bluuebunny/crossref_metadata_2025 huggingface dataset:
Parameters affecting the immunogenicity of a liposome‐associated synthetic hexapeptide antigen
Authors: Frisch Benoit , Muller Sylviane , Briand Jean Paul , Van Regenmortel Marc H. V. , Schuber Francis
Published: March 2007

The effect of liposome association on the immunogenicity of the hexapeptide IRGERA was investigated. When administered in the absence of a carrier and adjuvant this peptide, which corresponds to a linear epitope located at the C‐terminus of histone H3, was not immunogenic. When mice were immunized with the peptide covalently linked to the surface of small unilamellar vesicles containing monophosphoryl lipid A as adjuvant, a relatively long‐lasting response with memory cell induction was observed. The anti‐peptide antibodies raised in this way reacted with the cognate sequence in the native histone. In contrast, coupling of the peptide to the surface of large vesicles yielded both an IgM and IgG response of short duration whereas encapsulation of the free peptide in large vesicles was ineffective. These results indicate that with short synthetic peptides, liposomes provide a substitute for a carrier protein. However, an adjuvant has to be incorporated in the vesicles in order to obtain an efficient immune response. Such an approach may be useful for designing synthetic vaccines.
A single amino acid substitution in the Ak molecule fortuitously provokes an alloresponse
Authors: Dellabona Paolo , Wei Bing‐Yuan , Gervois Nadine , Benoist Christophe , Mathis Diane
Published: March 2007

We discovered by chance that the R28 T cell hybridoma has dual specificity. It responds to a peptide derived from ribonuclease presented by cells displaying Ak molecules and it reacts, in the absence of added antigen, to cells expressing Ak complexes with a single amino acid substitution at position 69 of the α chain. Modelling and functional studies suggest that residue 69 is a peptide contact residue, prompting the hypothesis that R28′s alloreactivity is a cross‐reactive response to an unknown peptide bound in the ‘groove’ of the mutant Ak complex. In this report, we employ a competition assay to confirm that this alloresponse involves a groove‐binding peptide, demonstrate that this peptide derives from or depends on fetal calf serum and exploit a panel of antigen‐presenting cell lines – each displaying an Ak complex with a different position 69 substitution – to establish that the alloresponse is not just a heteroclitic response to ribonuclease, itself. We speculate that much of the alloreactivity against murine class II molecules that is revealed in vitro may prove to be directed at bovine serum‐derived peptides, suggesting that in this context, alloreactivity is a misnomer.
Cyclosporin A renders target cells resistant to immune cytolysis
Authors: Hudnall S. David
Published: March 2007

Exposure of cytolytically susceptible human target cells with therapeutic concentrations of the immunosuppressive drug cyclosporin A renders these cells highly resistant to T cell‐mediated, natural killer (NK) cell‐mediated, and complement‐mediated cytolysis. The resistance is dose dependent, time dependent and reversible. The resistance is accompanied by target cell growth inhibition as measured by thymidine uptake. Surprisingly, target cell growth inhibition induced by serum depletion is associated with cell‐mediated cytolytic resistance. These data suggest that cyclosporin A (CsA) may block some target cell biochemical pathway(s) important in the suicidal cytolytic process which is (are) linked to some G0/G1 cell cycle events. In addition, these results suggest that the increased risk of Epstein‐Barr virus (EBV)‐associated lymphoproliferative disease in human organ transplant recipients may be contributed to by CsA‐induced resistance of EBV‐transformed B lymphocytes to immune cytolysis.

In the post‐transplant setting, CsA probably blocks T cell‐dependent responses to EBV‐transformed B lymphocytes (Bird, A. G., McLachlan, S. M. and Britton, S., Nature 1981. 289: 300) yet leaving the NK cell and antibody‐dependent responses intact (Shao‐Hsien, C. et al., Transplantation 1983. 35: 127). However, given the direct effect of CsA upon EBV‐transformed B lymphocytes, these cells would be rendered resistant to nearly all forms of cytolytic immune control (cytotoxic T lymphocyte, natural killer, antibody‐dependent cell‐mediated cytotoxicity, complement). Unregulated EBV‐transformed B lymphocytes may then proliferate in the CsA‐treated host thus leading to a polyclonal B cell hyperplasia. Our data would suggest that this early pre‐malignant process is likely to be reversible following CsA dose reduction. Indeed, EBV‐dependent polyclonal B cell hyperplasia is seen in early post‐transplant lymphoproliferative disorders (Hanto, D. W., et al., Transplantation 1989. 47: 458). Furthermore, in some cases CsA dose reduction does lead to disease regression (Starzl, T., et al., Lancet 1984. i: 583). However, further progression of the disease probably occurs following chromosomal changes leading to oncogene activation and might be resistant to CsA dose reduction.
Cytokine interactions in experimental cutaneous leishmaniasis. Interleukin 4 synergizes with interferon‐γ to activate murine macrophages for killing of Leishmania major amastigotes
Authors: Bogdan Christian , Stenger Steffen , Röllinghoff Martin , Solbach Werner
Published: March 2007

We investigated the effect of recombinant murine interleukin 4 (IL 4) in the absence or presence of recombinant murine interferon‐γ (IFN‐γ) on adherent bone‐marrow macrophages (MΦ), peritoneal exudate and resident peritoneal MΦ from susceptible BALB/c MΦ, which were pulse‐infected with Leishmania major amastigotes (AM). IL 4 (5–100 U/ml) failed to activate any of these MΦ populations for killing of intracellular AM. However, in the presence of low concentrations of IFN‐γ (10–20 U/ml), which alone caused only a slight or intermediate reduction of the number of intracellular parasites, IL 4 led to a dramatic increase of the parasite elimination by all MΦ populations. In the case of resident peritoneal MΦ, the synergism of IFN‐γ and IL 4 required the incubation of the MΦ with both cytokines or with IFN‐γ alone for at least 10 h prior to infection; adding both cytokines after infection of the MΦ did not cause a significant reduction of the intracellular parasite burden. The synergistic effect of IL 4 and IFN‐γ was completely abrogated in the presence of anti‐IL 4 antibodies. Furthermore, there was no significant difference between MΦ derived from either susceptible BALB/c or from resistant C57BL/6 mice. Evidence is presented that the synergistic action of IL 4 and IFN‐γ occurs via an L‐arginine‐dependent killing pathway. From these data we conclude that IL 4 provides a strong stimulus for the killing of intracellular L. major AM provided low concentrations of IFN‐γ are present. Also, IFN‐γ is apparently an important priming signal for the activation of resident MΦ to eliminate intracellular AM.
Evidence for differential responsiveness of human CD5+ and CD5− B cell subsets to T cell‐independent mitogens
Authors: Zupo Simona , Dono Mariella , Azzoni Livio , Chiorazzi Nicholas , Ferrarini Manlio
Published: March 2007

Tonsillar resting B cells were separated into CD5+ and CD5− cell subsets and stimulated with the thymus‐independent mitogens, Staphylococcus aureus Cowan strain I (SAC) or insolubilized anti‐μ monoclonal antibodies (aμAb). CD5+ cells incorporated [3H]thymidine more efficiently than unfractionated cells when stimulated with SAC and their response was augmented by the addition of interleukin (IL)2 to the cultures. CD5+ cells also proliferated in response to aμAb provided that IL 2 was present. SAC‐, but not aμAb‐stimulated CD5+ cells produced IgM and IgG molecules when IL 2 was added to the cultures and also secreted autoantibodies with rheumatoid factor activity and sometimes also with anti‐single‐stranded, but not double‐stranded, DNA activity. The efficient response of CD5+ cells was not explained by the fact that they contained cells already activated in vivo. Thus, they did not express the CD23, CD69, CD71 and CD39 activation markers, failed to incorporated [3H]thymidine and to secrete Ig spontaneously or in response to IL 2 and were found to be in a quiescent state by cell cycle flow cytometric analysis.

In contrast to CD5+ cells, CD5− cells displayed very little or no [3H]thymidine incorporation in response to SAC or to aμAb and their poor responsiveness was not altered by changing either the doses of the stimulants, the timing of the cultures, by co‐culturing the cells together with CD5+ cells, or by adding IL 2 or IL4. Immunofluorescence studies showed that freshly prepared CD5− cells did not have surface activation markers but that they expressed them following SAC stimulation. Thus, unlike that observed for CD5+ cells, SAC seems to be capable of activating CD5− cells but does not appear to be a sufficient stimulus for driving the cells into the subsequent phases of the cell cycle.

The above findings, that demonstrate marked differences in the response to CD5+ and CD5− cells to thymus‐independent stimuli, may bear relevance for the understanding of the normal clonal expansion of CD5+ cells as well as for the pathogenesis of autoimmune diseases.
Suppressor cells of popliteal lymph node origin are involved in the in vivo and in vitro control of experimental allergic encephalomyelitis effector cells in the Lewis rat
Authors: Chabannes Dominique , Borel Jean F.
Published: March 2007

Lewis rats immunized in the hind footpads with total guinea pig spinal cord tissue in mycobacteria‐enriched complete Freund's adjuvant develop chronic relapsing experimental allergic encephalomyelitis. It was previously shown that popliteal lymph node cells (LNC) isolated at the time of the first recovery (day 16) and transferred into naive syngeneic recipients protect from active induction of the disease. On the other hand, inguinal LNC taken at the onset of the disease (day 11) induce under similar conditions an acceleration of the appearance of the clinical symptoms. In this report, we show that the in vivo suppressive activity of popliteal LNC is associated with the absence of production of interleukin 2 in this compartment. The lack of production of this lymphokine and the suppressive activity can be detected only in the popliteal compartment and appear as early as day 11 after immunization. We show that this suppressive population displays in vitro inhibitory activity on the proliferative response of the disease effectors (inguinal LNC) to guinea pig myelin basic protein. This suppressive activity is not abrogated by addition of interleukin 2, suggesting that these suppressor cells do not inhibit the proliferation by absorption of the released lymphokine or by inhibition of its production.
Mycobacterial proliferation in macrophages is prevented by incubation with lymphocytes activated in vitro with a mycobacterial antigen complex
Authors: Beschin Alain , Cocito Carla , Brijs Lea , de Baetselier Patrick
Published: March 2007

Antigen A60 from Mycobacterium bovis bacillus Calmette Guérin was shown to trigger both humoral and cellular immune reactions. We explored the ability of A60 to block intracellular proliferation of phagocytosed mycobacteria with a model system involving peritoneal murine macrophages infected with Mycobacterium avium. Mixed lymphocytes from lymph nodes of mice inoculated with A60 hindered intracellular proliferation of this mycobacterium, owing to A60‐specific cells, proliferation of which was induced in vitro in an antigen concentration‐dependent manner. The lymphokines released by A60‐stimulated T lymphocytes in vitro were identified as interleukin 2 (IL 2) and interferon‐γ (IFN‐γ) : their production showed a clear A60 dose dependence. When supernatants of such induced lymphocyte cultures were incubated with anti‐IFN‐γ antibodies, macrophage activation was prevented, whereas anti‐IL 2 immunoglobulin had little effect. Treatment of infected macrophages with recombinant IFN‐γ reduced intracellular proliferation of mycobacteria, while exogenous IL 2 and tumor necrosis factor were ineffective. Therefore, A60 elicits in vitro proliferation of T lymphocytes responding specifically to this antigen with production of IFN‐γ, which in turn activates macrophages and prevents mutiplication of phagocytosed mycobacteria.
Characterization of immature CD4+CD8−CD3− thymocytes
Authors: Hugo Patrice , Waanders Gary A. , Boyd Richard L. , Scollay Roland , Petrie Howard T.
Published: March 2007

Previously we have described (Hugo, P. et al., Int. Immunol. 1990. 2: 209) an immature CD4+CD8−CD3− thymocyte subset which is thought to be the counterpart of the CD4−CD8+CD3− subset. In this study we show that the ontogeny of these two subsets is parallel in fetal thymic organ culture. Extensive phenotypic characterization of CD4+CD8−CD3− cells reveals that they closely resemble CD4−CD8+CD3− thymocytes being: HSAhigh, Thy‐1high, interleukin 2 receptor α chain negative, CD44−, H2K+/−, CD5low, MEL‐14low/intermediate, CD2+, LFA‐1+ and MTS 35+. Finally, we show that the proportion of CD4+CD8−CD3− thymocytes is highly variable between mouse strains.
Lack of anti‐DNA precursors in λ chain‐bearing B Iymphocytes in (NZB × NZW)F1 mice. Evidence for the contribution of Vχ genes to anti‐DNA specificity
Authors: Lacour Mare , Izui Shozo
Published: March 2007

We have analyzed by the limiting dilution assay on spleen cells from (NZB × NZW)F1 hybrid mice the repertoire of lipopolysaccharide‐responsive murine χ‐ and λ1‐secreting B cells committed to the production of anti‐DNA autoantibodies to determine the contribution of the heavy and light chains to anti‐DNA specificities. Our results demonstrated that anti‐DNA precursors were predominantly found in the χ‐secreting B cell population, but not in λ‐secreting B cells, while anti‐hapten, dinitrophenyl, and anti‐tetanus toxoid activities were distributed fairly well in both populations of B cells. This suggests that the Vχgene segments are critically involved in the generation of anti‐DNA specificities, and that at least at the germ‐line gene level, the heavy chain V region genes by themselves are not able to confer the anti‐DNA autoreactivity.
Islet‐infiltrating T cell clones from non‐obese diabetic mice that promote or prevent accelerated onset diabetes
Authors: Pankewycz Oleh , Strom Terry B. , Rubin‐Kelley Vicki E.
Published: March 2007

In humans and non‐obese diabetic mice (NOD), insulin‐dependent diabetes mellitus (IDDM) results from a spontaneous T cell‐dependent autoimmune destruction of insulin‐producing pancreatic β cells. Previous data suggest that a delicate balance between autoaggressive T cells and suppressor‐type immune phenomena determine whether expression of autoimmunity is limited to insulitis or progresses to IDDM. To resolve the cellular basis of this intricate network of pathogenic CD4+ and CD8+ T cells and the role of T cells in suppressive immune phenomena, T cell clones were propagated directly from islets of NOD mice at the onset of insulitis. Insofar as insulitis, but not IDDM, is universal in NOD mice, we have screened for the in vivo effects of the islet‐infiltrating T cell clones upon expression of IDDM, not insulitis. A CD4+ T cell clone, IS‐3S7D, proliferates in response to islet antigen(s) and its transfer into prediabetic NOD mice promotes the rapid onset of IDDM. An interleukin 2 (IL 2)‐dependent noncytolytic, Vβ11+ CD8+, T cell clone, IS‐2.15, prevents an accelerated onset diabetes in two distinct models. The present study, which documents the presence of CD4+ diabetogenic T cell clones and CD8+ T cell clones that dampen autoimmunity, gives tangible evidence that opposing autoimmune processes may determine whether an autoimmune‐prone host develops frank disease.
Differential activation of human basophils by anti‐IgE and formyl‐methionyl‐leucylphenylalanine. Indications for protein kinase C‐dependent and ‐independent activation pathways
Authors: Knol Edward F. , Koenderman Leo , Mul Frederik P. J. , Verhoeven Arthur J. , Roos Dirk
Published: March 2007

Upon activation, basophilic granulocytes release inflammatory mediators such as histamine. We studied histamine release of human basophils (64.1 ± 9.6% pure) after cross‐linking of membrane‐bound IgE via anti‐IgE, or after binding of the chemoattractant formyl‐methionyl‐leucyl‐phenylalanine (fMLP).

A variability in the extent of histamine release upon stimulation by either anti‐IgE or fMLP was found between donors. Kinetic studies revealed that the histamine release induced by anti‐IgE (t1/2 > 240 s) was more than 20‐fold slower than the almost instantaneous release upon stimulation with fMLP (t1/2 < 10 s). Differences in the cell activation pathways triggered by these stimuli were further analyzed with staurosporine, an inhibitor of protein kinase C (PKC) and with wortmannin, an inhibitor of a PKC‐independent pathway. Inhibition of PKC resulted in a partial inhibition of the anti‐IgE‐induced release, whereas the fMLP‐induced release was slightly potentiated. The anti‐IgE‐induced release was completely inhibited in the presence of wortmannin. This inhibitor too, had no effect on the fMLP‐induced release. We conclude that major differences exist in the signal‐response coupling between the anti‐IgE and fMLP‐induced histamine release in human basophils. The so‐called releasability of human basophils may be due to the availability of different cell activation pathways.
Vλ‐Jλ rearrangements are restricted within a V‐J‐C recombination unit in the mouse
Authors: Sanchez Pierre , Nadel Bertrand , Cazenave Pierre‐André
Published: March 2007

The murine λ gene locus is organized as follows: Vλ2‐Vλx‐Jλ2Cλ2‐ΨJλ4Cλ4‐Vλ1‐Jλ3Cλ3‐Jλ1Cλ1 where all segments have the same transcriptional orientation. The combinatorial process of gene recombination should allow the generation of eight distinct immunoglobulin light chains. We have therefore investigated the probability of obtaining such chains among the mature λ B cell repertoire. We analyze serum λ immunoglobulins and λ B cell clones induced by treatment with rabbit anti‐Δ antibodies coupled to LPS. Confirming previous data obtained by others, our results indicate that the rearrangements of Δ segments take place within each Vλ‐Jλ‐Cλ cluster, thereby defining a unit of recombination. Our results also provide no evidence for the use of undescribed segments as has been recently suggested by the finding of the Vλx segment.
Studies on the human T cell receptor α/β variable region genes. II. Identification of four additional Vβ subfamilies
Authors: Ferradini Laurent , Roman‐Roman Sergio , Azocar José , Michalaki Hélène , Triebel Frédéric , Hercend Thierry
Published: March 2007

The human T cell receptor (TcR) β chain gene segment diversity has been studied using the anchored‐polymerase chain reaction. Three hundred and fifty Cβ‐specific transcripts derived from peripheral lymphocytes were analyzed. Transcripts including V‐Dβ1‐Jβ2‐C2 sequences were found with a high frequency (> 10%), suggesting that “illegitimate” joinings may constitute a cis‐complementing rearrangement mechanism capable of substantially increasing the TcR β chain combinatorial diversity. Twelve previously undescribed Vβ gene segments have been identified. Five of them delineate four novel Vβ subfamilies (Vβw21: two members, Vβw22, 23, 24: one member) which all have a murine homologue. The additional seven gene segments belong to the Vβ5, Vβ6, Vβ12 and Vβ13 subfamilies. In addition, the sequences of two known Vβ7 and Vβ9 gene segments have been extended. Together, the present data support the view that the contribution of the β chain combinatorial diversity to the TcR α/β variability has not yet been fully appreciated.
Complement enhances the clearance of large‐sized soluble IgA aggregates in rats
Authors: Bogers Willy M. J. M. , Stad Robert Kees , Janssen Danieulle J. , Rits Michel , Bazin Hervé , van Es Leendert A. , Daha Mohamed R.
Published: March 2007

In the present study the involvement of the complement system (C) in the clearance of soluble IgA aggregates in the rat was studied. Monoclonal monomeric IgA (mIgA) antibody (which does not activate C) or aggregated polymeric IgA (aIgA; which activates C) were administered intravenously to phosphate‐buffered saline‐treated and complement‐depleted [Cobra venom factor (CVF)‐treated] rats and assessed for clearance from the circulation. In control rats, mIgA was cleared in a biphasic fashion with a first half‐life (T1/2) of 29.5 ± 14.2 min and a second T1/2 of 230 ± 176 min. No differences were observed in clearance of mIgA in CVF‐treated rats as compared to PBS‐treated rats. In PBS‐treated rats, aIgA with a size between 20 S and 150 S disappeared very rapidly from the circulation with a first T1/2 of 1.1 ± 0.4 min and a second T1/2 of 23.2 ± 11.3 min. In CVF‐treated rats the clearance of aIgA was significantly delayed as compared to that in control rats, namely with a first T1/2 of 7.3 ± 2.6 min and a second T1/2 of 64.2 ± 19.4 min.

Immunohistochemical studies of the liver (which is the main site of clearance of aIgA) revealed that Kupffer cells (KC) are mainly responsible for the uptake of aIgA. Furthermore, in PBS‐treated rats aIgA deposition was accompanied by C3 deposition in the KC. In CVF‐treated rats, the percentage of KC containing aIgA was significantly lower during the first 16 min after aIgA administration as compared to PBS treated rats. In addition no detectable C3 was found in KC of CVF‐treated rats. These results indicate that KC play an important role in the clearance of large molecular weight IgA in rats and that C facilitates the clearance of these complexes from the circulation.
CD77: an antigen of germinal center B cells entering apoptosis
Authors: Mangeney Marianne , Richard Yolande , Coulaud Dominique , Tursz Thomas , Wiels Joëlle
Published: March 2007

We have previously reported that a neutral glycolipid (globotriaosylceramide; Gb3) was specifically expressed on Burkitt's lymphoma cells and on a subset of germinal center tonsillar B lymphocytes. Recently the Gb3 molecule was recognized as a new B cell differentiation antigen and now defines the CD77 cluster. Here we report an extensive phenotypic and functional characterization of the tonsillar CD77+ B lymphocytes. These cells have a low buoyant density and are thus purified using a Percoll gradient. They express various B cell antigens such as CD19, CD20, CD21, CD22 and CD40, as well as the adhesion molecules LFA‐1, LFA‐3 and CD44. They are positive for surface IgM and negative for surface IgD. Although these results suggest a phenotype of activated B cells, the CD77+ cells are negative for the classical activation antigens: CD23 (the low‐affinity Fc receptor for IgE), CD25 [the interleukin (IL) 2 receptor α chain] and CD71 (the transferrin receptor). Proliferation and protein synthesis of CD77+ cells was measured after stimulation with a range of mitogens and IL. None of the agents tested are able to induce proliferation and protein synthesis with the exception of a combination of recombinant IL 4 plus anti‐CD40 antibody. When examined by electron microscopy, CD77+ B lymphocytes present a morphology similar to that of cells undergoing programmed cell death, also called apoptosis (i.e. chromatin condensation, nuclear fragmentation, membrane blebbing). As shown by direct examination of DNA, these CD77+ cells are indeed in the process of apoptosis. Treatment of the CD77+ cells by recombinant IL 4 and anti‐CD40 antibody prevents apoptosis. All these results suggest that the CD77 molecule defines a B lymphocyte maturation pathway, specific for germinal center, where the cells undergo programmed cell death.
Normal serum immunoglobulins influence the numbers of bone marrow pre‐B and B cells
Authors: Sundblad Anne , Marcos Miguel , Huetz Francois , Freitas António , Heusser Christoph , Portnoi Denis , Coutinho António
Published: March 2007

The homeostatic mechanisms controlling B lymphocyte output from bone marrow are not well understood. The present experiments evaluated putative influences of circulating immunoglobulins (Ig) on bone marrow (BM) pre‐B and B cell populations. Injections into normal mice of Ig isolated from normal mouse serum, resulted in a dose‐dependent and reversible reduction in numbers of BM B lineage cells, in particular of small B220+ surface IgM− cells. Maximal effects were observed upon injection of isologous polyclonal Ig and were independent of mature T cells. These results suggest a feedback modulation of peripheral Ig on cellular activities in BM B lineage compartments, mediated by mechanisms that seem to involve the variable regions of the Ig molecule.
Direct involvement of CD7 (gp40) in activation of TcRγ/δ+ T cells
Authors: Carrel Stefan , Salvi Suzanne , Rafti Fiorella , Favrot Marie , Rapin Christophe , Sekaly Rafick Pierre
Published: March 2007

In this study we reported that on T cell receptor (TcR)γ/δ+ cells from three cell lines Peer, MOLT‐13 and ICRF‐1, the T cell antigen CD7 (gp40) can be directly involved in the activation process. This is shown by a rapid increase in cytoplasmic free calcium after stimulation of these cells with an anti‐CD7 monoclonal antibody (mAb). Activation through CD7 was further confirmed by measuring the production of interleukin 2 in ICRF‐1 cells stimulated with anti‐CD7 mAb. In addition induction of mRNA for tumor necrosis factor (TNF)‐α and TNF‐β in Peer and for granulocyte‐macrophage‐colony‐stimulating factor in MOLT‐13 was observed in these anti‐CD7‐stimulated cells. The same anti‐CD7 antibody was unable to activate TcR α/β+ Jurkat cells or normal resting peripheral blood T lymphocytes. We further showed that normal resting TcR γ/δ+ cells were likewise activated via the CD7 molecule. TcR γ/δ+ cells obtained from a patient with acute lymphoblastic leukemia 3 months after autologous bone marrow transplantation were induced to proliferate, as measured by [3H]thymidine incorporation after stimulation with anti‐CD7 mAb but not with anti‐CD3 mAb. Interestingly TcR α/β+ cells from the same donor tested in parallel were not stimulated by anti‐CD7 but by anti‐CD3 mAb. In essence these findings contribute to the idea that on TcR γ/δ+ cell, the CD7 antigen could play an important role during T cell differentiation.
Increased frequency of interleukin 4‐producing T cells as a result of polyclonal priming. Use of a single‐cell assay to detect interleukin 4‐producing cells
Authors: Seder Robert A. , le Gros Graham , Ben‐Sasson S. Z. , Urban Joseph , Finkelman Fred D. , Paul William E.
Published: March 2007

A limiting dilution assay capable of detecting interleukin 4 (IL 4) production by a single cell has been developed. This assay is based on the stimulation of T cells, in the presence of IL 2 (5 U/ml), with anti‐CD3 antibody bound to the surface of Terasaki wells. Cells of the IL 4‐selective indicator line, CT.4S, are added 24–36 h later and IL 4 production is determined based on their survival 24–48 h thereafter. A frequency of 0.98 was obtained for IL 4 production by T cells of the D10.G4 cell line. T cells from naive donors capable of producing IL 4 in response to anti‐CD3 plus IL 2 were quite rare, with a frequency in four experiments ranging between 0.0003 and 0.0018. Treatment of mice with polyclonal activators known to increase the IL 4‐producing capacity of T cells when assayed in bulk culture caused striking increases in the frequency of IL 4‐producing cells. Similarly, culturing cells in vitro with anti‐CD3, IL 2 and IL 4 for 5 days caused a marked increase in the frequency of cells capable of producing IL 4, to 0.031.IL 4 production by individual T cells is dependent upon IL 2. Thus, in naive T cell populations, the frequency of IL 4‐producing cells in response to stimulation with anti‐CD3 in the absence of IL 2 was below the limit of detection. T cells from primed donors showed a striking inhibition in the frequency of IL 4‐producing cells in response to anti‐CD3 when IL 2 was not present. The availability of a simple assay to measure the frequency of cells capable of producing IL 4 should have substantial utility in allowing the evaluation of conditions that regulate IL 4 production in vivo and in vitro.
Analysis of T cell reactivities to phosphorylcholine‐conjugated hen egg lysozyme in C57BL/6 mice: hapten‐conjugate specificity reflects an altered expression of a major carrier epitope
Authors: Jang Yong‐Suk , Lim Kwang H. , Kim Byung S.
Published: March 2007

We have selected several different T cell hybridoma clones reactive to hen egg lysozyme (HEL) conjugated to phosphorylcholine (PC) after fusion of PC‐HEL‐primed C57BL/6 lymphocytes with BW5147 parent cells. These hybridoma clones preferentially recognize PC‐HEL over unconjugated HEL, but not other carrier molecules conjugated with the same hapten. All the PC‐HEL‐reactive clones are similarly responsive to not only p‐azobenzenearsonate (ABA)‐conjugated HEL (ABA‐HEL) but also to a variety of other diazotized hapten‐HEL conjugates. However, these clones are not stimulated by fluoresceinated or dinitrophenylated HEL beyond the level of HEL carrier alone. Therefore, the type of hapten linkage (diazonium) to the carrier molecule appears to affect T cell recognition. The hybridoma clones apparently recognize the carrier molecule alone, although the level of stimulation is relatively low compared to that induced by either PC‐HEL or ABA‐HEL. Interestingly, HEL unfolded by S‐carboxymethylation is capable of stimulating the hybridomas to a level comparable to that obtained with PC‐HEL. T cell recognition of the unfolded HEL is independent of antigen processing, which is different from that of PC‐HEL. The peptide sequence corresponding to the amino acids 81–93 of HEL appears to contain the epitope region for the hybridoma clones based on testing stimulation activity with synthetic peptides. Previously, the peptides including this region (81–96) have been reported as the determinant recognized by T cells derived from C57BL/6 mice after immunization with an HEL peptide (HEL 13–105) but not with native HEL. These results suggest that a hapten conjugation via diazonium linkage modifies antigen presentation and consequently the presentation of the major T cell epitopes similar to that of the HEL fragment.
Immunotherapeutic elimination of syngeneic tumors in vivo by cytotoxic T lymphocytes generated in vitro from lymphocytes from the draining lymph nodes of tumor‐bearing mice
Authors: Lynch David H. , Miller Robert E.
Published: March 2007

The draining lymph nodes (LN) of mice injected with viable syngeneic tumor cells contain populations of lymphoid cells which are capable of generating functional cytotoxic T lymphocytes (CTL) during an in vitro culture period without the need of secondary stimulation with irradiated tumor cells or addition of exogenous cytokines. Intravenous injection of as few as 2.5 × 106 tumor‐reactive effector cells generated using this protocol effectively eliminated challenges of tumor cells at distal skin sites, whereas injection of cultured control lymphoid cells was ineffective at rejecting tumor challenges. The anti‐tumor CTL generated from the draining LN of C57BL10 mice immunized with viable syngeneic tumor cells were found to be specific for the tumor used for immunization both in vitro and in vivo. Phenotypic analysis of the cells responsible for tumor elimination (both in vitro and in vivo) demonstrated the anti‐tumor effector cells to be CD4−CD8+ asialo GM NK1.1−. Finally, anti‐tumor CTL generated during the short in vitro culture period were able to effectively reject pre‐existing tumors in vivo in a systemic fashion. These data indicate that the generation of tumor‐reactive CTL from the lymphoid cells contained in the LN draining the site of a tumor may provide a valuable source of effector cells which can be used in the immunotherapeutic treatment of cancer.
Class II‐restricted IgG2ab‐specific T cells recognize a signal‐minus form of the V‐CH3b antigen
Authors: Bikoff Elizabeth K.
Published: March 2007

To study the question when and where self peptides become associated with major histocompatibility complex class II molecules for tolerance induction, we recently developed a system in which the intracellular site(s) of antigen expression could be manipulated using gene cloning techniques. We previously constructed a truncated IgG2a gene comprising a variable (V) domain and the C domain (not including the membrane exons) from the IgG2ab heavy (H) chain. The secreted form of the V‐CH3b protein was expressed at high levels under control of the Ig H chain enhancer in Ia+ B lymphoma cells and was efficiently recognized by class II‐restricted IgG2ab‐specific T cell hybrids. Here we describe a modified V‐CH3b gene construct in which the sequences encoding the signal peptide were deleted. A strong argument can be made that the signal‐less V‐CH3b protein is predominantly expressed in the cytosol. We show that transfected L cell lines expressing the signal‐less form of the V‐CH3b protein can stimulate class II‐restricted IgG2ab‐specific T cells. Cell mixing experiments indicate that this response cannot be due to passive uptake of soluble antigenic peptides released into culture supernatants. These experiments demonstrate that a cytoplasmic protein having no obvious means of reaching the cell surface can be presented to class II‐restricted T cells.
The mouse IgH 3′‐enhancer
Authors: Dariavach Piona , Williams Gareth T. , Campbell Kathryn , Pettersson Sven , Neuberger Michael S.
Published: March 2007

A lymphoid‐specific transcription enhancer element has recently been identified at the far 3′ end of the rat immunoglobulin heavy chain (IgH) locus. Sequence analysis presented here reveals that this enhancer is flanked by a 350‐bp invert repeat, giving a structure reminiscent of a transposable element. We therefore screened for the equivalent enhancer in the mouse to determine whether its presence was conserved during evolution. A mouse homologue was indeed identified and is located 16 kb downstream of the Cα1 exon. It is also flanked by invert repeats and these are not repeated throughout the genome. The mouse and rat enhancers retain high sequence homology. As regard activity, the IgH 3′‐enhancer is lymphoid specific. However, this activity was detected in two plasmacytoma lines tested but not in two B cell lymphomas nor in HeLa cells suggesting that the enhancer may only play a stage‐specific role during lymphocyte differentiation. As regards function within the IgH locus, we found that inclusion of the mouse IgH 3′‐enhancer (in addition to the intron‐enhancer) on μ gene expression plasmids effected a small increase in μ mRNA levels in stable plasmacytoma transfectants.
Vaccination of class I major histocompatibility complex (MHC)‐restricted murine CD8+ cytotoxic T lymphocytes towards soluble antigens: immunostimulating‐ovalbumin complexes enter the class I MHC‐restricted antigen pathway and allow sensitization against the immunodominant peptide
Authors: Heeg Klaus , Kuon Wolfgang , Wagner Hermann
Published: March 2007

In vivo induction of anti‐ovalbumin (OVA) cytotoxic T cell responses was brought about in an MHC class I‐restricted fashion by immunizing H‐2b mice with OVA in immunostimulating complexes (ISCOM). ISCOM formation with the hydrophilic soluble protein OVA was achieved upon unmasking hydrophobic protein domains by treatment at low pH values. The effector cells induced were MHC restricted, specific for the immunodominant peptide of OVA (258–276), and expressed the CD8+CD4− phenotype. These results suggest that ISCOM‐based vaccines may be useful to direct hydrophilic soluble antigens into the MHC class I presentation pathway in order to vaccinate CD8+ T lymphocytes.
Transcripts encoding mouse CD44 (Pgp‐1, Ly‐24) antigen: strain variation and induction by mitogen
Authors: Haegel Hélène , Ceredig Rhodri
Published: March 2007

The cell surface molecule CD44, thought to be a cell surface receptor for hyaluronic acid, is expressed by thymocytes and peripheral T cells. Following T cell activation with mitogens or antigens, surface expression of CD44 increases. This increase in CD44 cell surface expression has been used as a means of identifying memory T cells in vivo. In this report, using Northern analysis, we have quantitated mouse CD44 transcripts in thymocytes and a cloned T hybridoma cell line. In contrast to a recent report by Nottenburg et al. (Proc. Natl. Acad. Sci. USA 1989. 86: 8521), we show that in all instances CD44 transcripts are polyadenylated. In all cells studied, three major mRNA species of approximately 4.5, 3.5 and 1.6 kb are detected. After stimulation with phorbol 12‐myristate 13‐acetate (PMA), there is a de novo protein synthesis‐dependent increase in the level of CD44 transcripts. The increase in CD44 transcripts precedes the increase in cell surface expression. In contrast to CD44, PMA stimulation results in a decrease in the level of Thy‐1 transcripts. Thymocytes from different mouse strains vary in their level of CD44 cell surface expression and Northern analysis indicates that this mouse strain variation correlates with a corresponding difference in the level of CD44 transcripts.
Induction of the interleukin 1 receptor antagonist protein by transforming growth factor‐β
Authors: Turner Martin , Chantry David , Katsikis Peter , Berge Ann , Brennan Fionula M. , Feldmann Marc
Published: March 2007

Transforming growth factor‐β1 (TGF‐β1) mediates many immunosuppressive effects on immune cells and can inhibit the production of tumor necrosis factor and interleukin 1 (IL 1). However, TGF‐β1 can stimulate the production of IL6 and platelet‐derived growth factor, indicating that TGF‐β1 initiates complex effects on the production of cytokines. In this report we show that treatment of peripheral blood monocytes with TGF‐β1 leads to the induction of a recently described IL1 receptor antagonist protein (IRAP). The effect of TGF‐β was both dose and time dependent. TGF‐β1 induced de novo synthesis of IRAP, as Northern blotting experiments indicated a rapid and transient induction of the mRNA encoding IRAP. The induction of IRAP suggests a potential mechanism by which some of the inhibitory effects of TGF‐β1 are mediated.
Characterization of the CAMPATH‐1 (CDw52) antigen: biochemical analysis and cDNA cloning reveal an unusually small peptide backbone
Authors: Xia Meng‐Qi , Tone Masahide , Packman Len , Hale Geoff , Waldmann Herman
Published: March 2007

The CAMPATH‐1 (CDw52) antigen has been purified from human spleen. The antigenic epitope is heat stable but sensitive to mild alkali treatment. Experiments with phosphatidylinositol‐specific phospholipase C indicate that it is anchored by a glycosylphosphatidylinositol (GPI) anchor. An N‐terminal sequence of 11 amino acids was determined, followed by an abrupt stop. Using short overlapping mixed oligonucleotide primers, cDNA synthesized from the mRNA of a human B cell line was amplified by the polymerase chain reaction. The product was used to isolate cDNA clones and the full amino acid sequence of the CAMPATH‐1 antigen was deduced. It consists of 37 amino acid residues plus a 24‐residue signal peptide. It has all the features expected for a GPI‐anchored membrane protein except that the predicted mature protein is remarkably short, comprising no more than 18 residues and possibly as few as 12 (depending on the GPI linkage site). Potential attachment sites for carbohydrate are present and it is shown that the antigen contains N‐linked oligosaccharide(s). This structure accounts for the known properties of the antigen, though the exact reasons why it is such a good target for cell lysis in vitro and in vivo are not yet clear.
Remote T cell co‐stimulation via LFA‐1/ICAM‐1 and CD2/LFA‐3: Demonstration with immobilized ligand/mAb and implication in monocyte‐mediated co‐stimulation
Authors: Van Seventer Gijs A. , Shimizu Yoji , Horgan Kevin J. , Luce Gale E. Ginther , Webb Deborah , Shaw Stephen
Published: March 2007

Proliferative response of resting T cells generally requires not only cross‐linking of the T cell receptor (TcR) but also co‐stimulatory signals from accessory molecules. We here have used a “three‐cell” model consisting of: (a) resting human CD4+ T cells as responders; (b) CD3 monoclonal antibody (mAb) OKT3 on latex beads as surrogate stimulators; (c) autologous monocytes as source of co‐stimulation. As described by Kawakami et al. (J. Immunol. 1989. 142: 1818), T cell proliferation in this system is observed with paraformaldehyde‐fixed monocytes if they have been activated and interleukin (IL) 1β/IL6 is supplied. Since this three‐cell system provides TcR cross‐linking at a site spatially “remote” from co‐stimulation, they help distinguish adhesion from signal transduction but the molecules that mediate co‐stimulation in this system have not been identified. Our studies now demonstrate that co‐stimulation by the monocytes is dependent on each of two receptor/ligand pathways CD2/LFA‐3 and LFA‐1/ICAM‐1 since it is inhibited by each relevant mAb but not a variety of control mAb. The hypotheses that CD2 and LFA‐1 could each mediate co‐stimulation was tested in simplified model systems in which the monocyte was replaced with immobilized CD2 mAb or purified ICAM‐1 presented on a separate surface from the CD3 mAb. The results in these simplified models demonstrate that on resting T cells either CD2 or LFA‐1 molecules alone can mediate “remote” co‐stimulation unlike most other T cell surface molecules. Co‐stimulation requires IL 1β/IL6 both in the weaker LFA‐1 ligand‐mediated co‐stimulation and at lower CD2 mAb concentrations in the stronger CD2 mAb‐mediated co‐stimulation. Thus: (a) the accessory cell function of stimulated fixed monocytes in T cell proliferation requires both the LFA‐1/ICAM‐1 and CD2/LFA‐3 pathways; and (b) the T cell molecules CD2 and LFA‐1 can give co‐stimulatory signals that can act in a “remote” fashion.
Ontogeny of murine T cells: Thymus‐regulated development of T cell receptor‐bearing cells derived from embryonic yolk sac
Authors: Liu Chih‐Pin , Auerbach Robert
Published: March 2007

Pre‐thymic stem cells which arise extrinsically to the thymus differentiate into mature T cells after colonizing the fetal thymus, but the origin of the stem cells during mammalian embryogenesis remains unknown. More than one potential site has been proposed, including the liver, the omentum and the embryonic yolk sac (YS). Since the murine YS appears earliest in gestation and is the first site of hematopoiesis, our studies focus on the mouse YS as the early major source of pre‐thymic stem cells. Previous studies showed that freshly obtained YS cells expressing no known major T cell marker can develop into Thy‐1+ cells in vitro either transiently or stably. However, the critical steps leading to functional maturation of T cells involve the rearrangement and expression of the T cell receptor (TcR) genes. We report here that YS cells grown in short‐term cell culture show no detectable rearrangement or transcription at the TcRγ gene locus, although the TcRγ gene of in vitro transformed YS cells rearranges in the absence of the thymus. However, after seeding lymphocyte‐depleted thymuses in vitro, YS cells can differentiate into various subsets of cells appearing in the thymus, including CD3/TcR α/β‐ and γ/δ‐bearing cells. TcR Vγ3 which is expressed mainly on early fetal thymocytes and on Thy‐1+ dendritic cells can be detected on YS‐derived cells. TcR Vβ8‐expressing cells which appear later in thymic ontogeny are also detected. The kinetics for reconstituting the thymus varies among YS cells of different ages (day 10 to 13 gestation). These results indicate that a single wave of early mouse YS cells can undergo the molecular and cellular changes associated with T cell differentiation and acquire mature T cell characteristics in the absence of continuous inflow of other stem cells. The thymus acts in an inductive/regulatory role for that differentiation.
Diabetes‐prone BB rats express the RT6 alloantigen on intestinal intraepithelial lymphocytes
Authors: Fangmann Josef , Schwinzer Reinhard , Hedrich Hans‐Jürgen , Klöting Ingrid , Wonigeit Kurt
Published: March 2007

The RT6 alloantigen of the rat is expressed on most peripheral T cells but not on thymocytes and thus represents a marker for postthymic T lymphocyte maturation in this species. Diabetes‐prone (DP) BB rats exhibit a genetically determined T cell lymphopenia associated with a deficiency of RT6+ T cells. In this study we have analyzed the expression of RT6 on lymph node (LN) cells and intestinal intraepithelial lymphocytes (IEL) in two DP BB strains (BB/OK and BB/Mol) and two control strains (non‐lymphopenic BB/PhiK and LEW) by flow cytometry. In the DP BB rats the number of LN T cells was substantially reduced (< 25% TcR2+ cells) and completely lacked RT6 expression. The IEL population was also reduced in number and in marked contrast to normal rats consisted predominantly of CD4+ cells. The majority of IEL, however, clearly expressed RT6. Treatment with a phosphatidylinositol (PI)‐specific phospholipase C markedly reduced the RT6 density showing that PI‐mediated anchoring of RT6 in the cell membrane also applies to IEL of DP BB rats. The results demonstrate that the DP BB strains possess a functional RT6 gene and are also able to generate the PI anchor. The defect in RT6 expression is thus unlikely to be the primary cause of the T cell lymphopenia.
The binding affinity and dissociation rates of peptides for class I major histocompatibility complex molecules
Authors: Cerundolo Vincenzo , Elliott Timothy , Elvin John , Bastin Judy , Rammensee Hans‐Georg , Townsend Alain
Published: March 2007

Peptides of various lengths derived from the influenza nucleoprotein (NP) bind to H‐2Db class I molecules with affinities at 4 °C between ∼3 × 105‐∼3 × 107 M−1. The peptide with the highest affinity corresponds to the sequence of nine amino acids (NP366–374) recently isolated from cells infected with influenza. This peptide forms stable complexes with half‐lives > 110 h at 4 °C, 39 h at 22 °C and 3 h at 37 °C. Small increases in length of the peptide greatly reduce the stability of the complex (t1/2 ∼1–10 h at 4 °C). These results may explain the homogeneous length of peptides isolated from class I molecules formed in vivo, and suggest that class I and II may differ in their dependence on the length of peptides for the formation of stable complexes.
Structure‐function studies on human C4b‐binding protein using monoclonal antibodies
Authors: Hessing Martin , Heijnen Harry F. G. , Hackeng Tilman M. , Sixma Jan J. , Bouma Bonno N.
Published: March 2007

Human C4b‐binding protein (C4BP) is a multimeric regulatory complement component interacting with vitamin K‐dependent protein S and complement C4b. Using hybridoma technology, a panel of monoclonal antibodies (mAb) specific for intact human C4BP and its 160‐kDa chymotryptic central core fragment were prepared to study the structure‐function relationships of C4BP. By Western blot analysis and competition experiments, four distinct groups of mAb were identified and mapped on the C4BP molecule. By‐rotary shadowing, spider‐like images of C4BP‐antibody complexes were obtained and immunoelectron microscopy provided some information on the stoichiometry of the antibody‐C4BP interaction. Certain antibodies interacted with C4BP molecules only at a ratio of 1:1. Others formed complexes of two or more antibodies bound to homologous sites on the C4BP molecule. Using an enzyme‐linked immunosorbent sandwich assay for the measurement of the complex formation between protein S and C4BP, mAb against the central core and the disulfide‐linked β chain of C4BP were identified that inhibited the binding of protein S to C4BP. In a binding assay using 125I‐labeled C4BP and soiid‐phase C4b, the inhibitory effect of one group of anti‐C4BP mAb on the binding of C4BP to C4b was demonstrated.