scholarly journals GENERATION OF CYTOTOXIC T LYMPHOCYTES IN VITRO

1974 ◽  
Vol 140 (6) ◽  
pp. 1511-1521 ◽  
Author(s):  
H. Robson MacDonald ◽  
Jean-Charles Cerottini ◽  
K. Theodor Brunner
Keyword(s):  
T Lymphocytes ◽  
Physical Properties ◽  
Cell Lineage ◽  
Kinetic Studies ◽  
Large Cell ◽  
Velocity Sedimentation ◽  
Cell Fractionation ◽  
Anamnestic Response ◽  
Cell Fractions

Separation of cells by velocity sedimentation at unit gravity was utilized to investigate the physical properties of cytotoxic thymus-derived lymphocytes (CTL) generated in long-term mixed leukocyte cultures (MLC). In kinetic studies, CTL were found almost exclusively in the large cell fractions at the peak of the response on day 4, whereas the majority of CTL in day 14 MLC had the sedimentation properties of small lymphocytes. Reculture until day 14 of cells fractionated on the basis of size on day 4 indicated that the small CTL were derived exclusively from cells which had been large on day 4. Re-exposure of day 14 MLC cells to the original stimulating alloantigens resulted in significant cell proliferation and rapid regeneration of CTL activity. Cell fractionation experiments demonstrated that the cells in the day 14 MLC population which responded to the secondary allogeneic stimulus were small T lymphocytes, and that these cells rapidly developed into large, highly cytotoxic CTL following stimulation. Moreover, by restimulating on day 14 fractions which were selected on the basis of size on day 4, it was found that the responding small lymphocytes were themselves the progeny of cells which were large at the peak of the response. Since CTL and CTL progenitors showed concomitant changes in physical properties with time, the possibility exists that they belong to the same cell lineage, and hence that CTL can differentiate into cells which are no longer cytotoxic, but capable of mounting an anamnestic response.

scholarly journals BCL-6 expression during B-cell activation

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5257-5268 ◽  
Author(s):  
D Allman ◽  
A Jain ◽  
A Dent ◽  
RR Maile ◽  
T Selvaggi ◽  
...  
Keyword(s):  
B Cells ◽  
T Lymphocytes ◽  
Germinal Center ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Large Cell ◽  
Cd40 Ligand ◽  
Mrna Levels ◽  
Coding Region

Translocations involving the BCL-6 gene are common in the diffuse large cell subtype of non-Hodgkin's lymphoma. Invariably, the BCL-6 coding region is intact, but its 5′ untranslated region is replaced with sequences from the translocation partner. The present study shows that BCL-6 expression is regulated in lymphocytes during mitogenic stimulation. Resting B and T lymphocytes contain high levels of BCL-6 mRNA. Stimulation of mouse B cells with anti-IgM or IgD antibodies, bacterial lipopolysaccharide, phorbol 12-myristate 13-acetate plus ionomycin, or CD40 ligand led to a five-fold to 35-fold decrease in BCL-6 mRNA levels. Similar downregulation of BCL-6 mRNA was seen in human B cells stimulated with Staphylococcus aureus plus interleukin-2 or anti-IgM antibodies and in human T lymphocytes stimulated with phytohemagglutinin. BCL-6 mRNA levels began to decrease 8 to 16 hours after stimulation, before cells entered S phase. Although polyclonal activation of B cells in vitro invariably decreased BCL-6 MRNA expression, activated B cells from human germinal centers expressed BCL-6 mRNA at levels comparable to the levels in resting B cells. Despite these similar mRNA levels, BCL-6 protein expression was threefold to 34-fold higher in germinal center B cells than in resting B cells, suggesting that BCL-6 protein levels are controlled by translational or posttranslational mechanisms. These observations suggest that the germinal center reaction provides unique activation signals to B cells that allow for continued, high-level BCL-6 expression.

scholarly journals Induction of specific immune unresponsiveness using purified mixed leukocyte culture-activated T lymphoblasts as autoimmunogen. I. Demonstration of general validity as to species and histocompatibility barriers.

1977 ◽  
Vol 146 (4) ◽  
pp. 1124-1137 ◽  
Author(s):  
L C Andersson ◽  
M Aguet ◽  
E Wight ◽  
R Andersson ◽  
H Binz ◽  
...  
Keyword(s):  
Side Effects ◽  
T Lymphocytes ◽  
Guinea Pig ◽  
Third Party ◽  
Velocity Sedimentation ◽  
General Validity ◽  
Negative Side Effects ◽  
T Lymphoblasts ◽  
General Tool

Normal immunocompetent T lymphocytes can be induced into specific proliferation if confronted with the relevant alloantigen in vitro. Such mixed leuko-cyteculture-activated T lymphoblasts carring idiotypic receptors on their surface can be purified using velocity sedimentation and serve as immunogen if administered in adjuvant to the autologous host. Autoblast immunization can be shown to lead to specific, long-lasting unresponsiveness against the relevant alloantigens, while leaving reactivity against third-party antigens intact. When tested as to general validity, it could be shown to function in all species analyzed (mouse, rat, and guinea pig) as well as across both major and minor histocompatibility barriers. No negative side effects have been noted so far. It would thus seem clear that autoblast immunization using the above described scheme may serve as a general tool in inducing long-lasting, specific unresponsiveness in any species and across any histocompatibility barrier.

scholarly journals Novel Long Non Coding RNA Blackmamba Is Associated to ALK- anaplastic Large Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 461-461 ◽  
Author(s):  
Valentina Fragliasso ◽  
Akanksha Verma ◽  
Rohan Bareja ◽  
Tayla Heavican ◽  
Javeed Iqbal ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Board Of Directors ◽  
Cell Lines ◽  
Cellular Localization ◽  
Large Cell ◽  
Cell Fractionation ◽  
Rt Pcr ◽  
Advisory Committees ◽  
Neoplastic Phenotype ◽  
Non Coding Rna

Abstract Systemic Anaplastic Large Cell Lymphomas (ALCLs) is among the common subtypes of T-cell lymphomas. The prognosis of ALCLs patients has been proven to be associated with translocations of the anaplastic lymphoma kinase (ALK) gene, having ALK negative patients (ALK-ALCL) a poorer 5-years survival rate (30-40%). We now recognize that ALK- ALCL includes multiple subsets some of which are bear unique genetic defects and unique clinical outcomes. Long non coding RNAs (lncRNAs) are regulators of gene expression controlling critical processes (epigenetic regulation, imprinting, cell cycle, apoptosis) frequently deregulated in cancer. In T-cells, more than 500 lncRNAs are associated with lymphocyte signatures, control cell differentiation and cell identity. While lncRNAs are found to be differentially expressed in solid cancer and directly linked to the different stages of carcinogenesis, their role in lymphoid neoplasms is largely unknown. To identify novel lncRNAs maintaining the neoplastic phenotype of ALCL, we prepared cDNA libraries representative of coding and non-coding RNA. High coverage and directional RNA-sequencing was performed in 30 purified human T lymphocytes from multiple healthy donors, corresponding to different stages of differentiation, 22ALK+ ALCL and 20 ALK-ALCL patients' samples. Seven bona fide ALCL cell lines were included. By de novo transcriptome reconstruction and using a new bioinformatic pipeline, we identified 83lncRNAs exclusively expressed in ALCL patient's samples. Among them, 82 lncRNAs were coshared between ALK+ and ALK- ALCL and only one was exclusively expressed in ALK-ALCL samples. We named this new lncRNA BlackMamba. We selected the 10 top-scoring ALCL-associated lncRNAs and BlackMamba and determined their expression by RT-PCR, in a validation set of ALCL (ALK+: n=3, ALK-: n=3), T-ALL (n=8), PTCL (n=9), and AITL (n=9) samples. Resting and activated PBMCs from two additional donors were included. Our data confirmed that the 10 top-scoring ALCL-associated lncRNAs were expressed exclusively in ALCLs without differences in the expression among ALCL subtypes. Moreover, BlackMamba was detected only in ALCL samples with a preferential expression in ALK-ALCL samples. All lncRNAs were not expressed in normal T-lymphocytes. We next determine the BlackMamba expression in ALCL (n=6), T-ALL (n=3), cutaneous T-lymphomas (n=3) cell lines by RT-PCR. FedP, MAC1 and MAC2a (ALK-ALCL) cell lines showed the highest expression. Because the cellular localization of lncRNAs affects their function, we assessed the sub-cellular localization of BlackMamba by nucleo/cytoplasm cell fractionation and RT-PCR in FedP and MAC2A cell lines. BlackMamba displayed a preferential nuclear/chromatin-associated localization. Having proven that JAK/STAT signaling pathway plays a key role in both ALK+ and ALK-ALCL, we test whether the expression of BlackMamba could be modulated after treatment with JAK1/2 (ruxolitinib) TKi. Our data showed that, in FedP and MAC2A cell lines, BlackMamba expression was 50% down-regulated relative to control. Next, to identify whether BlackMamba may have a unique associated to specific chromatin remodel proteins, we verified its association with several candidates and found the physical interaction with the histone-lysine N-methyltransferase enzyme EZH2 by RIP in the FedP cells. Notably, after BlackMamba knock down, the proliferation of FedP cells was reproducibly reduced (25% at 84hrs using multiple RNAi oligonucleotides). Collective, our data indicate that ALCL aberrantly express novel and uncharacterized lncRNAs and that BlackMamba is a novel lncRNA associated with ALK-ALCL. Moreover, BlackMamba may contributes to the maintenance of ALK-ALCL neoplastic phenotype. Disclosures Merli: Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Teva Pharmaceuticals Industries: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Induction of human granulocyte differentiation in vitro by ubiquitin and thymopoietin

Blood ◽  
1977 ◽  
Vol 50 (2) ◽  
pp. 275-288 ◽  
Author(s):  
WA Kagan ◽  
GJ O'Neill ◽  
GS Incefy ◽  
G Goldstein ◽  
RA Good
Keyword(s):  
Bone Marrow Cells ◽  
Functional Differentiation ◽  
Velocity Sedimentation ◽  
Differentiation Markers ◽  
Band Form ◽  
Immature Granulocyte ◽  
Differentiation Marker ◽  
Cell Fractions ◽  
Human And Mouse

Abstract Human bone marrow cells were separated according to density by centrifugation on Ficoll-Hypaque gradients and then according to size by velocity sedimentation. This procedure resulted in fractions enriched for immature granulocytes, mature granulocytes, and lymphocytes. Cells in these fractions were analyzed for their expression of certain surface and functional differentiation markers and for their ability to respond to thymopoietin and ubiquitin with the expression of additional differentiation markers. A higher percentage of band form and segmented granulocytes than of more immature granulocytes expressed complement receptors on their surfaces. Thymopoietin and ubiquitin induced a significant percentage of the cells in the immature granulocyte fraction to express this marker. These data suggested that the complement receptor may be viewed as a differentiation marker on human granulocytes, the expression of which can be induced in vitro by thymopoietin and ubiquitin. Furthermore, fractions containing predominantly band form granulocytes were induced by ubiquitin (but not thymopoietin) to develop the capacity to respond to chemotactic agents, and cell fractions containing predominantly myelocytes and metamyelocytes were induced by thymopoietin and ubiquitin to develop the capacity to phagocytose latex particles. These findings indicated that thymopoietin and ubiquitin, two agents known to induce a number of stages of human and mouse lymphocyte differentiation, are also capable of inducing some stages of human granulocyte differentiation in vitro.

scholarly journals Heterogeneity of human colony-forming cells

Blood ◽  
1978 ◽  
Vol 51 (2) ◽  
pp. 195-206
Author(s):  
S Inoue ◽  
MJ Ottenbreit
Keyword(s):  
Fibroblast Cell ◽  
Velocity Sedimentation ◽  
Cell Fractionation ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Distinct Cell ◽  
Human Fibroblast Cell ◽  
Culture Supernatants ◽  
Fibroblast Cell Lines

Culture supernatants from established human fibroblast cell lines (FCM) and from phytahemagglutinin-stimulated peripheral blood leukocytes (LCM) were compared with respect to their stimulatory effects on the colony formation by human marrow leukocyte precursors in vitro using the methylcellulose culture system. The cultures were performed using specimens from children with a variety of disorders. LCM stimulation consistently produced a significantly higher proportion of macrophage colonies than did FCM stimulation, whereas FCM stimulation resulted in a significantly higher number of number of neutrophil colonies. Based on the sequential examination of colony numbers in 35 FCM-and 11 LCM- stimulated cultures, the colonies in LCM-stimulated cultures (predominantly macrophage colonies) survived much longer than those in FCM-stimulated cultures (predominantly neutrophil colonies). Cell fractionation experiments by velocity sedimentation on four different specimens revealed that the majority of FCM-responsive cells were large and formed pure neutrophil colonies, while the majority of LCM- responsive cells were smaller and formed either pure macrophage or mixed neutrophil-macrophage colonies. These observations suggest that human colony-forming cells consist of at least two distinct cell populations different in (1) cell size, (2) response to two different conditioned media, LCM and FCM, and (3) type of colonies each population forms.

scholarly journals Leukemic blast cell colony formation in semisolid culture with erythropoietin: a case report of acute poorly differentiated erythroid leukemia

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 546-552
Author(s):  
M Tomonaga ◽  
I Jinnai ◽  
M Tagawa ◽  
T Amenomori ◽  
K Nishino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Cell Lineage ◽  
Erythroid Differentiation ◽  
Large Cell ◽  
Leukemic Cells ◽  
Poorly Differentiated ◽  
A Minor

The bone marrow of a patient with acute undifferentiated leukemia developed unique colonies after a 14-day culture in erythropoietin (EPO)-containing methylcellulose. The colonies consisted of 20 to 200 nonhemoglobinized large blast cells. Cytogenetic analysis of single colonies revealed hypotetraploid karyotypes with several marker chromosomes that were identical to those found in directly sampled bone marrow. The concurrently formed erythroid bursts showed only normal karyotypes. No leukemic colony formation was observed in other culture systems with either colony-stimulating activity (CSA) or phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). The leukemic colonies exhibited a complete EPO-dose dependency similar to that of the patient's normal BFU-E. Although cytochemical and immunologic marker studies of the bone marrow cells failed to clarify the cell lineage of the leukemic cells with extraordinarily large cell size, ultrastructural study revealed erythroid differentiation such as siderosome formation in the cytoplasm and ferritin particles in the rhophecytosis invaginations. These findings indicate that the patient had poorly differentiated erythroid leukemia and that some of the clonogenic cells might respond to EPO in vitro. Corresponding to this biological feature, the leukemic cells were markedly decreased in number in response to repeated RBC transfusions, and partial remission was obtained. These observations suggest that erythroid leukemia distinct from erythroleukemia (M6) with a myeloblastic component, can develop as a minor entity of human acute leukemia.

scholarly journals Generation of cytotoxic T lymphocytes in vitro. IV. Functional activation of memory cells in the absence of DNA synthesis.

1975 ◽  
Vol 142 (3) ◽  
pp. 622-636 ◽  
Author(s):  
H R MacDonald ◽  
B Sordat ◽  
J C Cerottini ◽  
K T Brunner
Keyword(s):  
T Lymphocytes ◽  
Dna Synthesis ◽  
Cell Number ◽  
Primary Response ◽  
Precursor Cells ◽  
Lymphoid Cells ◽  
Velocity Sedimentation ◽  
Secondary Response ◽  
Ctl Activity

Re-exposure of day 14 mixed leukocyte culture (MLC) cells to the original stimulating alloantigens (secondary response) has previously been shown to result in significant proliferation and in rapid reappearance of high levels of cytolytic T-lymphocyte (CTL) activity within the next 4 days. Moreover, evidence has been presented that CTL precursor cells in day 14 MLC populations, while they derived from cells were large at peak of the primary response (day 4) were themselves small lymphocytes which developed into large CTL after restimulation. In this study, inhibition of DNA synthesis by cytosine arabinoside (ARA-C) was used to investigate whether CTL formation could be dissociated from proliferation during the secondary response. It was found that within the first 24 h after restimulation (a) CTL activity increased 6-to-20-fold, (b) 60-70% of the small T lymphocytes became medium- to large-sized cells, and (c) both events were independent of DNA synthesis. By using two successive cell separations by velocity sedimentation at unit gravity, before and after stimulation of day 14 MLC cells for 24 h in the presence or absence of ARA-C, direct evidence was obtained that small CTL precursor cells developed into large CTL, irrespective of DNA synthesis. The presence of ARA-C for periods longer than 24 h inhibited any further increase in CTL activity, in contrast to a parallel increase in lytic activity and cell number from day 1 to day 4 in control restimulated cultures. Taken together with the finding that 90% of the medium- and large-sized lymphoid cells in control restimulated cultures underwent DNA synthesis within 24 h, these results thus suggest that during a secondary MLC response there is initially a differentiation step leading to the formation of CTL which, although it can be clearly dissociated from DNA synthesis, is under normal conditions followed by proliferation of these effector cells.

scholarly journals Pre-emption of human cell-mediated lympholysis by a suppressive mechanism activated in mixed lymphocyte cultures

1975 ◽  
Vol 142 (6) ◽  
pp. 1606-1611 ◽  
Author(s):  
PM Sondel ◽  
MW Jacobson ◽  
FH Bach
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Positive Selection ◽  
Cytotoxic T Lymphocytes ◽  
Third Party ◽  
Velocity Sedimentation ◽  
Secondary Response ◽  
Suppressor Activity ◽  
Suppressor Mechanism

The regulation of B-cell and T-cell immune responses has been extensively examined and in the experimental animal appears to involve regulatory or suppressor T cells (1-4). The limitations of in vitro experimentation have made comparable study of nonpathological human suppression quite difficult (5). We report here an in vitro method that generates and quantitates suppressor activity in man after antigen-specific activation in mixed leukocyte culture (MLC). The one-way MLC induces both a proliferative response (6) and the generation of cytotoxic T lymphocytes (CTLs) (7). Both of these responses are mediated by antigen-specific T-cell subpopulations (8,9) and have been correlated with recognitive and destructive phases of allograft rejection. Recent reports have examined the antigen reactivity of mouse (10,11), rat (12), or human (13,14) lymphocytes obtained after proliferation in MLC. In all cases, after the primary MLC proliferative peak, the recovered lymphocytes rapidly differentiate upon re-exposure to the initial stimulating population, but do so only weakly when exposed to a presumably noncross-reactive third-party stimulating population. Velocity sedimentation separation studies have shown that the blast cells produced in a primary MLC revert to small lymphocytes that rapidly differentiate into proliferating and/or cytotoxic T lymphocytes upon restimulation with the initial antigen (15). These findings demonstrate that positive selection for the responding population in primary MLC does exist and may account for at least part of the specificity of the secondary response. However, this positive selection does not preclude possible involvement of a suppressor mechanism. In fact we have detected suppressor activity in primary MLC sensitization cultures at a time when the proliferation responsible for positive selection does not preclude possible involvement of a suppressor mechanism. In fact we have detected suppressor activity in primary MLC sensitization cultures at a time when the proliferation responsible for positive selection in not yet significant, suggesting that suppression may be overriding importance in the specificity of MLC-activated secondary responses.

scholarly journals Induction of human granulocyte differentiation in vitro by ubiquitin and thymopoietin

Blood ◽  
1977 ◽  
Vol 50 (2) ◽  
pp. 275-288
Author(s):  
WA Kagan ◽  
GJ O'Neill ◽  
GS Incefy ◽  
G Goldstein ◽  
RA Good
Keyword(s):  
Bone Marrow Cells ◽  
Functional Differentiation ◽  
Velocity Sedimentation ◽  
Differentiation Markers ◽  
Band Form ◽  
Immature Granulocyte ◽  
Differentiation Marker ◽  
Cell Fractions ◽  
Human And Mouse

Human bone marrow cells were separated according to density by centrifugation on Ficoll-Hypaque gradients and then according to size by velocity sedimentation. This procedure resulted in fractions enriched for immature granulocytes, mature granulocytes, and lymphocytes. Cells in these fractions were analyzed for their expression of certain surface and functional differentiation markers and for their ability to respond to thymopoietin and ubiquitin with the expression of additional differentiation markers. A higher percentage of band form and segmented granulocytes than of more immature granulocytes expressed complement receptors on their surfaces. Thymopoietin and ubiquitin induced a significant percentage of the cells in the immature granulocyte fraction to express this marker. These data suggested that the complement receptor may be viewed as a differentiation marker on human granulocytes, the expression of which can be induced in vitro by thymopoietin and ubiquitin. Furthermore, fractions containing predominantly band form granulocytes were induced by ubiquitin (but not thymopoietin) to develop the capacity to respond to chemotactic agents, and cell fractions containing predominantly myelocytes and metamyelocytes were induced by thymopoietin and ubiquitin to develop the capacity to phagocytose latex particles. These findings indicated that thymopoietin and ubiquitin, two agents known to induce a number of stages of human and mouse lymphocyte differentiation, are also capable of inducing some stages of human granulocyte differentiation in vitro.

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