bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of perforin in murine natural killer cells and cytotoxic T lymphocytes in vivo
Authors: Kawasaki Akemi , Shinkai Yoichi , Yagita Hideo , Okumura Ko
Published: March 2007

We have previously detected perforin expression in a subpopulation of asialo GM1+ natural killer (NK) cells and CD8+ T lymphocytes in murine spleen cells by immunocytochemical staining with an anti‐perforin monoclonal antibody. In the present study, more datailed analyses of perforin expression in murine cytotoxic lymphocyte subpopulations were performed. The expression of perforin in asialo GM1+ spleen cells was predominantly confined to the NK1.1+ subset, where all NK activity also resided. Perforin expression was also studied on alloreactive cytotoxic T lymphocyte (CTL) induced in vivo. The cells expressing perforin in peritoneal exudate lymphocytes predominantly resided in the CD8+ T cell subpopulation co‐expressing asialo GM1 where an allospecific CTL activity also resided. Furthermore, the percentage of perforin‐positive cells in this population was greatly reduced after stimulation with anti‐CD3 or anti‐T cell receptors antibodies, which induce serine esterase release from the cytoplasmic granules. These findings highly suggest that perforin is involved in in vivo NK cell and CTL‐mediated cytolysis.
Interleukin (IL)‐4 production by human T cells: differential regulation of IL‐4 vs. IL‐2 production
Authors: Van der Pouw‐Kraan Tineke , Van Kooten Cees , Rensink Irma , Aarden Lucien
Published: March 2007

We have examined the regulation of interleukin (IL)‐4 production by human peripheral blood T cells. Production of IL‐4 was shown to be regulated differently from IL‐2 and interferon(IFN)‐γ production. Stimulation of peripheral blood lymphocytes with anti‐CD3, anti‐CD2, anti‐CD28, Phorbol 12‐myristate 13‐acetate (PMA) or IL‐2 as a single stimulant did not induce IL‐4 production. However, combinations of anti‐CD2 with either anti‐CD28 or IL‐2 resulted in IL‐4 production, peaking at days 3–4. Stimulation with anti‐CD3 instead of anti‐CD2 gave similar results, but was less potent. After days 3–4, IL‐4 levels decreased, most likely due to consumption of IL‐4. PMA profoundly affected cytokine production, it enhanced IL‐2 production by at least tenfold, whereas, in the same cell population, IL‐4 production was almost completely inhibited. This was observed at the protein as well as at the mRNA level. In contrast, agents that increase intracellular cAMP levels inhibited IL‐2 production but left IL‐4 production unaffected. IFN‐γ production behaved similar to IL‐2 production but the effects were less outspoken.
Specificity of antigen processing for MHC class I restricted presentation is conserved between mouse and man
Authors: Falk Kirsten , Rötzschke Olaf , Rammensee Hans‐Georg
Published: March 2007

Alloreactive mouse cytotoxic T lymphocytes (CTL) specific for particular peptides presented by H‐2Kb molecules on mouse cells were found to recognize human cells transfected with Kb. The CTL‐recognized peptides (probably derived from conserved proteins) were extracted from Kb‐expressing human or mouse cells, respectively, and compared biochemically by high resolution high performance liquid chromatography. The results strongly suggest identity of peptides processed by cells from both species and thus indicate that the specificity of the processing machinery used in the major histocompatibility complex (MHC) class I‐restricted pathway, probably including enzymes, transport mechanisms, and chaperons, is highly conserved across species. The results are consistent with the notion that MHC molecules themselves have an instructive role in processing.
Secretion of interleukin‐8 following phagocytosis of Mycobacterium tuberculosis by human monocyte cell lines
Authors: Friedland Jon S. , Remick Daniel G. , Shattock Rubin , Griffin George E.
Published: March 2007

Tuberculosis is a major cause of mortality worldwide and incidence is increasing asaresult of the AIDS epidemic. Cytokines such as tumor necrosis factor (TNF) are important in the host response to Mycobacterium tuberculosis. TNF is involved in both granuloma formation and has direct anti‐mycobacterial activity. This study investigated the secretion of interleukin (IL)‐8 following phagocytosis of M. tuberculosis by a human monocytic cell line and by a more phenotypically mature macrophage‐like cell line. M. tuberculosis is shown to be a more potent inducer of IL‐8 but not of TNF than bacterial lipopolysaccharide invitro in bothcell types. IL‐8 production is partly a consequence of accumulation of mRNA coding for this cytokine. Secretion of IL‐8 is not a simple consequence of the phagocytic process but due to the specific interaction M. tuberculosis and the monocyte. IL‐8 production was independent of TNF and of virulence of the strain of M. tuberculosis. IL‐8 secretion following phagocytosis of M. tuberculosis suggests that this cytokine may be involved in granuloma formation in vivo, possibly acting, in part, as a T cell chemoattractant.
Engagement of class I major histocompatibility complex molecules by cell surface CD8 delivers an activation signal
Authors: Geppert Thomas D. , Nguyen Hoan , Lipsky Peter E.
Published: March 2007

Recent evidence has demonstrated that cross‐linking class I major histocompatibility complex (MHC) molecules on human T cells with monoclonal antibodies (mAb) triggers T cell activation. The only known natural ligand for MHC class I molecules is CD8.Therefore, the possibility that CD8+ T cells might provide activation signals to other T cells by engaging MHC class I molecules was examined by culturing CD4+ peripheral blood T cells with Chinese hamster ovary cells (CHO) cells that had been transfected with the α chain or α and β chains of CD8 and assessing interleukin (IL)‐2 production. CD4+ T cells did not secrete IL‐2 when cultured alone, with control or CD8+ CHO cells. In contrast, CD4+ T cells produced IL‐2 when cultured with CD8+ CHO cells and co‐stimulated with phorbol myristate acetate (PMA) or mAb to CD3 or CD28. PMA stimulated substantially less IL‐2 when control CHO cells were employed and the mAb to CD3 and CD28 did not stimulate IL‐2 production in the presence of control CHO cells. The co‐stimulatory activity of CD8+CHO cells was completelyeliminated by mAb to CD8 or MHC class I molecules. The data demonstrate that CD8 can interact with MHC class I molecules expressed on T cells and deliver a costimulatory signal that increases IL‐2 production. Thus, engagement of MHC class I molecules byits natural ligand, CD8, provides an activation signal to T cells. Under some circumstances, such interactions may amplify the responses of T cells.
Thymic epithelium induces neither clonal deletion nor anergy to Mls 1a antigens
Authors: Bandeira António , Coutinho António , Burlen‐Defranoux Odile , Khazaal Ibrahim , Coltey Monique , Jacquemart Frederic , Douarin Nicole Le , Salaün Josselyne
Published: March 2007

Grafting of thymic anlagen from day‐10 DBA/2 (H‐2d; Mls‐1a) embryos to newborn athymic BALB/c (H‐2d; Mls‐1b) mice leads to reconstitution of T cell populations in the recipients. Analysis of adult chimeras shows that their Vβ T cell receptor (TcR) repertoires, particularly Vβ6 and Vβ8.1, do not significantly differ in most animals (10 out of 13) from those scored in control chimeras that received syngeneic thymic anlagen. In all cases analyzed, such Mls‐1a−reactive T cells could be stimulated at levels comparable to control responses, both in vitro and in vivo. The few cases in which Mls‐1a reactive Vβ TcR were reduced seem to reflect the variability in TcR Vβ repertoires found inthis experimental system. In contrast, BALB/c mice, injected at birth with DBA/2spleencells show a marked, albeit variable, reduction in the frequencies of Vβ6− and Vβ8.1‐bearing CD4+ T cells, and lower frequencies of Mls‐1a−reactive T cells in limiting dilution analyses. It appears, however, that Vβ6− and Vβ8.1‐bearing T cells remaining in these mice are functionally competent. We conclude that Mls‐1antigens are not expressed by thymic epithelium.
Synthetic peptide libraries in the determination of T cell epitopes and peptide binding specificity of class I molecules
Authors: Schumacher Ton N. M. , Van Bleek Grada M. , Heemels Marie‐ThéRèSe , Deres Karl , Li Ka Wan , Imarai Monica , Vernie Leen N. , Nathenson Stanley G. , Ploegh Hidde L.
Published: March 2007

Major histocompatibility complex (MHC) class I molecules combine with short peptides of defined length and sequence. Here we describe an approach that may be used in the analysis of peptide preference of different allelic MHC class I molecules, and in the determination of T cell epitopes. We produced synthetic “peptide libraries” of limited complexity by standard peptide chemistry. Using these peptide mixtures we show that H‐2 Kb molecules can accomodate both 8‐and 9‐residue peptides, whereas Db molecules are unable to combine with peptides shorterthan 9 amino acids present in these libraries. When these peptide mixturesare used to provide “fingerprints” of Db molecules and mutants thereof, both loss and gain of the ability to combine with certain peptides is observed. For the Kbm1 mutant a strong influence of amino acid substitutions in class I molecules on the peptides selected is observed. In these synthetic peptide mixtures, the presence ofa specific T cell epitope, known to be represented once, can be detected. This approachmay be extended to the identification of new T cell epitopes from largerpeptide libraries.
Cell surface display of rat invariant γ chain: detection by monoclonal antibodies directed against a C‐terminal γ chain segment
Authors: Fisch Andreas , Reske Konrad
Published: March 2007

A series of 14 monoclonal antibodies (mAb) directed against the C‐terminal part of the rat invariant γ chain (amino acid 142–216) was generated using distinct fusion proteins that contain this γ segment for immunization and hybridoma screening. Additional fusion proteins were prepared carrying discrete regions of the γ chain. Employing these reagents confirmed that the obtained mAb do indeed recognize the C‐terminal portion of the invariant chain, as demonstrated by Western blot analysis. All mAb established recognize epitopes present on the native γ chain, as revealed by immunoprecipitation analysis using nonionic detergent extracts of metabolically labeled Lewis rat splenocytes combined with two‐dimensional gel electrophoresis. However, while the majority of the γ chain‐specific mAb precipitated γ chain‐containing polypeptide chain complexes in which immature, sialic acid‐deficient and mature, terminally sialylated forms of the γ chain were predominantly represented, a fraction of the antibodies preferentially precipitated the immature γ forms. Cell surface binding of these two groups of mAb correlated with the immunoprecipitation data in that the former group of antibodies did bind to intact Lewis rat spleen cells, while essentially no binding was observed with the antibodies of the latter group. Double‐fluorescence staining with the class II‐specific fluorescein isothiocyanate‐conjugated mAb OX3 andOX6, respectively, as well as a representative γ chain‐specific mAb visualized with phycoerythrin‐coupled secondary antibody shows coexpression of class IIdeterminants and the invariant chain at the cell surface.
Human‐mouse lymphoid chimeras: host‐vs.‐graft and graft‐vs.‐host reactions
Authors: Murphy William J. , Bennett Michael , Anver Miriam R. , Baseler Michael , Longo Dan L.
Published: March 2007

Human peripheral blood lymphocytes (huPBL) were injected into mice with severe combined immune deficiency (SCID). It was ascertained that murine natural killer (NK) cellswere capable of affecting engraftment of human lymphocytesin SCID mice. The presence of host NK cells resulted in the clearance of the human lymphocytes. Human T lymphocyteswere the primary cell to engraft in the SCID recipients, with human T cells being detected in the spleen, lymph nodes, bone marrow and peritoneal cavity of the mice up to several months after transfer. No human T cells were detected in the murine thymus and thelevel of engraftment in the periphery could be highly variable. Additionally, there appeared to be significant reactions between the human lymphocytes and the murine host resulting in a xenogeneic graft‐vs.‐host reaction (XGVHR). The predominant manifestation involved splenomegaly resulting from an expansion of murine hematopoietic cells in the spleens of these xenogeneic chimeras. The severity of the XGVHR could be correlated with the extent of human T cell engraftment and the recovered human Tcells were found to be in a proliferative state. Thus, there appear to be significant host‐vs.‐graft and graft‐vs.‐host interactionsoccurring in human/mouse lymphocyte chimeras.
Changes in expression of CD45R during the development of Th1 and Th2 cell lines
Authors: Lee William T. , Vitetta Ellen S.
Published: March 2007

Previous studies using anti‐CD45R monoclonal antibodies (mAb) have shown that normal CD4+ T cells can be separated into virgin and memory cells basedon their level of expression of CD45R. CD45Rhi (virgin)T cells secrete interleukin (IL)‐2 whereas CD45Rlo (memory) T cells secrete both IL‐2 and IL‐4. In contrast to these results, studies using cultured T cell lines haveshown that IL‐2‐secreting T helper cell type 1 (Th1) cells are CD45Rlo and IL‐4‐secreting Th2 cells are CD45Rhi. To resolve this discrepancy, and to determine how the Th1 and Th2 cell lines relate to memory CD45Rlo and virgin CD45Rhi CD4+ T cells, we have isolated CD45Rlo cells from alloantigen‐primed mice and developed allospecific Th1 and Th2 cell lines. The lymphokinessecreted by these lines were then evaluated. Our results show that as CD45Rlo T cells develop into in vitro cell lines which secrete IL‐4, they become CD45Rhi. In contrast, CD45Rlo cells that develop into lines which secrete IL‐2 maintain their CD45Rlo phenotype. Thus, although both Th1 and Th2 cells arise initially from CD45Rlo cells, Th2 cells become CD45Rhi during prolonged in vitro culture but, Th1 cells do not.
Staphylococcal enterotoxin B as a potent suppressant of T lymphocytes: trace levelssuppress T lymphocyte proliferative responses
Authors: Ben‐Nun Avraham , Yossefi Sara
Published: March 2007

Staphylococcal enterotoxins have long been known to be powerful stimulators of T lymphocytes in mouse and man. In a previous study we showed that high concentrations of staphylococcal enterotoxin serotype B (SEB) failed to stimulate strong proliferative responses by Lewis rat T lymphocytes. Moreover, concentrations of SEB (10–50 μg/ml) that stimulated optimal mouse T lymphocyte proliferative responses suppressed a mitogen‐ or antigen‐induced rat T lymphocytes proliferative responses. The present study shows that SEB at low concentrations (as low as 10−3−10−4 μg/ml) and often also trace levels (about 10−610−7 μg/ml) suppresses both rat and mouse T lymphocytes proliferative responses to mitogen or antigen. Furthermore, under different circumstances, SEB may have conflicting effects on the same T cells. While high concentrations (1–50 μg/ml) of SEB stimulate certain mouse T cell clones, low concentrations or trace levels havea potent suppressive effect on the same clones. The results indicate that the in vitro conflicting effects of SEB on the same T cells are concentration dependent and may reflect itsin vivo effects on SEB‐reactive T lymphocytes. The suppression of the mitogen‐ or antigen‐induced stimulation of T cell clones by SEB was direct and did not require the agency of suppressor cells. Furthermore, the suppression by low amounts of SEB was not major histocompatibility complex restricted and affected a largeproportion of both rat and mouse T lymphocyte subpopulations, regardless of their antigenic specificity. The concomitant suppressogenic and stimulatory characteristics of SEB support the conclusion that, under different conditions, SEB can be considered a “super‐suppressogen” as well as a “super‐antigen”. Overall, the results suggest that SEB, and possibly other bacterial toxins, could be useful in immunomodulation of specific T cell responses.
Characterization of two human monoclonal IgM antibodies that recognize nuclear lamins
Authors: Lassoued KaïS , André Chantal , Danon Francoise , Modigliani Robert , Dhumeaux Daniel , Clauvel Jean‐Pierre , Brouet Jean‐Claude , Courvalin Jean‐Claude
Published: March 2007

Using immunofluorescence and immunoblotting techniques, we have identified monoclonal IgM λ from two patients that are specific for lamins A and C and lamin B, respectively. Lamins A, B, and C are peripheral membrane proteins of the nuclear envelope with structural similarities to cytoplasmic intermediate filament proteins. When studiedby indirect immunofluorescence on rat tissues, the serum containing anti‐laminB IgM stained smooth and striated muscles in addition to nuclear envelopes. Lamin B antibodies affinity purified from this serum were able to label muscle cells, suggesting that lamin B shares an epitope(s) with an unidentified muscular component(s). Since in an enzyme‐linked immunosorbent assay there was no reactivity with a panel of proteins which are frequent targets of “natural” antibodies, these monoclonal IgM appear to belong to the rare category of IgM that possess a restricted specificity.
Spontaneous and ligand‐induced endocytosis of CD23 (Fcϵ receptor II) from the surface of B lymphocytes generates a 16‐kDa intracellular fragment
Authors: Grenier‐Brossette Nicole , Bourget Isabelle , Akoundi CaméLia , Bonnefoy Jean‐Yves , Cousin Jean‐Louis
Published: March 2007

It has been reported that the 45‐kDa low‐affinity Fcϵ receptor (FcϵRII) on B cells is cleaved spontaneously from the cell surface to release soluble fragments. This study demonstrates an additional fate of the FcϵRII. 125I‐labeled CD23+ B cells were cultured for 24 h at 37°C. After lysis, cell extracts were immunoprecipitated with CD23 monoclonal antibodies. Using this methodology, we demonstrated that an increasing amount of the labeled FcϵRII becomes progressively resistant to externally applied trypsin, indicating that a fraction of the cell surface receptors are internalized. In parallel, a labeled 16‐kDa material, recognized by CD23 monoclonal antibodies directed to the lectin‐like domain of the Fcϵ‐RII appears inside the cells. Chloroquine does not affect internalization of the FcϵRII, but completely abolishes the formationof the intracellular fragment, suggesting that the receptor is processed by proteolyticcleavage inacidic organelle. In addition, the internalization is enhanced in the presence of CD23 monoclonal antibodies. These data demonstrate that FcϵRII can be internalized by ligand‐induced endocytosis and subsequently cleaved in an intracellular compartment. These results also support the view that the FcϵRII is involved in antigen focusing and antigen presentation.
Thymic hyperplasia in transgenic mice caused by immortal epithelial cells expressing c‐kit ligand
Authors: Moll JüRgen , Eibel Hermann , Schmid Peter , Sansig Gilles , Botteri Florence , Palacios Ronald , Putten Herman Van Der
Published: March 2007

To dissect mechanisms that co‐ordinate specific events in thymopoiesis we have characterized alterations in thymic structure and function caused by expression of atransgene. This gene encodes SV40Tag and is specifically expressed in a subset of thymicepithelial (TE) cells around birth. As a result the number of immortal TE cells increases, thymic mass increases (up to 3 g), and thymopoiesis is expanded. The latter is reflected by a ˜ 100‐fold increase of the major thymocyte subsets and increased peripheral T cell counts. Grossly hyperplastic thymi retain many but not all morphological features of a normal thymus. Also in grafts, SV40Tag+ TE cells steer expansion (up to 8 g) and organize a tissue with mainly cortex‐like featuresthat includes mainly SV40Tag+ TE cells, thymocytes, and macrophages. To investigate expression of specialized gene functions in the immortal TE cells, a cell line was derived. The Epi‐A1 cell line expresses the genes for major histocompatibility complex class I and II, Thy‐1, interleukin (IL)‐6, IL‐7, macrophage‐colony‐stimulating factor, and transforming growth factor‐β3. Most importantly, Epi‐A1 cells also express the IL‐4 receptor and the c‐kit ligand (KL), a factor that, in concert with commitment factors, channels progenitors into hemopoietic lineages. The expression of low constitutive levels of KL mRNA does not require IL‐4, but KL mRNA levels are increased dramatically in response to IL‐4. Since constitutive expression of KL mRNA in vivo is restricted to a small subset of TE cells in the thymus, our findings reveal a novel specific interaction between thymocytesand a specialized subset of TE cells.
Specific epitope‐induced conversion of CD8+ memory cells into effector cytotoxic T lymphocytes in vitro: presentation of peptide antigen by CD8+ T cells
Authors: Kos Ferdynand J. , Müllbacher Arno
Published: March 2007

The requirements for the conversion of CD8+ memory T cells into effector class I major histocompatibility complex (MHC) Kd−restricted cytotoxic T (Tc) cells in vitro have been studied. Purified CD8+splenocytes from influenza A/WSN‐primed BALB/c (H‐2d) mice stimulated with a synthetic nucleoprotein peptide 147–158 R156− (NPP) alone generated Tc cells specific for influenza virus‐infected target cells. No additional requirements for accessory cells or their lymphokine products were necessary indicating that peptide antigen (Ag) in association with Kd was presented on CD8+ T cells. The evidence for presentation of NPP by CD8+ T cells was supported by the use of CD8+ memory T cells from semiallogeneic bone marrow radiation chimeras of P1 → F1 type (H2b→ [H‐2d x H‐2b]F1). Memory CD8+ splenocytes from A/WSN‐immune chimeras did not develop into secondary effector Tc cells as a result of a 4‐day culture with NPP alone, however, were able to do so if NPP was presented by Kd−bearing Ag‐presenting cells. In addition, these results exclude the possibility of direct recognition of free NPP molecules by the specificT cell receptor of CD8+ memory T cells. CD8+ memory splenocytes (H‐2b) from chimeras were also able to develop into functionally active Tc cells as a result of presentation of Db−restricted synthetic peptide (NP 366–374) with a sequence derived from influenza virus nucleoprotein with high affinity for Db MHC class I molecules. Blockade of endogenously produced interleukin 2 (IL‐2) activity by anti‐IL‐2 or anti‐IL‐2 receptor monoclonal antibody in the culture of CD8+ memory T cells during a 4‐day NPP stimulation completely abolished Tc cell generation, indicating that the utilization of this lymphokine is absolutely required for the secondary Tc cell development. These findings demonstrate that CD8+ memory T cells per se are able to recognize the restimulating epitope as a result of its presentation by CD8+ T cells and develop into cytolytically active and highly specific Tc cells with no requirements for other cellular helper components or their lymphokine products.
The fate of the three subunits of major histocompatibility complex class I molecules
Authors: Neefjes Jacques J. , Smit Linda , Gehrmann Matthias , Ploegh Hidde L.
Published: March 2007

At the surface of the murine T lymphoma cell line RMA‐S, the expression of “empty” class I molecules can be dramatically enhanced by culture at 26°C. These class I molecules are unstable following transfer to 37°C unless they are loaded with exogenously added peptides. Class I heterodimers that have failed to bind peptide (“empty” class I molecules) dissociate and the class I heavychains are degraded. Internalization, if it precedes breakdown, would be the rate‐limiting step.

Radioiodinated peptides (VSV NP8‐mer or Sendai NP9‐mer) dissociate from the class I molecules in the absence of exogenous peptide or β2 microglobulin and appear in the medium. Release of the iodinated peptides does notresult in a reduction in the quantity of stable assembled class I molecules. This paradox may be explained by a more rapid off‐rate for radioiodinated peptides, when compared with their unlabeled counterparts, which constitute about 99% of the total in our radiolabeled preparations. In the medium the peptide is rapidly modified by serum‐ and cell‐derived proteases. The short half‐life of empty class I molecules and offree ligand would effectively preclude sensitization of innocent bystanders for lysis by cytotoxic T lymphocytes.
Aberrant interleukin (IL)‐4 and IL‐5 production in vitro by CD4+ helper T cells from atopic subjects
Authors: Parronchi Paola , de Carli Marco , Manetti Roberto , Simonelli Cecilia , Piccinni Marie‐Pierre , Macchia Donatella , Maggi Enrico , Prete Gianfranco Del , Ricci Mario , Romagnani Sergio
Published: March 2007

The cytokine secretion profiles of T cell lines (TCL) specific for purified proteinderivative (PPD) or streptokinase (SK), contemporarily derived from nine atopic and nine nonatopic individuals, were compared. Upon stimulation with phorbol myristate acetate(PMA) plus anti‐CD3 monoclonal antibody (mAb), all TCL from both atopics and nonatopics produced interleukin (IL)‐2 and interferon (IFN)‐γ. The mean IL‐2 production by PPD‐ or SK‐specific TCL from both atopics andnonatopics was similar, whereas the mean IFN‐γ production by TCL derived from atopics was significantly lower. In addition, both PPD‐ and SK‐specific TCL from atopics produced detectable amounts of IL‐4 and IL‐5, whereasthe corresponding TCL derived from nonatopics did not.

A total number of 107 and 99 PPD‐specific CD4+ T cell clones (TCC)were then derived from TCL of 4 atopic and 4 nonatopic donors and assessed for their profile of cytokine production in response to stimulation with either PMA plus anti‐CD3 mAb or the specific antigen. Under both these experimental conditions, virtually all PPD‐specific TCC from both atopic and nonatopic individuals produced IL‐2 and IFN‐γ. In contrast, the great majority of PPD‐specific TCCderived from nonatopic individuals did not produce IL‐4 and IL‐5, whereashigh proportions of PPD‐specific TCC derived from atopic donors displayed the ability to produce noticeable amounts of IL‐4 and IL‐5 besides IL‐2 and IFN‐γ.

These data indicate that CD4+ T cells from atopic individuals are able toproduce IL‐4 and IL‐5 in response to bacterial antigens, such as PPDand SK, that usually evoke responses with a restricted type‐1 T helper (Th1)‐like cytokine profile in nonatopic individuals. Aberrant IL‐4 production by Th cells may represent one of the immune alterations responsible for enhanced IgE antibody production in atopic people.
FcγRIII activation is different in CD16+ cytotoxic T lymphocytes and natural killer cells
Authors: Uciechowski Peter , Gessner J. Engelbert , Schindler Ralf , Schmidt Reinhold E.
Published: March 2007

The transmembrane protein CD16 (FcγRIII) is detected on activated macrophages, natural killer (NK) cells and a small subset of T lymphocytes. From CD3− CD56+ CD16+bright NK cells and CD3+ CD56+ CD16+dim non‐major histocompatibility complex (MHC)‐restricted cytotoxic T lymphocyte (CTL) clones were generated reflecting the stable, but different, CD16 expression of the respective peripheral blood subpopulations. To compare the role of CD16 on NK cells and non‐MHC‐restricted CTL, FcγRIII activation and its mechanisms were investigated using monoclonal antibodies (mAb). Cross‐linking of CD16 induced Ca2+ influx in CD16+bright NK clones. In contrast, there was no Ca2+ mobilization after CD16 activation in CD16+dim CTL, which revealed a good response to cross‐linking of CD3 antigen. Pretreatment with CD16 mAb alone or cross‐linked CD16 mAb did not block the CD3 response of CD16+dim CTL. Again, CD16 cross‐linking induced more interferon‐γ transcription in NK cell clones than in non‐MHC‐restricted CTL clones. Also a higher tumor necrosis factor‐α production of NK clones after CD16 cross‐linking compared to CD16+dim CTL could be observed. These data suggest that after CD16 activationCD16+dim CTL and CD16+bright NK cells use different second messengers. In addition, signal transduction via CD3 and CD16 appears to function independently in CD16+dim non‐MHC‐restricted CTL.
Inhibition of abnormal T cell development and autoimmunity in gld mice by transgenic T cell receptor β chain
Authors: Yui Katsuyuki , Bhandoola Avinash , Komori Shinji , Katsumata Makoto , Greene Mark I. , Radic Marko Z.
Published: March 2007

Mice homozygous for the gld (generalized lymphoproliferative disease) mutation develop systemic autoimmune disease and severe lymphadenopathy due to an age‐related accumulation in the peripheral lymphoid organs of polyclonal T cells bearing a unique phenotype (CD4−CD8−TCR αβ+B220+). These T cells overex‐press T cell receptor (TcR) αβ chain RNA, proto‐oncogenes c‐myb and fyn, and proliferate poorly in response to TcR‐mediated stimulation. The origin of these T cells is poorly understood. To study the influence of a functionally rearranged TcR β chain on the T cell developmental abnormality of the gld mutation and autoimmunity, we have backcrossed TcR Vβ8.1‐transgenic mice to C3H‐gld/gld to homozygosity (transgenic gld mice). In transgenic gld mice, lymphadenopathy was markedly inhibited and the accumulation of CD4−CD8− T cells did not occur, although the remaining T cells overexpressed c‐myb and proliferated poorly in response to TcR occupancy. These features indicate that the pattern of proto‐oncogene expression and abnormal function persist in phenotypically normal T cells in transgenic gld mice, and that these characteristics can be dissociated from the accumulation of CD4−CD8− T cells. The hypergammaglobulinemia and anti‐double‐stranded DNA (anti‐dsDNA) antibody production was partially improved in transgenic gld mice, supporting the critical role of T cells in abnormal B cell activation described in autoimmunity‐prone mice. To investigate further the mechanisms underlying the inhibition of CD4−CD8− T cell accumulation in transgenic gld mice, the fetal ontogeny of T cells in transgenic mice was compared with that of non‐transgenic mice. In transgenic thymus, development of TcR αβ+ cells was accelerated as detected by earlier expression of CD4, CD8 and TcR in fetal thymus. In contrast, the number of TcR γδ+ cells was reduced. We suggest that altered T cell development in transgenic mice directly or indirectly inhibits the accumulation of abnormal T cells in gld mice.
Characterization and measurement of CD5+ B cells in normal and Trypanosoma congolense‐infected cattle
Authors: Naessens Jan , Williams Diana J. L.
Published: March 2007

CD5+ B cells in cattle are present in peripheral blood and spleen, but not in lymph nodes, tonsils or Peyer's patches. Compared to classical B cells, they express similar levels of B cell surface markers, but have higher levels of surface IgM. We failed to find evidence for IgD on bovine B lymphocytes. The CD5+ B cells expressed CD11b (Mac‐1). Another small subpopulation of B cells carried CD11b but not CD5. In cattle infected with Trypanosoma congolense, a dramatic increase in the percentage of CD5+ B cells in blood and spleen was observed. This increase occurred 7‐10 days after parasites were first detected in the blood and correlated with the increase in serum IgM and the increase in the absolute number of B cells that is typical to trypanosome‐infected animals. The increase in B cells was found to be due mainly to the expansion of the CD5+ B cell subpopulation. The cause of the amplification of the CD5+ B cells and their possible involvement in the production of autoantibodies and non‐parasite‐specific antibodies which have been described in trypanosome‐infected animals are discussed.
Human peripheral blood leukocytes transplanted on CB17 scid‐scid mice are transferred to their offspring
Authors: Ladel Christoph H. , Püschner Hubert , Kaufmann Stefan H. E. , Bamberger Uwe
Published: March 2007

Severe combined immunodeficient (scid) mice are deficient in functional T cells and B cells. Hence, scid mice reconstituted with human peripheral blood leukocytes (scid‐huPBL) provide an excellent model for analysis of the human immune response under in vivo conditions. We have investigated this model further by analyzing human immune responses in the progeny of scid‐huPBL (termed scid‐humo). We find markedly elevated levels of human immunoglobulins (Ig) in the serum of scid‐humo for more than 12 weeks indicating materno‐fetal transfer of human B lymphocytes. Consistent with this finding we obtained evidence for the existence of human lymphocytes in scid‐humo. Murine Ig levels in scid‐humo were also elevated and surface Ig‐expressing cells (probably B cells) were demonstrable. In this respect scid‐humo resembled “leaky” scid. In contrast to “leaky” scid, scid‐humo accepted transfer of human blood leukocytes. Not only leukocytes from autologous but also those from heterologous donors were accepted. Human Ig levels in scid‐humo increased more rapidly as compared to normal scid mice. Thus, despite these increased B cell activities in scid‐humo, transferred human leukocytes were not affected indicating that materno‐fetal transfer of human cells had caused tolerization or conditioning. This is in contrast to scid mice in which elevated Ig levels correlate with increased failure rates of reconstitution with human blood leukocytes. We propose that scid‐humo provide an improved model for studying the human immune responses in an in vivo setting.
Restoration of the LFA‐3 adhesion pathway in Burkitt's lymphoma cells using an LFA‐3 recombinant vaccinia virus: consequences for T cell recognition
Authors: Griffin Heather , Rowe Martin , Murray Ruth , Brooks Jill , Rickinson Alan
Published: March 2007

Conjugate formation between cytotoxic T lymphocytes (CTL) and target B cells, as observed in vitro, is mediated by interactions between adhesion molecules on the two cell surfaces rather than involving immune recognition through the T cell receptor. It is still not clear to what extent such adhesive contacts facilitate the process of immune recognition and target cell lysis. However, work on the Epstein‐Barr virus (EBV)‐associated malignancy Burkitt's lymphoma (BL) has suggested that down‐regulation of one particular adhesion molecule, the lymphocyte function‐associated antigen LFA‐3, on the tumor cell surface is a key factor in allowing these target cells to escape EBV‐speficic T cell surveillance. To examine this directly, we used a cDNA for the full‐length transmembrane form of LFA‐3 to construct a recombinant vaccinia virus (Vacc‐LFA 3), which is capable of restoring surface LFA‐3 in adhesion molecule‐negative BL cell lines to levels as high as seen in EBV‐transformed lymphoblastoid cell lines (LCL); biochemical studies confirmed expression of the authentic N‐glycosylated protein. The recombinant vaccinia‐encoded LFA‐3 was functional as an adhesion molecule since BL cells acutely infected with Vacc‐LFA‐3 then acquired the ability to form conjugates with activated T cells in vitro. However, there was no clear dependence upon LFA‐3 when such BL cell lines were tested as targets for cytotoxic T lymphocytes (CTL). Firstly, LFA‐3− BL cells could be killed by allospecific CTL recognizing HLA class I alloantigens, in some cases as efficiently as the corresponding LCL. In other cases where lysis was slightly below that of the LCL, Vacc‐LFA‐3 infection of the BL cells increased lysis up to, but never beyond, LCL values. Secondly, we studied the sensitivity of BL to EBV‐specific HLA class I‐restricted CTL using a BL target line which was LFA‐3− but which expressed the same spectrum of EBV target proteins as an LCL. This line was not recognized by appropriately HLA‐matched effectors, even after restoration of LFA‐3 expression. We conclude that the LFA‐3 status of BL cells influences their conjugate forming ability in in vitro assays but not necessarily their sensitivity to immune T cell‐mediated cytolysis.
Selection of murine T cell receptor αβ and γδ cells in organ cultures established from 14‐day embryos
Authors: Fisher Amanda G. , Waltzinger Caroline , Ceredig Rhodri
Published: March 2007

The expression of minor lymphocyte stimulatory locus (Mls) determinants in combination with murine major histocompatibility complex (MHC) class II molecules, leads to the destruction of lymphocytes bearing specific V region‐encoded T cell receptor (TcR) products. A much studied example is the elimination of Vβ6+ cells in IE+/Mls‐1a mice, in which deletion can be detected 7‐10 days after birth but is not fully operational earlier in embryonic life. Here we investigate this transitional period in development and show that selective deletion of Vβ6 occurs in vitro, approximatively 1 week after organ cultures are established from 14 day embryos. These unmanipulated organ cultures receive no additional cell immigrants after day 14, suggesting that the cellular elements mediating negative selection (or their direct precursors), are already resident in the fetal thymus by day 14 of gestation. Hence, the developmental timing of the outset of rigorous negative selection of Vβ6 is not dictated by the postnatal entry of deleting elements into the thymus, but perhaps by the maturation of the pre‐existing environment. Using a parallel organ‐culture approach we have looked at the development of Vδ4 and Vγ3, TcR γδ+ cells in a variety of mouse strains. These receptors have recently been reported to be subject of MHC and non‐MHC linked selection, respectively. We find that after an initial period of expansion, the number of Vγ3‐expressing cells dramatically declines. However, this selective loss of Vγ3 cells is not contingent on the C57BL/6 mouse strain (in contrast to a previous report). These findings are discussed in the context of current models of ontogeny and repertoire selection.
Molecular analysis of the VxIII‐Jx junctional diversity of polyclonal rheumatoid factors during rheumatoid arthritis frequently reveals N addition
Authors: Martin Thierry , Blaison Gilles , Levallois Honey , Pasquali Jean Louis
Published: March 2007

Much interest was stirred in recent years by the evidence that rheumatoid factors (RF) variable regions are encoded by a restricted set of V genes, with little or no somatic mutations, that are often overexpressed in the fetal repertoire. This is reminiscent of what has been observed for natural autoantibodies. However, these data come from studies of monoclonal RF (mRF) isolated from patients with lymphoproliferative disorders who usually do not present autoimmune symptoms. The molecular characterization of RF during autoimmune diseases such as rheumatoid arthritis (RA) has been hampered for some time because of their polyclonality; recently using the polymerase chain reaction method, we have demonstrated that RFχ variable regions from a patient with RA were encoded by VχIII genes known to code for mRF but that these genes had undergone somatic mutations with a pattern suggesting an antigen‐driven maturation. Because an important role of the light chain third complementarity‐determining region (CDR3) in anti‐IgG reactivity and idiotype expression has already been suspected for RF, we now report the molecular characterization of the junction regions of these rearranged Vχ genes. Surprisingly, our data show that in 55% of the cases there is addition of a proline and/or glycine amino acid residue at the recombination site between Vχ and Jχ. The sequence analysis of our patients' germ‐line Vg and Jχ4 gene segments and their flanking regions demonstrates that the additional codons are not readily explicable by recombination between germ‐line sequences and probably result from an N addition process. Since we could not find such an additional codon in 15 previously published mRF χ chains we suggest that “pathogenic” RF during RA and mRF derive from different, although overlapping, B cell subsets. Moreover, since additional codons at the recombination site of Vχ and Jχ seem exceptional in expressed human χ chains and because the resulting amino acid residue is a proline in most cases, we think that RFχ chain CDR3 is under a very strong selective pressure during RA.
Immunosuppression by lymphocytic choriomeningitis virus infection: competent effector T and B cells but impaired antigen presentation
Authors: Althage Alana , Odermatt Bernhard , Moskophidis Demetrius , Kündig Thomas , Hengartner Urs Hoffman‐Rohrer Hans , Zinkernagel Rolf M.
Published: March 2007

Lymphocytic choriomeningitis virus (LCMV) may cause a severe immunosuppression in mice. Its pathogenesis is apparently dependent on LCMV‐specific CD8 effector T cells that mediate the destruction of virus‐infected cells which are normally essentially involved in immune responses. Evaluation of various LCMV isolates in this study established a general correlation between their tropism for lymphohemopoietic cells and immunosuppression. When immune responses were assessed as the capacity of mice to mount an anti‐vaccinia virus cytotoxic T cell response or an IgG response to vesicular stomatitis virus (VSV), after a primary LCMV infection, LCMV‐Armstrong, WE, Clone 13 and Docile were increasingly immunosuppressive in a dose‐dependent fashion with respect to both extent and duration. Analysis of lymphocyte subpopulations showed variable effects of the various LCMV isolates that did not reveal patterns readily explaining immunosuppression. To evaluate whether LCMV infection affected T and/or B cell functions directly or whether antigen presentation was impaired, adoptive transfer experiments were performed. Untreated or irradiated but uninfected normal recipient mice receiving adoptively transferred T or B cells from LCMV‐WE or Docile‐infected immunosuppressed donor mice responded within 30%–100% of normal ranges in both assay systems. In contrast, when T or B cells from normal donors were transferred to irradiated or non‐irradiated LCMV‐immunosuppressed recipients, they failed to mount a significant cytotoxic T cell response against vaccinia virus or an IgG response to VSV. Thus, the T and B cells from LCMV‐immunosuppressed mice were able to function within normal ranges; in contrast, histologically and functionally, antigen presentation was severely impaired in LCMV‐immunosuppressed mice.
Identification of an immunodominant type‐II collagen peptide recognized by T cells in H‐2q mice: self tolerance at the level of determinant selection
Authors: Michaëlsson Erik , Andersson Mikael , Holmdahl Rikard , Engström Åke
Published: March 2007

The T cell recognition of type‐II collagen (CII) in H‐2q mice, susceptible to CII‐induced arthritis, was analyzed. With the use of T cell hybridomas derived from rat CII‐immunized mice, a peptide corresponding to amino acids 245–270 on chick CII was found to harbor a T cell epitope which is present on heterologous CII (chick, rat, human, and bovine CII) but not on autologous CII. It was shown that this epitope was located within amino acids 260–270, although flanking regions in either direction were necessary for proper recognition. A peptide corresponding to human CII (256–270) was used for further studies. A single amino acid difference at position 266 between mouse CII (aspartic acid) and heterologous CII (glutamic acid) strongly influenced recognition of this peptide. No response towards the mouse peptide was seen with any of the T cell hybridomas. Inhibition studies revealed that the mouse peptide did not bind as well to major histocompatibility complex as the corresponding heterologous peptide. Both peptides gave rise to a T cell response after immunization. However, immunization with the heterologous peptide resulted in a response strictly directed to rat CII and the immunogen while immunization with the autologous peptide elicited T cells which reacted in a heteroclitic fashion, with a stronger response to the heterologous peptide than to the autologous peptide, and did respond to rat CII but not to mouse CII. We suggest that aspartic acid in position 266 results in a cryptic determinant in mouse CII which is neither recognized after CII immunization nor capable of tolerance induction. A glutamic acid at position 266, however, gives rise to an immunodominant epitope which is recognized by a large proportion of the T cells activated after immunization with heterologous CII.
The murine IgG1/IgE class switch program
Authors: Siebenkotten Gregor , Esser Charlotte , Radbruch Andreas , Wabl Matthias
Published: March 2007

Immunoglobulin class switching is controlled by cytokines. Thus, interleukin‐4 (IL‐4) directs class switching to both IgG1 and IgE. Consistent with this are the results reported here on restriction endonuclease analysis of active and inactive alleles of the IgH locus in IgE‐producing cells. In cells that were stimulated in vitro by lipopolysaccharide and IL‐4 the silent alleles preferentially switched to γ1, whereas in cells that were stimulated by antigen in vivo both active and inactive alleles switched to γ1. Thirty percent of the recombined switch regions (Sμ/Sϵ) contain Sγ1 sequences, which we interpret as footprints of a previous switch to γ1. Since this percentage is a minimum estimate, between 30% and 100% of switching to ϵ must occur sequentially via γ1.
T cell clones with normal or defective O‐galactosylation from a patient with permanent mixed‐field polyagglutinability
Authors: Thurnher Martin , Berger Eric G. , Clausen Henrik , Fierz Walter , Lanzavecchia Antonio
Published: March 2007

To delineate the extent of O‐galactosyltransferase deficiency within the lymphoid lineage, monoclonal antibody specific for the Thomsen‐Friedenreich (TF) antigen (Galβ1 → 3GalNAcα1‐O‐Ser/Thr) and its precursor the Tn antigen (GalNAcα1‐O‐Ser/Thr) were applied to the flow cytometric analysis of peripheral blood lymphocytes from a patient with permanent mixed‐field polyagglutinability (PMFP). We show that only a minor population of 4% expressed the Tn antigen which is in contrast to 93% of the patient's erythrocytes carrying the defect. Tn+ lymphocytes mainly belonged to the CD3+ subset, but were also CD19+ or CD16+. Both Tn+ and TF+ T cell clones from patient R. R. were established and shown to belong to the CD4+ or CD8+ antigenic subset. Three glycosyltransferase activities were determined in lysates from these clones: all Tn+ clones were deficient in UDP‐Gal: GalNAcα1‐O‐Ser/Thr β1 → 3 galactosyl‐transferase (β3Gal‐T) activity; by contrast this activity was present in all lysates from TF‐expressing clones. UDP‐GalNAc:polypeptide α‐N‐acetyl‐galactosaminyltransferase (GalNAc‐T) and UDP‐Gal:GlcNAc‐Rβ1 → 4 galactosyl‐transferase (β4Gal‐T) exhibited similar activities in both Tn+ and TF+ T cell clones. As a consequence of defective O‐galactosylation in Tn+ T cells, cell surface sialic acid of Tn+ clones was reduced by > 50% when compared to TF+ clones as demonstrated by sialic acid‐specific labeling using fluoresceinated Limax flavus agglutinin(LA) and flow cytometry. The Tn phenotype of T cell clones was stable for more than 1 year of continuous expansion in vitro. These data demonstrate that in PMFP, T cells may also be affected by the O‐galactosyltransferase deficiency which is accompanied by a substantial loss of cell surface sialic acid. However, the frequency of Tn+ lymphocytes in peripheral blood from patient R.R. was strikingly low. These T cell clones should be useful to study the defect at a genetic level and the importance of O‐linked carbohydrates for proper T cell function.