, up‐regulation of integrin activity mediated by CD7 cross‐linking was completely inhibited by the tyrosine kinase inhibitor herbimycin A. Third, antiphosphotyrosine immunoblotting demonstrated that antibody engagement of CD7 results in a rapid but transient increase in tyrosine phosphorylation in human T cells. Finally, CD7 immunoprecipitates contain in vitro kinase activity, as demonstrated by phosphorylation of a predominant band of 80 kDa and multiple other bands. Phosphoamino acid analysis of the 80‐kDa substrate revealed phosphorylation on tyrosine as well as serine and threonine residues. Together, our results suggest that CD7 is associated with tyrosine kinase activity and that this tyrosine kinase activity correlates with the ability of CD7 to regulate T cell integrin functional activity.
Functional cell surface expression by a recombinant single‐chain class I major histocompatibility complex molecule with a cis‐active β2‐microglobulin domain
Authors: Lee Li , McHugh Louise , Ribaudo Randall K. , Kozlowski Steven , Margulies David H. , Mage Michael G.
Published: March 2007

As a preliminary step towards the use of cell surface single‐chain class I major histocompatibility complex (MHC) molecules as T cell immunogens, we have engineered a recombinant gene encoding a full‐length cell surface single‐chain version of the H‐2Dd class I MHC molecule (SCβDdm) which has β2‐microglobulin (β2m) covalently linked to the amino terminus of a full‐length H‐2Dd heavy chain via a peptide spacer. The single‐chain protein is correctly folded and stably expressed on the surface of transfected L cells. It can present an antigenic peptide to an H‐2Dd‐restricted antigen‐specific T cell hybridoma. When expressed in peptide‐transport‐deficient cells, SCβDdm can be stabilized and pulsed for antigen presentation by incubation with extracellular peptide at 27° or 37 °C, allowing the preparation of cells with single‐chain molecules that are loaded with a single chosen antigenic peptide. SCβDdm can be stably expressed in β2m‐negative cells, showing that the single‐chain molecule uses its own β2m domain to achieve correct folding and surface expression. Furthermore, the β2m domain of SCβDdm, unlike transfected free β2m, does not rescue surface expression of endogenous class I MHC in the β2m‐negative cells. This strict cis activity of the β2m domain of SCβDdm makes possible the investigation of class I MHC function in cells, and potentially in animals, that express but a single type of class I MHC molecule.
Interaction of HIV gp120 and anti‐CD4 antibodies with the CD4 molecule on human CD4+ T cells inhibits the binding activity of NF‐AT, NF‐χB and AP‐1, three nuclear factors regulating interleukin‐2 gene enhancer activity
Authors: Jabado Nada , Le Deist Françoise , Fischer Alain , Hivroz Claire
Published: March 2007

The membrane glycoprotein CD4 is required for optimal antigen‐mediated activation of CD4+ T cells restricted by class II molecules of the major histocompatibility complex (MHC). CD4 cross‐linking by anti‐CD4 antibodies or binding by human immunodeficiency virus (HIV) gp120 has been shown to inhibit antigen‐dependent and ‐independent T cell activation, abrogating T cell proliferation, IL‐2 synthesis and the increase in the intracellular calcium concentration. The molecular basis of these opposing phenomena is ill‐defined. To characterize further the inhibitory role of the CD4 molecule, we investigated the effects of CD4 ligands on the transcription factors regulating the IL‐2 gene enhancer and IL‐2 synthesis. We first confirmed that pre‐treatment of peripheral human CD4+ T lymphocytes by CD4 ligands, HIV gp120 or anti‐CD4 monoclonal antibodies inhibited IL‐2 production and cell proliferation, which was normally induced by an anti‐CD3 antibody (UCHT1) plus a protein kinase C activator (PMA). Moreover, these CD4 ligands inhibited the proliferation and synthesis of IL‐2 induced by activators bypassing membrane events, i.e. PMA and calcium ionophore, pointing to an active signaling pathway triggered by the CD4 molecule. Gp120 and anti‐CD4 antibodies induced a specific, significant decrease in the binding activity of NF‐AT, NF‐χB and AP‐1, three transcription factors regulating IL‐2 gene enhancer activity, as demonstrated by electrophoretic mobility shift assays. Inhibition was similarly observed following cell activation by activators involving membrane events and those bypassing them. These results strongly suggest that the inhibition mediated by cross‐linking of the CD4 molecule is at least partly due to a negative signal down‐regulating the availability of nuclear factors necessary for the regulation of IL‐2 gene transcription.
Activation of 70‐kDa S6 kinase, induced by the cytokines interleukin‐3 and erythropoietin and inhibited by rapamycin, is not an absolute requirement for cell proliferation
Authors: Calvo Victor , Wood Michael , Gjertson Carl , Vik Terry , Bierer Barbara E.
Published: March 2007

The cytokines interleukin (IL)‐3 and erythropoietin (EPO) are critical regulators of the proliferation and differentiation of cells of the hematopoietic system, but their intracellular mechanisms of action are not fully understood. Binding of IL‐3 to the IL‐3 receptor (IL‐3R) and binding of EPO to the EPOR both induce changes in intracellular tyrosine and serine/threonine phosphorylation; the phosphorylation of a number of polypeptides appears to be a shared response upon cytokine stimulation. We have previously shown that binding of IL‐2 to the IL‐2R activates the 70‐kDa (p70) S6 kinase, a serine/threonine kinase whose activity is regulated by serine/threonine phosphorylation; the immunosuppressant rapamycin inhibits IL‐2‐dependent proliferation and IL‐2‐triggered activation of p70 S6 kinase. We, therefore, sought to examine whether induction of p70 S6 kinase activity is a conserved response upon cytokine triggering, and whether this activity is essential for cell proliferation. Proliferation of the IL‐3‐dependent pro‐B cell line Ba/F3 transfected with the EPOR (Ba/F3‐EPOR) can be supported by either IL‐3 or EPO. In this cell line, both IL‐3 and EPO induced p70 S6 kinase activity; rapamycin inhibited both the IL‐3 and EPO‐induced activation of the 70‐kDa S6 kinase as well as cellular proliferation. Thus, p70 S6 kinase activation appears to be a common intermediate triggered by the stimulation of IL‐3, EPO, and IL‐2 receptors. The Friend spleen focus‐forming virus gp55 renders the EPOR constitutively active, and confers growth factor independence on cells expressing EPOR. Ba/F3‐EPOR cotransfected with gp55 (Ba/F3‐EpoRgp55) and the erythroleukemia cell line MEL, which also expresses both the EPOR and gp55, were analyzed. Rapamycin inhibited the activation of p70 S6 kinase in both cell lines. However, rapamycin inhibited proliferation of Ba/F3‐EpoRgp55 but not of MEL cells despite inhibition of p70 S6 kinase activity in both cells. Thus, p70 S6 kinase activation is not an absolute requirement for cell proliferation. These results are discussed in relation to the role of the activation of the 70‐kDa S6 kinase activation pathway in the regulation of cell cycle progression.
Properties of mouse CD40: the role of homotypic adhesion in the activation of B cells via CD40
Authors: Klaus Gerry G. B. , Holman Mary , Hasbold Jhagvaral
Published: March 2007

Stimulation of human B cells via CD40 is known to induce their homotypic aggregation. We show here that anti‐mouse CD40 monoclonal antibodies (mAb) also induce B cells to form large, spherical, extremely stable clusters. This clustering is markedly enhanced by co‐stimulation with either interleukin‐4 (IL‐4) or anti‐immunoglobulin (Ig). The aggregation is slow in onset, and is largely (but not completely) abrogated by anti‐LFA‐1 mAb, but not by mAb directed against other potentially important adhesion molecules on B cells. Anti‐LFA‐1 mAb also partially suppressed DNA synthesis induced by anti‐CD40, but not by other B cell mitogens, suggesting that clustering is an important component of B cell activation via CD40. This concept is supported by analyses of the phenotype of clustered B cells: the cells within clusters express higher levels of various activation markers, and also more of them are in cell cycle than non‐clustered cells. These results therefore suggest that CD40 stimulation may either induce B cells to secrete soluble factors which act in an autocrine way to promote Bcell activation, or that clustering generates cell contact‐mediated signals which are important in the activation cascade.
CD28 signal transduction: tyrosine phosphorylation and receptor association of phosphoinositide‐3 kinase correlate with Ca2+‐independent costimulatory activity
Authors: Lu Yiling , Phillips Catherine A. , Bjorndahl Jay M. , Trevillyan James M.
Published: March 2007

The interaction of CD28 with its counter‐receptor, B7, induces a cosignal in T cells required to prevent clonal anergy and to promote antigen‐dependent interleukin‐2 production. The molecular basis of the CD28 cosignal is not well understood but involves the activation of protein tyrosine kinase(s) (PTK). In this report we demonstrate that CD28 cross‐linking on Jurkat T leukemic cells causes the activation of at least two PTK pathways. A CD28‐induced, p56lck kinase‐independent pathway causes tyrosine‐phosphorylation of a 110‐kDa substrate while recruitment of p56lck kinase activity is apparently required for CD28‐induced tyrosine‐phosphorylation of 97‐ and 68‐kDa substrates as well as CD28‐induced increases in intracellular calcium. The tyrosine phosphorylation of p110, but not p97 or p68, correlated with CD28 calcium‐independent costimulatory activity. The pp110 molecule was tentatively identified as the catalytic subunit of phosphoinositide (PI)‐3 kinase based upon its coimmunoprecipitation with the p85 regulatory subunit of PI‐3 kinase. PI‐3 kinase protein and catalytic activity were found complexed with the CD28 receptor if the receptor was “activated” by cross‐linking on the surface of intact cells prior to detergent solubilization. The kinetics of association of PI‐3 kinase with the “activated” CD28 receptor was rapid, occurring within 30 s of receptor cross‐linking and was stable for at least 30 min. Analysis of the CD28 cytoplasmic peptide sequence revealed a putative PI‐3 kinase src homology 2 binding motif and CD28 tyrosine phosphorylation site, DYMNM. Tyrosine phosphorylation of CD28 was detected in pervanadate‐treated Jurkat B2.7 cells, but not untreated cells. Pervanadate‐induced tyrosine phosphorylation of CD28 correlated with receptor association of PI‐3 kinase in the absence of CD28 cross‐linking, suggesting that CD28 association with PI‐3 kinase uses a tyrosine phosphorylation‐dependent mechanism. These data provide a model for CD28 signal transduction and support a role for PI‐3 kinase in mediating the CD28 calcium‐independent, cyclosporin A‐insensitive costimulatory signal.
Surface immunoglobulins mediate efficient transport of antigen to lysosomal compartments resulting in enhanced specific antigen presentation by B cells
Authors: Liu Ko‐Jiunn , Parikh Vandana S. , Tucker Philip W. , Kim Byung S.
Published: March 2007

A BCL1 immunoglobulin (Ig) transfectant, expressing wild‐type surface (s)IgM with the TEPC‐15 idiotype (T15‐Id) and anti‐phosphorylcholine (PC) specificity, was previously shown to present PC‐conjugated hen egg‐white lysozyme (PC‐HEL) to a HEL‐specific T cell hybridoma at a lower antigen (Ag) concentration than that required for native HEL. Two variant Ig transfectants, expressing T15‐Id sIgM with substitutions either in the entire spacer, transmembrane (TM) domain and cytoplasmic tail (B186 variant) or in the NH2‐terminal third of TM domain only (TM2 variant), failed to display this sIgM‐mediated, enhanced presentation of PC‐HEL at low concentrations. However, prolonged treatment with anti‐T15‐Id monoclonal antibody (mAb) led to a reduction of surface expression of the T15‐Id sIgM in the wild‐type and TM2 variant, but not in the B186 variant sIgM transfectants. Treatment with anti‐T15‐Id mAb also resulted in an increased intracellular accumulation of T15‐Id sIgM in the wild‐type transfectant, but not in the B186 variant. Subcellular fractionation analysis revealed that the ligands bound to the T15‐Id sIgM are not efficiently transported to the dense lysosomal compartments in both B186 and TM2 transfectants, as compared to the wild‐type sIgM transfectant. A significant increase in tyrosine phosphorylation after cross‐linking of the T15‐Id sIgM was observed only in the wild‐type sIgM transfectant. These results suggest that, while the NH2‐terminal third of the TM region is not involved in the process responsible for the ligand‐induced reduction of surface expression of sIgM, it appears to be essential for subsequent transport of sIgM/ligand complexes to the lysosomal compartments, as well as efficient activation of tyrosine kinases. These results strongly suggest that sIg‐mediated enhancement of specific antigen presentation reflects the ability of sIg to efficiently transport antigen to the lysosomal compartments, and possibly the activation of protein tyrosine kinases.
T cell regulation of collagen‐induced arthritis in mice. III. Is T cell vaccination a valuable therapy?
Authors: Chiocchia Gilles , Manoury‐Schwartz Bénédicte , Boissier Marie‐Christophe , Gahery Hanne , Marche Patrice N. , Fournier Catherine
Published: March 2007

Since T cells play a critical role in collagen‐induced arthritis (CIA), CD4+ T cell hybridomas were derived from DBA/1 mice immunized with bovine type II collagen (CII). The hybrid clones selected were Thy‐1‐2+, CD4+, CD8−, T cell receptor (TcR) αβ+ and produced interleukin‐2 in response to CII peptides presented by I‐Aq molecules. The clones were collagen type‐specific and recognized CII from many species except the mouse. More precisely, the reactivity was directed against the immunodominant cyanogen bromide‐cleaved fragment CB11(II). Analysis of the TcR carried by the T cell hybridomas showed that they used identical Vα and Jα (VαBMB, Jα20) gene segments and two distinct Vβ (Vβ1 and Vβ4) associated with the Jβ2.5 gene segment. Interestingly, the junctional regions were highly conserved in structure and length. These findings may indicate a strong in vivo selection by the antigen for a particular combination of both α and β chains of the TcR. Inoculation of irradiated anti‐CII T cell hybrids into DBA/1 mice, before priming with CII, altered the course of the disease resulting in either a long‐lasting suppression or an exacerbation of CIA whereas a control CD4+ hybridoma with an unrelated specificity did not influence the development of arthritis. However, the regulatory effect of anti‐CII T cell clones was unpredictable, suggesting that the TcR structure may not solely account for the modulation of CIA and that T cell vaccination is not a reliable method for inducing suppression of CIA.
The mixotope: a combinatorial peptide library as a T cell and B cell immunogen
Authors: Estaquier Jérǒme , Gras‐Masse Hélène , Boutillon Christophe , Ameisen Jean‐Claude , Capron André , Tartar André , Auriault Claude
Published: March 2007

We report a new approach in peptide vaccine strategy based on combinatorial synthesis. A library of 7.5 × 105 related peptides, termed mixotope, was derived from the sequence of the third hypervariable domain (V3 loop) of the human immunodeficiency virus (HIV) envelope protein.

This preparation induced a strong immune response in all syngeneic and outbred rodents tested. The response directed against the mixotope included antibodies, CD4+ T helper cells (TH1 and TH2) and CD8+ T cells. In rodents immunized with the mixotope, the T cell response directed against individual V3 peptide sequences (BRU, MN, RF, SF2, and ELI) as measured by T cell proliferation and interleukin (IL)‐2 production, was found to be major histocompatibility complex haplotype‐dependent. However, additional experiments performed in mice indicated that selectivity was less restrictive when using IL‐3 secretion to explore T cell activation.

This combinatorial antigen could be considered as a series of agretopic motifs framing a multiplicity of closely related epitopes for T cell recognition and able to elicit a T cell and B cell repertoire. This new construct may therefore provide a basis for the design of future vaccine strategies.
Distinct structural and functional epitopes of the αEβ7 integrin
Authors: Russell Gary J. , Parker Christina M. , Cepek Karyn L. , Mandelbrot Didier A. , Sood Arvind , Mizoguchi Emiko , Ebert Ellen C. , Brenner Michael B. , Bhan Atul K.
Published: March 2007

Intestinal intraepithelial lymphocytes (iIEL) are predominantly CD3+, CD8+ T lymphocytes located above or adjacent to the mucosal basement membrane. Although they are positioned to interact with intercellular luminal antigen or with enterocytes, the function of iIEL remains unknown. Most (> 85%) of the iIEL express the αEβ7 integrin which appears to be involved in the adhesion of lymphocytes to epithelial cells. We report the characterization of three monoclonal antibodies (mAb) termed αE7‐1, αE7‐2, and αE7‐3, that react with the αEβ7 integrin recognized by the previously described mAb HML‐1 as demonstrated by identical sodium dodecyl sulfate – polyacrylamide gel electrophoresis mobility and charge. Flow cytometric analysis of antibody cross‐blocking indicated that these mAb recognize distinct epitopes of αEβ7. While all of the mAb were capable of blocking the adhesion of cultured iIEL to a breast epithelial cell line, only HML‐1 and αE7‐1 (which recognize an identical or closely related epitope) were co‐stimulatory with suboptimal concentrations of anti‐CD3 mAb in inducing proliferation of cultured iIEL. Thus, these mAb appear to recognize functionally distinct epitopes of αEβ7 and will be useful to study relationships between the structure and function of this integrin.
Genetic control of the human Vβ13.2 T cell repertoire: importance of allelic variation outside the coding regions of the TCRBV13S2 gene
Authors: Kay Richard A. , Snowden Neil , Hajeer Ali H. , Boylston Art W. , Ollier William E. R.
Published: March 2007

In humans, the T cell repertoire is influenced by HLA, T cell receptor null alleles and antigen. Here, we describe a novel mechanism, independent of superantigen or T cell receptor structure which influences the T cell repertoire in a Vβ‐dependent manner. We have identified a biallelic locus, the TCRBV13S2 T cell receptor gene, where allelic differences predominate in the non‐coding regions including transitions, transversions and frameshift deletions. The expressed protein is non‐polymorphic at this locus. The TCRBV13S2 genotype profoundly influences the circulating levels of Vβ13.2 CD4 T cells but does not affect T cell receptor expression or function.
Selective regulation of ICAM‐1 and major histocompatibility complex class I and II molecule expression on epidermal Langerhans cells by some of the cytokines released by keratinocytes and T cells
Authors: Chang Chung‐Hsing , Furue Masutaka , Tamaki Kunihiko
Published: March 2007

Epidermal Langerhans cells (LC) are major histocompatibility complex (MHC) class II (Ia)‐positive dendritic cells that act as potent antigen‐presenting or accessory cells for primary and secondary T cell‐dependent immune responses. Recent studies have disclosed that the morphological, functional, and phenotypic characteristics of LC are variably and drastically modulated by external stimuli both in vivo and in vitro. However, little is known of the biological significance of diverse cytokines in regulating the surface molecules of LC. To determine the regulatory properties of ICAM‐1, Ia, and MHC class I (H‐2K) molecules in LC, we have examined the effects of interleukin (IL)‐1α, IL‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐7, IL‐10, interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α), and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) on the expression of these molecules. Among the cytokines examined, IFN‐γ markedly and reproducibly up‐regulates the expression of H‐2K, but not ICAM‐1, in Ia+ LC in a time‐ and dose‐dependent manner. TNF‐α consistently up‐regulates the expression of ICAM‐1, but not H‐2K, in a time‐ and dose‐dependent manner. IL‐10 slightly but reproducibly inhibits the expression of ICAM‐1, but not H‐2K, in a time‐ and dose‐dependent manner. IL‐10 potently inhibits the TNF‐α‐induced ICAM‐1 up‐regulation, but not the IFN‐γ‐induced H‐2K up‐regulation. Moreover, no cytokine consistently affects the Ia expression of LC. In addition, slight enhancing effects have been observed on H‐2K expression by IL‐4, and on ICAM‐1 expression by IL‐1α, IL‐1β, or GM‐CSF. The present data suggest that the selective regulation is operative in a certain cell surface moiety of LC by various cytokines. These results further facilitate our understanding of immunobiology of LC.
CD8high(CD57+) T cells in normal, healthy individuals specifically suppress the generation of cytotoxic T lymphocytes to Epstein‐Barr virus‐transformed B cell lines
Authors: Wang Eddie Chung Yern , Lehner Paul Joseph , Graham Shek , Borysiewicz Leszek Krysztof
Published: March 2007

We have previously identified two subsets of CD8+, CD57+ lymphocytes in normal peripheral blood: i) T cells expressing high levels [CD8high(CD57+)] and ii) natural killer cells expressing low levels of surface CD8 [CD8low(CD57+)]. We investigated the cytotoxic and suppressive function of CD8high(CD57+) T lymphocytes from normal, healthy individuals using standard chromium‐release assays and limiting dilution analysis. In normal, healthy subjects, this cell subset suppressed the generation of cytotoxic T lymphocytes (CTL) to autologous, Epstein‐Barr virus (EBV)‐transformed B cell lines (BCL). Depletion of CD8high(CD57+) T lymphocytes from peripheral blood mononuclear cells (PBMC) resulted in a three‐ to sevenfold rise in CTL precursor frequency to autologous EBV‐transformed BCL, but not allogeneic PBMC or BCL by LDA. Replacement of CD8high(CD57+) T lymphocytes in limiting dilution cultures led to the dose‐dependent suppression of EBV‐specific, but not allogeneic, CTL generation. Supernatant from CD8high(CD57+) T lymphocytes cultured with autologous BCL did not exhibit suppression, suggesting that soluble factors were not responsible. As CD8high(CD57+) T lymphocytes did not, themselves, exhibit cytotoxicity against autologous BCL, removal of BCL stimulator cells in co‐culture was not the mechanism of suppression. Furthermore, while the CD8high(CD57+) T lymphocytes from healthy subjects suppressed the generation of CTL to autologous BCL, they did not suppress the cytotoxic activity of established mixed lymphocyte reactions or peptide‐specific CTL clones, as has been reported in bone marrow transplant recipients and human immunodeficiency virus patients. This suggests that CD8high(CD57+) T lymphocytes from healthy subjects suppress the generation of, rather than killing by, CTL in a contact‐dependent manner. To our knowledge, this is the first identification of a phenotypically distinct subset of human CD8+ T cells that can suppress generation of antigen‐specific major histocompatibility complex class I‐restricted CTL.
Heterogeneity in P‐glycoprotein (multidrug resistance) activity among murine peripheral T cells: Correlation with surface phenotype and effector function
Authors: Bommhardt Ursula , Cerottini Jean‐Charles , MacDonald H. Robson
Published: March 2007

P‐glycoprotein (P‐gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells. Functional P‐gly activity can be conveniently assessed microfluorometrically using the fluorescent dye rhodamine 123 (Rh123), which is an artificial substrate for the P‐gly transporter. Here we assess P‐gly activity in subsets of mouse peripheral T lymphocytes using the Rh123 efflux assay. Our data indicate that virtually all CD8+ cells extrude Rh123 efficiently, whereas only a subset of CD4+ cells exhibit P‐gly activity. Correlation of P‐gly activity in CD4+ cells with the expression of a panel of surface markers revealed that cells bearing an “activated/memory” phenotype (CD45RB−, CD44hi, CD62L−, CD25+, CD69+) were exclusively found in the fraction that can extrude Rh123. In contrast “naive” phenotype CD4+ cells (CD45RB+, CD44lo, CD62L+, CD25−, CD69−) could be further subdivided into two major subsets based on P‐gly activity. In functional studies of sorted cell populations the Rh123‐extruding subset of “naive” CD4+ cells proliferated more strongly and secreted higher levels of interleukin (IL)‐2 than its Rh123‐retaining counterpart when activated by a variety of polyclonal stimuli. Furthermore, this subset produced detectable levels of interferon (IFN)‐γ upon stimulation but no IL‐4 or IL‐10. As expected, the Rh123‐retaining “naive” subset produced only IL‐2 after stimulation, whereas the “memory” subset produced IFN‐γ, IL‐4 and IL‐10 in addition to low levels of IL‐2. Collectively, our data indicate that P‐gly activity is a novel parameter that can be used to distinguish a subset of “preactivated” CD4+ cells that would be considered as naive on the basis of their surface phenotype.
CD23/CD21 interaction is required for presentation of soluble protein antigen by lymphoblastoid B cell lines to specific CD4+ T cell clones
Authors: Grosjean Isabelle , Lachaux Alain , Bella Chantal , Aubry Jean‐Pierre , Bonnefoy Jean‐Yves , Kaiserlian Dominique
Published: March 2007

Previous studies have documented a role for membrane‐bound CD23 (the low affinity FcεRII) in presentation of alloantigens by B cells. The aim of the present study was to examine the involvement of cell surface CD23 in presentation of more conventional soluble protein antigens to T cells. We show that antibodies to CD23 and to its lymphocyte‐associated second ligand, CD21, inhibit presentation of the cow's milk allergen casein, by autologous CD23+CD21+ B‐EBV cell lines to casein‐specific HLA‐DP‐restricted CD4+ T cell clones obtained from patients with either reaginic or enterophatic forms of cow's milk protein intolerance. Maximal inhibition was achieved when the antibodies were added at the initiation of the culture. The absence of specific inhibition by an anti‐DRα monoclonal antibody (mAb) argues against a steric hindrance phenomenon impeding access of the T cell receptor to major histocompatibility complex class II molecules. Rather, anti‐CD23 and anti‐CD21 mAb‐induced inhibition of antigen presentation seems to affect at least partly, heterotypic conjugate formation through CD23/CD21 interaction. Double immunofluorescence labeling of the T cell clones and antibody inhibition of T/B conjugate formation shows that functional CD23 and CD21 molecules are induced on T cells following contact with B‐EBV cell lines. Taken together, these data indicate that CD23/CD21 interactions between T and B cells are required for presentation of soluble protein antigens by B‐EBV cell lines to specific CD4+ T cells. The potential implications of these findings for allergen‐specific T cell activation are discussed.
The murine interleukin‐2 receptor γ chain gene: Organization, chromosomal localization and expression in the adult thymus
Authors: Disanto James P. , Certain Stéphanie , Wilson Anne , MacDonald H. Robson , Avner Phillip , Fischer Alain , de Saint Basile Geneviève
Published: March 2007

Defects in the interleukin‐2 receptor γ (IL‐2Rγ) chain in man result in an X‐linked severe combined immunodeficiency, SCIDX1, characterized by an absence of T‐cell differentiation. This phenotype may result from pertubations in IL‐2‐, IL‐4‐, IL‐7‐ or IL‐15‐mediated signaling, as the IL‐2Rγ chain forms an integral component of these receptor systems. We have isolated and characterized cDNA and genomic clones for the murine IL‐2Rγ. The gene(Il2rg)is well conserved between mouse and man with respect to overall structure and size, and contains regions of high conservation in the promoter region as well.Il2rgmaps to mouse X chromosome region 40, in a region of synteny with human Xq12‐13.1. We have also explored the expression of the IL‐2Rγ during thymocyte development. IL‐2Rγ transcripts are detected in the earliest thymocyte precursor cells and persist throughout intrathymic development into the mature peripheral compartment. Genomic clones for the murine IL‐2Rγ will allow for further studies on the regulation and function of this genein vivo.
Only simultaneous blocking of the L‐ and P‐selectin completely inhibits neutrophil migration into mouse peritoneum
Authors: Bosse Roland , Vestweber Dietmar
Published: March 2007

We have examined the inhibitory effect of monoclonal antibodies against mouse P‐, E‐ and L‐selectin on the migration of neutrophils into the chemically inflamed peritoneum of the mouse. For this purpose, monoclonal antibodies were raised against mouse P‐ and E‐selectin, which block cell adhesion. We found that blocking of each selectin alone inhibited neutrophil migration to a similar degree ranging from 63 % to 72 %. Of the three possible combinations of antibodies against two different selectins only the combination of anti‐P‐ and anti‐L‐selectin antibodies caused an essentially complete blockade of neutrophil emigration. Only the effects of these two antibodies were additive, while the effect of anti‐E‐selectin antibodies did not add to the effect of antibodies against P‐ or L‐selectin. Thus, although E‐selectin is involved in neutrophil migration into the inflamed peritoneum of the mouse, it cannot compensate the block of the other two selectins which seem to play the dominant role in this process.
T cell receptor γδ repertoire in HIV‐1‐infected individuals
Authors: Hinz Thomas , Wesch Daniela , Friese Klaus , Reckziegel Anne , Arden Bernhard , Kabelitz Dieter
Published: March 2007

While Vγ9Vδ2 cells dominate among peripheral blood γδ T cells in healthy adults, the majority of γδ T cells in most HIV‐1‐infected individuals express Vδ1. We asked whether these elevated levels of Vδ1 T cells were due to clonal expansion. Three‐color flow cytometry with monoclonal antibodies against Vγ2/Vγ3/Vγ4, Vγ4 and Vγ9 was used to investigate Vγ usage in 27 patients with elevated numbers of Vδ1 T cells. While the relative proportion of Vγ9 cells among γδ T cells was significantly reduced in HIV‐1+ inviduals (10 ± 11% vs. 80 ± 17%, p < 0.001), the fraction of γδ T cells using Vγ5 or Vγ8 was significantly increased (54 ± 15% vs. 7 ± 11%, p < 0.001). In 1 patient, 76% of the Vδ1 cells expressed Vγ2 or Vγ3, suggesting clonality of the Vδ1 population. In line with this assumption, analysis of the Vδ1‐Jδ junctional regions by reverse transcription‐polymerase chain reaction (RT‐PCR) resulted in products of only one junctional length, as demonstrated by electrophoresis on denaturing gels, and 12 out of 16 (75%) in‐frame junctional sequences were identical in this patient. In other HIV‐1+ patients, RT‐PCR resulted in products of several distinct sizes, also indicating a highly restricted repertoire. After sequencing the Vδ1‐Jδ junctional regions of 3 additional patients, we found repeated but patient‐specific in‐frame junctions accounting for 10–30% of the sequenced clones. However, limited Vδ1‐Jδ junctional diversity was also seen in healthy donors. RT‐PCR products from 10 healthy individuals resulted in distinct bands on denaturing gels. In 1 of them exhibiting a single prominent band, 10 out of 17 (58%) sequenced junctions were identical. Two other healthy donors displayed 2/14 and 5/18 identical junctional sequences, respectively. Taken together, our results reveal significant alterations of Vγ usage in HIV‐1+ patients, while the Vδ1 junctional repertoire is similarly restricted in HIV‐1+ and HIV‐1− individuals. Therefore, these data argue against an obligatory clonal expansion of Vδ1‐expressing cells during HIV‐1 infection.
T cell receptor Vβ8.2 gene germ‐line transcription: An early event of lymphocyte differentiation
Authors: Candéias Serge , Hardy Richard R. , Li Yue Sheng , Staerz Uwe D.
Published: March 2007

Rearrangement of the T cell antigen receptor (TCR) β chain genes is highly regulated in both a developmental and a tissue‐specific manner. T cell precursors originate from the yolk sac or fetal liver during gestation and from the bone marrow during adulthood. They initiate the recombination of TCR genes primarily during differentiation in the thymus. It has previously been suggested that transcription of immunoglobulin genes in germ‐line configuration is linked to recombination events within these loci. Here, we examine whether germ‐line transcription of TCR variable genes coincides with their rearrangement or whether it marks even earlier stages of T lymphocyte development. During gestation, we found Vβ8.2 germ‐line transcripts in the fetal liver and the fetal thymus, but not in the yolk sac. This transcription precedes Vβ8.2 to DβJβ rearrangement. In adult animals, we found these transcripts in the thymus, the spleen, the liver and the bone marrow. However, in the liver, this transcription is dependent on the presence of mature lymphocytes. This transcription does not happen in non‐lymphoid cells. In the B lymphocyte lineage, Vβ8.2 germ‐line transcripts are detected only in the earliest stages of differentiation (pre‐pro‐ and pro‐B cells), but not in pre‐B cells and mature B lymphocytes. Altogether, our results show that transcription of the unrearranged Vβ8.2 gene is an early event of lymphocyte development, taking place in lymphocyte precursors, long before Vβ8.2 rearrangement.
Peyer's patch CD8+ memory T cells secrete T helper type 1 and type 2 cytokines and provide help for immunoglobulin secretion
Authors: Lagoo Anand S. , Eldridge John H. , Lagoo‐Deenadaylan Sandhya , Black C. Allen , Ridwan Ben U. , Hardy Kenneth J. , McGhee Jerry R. , Beagley Kenneth W.
Published: March 2007

Analysis of cytokine gene expression by reverse transcription‐polymerase chain reaction (RT‐PCR) demonstrated high spontaneous levels of transcripts for multiple cytokines in murine Peyer's patches (PP) compared to spleen and peripheral lymph nodes. This is consistent with the presence of active germinal centers in PP and their continuous exposure to lumenal antigen including bacterial endotoxin. RT‐PCR analysis of cytokine transcripts in purified PP T cell populations revealed the presence of transcripts for interleukin‐4 (IL‐4), IL‐5 and IL‐10 in addition to interferon‐γ (IFN‐γ) in CD8+ cells purified by flow cytometry. The majority of PP CD8+ T cells were also CD45RB10 (MB23G2−), suggesting that these cells were activated/memory cells. CD8+ cells in spleen and mesenteric lymph nodes (MLN) were predominantly CD45RBhi (MB23G2+) consistent with a resting/naive phenotype. PP and MLN CD8+ T cells also secreted IL‐5 and IL‐10 when stimulated with anti‐CD3 monoclonal antibody and when co‐cultured with PP B cells enhanced secretion of both IgG and IgA. These studies suggest that CD8− T cells at mucosal sites secrete T helper type 2 cytokines and can provide functional help for B cells in these tissues.
Recirculation, phenotype and functions of lymphocytes in mice treated with monoclonal antibody MEL‐14
Authors: Lepault Françoise , Gagnerault Marie‐Claude , Faveeuw Christelle , Boitard Christian
Published: March 2007

The effect of a single injection of an antibody against the peripheral lymph node (PLN) homing receptor or L‐selectin (gp90MEL‐14) was studied in vivo in C57BL/6 mice.

L‐selectin is known to be rapidly shed from leukocytes in humans and in mice following activation or cross‐linking in vitro. Here we demonstrate that in vivo a single injection of MEL‐14 antibody induces a rapid, almost complete and reversible down‐regulation of L‐selectin expression on both T and B cells. This modulation is dose dependent, specific for L‐selectin and lasts for 10 days. On neutrophils. L‐selectin expression was moderately decreased, and the injected antibody was detectable on the cell surface for several days. Thus, L‐selectin expression after antibody binding in vivo was affected differently on neutrophils and lymphocytes.

MEL‐14 treatment induces profound alterations of cell traffic. Loss of L‐selectin on lymphocytes leads to drastic PLN depletion and increased spleen cellularity. Depleted PLN were highly enriched in MEL‐14−/lo, CD44hi and CD11ahi cells, which may represent transiently sessile memory/activated cells.

The unresponsiveness in mixed lymphocyte reaction of PLN cells from treated animals and of purified L‐selectin− PLN T cells from normal mice supports this view. However, PLN and spleen cells from treated animals responded normally to non‐antigen‐specific stimuli.
Effect of interferon‐γ on antigen processing in human monocytes
Authors: Nadler Steven G. , Rankin Bruce M. , Moran‐Davis Patty , Cleaveland Jeffrey S. , Kiener Peter A.
Published: March 2007

The effect of interferon‐γ (IFN‐γ) on the ability of human monocytic cells to process exogenous (major histocompability complex class II) antigens was investigated. The processing (i.e. protein degradation) of antigens that were internalized via Fcγ receptor (FcγR) was followed for various times after treatment of cells with IFN‐γ. THP‐1 cells that had been treated with IFN‐γ for 4 h degraded antigen, internalized as an immune complex, at an enhanced rate. After 24 h of IFN‐γ treatment the rate of processing was similar to untreated cells. Unexpectedly, in cells which had been treated for 48–72 h there was a significant decrease in the rate of processing of the exogenous antigen. These effects were not due to changes in the rate of internalization of immune complex. The inhibition of the rate of processing was independent of the type of antigen, was dependent on the dose of IFN‐γ, and also occurred with normal human peripheral monocytes. Analysis of the degraded peptides by high‐pressure liquid chromatography indicated that some of the peptides generated in the IFN‐γ‐treated cells were both quantitatively and qualitatively different from the peptides generated in untreated cells. These data suggest that IFN‐γ modulates the way in which antigens, internalized through Fc receptors as immune complexes, are processed. Additionally, the results imply that decreases in the rate of antigen processing may lead to more efficient antigen presentation.
Role of the CD40‐CD40 ligand interaction in CD4+ T cell contact‐dependent activation of monocyte interleukin‐1 synthesis
Authors: Wagner David H. , Stout Robert D. , Suttles Jill
Published: March 2007

Most studies of the induction of cytokine synthesis in monocytes have employed an exogenous triggering agent such as lipopolysaccharide. However, in nonseptic inflammatory responses (e.g. rheumatoid arthritis) monocyte activation occurs as a result of T cell‐generated signals. In previous reports, we and others have demonstrated that contact‐dependent T cell‐generated signals are capable of contributing to macrophage activation. We have shown that plasma membranes from anti‐CD3 activated purified peripheral CD4+ T cells (TmA) but not from resting CD4+ cells (TmR) induce monocytes to synthesize interleukin (IL)‐1 in the absence of co‐stimulatory cytokines. Studies to determine the expression kinetics of the molecule(s) unique to activated CD4+ T cells which interact with monocytes to induce IL‐1 revealed that optimal expression occurred at 6 h post activation. This matched the previously reported kinetics of expression of CD40 ligand (CD40L) on activated peripheral T cells, implicating the CD40‐CD40L interaction as a candidate for the initiator of the IL‐1 signaling event. The ability of TmA to induce IL‐1 synthesis in resting monocytes could be markedly reduced by addition of a monoclonal anti‐CD40L antibody, 5c8. In addition, a monoclonal anti‐CD40 IgM (BL‐C4) proved dramatic in its ability to induce resting monocytes to synthesize IL‐1. In summary, these results demonstrate that the CD40‐CD40L interaction provides a critical component of CD4+ T cell contact‐dependent activation of monocyte IL‐1 synthesis.
Identification of a naturally processed HLA A0201‐restricted viral peptide from cells expressing human papillomavirus type 16 E6 oncoprotein
Authors: Bartholomew Jennifer S. , Stacey Simon N. , Coles Brian , Burt Deborah J. , Arrand John R. , Stern Peter L.
Published: March 2007

Human papillomavirus (HPV) DNA encoding the oncogenic proteins E6 and E7 is usually retained in cervical carcinomas, implicating these proteins as potential target antigens for immune recognition in this virally associated tumor. We have characterized endogenously processed peptides eluted from major histocompatibility complex class I molecules in cells infected with a recombinant vaccinia expressing the HPV‐16 E6 oncoprotein. The reverse‐phase chromatography profile of peptides eluted from isolated HLA‐A0201 molecules in cells expressing the E6 oncoprotein differs from that of cells not expressing E6. Sequential Edman degradation of novel peaks found in the peptide profiles from cells expressing HPV‐16 E6 led to the identification of a naturally processed HLA‐A0201‐restricted E6 peptide of sequence KLPQLCTEL. This approach has allowed the identification of a viral peptide which is processed and presented by cells expressing the E6 oncoprotein and is a likely target for cytotoxic T lymphocyte recognition in HLA‐A0201‐positive patients.
Identification of the core residues of the epitope of a monoclonal antibody raised against glycoprotein D of herpes simplex virus type 1 by screening of a random peptide library
Authors: Schellekens Gerard A. , Lasonder Edwin , Feijlbrief Matty , Koedijk Danny G. A. M. , Drijfhout Jan Wouter , Scheffer Albert‐Jan , Welling‐Wester Sytske , Welling Gjalt W.
Published: March 2007

Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules. We have used a 15‐amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal antibody (mAb) A16 which interacts with a continuous epitope restricted to amino acid residues 9 to 19 in the N‐terminal region of glycoprotein D of herpes simplex virus type 1 (gD‐1). The single peptide sequence obtained after three rounds of selection contained identical residues at three positions compared to the authentic gD‐1 sequence. Synthetic peptides were prepared based on the sequence of the original epitope and the phage‐derived epitope. The binding constants (Ka) with mAb A16 were determined using surface plasmon resonance (SPR) biosensor technology. The RPL‐derived peptide and peptide 9–19 of gD‐1 had approximately the same affinity for mAb A16. This suggests that those residues within the epitope that are essential for binding were identified. The synthesis of shorter versions of the RPL‐derived peptide restricted the binding region to seven amino acid residues. These results show that minimal information retrieved from the screening of an RPL combined with peptide synthesis can characterize the epitope of an mAb with high resolution. Immunization of mice with the phage‐derived peptide protected against a challenge with a lethal dose of herpes simplex virus type 1 equally well as the gD‐1 derived peptide.
Lymphocyte‐activation gene 3/major histocompatibility complex class II interaction modulates the antigenic response of CD4+ T lymphocytes
Authors: Huard Bertrand , Tournier Muriel , Hercend Thierry , Triebel Frédéric , Faure Florence
Published: March 2007

The activation requirements for antigen‐dependent proliferation of CD4+ T cells are well documented, while the events leading to the inactivation phase are poorly understood. Here, we tested the hypothesis that the lymphocyte‐activation gene 3 (LAG‐3), a second major histocompatibility complex (MHC) class II ligand, plays a regulatory role in CD4+ T lymphocyte activation. CD4+ class II‐restricted T cell clones were stimulated by their relevant antigen (hemagglutinin peptide or diphteria toxoid) and antigen‐presenting cells with or without anti‐LAG‐3 monoclonal antibody (mAb). Kinetic studies were performed to monitor different activation parameters, including the measurement of thymidine incorporation, expression of activation antigens and cytokine secretion. Results showed that the time course from the initial time points up to the peak time point was not modified in the presence of anti‐LAG‐3 mAb. However, addition of these antibodies, either as whole IgG or as Fab fragments, led to increased thymidine incorporation values for late time points and, hence, to a shift in the decreasing proliferation curve. We also showed that expression of activation antigens, such as CD25, was higher in the presence of anti‐LAG‐3 mAb, and that cytokine concentrations, i.e. of interferon‐γ or interleukin‐4, were higher in the corresponding culture supernatants. In addition, we tested whether the effects of anti‐LAG‐3 mAb were limited to antigen‐dependent. MHC class II‐restricted responses. The proliferative responses of CD4+ T cell clones following stimulation with either interleukin‐2, mitogens, a combination of anti‐CD2 mAb, immobilized anti‐CD3 or anti‐T cell receptor mAb were not altered by anti‐LAG‐3 mAb. The allogeneic proliferative response of a CD8+ T cell clone was also not affected. Overall, the present analysis reveals a modulating effect of anti‐LAG‐3 mAb, mediated specifically on antigen‐dependent, MHC class II‐restricted responses of CD4+ T cell lines. These results support the view that LAG‐3/MHC class II interaction down‐regulates antigen‐dependent stimulation of CD4+ T lymphocytes.