scholarly journals A peripheral blood-derived monolayer supports long-term cultures of human CD4+ and CD8+ T lymphocytes

Blood ◽  
1995 ◽  
Vol 85 (11) ◽  
pp. 3213-3222 ◽  
Author(s):  
N Sutkowski ◽  
ML Kuo ◽  
PS Amenta ◽  
JP Dougherty ◽  
Y Ron
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Retroviral Vector ◽  
Target Cells ◽  
Adherent Cells ◽  
Lacz Gene ◽  
Cd8 Cells

An in vitro culture system has been developed for the long-term maintenance of primary, human peripheral blood and umbilical cord blood T lymphocytes, which does not rely on the use of stimulatory cytokines, antigen, or mitogens. In these cultures, a monolayer of adherent cells, some spindle-shaped and some resembling macrophages, developed within a week. All adherent cells were positive for the extracellular matrix proteins laminin and fibronectin, the intermediate filament vimentin, and for the surface markers major histocompatibility complex class II, platelet-endothelial cell adhesion molecule l (CD31), and E-Selectin (ELAM-1; CD62E). They were negative for the leukocyte common antigen (CD45), the macrophage marker MO-2 (CD14), muscle-specific actin, and Factor VIII-related antigen. These monolayers supported the maintenance of nonadherent, resting, mature T cells for up to 3 months, and these cells retained their ability to respond to mitogens and allogeneic cells. Both CD4+ and CD8+ cells were supported. The proportion of CD4+ and CD8+ cells remained unchanged after 3 months in culture. We have also used T cells from 2-month-old cultures as target cells for retroviral vector-mediated gene transfer. Up to 30% of the long-term T cells expressed the transferred lacZ gene after infection with a retroviral vector. The infection efficiency was similar to that obtained for fresh peripheral blood T cells, indicating that the long-term-cultured cells might be suitable for certain gene therapy applications.

scholarly journals Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity.

1983 ◽  
Vol 157 (2) ◽  
pp. 743-754 ◽  
Author(s):  
A Moretta ◽  
G Pantaleo ◽  
L Moretta ◽  
J C Cerottini ◽  
M C Mingari
Keyword(s):  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Cell Populations ◽  
Target Cells ◽  
Flow Cytofluorometry ◽  
Hla Dr ◽  
Clonogenic Potential ◽  
Precursor Frequency

In an attempt to determine the clonogenic properties of human peripheral blood T cells, we have developed a limiting dilution microculture system using phytohemagglutinin (PHA) as T cell activator and supernatant from PHA-stimulated spleen cultures as a source of T cell growth factors. The frequencies of cells capable of extensive proliferation under these culture conditions were 0.52-0.73, 0.98-1.11, and less than 0.02 in peripheral blood mononuclear, E-rosette-positive, and E-rosette-negative cell populations, respectively. The clonogenic potential of virtually all T cells was confirmed in experiments using single cells isolated by micromanipulation. Clone size ranged between 5 and 30 X 10(4) cells on day 14 of culture. The same microculture system was used to determine the precursor frequency of all cytolytic T lymphocytes (CTL-P). As assessed by a lectin-dependent 51Cr release assay, the CTL-P frequency in purified T cell populations ranged between 0.30 and 0.34. In comparison, the precursor frequency of T cells capable of lysing K562 target cells was ranging between 0.14 and 0.16. Parallel analysis of individual clonal cultures for both lytic activities showed that 50% of the clones exhibiting lectin-dependent lysis were also active against K562 target cells. All of the proliferating clones expressed HLA-DR antigens, although to a varying degree as assessed by flow cytofluorometry. Given the high cloning efficiency of this culture system, it appears now possible to determine the precursor frequencies of the various classes of functional cells in T cell populations.

scholarly journals Peripheral blood lymphocyte receptors for B-lymphoblastoid cell lines (B-LCL)

Blood ◽  
1980 ◽  
Vol 56 (4) ◽  
pp. 690-695
Author(s):  
J Zighelboim ◽  
A Lichtenstein
Keyword(s):  
Immune Response ◽  
T Cells ◽  
B Cells ◽  
T Lymphocytes ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Wide Distribution ◽  
Lymphoblastoid Cell Lines ◽  
Surface Receptor

Because interactions between B cells and T lymphoyctes are of fundamental importance in the generation of the immune response to most antigens, we attempted to identify the cells capable of binding B- lymphoblastoid cell lines (B-LCL), their tissue distribution, and their presence in other species. Cells bearing a surface receptor for B-LCL were found in human peripheral blood, tonsil, and bone marrow, as well as mouse and rat spleen. Binding cells were phenotypically heterogeneous. The majority are T cells as defined by their ability to bind sheep red blood cells (E-rosettes). However, a subpopulation of non-T-lymphocytes were capable of binding B-LCL. This was demonstrated by depleting T cells with an E-rosette centrifugation technique and then performing a double rosette assay. The wide distribution of T lymphocytes with receptors for B-lymphoblastoid cells within peripheral lymphoid organs and their presence in several species suggest that these surface molecules may represent one of the means by which T cells and B cells interact in the induction of the immune response to T- dependent antigens.

scholarly journals Constitutive expression of pore-forming protein in peripheral blood gamma/delta T cells: implication for their cytotoxic role in vivo.

1990 ◽  
Vol 172 (6) ◽  
pp. 1877-1880 ◽  
Author(s):  
M Nakata ◽  
M J Smyth ◽  
Y Norihisa ◽  
A Kawasaki ◽  
Y Shinkai ◽  
...  
Keyword(s):  
T Cells ◽  
Cytotoxic Activity ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Constitutive Expression ◽  
Immunocytochemical Staining ◽  
Target Cells ◽  
Gamma Delta T Cells ◽  
Gamma Delta

The cytotoxic activity and pore-forming protein (PFP) expression of human peripheral blood (PB) gamma/delta T cells were examined. Fresh gamma/delta T cells isolated from PB lymphocytes by fluorescence-activated cell sorting exhibited a substantial natural killer-like cytotoxic activity against K562 target cells and had a high cytotoxic potential triggered by anti-CD3 monoclonal antibody (mAb) against P815 target cells bearing Fc gamma R. Immunocytochemical staining with an anti-PFP mAb revealed that virtually all PB gamma/delta T cells are granular lymphocytes with abundant PFP in their cytoplasmic granules. Constitutive expression of PFP in PB gamma/delta T cells was also demonstrated by Northern blot analysis. These observations support the proposed role of gamma/delta T cells in cytolytic immune surveillance in vivo.

scholarly journals Induction of Phosphodiesterases 3B, 4A4, 4D1, 4D2, and 4D3 in Jurkat T-cells and in Human Peripheral Blood T-lymphocytes by 8-Bromo-cAMP and Gs-coupled Receptor Agonists

1998 ◽  
Vol 273 (32) ◽  
pp. 20575-20588 ◽  
Author(s):  
Joachim Seybold ◽  
Robert Newton ◽  
Lyndon Wright ◽  
Paul A. Finney ◽  
Norbert Suttorp ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Jurkat T Cells ◽  
Receptor Agonists

scholarly journals Generation and characterization of ex vivo propagated autologous CD8+ cells used for adoptive immunotherapy of patients infected with human immunodeficiency virus

Blood ◽  
1993 ◽  
Vol 81 (8) ◽  
pp. 2085-2092 ◽  
Author(s):  
TL Whiteside ◽  
EM Elder ◽  
D Moody ◽  
J Armstrong ◽  
M Ho ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Lymphocytes ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Specific Activity ◽  
Interleukin 2 ◽  
Cd8 Cells ◽  
Immunodeficiency Virus ◽  
Anti Hiv

Abstract Cytolytic T lymphocytes play an important role in host defense against viral infections, including human immunodeficiency virus (HIV). In a phase I clinical trial (protocol 080 of the AIDS Clinical Trials Group), generation of CD8+ effector cells from peripheral blood of patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC) or AIDS and safety of autologous adoptive transfer of these cells were evaluated. For therapeutic infusions, CD8+ T cells were purified by positive selection on anti-CD8 monoclonal antibody-coated flasks from leukapheresed peripheral blood of seven patients. These CD8+ T cells were cultured in the presence of interleukin-2 and phytohemagglutinin for up to 3 weeks to obtain cells sufficient for therapeutic infusions (10(8) to 10(10)). All 31 cell cultures established from the seven patients and used for therapy were highly enriched in CD8+ (mean, 97%), CD8+HLA-DR+ (50%), cytotoxic CD8+CD11b- (82%), and memory CD29+ (78%) T lymphocytes. In vitro expanded CD8+ cells had excellent cytotoxic function at the time they were used for therapy, including HIV-specific activity against autologous targets infected with vaccinia vectors expressing HIV-IIIb antigens, gag, pol, and env. Anti-HIV activity of cultured CD8+ cells was significantly higher than that of autologous fresh peripheral blood lymphocytes. Our results show that CD8+ T lymphocytes obtained from peripheral blood of symptomatic HIV-infected patients can be purified, cultured to obtain large numbers of cells with enhanced anti-HIV activity, and safely infused into patients with AIDS as a form of immunotherapy.

scholarly journals Specific targeting of human peripheral blood T cells by heteroaggregates containing anti-T3 crosslinked to anti-target cell antibodies.

1986 ◽  
Vol 163 (1) ◽  
pp. 166-178 ◽  
Author(s):  
P Perez ◽  
R W Hoffman ◽  
J A Titus ◽  
D M Segal
Keyword(s):  
T Cells ◽  
Target Cell ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Target Cells ◽  
Ifn Gamma ◽  
Effector Cells ◽  
Infected Cells ◽  
Covalently Linked

Antibody heteroaggregates have been used to render human peripheral blood T cells lytic for specified targets. The heteroaggregates contain anti-T3 covalently linked to antibodies against nominal target cell antigens. Such heteroaggregates bind target cells directly to T3 molecules on effector cells and trigger target cell lysis. Freshly prepared human PBL, when coated with anti-T3-containing heteroaggregates, are lytic without further stimulation, although brief exposure to crude lymphokine-containing supernatants or recombinant IL-2, but not recombinant IFN-gamma, enhances the activity. The effector cells are T8+, and when fully stimulated, their lytic activity approaches that of some cloned CTL. When T cells are treated with heteroaggregate, washed, and incubated at 37 degrees C in medium not containing heteroaggregate, they retain activity for at least 24 h. The results of this study suggest a strategy in which heteroaggregate-coated T cells could be used in vivo to mount a lytic response against pathogenic cells such as tumor cells or virus-infected cells.

HLA-A*0201 Restricted Killing Activity of WT1-Specific CTL Generated From the Peripheral Blood Lymphocytes of Normal Healthy Donors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3026-3026
Author(s):  
Deepa Kolaseri Krishnadas ◽  
Mindy Stamer ◽  
Kim Dunham ◽  
Lei Bao ◽  
Kenneth Lucas
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Peripheral Blood Lymphocytes ◽  
Target Cells ◽  
Cd8 Cells ◽  
Blood Lymphocytes ◽  
Healthy Donors ◽  
Ifn Γ

Abstract Abstract 3026 Poster Board II-1002 The Wilms' tumor antigen (WT1) is over-expressed on several human leukemia and solid tumors, and thus is considered as a potential target for cancer immunotherapy. Combating leukemia by targeting WT1 expressing leukemic cells using in vitro generated WT1-specific CTL is one potential approach, but it is difficult to generate an immune response against WT1 due to low T cell precursor frequency in normal healthy individuals. Earlier studies have shown the generation of WT1-A*0201 peptide specific CTL from CD8+ T cells by cloning. Another study reported the production of IFN- γ by WT-1 specific CD8+ T cells. However, the cytolytic killing ability of these IFN- γ producing cells was not further characterized. Here, we demonstrate the generation of WT1-A*0201 specific CTL from the peripheral blood lymphocytes (PBL) of normal healthy donors using CD137 selection. The PBL were stimulated once with RMFPNAPYL (WT1-A*0201 peptide) pulsed autologous dendritic cells and twice with WT1-A*0201 peptide pulsed irradiated peripheral blood mononuclear cells (PBMC). Following three stimulations, the PBL were selected for CD137+ expression and rapidly expanded with OKT3 and IL-2. The WT1-A*0201 specific CTL showed killing of target cells and production of IFN-γ in an antigen-specific manner. The percent killing of WT1-A*0201 peptide pulsed T2 cells (TAP−, HLA- A2+) and autologous B blast (BB) were significantly higher when compared with their control targets. T2 cells and BB either pulsed with an irrelevant A*0201 peptide or un-pulsed served as the control. We have observed similar results with WT1-A*0201 specific CTL generated from normal donor CD8+ cells. However, the efficiency of WT1-A*0201 CTL generated from PBL to kill target cells and produce IFN- γ was higher than CTL from CD8+ cells. The CTL generated from PBL killed BA25, a WT1 expressing A2+ leukemia cell line but failed to kill Molt-4, a WT1 expressing A2− cell line, clearly indicating HLA-A2 restricted CTL activity. The specificity of the generated CTL were further confirmed by staining with WT1-HLA-A*0201 tetramer. The percentage of WT1-specific CD3+CD8+Tetramer+ cells either remained same or higher in CTL generated from PBL when compared with those generated from CD8+ cells. CD137 selection leads to the generation of significant number of CTL in a shorter time when compared to conventional cloning methods. In addition, generation of WT1-A*0201 specific CTL from PBL avoids CD8+ selection. Currently, we are aiming to generate WT1-specific CTL using an overlapping WT1 peptide-mix in order to widen our ability to treat patients with different HLA types. This study has implications for cellular immunotherapy in leukemia patients who relapse following allogeneic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Peripheral blood lymphocyte receptors for B-lymphoblastoid cell lines (B-LCL)

Blood ◽  
1980 ◽  
Vol 56 (4) ◽  
pp. 690-695 ◽  
Author(s):  
J Zighelboim ◽  
A Lichtenstein
Keyword(s):  
Immune Response ◽  
T Cells ◽  
B Cells ◽  
T Lymphocytes ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Wide Distribution ◽  
Lymphoblastoid Cell Lines ◽  
Surface Receptor

Abstract Because interactions between B cells and T lymphoyctes are of fundamental importance in the generation of the immune response to most antigens, we attempted to identify the cells capable of binding B- lymphoblastoid cell lines (B-LCL), their tissue distribution, and their presence in other species. Cells bearing a surface receptor for B-LCL were found in human peripheral blood, tonsil, and bone marrow, as well as mouse and rat spleen. Binding cells were phenotypically heterogeneous. The majority are T cells as defined by their ability to bind sheep red blood cells (E-rosettes). However, a subpopulation of non-T-lymphocytes were capable of binding B-LCL. This was demonstrated by depleting T cells with an E-rosette centrifugation technique and then performing a double rosette assay. The wide distribution of T lymphocytes with receptors for B-lymphoblastoid cells within peripheral lymphoid organs and their presence in several species suggest that these surface molecules may represent one of the means by which T cells and B cells interact in the induction of the immune response to T- dependent antigens.

scholarly journals Inhibition of human immunodeficiency virus (HIV-1/HTLV-IIIBa-L) replication in fresh and cultured human peripheral blood monocytes/macrophages by azidothymidine and related 2',3'-dideoxynucleosides.

1988 ◽  
Vol 168 (3) ◽  
pp. 1111-1125 ◽  
Author(s):  
C F Perno ◽  
R Yarchoan ◽  
D A Cooney ◽  
N R Hartman ◽  
S Gartner ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Target Cells ◽  
Blood Monocytes ◽  
Deoxynucleoside Triphosphate ◽  
Dideoxynucleoside Triphosphate ◽  
Hiv 1

Because of the probable role of HIV-infected monocyte/macrophages in the pathogenesis and progression of AIDS, it is essential that antiretroviral therapy address viral replication in cells of this lineage. Several dideoxynucleosides have been shown to have potent in vitro and, in the case of 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC), in vivo activity against HIV. However, because these compounds must be phosphorylated (activated) in target cells, and because monocyte/macrophages may have levels of kinases that differ from those in lymphocytes, we investigated the capacity of these drugs to suppress HIV replication in monocyte/macrophages using HIV-1/HTLV-IIIBa-L (a monocytotropic isolate). In the present study, we observed that HTLV-IIIBa-L replication in fresh human peripheral blood monocyte/macrophages was suppressed by each of three dideoxynucleosides: 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-dideoxycytidine (ddC), and 2',3'-dideoxyadenosine (ddA). Similar results were observed in 5-d-cultured monocyte/macrophages, although higher concentrations of the drugs were required. We then studied the metabolism of AZT and ddC in such cells. The phosphorylation of ddC to a triphosphate moiety was somewhat decreased in monocyte/macrophages as compared with H9 T cells. On the other hand, the phosphorylation of AZT in monocyte/macrophages was markedly decreased to 25% or less of the level in T cells. However, when we examined the level of the normal endogenous 2'-deoxynucleoside triphosphate pools, which compete with 2',3'-dideoxynucleoside triphosphate for viral reverse transcriptase, we found that the level of 2'-deoxycytidine-triphosphate (dCTP) was six- to eightfold reduced, and that of 2'-deoxythymidine-triphosphate (dTTP) was only a small fraction of that found in T cell lines. These results suggest that the ratio of dideoxynucleoside triphosphate to normal deoxynucleoside triphosphate is a crucial factor in determining the antiviral activity of dideoxynucleosides in HIV target cells, and that the lower levels of dTTP may account for the antiretroviral activity of AZT in the face of inefficient phosphorylation of this compound.

Sign in / Sign up

Export Citation Format

Share Document