bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell antigen CD28 binds to the GRB‐2/SOS complex, regulators of p21ras
Authors: Schneider Helga , Cai Yun‐Cai , Prasad K. V. S. , Shoelson Steven E. , Rudd Christopher E.
Published: March 2007

The T cell molecule CD28 provides a co‐stimulatory signal that is required for T cell proliferation, and has been implicated in the control of T cell anergy. An important clue to the signaling mechanism of CD28 is the finding that CD28 can bind to phosphatidylinositol 3‐kinase (PI 3‐kinase) by means of a cytoplasmic phospho‐YMNM (pYMNM) motif. A remaining issue concerns whether CD28 can recruit other intracellular signaling molecules. In this study, we show that CD28 uses the same pYMNM motif to recruit a second intracellular protein, GRB‐2. CD28‐associated GRB‐2, as detected by anti‐GRB‐2 immunoblotting, was found in human peripheral T cells, HPB‐ALL and Jurkat cells. As in the case of PI 3‐kinase, antibody‐induced cross‐linking of CD28 induces a time‐dependent recruitment of GRB‐2. Likewise, mutation of the pY‐191 residue within the pYMNM motif reduces GRB‐2 binding. Peptide binding studies show that the SH2 domain of GRB‐2 binds to the pYMNM motif with an affinity comparable to GRB‐2/SHC, but some 10‐ to 100‐fold lower than the CD28/PI 3‐kinase. Despite this, CD28/GRB‐2 and CD28/PI 3‐kinase complexes are found to co‐exist in peripheral T cells. Finally, immunoblotting shows that CD28 also associates with the gene product of the human homolog of the Drosophila Son of sevenless gene (SOS), a GRB‐2‐complexed guanine nucleotide exchange factor responsible for converting p21ras to a GTP‐bound active state. CD28‐associated GRB2/SOS is likely to serve an important link in the regulation of p21ras and lymphokine expression mediated by CD28.
Epitope specificity, cytokine production profile and diabetogenic activity of insulin‐specific T cell clones isolated from NOD mice
Authors: Daniel Dylan , Gill Ronald G. , Schloot Nanette , Wegmann Dale
Published: March 2007

T cells are known to play an important role in beta cell destruction in the nonobese diabetic (NOD) mouse model of Type I diabetes and islet‐specific T cell clones have been demonstrated to be capable of adoptive transfer of diabetes. One important issue involves the identity of beta cell antigens that are recognized by nominally islet cell‐specific T cell clones. We have previously reported that insulin‐specific T cells are a predominant component of islet‐specific T cells isolated from islet infiltrates of pre‐diabetic NOD mice. In this report we examine six independently derived insulin‐specific T cell clones established from islet infiltrates of pre‐diabetic NOD mice in detail. All six clones were found to be specific to a region of the insulin molecule defined by a synthetic peptide encompassing residues 9–23 of the B chain. Despite this restricted specificity, each member of this panel exhibited a distinct receptor specificity defined either by Vβ usage or antigen fine specificity. Five clones produced interferon (IFN)‐γ but not interleukin (IL)‐4, placing them in the T helper type 1 (TH1)‐like category whereas one clone produced both IL‐4 and IFN‐γ, a characteristic of TH0 cells. All six clones were capable of either acceleration of diabetes in young NOD mice or adoptive transfer to NODscid mice. Taken together, these results suggest that spontaneously arising insulin‐specific T cells participate in beta cell destruction during development of diabetes in NOD mice.
Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow‐derived macrophages by TH 1 and TH 2 cytokines
Authors: Modolell Manuel , Corraliza Ines M. , Link Franz , Soler Germán , Eichmann Klaus
Published: March 2007

Activation with lipopolysaccharide induces macrophages to produce the enzymes arginase and nitric oxide (NO) synthase. Both enzymes use as a substrate the amino acid L‐arginine, which can be either hydrolyzed by arginase to urea and ornithine or oxidized by NO synthase to NO and citrulline. NO is important in the bactericidal and cytotoxic activities of macrophages. An equivalent functional role of arginase and its products is not known. We tested the induction of arginase in bone marrow‐derived macrophages by endogenous mediators that are known to induce NO synthase, such as interferon‐γ (IFN‐γ), or suppress the induction of this enzyme, such as interleukin (IL)‐4, IL‐10, and prostaglandin E2 (PGE2). We find that PGE2 and the TH2 cytokines IL‐4 and IL‐10 are potent inducers of arginase. In contrast, the TH 1 cytokine IFN‐γ does not induce arginase. Simultaneous application of both types of mediators leads to reduced induction of both arginase and NO synthase. Exposure of macrophage cultures to inducers of NO synthase exhausts their ability to respond subsequently to inducers of arginase. Conversely, exposure of the cells to inducers of arginase exhausts their ability to respond subsequently to inducers of NO synthase. The results are consistent with a competition of both enzymes for their substrate, L‐arginine, with a reciprocal inhibition in the induction of both enzymes, or a combination of both phenomena. The enzymes NO synthase and arginase appear to define two alternate functional states of macrophages, induced by TH 1 and TH 2 cytokines, respectively.
Activation of murine epidermal γδ T cells through surface 2B4
Authors: Schuhmachers Georg , Ariizumi Kiyoshi , Mathew Porunelloor A. , Bennett Michael , Kumar Yinay , Takashima Akira
Published: March 2007

Dendritic epidermal T cells (DETC) are γδ T cells that normally reside in murine skin. They express on their surface the 2B4 molecule, a 66‐kDa glycoprotein of the immunoglobulin gene superfamily thought to be associated with anti‐tumor cytotoxicity by natural killer and lymphokine‐activated killer cells. Here, we show that ligation of surface 2B4 transduces cell activation signals in DETC. Treatment with anti‐2B4 monoclonal antibodies triggers the secretion of interferon‐γ and interleukin‐2 by DETC lines, induces proliferation of resting DETC lines, amplifies anti‐CD3‐dependent proliferation of DETC freshly isolated from mouse skin; and up‐regulates egr‐1 and c‐fos mRNA expression. These results indicate a unique pathway for DETC activation.
Activated T cells induce interleukin‐12 production by monocytes via CD40‐CD40 ligand interaction
Authors: Shu Uno , Kiniwa Mamoru , Wu Chang You , Maliszewski Charles , Vezzio Nadia , Hakimi John , Gately Maurice , Delespesse Guy
Published: March 2007

Previous studies on the production of interleukin‐12 (IL‐12) have shown that it is released, together with other proinflammatory cytokines, shortly after exposure of phagocytic cells to a variety of pathogens. We here report that IL‐12 is also released during the recall response to soluble antigen (Ag) devoid of intrinsic adjuvant activity. We show that activated T cells induce the production of IL‐12 by monocytes via a mechanism involving the interaction of T cell‐associated CD40 ligand with CD40 on monocytes. The data suggest that Ag presentation on monocytes favors the persistence of type 1 responses.
RAG1 and RAG2 expression in human intestinal epithelium: evidence of extrathymic T cell differentiation
Authors: Lynch Sara , Kelleher Dermot , McManus Ross , O'Farrelly Cliona
Published: March 2007

We examined the hypothesis that the adult human gastrointestinal tract is a site of extrathymic T cell differentiation. When T lymphocytes undergo gene rearrangement, the products of both RAG1 and RAG2 genes are expressed; RAG mRNA is present only in tissue governing lymphocyte maturation. In this study, reverse transcription polymerase chain reaction (RT‐PCR) was used to detect RAG1‐ and RAG2‐specific mRNA. Total RNA was purified from small intestinal samples from five adults. Peripheral blood mRNA from the same patient was used as a negative control in two cases. Bone marrow RNA preparations from two healthy donors were used as positive controls. cDNA synthesis was carried out using random hexamers. Primers for first round and nested PCR of RAG1 and RAG2 were synthesized. RAG1 and RAG2 mRNA was detected in all bone marrow preparations but was absent in all peripheral blood samples. RAG1 and RAG2 mRNA was detected in the small intestine of four of the five patients studied. RAG1 and RAG2 expression was localized in the epithelial layer and absent in the lamina propria. RAG1 and RAG2 expression in the epithelial layer is strong evidence that T cell differentiation occurs in the adult human intestine.
Evidence for the stochastic acquisition of cytokine profile by CD4+ T cells activated in a T helper type 2‐like response in vivo
Authors: Kelso Anne , Groves Penny , Troutt Anthony B. , Francis Kari
Published: March 2007

The diversity of cytokine production patterns displayed by T cells activated in vivo was investigated by analyzing short‐term antigen‐specific CD4+ T cell clones and single CD4+ T cells derived from draining lymph nodes of mice undergoing a T helper 2 (Th2)‐like response to keyhole limpet hemocyanin (KLH). On average, 2.7% of CD4+ lymph node cells gave rise to clones in the presence of the immunizing antigen and, of these, about 90% secreted interleukin‐4 (IL‐4) and 20% secreted interferon‐γ (IFN‐γ) when restimulated after 2 weeks in vitro. Almost all IFN‐γ‐producing clones co‐produced IL‐4. The definition of clones as positive or negative for cytokine synthesis depended on assay sensitivity, however, since their titers were distributed continuously from the threshold of detection over at least a 1000‐fold range.

Reverse‐transcription polymerase chain reaction analysis of 59 clones revealed multiple patterns of co‐expression of IL‐2, IL‐3, IL‐4, IL‐6, IFN‐γ and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) mRNA. Although most clones contained detectable IL‐4 and IL‐6 mRNA and a minority contained IFN‐γ mRNA, only 1 clone expressed the canonical Th2 cytokine profile. The observed frequencies of mRNA co‐expression for most of the six cytokines (including IL‐4 with IFN‐γ), and the frequency of co‐secretion of IL‐4 and IFN‐γ, were not significantly different from those predicted for random association. Independent regulation of IL‐4 and IFN‐γ mRNA expression was confirmed at the single‐cell level in a polyclonal population of KLH‐primed CD4+ cells, among which co‐expression of these cytokines again occurred at the frequency predicted for a random event. The data suggest that the polarization of this immune response towards a Th2 cytokine profile is achieved by altering the probabilities of expression of the IL‐4, IFN‐γ and other cytokine genes at the population level, rather than by selective expansion of a distinct T cell subset.
Biphasic effect of interferon‐γ in murine collagen‐induced arthritis
Authors: Boissier Marie‐Christophe , Chiocchia Gilles , Bessis Natacha , Hajnal Julia , Garotta Gianni , Nicoletti Ferdinando , Fournier Catherine
Published: March 2007

Interferon‐γ (IFN‐γ) exerts both enhancing and suppressing influences on collagen‐induced arthritis (CIA), depending on the route and protocol of administration. To study the role of IFN‐γ on the autoimmune process of CIA, we treated DBA/1 mice with two different rat monoclonal antibodies (mAb) to murine IFN‐γ. Treatments, given twice weekly for 4 weeks, consisted of intraperitoneal injections of either mAb. In early treatments, starting from the day of immunization with type II collagen (CII), the severity of arthritis was reduced in both groups of anti‐IFN‐γ‐treated mice compared with control groups. Moreover, anti‐CII antibody levels decreased in the sera of these mice. CIA was also down‐regulated in mice treated from days 14 or 28 post immunization. In contrast, late treatments with anti‐IFN‐γ mAb either induced aggravating effects, or did not affect the course of the disease. On the other hand, administration of high doses (8 × 104 U three times/week) of rat recombinant IFN‐γ exerted a transient increase of CIA severity. These findings suggest that IFN‐γ may play a critical role during both the induction and the course of CIA, first enhancing the immune response, and then regulating the arthritis process.
Orally induced bystander suppression in experimental autoimmune uveoretinitis occurs only in the periphery and not in the eye
Authors: Wildner Gerhild , Thurau Stephan R.
Published: March 2007

Oral administration of retinal soluble antigen (S‐Ag) suppresses the induction of S‐Ag‐mediated experimental autoimmune uveitis (EAU) in Lewis rats. EAU induced with interphotoreceptor retinoid‐binding protein (IRBP), another retinal autoantigen, can also be suppressed by oral administration of IRBP. It has been speculated that feeding with one retinal autoantigen could suppress induction of uveitis with the other retinal protein by means of bystander suppression. Both uveitogenic effector and suppressor cells should find their antigens within the retina, where the suppressor cells would be expected to act on the effector cells. However, reciprocal combinations of antigens used for induction and suppression of uveitis failed to prevent onset of disease, demonstrating that bystander suppression obviously does not occur in the eye. To investigate further the localization of suppressor mechanisms, we fed Lewis rats either with retinal S‐Ag or with ovalbumin (OVA) and then immunized the animals either with a mixture of S‐Ag and OVA or with each antigen separately, injected into contralateral hind legs. Feeding of S‐Ag prior to immunization led to suppression of uveitis, whereas feeding of OVA had no tolerizing effect when S‐Ag and OVA were injected into different legs. Nevertheless, immunizing rats with a mixture of S‐Ag and OVA after OVA feeding suppressed uveitis to a high degree. These findings suggest that orally induced bystander suppression might not occur in the target organ, but rather peripherally at the site of induction of the autoimmune T cells.
Toxoplasma gondii‐specific CD4+ T cell clones from healthy, latently infected humans display a ThO profile of cytokine secretion
Authors: Prigione Ignazia , Facchetti Paola , Ghiotto Fabio , Tasso Paola , Pistoia Vito
Published: March 2007

Human Toxoplasma gondii (Tg)‐specific T cell clones were raised by infecting peripheral blood mononuclear cells (MNC) from two healthy, latently infected individuals with Tg trophozoites. All of the clones had a CD4+ immunophenotype and produced simultaneously interleukin (IL)‐2, interferon (IFN)‐γ, IL‐4 and IL‐5 upon mitogen or antigen stimulation. Tg‐specific T cell clones were classified as T helper of type 0 (ThO) since most of them released roughly comparable amounts of IFN‐γ and IL‐4. In some clones, a trend to an increased production of IFN‐γ following antigen‐specific as compared to non‐specific stimulation was observed. The ThO phenotype was also expressed by T cell clones that had been raised from bulk cultures performed in the presence of IL‐4 or IFN‐γ. All of the Tg‐specific T cell clones were cytolytic in a non‐specific assay which involves the triggering of the CD3‐T cell receptor (TcR) complex. Some clones specifically lysed an autologous lymphoblastoid cell line (LCL) that had been infected with Tg trophozoites. Finally, most of the Tg‐specific T cell clones produced IL‐10, irrespective of whether they had been raised from bulk cultures incubated in the presence or absence of IL‐4 or IFN‐γ. Taken together, these findings suggest that Tg‐specific ThO helper cell clones from healthy, latently infected individuals, beside activating toxoplasmacidal mechanisms through IFN‐γ release, might limit the magnitude of the immune response to the parasite by killing Tg‐infected antigen‐presenting cells and by releasing IL‐10.
An interleukin‐6 transgene expressed in B lymphocyte lineage cells overcomes the T cell‐dependent establishment of normal levels of switched immunoglobulin isotypes
Authors: Oka Yoshihiro , Rolink Antonius G. , Suematsu Sachiko , Kishimoto Tadamitsu , Melchers Fritz
Published: March 2007

Long‐term proliferating, stromal cell/interleukin (IL)‐7‐reactive precursor B cell lines established from fetal liver and bone marrow of human IL‐6‐transgenic B6Ld46 mice produce and secrete human IL‐6. When transplanted into severecombined immunodeficient (SCID) or Rag2 knockout (Rag2‐T) mice, these pre‐B cell lines establish a part of the B cell compartment but yield no T cells, as do pre‐B cell lines from genetically matched non‐transgenic mice. Within 2 to 3 months after transplantation, the serum of mice transplanted with pre‐B cells from normal mice contains normal levels of IgM (200–600 μg/ml) but 10–100‐fold lower levels of the IgG subclasses and of IgA. In contrast, the sera of mice transplanted with IL‐6 transgenic pre‐B cells contain not only IgM, but also IgG and IgA at nearly normal levels. The results indicate that at least a part of the plasmacytosis and elevated IgG production observed previously in the IL‐6‐transgenic mice appears to be due to a T cell‐independent activation of IgG and IgA production by the IL‐6‐secreting pre‐B cells and their differentiated progeny in the immunodeficient hosts.
Signaling through murine CD38 is impaired in antigen receptor‐unresponsive B cells
Authors: Lund Frances E. , Solvason Nanette W. , Cooke Michael P. , Heath Andrew W. , Grimaldi J. Christopher , Parkhouse R. M. E. , Goodnow Christopher C. , Howard Maureen C.
Published: March 2007

CD38 is a 42‐kDa membrane associated enzyme which converts NAD into cyclic ADP‐ribose (cADPR), a Ca2+‐mobilizing second messenger, and ADP‐ribose (ADPR). Agonistic antibodies to murine CD38 deliver a potent growth co‐stimulus to mature splenic B lymphocytes. In this report we demonstrate a striking relationship between CD38‐mediated mitogenesis and the ability of surface IgM to promote B cell proliferation. Tolerized B lymphocytes obtained from a double‐transgenic mouse model of B cell tolerance do not proliferate in response to antigen stimulation through the Ig receptor or to agonistic anti‐CD38 antibodies. Similarly, B‐1 cells isolated from the peritoneal cavity of normal mice, and splenic B cells isolated from newborn mice were also unresponsive to both anti‐IgM and anti‐CD38 stimulation. All of these CD38‐unresponsive B cells expressed normal levels of cell surface CD38 and responded to numerous other stimuli. CD38 immunoprecipitated from these B cell populations was normal in size and effectively hydrolyzed NAD, suggesting that the defect in CD38 signaling likely occurs downstream of CD38 itself. Signaling through CD38 and IgM does not always have identical effects on B cells since anti‐CD38 cannot deliver inhibitory growth or differentiation signals to normal B cells or immature B cell lines. Nevertheless, the correlative data with these multiple B cell models of unresponsiveness suggests that the signaling pathway utilized by CD38 and IgM intersect, possibly sharing at least one of the crucial components of the Ig receptor signaling cascade.
Increased dexamethasone sensitivity of neonatal leukocytes: different mechanisms of glucocorticoid inhibition of T cell proliferation in adult and neonatal cells
Authors: Kavelaars Annemieke , Zijlstra Jitske , Bakker Joost M. , Van Rees Emmelien P. , Visser Gerard H. A. , Zegers Ben J. M. , Heijnen Cobi J.
Published: March 2007

Glucocorticoids (GC) are known to inhibit the proliferative response of leukocytes after mitogenic activation. Until now, the effects of GC on the immune system have been studied predominantly in adults. However, GC are frequently administered to human fetuses and newborns for the prevention and treatment of respiratory distress syndrome. The immune system of human newborns is still a functionally immature system. Therefore, we wondered whether the immaturity is also reflected by altered responses to hormonal signals such as glucocorticoids. We studied the effects of the GC dexamethasone (DEX) on the proliferation of peripheral blood mononuclear cells and T cells in vitro after stimulation with phytohemagglutinin, anti‐CD3, anti‐CD3/anti‐CD28 or anti‐CD2/anti‐CD28. Our data demonstrate that neonatal cells are much more sensitive to inhibition of the proliferative response by DEX than adult cells (ED50 1 ± 0.8 nM vs. 221 ± 135 nM). This difference in sensitivity is not related to differences in affinity and capacity of binding of [3H] DEX. Moreover, we show that the mechanisms of GC inhibition differ between adult and neonatal cells. In adult cells, addition of interleukin (IL)‐2 does not restore DEX inhibition of the proliferative response. In contrast, the proliferative response of neonatal cells can be restored completely by the addition of IL‐2. These data suggest that the primary target of GC in neonatal cells is inhibition of IL‐2 production. In adult cells, other mechanisms are responsible for inhibition of T cell proliferation.
Role of T helper cell precursor frequency on vesicular stomatitis virus neutralizing antibody responses in a T cell receptor β chain transgenic mouse
Authors: Freer Giulia , Burkhart Christoph , Rülicke Thomas , Ghelardi Emilia , Rohrer Urs Hoffmann , Pircher Hanspeter , Zinkernagel Rolf M. , Hengartner Hans
Published: March 2007

During most immune responses, T cells help antigen‐specific B cells to make antibodies against the antigen. One of the contributions of T cells to antibody production is the induction of isotype switching from IgM to IgG, which is the most abundant isotype in blood serum during recall responses. Other features of memory responses are faster kinetics and higher titers of antibody in the serum. What causes a primary immune response to be different from a secondary is not yet very clear and, particularly, the influence of precursor frequencies of T and B cells on memory responses still remains to be answered. To address this issue, a transgenic (tg) mouse line (ADA) was developed; it expresses the β chain (Vβ2) of a major histocompalibility complex class II‐restricted T cell receptor (TcR) specific for the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana (VSV‐IND). These mice exhibit an increased precursor frequency of VSV‐specific CD4+ T cells that leads to enhanced neutralizing IgG production against VSV in vivo in unprimed mice. The data indicate that increased frequency of naive specific helper T cells alone may account for features of a memory phenotype such as high titer of antibodies and isotype switching.
Antibody responses to Toxoplasma gondii antigen in human peripheral blood lymphocyte‐reconstituted severe‐combined immunodeficient mice reproduce the immunological status of the lymphocyte donor
Authors: Walker William , Roberts Craig W. , Brewer James M. , Alexander James
Published: March 2007

These studies describe the production of specific antibodies in human peripheral blood lymphocyte‐reconstituted severe‐combined immunodeficient (PBL‐SCID) mice following vaccination with antigen from the protozoan parasite Toxoplasma gondii. To determine the effect of previous exposure of the lymphocyte donor to antigen, human‐PBL‐SCID animals were created by transferring peripheral blood lymphocytes from either a single T. gondii‐seronegative or a single seropositive donor. These reconstituted animals were subsequently inoculated with T. gondii soluble tachyzoite antigen (STAg) entrapped within non‐ionic surfactant vesicles as an immunological adjuvant. Animals were bled at pre‐determined time points post‐vaccination and the expression of human anti‐STAg antibodies in the plasma determined by enzyme‐linked immunosorbent assay. Human antibodies specific for STAg were readily inducible in both groups of reconstituted animals, although the pattern of isotype production differed markedly between groups. The response in animals reconstituted with lymphocytes from the T. gondii‐seronegative donor consisted primarily of IgM and subsequently of IgG (predominantly IgG1). In animals reconstituted with lymphocytes from the seropositive donor, no parasite‐specific IgM could be demonstrated. The detectable response to STAg consisted entirely of human antibodies of the IgG isotype (IgG1), indicative of a memory‐type response. These results mimicked exactly the antibody responses that would be expected had the lymphocyte donors been directly challenged with either the antigen or the live infectious agent, demonstrating that the immune system within these animals is functional and reproducible with regard to both the primary and secondary responses of the human donors.
The role of γδ T cells in priming macrophages to produce tumor necrosis factor‐α
Authors: Nishimura Hitoshi , Emoto Masashi , Hiromatsu Kenji , Yamamoto Shunsuke , Matsuura Keiko , Gomi Hiroshi , Ikeda Toshio , Itohara Shigeyoshi , Yoshikai Yasunobu
Published: March 2007

The secretion of tumor necrosis factor (TNF)‐α from macrophages is regulated by both priming and triggering signals. We found that macrophages from mice lacking γδ T cells [T cell receptor (TCR) δ−/‐ mice], which lack the gene encoding the δ chain, produced only small amounts of TNF‐α in response to lipopolysaccharide (LPS) and showed a reduced level of expression of CD14. Pre‐incubation of macrophages from TCR δ‐/‐ mice with γδ T cells from their TCR δ+/‐ littermates restored their capacity to produce TNF‐α in response to LPS. The priming activity of γδ T cells was in part inhibited by neutralizing anti‐interferon (IFN)‐γ monoclonal antibodies. Collectively, these results suggest that γδ T cells play a role in priming macrophages to a steady state of activation via IFN‐γ secretion, which allows them to produce TNF‐α when exposed to LPS.
CD38 ligation induces discrete cytokine mRNA expression in human cultured lymphocytes
Authors: Ausiello Clara M. , Urbani Francesca , la Sala Andrea , Funaro Ada , Malavasi Fabio
Published: March 2007

Human CD38 is a surface glycoprotein expressed by different immuno‐competent cells such as immature and activated lymphocytes, plasma cells and natural killer cells. It has recently been reported that the CD38 molecule exerts adenosine diphosphate ribosyl cyclase activity and is associated with distinct transmembrane signaling molecules. This study reports that ligation of CD38 by specific monoclonal antibodies (mAb) induces multiple cytokine mRNA expression in cultured peripheral blood mononuclear cells (PBMC). The mRNA for tumor necrosis factor‐α, interleukin (IL)‐1β, IL‐6, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and IL‐12 were always detected, whereas interferon‐γ and IL‐10 mRNA expression were seen in most, but not all PBMC cultures. Low levels of IL‐2, IL‐4 and IL‐5 mRNA were also found. The key observation of this work is that CD38 ligation in PBMC induces a large spectrum of cytokines, many of which overlap with those induced via CD3 activation. The main differences between CD38 and CD3 activation are the low to undetectable levels of IL‐2 mRNA, and the sustained IL‐1β and IL‐6 mRNA accumulation found in PBMC cultures following treatment with anti‐CD38 mAb. Furthermore, PBMC proliferation was not found to be a prerequisite for CD38‐mediated cytokine induction. Together, these results suggest that human CD38 activates a signaling pathway which leads to the induction of a discrete array of cytokines, and that this pathway only partially overlaps with that controlled by T cell receptor CD3.
Covalent linkage of C3 to properdin during complement activation
Authors: Whiteman Leslie Y. , Purkall Donald B. , Ruddy Shaun
Published: March 2007

Activation of the alternative complement pathway of serum produces complexes of properdin (P) and C3 as measured in a double antibody enzyme‐linked immunosorbent assay. When purified from serum, these complexes decrease factor B hemolytic activity in serum and do not restore the alternative pathway hemolytic activity of serum deficient in P. Sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of activated serum containing biotinylated P, followed by blotting to nitrocellulose and development with streptavidin‐alkaline phosphatase revealed a band at 53 kDa for monomeric P and an additional band at 160 kDa. P samples eluted from zymosan and purified from activated serum revealed a band at 116 kDa for C3α and 74 kDa for C3β, and an additional band at 160 kDa when analyzed by SDS‐PAGE, Western blotting and development with antibody to C3. The appearance of a 160 kDa band containing P and C3 indicates that these proteins are contained in a complex formed during activation of the alternative pathway. Activation of a purified reagent mixture containing factors B, D, and H, and 125I‐labeled P or 125I‐labeled C3, followed by SDS‐PAGE and autoradiography confirmed the presence of a 160‐kDa band which disappeared following hydroxylamine treatment of the sample. These data are consistent with a covalent linkage of C3 to P via the C3α chain, producing the 160‐kDa complex.
Selective variations in vivo of VH3 and VH1 gene family expression in peripheral B cell IgM, IgD and IgG during HIV infection
Authors: David Denis , Demaison Christophe , Bani Lynda , Zouali Moncef , Thèze Jacques
Published: March 2007

We have analyzed the expression of VH gene families in IgM, IgD and IgG of peripheral blood B cells from a group of HIV‐infected patients. CD19+CD20+ cells were purified and anchored reverse transcriptase‐polymerase chain reaction products were hybridized with VH gene family probes. IgM, IgD and IgG that expressed a VH3 gene family segment, were decreased in patients with low CD4 counts and to a greater extend in patients with AIDS symptoms (up to 85% for IgG) compared to adult healthy donors. This was correlated with elevated levels of IgM and IgG encoded by a VH1 gene family segment (around 60% for IgG). These results confirm and extend previous work that has detected the VH3 gene family under‐representation in HIV infection. Here, we show that, in vivo, this phenomenon actually affects the different B cell populations of the peripheral blood: IgM+ or IgG+ B cells and also IgM+IgD+ naive B cells. In the course of HIV infection, this results in their gradual depletion. Data presented here strengthen the hypothesis that a B cell superantigen exists in HIV infection. These pronounced variations of the normally most‐expressed VH gene family may be related to B cell abnormalities detected in HIV‐infected patients.
Altered T lymphocyte signaling in rheumatoid arthritis
Authors: Allen Maggie E. , Young Stephen P. , Michell Robert H. , Bacon Paul A.
Published: March 2007

Synovial and peripheral blood T cells from patients with rheumatoid arthritis are functionally deficient. This may be secondary to their reduced cytokine (e.g. interleukin‐2) synthesis. We have investigated the possibility of an alteration in pathways common to interleukin‐2 production and proliferation in peripheral blood T cells from patients with active rheumatoid arthritis. Intracellular calcium levels ([Ca2+]i) were analyzed by flow cytometric methods in Indo 1‐loaded T cells. These were purified by negative selection from patients or age/sex‐matched controls, and stimulated with phytohemagglutinin‐P or anti‐CD3. Rheumatoid [Ca2+]i responses to both stimuli were reduced (p < 0.005). Patient cell samples included a larger proportion of non‐responding cells, but even in the responsive population the magnitude of the response in rheumatoid cells was impaired compared with those in normal cell samples (p < 0.0001) for both stimuli. Proliferation responses were also impaired (p < 0.005), and there was a positive correlation between the paired [Ca2+]i elevation and proliferative responses for both stimuli. CD2 and CD3 expression were normal, and the proportions of CD4, CD8 and CD45RO and CD45RA subsets were also unaffected by disease. Thus a signaling defect downstream of CD2 or CD3 surface molecules may contribute to functional deficiencies in rheumatoid T lymphocytes. This effect is not due to non‐steroidal anti‐inflammatory drugs which some patients were taking. We have demonstrated similar alterations in [Ca2+]i responses and proliferation in a smaller study of patients with inflammatory bowel disease, indicating that such changes might be present in other chronic inflammatory states.
Co‐expression of CD8α in CD4+ T cell receptor αβ+ T cells migrating into the murine small intestine epithelial layer
Authors: Reimann Jörg , Rudolphi Angelika
Published: March 2007

We investigated the surface phenotype of CD3+CD4+ T cell receptor (TCR) αβ+ T cells repopulating the intestinal lymphoid tissues of C.B‐17 scidlscid (severe‐combined immunodeficient; scid) (H‐2d, Ld+) mice. CD4+ CD8− T cells were cell sorter‐purified from various secondary and tertiary lymphoid organs of congenic C.B‐17 +/+ (H‐2d, Ld+) or semi‐syngeneic dm2 (H‐2d, Ld−) immunocompetent donor mice. After transfer of 105 cells into young scid mice, a mucosa‐homing, memory CD44hi CD45RBlo CD4+ T cell population was selectively engrafted. Large numbers of single‐positive (SP) CD3+ CD2+ CD28+ CD4+ CD8− T cells that expressed the α4 integrin chain CD49d were found in the spleen, the mesenteric lymph nodes, the peritoneal cavity and the gut lamina propria of transplanted scid mice. Unexpectedly, large populations of donor‐type doublepositive (DP) CD4+ CD8α+ CD8β− T cells with high expression of the CD3/TCR complex appeared in the epithelial layer of the small intestine of transplanted scid mice. In contrast to SP CD4+ T cells, the intraepithelial DP T cells showed low expression of the CD2 and the CD28 co‐stimulator molecules, and of the α4 integrin chain CD49d, but expressed high levels of the αIEL integrin chain CD103. The TCR‐Vβ repertoire of DP but not SP intraepithelial CD4+ T cells was biased towards usage of the Vβ6 and Vβ8 viable domains. Highly purified populations of SP and DP CD4+ T cell populations from the small intestine epithelial layer of transplanted scid mice had different abilities to repopulate secondary scid recipient mice: SP CD4+ T cells repopulated various lymphoid tissues of the immunodeficient host, while intraepithelial DP CD4+ T cells did not. Hence, a subset of CD3+ CD4+ TCRαβ+ T cells apparently undergoes striking phenotypic changes when it enters the microenvironment of the small intestine epithelial layer.
Leishmania infantum‐specific T cell lines derived from asymptomatic dogs that lyse infected macrophages in a major histocompatibility complex‐restricted manner
Authors: Pinelli Elena , Gonzalo Rosa M. , Boog Claire J. P. , Rutten Victor P. M. G. , Gebhard Douglas , Del Real Gustavo , Ruitenberg E. Joost
Published: March 2007

Protective immunity to leishmaniasis has been demonstrated in murine models to be mediated by T cells and the cytokines they produce. We have previously shown that resistance to experimental Leishmania infantum infection in the dog, a natural host and reservoir of the parasite, is associated with the proliferation of peripheral blood mononuclear cells (PBMC) to parasite antigen and to the production of interleukin‐2 and tumour necrosis factor. In this study we show that PBMC from asymptomatic experimentally infected dogs produce interferon‐γ upon parasite antigen‐specific stimulation, whereas lymphocytes from symptomatic dogs do not. In addition, we report for the first time the lysis of L. infantum‐infected macrophages by PBMC from asymptomatic dogs and by parasite‐specific T cell lines derived from these animals. These T cell lines were generated by restimulation in vitro with parasite soluble antigen and irradiated autologous PBMC as antigen‐presenting cells. We show that lysis of infected macrophages by T cell lines is major histocompatibility complex restricted. Characterization of parasite‐specific cytotoxic T cell lines revealed that the responding cells are CD8+. However, for some animals, CD4+ T cells that lyse infected macrophages were also found. In contrast to asymptomatic dogs, lymphocytes from symptomatic dogs failed to proliferate and produce interferon‐γ after Leishmania antigen stimulation in vitro and were not capable of lysing infected macrophages. These results suggest that both the production of interferon‐γ and the destruction of the parasitized host cells by Leishmania‐specific T cells play an important role in resistance to visceral leishmaniasis.
Expression of two T cell receptor α chains on the surface of normal murine T cells
Authors: Heath William R. , Carbone Francis R. , Bertolino Patrick , Kelly Jannice , Cose Steve , Miller Jacques F. A. P.
Published: March 2007

We have previously reported that a subset of T cells in T cell receptor (TCR)‐transgenic mice may express two different α chains on their surface. The expression of two functional α chains has also been demonstrated for human peripheral blood T cells. In this report, we show that a proportion of normal murine lymph node T cells express two functional α chains on their surface. The extrapolated frequency of these cells present in the normal repertoire ranges from 7–21%, with an average of 15%. Our analysis of a small number of antigen‐specific T cell clones suggests that the frequency of antigen‐responsive cells expressing two surface α chains is relatively low. This raises the possibility that dual α chain T cells may have a selective disadvantage in responding to specific antigen.
Mapping T cell recognition: the identification of a T cell receptor residue critical to the specific interaction with an influenza hemagglutinin peptide
Authors: Wedderburn Lucy R. , Searle Stephen J. M. , Rees Anthony R. , Lamb Jonathan R. , Owen Michael J.
Published: March 2007

The fine specificity of T cell receptor (TCR) interaction with the influenza hemagglutinin peptide HA 307–319 in the context of the DR1 (DRA, DRB1*0101) and DR4 (DRA, DRB1*0404) was studied in two human T cell clones (HA1.7 and Cl‐1) derived from different individuals. Sequencing of amplified TCR transcripts revealed that these two clones express highly related TCRα chains, with a conserved junctional motif, but very different TCRβ chains. Modeling studies led to the prediction that the conserved glutamic acid residue in the TCRα chain could interact with the lysine at position 316 in the peptide, a known TCR contact residue. HA1.7 TCR‐CD3ζ, chimeric constructs were expressed in the rat basophil line (RBL) and shown to confer specific antigen recognition. In two TCRα chain mutants, with the conserved glutamic acid residue altered to alanine and lysine, respectively, peptide recognition was lost. Specific recognition was not rescued by altered peptide ligands. Furthermore, Jurkat derivatives expressing the related Jurkat TCRα chain paired with the HA 1.7 TCRβ chain did not recognize the HA 307–319 /DR1 complex. These data provide evidence for the critical interaction of a TCR residue with antigenic peptide.
Vitamin A deficiency results in a priming environment conducive for Th1 cell development
Authors: Cantorna Margherita T. , Nashold Faye E. , Hayes Colleen E.
Published: March 2007

Certain infections, like that with the human immunodeficiency virus‐1, deplete vitamin A, and when vitamin A levels are low, immune dysfunctions establish susceptibility to further infection. Our research has focused on the immune dysfunctions that are a consequence of vitamin A deficiency and that predispose to further infection. We previously studied a helminth infection in mice, and showed that when vitamin A levels are low, the immune response develops a strong regulatory T cell imbalance with excessive T helper type‐1 cell interferon (IFN)‐γ synthesis and insufficient T helper type‐2 cell development and function. Here, we studied the T cell priming environment in vitamin A‐deficient mice to learn how that priming environment might produce a regulatory T cell imbalance and consequently distort the ability of the immune system to respond to an infection. Our results show that during vitamin A deficiency, the priming environment included constitutive interleukin (IL)‐12 and IFN‐γ transcripts, but it was devoid of constitutive IL‐4 and IL‐10 transcripts. Dietary all‐trans‐retinoic acid supplementation down‐regulated the level of constitutive IL‐12 and IFN‐γ transcripts. Furthermore, when T cells from naive vitamin A‐deficient animals were stimulated through the T cell receptor, they produced excess IFN‐γ protein compared to T cells from control animals. In contrast, T cell stimulation failed to induce IL‐4 or IL‐10 secretion. The inducible IFN‐γ was largely from CD8+ T cells and all‐trans‐retinoic acid addition in vitro inhibited IFN‐γ production at the transcript level. Retinoic acid addition in vitro also decreased natural killer cell IFN‐γ synthesis at the transcript level. Taken together, the distorted constitutive and inducible cytokine gene expression patterns that occurred when vitamin A levels were low would be expected strongly to favor T helper type‐1 development and limit T helper type‐2 cell growth and differentiation, thereby limiting the animal's humoral immune response capability.
Expression in vivo of CD45RA, CD45RB and CD44 on T cell receptor‐transgenic CD8+ T cells following immunization
Authors: Pihlgren Maria , Lightstone Liz , Mamalaki Clio , Rimon Galia , Kioussis Dimitris , Marvel Jacqueline
Published: March 2007

We used mice transgenic for a major histocompatibility complex class I‐restricted T cell receptor to study the changes of phenotype in vivo which follow priming by antigen of CD8 T cells. We show that following priming with peptide, CD44 on CD8 T cells is up‐regulated. The change of phenotype was relatively stable, as primed CD8 cells isolated from thymectomized mice 6 weeks after priming still expressed increased levels of CD44. CD8 T cells in these mice are still responsive to peptide and could represent long‐lived primed cells. No downregulation in vivo of the CD45RA or CD45RB isoforms was found, indicating that there is a differential regulation of the expression of CD44 and CD45RB by activated CD8 transgenic T cells. These results contradict earlier studies in vitro which showed that CD8 T cells which have been primed earlier belong to the CD45RA− or CD45RB− subset.
Primary proliferative T cell response to wild‐type p53 protein in patients with breast cancer
Authors: Tilkin Anne‐Françoise , Lubin Richard , Soussi Thierry , Lazar Vladimir , Janin Nicolas , Mathieu Marie‐Christine , Lefrère Isabelle , Carlu Christiane , Roy Marguerite , Kayibanda Michèle , Bellet Dominique , Guillet Jean‐Gérard , Paillerets Brigitte Bressac‐De
Published: March 2007

Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human tumors. These are mainly point mutations that lead to single amino acid substitutions. The mutated proteins have a longer half‐life than wild‐type p53 and accumulate in the nucleus of tumor cells. Anti‐p53 antibodies have been found in sera of patients with several types of cancers including breast cancer. This report describes a T cell immune response in three patients with breast tumors who had mutated p53 gene and accumulated p53 protein. All showed a humoral response to p53 protein and the T cells of these patients recognized the wild‐type p53 protein and proliferated in response to it. The data reported here are relevant to the immune processes leading to autoimmunity and have a bearing on anti‐p53 vaccine development in tumor immunology.
Induction of peptide‐specific primary cytotoxic T lymphocyte responses from human peripheral blood
Authors: Plebanski Magdalena , Allsopp Catherine E. M. , Aidoo Michael , Reyburn Hugh , Hill Adrian V. S.
Published: March 2007

Various protocols were developed and compared for eliciting specific cytotoxic T lymphocyte (CTL) cell lines from the unselected human peripheral blood mononuclear cells of naive donors. Interleukin‐7 and CD4+ T cells primed in vitro by keyhole limpet hemocyanin were shown to act together in the generation of these responses. Primary responses were consistently induced with a variety of different HLA class I‐binding malarial peptides. Primary CTL responses could be induced from unselected CD8+ and from CD45RA+CD8+ T cells. The CTL lines derived from these naive donors were CD8+ and demonstrated a high level of HLA class I‐restricted killing for > 3 months after priming in vitro. They were also able to recognize and kill targets infected with a recombinant vaccinia virus containing the full‐length antigen. In addition, this same protocol enhanced up to fourfold the levels of secondary CTL responses induced. The optimal method presented for naive cytotoxic T cell stimulation is simple, rapid and generally applicable and should provide a useful tool for both basic research and human therapy.
CD40 ligand‐independent B cell activation revealed by CD40 ligand‐deficient T cell clones: evidence for distinct activation requirements for antibody formation and B cell proliferation
Authors: Lane Peter , Burdet Corinne , McConnell Fiona , Lanzavecchia Antonio , Padovan Elisabetta
Published: March 2007

We report the capacity of CD40 ligand (CD40L)‐negative T cell clones to activate human B cells. CD40L‐negative T cells induce a level of B cell proliferation 10–20% of that seen with normal T cells. The signal provided by the negative clones is synergistic with that derived from a CD40L transfectant, and restores B cell proliferation to normal levels, showing that CD40L‐negative T cell clones are not inherently inhibitory for B cells. Although their capacity to induce proliferation was much reduced, CD40L‐negative T cell clones were still strong inducers of B cell differentiation to plasma cells. This differentiation to plasma cells was inhibited by a CD40L transfectant. The data are discussed with regard to the normal in vivo mechanism for maintaining B cell memory and memory antibody responses to T‐dependent antigens.
The self antigen heme evades immune recognition by sequestration in some hemoproteins
Authors: Sutherland Robyn M. , Brassell Stephen , Liu Qiang , Paterson Yvonne
Published: March 2007

Heme is a non‐protein autoantigen which is ubiquitous in vivo, primarily complexed in various hemoproteins or bound to specialized carrier molecules. Nevertheless, heme is able to stimulate a high frequency of CD4+, class II‐restricted T cells, freshly explanted from unprimed mice, to proliferate in vitro. In this study, we show that heme incorporated into various species of mammalian cytochrome c (cyt c), including murine cyt c, represents a facultative cryptic determinant, able to be recalled only at high doses of native cyt c. By contrast, avian cyt c is of comparable antigenicity to free heme. Artificially denatured carboxymethylated (CM) mammalian cyt c exhibited greatly increased antigenicity, comparable to that of heme and avian cyt c, indicating that the crypticity of heme in native mammalian cyt c is due to the resistance of the native conformation of this molecule to antigen processing within murine antigen‐presenting cells. Thus, tolerance to the heme group of at least some hemoproteins, may be maintained by the crypticity of the heme, rather than by deletion of hemereactive T cells. Given the high frequency of heme‐reactive T cells in unprimed mice, these findings suggest that heme may become an important modulator during an inflammatory response.
Destructive proteolysis by cysteine proteases in antigen presentation of ovalbumin
Authors: Rodriguez G. Marcela , Dimen Stephanie
Published: March 2007

Most native antigens require digestion by acidic proteases in order to be recognized in the context of major histocompatibility complex class II by T helper cells (Th). We have studied the roles of three different acidic proteases, cathepsin D, cathepsin B and cathepsin L, in the processing of ovalbumin (OVA) for presentation in the context of I‐Ad. We report that digestion of OVA in vitro with the aspartyl protease cathepsin D generates the epitope OVA322–336, which is recognized by I‐Ad‐restricted OVA‐specific Th in the presence of paraformaldehyde‐fixed antigen‐presenting cells (APC). In contrast, digestion of OVA with the cysteine proteases cathepsin B and L not only failed to generate an epitope, but also destroyed OVA322–336. In the presence of fixed APC expressing I‐Ad, OVA322–336 was protected from destructive proteolysis by cathepsin L. These results illustrate the dependence of epitope selection on the intracellular proteolytic environment in APC, and suggest that mechanisms must exist for protection of epitopes from destructive proteolysis in the processing compartments.