c-myc gene expression and interleukin-2 receptor levels in cloned human CD2+,CD3+ and CD2+,CD3- lymphocytes

The levels of c-myc mRNA and interleukin-2 receptors (IL-2 Rec) were studied in human peripheral blood lymphocytes (PBL); mature CD2+,CD3+ T cell clones and CD2+,CD3- natural killer (NK) cell clones, and CD2+,CD3+ and CD2-,CD3- T lymphoma cell lines. A transient induction of the expression of c-myc and IL-2 Rec was observed in PBL after activation with phytohemagglutinin (PHA). Expression of c-myc and IL-2 Rec was also found in the CD2+,CD3+ and CD2+,CD3- clones. The CD2+,CD3+ showed higher levels of c-myc mRNA and IL-2 Rec than the CD2+,CD3- clones. In three T lymphoma cell lines constitutively high levels of c-myc mRNA but no IL-2 Rec were found. Only in JURKAT (CD2+,CD3+), c-myc mRNA levels could be further enhanced by PHA. These results suggest that in the presence of PHA, expression of c-myc and IL-2 Rec is induced via the CD3 receptor, and in the absence of PHA and/or the CD3 receptor alternative routes of induction are involved.     

NK1.1+ thymocytes. Adult murine CD4-, CD8- thymocytes contain an NK1.1+, CD3+, CD5hi, CD44hi, TCR-V beta 8+ subset.

CD4-, CD8- thymocytes were purified from thymi obtained from normal C57BL/6 mice. By flow cytometry analysis, 5 to 10% of these double negative (DN) thymocytes were found to express NK1.1 on their surface. The NK1.1+ DN thymocytes were demonstrated, by two-color fluorescence, to be CD3lo, CD5hi, CD44hi, J11d-, B220-, MEL 14-, IL2R- with 60% expressing TCR-V beta 8 as determined by the mAb F23.1. In contrast, splenic and peripheral blood NK cells were NK1.1+, CD3-, CD5-, TCR-V beta 8- with 40 to 60% being MEL 14+. Unlike peripheral NK cells, fresh DN thymocytes enriched for NK1.1+ cells were unable to kill YAC-1, the classical murine NK cell target. However, these cells were able to mediate anti-CD3 redirected lysis even when they were assayed immediately after purification, i.e., with no culture or stimulation. These data demonstrate that adult murine thymocytes contain NK1.1+ cells which are distinct, both by function and phenotype, from peripheral NK cells. These data also raise the issue of a possible NK/T bipotential progenitor cell.     

Lymphokine-activated killer (LAK) cells. VI. NK1.1+, CD3+ LAK effectors are derived from CD4-, CD8-, NK1.1- precursors.

Normal murine splenocytes cultured with IL2 for 6, but not 3, days contained an NK1.1+, CD3+ lytically active subset. These lymphocytes were not derived from NK1.1+ precursors since NK1.1+ cells, purified by flow cytometry, failed to express CD3, as determined by the 145-2C11 mAb, on their surface even after culture with IL2 for 6 days. Instead, the precursors of the NK1.1+, CD3+ effectors were contained in a B cell-depleted CD4-, CD8-, NK1.1- splenic subset. Freshly obtained CD4-, CD8-, NK1.1- splenocytes were mostly CD3+, CD5+, B220-, had no spontaneous lytic activity against YAC-1, and were unable to mediate anti-CD3 directed lysis against FcR-bearing target cells. Culture of the CD4-, CD8-, NK1.1- splenocytes with IL2, for 6 days, resulted in the development of NK1.1+, CD3+, B220+ effectors 40% of which were CD5dim and 20-25% of which expressed TCR-V beta 8 as determined by the F23.1 mAb. The acquisition of NK1.1, B220, and lytic activity by this triple-negative subset was readily inhibited by cyclosporine A (CSA). On the other hand, CSA had no effect on the acquisition of B220 or lytic activity by NK1.1+ precursors obtained by flow cytometry sorting. Moreover, all of the NK1.1+ cells generated by IL2 culture of splenocytes obtained from mice depleted of NK1.1+ lymphocytes (by in vivo injection of anti-NK1.1 mAb) coexpressed CD3 on their surface and were thus distinct from classical NK cells. These findings demonstrate that splenic NK cells do not express or acquire CD3; that the NK1.1+, CD3+ LAK effectors are derived from an NK1.1- precursor; and that CSA is exquisitely selective in its inhibitory effect on LAK generation.     

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+renal-cell-carcinoma-infiltrating lymphocytes

The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3⁺ tumor-infiltrating lymphocytes (CD3⁺ TIL) and in autologous CD3⁺ peripheral blood lymphocytes (CD3⁺ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4⁺ TIL and CD8⁺ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3⁺ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3⁺ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4⁺ TIL and simultaneously obtained CD8⁺ TIL revealed a significantly higher expression of IFN in the CD8⁺ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8⁺ TIL. Received: 14 January 1999 / Accepted: 30 April 1999     

Regulation of activities of NK cells and CD4 expression in T cells by human HNP-1, -2, and -3

Human neutrophil defensin alpha (HNP) is a group of cationic peptides of diverse physiological roles. Recent studies revealed the nature of HNPs as the dominant HLA-DR binding peptides on malignant cancer cells, which may block the major histocompatibility complex for antigen presentation. Here we show that HNPs may inhibit T cells by downregulating CD4 expression, a molecule of critical importance for T cell's interaction with the target cell. HNPs also inhibited tumor-cell-lysis activities of NK cells by downregulating CD16-CD56 expression. More importantly, HNPs were markedly elevated in 14 cancer tissues out of 15 self-paired human colorectal cancers and their adjacent noncancerous tissues. The subset compositions of HNPs extracted from cancer tissues and neutrophils were identical. Immunohistochemical studies indicated that HNPs mainly distributed in the infiltrated neutrophils in the interstitium. The elevated HNPs in cancer tissues may create a microenvironment unfavorable for adaptive immune reaction, implicating the cancer evasion.     

Induction and blocking of cytolysis in CD2+, CD3- NK and CD2+, CD3+ cytotoxic T lymphocytes via CD2 50 KD sheep erythrocyte receptor

The 50 KD sheep red blood cell antigen receptor CD2 is the earliest T cell differentiation marker and is present on all blood-derived T cells, including natural killer (NK) cells. The CD2 antigen is also known to serve as an important activation site regulating various T cell functions. We report that anti-CD2 monoclonal antibodies (MAb) block MHC-restricted class I- and class II-specific cytolysis by CD2+, CD3+ clones of the relevant target cells, irrespective of whether lysis by these clones is blocked by anti-CD3 or anti-CD8 MAb. Moreover, anti-CD2 MAb (but not anti-CD3 MAb) are able to reduce MHC-nonrestricted, nonspecific cytolysis: a) by CD2+, CD3+ clones of K562 target cells; and b) by CD2+, CD3 NK clones of K562 as well as Daudi cells. Different preparations of anti-CD2 MAb vary in their capacity to inhibit cytolysis. For cloned effector cells, the percent inhibition of lysis by CLB-T11 greater than Lyt-3 MAb, whereas with "fresh" NK cells, the lysis inhibitory ability of Lyt-3 greater than CLB-T11. The antibody-dependent cellular cytotoxicity by "fresh" and cloned NK cells is not inhibited by anti-CD2 MAb. Anti-CD2 MAb also prevent the induction of lysis by cross-linked anti-CD3 MAb, e.g., by CD2+, CD3+ cloned cloned cells against (IgG-FcR+) Daudi cells. Anti-CD2 MAb can also induce cytolysis in some, but not all, CD2+, CD3- NK clones against xenogeneic P815 mouse mastocytoma cells. Anti-CD2 MAb, in combination with lectins (PHA or Con A: pretreatment of effector cells), can also induce cytolytic activity by CD2+, CD3+ clones against Daudi cells. Our data therefore support the concept that the CD2 antigen is an important activation site regulating a wide variety of T cell functions including cytolysis. Whether ligand interaction with the CD2 antigens results in augmentation or inhibition of T cell functions may very well depend on the type of CD2 antigen-ligand interaction, e.g., cross-linked ligand-receptor interaction may, in general, enhance the various T cell functions, whereas noncross-linked ligand-receptor interactions may inhibit such functions, as we and other investigators demonstrated earlier for the CD3/Ti antigen-receptor complex activation site.     

In vitro and in vivo analysis of bone marrow-derived CD3+, CD4-, CD8-, NK1.1+ cell lines

The development of methods of avoiding graft-versus-host disease (GVHD) while retaining the alloengraftment-promoting and anti-leukemic effects of allogeneic T cells is a major goal of research in bone marrow transplantation (BMT). We have recently obtained evidence suggesting that natural suppressor (NS) cells derived from T cell-depleted (TCD) syngeneic marrow can protect against GVHD while permitting alloengraftment. We have now attempted to enrich and then propagate NS cells in vitro, with the goal of obtaining an enhanced anti-GVHD effect by adoptive transfer in vivo. Two long-term cell lines were generated culturing BMC depleted of Mac1-positive cells and of Mac1-positive plus Thy1-positive cells in high concentrations of IL-2. Both cell lines showed anti-GVHD effects when administered along with a GVHD-producing inoculum, while permitting complete allogeneic reconstitution. A clone derived from Mac1-depleted BMC protected completely against a more chronic pattern of GVHD. These cell lines demonstrated suppressive activity in vitro, cytolytic activity against a broad range of natural killer (NK)-sensitive and NK-resistant targets, and a novel cell surface phenotype, with characteristics of both alpha beta-TcR-bearing T cells and of NK cells. In some respects, these cells resemble LAK cells and differ from fresh NS cells, and from the cloned NS cells derived from spleens of total lymphoid irradiation (TLI)-treated mice and neonatal mice. To our knowledge, this is the first detailed phenotypic analysis of cell lines with in vivo anti-GVHD activity. If applicability can be demonstrated in large animal models, the ability to use bone marrow as a source of such protective cell lines might also have potential utility in clinical BMT.     

Peripheral murine CD3+, CD4-, CD8- T lymphocytes express novel T cell receptor gamma delta structures

A mAb directed against the CD3 molecule was used to identify a subset of CD3+, CD4-, CD8- T cells previously undefined in the peripheral lymphoid organs of the mouse. Biochemical analysis of CD3+, CD4-, CD8- splenocytes revealed that the vast majority of these cells express one of at least two distinct CD3-associated TCR gamma delta heterodimeric structures, but no detectable TCR alpha beta. One disulfide-linked heterodimer (77 kDa) is composed of two chains of 45 to 46 and 32 kDa. The latter chain was immunoprecipitated with an anti-TCR C gamma 1/C gamma 2 antiserum and was not glycosylated. An antiserum produced against a peptide corresponding to the C-terminal region of the predicted C gamma 4 gene product immunoprecipitated additional heterodimers (80 to 90 kDa). One heterodimer, composed of disulfide-linked 41- to 45-kDa protein (including a V gamma/C gamma 4 component), is expressed on a T cell hybridoma, DN-1.21, which was derived from fused splenic CD3+, CD4-, CD8- T cells. Another V gamma/C gamma 4-containing heterodimer is composed of disulfide-linked 46- to 47-kDa glycoproteins. These findings demonstrate that CD3+, CD4-, CD8- T cells present in the peripheral lymphoid organs express a variety of paired TCR gamma delta proteins. Unlike CD3+, CD4-, CD8- thymocytes, these cells express high levels of C gamma 4, but little, if any TCR alpha beta.     

Phenotypic and functional analysis of murine CD3+,CD4-,CD8- TCR-gamma delta-expressing peripheral T cells

Murine CD3+,CD4-,CD8- peripheral T cells, which express various forms of the TCR-gamma delta on their cell surface, have been characterized in terms of their cell-surface phenotype, proliferative and lytic potential, and lymphokine-producing capabilities. Three-color flow cytofluorometric analysis demonstrated that freshly isolated CD3+,CD4-, CD8- TCR-gamma delta lymph node cells were predominantly Thy-1+,CD5dull,IL-2R-,HSA-,B220-, and approximately 70% Ly-6C+ and 70% Pgp-1+. After CD3+,CD4-,CD8-splenocytes were expanded for 7 days in vitro with anti-CD3-epsilon mAb (145-2C11) and IL-2, the majority of the TCR-gamma delta cells expressed B220 and IL-2R, and 10 to 20% were CD8+. In comparison to CD8+ TCR-alpha beta T cells, the population of CD8+ TCR-gamma delta-bearing T cells exhibited reduced levels of CD8, and about 70% of the CD8+ TCR-gamma delta cells did not express Lyt-3 on the cell surface. Functional studies demonstrated that splenic TCR-gamma delta cells proliferated when stimulated with mAb directed against CD3-epsilon, Thy-1, and Ly-6C, but not when incubated with an anti-TCR V beta 8 mAb, consistent with the lack of TCR-alpha beta expression. In addition, activated CD3+,CD4-,CD8- peripheral murine TCR-gamma delta cells were capable of lysing syngeneic FcR-bearing targets in the presence of anti-CD3-epsilon mAb and the NK-sensitive cell line, YAC-1, in the absence of anti-CD3-epsilon mAb. Finally, activated CD3+, CD4-,CD8-,TCR-gamma delta+ splenocytes were also capable of producing IL-2, IL-3, IFN-gamma, and TNF when stimulated in vitro with anti-CD3-epsilon mAb.     

胎盘多肽对子宫肌瘤患者T 淋巴细胞亚群、NK细胞及炎症因子水平的影响

目的:探讨胎盘多肽对子宫肿瘤切除术后患者血清炎症因子变化和免疫功能的影响。方法:收集我院收治的子宫肿瘤切除术后患者57例,根据就诊先后顺序不同分为对照组和实验组,实验组采用胎盘多肽治疗,对照组采用常规治疗。检测两组患者CD3~+、CD4~+、CD8~+、NK细胞、CRP及IL-6水平,并比较两组的临床效果。结果:治疗后,两组患者血清CRP及IL-6水平均低于治疗前,且实验组明显低于对照组,差异具有统计学意义(P0.05)。治疗后,两组患者CD3~+、CD4~+、CD4~+/CD8~+、NK细胞均高于治疗前,而CD8~+低于治疗前,差异具有统计学意义(P0.05);实验组患者治疗后CD3~+、CD4~+、CD4~+/CD8~+、NK细胞显著高于对照组,而CD8~+低于对照组,差异具有统计学意义(P0.05)。与对照组相比,实验组患者的首次排气和排便时间明显较短,差异具有统计学意义(P0.05)。结论:胎盘多肽能够明显改善子宫肌瘤切除患者的免疫功能,降低炎症反应,有助于患者术后康复。     

Apoptotic human lymphocytes have diminished CD4 and CD8 receptor expression

We used quantitative multiparameter flow cytometric assays to simultaneously detect viable, apoptotic, and necrotic human peripheral blood mononuclear cells (PBMC) and immunophenotyped lymphocyte subsets within the PBMC. Apoptosis was induced by a spectrum of treatments, including camptothecin, cisplatin, dexamethasone, hyperthermia, staurosporine, and etoposide in anti-CD3 mAb-stimulated cells and by cyclohexamide in both quiescent and stimulated cells; apoptosis in the latter was augmented by anti-fas mAb. We found that CD4(+) and CD8(+) cells were significantly underrepresented in the apoptotic PBMC and that the percentage of CD4(+) and CD8(+) PBMC each markedly decreased as apoptosis increased. This suggested that surface expression of these receptors was lessened on apoptotic CD4(+) and CD8(+) cells. This was directly confirmed by observation of sorted CD4(+) PBMC. This analysis of a wide variety of apoptotic stimuli demonstrates that diminished CD4 and CD8 surface receptor expression is a common feature of human T lymphocyte apoptosis.     

Lysis of tumor cells by CD3+4-8-16+ T cell receptor alpha beta- clones, regulated via CD3 and CD16 activation sites, recombinant interleukin 2, and interferon beta 1

A small subpopulation (about 2%) of normal CD3+ human T lymphocytes lacks both CD4 and CD8 antigens. We have cloned these cells from peripheral blood lymphocytes (PBL) obtained from healthy individuals and from a patient with severe combined immunodeficiency. Six out of seven CD3+4-8-clones exert strong cytolytic activity against a variety of so-called NK-susceptible and -nonsusceptible tumor target cells. Their target cell specificity spectrum can virtually be as wide as that of CD3-NK cell-derived clones, with strong lytic capacity. Some of these clones also exert antibody-dependent cellular cytotoxicity (ADCC), a characteristic of NK cell-derived clones but not of CD3+4+ or CD8+ mature T cell-derived clones. Such CD3+ T cell clones do not express the CD16 (IgG Fc receptor) antigen, but as we demonstrate here, the CD16 antigen can be identified on CD3+4-8-clones. Both ADCC activity and CD16 antigen expression are lower in CD3+4-8- than in CD3- NK cell clones. Lytic activity of mature CD3+4+ or CD8+ and CD3- NK cell clones can be augmented, respectively, by anti-CD3 or anti-CD16 monoclonal antibodies (MAb), but that of CD3+4-8- clones are augmented by both MAb. Lytic activity of CD3+4+ or CD8+ clones is considerably enhanced after 3 hr of incubation with recombinant IL 2, as found for CD3- NK cells. Enhancement of lytic activity of allospecific CD3+4+ or CD8+ clones requires 18 hr of incubation. Thus, CD3+4-8-16+ cells share several features with CD3- NK cells. However, they express the CD3 antigen, which is characteristic for CD4+ or CD8+ mature T cells. Our results also indicate that although CD3+4-8- clones react with five preparations of anti-CD3 MAb tested, these clones do not express a classical CD3+/Ti alpha, beta antigen receptor complex. This is suggested by the finding that the CD3+4-8- clones do virtually not express the common epitope of the T cell receptor alpha, beta-chains as identified by the WT31 MAb. These CD3+4-8- lymphocytes may represent functionally mature lymphocytes of a distinct T cell subpopulation having a particular immune function.     

The role of tumor cells in the modification of T lymphocytes activity--the expression of the early CD69+, CD71+ and the late CD25+, CD26+, HLA/DR+ activation markers on T CD4+ and CD8+ cells in squamous cell laryngeal carcinoma. Part I

The role of interactions between tumor cells and autologous immunocompetent cells, the impact on the modulation of the activity of T CD4(+) and CD8(+) lymphocytes, as well as the influence on the regulation and determination of antitumor cellular immune response in patients with head and neck squamous cell carcinomas (HNSCC) is not completely clear. The aim of this study was to analyze early and late activation antigens expression on T cells subpopulations modified under the influence of the presence of cancer cells to investigate the regulatory mechanisms of the local cellular immune response in carcinoma of the larynx. Cytofluorymetric analysis of the early (CD69(+), CD71(+)) and late activation markers (CD25(+) (high), CD26(+), HLA/DR(+)) expression on T CD3(+)CD4(+) and CD3(+)CD8(+) cells subpopulations in mixed cellular cultures of freshly isolated tumor cells (MLTMC) and non-cancerous normal epithelial cells (MLNCC) with immunocompetent cells was performed in 55 cases of squamous cell laryngeal carcinoma. The whole peripheral blood concentrations of IL-10 and IFN-γ in 21 h and 72 h of experiments were also measured by ELISA. The relationships between the activation markers expression depending on the type of cells used in co-cultures, as well as the level of secreted cytokines, were investigated. Our work has revealed a statistically significant dependence of cytofluorymetric results on the presence of TMC or NCC in mixed cellular cultures. Increased expression of CD69(+), CD71(+) and CD25(+) (high), CD26(+), HLA/DR(+) antigens on T CD3(+)CD4(+) and CD3(+)CD8(+) cells was higher in MLTMC cultures, in comparison with MLNCC. We demonstrated negative significant relationships of IFN-γ and IL-10 secretion with regard to CD4(+)CD69(+), CD8(+)CD69(+), CD4(+)CD71(+), CD8(+)CD71(+) antigens expression in 21 h of experiments without mitogenic stimulation. Furthermore, this study revealed negative significant relationships of IFN-g secretion with regard to CD4(+)HLA/DR(+) and CD8(+)HLA/DR(+) as well as between IL-10 concentration and CD4(+)HLA/DR(+) in trials without PHA stimulation. Our findings have confirmed a key role for tumor cells in determining the function of T cells involved in the immunological processes and impact of neoplastic cells on modulating the activity of T CD4(+) and CD8(+) lymphocytes in laryngeal carcinoma.     

Immunoregulatory function of CD3+, CD4-, and CD8- T cells. Gamma delta T cell receptor-positive T cells from nude mice abrogate oral tolerance

Previous work has shown that abrogation of oral tolerance is mediated by T cells which are found in the CD3+, L3T4- (CD4-), and Lyt-2- (CD8-) subset (termed double-negative; DN) in mice. Inasmuch as it is known that athymic, nude (nu/nu) mice possess Thy 1+, CD4-, and CD8- T cells which also exhibit a functionally rearranged TCR gamma-chain, we investigated whether this subset of nude T cells contained functional immunoregulatory cells. In this report, we examined the phenotype and distribution of CD3+ T cells in the spleen and in the mesenteric and peripheral lymph nodes of BALB/c nu/nu mice in comparison with normal mice (+/+). In the spleens of nude mice, the predominant CD3+ T cell subpopulation was DN. Further, in mesenteric and peripheral lymph nodes, approximately one-third and one-half of the CD3+ T cells were double negative, respectively. In contrast, CD3+, DN T cells represent a small subpopulation in normal (+/+) mice. We next showed that functional regulatory T cells which possess the ability to abrogate oral tolerance were induced in nu/nu mice by Ag priming. BALB/c nude mice were immunized with SRBC, and the splenic CD3+, Vicia villosa-adherent cells were obtained by panning. Adoptive transfer of CD3+, V. villosa-adherent T cells to orally tolerant BALB/c mice restored responsiveness to SRBC, whereas V. villosa nonadherent cells were without effect. In other experiments, CD3+ T cells from the spleens of SRBC-primed mice were further enriched for the CD5+, DN phenotype and adoptive transfer of this subset completely abrogated oral tolerance to SRBC. To characterize the nature of the TCR expressed on these CD3+, DN T cells, we developed a rabbit antibody to a synthetic peptide (residues 209-218: Tyr-Ala-Asn-Ser-Phe-Asn-Asn-Glu-Lys-Leu) which was synthesized from a deduced sequence of the murine delta-gene. Immunoprecipitation of a cell membrane fraction from CD3+, DN T cells with anti-delta TCR antibody isolated a 45-kDa band. Furthermore, immunoprecipitation of these cells with anti-CD3 (145-2C11) revealed bands at 45 and 35 kDa (corresponding to delta- and gamma-chains, respectively). Taken together, these results are the first to show that gamma delta-TCR bearing CD3+, CD4-, and CD8- T cells are functional and reverse oral tolerance when adoptively transferred.     

Cutting edge: intravenous soluble antigen is presented to CD4 T cells by CD8- dendritic cells, but cross-presented to CD8 T cells by CD8+ dendritic cells

Mouse spleen contains three distinct mature dendritic cell (DC) populations (CD4(+)8(-), CD4(-)8(-), and CD4(-)8(+)) which retain a capacity to take up particulate and soluble AGS: Although the three splenic DC subtypes showed similar uptake of injected soluble OVA, they differed markedly in their capacity to present this Ag and activate proliferation in OVA-specific CD4 or CD8 T cells. For class II MHC-restricted presentation to CD4 T cells, the CD8(-) DC subtypes were more efficient, but for class I MHC-restricted presentation to CD8 T cells, the CD8(+) DC subtype was far more effective. This differential persisted when the DC were activated with LPS. The CD8(+) DC are therefore specialized for in vivo cross-presentation of exogenous soluble Ags into the class I MHC presentation pathway.     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

Abstract. The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry.
Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lumphocytes on the basis of their nucleolar antigen content.     

IL-2 upregulates CD86 expression on human CD4(+) and CD8(+) T cells

The glycoprotein CD86 is an important costimulatory molecule that has been shown to be predominantly expressed on APCs, such as dendritic cells, macrophages, and B cells. More recently, CD86 was also detected on T cells in specific pathological conditions. The mechanisms of how CD86 might be induced and its functional role in T cells are not well understood. In the present study, we showed that treatment with IL-2 markedly upregulated CD86, but not CD80, in human CD4(+) and CD8(+) T cells. This upregulation occurred in the absence of bystander cells, and isolated naive CD4(+) or CD8(+) T cells exhibited different time-dependent CD86-expression patterns in response to IL-2. Upregulation of CD86 on activated T cells was reduced by Abs that block IL-2 and IL-2Rα (CD25), indicating a receptor-mediated mechanism. IL-2-dependent CD86 upregulation was blocked by pharmacological inhibitors of the NFAT and mammalian target of rapamycin pathways and was largely reduced by simultaneous exposure to IFN-α. Importantly, a marked increase in CD86 on T cells was also observed in vivo in IL-2-treated patients. In conclusion, IL-2 upregulates CD86 expression on human CD4(+) and CD8(+) T cells via a receptor-dependent mechanism that involves the NFAT and mammalian target of rapamycin pathways.     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry. Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lymphocytes on the basis of their nucleolar antigen content.