bluuebunny/crossref_metadata_2025 huggingface dataset:
Thymic and peripheral apoptosis of antigen‐specific T cells might cooperate in establishing self tolerance
Authors: D'Adamio Luciano , Awad Katherine M. , Reinherz Ellis L.
Published: March 2007

Aside from CD4+CD8+ double‐positive (DP) thymocytes, the subpopulations of T lineage cells affected by negative selection are unknown. To address whether this process occurs in more mature cell types, we have compared the responses of purified single‐positive (SP) murine thymocytes and peripheral T cells to the superantigen staphylococcal enterotoxin B (SEB) utilizing as antigen‐presenting cells (APC) a fibroblast cell line expressing transfected I‐Ek class II molecules. Whereas ∽ 70% of SEB‐reactive SP thymocytes, either CD4+ or CD8+, undergo programmed cell death (apoptosis) and, therefore, negative selection, CD4+ and CD8+ antigen‐specific peripheral T cells are predominantly activated and proliferate to APC+SEB. Thus, mature thymocytes and peripheral T cells, with identical patterns and levels of expression of CD4, CD8 and T cell receptor (TCR), are programmed to elicit different responses followingTCR stimulation. Unexpectedly, however activation of peripheral T cells was preceded by deletion of a large fraction of Vβ8+ T lymphocytes (SEB specific). This surprising phenomenon was also observed in in vivo studies: in fact, administration of SEB to adult mice resulted in depletion of the majority of antigen‐specific T cells from the peripheral lymphoid tissues analyzed (lymph nodes and spleen). This depletion is the consequence of deletion as indicated by program cell death of Vβ8+ T cells and is followed by proliferation of the remaining SEB‐reactive T cells. Clonal elimination of peripheral T cells may represent a mechanism by which tolerance to self antigens never expressed in and/or exported to the thymus is achieved.
Role of environmental antigens in B cell proliferation in the bursa of Fabricius at neonatal stage
Authors: Ekino Shigeo
Published: March 2007

Effect of environmental antigens on S phase entry rate of bursa cells was studied by utilizing in vivo incorporation of bromodeoxyuridine (BrdUrd). S phase entry rate of bursa cells in chick embryo was determined to be 6.4 %/h, this rate increased to 10.0 %/h at neonatal stage and decreased to 6.4 %/h 6 weeks after hatching. Evidence is presented that isolation of the bursa from the gut before hatching suppressed S phase entry rate of bursa cells after hatching, whereas S phase entry rates of thymocytes were not affected. On tissue sections, a decrease of BrdUrd+ cells was observed in medullary areas of the isolated bursa. It is proposed that in situ stimulation of bursa cells by environmental antigens to the bursa accelerates S phase entry rates, and that medullary areas are the major sites for antigen‐driven B cell proliferation after hatching.
Effects of intrathymic injection of organ‐specific autoantigens, parietal cells, at the neonatal stage on autoreactive effector and suppressor T cell precursors
Authors: Murakami Kyoko , Maruyama Hidenori , Nishio Akiyoshi , Kuribayashi Kagemasa , Inaba Muneo , Inaba Kayo , Hosono Masamichi , Shinagawa Kunihiro , Sakai Masahiko , Masuda Tohru
Published: March 2007

Thymectomy on day 3 after birth (3d‐Tx) induces autoimmune gastritis (AIG) in 81%, and oophoritis (AIO) in 25% of BALB/c mice at the age of 2 to 3 months. Intrathymic, but not intraperitoneal injection of syngeneic parietal cells into sex‐matched BALB/c mice within 24 h of birth resulted in almost complete prevention of the development of AIG in these mice in which 3d‐Tx was performed. The prevention induced was parietal cell specific, since the development of AIO was not inhibited in female mice. Moreover, the injection of BALB/c liver cells, Mls‐matched (BALB/c) and ‐disparate (DBA/2) B blasts which resulted in Vβ6 T cell deletion, as well as the injection of staphylococcal enterotoxin B failed to prevent the diseases. These findings suggested that recognition of an autoantigen in the thymus is necessary for the induction of tolerance, and that involvement of Mls‐1 antigens in the pathogenesis of AIG, as has been suggested previously (Schwartz, R. H., Cell 1989. 57: 1073), was unlikely.

T cells that suppress the development of organ‐specific autoimmune diseases in 3d‐Tx mice seem to maintain the unresponsiveness of autoreactive T cells at the periphery in normal mice. In agreement with our previous observations, we found that intraperitoneal (i.p.) injection of spleen cells from 3‐month‐old normal mice into 3d‐Tx mice on day 10 after birth prevented the development of AIG, whereas spleen cells from age‐matched AIG+ (mice with AIG) or AIG− (mice without AIG) 3d‐Tx mice failed to do this. This implies that the suppressor cells probably affect the differentiation of effector‐precursor to effector. In fact, these suppressor cells did not inhibit the adoptive transfer of AIG to nu/nu BALB/c mice by spleen cells from 3d‐Tx mice manifesting AIG. By negative selection using monoclonal antibody and complement, it was confirmed that the phenotype of the suppressor cell was CD4. In contrast to 3d‐Tx, 10d‐Tx did not induce AIG, indicating the peripheralization of the suppressor cell by that time. On the other hand, intrathymic injection of parietal cells immediately after birth did not affect suppressor cell generation, implying that some T cells, including suppressor cells, escape thymus selection. We postulate that these cells correspond to the precursors of the autoreactive effector T cells and suppressor T cells that are present in normal mice.
Clonal deletion of thymic mature T cells induced by staphylococcal enterotoxin B in murine fetal thymus organ culture
Authors: Aiba Yuichi , Mazda Osam , Matsuzaki Yumi , Nakauchi Hiromitsu , Muramatsu Shigeru , Katsura Yoshimoto
Published: March 2007

The present study aims at investigating the intrathymic maturational stage of T cells at which clonal deletion can be induced. Staphylococcal enterotoxin B (SEB) was added to organ cultures of murine fetal thymus lobes at various time points of culture, and Vβ8‐expressing cells were assayed on day 14. Vβ8 low‐expressing (Vβ8′°) cells were reduced to 40‐60 % of the control receiving no SEB, though the reduction was ambiguous when SEB was given on day 13. In marked contrast, Vβ8 high‐expressing (Vβ8hi) cells were virtually completely deleted in all groups including the group given SEB on day 13. Most of the Vβ8hl cells that were deleted by 24 h of treatment with SEB were shown to be of the CD4+8 mature phenotype, though CD4−8+Vβ8hi cells were also deleted. It was further shown that the thymic Vβ8hi CD4+8− cells recovered from organs cultured for 14 days without SEB responded to immobilized anti‐Vβ8 monoclonal antibody, indicating that Vβ8hi cells, which were highly sensitive to clonal deletion, were functionally competent mature T cells. These results strongly suggest that the thymus is capable of eliminating all T cells recognizing antigen present in the thymus regardless of the maturational stage of T cells.
Large, detergent‐resistant complexes containing murine antigens Thy‐1 and Ly‐6 and protein tyrosine kinase p56lck
Authors: Bohuslav Jan , Cinek Tomáš , Hořejší Václav
Published: March 2007

A number of human and mouse leukocyte surface (glyco)proteins anchored in a membrane via glycosylphosphatidylinositol (GPI) moiety have been previously shown to be noncovalently associated with protein tyrosine kinases (Science 1991. 254: 1016; J Biol. Chem. 1992. 267:12317). Here we show that two murine antigens of this group, Thy‐1 and Ly‐6, implicated in the activation of the T cells, are associated with each other, with the kinase p56lck and with several of potential kinase substrates in very large, detergent‐resistant complexes, the size of which is between 50 and 200 nm, as determined by ultrafiltration and gel chromatography. Experiments on simultaneous solubilization of mixed human and mouse cells rule out that the observed complexes are artifacts induced by the detergent. Complexes of similar composition and properties were obtained when either detergents Brij‐58, Nonidet‐P40 or 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane‐sulfonate (Chaps) were used for solubilization of the cells, while octylglucoside at least partially dissociated them. These “GPI‐complexes” may be essential for the well‐known signal‐transducing capacity of Thy‐1 and Ly‐6.
The human immunoglobulin VH7 gene family consists of a small, polymorphic group of six to eight gene segments dispersed throughout the VH locus
Authors: van Dijk Ko Willems , Mortari Frank , Kirkham Perry M. , Schroeder Harry W. , Milner Eric C. B.
Published: March 2007

In this report we describe the analysis and mapping of members of the human immunoglobulin VH7 gene family. VH7 and VH1 gene segments are closely related, with individual gene segments sharing between 78% and 82% sequence identity. Divergence from VH1 gene sequence occurs as an abrupt event at the boundary between framework region (FR) 2 and complementarity‐determining region (CDR) 2 and continues through a major portion of FR 3. We used polymerase chain reaction amplification to create a 162‐base pair probe spanning the family‐specific region of CDR 2 and FR 3 that proved suitable for standard Southern analysis of genomic DNA. The VH7 gene family was found to be a small but discrete VH gene family consisting of five to eight germ‐line elements, of which at least three are polymorphic. Four different VH7 gene segments were cloned from the germ line of a single individual, and assigned to specific restriction fragments by sequence‐specific hybridization. Two of the four VH7 elements were pseudogenes. The pattern of sequence variation in these and other known pseudogenes suggests that these nonfunctional elements may play a role in the evolution of novel VH families. A combination of one and two‐dimensional pulsed field gel electrophoresis was employed to map the chromosomal location of all of these VH7 elements. Individual VH7 gene segments were found to be dispersed over a region of at least 940 kb of DNA, and interspersed with members from other VH gene families. The polymorphism of the VH7 gene segments and their scattered location throughout the VH locus makes them potentially useful markers for mapping and linkage studies.
The effect of anchor residue modifications on the stability of major histocompatibility complex class I‐peptide interactions
Authors: Neefjes Jacques J. , And John Dierx , Ploegh Hidde L.
Published: March 2007

Anchor residues in peptides determine the specificity of binding to major histocompatibility complex class I molecules through interactions of their side chains with pockets in the peptide‐binding groove. We have compared the kinetics of association of a Sendai virus nucleoprotein‐derived peptide (FAPGNYPAL, termed SV9) with H‐2Kb class I molecules, and the same peptide iodinated on the anchor residue tyrosine (125I‐SV9). Even though the association rates were too rapid for direct measurements, competition studies indicated that they were similar for SV9 and 125I‐SV9. To measure the binding of non‐radioactive SV9 directly, SV9 was tritiated (3H‐SV9). 3H‐SV9 remained stably associated with H‐2Kb molecules, whereas 125I‐SV9 dissociated in a temperature‐dependent fashion. Thus, modifications on anchor residues do not necessarilly have to affect the specificity and association kinetics of peptide binding to class I molecules but can affect the stability of the resulting class I‐peptide interaction. The dissociation of peptides with modified and, more generally, suboptimal anchor residue side chains may explain the presence of empty class I molecules and free class I heavy chains at the cell surface.
Differential induction of T cell cytokine mRNA in Epstein‐Barr virus‐transformed B cell clones: constitutive and inducible expression of interleukin‐4 mRNA
Authors: Tang Huayang , Matthes Thomas , Carballido‐Perrig Nicole , Zubler Rudolf H. , Kindler Vincent
Published: March 2007

Clones of human B lymphocytes, obtained after immortalization with Epstein‐Ban virus (EBV) of single CD 19+ B cells and expansion in the absence of human T lymphocytes, produced mRNA for the T cell cytokines interleukin(IL)‐2, IL‐4, and interferon(IFN)‐γ. As detected by reverse transcriptase‐polymerase chain reaction, IL‐2 mRNA was expressed only after stimulation with the combination of phorbol 12‐myristate 13‐acetate (PMA) plus ionomycin. IL‐4 mRNA was constitutively detectable in all (10/10) EBV‐transformed B cell clones, and the mRNA for IFN‐γ was constitutively present in half of the clones. In contrast to IL‐2 mRNA, the expression of IL‐4 and IFN‐γ mRNA could be increased by PMA alone. Most of the clones produced IL‐2 bioactivity and immunoreactive protein, but neither IL‐4 nor IFN‐γ protein secretion was detected. The intriguing question raised by these results is whether IL‐2 secretion could contribute to the immune control of EBV‐infected B lymphocytes by cytolytic T cells, and whether normal B lymphocytes can potentially be induced to express certain cytokines including IL‐4 in response to the appropriate activation signals.
Mutation of Asp20 of human interleukin‐2 reveals a dual role of the p55 α chain of the interleukin‐2 receptor
Authors: Flemming Claudia L. , Russell Stephen J. , Collins Mary K. L.
Published: March 2007

Mutation of Asp20 in human interleukin‐2 (IL‐2) to Lys is known to result in an IL‐2 molecule with unchanged binding to the p55 subunit of the IL‐2 receptor, but with greatly decreased affinity for the p75 subunit (Collins, L., Tsien, W.‐H., Seals, C. et al. Proc. Natl. Acad. Sci USA 1988. 85: 7709). Here we demonstrate that Lys20 IL‐2 competed with a reduced (10‐fold) affinity for high‐affinity IL‐2 receptors on two murine cell lines HT2 and CTLL. In parallel with this difference in receptor interaction, Lys20 IL‐2 stimulated half‐maximal HT2 cell proliferation at a 10‐fold higher concentration than wild‐type IL‐2. However, half‐maximal stimulation of CTLL cells required a 100‐fold higher concentration of Lys20 IL‐2. A similar 100‐fold reduction in bioactivity of Lys20 IL‐2 was observed for primary, activated, human or murine lymphocytes. Anti‐p55 antibodies increased the concentration of Lys20 IL‐2 required to stimulate HT2 cells to that required for CTLL cells. These data suggest that CTLL cells, while able to bind Lys20 IL‐2 with high affinity, are lacking a p55‐dependent function necessary for optimal stimulation. Therefore, p55 has a dual role, being important both for high‐affinity IL‐2 binding and for optimal cell triggering.
Analysis of cytokine gene expression in subpopulations of freshly isolated thymocytes and thymic stromal cells using semiquantitative polymerase chain reaction
Authors: Moore Nel C. , Anderson Graham , Smith Christopher A. , Owen John J. T. , Jenkinson Eric J.
Published: March 2007

Using a semi‐quantitative polymerase chain reaction (PCR) technique we have examined the expression of a panel of cytokines during thymus development, localizing the expression to individual components of the thymic stroma and thymocytes at different maturational stages. The expression of interleukin (IL)‐7, stem cell factor (SCF), IL‐1α and granulocyte‐monocyte‐colony‐stimulating factor (GM‐CSF) mRNA was mapped to individual stromal cell types, while the expression of IL‐1α and GM‐CSF, along with interferon (IFN)‐γ and IL‐4 was detected in the lymphoid compartment of fetal day (Fd) 14 thymus. The expression of lymphoid‐specific cytokine genes was selectively down‐regulated in thymocytes undergoing maturation. CD3−/104+8+ cells, representing an intermediate stage of thymocyte maturation, were devoid of cytokine gene expression. Their CD3+ progeny, on the other hand, expressed IFN‐γ mRNA, supporting the notion that positive selection of cells for further maturation induces the reexpression of some cytokine genes. The cytokine profiles of the various stromal components differed. Purified major histocompatibility complex class II+ cortical epithelial cells strongly expressed IL‐7 and SCF, but only limited expression of IL‐lα and GM‐CSF could be detected. Fetal mesenchyme, on the other hand, expressed SCF, IL‐lα and GM‐CSF but not IL‐7. The importance of these cytokine profiles in relation to T cell development is discussed.
Direct evidence for anergy in T lymphocytes tolerized by oral administration of ovalbumin
Authors: Melamed Doron , Friedman Aharon
Published: March 2007

The present study investigated bystander suppression, specific suppression and anergy as mechanisms for oral tolerance. Oral tolerance was induced in mice by a single gastric intubation of 20 mg ovalbumin (OVA) and was evaluated in vitro by the absence of T lymphocyte proliferative responses to OVA after priming by OVA‐complete Freund's adjuvant (CFA). T lymphocyte unresponsiveness was antigen specific, systemic and was not affected by the vehicle used for immunization. T lymphocytes derived from tolerant popliteal lymph nodes (PLN) responded to an acetone precipitate (AP) of mycobacteria present in CFA; this response was not suppressed by co‐culture with OVA, thereby arguing against a mechanism of bystander suppression in our system. Responses of PLN T lymphocytes derived from OVA‐CFA primed, non‐tolerant mice, or those of an OVA‐specific T lymphocyte line, were not suppressed by PLN or spleen cells derived from OVA tolerant mice. These results excluded the possibility that oral tolerance was induced and maintained by a mechanism of specific suppression. At the cellular level, we found that OVA‐tolerant T lymphocytes did not produce interleukin‐2 (IL‐2) nor express IL‐2 receptor in response to OVA stimulation in vitro; both observations are indicative of a state of anergy. Incubation of OVA‐tolerant PLN T lymphocytes together with murine recombinant IL‐2 for 5 days, released anergic T lymphocytes and a concomitant OVA‐specific proliferative response of CD4+ T cells was detected. Taken together, our experimental system excludes the involvement of bystander or specific suppression in the induction of oral tolerance to OVA, and provides direct evidence to show that oral tolerance results from specific T lymphocyte anergy.
Expression of a 32‐kDa ligand for the CD40 antigen on activated human T lymphocytes
Authors: Hermann Patrice , Blanchard Dominique , de Saint‐Vis Blandine , Fossiez François , Gaillard Claude , Vanbervliet BÉAtrice , Briére Francine , Banchereau Jacques , Galizzi Jean‐Pierre
Published: March 2007

To identify the ligand(s) of the human CD40 antigen, a cDNA encoding the extracellular domain of the CD40 antigen was fused to a cDNA encoding the constant region (Fc) of human IgGl. The CD40‐Fc fusion protein was able to specifically bind to CD4+ and various CD8+ T cell clones activated with immobilized anti‐CD3. The 125I‐labeled CD40‐Fc fusion protein bound anti‐CD3 activated CD4+ T cell clone (MT9) with an equilibrium dissociation constant (Ka) of 10‐20 nM. The human CD40‐binding protein expressed on the cell surface of activated T lymphocytes is a monomeric protein of ≈ 32 kDa. Minor components of 29 kDa and 17 kDa were also detected. A small proportion of CD4+ and CD8+ blood mononuclear T cells activated by anti‐CD3 expressed the CD40 ligand but its detection was best observed following depletion of B cells. Addition of B cells to purified T cells abolished the binding of CD40‐Fc obtained after anti‐CD3 activation.
Active suppression induced by anti‐CD4
Authors: Hutchings Patricia R. , Cooke Anne , Dawe Kim , Waldmann Herman , Roitt Ivan M.
Published: March 2007

Treatment with a non‐depleting monoclonal antibody to CD4 in the presence of mouse thyroglobulin (MTg) inhibits the development of murine autoimmune thyroiditis. This unresponsiveness was transferrable since such treatment generated a population of donor cells which could suppress the thyroiditis induced in lightly irradiated recipients by subsequent challenge with specific antigen. The suppression appears to be both antigen specific and antigen dependent and seems to discriminate between TH1 and TH2 helper subsets in that there is no significant effect on anti‐MTg autoantibodies after challenge.
Pathogenic autoimmunity to affinity‐purified mouse acetylcholine receptor induced without adjuvant in BALB/c mice
Authors: Jermy Andy , Beeson David , Vincent Angela
Published: March 2007

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are antibody‐mediated disorders in which anti‐acetylcholine receptor (anti‐AChR) antibodies cause loss of muscle AChR and subsequent weakness. Many species are susceptible to induction of EAMG with purified xenogeneic AChR in adjuvant, but injection of Torpedo AChR without adjuvants can also induce evidence of EAMG. To see whether pathogenic autoimmunity could be induced in mice by isolated mouse AChR we injected BALB/c mice with several doses (1 pmole; about 0.1 ug) of affinity‐purified AChR (from the BC3H1 cell line but thought to be identical with denervated mouse muscle) intraperitoneally, without adjuvant, over a period of 10‐22 weeks. Some of the mice became ill and died. High levels of serum anti‐mouse AChR, directed mainly towards the main immunogenic region, were found and, in the survivors, correlated with loss of muscle AChR. Thus BALB/c mice can mount an autoimmune response to minute amounts of mouse AChR, without the use of adjuvants, and this response is very similar to that found in MG. This novel finding has implications regarding the etiology of the human disease.
Reactive oxygen product formation after Fcγ receptor‐mediated neutrophil activation by monomeric mouse IgG2a: implications for the generation of first dose effects after OKT3 treatment
Authors: Zlabinger Gerhard J. , Rosenkranz Alexander R. , Schmaldienst Sabine , Stuhlmeier Karl , Böhmig Georg , Stöckl Johannes , Pohanka Erich , Kovarik Josef
Published: March 2007

In the present study, we provide evidence that IgG2a monoclonal antibody (mAb) OKT3 is able to induce reactive oxygen intermediate (ROI) formation in polymorphonuclear leukocytes (PMN) when FcγRIIIB as well as FcγRII are bound concomitantly. Inhibition of binding to either FcγR by specific mAb (3G8 or IV. 3, respectively) resulted in complete abrogation of the OKT3‐induced respiratory burst. The effect of OKT3 was independent from its specificity and thus also from its T cell‐activating property, since nonbinding IgG2a isotype controls induced similar amounts of ROI. The IgG2b mAb BMA031 as well as the respective nonbinding isotype control were only minimally effective. With regard to the potential role of PMN activation in inflammation and tissue damage, our findings offer an extended explanation for the generation of initial adverse reactions to OKT3. Thus, one might speculate that the concerted action of cytokines liberated after its administration, what may lead to margination of leukocytes, and activation of PMN via FcγR might produce first‐dose reactions to OKT3 by directing radical‐mediated damage against the endothelium.
Rat intestinal epithelial cell line IEC‐6 is activated by recombinant inferferon‐γ to inhibit replication of the coccidian Toxoplasma gondii
Authors: Dimier Isabelle H. , Bout Daniel T.
Published: March 2007

Toxoplasma gondii tachyzoites invaded and replicated in cultured rat small intestine cells of the line IEC‐6. Treatment of rat small intestine cells with murine recombinant interferon‐y resulted in a dose‐dependent inhibition of intracellular T. gondii replication. This could be of relevance as a first line defense against Toxoplasma infection.
Quantitative analysis of molecules which distinguish functional compartments within germinal centers
Authors: Hardie Deborah L. , Johnson Gerald D. , Khan Mahmood , MacLennan Ian C. M.
Published: March 2007

Five zones in the secondary follicles of human tonsils are described, which are distinguished by the phenotype of their constituent cells. Moving from the apex to the base of the follicle the zones are termed: follicular mantle, outer zone, apical light zone, basal light zone and dark zone. The dark zone contains proliferating, CD77high, centroblasts and thin, widely spaced processes of follicular dendritic cells (FDC). The apical and basal light zones on the other hand contain a dense network of FDC which express CD21 and CD54 strongly. The FDC of the apical light zone differ from those of the basal light zone by their high expression of CD23. Centroblasts of the dark zone give rise to non‐proliferating centrocytes which move apically through the light zone (Eur. J. Immunol. 1991. 21: 2951). The centrocytes of the basal light zone are more pyroninophilic, more closely‐packed and larger than those in the apical light zone. Consequently, by conventional histology the basal light zone appears to be part of the dark zone. The nomenclature adopted, however, adheres to the convention that the dark zone is filled with proliferating centroblasts. Cells undergoing apopotosis were identified both in the dark and light zones, but more than half of these cells were located in the basal light zone. This is consistent with the concept that the progeny of cells which undergo somatic mutation in their immunoglobulin variable region genes in the dark zone migrate to the light zone where they are selected on the basis of their capacity to bind to antigen held on FDC. Cells receiving an antigen‐dependent signal survive while those that do not kill themselves by apoptosis. The outer zone does not contain CD23high cells and in this way is distinguished from the adjacent follicular mantle and apical light zone. It contains small lymphoid cells, blasts and plasmacytoid cells. Many cells of the outer zone express CDw75 strongly. The outer zone also extends as a narrow band around the dark zone. Possible roles of FDC and T cells of the light zone and outer zone in inducing centrocytes to differentiate to memory cells or plasmablasts are discussed.
Molecular cloning of porcine T cell receptor α, β, γ and δ chains using polymerase chain reaction fragments of the constant regions
Authors: Thome Margot , Saalmüller Armin , Pfaff Eberhard
Published: March 2007

Fragments of the constant regions of porcine α, β, δ and two types of γ T cell receptor (TcR) chains were obtained by reverse transcription and polymerase chain reaction (PCR). Screening of a porcine peripheral T cell cDN A library with these PCR fragments led to the isolation of porcine TcR α, β, γ and δ chain clones. Sequence analysis of these clones and the respective PCR fragments demonstrated the existence of one α, one β, three γ and one δ chain isotype. Comparisons of the deduced amino acid sequences of the constant region with other species revealed a significant homology. For two of the three identified porcine γ chain insertions of 38 and 40 amino acids were found within the hinge region. In addition, our sequence data demonstrate a high variability in the cytoplasmic Cγ domain among the three porcine TcR γ isotypes as well as between species, which might be of structural and/or functional significance. Comparison with biochemical data indicate the existence of four porcine TcR γ isotypes.
Murine anti‐mouse T cell monoclonal antibodies elicit anti‐antibodies in mice: intra‐species immunization model for estimating potential patient sensitization against humanized anti‐T cell antibodies
Authors: Kremmer Elisabeth , Mysliwietz Josef , Lederer Raimund , Thierfelder Stefan
Published: March 2007

Humanization of immunosuppressive anti‐T cell monoclonal antibodies (mAb) raises the question as to how completely it helps to avoid formation of neutralizing anti‐antibodies (anti‐Ab) in patients. To get more information on intra‐species sensitization against anti‐T cell mAb, we produced two immunosuppressive mouse IgG2a anti‐mouse Thy‐1.2 mAb (MmT1 and MmT5) in AKR/J mice and measured the potential of MmT1 to elicit inhibitory anti‐Ab in AKR/J (H‐2k), C57BL/6 (H‐2b), congenic B10.BR (H‐2k) and DBA/2 (H‐2d) mice. After one injection once weekly for 4 weeks of 5 μg MmT1 (200 μg/kg) in C57BL/6 mice, without the use of any adjuvants, high concentrations of anti‐Ab directed against MmT1 (300 μg/ml) and MmT5 (100 μg/ml) were measured by enzyme‐linked immunosorbent assay. Similar concentrations of anti‐Ab were found in immunized DBA/2 and less in B10.BR mice. No syngeneic anti‐Ab could be produced in AKR/J. From the C57BL/6 mice, we raised anti‐MmT1+, MmT5+ idiotype (IDIO1) and anti‐MmT1+, MmT5+ allotype (ALLO1) mAb. An in vivo test system was adapted to measure the inhibitory effects of circulating poly‐ or monoclonal anti‐Ab. It revealed a reduction of in vivo depletion capacity not only of the sensitizing mAb (MmT1), but also of another anti‐Thy‐1.2 mAb (MmT5), with identical allotype but different idiotype.

From this we conclude that intra‐species immunization following injection of anti‐T cell mAb can produce high titer inhibitory anti‐idiotype and anti‐allotype antibodies. Implications for hyperchimeric or fully human anti‐T cell mAb are discussed.
Early appearance and activation of natural killer cells in tumor‐infiltrating lymphoid cells during tumor development
Authors: Kurosawa Shin , Matsuzaki Goro , Harada Mamoru , Ando Takashi , Nomoto Kikuo
Published: March 2007

We investigated NK cell infiltration into tumor developing lesions at early stage of tumor development after intraperitoneal inoculation of 3LL lung carcinoma into syngeneic C57BL/6 mice. Natural killer (NK) cells, which were detected by anti‐NK 1.1 monoclonal antibody (mAb), remarkably increased in number in tumor‐developing lesions (peritoneal cavity) as early as day 3 after inoculation of 3LL. The tumor‐infiltrating NK cells from 3LL‐inoculated mice produced a high level of interferon‐γ by co‐culture with 3LL and showed enhanced cytotoxic activities against both NK‐sensitive (YAC‐1) and NK‐resistant (3LL and P815) tumors. Furthermore, mice depleted of NK cells by injection of anti‐NK 1.1 mAb or antiasialo GM1 antibody showed shorter survival times after intraperitoneal inoculation of 3LL when compared with control mice. These results suggest that NK cells infiltrate the tumor‐developing lesion at an early stage and may participate in the early protection against tumors through production of a high amount of interferon‐γ and enhanced cytotoxicity at tumor‐bearing sites.
Recombinant CD40 ligand stimulation of murine B cell growth and differentiation: cooperative effects of cytokines
Authors: Maliszewski Charles R. , Grabstein Kenneth , Fanslow William C. , Armitage Richard , Spriggs Melanie K. , Sato Timothy A.
Published: March 2007

The ligand for the B cell surface antigen CD40 was recently cloned from a murine thymoma cDNA library and shown to be expressed on activated T cells. In this study, we investigate the biological effects of murine recombinant CD40 ligand. The recombinant CD40 ligand expressed on the CV‐1/EBNA monkey fibroblast cell line directly activated resting B cell to express elevated levels of cell surface class II major histocompatibility complex and CD23 molecules. CD40 ligand also stimulated B cell proliferation, reaching maximal levels on day 2 of culture and declining thereafter. This effect was positively regulated by other cytokines, most notably interleukin (IL)‐4 and IL‐5. By itself, CD40 ligand had no effect upon immunoglobulin secretion by B cells. However, when B cells were treated with CD40 ligand plus cytokines, immunoglobulin secretion was stimulated in a cytokine‐dependent and isotype‐specific manner. IL‐4 was a potent co‐stimulator of IgE and IgG1 in the presence of CD40 ligand, and IL‐5 acted synergistically with IL‐4 in these responses as well as in IgM and IgG3 production. Taken together, the results indicate that CD40 ligand is a potent regulatory molecule for B cell growth and differentiation, and its activities are potentiated in a cytokine‐specific manner.
B cell epitope on the U1 snRNP‐C autoantigen contains a sequence similar to that of the herpes simplex virus protein
Authors: Misaki Yoshikata , Yamamoto Kazuhiko , Yanagi Kazuo , Miura Hitoshi , Ichijo Hiroko , Kato Tomohiro , Mato Takashi , Welling‐Wester Systke , Nishioka Kusuki , Ito Koji
Published: March 2007

The mechanism of autoantibody production in autoimmune diseases is not well understood. In the present study we performed the B cell epitope mapping of the U1 small nuclear ribonucleoprotein (snRNP)‐C, one of the target molecules of anti‐nRNP autoantibody to investigate how B cells respond to the autoantigen. After cloning and expression of a full length complementary DNA (cDNA) encoding the U1‐C protein, several truncated mutants of the cDNA were constructed and expressed in E. coli. Although a few epitopes were distributed on the whole molecule, all anti‐C protein antibody‐positive patients' sera tested recognized the region between amino acid residues 102 and 125 of the coding sequence. This universal epitope region contains an amino acid sequence similar to that of the herpes simplex virus type 1 ICP4 protein. The peptides representing each molecule were cross‐reactive to each other. In addition this region cross‐reacted to the B/B′ protein. These observations suggest that molecular mimicry might be involved in the initiation of autoantibody production, followed by cross‐reactive events between the autoantigens and by antigen‐driven mechanisms to generate more complicated autoantibody patterns against the U1 snRNP complexes.
The structure of the μ/pseudo light chain complex on human pre‐B cells is consistent with a function in signal transduction
Authors: Brouns Gaby S. , de Vries Evert , van Noesel Carel J. M. , Mason David Y. , van Lier René A. W. , Borst Jannie
Published: March 2007

Prior to immunoglobulin (Ig) light (L) chain rearrangement, pre‐B cells can express μ heavy (H) chains at the cell surface in association with pseudo (ψ) L chains. This complex may be essential for B cell development. We have investigated the composition of the μ/ψL chain complex of a human pre‐B cell line, in view of its potential role in transmembrane signal transduction. The μ/λ. receptor of a mature B cell line was analyzed in comparison. The μ/ψL chain complex is associated with disulfide‐linked molecules that are homologous or identical to the mb‐1 and B29 proteins, known to be integral components of membrane Ig receptors on mature B cells. Both receptors contain tyrosine (Tyr) kinase activity. In the μ/λ receptor, the lyn and lck Tyr kinases could clearly be identified. The mb‐1 and B29 proteins in both μ/λ and μ/ψL chain receptors are substrates for in vitro phosphorylation on Tyr, but also on serine (Ser) and threonine (Thr) residues. The undefined μ‐associated Ser/Thr kinase also phosphorylates the sre‐related kinases in the μ/λ, receptor and a 43‐kDa μ‐associated protein that is present in both complexes. The 43‐kDa protein may be an integral part of both receptor types, or a transiently associated molecule instrumental in the signaling process. We conclude that the μ/ψL receptor on human pre‐B cells fulfills the presently known criteria to function as a signal transduction unit.
Structural motifs involved in human IgG antibody effector functions
Authors: Greenwood Judith , Clark Mike , Waldmann Herman
Published: March 2007

A humanized IgG antibody to CAMPATH‐1 antigen (CDw52) is known to be lympholytic both in vitro and in vivo. So as to improve therapeutic potency through protein engineering strategies, we wish to define the structural motifs underlying some of the documented differences in function between human (h) IgG1 and IgG4 forms of the antibody. By the creation of heavy chain domain‐switch and intra‐domain recombinant antibodies we have established an important role for the carboxy‐terminal half of the CH2 domain in determining differential behaviour in antibody‐dependent cytotoxicity (ADCC) and in complement lysis. If this same region were necessary for the effector mechanisms that operate in vivo, then it might be possible to improve antibody effector functions by construction of novel antibodies that possess within the one molecule multiple copies of the crucial hinge‐CH2 associated structures. Although our previous work suggested that the hIgG4 CAMPATH‐1 antibody was ineffective at ADCC, we found this to be so only in some individuals. In others, IgG4, and indeed all the IgG subclasses were able to mediate ADCC. Overall, though, hIgG1 remains the best choice isotype for lytic therapy in vivo.
Activation of uterine intraepithelial γδ T cell receptor‐positive lymphocytes during pregnancy
Authors: Meeusen Els , Fox Annette , Brandon Malcolm , Lee Chee‐Seong
Published: March 2007

Intraepithelial lymphocytes (IEL) of the uterus of non‐pregnant sheep were analyzed by single‐ and two‐color flow cytometry. Very few lymphocytes carrying classical B and T cell markers (CD5, surface immunoglobulin) were detected in the uterine epithelial cell suspensions and all IEL expressed the CD8 surface marker although with varying intensities. Three distinct subpopulations were identified including a major (46‐56%) population of CD8+CD45R−γδ T cell receptor (TcR)‐negative cells and approximately equal numbers of CD8+CD45R+γδTcR− and CD8+CD45R+γδTcR+ lymphocytes. The same three subpopulations were also present in the interplacentomal areas of the uterus of ewes at a late stage of pregnancy but there was a dramatic increase (60‐70%) in the γδ TcR+ subpopulation. In addition, a pronounced increase in both size and granularity was observed in the IEL population of pregnant uteri and this was attributed to the γδ TcR+ cells. Light and electron microscopic examination of these γδ TcR+ IEL revealed an increase in metabolic activity and the formation of exceptionally large cytoplasmic granules and confirmed their restricted localization within the uterine epithelium close to the trophoblast. These results represent for the first time, a clear example of the activation of γδ TcR+ cells which is not associated with an ongoing disease process or infection, γδ TcR+ cells have recently been observed in the epithelium of the murine reproductive tract and were characterized by their unique homogeneous receptor structure. The present results indicate that these cells may play an important physiological role during pregnancy.
Interleukin‐9 stimulates in vitro growth of mouse thymic lymphomas
Authors: Vink Anne , Renauld Jean‐Christophe , Warnier Guy , Van Snick Jacques
Published: March 2007

The ability of interleukin (IL)‐9 to stimulate the in vitro proliferation of freshly collected mouse thymic lymphoma cells was tested on a panel of 45 tumors, induced by N‐methyl‐N‐nitrosourea (MNU) or by X‐ray irradiation. IL‐9 significantly stimulated the proliferation of 26 of these tumors. Out of 11 other factors tested, only IL‐2, IL‐4 and IL‐7 showed similar activities. In addition to the responses to IL‐9 alone, a potent synergy was often observed between IL‐9 and IL‐2 for MNU‐induced tumors. Synergies between IL‐9 and IL‐7 or IL‐9 and IL‐4 were also observed but less frequently. The growth‐promoting activity of IL‐9 and the synergistic activities of IL‐2 and IL‐9 for thymic lymphoma cells were confirmed with cell lines established from the fresh tumors.
Chicken major histocompatibility complex class II B genes: analysis of interallelic and inter‐locus sequence variance
Authors: Zoorob Rima , Bernot Alain , Renoir Dominique Martel , Choukri Fatima , Auffray Charles
Published: March 2007

Five different chicken B‐LB genes were cloned and sequenced. The comparison of these sequences shows that they can be classified as members of two different families, the B‐LBII family (containing the B‐LBI and B‐LBII genes) and the B‐LBIII family (containing the B‐LBIII, B‐LBIV, and B‐LBV genes). The extent of polymorphism within each of these families was assessed by in vitro amplification of DNA fragments encompassing exon 2 in several haplotypes. The nucleotide sequences were determined, and pairwise relationships were evaluated. In the course of this work, a sixth gene termed B‐LBVI was identified, defining a third family (B‐LBVI family). Polymorphism of the B‐LBIII or B‐LBVI families is far less extensive than that of the B‐LBII family. In this latter, the distribution of conserved and polymorphic residues is similar to what has been described in mammals. These families seem to have been generated by gene duplication events giving rise to several isotypes, as observed in mammals. However, phylogenetic analyses indicate that these families are not homologous to their mammalian counterparts. Evaluation of the level of transcription of these different genes showed that genes from the B‐LBII family are predominantly transcribed over those of the other families.
Autoantibodies from patients with systemic lupus erythematosus bind a shared sequence of SmD and Epstein‐Barr virus‐encoded nuclear antigen EBNA I
Authors: Sabbatini Alessandra , Bombardieri Stefano , Migliorini Paola
Published: March 2007

SmD is one of the small nuclear ribonucleoproteins frequently targeted by autoantibodies in systemic lupus erythematosus. We isolated and characterized the antibodies present in lupus sera that are specific for the C‐terminal region of SmD (sequence 95–119). This region is highly homologous to sequence 35‐58 of the EBNA I antigen, one of the nuclear antigens induced by infection with Epstein‐Barr virus. Antibodies affinity purified over a peptide 95–119 column were able to recognize this sequence in the context of the whole SmD molecule, as they reacted with blotted recombinant SmD. Anti‐SmD 95‐119 antibodies bound also the EBNA I 35–58 peptide and detected the EBNA I molecule in a total cell extract from Epstein‐Barr virus‐infected lines. A population of anti‐SmD antibodies is, therefore, able to bind an epitope shared by the autoantigen and the viral antigen EBNA I. To investigate the involvement of this shared epitope in the generation of anti‐SmD antibodies, we immunized mice with the EBNA I 35‐58 peptide. Sera from immunized animals displayed the same pattern of reactivity of spontaneously produced anti‐SmD antibodies. They reacted in fact with the EBNA peptide as well as with SmD 95‐119 and recombinant SmD. These data suggest that molecular mimicry may play a role in the induction of anti‐SmD autoantibodies.
Characterization of the human T cell receptor α‐chain repertoire and demonstration of a genetic influence on Vα usage
Authors: Moss Paul A. H. , Rosenberg William M. C. , Zintzaras Elias , Bell John I.
Published: March 2007

Despite considerable knowledge of the determinants and profile of the Tcell receptor (TCR) β‐chain repertoire, very little is known about the repertoire of the Vα and Jα segments of the TCR in man or mouse. We have used the anchored polymerase chain reaction (PCR) to analyze the human TCR α‐chain repertoire at the mRNA level in peripheral blood lymphocytes. We find marked differences in the usage of individual Vα segments. The Vα repertoire is determined both by differences in the use of individual Vα gene segments at the time of gene rearrangement and by genetic influences on the selection of the expressed Vα repertoire, presumably during thymic maturation. Analysis of the Jα repertoire shows non‐random usage of individual Jα segments but no obvious relationship to chromosomal position. We also report three new Vα segments, three new Jα segments and possible allelic variation in Vα gene segments. Two unusual TCRα transcripts are described which add new information on the nature of TCRα transcript assembly.
Essential role of tumor necrosis factor‐α in the differentiation of human tonsil in vivo induced B cells capable of spontaneous and high‐rate immunoglobulin secretion
Authors: Rodríguez Carmen , Roldán Ernesto , Navas Gloria , Brieva José A.
Published: March 2007

Human tonsils contain B cells capable of spontaneous and high‐rate immunoglobulin (Ig) secretion in vitro. These cells are in vivo induced mature B cells, and, as such, they provide an adequate model for studying tonsil B cell differentiation. The present report analyzes the effect of a variety of factors on purified tonsil B cells capable of spontaneous IgG secretion in fetal calf serum (FCS)‐containing and serum‐free supplemented cultures. Tumor necrosis factor‐(TNF)α was found to be important for these B cells to reach the high‐rate IgG‐secreting stage, as is indicated by the following findings: (a) none of the factors used modified tonsil B cell IgG secretion in FCS‐containing cultures; (b) TNF‐α (5‐20 ng/ml), but not other cytokines or factors including interleukin (IL)‐6, was capable of restoring missing IgG production in serum‐free supplemented cultures of tonsil B cells; and (c) IgG secretion in FCS‐containing cultures was inhibited by the addition of blocking anti‐TNF‐α antibodies, but not anti‐IL‐6 antibodies, and this inhibition could be specifically reversed by exogenous TNF‐α. TNF‐α was actively produced by tonsil B cells (range 120‐750 pg/ml) in the presence, but not in the absence, of FCS. The TNF‐α inductive effect occurred during the first 12 h of culture and did not require DNA synthesis. These results indicate that the early and endogenous generation of TNF‐α seems to be essential for tonsil in vivo induced B cells to differentiate into the high‐rate Ig‐secreting stage.
Engagement of CD40 lowers the threshold for activation of resting B cells via antigen receptor
Authors: Wheeler Kay , Pound John D. , Gordon John , Jefferis Roy
Published: March 2007

Cross‐linking of surface Ig (sIg) on resting B cells can generate intracellular signals; however, for T‐dependent antigens to promote growth and differentiation additional surface receptors must be engaged. Ligation of CD40 can stimulate B cell proliferation in the presence of interleukin‐4. A recently identified counterstructure for CD40 is found on T helper cells and is believed to represent the cognate ligand for B cell activation. This study investigates the role of CD40 as an accessory molecule in sIg‐dependent B cell activation. Simultaneous ligation of sIg and CD40 by monoclonal antibodies (mAb) in the presence of mouse L cells which express human Fey receptor type II (FcγRII‐L cells) results in potent stimulation of small resting B cells. When CD40 is co‐ligated, picomolar concentrations of mouse IgG1 anti‐μ, and anti‐δ mAb can stimulate B cell proliferation. This requires interaction of the anti‐Ig mAb with the FcγRII‐L cells: a mouse IgG2a anti‐μ, mAb which is not recognized by FcγRII, was ≥ 1000‐fold less effective. These findings suggest a mechanism for B cell activation whereby engagement of T cells via CD40 and its cognate ligand provides potent enhancement of signals delivered through sIg. Based on these observations, models for the activation of B cells by T‐dependent antigens are presented.
T cell response in murine Leishmanial mexicana amazonensis infection: production of interferon‐γby CD8+ cells
Authors: Chan Marion Man‐Ying
Published: March 2007

The immune response to Leishmania major has been the subject of many investigations. However, Leishmania includes many species with different clinical manifestations. In this report, we studied the Tcell response to L. mexicana amazonensis, a New World species, in a murine model. We found that, similar to L. major, an Old World species, resistant C57BL/6 mice produced a high level of IFN‐γ and a low level of IL‐4. Conversely, susceptible BALB/c mice produced a much lower level of IFN‐γ and higher level of IL‐4.

Although IFN‐γ is one of the important lymphokines that mediate macrophage activation and thus the destruction of the intracellular parasites, which lymphocyte subsets are producing the IFN‐γ is still a controversy. Much evidence including the isolation of protective, IFN‐γ‐producing, CD4+ cell lines have confirmed the participation of CD4+ Thl cells unequivocally. However, both CD4+ and CD8+ cells produced IFN‐γ. Recently, an increasing body of evidence has appeared suggesting that CD8+ cells also play a role in the resolution of murine L. major infection. We found that in the L. m. amazonensis model, when CD8+ lymphocytes from resistant C57BL/6 mice were eliminated by anti‐CD8 antibody and complement‐mediated lysis, the IFN‐γ production was reduced by 77%. This indicated that CD8+ cells produced a significant amount of the IFN‐γ. However, our results also indicate that IFN‐γ production by CD8+ cells was dependent on CD4+ cells.