bluuebunny/crossref_metadata_2025 huggingface dataset:
CD45 modulates T cell receptor/CD3‐induced activation of human thymocytes via regulation of tyrosine phosphorylation
Authors: Turka Laurence A. , Kanner Steven B. , Schieven Gary L. , Thompson Craig B. , Ledbetter Jeffrey A.
Published: March 2007

Stimulation of thymocytes or mature T cells via the T cell receptor (TcR)/CD3 complex activates a cascade of processes inducing cells to enter the cell cycle. A key step is the activation of phosphatidylinositol‐specific phospholipase C (PI‐PLC) within seconds following TcR/CD3 stimulation, an event which is strongly enhanced by co‐ligation of the CD4 (or CD8) accessory molecule with TcR/CD3. In contrast, co‐ligation of CD45 inhibits the same TcR/CD3 responses. The machinery which couples the TcR/CD3 complex, CD4, and CD45 to PI‐PLC appears to involve regulation of tyrosine phosphorylation, as the TcR/CD3 and CD4 receptors are associated with the tyrosine kinases p59fyn and p56lck, respectively, and CD45 has intrinsic tyrosine phosphatase activity. Here, we have examined the ability of CD45 to regulate signal transduction via TcR/CD3 in human thymocytes. Co‐cross‐linking CD45 to the TcR/CD3 complex strongly suppressed the tyrosine phosphorylation of several intracellular substrates normally seen following TcR/CD3 stimulation. This effect of CD45 was associated with inhibition of a rise in intracellular calcium following TcR/CD3 ligation. Since TcR/CD3 stimulation of mature T cells induces tyrosine phosphorylation of PLCγ1, we investigated this phenomenon in thymocytes, and asked whether ligation of CD45 might regulate this process. By immunoprecipitation we found that TcR/CD3 stimulation induced tyrosine phosphorylation of PLCγ1, an effect which was enhanced by co‐cross‐linking CD4 to TcR/CD3. In contrast, co‐ligation of CD45 strongly blocked PLCγ1 phosphorylation induced by either stimulus. Consistent with previous findings in mature T cells, CD45 cross‐linking was able to partially inhibit TcR/CD3‐induced thymocyte proliferation when interleukin 2 was used as a second signal, but almost completely (80%–90%) blocked proliferation when anti‐CD28 mAb was used as the second signal, suggesting that CD45 cross‐linking may be able to block interleukin 2 production via the CD28 pathway. These effects of CD45 on TcR/CD3 signaling and proliferation in thymocytes point towards a potential role for this pathway in thymic selection.
T cell receptor diversity and activation markers in the Vδ1 subset of rheumatoid synovial fluid and peripheral blood T lymphocytes
Authors: Bucht Anders , Söderström Kalle , Hultman Thomas , Uhlén Mathias , Nilsson Ethel , Kiessling Rolf , Grönberg Alvar
Published: March 2007

In the present study we have characterized the γ/δ T cell receptor (TcR) population in synovial fluid (SF) and peripheral blood (PB) of patients with chronic inflammatory arthritis. By double staining we have shown that (a) synovial Vδ1+ cells have a high expression of activation markers CD45R0 (“memory cells”) and HLA‐DR as compared to PB, indicating a preactivated population of Vδ1‐carrying T cells in vivo and (b) interleukin 2‐induced expansion of synovial cells yields a high proportion of γ/δ in most samples expressing predominantly the Vδ1 TcR. Junctional sequence analysis of the TcR δ chain from interleukin 2‐expanded PB cell lines demonstrated a polyclonal Vδ1 population in three out of three samples. In SF cell lines three out of four samples were polyclonally expanded. In SF from one patient, however, a limited repertoire of expressed Vδ1 genes was found. Altogether, our data demonstrate the presence of preactivated Vδ1‐expressing cells in the synovial compartment. This Vδ1 population is predominantly polyclonal, except in one patient where oligoclonally expanded Vδ1 cells were detected.
Differential sensitivity of freshly isolated and cultured murine Langerhans cells to ultraviolet B radiation and chemical fixation
Authors: Tang Aimin , Udey Mark C.
Published: March 2007

Previous studies have demonstrated that low doses of ultraviolet B (UVB) radiation (100 J/m2) abrogate the accessory function of freshly isolated murine epidermal Langerhans cells (fLC) and cause a parallel decrease in the ability of LC to express increased amounts of ICAM‐1 (CD54) in vitro. We have subsequently observed that the accessory cell function of cultured LC (cLC), as assessed by their ability to support anti‐CD3 monoclonal antibody (mAb)‐induced T cell mitogenesis, was not inhibited by levels of UVB exposure (100 J/m2) that completely inhibited the function of fLC, although exposure of cLC to UVB radiation (100 J/m2) resulted in a decrease in the level of ICAM‐1 expression on most cLC and a concomitant decrease in cLC survival during a subsequent 24‐h incubation. Time course studies revealed that T cells stimulated with anti‐CD3 mAb in the presence of cLC became committed to proliferate 4‐8 h after culture initiation, while 24–30 h of co‐culture was required for irreversible T cell activation when fLC were utilized as accessory cells. In addition, paraformaldehyde (PFA)‐fixed (non‐viable) cLC supported anti‐CD3 mAb‐induced T cell proliferation, whereas PFA‐fixed fLC were ineffective. We propose that cLC are functionally resistant to low doses of UVB radiation and chemical fixation because cLC express sufficient levels of the adhesion or co‐stimulatory molecules [including ICAM‐1 and Mac‐1 (CD11b/CD18)] required to induce T cell activation. Conversely, fLC are sensitive to the effects of UVB radiation and chemical fixation because these physicochemical agents prevent acquisition of critically important surface molecules in culture.
Mapping of the human VLA‐α4 gene to chromosome 2q31‐q32
Authors: Fernández‐Ruiz Elena , de Villena Fernando Pardo‐Manuel , Rubio Miguel Angel , Corbi Angel L. , De Córdoba Santiago Rodriguez , Sánchez‐Madrid Francisco
Published: March 2007

The integrin VLA‐4 (α4: β1; CD49d/CD29) is involved in both cellular adhesion to extracellular matrix and cell‐cell interactions. We have determined that the human gene coding for VLA‐α4 is located on the long arm of chromosome 2 by using fluorescence in situ hybridization. The VLA‐α4 gene has been more precisely mapped to the 2q31‐q32 region after GTG banding (G‐bands by trypsin using Giemsa). These data suggest that the VLA‐4 gene belongs to the COL3A1‐(ELN‐FN)‐COL6A3 linkage group and establishes a potential genetic relationship between the α4 and αv integrin subunits.
Characterization of rat encephalitogenic T cells bearing non‐Vβ8 T cell recepotors
Authors: Sun Deming , Gold Daniel P. , Smith Lawrence , Brostoff Steven , Coleclough Christopher
Published: March 2007

In this study, we demonstrate that T cell lines specific for a synthetic peptide representing sequence 87 to 99 of myelin basic protein (MBP) are encephalitogenicin Lewis rats. However, unlike syngeneic T cells specific for MBP residues 68 to 88 which exclusively use Vβ8 in their antigen receptors, these cells do not. None of the 10 T cell lines and T hybridomas specific for MBP (87–99) used Vβ8 in their T cell receptors. Our results document for the first time that rat encephalitogenic T cells do not exclusively use Vβ8 in T cell receptors that rat encephalitogenic T cells specific for MBP (87–99) are heterogeneous and that MBP (87–99) contains at least two epitopes for rat T cells.
Extrathymic origin of Vγ1/Vδ6 T cells in the skin
Authors: Ota Yasuo , Kobata Tetsuji , Seki Makiko , Yagita Hideo , Shimada Shinji , Huang Ying‐Ying , Takagaki Yohtaroh , Okumura Ko
Published: March 2007

The dendritic epidermal cells (dEC) from young (8‐week‐old) athymic mice were found to express mRNA for γ/δ T cell receptors (TcR). The mRNA found in the dEC from athymic mice was expressed only for Vγ1‐Jγ4‐Cγ4 and Vδ6‐Jδ‐Cδ with both chains having productive rearrangements and junctional diversity. On the other hand, the dEC from euthymic mice carried Vγ5‐Jγ1‐Cγ1 and Vδ1‐Jδ2‐Cδ in addition to the Vγ1/Vδ6 subset found in athymic mice. Therefore, this Vγ1/Vδ6 subset, which has been implicated in the murine γ/δ T cell responses to mycobacterial antigens and self heat‐shock proteins, is the product of extrathymic maturation in the dEC from both athymic and euthymic mice.
Interleukin 6‐induced differentiation of a human B cell line into IgM‐secreting plasma cells is mediated by c‐fos
Authors: Körholz Dieter , Gerdau Stefan , Enczmann Jürgen , Zessack Norbert , Burdach Stefan
Published: March 2007

The role of the protooncogene c‐fos in interleukin (IL) 6‐induced B cell differentiation was assessed. Treatment of SKW 6.4 cells with IL 6 induced a transient and early stimulation of c‐fos sense mRNA expression. The effect appeared within 30 min and returned to basal levels after 2 h. The addition of antisense oligonucleotides to c‐fos significantly inhibited IL 6‐induced IgM production by SKW 6.4 cells (p < 0.001), whereas control oligonucleotides had no inhibitory effect. These results indicate that activation of c‐fos is involved in IL 6‐induced differentiation of SKW 6.4 cells into IgM‐secreting cells.
Gene transfer experiments imply instructive role of major histocompatibility complex class I molecules in cellular peptide processing
Authors: Wallny Hans‐Joachim , Rötzschke Olaf , Falk Kirsten , Hämmerling Günther , Rammensee Hans‐Georg
Published: March 2007

DBA/2‐derived mouse tumor cells were transfected with the H‐2Kb gene. Naturally processed minor histocompatibility (H) peptides were extracted from both transfected and non‐transfected cells by acid elution, and were separated by high‐performance liquid chromatography. Kb‐restricted minor H epitopes corresponding to H‐4b and mapki, both encoded by non‐major histocompatibility complex genes of DBA/2, were readily detected by the respective cytotoxic T lymphocyte in peptides extracted from Kb‐transfected, but not from non‐transfected or Db‐transfected cells. Titration experiments indicated at least 3000‐fold less copies of correctly processed Kb‐restricted epitopes in cells without Kb as compared to cells with Kb. Since we estimate the copy number of Kb‐restricted H‐4b epitopes in Kb‐expressing transfectants to be less than 1000 per cell, the pool size of H‐4b epitopes correctly processed in the absence of Kb should be less than 1/3 copy per cell.
High frequency of malaria‐specific T cells in non‐exposed humans
Authors: Zevering Yinka , Amante Fiona , Smillie Anne , Currier Jeff , Smith Gale , Houghten Richard A. , Good Michael F.
Published: March 2007

A major goal of current candidate malaria vaccines is to stimulate the expansion of clones of malaria‐specific lymphocytes. We have examined the in vitro T cell responses of a group of malaria exposed and non‐exposed adult Caucasian donors to recombinant circumsporozoite (CS) proteins, one of which is undergoing clinical trials, to blood‐stage parasites, and to synthetic peptides copying the CS protein and defined blood‐stage proteins. In nearly all individuals tested, CD4 T cell proliferation or lymphokine production occurred in response to whole parasite or CS protein stimulation, and T cells from many individuals responded to synthetic peptides. T cell responses were major histocompatibility complex‐restricted, and stimulation of T cells with malaria parasites or CS protein did not appear to expand a population of T cell receptor γ/δ cells. Malaria‐specific responses were independent of prior malaria exposure, and in some cases exceeded the magnitude of response to tetanus toxoid. Specific T cells are present in high frequency in the peripheral blood of many donors who have never been exposed to malaria. Although malaria‐specific CD4 T cells play an important role in immunity, these data question whether vaccines need to stimulate such cells, and focus attention on other aspects of malaria immunity which may be more critical to a successful vaccine.
Complement‐mediated follicular localization of T‐independent type‐2 antigens: the role of marginal zone macrophages revisited
Authors: van den Eertwegh Alfons J. M. , Laman Jon D. , Schellekens Marc M. , Boersma Wim J. A. , Claassen Eric
Published: March 2007

In this study we demonstrate a hitherto undescribed phenomenon, namely that thymus‐independent type‐2 antigens (TI‐2 Ag) localize in splenic follicles within 1 h after administration. The follicular localization of 2,4,6‐trinitrophenyl (TNP)‐Ficoll was not antibody mediated. In addition in case of high‐dose administration we observed a relatively large amount of TI‐2 Ag in marginal zone macrophages. However, after low‐dose administration we observed a preferential localization of TNP‐Ficoll in the splenic follicles. Detection of TNP‐haptenated Ag in cryostat sections of murine spleens was performed with a high‐affinity TNP‐specific monoclonal antibody conjugated to β‐galactosidase. Within minutes after injection the TI‐2 Ag localized in the marginal zone, attached to marginal zone macrophages and B cells. Twenty minutes after injection the Ag was also detected in the follicles and gradually accumulated there until 7 h after injection. Thereafter, the amount of follicular Ag gradually decreased but was still detectable up to 14 days after immunization. The follicular localization of TNP‐Ficoll was complement dependent in contrast to the binding to and uptake by marginal zone macrophages. Double staining revealed that Ag was bound by macrophages, B cells and follicular dendritic cells. Haptenated thymus‐dependent (TD) Ag localized exclusively in the red pulp macrophages. In vivo macrophage elimination drastically increased the amount of TNP‐Ficoll in the follicles, and enhanced the humoral immune response at low doses of Ag. Moreover, complement deprivation of mice abrogated the localization of TI‐2 Ag in the follicles, and led to a decreased humoral TI‐2 immune response. In conclusion, we demonstrate for the first time that TI‐2 Ag localize in follicles. Moreover, the presented results provide further evidence that B cells and follicular localized Ag play an important role in the induction of humoral TI‐2 immune responses.
Suppression of interleukin 2 secretion and interleukin 2 receptor expression during tsetse‐transmitted trypanosomiasis in cattle
Authors: Sileghem Maarten , Norman Flynn J.
Published: March 2007

Infection with Trypanosoma congolense in cattle was found to be associated with a profound suppression of the host's immune system. Lymph node cells from infected cattle were unable to secrete interleukin 2 (IL 2) in vitro following mitogenic stimulation and the exogenous supply of IL 2 did not restore T cell proliferative responses. This was associated with an impaired expression of the α chain of the IL 2 receptor (IL 2R α). Co‐culture experiments, where cells from an infected animal were mixed with cells from a major histocompatibility complex‐matched normal animal, demonstrated the presence of suppressor cells capable of blocking both IL 2 secretion and IL 2Rα expression. Removal of macrophages by fluorescence‐activated cell sorting abrogated suppression in such co‐cultures. Following depletion of macrophages, lymph node cells from an infected animal expressed IL 2Rα at a normal level, but remained incapable of producing IL 2. Hence, the unresponsiveness was associated with macrophage‐like suppressor cells which operated at the level of both IL 2 secretion and IL 2Rα expression, and to an intrinsic unresponsiveness of the T cells which was restricted to IL 2 secretion.

Inhibition of prostaglandin synthesis by addition of indomethacin failed to abrogate suppression of either IL 2 secretion or IL 2Rα expression. This revealed a major difference between the regulation of suppression in murine model infections where the suppression of IL 2 secretion is due to prostaglandin secretion, and the situation in cattle where prostaglandins would not appear to be involved.
Correlation of antibody multireactivity with variable region primary structure among murine anti‐erythrocyte autoantibodies
Authors: Conger John D. , Sage Harvey J. , Corley Ronald B.
Published: March 2007

A high proportion of the antibodies in the preimmune repertoire bind to several unrelated antigens and are considered to be multireactive. This property is reportedly associated with the antibodies produced by CD5+ B lymphocytes. Because many antibodies specific for bromelain‐treated mouse red blood cells (BrMRBC) derive from CD5+ B cells, we tested monoclonal antibodies of this specificity for multireactivity. Two variable region combinations, VH11/Vϰ9 and VH12/Vϰ4, account for >80% of this repertoire, but none of these antibodies exhibited a multireactive phenotype. In contrast, three anti‐BrMRBC binding antibodies belonging to the J558 family (BrM1, BrM8, and CH12) showed varying degrees of multireactivity, and bound both highly negatively and positively charged antigens. The amino acid sequences of the VH regions of these antibodies are highly homologous (greater than 85 % identical) and they possess large VH‐D‐J junctions with extensive N‐region insertions. The ϰ chains of two of these antibodies utilize an identical Vϰ gene segment, while the third uses a very different Vϰ with only 50 % homology. The entire H chain V regions of these antibodies are unusually basic, with isoelectric points of 9.5–10, a feature which might be important in promoting interactions with acidic epitopes. The multi‐reactive antibodies also contain regions with a high concentration of hydroxylside chain amino acids, especially in their VH‐D‐J junctions. This region also contains acidic amino acid residues, which may be important in binding of positively charged epitopes. We propose that an open, accessible binding site and a charge polarity may be features which facilitate the binding of charged epitopes, providing a structural basis for multireactivity of at least some antibodies.
Expression of the murine interleukin 6 receptor in hepatoma cells: the intracytoplasmic domain is not required for interleukin 6 signal transduction
Authors: Fiorillo Maria Teresa , Toniatti Carlo , Van Snick Jacques , Ciliberto Gennaro
Published: March 2007

This paper reports on cDNA coding for the 80‐kDa murine IL 6 receptor (mIL 6R) that was cloned from a mouse liver cDNA library. Human hepatoma Hep3B cells transfected transiently or stably with an expression vector carrying the entire coding region for mIL6R become responsive to mouse IL 6 (mIL 6). We monitored response to the cytokine through the transcriptional activation of a co‐transfected IL 6‐inducible human C‐reactive protein (CRP) promoter; response to mIL 6 is lost upon treatment of the cells with increasing amounts of a monoclonal antibody to mIL 6R. mIL 6R mutants have been generated in the carboxy‐terminal portion of the molecule. Their functional analysis in hepatoma cells shows that the intracytoplasmic domain of the receptor is not absolutely essential to IL 6 signal transduction (i.e. CRP promoter activation), but that the last 40 amino acids contribute to maximal IL 6 response in these cells.
Tolerance in the mouse to major histocompatibility complex‐mismatched heart allografts, and to rat heart xenografts, using monoclonal antibodies to CD4 and CD8
Authors: Chen Zonghua , Cobbold Stephen , Metcalfe Su , Waldmann Herman
Published: March 2007

Mice given a single short course of anti‐CD4 and anti‐CD8 monoclonal antibodies (mAb) became tolerant of major histocompatibility complex (MHC)‐incompatible vascularized heart allografts in a donor‐ and organ‐specific manner. T cell depletion was not important, as blocking antibodies were equally effective. Anti‐CD4 antibody therapy alone was sufficient to establish tolerance. Anti‐CD8 mAb therapy alone was associated with poor recipient survival, although survivors were rendered tolerant. A second donor‐type heart allograft to the neck was always accepted in recipients which had carried the first abdominal heart allograft for over 120 days, with the first heart also continuing to function. However, donor‐type skin grafted at 100 days was sometimes rejected, albeit chronically. Those recipients that accepted the donor‐type skin were able to reject third‐party skin grafts mismatched at the MHC, or over multiple minor differences alone. Thus, the tolerance induced appeared to be donor and tissue specific, although spreading in some cases to tolerance of skin. Similarly donor‐specific tolerance was found when xenogeneic PVG rat hearts were grafted into CBA/Ca mice using the same protocols of anti‐CD4 plus anti‐CD8 mAb. Resolution of the mechanisms underlying these forms of tolerance may permit the design of improved immunosuppressive protocols for human organ transplantation.
The glycosylphosphatidylinositol‐linked Fcγ receptor III represents the dominant receptor structure for immune complex activation of neutrophils
Authors: Hundt Matthias , Schmidt Reinhold E.
Published: March 2007

Polymorphonuclear neutrophils (PMN) express constitutively two low‐affinity Fcγ receptors, FcγRII and FcγRIII. FcγRII is a transmembrane molecule, and FcγRIII is linked via a glycosylphosphatidylinositol (GPI) anchor to the membrane. The role of each of these receptors in activation of PMN is still unclear. We used specific cross‐linking of FcγRII via Fab fragments of IV.3 (anti‐FcγRII, CDw32) and of FcγRIII using F(ab')2 fragments of 3G8 (anti‐FcγRIII, CD16) to activate PMN. Stimulation of FcγRIII was significantly more effective in inducing a respiratory burst than cross‐linking of FcγRII. A synergistic effect was observed after simultaneous activation of FcγRIII. We could demonstrate that both FcγR mobilize calcium as intracellular signal in spite of their different membrane linkage. The kinetic of calcium mobilization after FcγR stimulation is delayed in comparison to formyl‐methionyl‐leucyl‐phenylalanine activation. In addition FcγR‐induced increase of cytoplasmic calcium is pertussis toxin insensitive. When monoclonal IgG1χ complexes were used for stimulation calcium mobilization and hydrogen peroxide (H2O2) production could also be demonstrated. Inhibition studies of this activation using monoclonal antibodies suggested that this immune complex activation was predominantly mediated via FcγRIII. Only in FcγRIII‐deficient PMN from paroxysmal nocturnal hemoglobinuria patients could a decreased H2O2 production be demonstrated to be FcγRII dependent. In normal PMN the GPI‐anchored FcγRIII structure is the predominant receptor.
Interleukin (IL)‐2 activation of p21ras in murine myeloid cells transfected with human IL‐2 receptor β chain
Authors: Izquierdo Manuel , Downward Julian , Otani Hiroki , Leonard Warren J. , Cantrell Doreen A.
Published: March 2007

The T cell growth factor interleukin‐2 (IL‐2) induces p21ras activation in T lymphocytes. To determine whether the IL‐2 receptor (IL‐2R) can regulate p21ras when expressed in a non‐T cell environment we have examined the ability of IL‐2 to activate p21ras in 32D murine myeloid progenitor cells transduced with human IL‐2R β chains. These cells are denoted β53 cells. 32D cells normally proliferate in response to IL‐3 but the expression of the IL‐2R β chain confers IL‐2 responsiveness to the cells. Our data show that IL‐3 is able to activate p21ras in the parental 32D cells and both IL‐2 and IL‐3 can stimulate p21ras in the IL‐2R‐expressing β53 clone of 32D. In T lymphocytes, activation of protein kinase C (PKC) with phorbol esters is sufficient to stimulate p21ras. However, in 32D and β53 cells activation of PKC with phorbol esters does not result in p21ras activation even though these cells express functional PKC. It appears, therefore, that a PKC‐mediated pathway for p21ras regulation exists in T lymphocytes but not in 32D cells. The IL‐2R can couple to p21ras independently of the concomitant presence of the PKC pathway for p21ras regulation. These data imply that multiple intracellular mechanisms may exist to regulate p21ras and that cells of different lineages may differ with regard to p21ras regulation.
T cells from atopic individuals produce IgE‐inducing activity incompletely blocked by anti‐interleukin‐4 antibody
Authors: Zhang Xiaohong , Polla Barbara , Hauser Conrad , Zubler Rudolf H.
Published: March 2007

We investigated peripheral blood B and T lymphocyte functions in atopic individuals. B cells were co‐cultured with mutant EL4 thymoma cells in the presence of a standard T cell supernatant (T‐SN) with or without exogenous interleukin (IL)‐4. IgE secretion in this assay was found to be IL‐4 dependent, but not significantly different for atopic patients (n = 25) vs. normal controls (n = 25). Phytohemagglutinin plus phorbol 12‐myristate 13‐acetate (PHA+PMA)‐induced T‐SN from patients or controls was tested on normal B cells in the same assay system (in the absence of exogenous IL‐4). Compared to the controls, the IgE‐inducing activity was significantly increased for patients with asthma or allergic rhinitis (n = 12; p<0.005) but not for patients with atopic dermatitis (n = 13). The difference between the asthma or allergic rhinitis vs. the atopic dermatitis groups was significant (p>0.05). Since the assay was not inhibited by interferon (IFN)‐γ, this difference can not be attributed to IFN‐γ concentrations. Other T cell activities may be different between the patient groups or atopic T cells from the respiratory mucosa may recirculate more than those from the skin. In any case, the T cells rather than the B cells were found to be abnormal in atopic individuals.

If atopic T cells were stimulated with PHA + PMA not as immediately but after a resting period of 48 h in culture medium alone, the IgE‐inducing activity, but not the total Ig‐inducing activity or the IL‐2 secretion, disappeared. In addition, a mean of 37 % of the IgE‐inducing activity (range of 13 % to 79 % for five very active T‐SN) was not inhibited by an anti‐IL‐4 antibody which neutralized exogenous IL‐4, indicating a participation of factors capable of bypassing the requirement for IL‐4 for the IgE response.
Antigen contact sites in class I major histocompatibility complex‐restricted, trinitrophenyl‐specific T cell receptors
Authors: Weltzien Hans Ulrich , Hebbelmann Sabine , Pflugfelder Ulrike , Ruh Helga , Ortmann Bodo , Martin Stefan , Iglesias Antonio
Published: March 2007

Cloned trinitrophenyl (TNP)‐specific cytotoxic T cells (CTL) were obtained from mice transgenic for the β chain of the antigen‐specific receptor (TcR) of a Kb‐restricted, TNP‐specific CTL clone (BT7.4.1). The transgene‐expressing CTL, specific for TNP/Kb were found to select for TcR α chains highly similar to that of the transgene donor clone BT7.4.1. In that way, two clones (II/7 and III/1) were identified whose TcR differed from the BT7.4.1 receptor only in their Nα‐ and Jα‐sequences, i.e. within the third complementarity‐determining regions of their α chains (CDR3α). Moreover, the TcR of clones II/7 and III/1 had both rearranged the same Jα element, thus differing from each other by only two amino acids in their Vα/Jα junctional regions. Functionally, however, clone III/1 exhibited unique cytolytic specificities for synthetic, Kb‐binding TNP‐peptides as well as for chemically TNP‐modified allogeneic (H‐2k) target cells. These findings demonstrate that (a) similar to “conventional” peptide antigens, synthetic hapten‐peptide determinants are contacted by CDR3α‐determined amino acids of the TcR and (b) in contrast to current models, CDRα also appears to influence the major histocompatibility complex restriction specificity of a given TcR.
Cell selection strategies for making antibodies from variable gene libraries: trapping the memory pool
Authors: Hawkins Robert E. , Winter Greg
Published: March 2007

The B cells of immunized animals can be used as a source of variable region (V) gene libraries. Such libraries offer a way of making antibodies directly in bacteria: rearranged V genes are amplified using the polymerase chain reaction, cloned and expressed as soluble fragments in bacteria, and then screened for antigen binding. Here we have used a model system to investigate antigen‐selected B cells as a source of V gene libraries. Mice were immunized with (4‐hydroxyl‐3‐nitrophenyl)acetyl (NP)‐chicken gammaglobulin, and the splenocytes harvested seven days after primary immunization. We prepared a heavy chain variable (VH) gene library from the DNA of cells selected on antigen‐coated magnetic beads, and two other libraries from the DNA or mRNA of unselected cells. The VH gene libraries were combined with the Vλ1 gene (as this light chain dominates the primary response to NP), expressed as Fv fragments in Escherichia coli and screened for binding to (4‐hydroxy‐3‐iodo‐5‐nitrophenyl)acetyl‐bovine serum albumin. The frequency of antigen‐binding clones was much greater (>50 fold) in the library from the DNA of antigen‐selected cells (17/282) or from the mRNA of unselected cells (29/282) compared to the DNA from unselected cells (0/940). Sequencing of the antigen‐binding clones revealed that they almost invariably used the V‐186.2 heavy chain, as expected from analysis of primary response hybridomas. The D segments from the mRNA library were entirely DFL16.1 (29/29), as in primary response hybridomas, whereas those from the DNA of selected cells were more diverse, using in addition to DFL16.1, other D segments (5/17) as in later response hybridomas. This suggests that the DNA library from selected cells is derived at least in part from cells destined for the memory compartment. Given the long life of memory cells, they may prove a useful source of antibody libraries in the absence of recent immunization.
Existence of mature human CD4+ T cells with genuine class I restriction
Authors: de Bueger Marleen , Bakker Astrid , Goulmy Els
Published: March 2007

A human T cell receptor (TcR) α/β CD4+CD8− T cell clone (R416) is reactive with the minor histocompatibility antigen H‐Y in the context of major histocompatibility complex (MHC) class I and not class II molecules. Therewith clone R416 violates the so‐called specificity association of mature TcR α/β+ T cells. R416 displays H‐Y‐specific, HLA‐A2‐restricted proliferation as well as cytotoxicity in vitro. Its fine specificity is identical to that of a classical H‐Y‐reactive CD4−CD8+ MHC class I‐restricted CTL clone, showing that CTL expressing either CD4 or CD8 can display identical antigenic specificities. Exploiting the MHC class I restriction of this CD4+ T cell clone, it was found that interaction of CD4 with non‐TcR‐bound MHC class II molecules does not contribute to antigen specific activation of these CD4+ T cells. This coreceptor‐mismatched T cell clone was not generated in vitro but obtained by expansion of CD8‐depleted peripheral blood mononuclear cells of a female who had been immunized against H‐Y. The existence of such MHC class I‐restricted mature TcR α/β+ T cells expressing CD4 and not CD8 is relevant because it indicates that the generally accepted model for thymic selection, in which the TcR specificity alone determines CD4/CD8 expression of mature thymocytes, may not be absolute.
Biochemical analysis of the mouse mammary tumor virus long terminal repeat product. Evidence for the molecular structure of an endogenous superantigen
Authors: Knight Andrew M. , Harrison Georgina B. , Pease Richard J. , Robinson Peter J. , Julian Dyson P.
Published: March 2007

Recent reports have shown that both exogenous and endogenous mouse mammary tumor viruses (MMTV) can encode superantigens. Transfection and transgenic studies have identified the open reading frame (ORF) present in the 3′ long terminal repeat (LTR) as encoding superantigen function. In this study, we have used an in vitro translation system in an attempt to characterize the molecular nature of the protein encoded by the 3′ ORF of Mtv‐8. Using various constructs encoding full‐length and truncated versions of the ORF product, we report that the hydrophobic region close to the amino terminus of the 36‐kDa protein can function as a transmembrane domain. Protease digestion experiments also demonstrate that the protein has a type‐II transmembrane conformation with an extra‐cytoplasmic carboxy terminus. Since this hydrophobic region is conserved between all known MMTV, we speculate that LTR ORF, including those proposed to encode the minor lymphocyte stimulatory antigens, are also capable of encoding type‐II transmembrane glycoproteins. The polymorphism between MMTV LTR ORF products, which correlates with deletion phenotypes, is predominantly in the carboxy‐terminal extracellular region, consistent with a major role in interaction with the T cell receptor.
Identification of the nonamer peptide from influenza A matrix protein and the role of pockets of HLA‐A2 in its recognition by cytotoxic T lymphocytes
Authors: Morrison Joanne , Elvin John , Latron France , Gotch Frances , Moots Robert , Strominger Jack L. , McMichael Andrew
Published: March 2007

Influenza matrix peptide 58–66 is shown to be the optimal nonamer for binding to HLA‐A2 and presentation to cytotoxic T lymphocytes (CTL). If titered out to 2 × 10−10 – 4 × 10−10M in CTL‐mediated lysis assays and to 3 × 10−9M in an HLA‐A2 assembly‐stabilization assay in cell lysates. The peptide was shown to make probable contacts with its carboxy terminus close to residue 116 in the floor of the cleft of HLA‐A2, close to the F pocket. The side chain of the amino‐terminal amino acid was unimportant, but its free amino and carbonyl groups in the A pocket appeared important in optimizing peptide presentation. The B pocket probably accommodates the side chain of residue 2 (isoleucine) and was shown to be critical in peptide presentation.
Human U937 cell surface peptidase activities: characterization and degradative effect on tumor necrosis factor‐α
Authors: Bauvois Brigitte , Sancéau Josiane , Wietzerbin Juana
Published: March 2007

Surface peptidase activities on the human monocytic lineage cell line U937 were characterized. Two diisopropyl phosphofluoridate (DFP)‐inhibitable serine peptidases were identified by differences in their hydrolytic activities on chromogenic peptides: one removed tripeptides from the free NH2‐terminal end of the synthetic peptide Ala‐Ala‐Phe‐p‐nitroanilide (pNA) and was not inhibited by inhibitors of metallo‐, cysteic‐, and aspartic‐proteinases, or by those of elastase‐, trypsin‐ and chymotrypsin‐like enzymes, suggesting the presence of a hitherto unidentified serine tripeptidyl endopeptidase; the other peptidase catalyzed the release of Gly‐Pro from Gly‐Pro‐pNA and was inhibited by DFP, phenylmethyl sulfonyl fluoride and diprotin A, thus resembling dipeptidyl peptidase IV (DPP IV) with respect to its substrate specificity and inhibitor profile. A group of N‐exoaminopeptidase activities specifically inhibited by bestatin, was also detected when Ala‐, Leu‐, Arg‐ and Lys‐pNA were used as substrates. The activities were surface associated and not secreted as determined by extracellular location of product and enzymatic recovery in highly purified U937 cell membranes. Peripheral monocytes and macrophages were found to virtually exhibit identical levels of these two classes of peptidase activities when compared to those detected on U937 cells. The relative contributions of these hydrolytic enzymes to the cleavage of bioactive and radioiodinated cytokines including tumor necrosis factor‐α (TNF‐α), interleukin‐1α and interferon‐γ was next examined. The results indicated that N‐aminopeptidases do not appear to participate in the catabolism of any tested cytokine. In contrast, the most interesting finding was that both serine peptidases participate in TNF‐α degradation. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native 17‐kDa molecule TNF‐α, and the concomitant release of biologically inactive fragments of ≤2 kDa. Together, these observations indicate new roles for both the DPP IV‐like enzyme and the tripeptidyl endopeptidase located at the surface of human monocytic cells, including the regulation of the extracellular TNF‐α concentration. Thus, the identification of functional ectopeptidases provides insight into their potential role in both normal and malignant monocytic function.
T cell epitope selection: dominance may be determined by both affinity for major histocompatibility complex and stoichiometry of epitope
Authors: Li Wan‐Fen , Fan Ming‐Dau , Pan Ching‐Biau , Lai Ming‐Zong
Published: March 2007

The majority of T cell hybridomas produced in the BALB/c mouse in response to immunization with λ, repressor cI recognize a peptide fragment comprising of residues 12 to 26 (P12–26). Some other parts of the cI (P1–14, P33–48 and P73–88) are defective in generating T cell responses in the BALB/c mouse. P73–88 may be converted into a T cell determinant if a few more amino acid residues are included (P67–88). Together with P46–67 and P80–102, most peptides derived from cI were capable of eliciting T cell responses by themselves in BALB/c mouse. The mechanisms underlying the selection of P12–26 over the other epitopes when λ repressor was used as immunogen were examined. The dominant response to P12–26 was attenuated by tolerizing with intravenous administration of P12–26. Under such treatment the T cell response to P12–26 was reduced by 80% but there was no enhancement on the responses toward other epitopes. The selection of P12–26 is, thus, unlikely to be due to a competition at the T cell level. It was also found that the dominance of P12–26 was not simply due to a higher affinity of P12–26 for major histocompatibility complex molecules. For example P12–26 binds better to I‐Ad molecule than P80–102, but co‐injection with equimole of P12–26 only slightly inhibited P80–102‐induced T cell response. Instead, it required a few molar excess of P12–26 to effectively block the association of P80–102 with I‐Ad molecules and to inhibit the T cell immunity to P80–102. Since epitopes such as P46–67, P67–88 and P80–102 were generated from λ repressor cI at a lower molar basis than that of P12–26, it is suggested that the dominance of P12–26 was probably generated by such stoichiometry difference, in addition to the higher affinity of P12–26 for I‐Ad molecules.
Selective inhibition of immune complex trapping by follicular dendritic cells with monoclonal antibodies against rat C3
Authors: van den Berg Timo K. , Döpp Ed A. , Daha Mohamed R. , Kraal Georg , Dijkstra Christine D.
Published: March 2007

The role of complement component C3 in the trapping of immune complexes by follicular dendritic cells (FDC) was studied in the rat, by means of the C3‐specific monoclonal antibody ED11. Immunocytochemistry revealed the presence of C3 on FDC, where it co‐localized with trapped peroxidase anti‐peroxidase complexes. Furthermore, C3 was detected on reticular cells occupying the T cell areas of peripheral lymphoid organs, which are not involved in the handling of immune complexes. The in vivo administration of anti‐C3 abolished the trapping of immune complexes in splenic follicles, but was unable to release preexisting complexes from the FDC. Trapping of immune complexes was also prevented by treatment of rats with cobra venom factor (CoVF). While CoVF caused massive depletion of C3 from serum, ED11 treatment had no such effect. The effect of anti‐C3 appeared at least in part to be due to an inhibition of complement activation by immune complexes. We also analyzed earlier stages of the trapping process, with respect to their C3 dependence. Upon systemic injection immune complexes are initially observed in the marginal zone. Administration of anti‐C3 reduced this localization, indicating a role for C3 in the entry of immune complexes into the spleen. Our results confirm experiments in CoVF‐treated animals and extend the evidence for a role of C3 in the follicular trapping process using anti‐C3 in vivo. The mechanism of immune complex trapping and the role of complement therein is discussed.
The glucocorticoid antagonist RU38486 mimics interleukin‐1 in its sensitization to the lethal and interleukin‐6‐inducing properties of tumor necrosis factor
Authors: Brouckaert Peter , Everaerdt Bart , Fiers Walter
Published: March 2007

Although tumor necrosis factor (TNF) is undoubtedly a major mediator of the antitumor and shock‐inducing activities of lipopolysaccharide (LPS), the outcome of a challenge with TNF is highly dependent on the presence or absence of other substances or conditions. We have previously shown that to obtain lethality in mice after TNF administration both TNF receptor (TNF‐R) types have to be triggered. This is illustrated by the fact that recombinant human (rh) TNF, which is a selective murine (m) TNF‐R55 agonist, is not lethal, whereas mTNF, which binds both mTNF‐R55 and mTNF‐R75, is lethal in mice. Triggering of TNF‐R75 is, however, no longer needed when sensitizers such as galactosamine or low doses of LPS or interleukin (IL)‐1 are also present. Here, we report that this selective species specificity of TNF is also reflected in patterns of induced IL‐6: both rmTNF and rhTNF could induce considerable IL‐6 peak levels in the plasma (up to 10 ng/ml) 2 to 3 h after TNF administration. However, only rmTNF was capable of inducing the same pattern of sustained IL‐6 levels previously observed after lethal LPS doses, while rhTNF only caused induction of transient IL‐6 levels, as found after nonlethal LPS doses. We also observed that the sensitizer IL‐1 could complement rhTNF to induce such a sustained IL‐6 induction. Since we were interested in sensitizers with a defined mechanism of action, we further investigated the effects of the glucocorticoid and progesterone inhibitor RU38486 on the lethal and IL‐6‐inducing properties of TNF. We observed that RU38486 closely mimicked IL‐1: both had similar effects on IL‐6 induction and sensitized mice to the lethal effects of TNF with comparable efficiency and kinetics. Using a monoclonal anti‐IL‐1R antibody, we finally observed that the effects of RU38486 were most probably not mediated by IL‐1. These observations suggest that a glucocorticoid‐antagonistic activity might be a key factor in the pathways leading to septic shock and that such activity could be a key target for the pharmacological manipulation of sepsis.
Peripheral tolerance to an islet cell‐specific hemagglutinin transgene affects both CD4+ and CD8+ T cells
Authors: Lo David , Freedman Jessica , Hesse Sabine , Palmiter Richard D. , Brinster Ralph L. , Sherman Linda A.
Published: March 2007

To study the basis for immunological tolerance of peripheral tissue‐specific antigens, a transgenic mouse line was established that expresses the influenza hemagglutinin (HA) on pancreatic islet β cells (Ins‐HA transgenic mice). When followed up to 14 months of age, Ins‐HA transgenic mice did not develop spontaneous autoimmune disease. Upon immunization with HA‐expressing viruses, high titers of HA‐specific circulating antibody were detected; however, T cell responses by both the T helper and T cytolytic compartment were markedly reduced as compared with transgene‐negative littermates, and no evidence could be found for islet infiltrates. Adoptive transfer of histocompatible lymphocytes from transgene‐negative mice plus virus into irradiated Ins‐HA hosts resulted in islet inflammation dominated by CD4+ T cells, indicating that the HA antigen was accessible to activated T cells. These results suggest that T cells can be rendered tolerant of antigens expressed outside the thymus.
Fluids from immune privileged sites endow macrophages with the capacity to induce antigen‐specific immune deviation via a mechanism involving transforming growth factor‐β
Authors: Wilbanks Garth A. , Wayne Streilein J.
Published: March 2007

The eye, brain, and fetoplacental unit within the pregnant uterus are immunologically privileged sites that contain unique fluids with suspected immunoinhibitory properties. Aqueous humor, which is normally present within the anterior chamber (AC) of the eye, has been shown to suppress antigen‐driven T cell activation, and to contain significant amounts of transforming growth factor β‐2 (TGF‐β). Antigens injected into the AC of normal mice induce a deviant form of systemic immunity, termed anterior chamber‐associated immune deviation (ACAID), which is characterized by a selective inability to display antigen‐specific delayed hypersensitivity. It has recently been reported that eye‐derived macrophages appear in the blood following AC injection of a soluble antigen, and that these cells can induce antigen‐specific ACAID when injected into naive, syngeneic recipients. Moreover, antigen‐pulsed peritoneally derived macrophages that are exposed in vitro to aqueous humor, supernatants of cultured iris and ciliary body cells (I/CD), or TGF‐β have been found to assume ACAID‐inducing properties. In the present experiments, amniotic fluid and cerebrospinal fluid from mice, rats and humans, as well as supernatants from cultured I/CB cells, have been examined for their capacity to confer ACAID‐inducing properties on peritoneal macrophages. It was found that each of these biologic fluids, but neither normal mouse serum nor rat thoracic duct lymph, was able to endow antigen‐pulsed peritoneal macrophages with ACAID‐inducing properties. Moreover, fluids from these immune privileged sites were found to contain latent and/or active TGF‐β, as determined by bioassay, using neutralizing anti‐TGF‐β antibodies. It is concluded (a) that the immune privileged states of the eye, the brain, and the fetoplacental unit share common features, and possess unique fluids with a similar capacity to force macrophages to present antigens in a “deviant” manner and (b) that this capacity is mediated, at least in part, by TGF‐β. These results are discussed in terms of the potential physiologic and pathophysiologic significance of immune privilege in these three specialized tissues.
Does co‐aggregation of the CD45 and CD3 antigens inhibit T cell antigen receptor complex‐mediated activation of phospholipase C and protein kinase C?
Authors: Shivnan Emer , Biffen Mark , Shiroo Masahiro , Pratt Edward , Glennie Martin , Alexander Denis
Published: March 2007

The binding of agonistic monoclonal antibodies (mAb) to the CD3 antigen in T cells induces a rapid increase in tyrosine phosphorylation, inositive phosphate (IP) production, a rise in intracellular calcium and protein kinase C (PKC) activation. These intracellular signals have been implicated in the control of interleukin‐2 and interleukin‐2R receptor gene expression, thereby regulating T cell proliferation. Previous studies have shown that co‐ligation of the CD45 and CD3 antigens inhibits CD3‐induced tyrosine phosphorylation, IP production, calcium signals and T cell proliferation. It has therefore been suggested that the CD45 antigen uncouples the T cell receptor (TcR) from mitogenic signal pathways. In this study co‐ligation of the CD3 and CD45 antigens with precisely constructed bispecific mAb did not inhibit CD3‐induced T cell proliferation, IP production, calcium signals, diacylglycerol production or PKC activation. Furthermore, co‐ligation of CD3 and CD45 antigens already cross‐linked with IgM mAb did not lead to inhibition of CD3‐induced calcium signals. Inhibitions of CD3‐induced intracellular signals were observed following co‐ligation of IgG CD45 and CD3 mAb with anti‐IgG F(ab')2 fragments. However, comparable inhibitions were also noted following co‐ligation of CD3 with other abundant cell‐surface antigens such as CD5 and LFA‐1, and inhibitions were only observed when the CD3 mAb used required cross‐linking to induce signals. These results suggested that the inhibitory effects of CD45 IgG mAb were not specific and were caused by the prevention of CD3‐CD3 cross‐linking following CD3 antigen co‐ligation with other cell surface molecules. These findings are inconsistent with a specific inhibitory role for the CD45 phosphotyrosine phosphatase in uncoupling the TcR from mitogenic signal pathways.