bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterial‐induced cytotoxic T cells as well as nonspecific killer cells derived from healthy individuals and leprosy patients
Authors: Kaleab Birhane , Ottenoff Tom , Converse Paul , Halapi Eva , Tadesse Genet , Rottenberg Martin , Kiessling Rolf
Published: March 2007

Little information is available about the generation and specificity of the cytotoxic cells that eliminate human monocytes/macrophages infected with mycobacteria. To address this we have developed a cytotoxicity assay in which 51Cr‐labeled monocytes pulsed with bacillus Calmette Guérin (BCG) or Myco‐bacterium leprae, were used as target cells in overnight cytotoxicity assays. As effector cells, peripheral blood mononuclear cells from healthy occupational contacts or from leprosy patients stimulated with antigen for 7 days were used. Cytotoxicity against antigen‐pulsed monocytes that could be induced by mycobacterial antigens was proportional to the degree of antigen responsiveness in each individual, as measured in lymphocyte transformation tests. The lepromatous leprosy patients tested were often poor responders to BCG as well as M. leprae, both with regard to induction of cytotoxicity as well as in lympho‐proliferation. Killing was significantly higher against antigen‐pulsed vs. non‐pulsed monocytes, although significant killing was induced against the latter as well and paralleled by induction of natural killer activity against the K‐562 target cell. Cross‐reactivity was observed between BCG and M. leprae, but not with unrelated antigen (tetanus toxoid) or with endogenous stress proteins induced by heat shock. M. leprae‐ and BCG‐activated cytotoxic cells were found in both the CD4−CD8+ and CD4+CD8− populations, whereas in contrast the soluble antigen, purified protein derivative of M. tuberculosis, generated cytotoxic cells that were exclusively of the CD4+ phenotype. The involvement of both specific T cells as well as nonspecific cells in the killing of human macrophages may be important with respect to protection and immunopathology induced by mycobacterial antigens.
CD27 expression by a distinct subpopulation of human B lymphocytes
Authors: Maurer Dieter , Holter Wolfgang , Majdic Otto , Fischer Gottfried F. , Knapp Walter
Published: March 2007

CD27 is present on the surface of a major subset of peripheral blood T lymphocytes. In this report we show that CD27 is also expressed on a subpopulation of the normal human B cell lineage which is absent from cord blood but present in tonsils and in the peripheral blood of adult individuals. CD27+ B lymphocytes are characterized by the following criteria: (a) in terms of physical properties, the CD27+ B cells form a population with an increased cell size combined with a decreased cell density; (b) the CD27 expression of tonsillar B lymphocytes is positively correlated with mIgA but negatively correlated with membrane IgM/membrane IgD positivity; (c) CD27 on B cells can be induced selectively by the combination of Staphylococcus aureus plus interleukin 2, but not by either treatment alone, and (d) CD27+ B lymphocytes express high levels of the adhesion structures LFA‐1 (CD11a), ICAM‐1 (CD54), LFA‐3 (CD58) and of the lymphocyte homing receptor CD44. These latter findings suggest that CD27+ B cells are predisposed to form cell‐cell interactions. Accordingly, within 3 h of cell culture CD27+, but not CD27−, B lymphocytes were found to form LFA‐1‐mediated homotypic B cell clusters.
A unique subset of normal murine CD4+ T cells lacking Thy‐1 is expanded in a murine retrovirus‐induced immunodeficiency syndrome, MAIDS
Authors: Holmes Kevin L. , Morse Herbert C. , Makino Masahiko , Hardy Richard R. , Hayakawad Kyoko
Published: March 2007

Some strains of mice inoculated with LP‐BM5 murine leukemia virus (MuLV) develop a syndrome, termed mouse acquired immunodeficiency syndrome (MAIDS), characterized by progressive lymphoproliferation and profound immunodeficiency. LP‐BM5 MuLV is a virus mixture that contains ecotropic (eco) and mink cell focus‐induced MuLV and a defective genome that is the proximal cause of disease. Flow cytometry analyses of spleen and lymph nodes from susceptible C57BL/6 mice infected with this virus mixture revealed the presence in spleen and peripheral lymph nodes of a previously unrecognized subset of CD4+CD3+ T cells that are Thy‐1−. The frequency of these cells increased with progression of disease, eventually comprising between 30% and 50% of all CD4+ cells. Infection of A/J mice, a strain which is genetically resistant to development of MAIDS, did not induce an increase of this T cell population, indicating that infection with the virus mixture was insufficient to induce its proliferation. A central role for the defective virus in this process was suggested by the finding that C57BL/6 mice infected with LP‐BM5 eco alone did not have increased frequencies of Thy‐1−CD4+ cells in spleen. Studies of spleen and peripheral lymph node cells from normal mice demonstrated the presence of Thy‐1−CD4+ cells at frequencies of 1%–2%. Studies using two anti‐T cell monoclonal antibodies, SM6C10 and SM3G11, that define four CD4+ subsets showed that Thy‐1−CD4+ T cells from normal and infected mice were present only in the 6C10− subsets.
Development of immature thymocytes: initiation of CD3, CD4, and CD8 acquisition parallels down‐regulation of the interleukin 2 receptor α chain
Authors: Petrie Howard T. , Pearse Martin , Scollay Roland , Shortman Ken
Published: March 2007

We have previously identified a developmental sequence among immature thymocytes, prior to their expression of the lineage markers CD3, CD4, and CD8. This sequence is marked by transient expression of the interleukin 2 receptor α chain (IL 2Rα). The most mature cells in this sequence (surface phenotype heat‐stable antigen (HSA)++ Pgp‐1− IL 2Rα−) are the immediate precursors to CD4+CD8+ small cortical thymocytes, and have by definition been considered to be CD4−CD8−. We now show that these cells display low levels of surface CD4 and CD8, but not CD3. This low‐level expression begins to appear immediately after the loss of IL 2Rα expression. Northern blot analysis for mRNA expression confirms that these IL 2Rα− cells are transcribing CD4 and CD8 mRNA, in contrast to their immediate (IL 2Rα+) precursor. Upon unstimulated culture, these IL 2Rα− cells gradually acquire high levels of CD4 and CD8, as well as low levels of CD3, whereas IL 2Rα− cells do not. These findings suggest that the IL 2Rα+ subset is the end of the true CD3−CD4−CD8− phase, and that the intracellular signals for CD3, CD4, and CD8 acquisition occur simultaneously with, or immediately prior to, the signal for down‐regulation of IL 2Rα.
T helper cell epitope of rabies virus nucleoprotein defined by tri‐ and tetrapeptides
Authors: Ertl Hildegund C. J. , Dietzschold Bernhard , Otvos Laszlo
Published: March 2007

T cell clones and subsequently hybridomas were generated from rabies virus‐immune C3H/He mice to an immunodominant epitope of the viral nucleoprotein, termed 31D, that had previously been identified by a 15‐amino acid‐long synthetic peptide. T cells to this epitope that by phenotypical and functional characteristics belonged to the T helper cell subset were shown to respond to most rabies and rabies‐related viruses. In order to define the minimal sequence needed to elicit a response from 31D‐specific T cell clones or hybridomas, a number of peptides of varied lengths, i.e. 3–32 amino acids long, were tested. The ability of the peptides to induce a response was inversely correlated in their lengths, i.e., short peptides (3–5 amino acids long) had to be used at 106 times higher concentrations as compared to long peptides (15 or 32 amino acids long). Conversely, the specificity of the T cell response was directly correlated to the length of the peptides, i.e., while the response to 15‐amino acid‐long peptides exhibited a high degree of specificity, the response to 3‐ to 5‐amino acid‐long peptides showed a high degree of flexibility. The long as well as the short peptides had to be presented in association with I‐Ek. We speculate that in this system the T cell receptor interacts predominantly with a peptide‐induced modification of the I‐Ek molecule.
Differential expression of the alternatively spliced exons of murine CD45 in Th1 and Th2 cell clones
Authors: Luqman Mohammad , Johnson Pauline , Trowbridge Ian , Bottomly Kim
Published: March 2007

Antigen‐specific murine CD4+ T cell clones can be divided into functionally distinct subsets known as Th1 and Th2. To date these cells have been indistinguishable by surface phenotype. This report identifies two anti‐CD45R monoclonal antibodies (14.8 and C363.16A) that bind preferentially to Th2 cells. Further analysis of the CD45‐specific mRNA in Th1 and Th2 cells shows clear differences between these two cell types. Th1 cell clones express mRNA for the two smallest forms of CD45 containing none or only one of the alternatively splices exons. In contrast, Th2 cell clones express predominantly the high molecular weight isoforms of CD45 containing two or three of the alternatively spliced exons.
Specific suppression of antibody responses by soluble protein‐specific, class II‐restricted cytolytic T lymphocyte clones
Authors: Shinohara Nobukata , Huang Yi‐Ying , Muroyama Akiko
Published: March 2007

Antigenic stimulation with exogenous soluble proteins induces cytotoxic T lymphocytes (CTL) recognizing antigenic peptides presented on class II major histocompatibility complex (MHC) molecules. Such CTL have been shown to lyse preferentially B cells expressing immunoglobulin receptors reactive with the relevant antigens, presumably because such B cells can efficiently trap and present the antigen. Therefore, possible involvement of soluble protein antigen‐specific CTL in specific suppression of antibody responses has been hypothesised. In this report, keyhole limpet hemocyanin and ovalbumin‐specific, class II‐restricted CD4+ CTL clones established from lymph nodes of immunized mice were examined for their suppressive activities on antibody production. When these CTL clones were added to in vitro secondary cultures, genetically restricted, carrier‐specific suppression of anti‐2, 4, 6‐trinitrophenyl antibody production was observed. These data therefore demonstrate that CTL directed toward soluble antigens are capable of mediating specific suppression of antibody responses. Furthermore, the antibody response of MHC‐heterozygous F1 lymphocytes was almost completely suppressed by a CTL clone restricted to one parental class II MHC antigen, indicating that the mechanism of suppression by these CTL is distinct from that by classical suppressor T cells.
Role of bone marrow cells in autoantibody production and lymphoproliferation in the novel mutant strain of mice, CBA/KIJms‐lprcg/lprcg
Authors: Kimura Mikio , Katagiri Takuya , Kikuchi Yoshimi , Shimada Kaoru , Nariuchi Hideo , Wakabayashi Tomo , Matsuzawa Akio
Published: March 2007

The novel mutant gene, lprcg, is allelic with lpr, but complements the gld gene in induction of lymphoproliferation. Mice with this autosomal recessive mutation, CBA/KlJms‐lprcg/lprcg (CBA‐lprcg), served in this study to analyze the abnormalities of bone marrow (BM) stem cells responsible for autoantibody production and lymphoproliferation by BM transfer experiments. Transferred CBA‐lprcg BM cells might have differentiated into so‐called double‐negative, anomalous lymphoid cells and caused production of autoantibodies such as anti‐DNA antibodies in the environment of normal CBA/KlJms−+/+ (CBA−+) mice. Macroscopic graft‐vs.‐host‐like disease as reported in lpr→non‐lpr BM transfer was not observed in these recipients. In this BM chimera, however, lymphoproliferation did not ensue and the host's lymph nodes became atrophic. The lymphoproliferation required the coexistence of lprcg BM cells and lprcg lymph nodes in CBA−+ mice. The results indicate that the functions of the lprcg gene are expressed at both BM and lymph node levels. Thus, this mutant strain of mice should provide an excellent model for analyzing aberrant lymphocyte differentiation from the BM cells leading to autoimmunity and lymphoproliferative disorder.
Prevention of lethal graft‐vs.‐host disease by a single low dose injection of anti‐T cell monoclonal antibody to the allograft recipients
Authors: Knulst André C. , Bril‐Bazuin Clara , Benner Robbert
Published: March 2007

We investigated the capacity of monoclonal antibody (mAb) treatment to prevent graft‐vs.‐host disease (GVHD) in lethally irradiated, allogeneically reconstituted mice, employing anti‐T cell (subset) mAb and a fully allogeneic strain combination. In this strain combination, purified CD4+ cells were able to induce a lethal GVH reaction, whereas purified CD8+ cells were not. In the same strain combination, a single intraperitoneal injection of IgG2b anti‐Thy‐1 mAb, one day after reconstitution, caused a dose‐dependent improvement of the survival. A single injection of a dose as low as 12.5 μg per mouse was already effective. Intravenous and intraperitoneal administration of the mAb appeared equally effective. For effective prevention of GVHD the treatment could be postponed until the 4th day after transplantation, but treatment delayed until day 6 was no longer effective. Treatment with IgG2b mAb specific for either helper or cytotoxic T cells also led to improvement of GVHD and survival, but was less effective than treatment with anti‐Thy‐1 mAb. Clinically, there was a difference in the effectiveness of anti‐CD4 and anti‐CD8 treatment, since symptoms of GVHD started earlier in the anti‐CD8 treated group and the survival was better in the anti‐CD4 treated group. These results press for prospective clinical studies employing anti‐T cell mAb treatment early after allogeneic bone marrow transplantation, especially in HLA mismatched cases.
A monoclonal antibody specific for a cytochrome c T cell stimulatory peptide inhibits T cell responses and affects the way the peptide associates with antigen‐presenting cells
Authors: Jemmerson Ronald , Johnson Julia G. , Burrell Edwin , Taylor P. Sean , Jenkins Marc K.
Published: March 2007

A monoclonal antibody (mAb) specific for the 93–104 segment of pigeon cytochrome c (cyt) was shown to block interleukin 2 production and proliferation by pigeon cyt‐specific T cells in response to the pigeon cyt 81–104 peptide using either the LK35.2 B cell hybridoma or normal splenocytes as antigen‐presenting cells (APC). The mAb inhibited the response to soluble peptide antigen presented by metabolically inactive paraformaldehyde‐fixed APC but not the response to APC that were pre‐pulsed with Ag. These results suggest that the mAb blocked the formation of peptide‐major histocompatibility complex (MHC) class II molecule complexes at the cell surface but did not displace the peptide once bound to the MHC class II molecule. As determined by direct binding experiments using labeled peptide, the major means of free peptide association with live APC was fluid‐phase endocytosis. No free peptide associated directly with the MHC class II molecule at the cell surface near 0°C since APC pulsed with peptide on ice did not activate cyt‐specific T cells. The mAb enhanced the association of the radiolabeled peptide with APC at 4°C apparently by binding of the peptide‐mAb complex to Fc receptors. By stripping molecules from the LK35.2 cell surface using a nonspecific protease it was shown that the peptide‐mAb complexes were not internalized either at 4°C or 37°C. Since the mAb was found to stably bind the peptide at pH levels below that of endosomes (pH 5.5–6.2) even if the peptide‐mAb complexes were taken up by fluid‐phase endocytosis, it is likely that the peptide would not be able to associate with MHC class II molecules inside the APC. This mAb appears to inhibit T cell activation by blocking the formation of peptide‐MHC class II molecule complexes at the cell surface and by interfering with uptake of the peptide into endosomes. Therefore, it is different from other antibodies that have been reported to block T cell receptor recognition of preformed peptide/MHC class II molecule complexes.
Cell‐type‐specific recognition of allogeneic cells by alloreactive cytotoxic T cells: A consequence of peptide‐dependent allorecognition
Authors: Heath William R. , Sherman Linda A.
Published: March 2007

Recently, we provided the first direct evidence that the determinant recognized by some alloreactive cytotoxic T lymphocytes (CTL) clones included an endogenous peptide antigen(s), which could be derived by cyanogen bromide cleavage of cytoplasmic proteins. The studies outlined in this report provide three important findings which confirm and extend our previous results. First, the description of peptide‐dependent Kb‐specific alloreactive CTL clones is now extended to clones isolated from several responders primed with allogeneic EL4 tumor cells. This indicates that the previously reported results were not unique to the original EL4‐primed responder. Second, we establish that peptide‐dependent Kb‐specific alloreactive CTL clones can be identified after priming with normal allogeneic spleen cells. Thus, induction of peptide‐dependent CTL clones is not a unique property of EL4 tumor cells. These results suggest that peptide plays an important role in formation of the allogeneic determinants recognized during graft rejection. Third, these studies reveal that a number of alloreactive CTL clones exhibit cell‐type‐specific recognition of allogeneic murine target cells. This is consistant with the possibility that some CTL clones recognize peptide antigens that are limited in their tissue distribution. The mechanisms and implications of these findings are discussed.
Cross‐linking of VLA/CD29 molecule has a co‐mitogenic effect with anti‐CD3 on CD4 cell activation in serum‐free culture system
Authors: Yamada Akira , Nojima Yoshihisa , Sugita Kanji , Dang Nam H. , Schlossman Stuart F. , Morimoto Chikao
Published: March 2007

In this study, we demonstrated that antibodies reactive with a common β1 subunit (CD29) of the VLA family of antigens could synergize with CD3 but not CD2 in inducing CD4 cell proliferation in a serum‐free culture system. The extent of activation was similar to that obtained when CD4 cells were cultured with anti‐CD3 plus fibronectin. The proliferative response induced via the CD3‐CD29 pathway was associated with the interleukin (IL) 2 autocrine pathway, including IL2 production and IL2 receptor expression. Specifically, cross‐linking of VLA‐5, but not other members of the VLA family of antigens with the CD3 molecule induced strong CD4 cell activation. However, cross‐linking of CD3 with other cell surface antigens such as CD4 did not induce CD4 cell activation in this serum‐free culture system. The above results strongly suggest that the VLA/CD29 family of antigens may play an important role in regulating CD4 T cell activation via the CD3‐T cell receptor pathway.
Selective migration of the human helper‐inducer memory T cell subset: confirmation by in vivo cellular kinetic studies
Authors: Pitzalis Costantino , Kingsley Gabrielle H. , Covelli Michele , Meliconi Riccardo , Markey Andrew , Panayi Gabriel S.
Published: March 2007

The vast majority of T cells present in chronic inflammatory lesions are of the helper‐inducer/memory (CD45RO+CD29+) phenotype; suppressor‐inducer/naive cells (CD45RA+) are virtually absent. Furthermore, CD4+ T cells are found more frequently than CD8+ cells. Previous in vitro studies have suggested that this may be, in part, due to the increased capacity of CD45RO+CD29+ T cells to bind to endothelium and, thus, enter inflammatory foci but no in vivo evidence for preferential migration exists. To investigate this, suction blisters were generated over a purified protein derivative of tuberculin‐induced delayed‐type hypersensitivity lesions in humans and the phenotype of the blister cells was studied. At all time points, a preponderance of CD45RO+CD29+ cells over CD45RA+ cells and of CD4 over CD8 cells was demonstrated. Because of the rapid kinetics, this appears to represent preferential migration of these cell types rather than in situ proliferation or phenotype conversion. In addition the expression of the CD45RO but not the CD45RA antigen was up‐regulated on blister T cells compared to blood T cells. Analysis of blister fluid showed high concentrations of interleukin 6 but not tumor necrosis factor‐α or lymphotoxin. This study shows for the first time directly in vivo that CD45RO+ T cells migrate preferentially into inflammatory lesions. Furthermore, it illustrates the potential usefulness of this system in the analysis of the early phases of the immune inflammatory response.
Human transferrin allows efficient IgE production by anti‐CD3‐stimulated human lymphocytes at low cell densities
Authors: van der Pouw‐Kraan Tineke , van Kooten Cees , van Oers Rien , Aarden Lucien A.
Published: March 2007

A solid‐phase‐coupled anti‐CD3 T cell activation system was used to study the regulation of human IgE production in vitro. Using 5000 peripheral blood lymphocytes from healthy donors, containing 10%–20% B lymphocytes and no monocytes, IgE was produced very efficiently on a per cell basis. A key observation was that apart from interleukin (IL)4, human transferrin was essential for IgE production. Furthermore it was found that IgE was produced at low densities only; at higher cell concentrations IgE production was completely abrogated, whereas IgM production increased with increasing cell density. This inhibition at higher cell densities is probably mediated by IL 2. Addition of low amounts (6 U/ml) of IL 2 strongly enhanced IgE and IgM production at low cell densities, but higher concentrations of IL 2 (50 U/ml) were strongly inhibitory for IgE production.
FcγRIII (CD16) on human macrophages is a functional product of the FcγRIII‐2 gene
Authors: Perussia Bice , Ravetch Jeffrey V.
Published: March 2007

The low‐affinity Fc receptor for immune‐complexed IgG (FcγRIII; CD16) present on in vitro cultured human monocytes are encoded by an FcγRIII‐2 gene that, by cDNA sequence analysis, is identical to that expressed on tissue macrophages and on natural killer cells. In macrophages, FcγRIII‐2 encodes a glycoprotein of 52‐62 kDa, with a peptide backbone of 33 kDa identical to that of the homologous receptor on natural killer cells. Like this and unlike in polymorphonuclear neutrophils, FcγRIII (CD16) on cultured monocytes is insensitive to phosphatidylinositol‐specific phospholipase C, is not allelic for the neutrophil NA alloantigens NA‐1/NA‐2, is not recognized by a monoclonal antibody (1D3) detecting an epitope present only on neutrophil FcγRIII (CD16) and functions to trigger cytotoxicity upon ligand binding.
Regulation of tumor necrosis factor (TNF) release by murine peritoneal macrophages: role of cell stimulation and specific phagocytic plasma membrane receptors
Authors: Stein Michael , Gordon Siamon
Published: March 2007

In spite of the physiologic and pathologic importance of tumor necrosis factor (TNF), the cellular factors that govern its release by macrophages (MΦ) are poorly understood, in comparison with other secretory products. We have studied the role of MΦ heterogeneity and of plasma membrane receptors in regulating TNF release in vitro. Resident and various exudate murine peritoneal MΦ populations were challenged with lipopolysaccharide (LPS) or different phagocytic particles, and TNF release assayed by cytotoxicity for L‐929 fibroblasts. Resident peritoneal MΦ (RPMΦ) released a small amount of TNF in response to LPS whereas thioglycollate‐elicited MΦ (TPMΦ) released high levels of TNF (5000 U/3 × 105 MΦ/ml). MΦ elicited by Bio‐Gel polyacrylamide beads (BgPMΦ), another nonspecific inflammatory stimulus, or early in the course of intraperitoneal Bacillus Calmette‐Guérin infection, before recruited cells become immunologically activated, released tenfold less TNF after the same stimulus. By contrast, TNF release in response to various phagocytic triggers was similar (∼300‐600 U/3 × 105 MΦ/ml) in all MΦ populations including RPMΦ. The response by BgPMΦ to LPS was enhanced by pre‐treatment in vitro with interferon‐γ or thioglycollate broth.

With respect to phagocytic receptor triggering we found that complement receptor type 3 (CR3) ligation or latex uptake did not mediate release of significant quantities of TNF (<48 U/3 × 105 MΦ/ml) by any MΦ, whereas ligation of the Fc receptor for IgG1/IgG2b subclasses or of receptors for zymosan particles sufficed, in the absence of ingestion. to induce release of circa 500 U/3 × 105 MΦ/ml TNF by all MΦ tested. Our studies show that MΦ vary in respect to priming for TNF release and that heterogeneity should be related to a particular triggering stimulus. Furthermore, the capacity of some MΦ populations to release unusually high levels of TNF depends on immune or nonspecific stimuli subsequent to the process of inflammatory recruitment.
The effect of the immunosuppressant FK‐506 on alternate pathways of T cell activation
Authors: Bierer Barbara E. , Schreiber Stuart L. , Burakoff Steven J.
Published: March 2007

Structurally unrelated, FK‐506 and cyclosporin (CsA) bind to and inhibit the action of distinct cytoplasmic receptors, FK‐506‐binding protein (FKBP) and cyclophilin (CyP), respectively. These receptors, termed immunophilins, share no sequence similarity, and yet both have been demonstrated to be capable of catalyzing the cis‐trans isomerization of peptidyl‐prolyl bonds (rotamase activity). Because FK‐506 and CsA bind to different intracellular target structures, we investigated the spectrum of action of FK‐506, in comparison to CsA, on T cell activation. We have shown that FK‐506, like CsA, is able to inhibit T cell activation mediated not only by the T cell receptor‐CD3 complex, but also via another surface molecule, CD2. T cell proliferation, stimulation of interleukin 2 production, and induction of apoptosis were all sensitive to inhibition by both FK‐506 and CsA. With each parameter of activation, FK‐506 is approximately 10‐100‐fold more effective than CsA. In contrast, FK‐506 did not affect T cell proliferation induced by anti‐CD28 monoclonal antibody in the presence of phorbol 12‐myristate 13‐acetate. This CD28 pathway, however, was inhibitable by a structural homolog of FK‐506, rapamycin, demonstrating that the mechanism of action of FK‐506 has specificity. These data suggest that immunophilins or the complex of drug coupled to immunophilin (i.e. FK‐506/FKBP, CsA/CyP) are involved in and regulate selective pathways of T cell stimulation.
Murine tumor cells expressing the gene for the human interferon αβ receptor elicit antibodies in syngeneic mice to the active form of the receptor
Authors: Uzé Gilles , Lutfalla Georges , Eid Pierre , Maury Chantal , Bandu Marie‐Thérèse , Gresser Ion , Mogensen Knud
Published: March 2007

The cellular receptor for the human α and β interferons (IFN) was expressed, by gene transfer, in a murine hepatoma‐derived cell line, BTG 9A. Injected subcutaneously into the syngeneic mouse (C57BL/6), the parental and the transfected cells grew and formed tumors which later regressed. More than half the mice bearing tumors derived from cells expressing the receptor, developed IgG antibodies capable of blocking the activity, on human cells, of human recombinant IFN‐αB, ‐αA, ‐αD and of natural human IFN‐β, but not of recombinant IFN‐γ. Cross‐reactivity of human IFN‐α on murine and bovine cells was unaffected by these antibodies. The binding of human IFN‐α to solubilized receptors from human lymphoid cell lines was also blocked and complexes of radiolabeled recombinant IFN‐αA or IFN‐αB, chemically cross‐linked to their human receptor could be immunoprecipitated by the antisera. IFN αβ receptor protein, purified by electrophoresis in sodium dodecylsulfate, was not recognized. We conclude that the antibodies are directed against the forms of the IFN αβ receptor actually expressed on the membrane.
Development of lymphocytes in interleukin 7‐transgenic mice
Authors: Samaridis Jacqueline , Casorati Giulia , Traunecker André , Iglesias Antonio , Gutierrez José C. , Müller Urs , Palacios Ronald
Published: March 2007

We have developed and established mouse transgenic lines in which the mouse interleukin 7 gene was targeted for expression in the lymphoid cell compartment. Northern blot analysis indicate that the transgene is expressed in bone marrow (BM), spleen and thymus, but not in kidney, liver, brain or heart. Both the frequency and absolute numbers of B cell precursors and mature B lymphocytes are increased in the BM and spleen of the transgenic mice. Although there is no expansion of the pro‐T lymphocyte population in the BM, the number of all major subsets of thymocytes and peripheral T lymphocytes is increased in the majority of the transgenic mice analyzed. The B and T cell lymphocytes in the transgenic mice are functionally competent. In contrast, the number of granulocytes and macrophages in the BM of transgenic mice is similar to that in control non‐transgenic littermates. Our results indicate that interleukin 7 plays an important role in vivo in the development of B and T lymphocytes.
Restricted expression of T cell receptor Vβ but not Vα genes in rheumatoid arthritis
Authors: Sottini Alessandra , Imberti Luisa , Gorla Roberto , Cattaneo Roberto , Primi Daniele
Published: March 2007

Synovial fluids of patients with rheumatoid arthritis contain activated T lymphocytes that may play an important role in the pathogenesis of the disease. Previous studies have suggested that the T cell receptor (TcR) repertoire of these cells is restricted, reflecting in vivo selection of a limited number of T cell specificities at the site of inflammation. To characterize better these T cell populations we used the polymerase chain reaction technology to estimate the proportion of TcR α and β RNA containing any particular V elements from transcripts directly isolated from the synovial fluid cells and from peripheral blood mononuclear cells of three patients with rheumatoid arthritis. Our data show that, in contrast to peripheral blood mononuclear cells, synovial fluid T cells expressed only few Vβ transcripts, one of which was overrepresented in two patients. Peripheral and joint fluid T cells, on the other hand, appeared to express the same set of non‐restricted Vα elements. These results suggest that a major antigen associated with the pathogenesis of rheumatoid arthritis may interact selectively with the Vβ component of the TcR.
A single‐chain murine class I major transplantation antigen
Authors: Mottez Estelle , Jaulin Christian , Godeau François , Choppin Jeanine , Levy Jean‐Paul , Kourilsky Philippe
Published: March 2007

Single‐chain mouse Kd molecules (SC‐Kd) were engineered by connecting residue 276 of Kd heavy chain to the first residue of β2‐microglobulin through spacers of various lengths, and expressed intracellularly in monkey COS‐1 cells. Labeled SC‐Kd molecules were found to react with several monoclonal antibodies which recognize native Kd molecules. SC‐Kd‐15 (with a spacer of 15 residues) was studied in more details. It could be purified and shown to regain a native‐like structure after treatment with denaturing agents. Purified SC‐Kd‐15 could bind certain peptides in a manner qualitatively similar to that of Kd.
Cytotoxicity and immunocyte markers in cells from the freshwater snail Planorbarius corneus (L.) (Gastropoda pulmonata): implications for the evolution of natural killer cells
Authors: Franceschi Claudio , Cossarizza Andrea , Monti Daniela , Ottaviani Enzo
Published: March 2007

The hypothesis that natural killer (NK) cells represent an important form of cell recognition and cytotoxicity leads to the prediction that NK‐like activity should be preserved throughout phylogenetic development. This was tested in the invertebrate Planorbarius corneus. Two types of cells can be identified and separated from the hemolymph of this mollusc, i.e. glass‐adherent macrophage‐like spreading hemocytes (SH) and nonadherent round hemocytes (RH). Only RH are able to lyse the K‐562 human target cell line in a short‐term NK cytotoxicity test. This NK‐like activity, severely reduced after 18 h incubation at 24 °C, is preserved by human recombinant interleukin 2. A further analysis of P. corneus hemocytes has been performed by using several mouse anti‐human monoclonal antibodies and cytofluorimetric analysis. Unexpectedly, both SH and RH react with several monoclonal antibodies, including those directed against epitopes typical of mammalian NK cells and cell adhesion molecules. On the whole, these data support the hypothesis that a primitive NK‐like activity appeared early in evolution and is not shared by phagocytic cells.
β1 Integrin‐mediated lymphocyte adherence to extracellular matrix is enhanced by phorbol ester treatment
Authors: Wilkins John A. , Stupack Dwayne , Stewart Steve , Caixia Shen
Published: March 2007

The interaction of the human T cell leukemia, Jurkat, with the extracellular matrix components collagen and fibronectin was examined. These cells displayed constitutive binding to fibronectin and low levels of adherence to collagen which were enhanced following stimulation with phorbol esters. The relevant binding structures were identified as members of the CD29/β1 integrin family of adhesion molecules. Adherence to collagen and to fibronectin was mediated by α2β1 and α5β1, respectively. The enhancement of adherence by phorbol esters did not involve up‐regulation of receptor expression but appeared to derive from the increased functionality of structures which were expressed on the cell surface.
Modulation of type II Fcγ receptor expression on activated human B lymphocytes
Authors: Sármay Gabriella , Rozsnyay Zoltán , Szabó Ildikó , Biró Anna , Gergely János
Published: March 2007

We have monitored FcγRII expression during the activation of human B lymphocytes by simultaneous analysis of monoclonal antibody (mAb) binding and EA resetting. The expression of FcγRII showed a biphasic time course. Initially, a transient increase of FcγRII with no ligand‐binding capacity was observed with mAb staining as early as 10 min after stimulation by the F(ab')2 fragment of anti‐human IgM or phorbol 12‐myristate 13‐acetate and then after 3 to 24 h a decrease in the number of FcγRII+ cells was seen. Trypsin‐like serine protease activity also appeared in the lysate of activated B cells at this time. On the 2nd day of activation a significant enhancement of FcγRII expression was observed, mainly on enlarged blast cells as monitored by both mAb and by ligand binding (EA rosette). At the same time, soluble fragments of FcγRII with the ability to bind human Fc were detected in the supernatant of activated B cells, probably as a result of proteolytic cleavage. These findings suggest that activated B cells are identical with the population of mononuclear cells which shed FcγR when incubated at 37°.

The ability of activated but not resting B cells to release FcγRII correlates with the expression of early activation markers and with the appearance of a trypsin‐like serine protease activity of the same cells; thus, the release of FcγRII occurs in the early G1 phase of cell cycle as a result of proteolysis. Later the release of FcγRII is accompanied by the enhancement of FcγRII expression before the cells reach the S phase.

The fragments of cleaved FcγRII had an apparent molecular mass of 33 and 14–18 kDa under nonreducing conditions, and upon reduction fragments of smaller size were observed.
Interleukin 2/interleukin 4‐independent T helper cell generation during an in vitro antigenic stimulation of mouse spleen cells in the presence of cyclosporin A
Authors: Motta Iris , Colle Jean‐Hervé , Shidani Babak , Truffa‐Bachi Paolo
Published: March 2007

Cyclosporin A (CsA), an immunosuppressive drug which completely suppresses the humoral response to thymus‐dependent antigens, does not affect the generation of T helper (Th) cells during an in vitro stimulation of murine spleen cells with sheep red blood cells. The appearance of Th cells depends on time and their development can be prevented by X‐ray irradiation performed at the onset of the culture and up to 3–4 days later; however, beyond this time, irradiation is ineffective, suggesting that cell proliferation is essentially completed by this time. The activity of Th cells generated in the presence of CsA is resistant to irradiation, indicating that the effector cells belong to a memory subset. Limiting dilution analysis has shown that the frequency of the Th cells recovered from 6‐day sheep red blood cell‐stimulated and CsA‐treated cultures is of approximately 1.4 × 10−3, representing an increment of about 500‐fold compared to naive spleen cells. The increased frequency of Th cells and the sensitivity to irradiation of the generation of these cells demonstrate that Th cell precursors proliferate in the presence of CsA. Lymphokine genes transcription analysis confirms that the inhibition of interleukin (IL) 2/IL 4 gene expression is one target of CsA action. That the generation of Th memory cells can nevertheless take place strongly argues in favor of the existence of an IL 2/IL 4‐independent pathway for murine T cell proliferation. Our finding that the transcription of the IL 7 gene is not inhibited by CsA raises the possibility for a role of this T cell growth factor in the generation of memory Th cells.
Conserved nucleotide sequences at the 5' end of T cell receptor variable genes facilitate polymerase chain reaction amplification
Authors: Broeren Chris P. M. , van Eden Willem , Kusters Johannes G. , Lenstra Johannes A. , Verjans Georges M. G. M. , Logtenberg Ton
Published: March 2007

Alignment of all available nucleotide sequences of mouse and rat α/β T cell receptor (TcR) variable (V) regions revealed the presence of relatively conserved sequences at the 5' end of the V gene segments. Based on these conserved sequences, degenerate primers were developed for use in the polymerase chain reaction (PCR). The degenerate primers developed on the basis of the conserved sequences at the 5' end of rat and mouse V gene segments are expected to enable the amplification of all mouse and rat TcR α/β chain V regions. To test their applicability, the primers were used for the amplification of the V region of the TcR α/β expressed by rat T cell lines. After amplification, the TcR V regions expressed were cloned and sequenced. The Zla T cell line was shown to use the same TcR V gene segments (Va2 and Vβ8.2), as most other experimental allergic encephalomyelitis associated T cell lines, but had different D and J segments. In spite of these differences at the nucleotide level, a remarkable conservation of the amino acid sequence at the VbTDβJβ junction was found.

Alignment of a large number of human Vα and Vβ gene segments revealed the presence of similarly conserved sequences. Degenerate primers based on these conserved sequences enabled the amplification of TcR V regions of human T cell lines.
Coding joint formation of endogenous T cell receptor genes in lymphoid cells from scid mice: unusual P‐nucleotide additions in VJ‐coding joints
Authors: Schuler Walter , Amsler Michaela , Ruetsch Norman R. , Bosma Melvin J.
Published: March 2007

The mouse mutation scid adversely affects the process of VDJ recombination. Attempts to form coding joints, that is, to join V or D to J gene segments generally fail in developing scid lymphocytes. It has been proposed that the scid mutation results a defective VDJ recombinase system. Here we describe five scid T cell lymphomas containing one or two TcRγ coding joints each, even though the majority of the multiple TcRγ chain gene rearrangements and all TcRβ rearrangements in these cells were abnormal with the deletions typically found in scid lymphoid cells. One of the five T cell lymphomas was shown to have an active VDJ recombinase system; however, this activity was defective indicating that the scid phenotype has been retained. We conclude that the scid VDJ recombinase system has not completely lost the ability to form coding joints. P‐nucleotide additions of unusual length or composition were found at the junctional border in five of the eight TcRγ coding joints. This might reflect a defect in the activity of a component of the VDJ recombinase system involved in the generation of P‐nucleotide additions. In one of the observed rearrangements, a Vγ5‐Jγ3 coding joint was formed. This establishes the transcriptional orientation of Jγ3‐Cγ3 and confirms a previously proposed organization of the TcRγ genes.
Self‐reactive T cells are activated by the 65‐kDa mycobacterial heat‐shock protein in neonatally thymectomized mice
Authors: Iwasaki Akinori , Yoshikai Yasunobu , Yuuki Hiroyuki , Takimoto Hiroaki , Nomoto Kikuo
Published: March 2007

To elucidate the mechanism of autoimmune disease in neonatally thymectomized (NTX) mice, we have investigated the responsiveness of the self‐reactive T cells which have not undergone clonal deletion in such animals. Consistent with a recent report (Yuuki et al., Eur. J. Immunol. 1990. 20: 1475), T cells bearing Vβ11‐gene products capable of recognizing I‐E‐encoded molecules were readily detected in the mature T cell pool of NTX BALB/c (I‐Ed, Mls‐2a) mice. The Vβ11‐bearing T cells in NTX mice expressed interleukin 2 receptors and responded normally to signals delivered through the T cell receptor. Notably, these T cells in NTX mice proliferated significantly after culture with the 65‐kDa mycobacterial heat‐shock protein, whose amino acid sequence is highly homologous to that in eukaryotes. These results suggest that self‐reactive T cells in NTX mice may be activated by heat‐shock proteins derived from various pathogens and/or stressed autologous cells, resulting in the development of autoimmune diseases in such animals.
Susceptibility to the induction of either autoimmunity or immunosuppression by mercuric chloride is related to the major histocompatibility complex class II haplotype
Authors: Aten Jan , Veninga Aletta , de Heer Emile , Rozing Jan , Nieuwenhuis Paul , Hoedemaeker Philip J. , Weening Jan J.
Published: March 2007

Mercuric chloride (HgCl2) induces in Brown Norway rats a CD4+ T lymphocyte‐dependent systemic autoimmune syndrome, involving synthesis of anti‐glomerular basement membrane autoantibodies and development of proteinuria. Lewis rats are resistant to HgCl2‐induced autoantibody production and, in contrast, develop immunosuppression, mediated by CD8+ T lymphocytes. In the present study, genetic requirements governing autoreactivity or immunosuppression in response to HgCl2 were further explored. Both major histocompatibility complex (MHC) and non‐MHC genes are involved in determining susceptibility to HgCl2‐induced autoimmunity. Both AO (RT1u) and DZB (RT1u) rats were found to develop a membranous autoimmune glomerulopathy upon exposure to HgCl2. Only the DZB strain, which differs in part of the non‐MHC background from AO, developed proteinuria. AO.1P (RT1.AuBlDlEu) rats, which are genetically identical to AO except for the Lewis haplotype at the MHC class II loci, appeared to develop immunosuppression upon exposure to HgCl2. It is concluded that autoreactivity and immunosuppression, induced by HgCl2, are both dependent on the MHC class II haplotype. In autoimmune responder strains the type of autoimmune glomerulopathy is influenced by non‐MHC genes.
Characterization of the rat leukocyte integrin, CD11/CD18, by the use of LFA‐1 subunit‐specific monoclonal antibodies
Authors: Tamatani Takuya , Kotani Masaharu , Miyasaka Masayuki
Published: March 2007

We have attempted to characterize the rat leukocyte integrin, CD11/CD18, by the use of newly generated monoclonal antibodies (mAb) WT.1 (anti‐CD11a) and WT.3 (anti‐CD18) in conjunction with an mAb, OX42, reactive with a rat integrin‐like molecule, with respect to the biochemistry, cellular distribution and function. The conclusion that the mAb WT.1 and WT.3 specifically recognize the rat CD11a and CD18, respectively, was based on: (a) their ability to inhibit homotypic aggregation of splenic concanavalin A (Con A) blasts; (b) sodium dodecyl sulfate‐polyacrylamide gel electrophoretic analysis of the antigens recognized; (c) their ability to inhibit binding of Con A blasts to the purified ligand, namely the ICAM‐1 antigen and (d) their blocking abilities in mixed leukocyte reaction.

In the rat, CD18 has an apparent molecular mass of 95–100 kDa and can associate with at least three distinct α subunits of 160–170 kDa (CD11a), 140–150 kDa and 120–130 kDa. The latter two are precipitated by OX42 from MΦ but not from unstimulated lymphocytes. They presumably represent the rat CD11b and CD11c, respectively. Rat thymocytes, PBL, thoracic duct lymphocytes, monocytes and neutrophils expressed differential levels of CD11a and CD18. Peritoneal MΦ showed virtually no CD11a expression, although CD18 was expressed at levels similar to those seen on blood monocytes, showing an interesting pattern of LFA‐1 expression regulation in this cell lineage. Both WT.1 (anti‐CD11a) and WT.3 (anti‐CD18) apparently recognize a “low‐affinity” as well as a “high‐affinity” form of LFA‐1 and do not discriminate between the two.
Molecular basis of IgG Fc receptor III defect in a patient with systemic lupus erythematosus
Authors: Enenkel Barbara , Jung Dirk , Frey Jürgen
Published: March 2007

By flow cytometry analysis we could show a decreased expression of Fcγ receptor type III (FcγRIII) on granulocytes of a patient with systemic lupus erythematosus (SLE). Therefore, we constructed a leukocyte cDNA library from this patient with the aim of investigating this defect on the molecular level. Using an FcγRIII cDNA probe we isolated 15 FcγRIII cDNA clones, which were all characterized by sequencing. Our sequence data revealed that the patient was heterozygous for FcγRIII (NA‐1/NA‐2). Only clone 5 (NA‐2) was a full‐length cDNA clone. In contrast to the wild‐type FcγRIII the signal sequence is mutated, lacking the hydrophobic region essential for co‐translational transport across the endoplasmic reticulum membrane. The predicted transport defect leading to the lack of membrane expression could be confirmed by immunofluorescence staining after expression of this cDNA clone in BHK cells. The cDNA clones 6 and 8 (NA‐1) lack the first 45 bp of the signal sequence, but considering the flow cytometry data the signal sequence must be functional allowing the membrane expression of this receptor allele. The part of the cDNA sequence of all isolated clones coding the mature FcγRIII is identical to the wild‐type sequence. Therefore, we conclude that the decreased expression of FcγRIII on granulocytes of this SLE patient is due to the transport defect of one of the receptor alleles.