Cell surface antigens on rat neural progenitors and characterization of the CD3 (+)/CD3 (-) cell populations

While the hematopoietic lineage has been extensively studied using cluster of differentiation (CD) antibodies, very few data are available on the extracellular epitopes expressed by rat neural progenitors (rNPC) and their derivatives. In the present study, we used flow cytometry to screen 47 cell surface antigens, initially known as immune markers. The quantitative analyses were performed on rat neurospheres and compared with primary cultures of astroglial cells or cerebellar neurons. Several antigens such as CD80 or CD86 were clearly undetectable while others, like CD26 or CD161, showed a weak expression. Interestingly, 10% and 15% of the cells were immunopositive for CD172a and CD200, two immunoglobulin superfamily members preferentially expressed by glial or neuronal cells, respectively. Over 40% of the cells were immunopositive for CD3, CD71, or MHCI. The biological significance of the latter markers in rNPC remains to be determined but analyses of the CD3(-)/CD3(+) populations isolated by magnetic cell separation revealed differences in their cell fate. Indeed, CD3(+) cells did not establish neurospheres and differentiated mostly into GFAP(+) cells while CD3(-) cells were able to generate neurospheres upon mitogen treatment and gave rise to GFAP(+), A2B5(+), Tuj-1(+), and RIP(+) cells under differentiating conditions. In contrast, CD71(-)/CD71(+) cells did not show any significant difference in their proliferating and differentiating potentials. Finally, it is worth noting that an subpopulation of cells in rat neurospheres exhibit an immunoreactivity against anti-CD25 (IL2 receptor) and anti-CD62L (L-selectin) antibodies. The results reveal particular surface antigen profiles, giving new perspectives on the properties of rat brain-derived cells.     

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.     

Prognostic value of the immunological phenomena and relationship with clinicopathological characteristics of the tumor--the expression of the early CD69+, CD71+ and the late CD25+, CD26+, HLA/DR+ activation markers on T CD4+ and CD8+ lymphocytes in squamous cell laryngeal carcinoma. Part II

One of the most important challenges in contemporary oncology is to find objective biomarkers of tumor aggressiveness, which help to identify more invasive phenotypes of the carcinoma. The purpose of this study was to investigate the relationships between the early and the late activation markers expression on T CD4(+) and CD8(+) cells subpopulations and certain clinicopathological characteristics of the neoplastic infiltration in order to determine their role as biomarkers for tumor behavior in squamous cell laryngeal carcinoma. Analysis of the early (CD69(+), CD71(+)) and the late activation antigens (CD25(+) (high), CD26(+), HLA/DR(+)) expression on T CD4+ and CD8(+) lymphocytes by cytofluorymetry in 55 patients treated for squamous cell laryngeal carcinoma was performed. Clinicomorphological analysis on the basis of TNM criteria and tumor front grading, which included tumor-related features and adjacent stroma-related characteristics of the peripheral edge of infiltration was carried out. The relationships between the activation markers expression and parameters of tumor aggressiveness were investigated. Our work revealed statistically significant differences in the expression of the studied activation markers on T cells with regard to certain clinicomorphological features. The expressions of CD69(+) and CD71(+) antigens on T CD3(+)CD4(+) and CD3(+)CD8(+) cells as well as CD4(+)HLA/DR(+) markers were higher for pT3 and pT4 tumors, in comparison with pT2 carcinomas. Moreover, tumors with the smallest number of TFG points were characterized by significantly lower values of the average expression of CD3(+)CD69(+) and CD3(+)CD71(+) as well as CD4(+)HLA/DR(+) markers on T lymphocytes. In addition, more aggressive and deeply infiltrating laryngeal carcinomas were most often characterized by significantly higher values of the average expression of CD69(+) and CD71(+) antigens on CD8(+) as well as HLA/DR(+) markers on CD4(+). Our study confirmed the implication of the early and the late activation antigens expression on CD4(+) and CD8(+) T lymphocytes in clinicomorphological parameters of the tumor, especially TFG total score and depth of invasion, and their importance as indicators of the invasive phenotype of laryngeal carcinoma.     

A new dynamic model of CD8+ T effector cell responses via CD4+ T helper-antigen-presenting cells

A long-standing paradox in cellular immunology has been the conditional requirement for CD4(+) Th cells in priming of CD8(+) CTL responses. We propose a new dynamic model of CD4(+) Th cells in priming of Th-dependent CD8(+) CTL responses. We demonstrate that OT II CD4(+) T cells activated by OVA-pulsed dendritic cells (DC(OVA)) are Th1 phenotype. They acquire the immune synapse-composed MHC II/OVAII peptide complexes and costimulatory molecules (CD54 and CD80) as well as the bystander MHC class I/OVAI peptide complexes from the DC(OVA) by DC(OVA) stimulation and thus also the potential to act themselves as APCs. These CD4(+) Th-APCs stimulate naive OT I CD8(+) T cell proliferation through signal 1 (MHC I/OVAI/TCR) and signal 2 (e.g., CD54/LFA-1 and CD80/CD28) interactions and IL-2 help. In vivo, they stimulate CD8(+) T cell proliferation and differentiation into CTLs and induce effective OVA-specific antitumor immunity. Taken together, this study demonstrates that CD4(+) Th cells carrying acquired DC Ag-presenting machinery can, by themselves, efficiently stimulate CTL responses. These results have substantial implications for research in antitumor and other aspects of immunity.     

MDR1 gene transfer using a lentiviral SIN vector confers radioprotection to human CD34+ hematopoietic progenitor cells

Tumor radiotherapy with large-field irradiation results in an increase in apoptosis of the radiosensitive hematopoietic stem cells (CD34(+)). The aim of this study was to demonstrate the radioprotective potential of MDR1 overexpression in human CD34(+) cells using a lentiviral self-inactivating vector. Transduced human undifferentiated CD34(+) cells were irradiated with 0-8 Gy and held in liquid culture under myeloid-specific maturation conditions. After 12 days, MDR1 expression was determined by the rhodamine efflux assay. The proportion of MDR1-positive cells in cells from four human donors increased with increasing radiation dose (up to a 14-fold increase at 8 Gy). Determination of expression of myeloid-specific surface marker proteins revealed that myeloid differentiation was not affected by transduction and MDR1 overexpression. Irradiation after myeloid differentiation also led to an increase of MDR1-positive cells with escalating radiation doses (e.g. 12.5-16% from 0-8 Gy). Most importantly, fractionated irradiation (3 x 2 Gy; 24-h intervals) of MDR1-transduced CD34(+) cells resulted in an increase in MDR1-positive cells (e.g. 3-8% from 0-3 x 2 Gy). Our results clearly support a radioprotective effect of lentiviral MDR1 overexpression in human CD34(+) cells. Thus enhancing repopulation by surviving stem cells may increase the radiation tolerance of the hematopoietic system, which will contribute to widening the therapeutic index in radiotherapy.     

Expression of CD24 on CD19- CD79a+ early B-cell progenitors in human bone marrow

CD24 is a surface marker expressed in immature and mature B cells and involved in cellular adhesion and apoptosis. There are no data, which delineate the stage in early development of human B cells, which marks the expression of CD24. We studied lymphopoiesis in normal pediatric bone marrow (BM) and found that 1.5+/-0.2% of WBC were CD24(+) lymphocytes which did not express CD19. A significant fraction of these cells expressed low levels of CD45 (CD19- CD24+ CD45low cells). Small numbers of CD19- CD24+ CD45low cells were found in the regenerating BM of children with acute lymphoblastic leukemia after the completion of chemotherapy and in normal adult BM. Flow cytometric analyses have shown that CD19- CD24+ CD45low lymphocytes express CD10, CD34, CD79a, CD179a (VpreB), and TdT markers, i.e., displayed antigenic properties of early B-cell progenitors. Our data indicate that CD19- early B-cell progenitors in human BM express CD24, and that the expression of CD24 in human B-cell development precedes the expression of CD19.     

Chemokine-guided CD4+ T cell help enhances generation of IL-6RalphahighIL-7Ralpha high prememory CD8+ T cells

CD4(+) T cells promote effective CD8(+) T cell-mediated immunity, but the timing and mechanistic details of such help remain controversial. Furthermore, the extent to which innate stimuli act independently of help in enhancing CD8(+) T cell responses is also unresolved. Using a noninfectious vaccine model in immunocompetent mice, we show that even in the presence of innate stimuli, CD4(+) T cell help early after priming is required for generating an optimal pool of functional memory CD8(+) T cells. CD4(+) T cell help increased the size of a previously unreported population of IL-6Ralpha(high)IL-7Ralpha(high) prememory CD8(+) T cells shortly after priming that showed a survival advantage in vivo and contributed to the majority of functional memory CD8(+) T cells after the contraction phase. In accord with our recent demonstration of chemokine-guided recruitment of naive CD8(+) T cells to sites of CD4(+) T cell-dendritic cell interactions, the generation of IL-6Ralpha(high)IL-7Ralpha(high) prememory as well as functional memory CD8(+) T cells depended on the early postvaccination action of the inflammatory chemokines CCL3 and CCL4. Together, these findings support a model of CD8(+) T cell memory cell differentiation involving the delivery of key signals early in the priming process based on chemokine-guided attraction of naive CD8(+) T cells to sites of Ag-driven interactions between TLR-activated dendritic cells and CD4(+) T cells. They also reveal that elevated IL-6Ralpha expression by a subset of CD8(+) T cells represents an early imprint of CD4(+) T cell helper function that actively contributes to the survival of activated CD8(+) T cells.     

Differentiation of human CD8(+) T cells from a memory to memory/effector phenotype

Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA(-)CD8(+) T cells with different CD28/CD27 phenotypes showed that CD28(+)CD45RA(-)CD8(+) and CD27(+)CD45RA(-)CD8(+) T cells have characteristics of memory T cells, whereas CD28(-)CD45RA(-)CD8(+) and CD27(-)CD45RA(-)CD8(+) T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8(+) T cells into memory/effector CD8(+) T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8(+) T cells. Three subsets of CD45RA(-)CD8(+) T cells were observed in both total CD8(+) T cells and EBV- or HCMV-specific CD8(+) T cells: CD27(+)CD28(+), CD27(+)CD28(-), and CD27(-)CD28(-). A significant number of the CD27(-)CD28(+) subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8(+) T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-)-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). This was supported by the observation that the frequency of CCR5(+) cells and CCR7(+) cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27(+)CD28(+) memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5(-)CCR7(+), CCR5(+)CCR7(+), and CCR5(+)CCR7(-). These findings suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-) (CCR5(-)CCR7(+)-->CCR5(+)CCR7(+)-->CCR5(+)CCR7(-))-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). The presence of a CD27(-)CD28(+) subset with a CCR5(+)CCR7(-) phenotype implies a specialized role for this subset in the differentiation of CD8(+) T cells.     

Influence of pre-ovulatory insemination and early pregnancy on the distribution of CD2, CD4, CD8 and MHC class II expressing cells in the sow endometrium

The aim was to investigate the distribution of CD2(+), CD4(+) and CD8(+) lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium following pre-ovulatory insemination and during early pregnancy. Crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire) were inseminated once at 15-20 h before ovulation. The sows were slaughtered at 5-6h (group I, n=4) after AI or at 20-25 h (group II, n=4) and 70 h (group III, n=4) after ovulation, day 11 (group IV, day 1=first day of standing oestrus, n=3) and day 19 (group V, n=3). Uterine horns were flushed to control for the presence of spermatozoa and neutrophils (groups I-IV) and/or for recovery of oocytes and/or embryos (groups II-IV, control of pregnancy). Cryofixed mesometrial uterine samples were analysed by immunohistochemistry with an avidin-biotin-peroxidase method using monoclonal antibodies to lymphocyte subpopulations and MHC class II molecules. The surface (SE) and glandular (GE) epithelia as well as connective tissue layers in subepithelial (SL) and glandular (GL) areas were examined by light microscopy. Taking all groups and different tissue layers together, the most commonly observed positive cells were CD2(+) cells (P相似文献   

Peripheral blood mononuclear cells immunophenotyping in pulmonary tuberculosis patients before and after treatment

Tuberculosis (TB) is a lung disease caused by Mycobacterium tuberculosis. The interaction between the bacillus and the host may lead to a protective cellular immune response. In the present study, we propose the "in vitro" evaluation of this cellular immune response in patients with tuberculosis before and after chemotherapic treatment. Eleven patients with TB and 9 asymptomatic subjects with tuberculin skin test negative (TST-) (purified protein derivative (PPD) 相似文献   

Relationships among murine CD11c(high) dendritic cell subsets as revealed by baseline gene expression patterns

The functional relationships and properties of different subtypes of dendritic cells (DC) remain largely undefined. To better characterize these cells, we used global gene analysis to determine gene expression patterns among murine CD11c(high) DC subsets. CD4(+), CD8alpha(+), and CD8alpha(-) CD4(-) (double negative (DN)) DC were purified from spleens of normal C57/BL6 mice and analyzed using Affymetrix microarrays. The CD4(+) and CD8alpha(+) DC subsets showed distinct basal expression profiles differing by >200 individual genes. These included known DC subset markers as well as previously unrecognized, differentially expressed CD Ags such as CD1d, CD5, CD22, and CD72. Flow cytometric analysis confirmed differential expression in nine of nine cases, thereby validating the microarray analysis. Interestingly, the microarray expression profiles for DN cells strongly resembled those of CD4(+) DC, differing from them by <25 genes. This suggests that CD4(+) and DN DC are closely related phylogenetically, whereas CD8alpha(+) DC represent a more distant lineage, supporting the historical distinction between CD8alpha(+) and CD8alpha(-) DC. However, staining patterns revealed that in contrast to CD4(+) DC, the DN subset is heterogeneous and comprises at least two subpopulations. Gene Ontology and literature mining analyses of genes expressed differentially among DC subsets indicated strong associations with immune response parameters as well as cell differentiation and signaling. Such associations offer clues to possible unique functions of the CD11c(high) DC subsets that to date have been difficult to define as rigid distinctions.     

Loss of CD127 expression defines an expansion of effector CD8+ T cells in HIV-infected individuals

The immunodeficiency that follows HIV infection is related to the virus-mediated killing of infected CD4(+) T cells, the chronic activation of the immune system, and the impairment of T cell production. In this study we show that in HIV-infected individuals the loss of IL-7R (CD127) expression defines the expansion of a subset of CD8(+) T cells, specific for HIV as well as other Ags, that show phenotypic (i.e., loss of CCR7 and CD62 ligand expression with enrichment in activated and/or proliferating cells) as well as functional (i.e., production of IFN-gamma, but not IL-2, decreased ex vivo proliferative potential and increased susceptibility to apoptosis) features of effector T cells. Importantly, in HIV-infected individuals the levels of CD8(+)CD127(-) T cells are directly correlated with the main markers of disease progression (i.e., plasma viremia and CD4(+) T cell depletion) as well as with the indices of overall T cell activation. In all, these results identify the expansion of CD8(+)CD127(-) effector-like T cells as a novel feature of the HIV-associated immune perturbation. Further studies are thus warranted to determine whether measurements of CD127 expression on CD8(+) T cells may be useful in the clinical management of HIV-infected individuals.     

A critical precursor frequency of donor-reactive CD4+ T cell help is required for CD8+ T cell-mediated CD28/CD154-independent rejection

Ag-specific precursor frequency is increasingly being appreciated as an important factor in determining the kinetics, magnitude, and degree of differentiation of T cell responses, and recently was found to play a critical role in determining the relative requirement of CD8(+) T cells for CD28- and CD154-mediated costimulatory signals during transplantation. We addressed the possibility that variations in CD4(+) T cell precursor frequency following transplantation might affect CD4(+) T cell proliferation, effector function, and provision of help for donor-reactive B cell and CD8(+) T cell responses. Using a transgenic model system wherein increasing frequencies of donor-reactive CD4(+) T cells were transferred into skin graft recipients, we observed that a critical CD4(+) T cell threshold precursor frequency was necessary to provide help following blockade of the CD28 and CD154 costimulatory pathways, as measured by increased B cell and CD8(+) T cell responses and precipitation of graft rejection. In contrast to high-frequency CD8(+) T cell responses, this effect was observed even though the proliferative and cytokine responses of Ag-specific CD4(+) T cells were inhibited. Thus, we conclude that an initial high frequency of donor-reactive CD4(+) T cells uncouples T cell proliferative and effector cytokine production from the provision of T cell help.     

BM cells giving rise to MSC in culture have a heterogeneous CD34 and CD45 phenotype

BACKGROUND: Mesenchymal stromal cells (MSC) isolated from adult human BM are characterized by their fibroblast-like morphology, adherent growth and capacity to differentiate into adipocytes, osteocytes, chondrocytes, cardiomyocytes and neuroprogenitors. After culturing these cells in vitro, they express the cell-surface molecules CD44, CD90, SH2 and SH3, and are negative for CD34 and the hematopoietic marker CD45. The aim of this study was to characterize the in vivo phenotype of MSC relative to the expression of CD34 and CD45. METHODS: BM mononuclear cells were stained with Ab against both molecules and separated into the CD34(+), CD34(-), CD45(+) CD34(+), CD45(high+) CD34(-), CD45(med,low+) CD34(-) and CD45(-) CD34(-) subpopulations, which were then cultured under the same conditions and analyzed for growth of MSC. RESULTS: A small population of MSC arose from the CD45(+) CD34(+) fraction, although the majority was obtained from the CD45(-) CD34(-) subpopulation. MSC from all fractions could be differentiated into adipocytes and osteocytes. In addition, MSC from the CD34(+) and CD34(-) fractions were shown to differentiate into chondrocytes. After in vitro culture, MSC from both fractions possessed the same phenotype, which was negative for CD34 and CD45. DISCUSSION: MSC from the CD45(+) CD34(+) fraction change their phenotype under in vitro conditions.     

Preferential proliferation and differentiation of double-positive thymocytes into CD8(+) single-positive thymocytes in a novel cell culture medium

The identification of factors that regulate the proliferation and differentiation of double-positive (DP) into CD4(+) and CD8(+) single-positive (SP) thymocytes has proven difficult due to the inability of DP thymocytes to proliferate, expand, and differentiate into SP thymocytes in available cell culture media. Here we report on the ability of DP thymocytes to differentiate in a novel conditioned medium, termed XLCM, derived from the supernatant of mitogen activated human cord blood mononuclear cells. During a 5-day culture in XLCM in the absence of thymic stromal cells, DP thymocytes from normal mice and MHC double knockout mice (lack SP thymocytes) proliferate, expand, and differentiate into several (alphabetaTCR(+), NK1.1(+)alphabetaTCR(+), and gammadeltaTCR(+)) subsets of CD4(+) and predominantly CD8(+) SP thymocytes. These studies suggest that the use of XLCM may aid in the characterization of factors that regulate the differentiation of DP thymocytes into CD8(+) SP thymocytes.     

CD30/CD30 ligand (CD153) interaction regulates CD4+ T cell-mediated graft-versus-host disease

CD30, a TNFR family member, is expressed on activated CD4(+) and CD8(+) T cells and B cells and is a marker of Hodgkin's lymphoma; its ligand, CD30L (CD153) is expressed by activated CD4(+) and CD8(+) T cells, B cells, and macrophages. Signaling via CD30 can lead to proliferation or cell death. CD30-deficient (-/-) mice have impaired thymic negative selection and increased autoreactivity. Although human alloreactive T cells preferentially reside within the CD30(+) T cell subset, implicating CD30 as a regulator of T cell immune responses, the role of CD30/CD153 in regulating graft-vs-host disease (GVHD) has not been reported. We used a neutralizing anti-CD153 mAb, CD30(-/-) donor mice, and generated CD153(-/-) recipient mice to analyze the effect of CD30/CD153 interaction on GVHD induction. Our data indicate that the CD30/CD153 pathway is a potent regulator of CD4(+), but not CD8(+), T cell-mediated GVHD. Although blocking CD30/CD153 interactions in vivo did not affect alloreactive CD4(+) T cell proliferation or apoptosis, a substantial reduction in donor CD4(+) T cell migration into the gastrointestinal tract was readily observed with lesser effects in other GVHD target organs. Blockade of the CD30/CD153 pathway represents a new approach for preventing CD4(+) T cell-mediated GVHD.     

Lymphocyte subpopulations in pulmonary tuberculosis patients

Protection against Mycobacterium tuberculosis is based on cell-mediated immunity, most importantly involving CD4(+) and CD8(+) T-cell subsets. The aim of this study was to evaluate CD4(+) and CD8(+) T-cell profiles and CD19(+) and CD3(+)CD(16 + 56)(+) populations in patients with pulmonary tuberculosis. CD4(+) and CD8(+) T cells, B-lymphocytes, and natural killer (NK) cells were evaluated in 75 active (APTB) and 25 inactive (IPTB) pulmonary tuberculosis cases and 20 healthy subjects (HCs). The results were compared at different stages of antituberculosis treatment in the APTB patients and also according to X-ray findings in the newly diagnosed APTB patients. The percentages of CD4(+) T cells were significantly lower (P < .01) and those of CD3(+)CD(16+56)(+) cells were significantly higher (P < .01) in APTB patients than in HCs. CD8(+) T cells were significantly decreased (P < .05), and CD3(-)CD(16+56)(+) cells were significantly increased (P < .01), in IPTB patients compared to HCs. The percentages of CD4(+), CD8(+), CD3(-)CD19(+), and CD3(-)CD(16+56)(+) cells showed no differences at different times of the antituberculosis regimen, and different stages of newly diagnosed APTB patients. APTB patients have a reduced percentage of circulating CD4(+) T cells and an increased percentage of NK cells compared with healthy individuals. These cells could play important roles in the immune response to M tuberculosis infection.     

CD4(+) T cell levels are decreased during active CMV infection in kidney transplant recipients

The numbers of T lymphocytes and T cell subsets (CD2(+), CD3(+), CD4(+), CD8(+)), activated T cells (CD26(+)), B cells (CD19(+)), granulocytes (CD15(+)) and natural killer cells (CD16/56) were monitored by flow cytometry in 79 kidney transplant recipients, 35 of whom had cytomegalovirus infection. The percentages of these cells were correlated with viral load, as determined by cytomegalovirus antigenemia. Development of cytomegaloviral infection coincided with a significant reduction in the percentages of CD4(+) (P < 0.005) and CD3(+) (P < 0.05) cells. Monitoring of lymphocyte subsets may provide useful information on immunological events during cytomegaloviral infection.     

CD133 and CD44 cell surface markers do not identify cancer stem cells in primary human gastric tumors

Emerging evidence suggests that tumors contain and are driven by a cellular component that displays stem cell properties, the so-called cancer stem cells (CSCs). CSCs have been identified in several solid human cancers; however, there are no data about CSCs in primary human gastric cancer (GC). By using CD133 and CD44 cell surface markers we investigated whether primary human GCs contain a cell subset expressing stem-like properties and whether this subpopulation has tumor-initiating properties in xenograft transplantation experiments. We examined tissues from 44 patients who underwent gastrectomy for primary GC. The tumorigenicity of the cells separated by flow cytometry using CD133 and CD44 surface markers was tested by subcutaneous or intraperitoneum injection in NOD/SCID and nude mice. GCs included in the study were intestinal in 34 cases and diffuse in 10 cases. All samples contained surface marker-positive cells: CD133(+) mean percentage 10.6% and CD133(+)/CD44(+) mean percentage 27.7%, irrespective of cancer phenotype or grade of differentiation. Purified CD133(+) and CD133(+)/CD44(+) cells, obtained in sufficient number only in 12 intestinal type GC cases, failed to reproduce cancer in two mice models. However, the unseparated cells produced glandular-like structures in 70% of the mice inoculated. In conclusion, although CD133(+) and CD133(+)/CD44(+) were detectable in human primary GCs, they neither expressed stem-like properties nor exhibited tumor-initiating properties in xenograft transplantation experiments.     

Human vascular endothelial cells stimulate a lower frequency of alloreactive CD8+ pre-CTL and induce less clonal expansion than matching B lymphoblastoid cells: development of a novel limiting dilution analysis method based on CFSE labeling of lymphocytes

We have previously shown that human endothelial cells (EC) are less efficient than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulating allogeneic CD8(+) T cells to develop into CTL. In this study we describe FACS-based limiting dilution analyses using the dilution of the intracellular dye CFSE as an indicator of CD8(+) T cell alloactivation and expansion with significantly increased sensitivity compared with conventional, cytotoxicity-based assays. In addition, this assay permits the relative size of clonal CTL populations that are generated in individual CD8(+) T cell cultures to be determined (clonal burst size). We have applied this method to quantitatively compare the generation of CTL at the clonal level following stimulation of allogeneic CD8(+) T cells by either BLC or HUVEC derived from the same donor. CD8(+) T cells expanded by allostimulation were identified as CD8(+), CFSE(low) cells and were categorized as CTL by the expression of intracellular perforin and IFN-gamma. Precursor frequencies for EC-stimulated CTL were 5- to 40-fold (mean, 7.5-fold) lower compared with BLC-stimulated CTL (p < 0.01). Concomitantly, the average clonal burst sizes in EC-stimulated CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes exclusively with BLC stimulation. Although EC-stimulated CTL were generated only from the memory subset of CD8(+) T cells, BLC-stimulated very large burst sizes of CTL were observed from both naive and memory CD8(+) T cell precursors. These data establish that both a lower frequency of reactive precursors and more limited clonal expansion, but not regulatory T cells, contribute to the reduced capacity of EC to promote alloreactive CTL differentiation compared with that of professional APC.