摘要:

AbstractVirus‐specific cytotoxic T lymphocytes (CTL) play a crucial role in modulating an immune response against human immunodeficiency type 1 (HIV‐1) infection. The generation of effector cytotoxic cells from CTL precursors involves intricate interactions with antigen via T cell receptors (TcR) and soluble cytokines. Interleukin (IL)‐7 can affect T cell maturation and differentiation. Here we report on a group of five HIV‐1‐positive individuals who tested negative forenv‐andgag‐specific CTL activity. When exogenous recombinant human IL‐7 was added as a stimulus to the cultures, none (0/5) of the CTL‐negative individuals exhibited a CTL response. Individuals that were negative for HIV‐1‐specific CTL activity were found to lack IL‐7 receptor (IL‐7R) on CD8+cells with a comparable reduction on CD4+cells. Increased shedding of IL‐7R in the culture supernatant was observed. A significant reduction in receptor number was detected by binding of125I‐labeled IL‐7 and Scatchard analysis. The lack of IL‐7R is probably not due to endogenous IL‐7, since it was not detectable in the culture supernatants of the patients studied. HIV‐1 proteins may cause down‐modulation of IL‐7R expression, either by producing an insufficient number of molecules or by rapid decay of IL‐7R on T cells. These changes may alter the cells' capability to respond to the IL‐7 growth signal, resulting in CTL fa

ISSN:0014-2980    

DOI:10.1002/eji.1830241202

出版商:WILEY‐VCH Verlag GmbH    

年代:1994

数据来源: WILEY

摘要:

AbstractThe question as to whether other cell types apart from helper T lymphocytes are capable of producing interleukin‐4 (IL‐4) has gained much interest over the last years. Recent studies indicate that human basophils also produce IL‐4, although direct proof is missing so far. In this study we demonstrate the presence of IL‐4 in the cytoplasm ofin vitroactivated human peripheral blood basophils derived from normal donors. Cytokine‐producing cells were revealed at the single‐cell level by intracellular immunofluorescence staining using IL‐4‐specific monoclonal antibodies. Basophils showed a characteristic, apparently granular staining pattern easily discerned from the eccentric dot‐shaped staining pattern in activated T cells used in control experiments. Cell counts following priming with IL‐3 and stimulation with polyclonal sheep anti‐IgE antibody or the anaphylatoxin C5a revealed a significant increase in IL‐4‐positive basophils to about 19% as compared with unprimed, unstimulated control cells (6%). The amount of IL‐4 in the supernatant of these cell preparations paralleled these observations with an at least five‐ to sevenfold increase following stimulation as compared with control cells (<5 ng/ml). Using confocal scanning laser microscopy, the intracellular presence of IL‐4 was confirmed, and the cells were identified as being basophils on terms of their characteristic multilobed nucleus. This observation was supported by double labeling studies using antibodies to IL‐4 and to the high‐affinity IgE receptor (FcεR1). Interestingly, stimulation of cells led to a decrease in the number of FcεR1‐positive cells. The above results show direct evidence that IL‐4

ISSN:0014-2980    

DOI:10.1002/eji.1830241203

出版商:WILEY‐VCH Verlag GmbH    

年代:1994

数据来源: WILEY

摘要:

AbstractWe isolated immunoglobulin (Ig) VH4 genes that were rearranged in the genomic DNA of 160 day human fetal spleen. Productively rearranged VH4‐21 genes were cloned into pRTM1, a human IgM expression vector. This allowed us to generate IgMϰK‐expressing transfectomas by co‐transfecting each of these constructs with pSVG‐VϰK3, an Ig ϰK light‐chain expression vector that has a variable region encodedHumkv325, a conserved VϰK gene that is frequently expressed early B cell ontogeny. We find that all transfectomas expressing IgMϰK encoded by VH4‐21 make IgM autoantibodies reactive with i, a linear poly‐N‐acetyllactosamine determinant present on neonatal red blood cells and a B cell‐restricted isoform of the CD45 surface molecule. In contrast, a transfectoma expressing pSVG‐VϰK3 and pRTM1 containing a rearranged VH4‐59 (V71‐4) gene isolated from a chronic lymphocytic leukemia B cell population, designated WIL, produced IgMϰK antibodies that had no detectable anti‐i binding activity. However, transfectomas expressing VH4‐21 fused onto the Ig heavy‐chain third complementarity determining region (CDR3) of WIL are found to make anti‐B cell autoantibodies with anti‐i activity. These studies indicate that VH4‐21 genes rearranged in human fetal B cell ontogeny can encode anti‐B cell autoantibodies with a binding specificity t

ISSN:0014-2980    

DOI:10.1002/eji.1830241204

出版商:WILEY‐VCH Verlag GmbH    

年代:1994

数据来源: WILEY

摘要:

AbstractMouse mammary tumor virus MMTV(C4) encodes a Vβ2‐specific superantigen. In Vβ2 transgenic (TG2) mice more than 98 % of peripheral T cells express Vβ2. Infection of Tg2 mice with MMTV(C4) at birth through their mothers' milk or at 6–8 weeks of age by intravenous injection resulted in massive deletion of peripheral CD4+T cells and suppressed thymopoiesis. The number of peripheral CD8+T cells was not affected in neonatally infected mice. In older mice injected with MMTV(C4), splenic CD8+T cells were significantly elevated. Suppressed thymopoiesis was observed in both neonatally infected and older mice injected with MMTV(C4). Thymocytes which expressed high level CD3 or Vβ2 were deleted. To determine if T cells or thymocytes were deleted through apoptosis, DNA fragmentation was examined by flow cytometry and diphenylamine (DPA) binding assay. Approximately 31 % of CD4+T cells from MMTV(C4)‐infected Tg2 mice as compared to 6% from normal Tg2 mice contained fragmented nuclear DNA by flow‐cytometric analysis. The DPA binding assay showed significantly increased total soluble DNA in lymph node cells and thymocytes from MMTV(C4)‐infected mice. The kinetics of T cell and thymocyte apoptosis correspond to their deletion, supporting apoptosis as the mechanism of T cell and thymocyte deletion. CD4+T cell and thymocyte deletion by MMTV(C4) in Tg2 mice provides a sensitive system for the analysis of retrovirus superantigen‐in

ISSN:0014-2980    

DOI:10.1002/eji.1830241205

出版商:WILEY‐VCH Verlag GmbH    

年代:1994

数据来源: WILEY

摘要:

AbstractIn vivo, natural killer (NK) cells dominate among the early invading cells in allografts and virus‐infected tissues, and they are followed later by an influx of T cells. The same sequence of events was seen in our modified Boyden chamber assay. The migration of both CD3+/CD4+and CD3+/CD8+cells through fibronectin‐coated filters increased after co‐culture with NK cells. The migratory response to a soluble factor from NK cells supernatants was predominantly chemotactic rather than chemokinetic. Endogenous NK cells, purified in the presence of human serum albumin, did not induce T cell chemotaxis, but NK cells which were purified in the presence of 10% fetal calf serum (FCS), or which were activated in the absence of FCS with 10−4Mhistamine, with 300 IU/ml interleukin (IL)‐2, or with a combination of 10 IU/ml IL‐2 and 10 μg/ml CD16 monoclonal antibody increased T cell migration by 30–70%. Both the random and chemotactic migration were dependent on fibronectin receptors VLA‐4 and VLA‐5 on T cells. About 60% of the chemotactic was neutralized by NAP‐1/IL‐8 polyclonal antibody. Northern blot analysis revealed IL‐8 mRNA expression in highly purified, stimulated NK cells; dimeric IL‐8 protein secreted by NK cells was detected by immunoblotting, and, in immunofluorescence staining IL‐8 was visualized in NK cells. These observations suggest that NK cells, early invaders in the foci of injury, participate in the initiation of a specific immune response by fac

ISSN:0014-2980    

DOI:10.1002/eji.1830241206

出版商:WILEY‐VCH Verlag GmbH    

年代:1994

数据来源: WILEY

摘要:

AbstractT cell tolerance is usually established by clonal deletion of self‐specific T cells in the thymus, or some times, in the periphery. Alternatively, tolerance may also be achieved by induction of clonal T cell unresponsiveness by a poorly understood mechanism called “anergy”. We found that transgenic mice expressing a soluble form of vesicular stomatitis virus (VSV) glycoprotein (G) predominantly in liver and kidney exhibited normal B cell responses. VSV‐G‐specific T help‐independent neutralizing IgM responses were within normal ranges, but no T help‐dependent neutralizing IgG antibodies were generated upon immunization with recombinant VSV‐G protein and recombinant vaccinia virus expressing VSV‐G. This demonstrated absence of B cell tolerance but presence of T helper cell unresponsiveness. After adoptive transfer of transgenic spleen cells into thymectomized immuno‐incompetent hosts, the unresponsive T helper cells regained function and switched the neutralizing IgM response to IgG, comparably to control T helper cells, within 7 days. Conversely, when naive non‐transgenic spleen cells were transferred into transgenic mice, VSV‐G‐specific T helper cells became unresponsive within 3–4 days. These results suggest that VSV‐G‐specific T helper cells are rendered unresponsive within a few days in the VSV‐G transgenic host also outside of the thymus and that this unresponsiveness was reversed by tran

ISSN:0014-2980    

DOI:10.1002/eji.1830241207

出版商:WILEY‐VCH Verlag GmbH    

年代:1994

数据来源: WILEY

摘要:

AbstractP‐glycoprotein (P‐gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells. Functional P‐gly activity can be conveniently assessed microfluorometrically using the fluorescent dye rhodamine 123 (Rh123), which is an artificial substrate for the P‐gly transporter. Here we assess P‐gly activity in subsets of mouse peripheral T lymphocytes using the Rh123 efflux assay. Our data indicate that virtually all CD8+cells extrude Rh123 efficiently, whereas only a subset of CD4+cells exhibit P‐gly activity. Correlation of P‐gly activity in CD4+cells with the expression of a panel of surface markers revealed that cells bearing an “activated/memory” phenotype (CD45RB−, CD44hi, CD62L−, CD25+, CD69+) were exclusively found in the fraction that can extrude Rh123. In contrast “naive” phenotype CD4+cells (CD45RB+, CD44lo, CD62L+, CD25−, CD69−) could be further subdivided into two major subsets based on P‐gly activity. In functional studies of sorted cell populations the Rh123‐extruding subset of “naive” CD4+cells proliferated more strongly and secreted higher levels of interleukin (IL)‐2 than its Rh123‐retaining counterpart when activated by a variety of polyclonal stimuli. Furthermore, this subset produced detectable levels of interferon (IFN)‐γ upon stimulation but no IL‐4 or IL‐10. As expected, the Rh123‐retaining “naive” subset produced only IL‐2 after stimulation, whereas the “memory” subset produced IFN‐γ, IL‐4 and IL‐10 in addition to low levels of IL‐2. Collectively, our data indicate that P‐gly activity is a novel parameter that can be used to distinguish a subset of “preactivated” CD4+cell

ISSN:0014-2980    

DOI:10.1002/eji.1830241208

出版商:WILEY‐VCH Verlag GmbH    

年代:1994

数据来源: WILEY

摘要:

AbstractPrevious studies have documented a role for membrane‐bound CD23 (the low affinity FcεRII) in presentation of alloantigens by B cells. The aim of the present study was to examine the involvement of cell surface CD23 in presentation of more conventional soluble protein antigens to T cells. We show that antibodies to CD23 and to its lymphocyte‐associated second ligand, CD21, inhibit presentation of the cow's milk allergen casein, by autologous CD23+CD21+B‐EBV cell lines to casein‐specific HLA‐DP‐restricted CD4+T cell clones obtained from patients with either reaginic or enterophatic forms of cow's milk protein intolerance. Maximal inhibition was achieved when the antibodies were added at the initiation of the culture. The absence of specific inhibition by an anti‐DRα monoclonal antibody (mAb) argues against a steric hindrance phenomenon impeding access of the T cell receptor to major histocompatibility complex class II molecules. Rather, anti‐CD23 and anti‐CD21 mAb‐induced inhibition of antigen presentation seems to affect at least partly, heterotypic conjugate formation through CD23/CD21 interaction. Double immunofluorescence labeling of the T cell clones and antibody inhibition of T/B conjugate formation shows that functional CD23 and CD21 molecules are induced on T cells following contact with B‐EBV cell lines. Taken together, these data indicate that CD23/CD21 interactions between T and B cells are required for presentation of soluble protein antigens by B‐EBV cell lines to specific CD4+T cells. The potential implications of these findings for allergen‐specific T ce

ISSN:0014-2980    

DOI:10.1002/eji.1830241209

出版商:WILEY‐VCH Verlag GmbH    

年代:1994

数据来源: WILEY

摘要:

AbstractThe purpose of this study was to assess the V‐(D)‐J junctional region of the T cell receptor (TCR), the CDR3 region, which is responsible for glioma‐specific antigen contact in αβ TCR‐mediated recognition. We sequenced the TCR α and β chians of Vα7, and Vβ13.1 cDNA derived from tumor‐infiltrating lymphocytes (TIL) of 12 glioma patients and also the corresponding clones from the patients' peripheral blood lymphocytes (PBL). A shared Vβ13.1 DJ sequence of the CDR3 region, NDβN, was demonstrated in 49 of 66 Vβ13.1+clones (74.2 %) from the glioma TIL, whereas only 4 of 33 clones (12.1 %) were observed in the Vβ13.1+clones from the PBL (p<0.001). A common VDJ sequence, FCASS (Vβ13.1)‐YRLPWGTSDS (NDβN)‐GELFF(Jβ2.2), was observed not only in the gliomas from each patient, but also among all the patients with a preference for Vβ13.1. In contrast, the amino acid sequences of the Vβ13.1+PBL clones were diverse and random. Next, we sequenced subclones from other Vβ subfamilies randomly selected to compare their VDJ region rearrangements (Vβ3 and Vβ5.1). In contrast to Vβ13.1, the amino acid sequences of these junctional regions were completely different in these subclones. The V‐J junctional region of the α chain is dominated by a few clones in some patients, and no shared amino acid sequences were detected in the TCR Vα junctional region. However, in the Nα region of the Vα7‐bearing TIL clones, arginine was used in 27 of 44 clones (61.4%) compared to only 3 of 12 clones from the PBL (p<0.05). These results are consistent with the hypothesis that a clonal expansion/accumulation of glioma lineage‐specific T cells occurredin vivoat the tumor site and that these T cells m

ISSN:0014-2980    

DOI:10.1002/eji.1830241210

出版商:WILEY‐VCH Verlag GmbH    

年代:1994

数据来源: WILEY

摘要:

AbstractDuring cognate B : T interactions, B cells encounter antigen (Ag) through surface immuno‐globulin (sIg) and present antigenic peptides to T helper (Th) cells. However, mostin vitrosystems used to study contact events involved in the delivery of T help for B cells circumvent the requirement for T cell Ag specificity by using anti‐CD3/T cell receptor (TcR) monoclonal antibodies (mAb) to activate T cells. To study the role of sIg engagement in the responsiveness of B cells to T help, we pre‐treated small resting B cells with soluble anti‐χ mAb prior to contact with an activated Th1 clone. By reducing the concentration of anti‐TcR mAb we obtained low levels of CD40 ligand (CD40Llow) on Th cells, comparable to those expressed by lymph node T cells activatedin vitro(ex vivoT cells). In contrast to untreated B cells, which did not respond to CD40LlowTh, anti‐Ig‐treated B cells responded strongly. Low buoyant density B cells also responded to CD40LlowTh cells. There was no B cell response to resting Th cells. mAb against CD54/intercellular adhesion molecule‐1 or major histocompatibility complex (MHC) class II completely inhibited B cell responses to CD40LlowTh1 cells, equivalent to the effects of blocking CD40 interactions. This contrasts with mAb blocking responses to CD40LhighTh, where CD40 effects predominate. Our data show that sIg engagement is necessary for the induction of B cell response to CD40LlowTh cells. Anti‐CD3‐activatedex vivoT cells that were also CD40Llowdid not provide help to small resting B cells, but did induce responses from sIg‐stimulated B cells. Thus, our data support a requirement for sIg signaling in physiological B cell activation, and further confirm previous work showing CD40 ligation to be necessary but not sufficient for deliver

ISSN:0014-2980    

DOI:10.1002/eji.1830241211

出版商:WILEY‐VCH Verlag GmbH    

年代:1994

数据来源: WILEY