T-cell Colony Formation Research Articles

Growth Hormone Induces Resistance to the Mitogenic Action of Insulin Through Local IGF-I: Studies in Normal and Pygmy T-Cell Lines

Growth hormone (GH) and insulin have both mitogenic and metabolic actions. The growth-promoting effects of GH in vivo are thought to be mediated by insulin-like growth factor-I (IGF-I), whereas the metabolic effects of GH are thought to be either direct or mediated by factors other than IGF-I. In previous studies using HTLV-II-transformed T-lymphoblast cell lines established from normal individuals, we have shown that GH preincubation induces resistance to the growth-promoting (mitogenic) action of insulin. In this study, using T-cell lines from 3 American control subjects, 1 African control subject, and 1 African Pygmy (the latter previously shown to be resistant to the growth-promoting actions of both IGF-I and GH), we examined the role of local IGF-I in the mediation of GH-induced resistance to the mitogenic action of insulin. In these studies, we quantified the stimulation of T-cell colony formation in response to insulin in the presence and absence of either GH or IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-specific human T-cell colony formation in a liquid culture system.

We have developed a limiting dilution microculture T-cell cloning technique to generate antigen-specific T-cell clones. Cultures contained peripheral blood T-cells, irradiated autologous mononuclear cells (feeders), human AB serum and recombinant IL-2. Colonies were enumerated at 14 days. Under these conditions the cloning efficiency expressed as the frequency of proliferating cells in response to PHA, ranged from 1/1.07 to 1/3.35. In response to tetanus toxoid, the frequency of proliferating cells ranged from 1/415 to 1/5655. Average colony size ranged from 0.7 x 10(5) to 6.2 x 10(5) cells with a mean T-cell recovery of 2.5 x 10(5) cells per microculture. Restimulation of 14-day tetanus toxoid T-cell colonies revealed marked proliferation to PHA and tetanus toxoid but only background responses to PPD. These studies document the development of a rapid, antigen-specific liquid culture T-cell cloning system which is likely to detect T-cell clones that are representative of the original T-cell population. The clones are of sufficient size to be useful in studies of antigen cross-reactivity.

The effect of changes in thiol subcompartments on T-cell colony formation and cell cycle progression: Relevance to AIDS

Recently, it has been shown that intra- and extracellular thiol levels are significantly lower than normal even in the relatively early stages of human immunodeficiency virus (HIV) infection. It is plausible that this deficiency could contribute both to the loss of T-cell function and the ability to replenish T cells associated with HIV infection. We had previously reported that the T-cell colony-forming cell (T-CFC) is impaired in HIV infection and that it can be enhanced with the thiol compounds 2-mercaptoethanol (2-ME) and N-acetylcysteine (NAC). In this study, the effect of the thiol-depleting reagents buthionine sulfoximine, cyclohexene-1-one, and copper phenanthroline on T-CFC formation and cell cycle progression was determined in HIV+ subjects and/ or controls. All three reagents inhibited T-CFC formation and cell cycle progression with a suggestion that colony formation by cells from HIV+ subjects was more sensitive to the effects of thiol depletion. 2-ME and NAC enhanced T-CFC formation in the presence of all thiol-depleting reagents. In preliminary experiments, the enhancing effect of NAC did not appear to involve increased protein kinase C translocation. Our results suggest that oxidation of membrane thiols, as well as depletion of intracellular glutathione, inhibits T-CFC formation as well as cell cycle progression for mitogen-stimulated cells in bulk culture.

Increased T-cell colony formation in AIDS patients treated with zidovudine

Treatment of AIDS patients with zidovudine is associated with an increase in lymphocyte counts. The mechanism for this increase is unclear and somewhat surprising in view of the myelosuppressive effect of zidovudine. To investigate this further, we measured lymphocyte numbers, T-cell subsets, and the ability of peripheral blood mononuclear cells (PBMC) to form T-cell colonies (T-CFC) in agar formation, in a group of patients with AIDS, before and during the first 6 months of zidovudine treatment. Eight patients were treated for an average of 11 weeks. There was a significant increase in T-CFC with zidovudine treatment (11.5 +/- 4.7% versus 29.8 +/- 6.9%, P less than 0.02 using a paired Student's t-test). There was a non-significant trend in the improvement of lymphocyte counts in these patients (872 +/- 117 versus 1102 +/- 204, NS). In vitro exposure of lymphocytes to zidovudine (200 mumol/l) resulted in modest suppression of T-CFC formation, suggesting that the effect of zidovudine treatment is indirect. Given that we have previously shown that inactivated HIV can inhibit T-CFC formation, we suggest that zidovudine treatment indirectly allows an increase in lymphocyte number by decreasing virus load, thereby permitting greater T-cell repopulation.

Effect of histamine on the T-cell colony formation of PHA-stimulated cells

The effect of histamine on T-cell colony formation was studied in human peripheral blood mononuclear cells. Histamine inhibited dose-dependently (10(-4)-10(-6) M) the colony formation of PHA-stimulated T-cells. The inhibition was similar in normal controls and rheumatoid arthritis (RA) patients in spite of the fact that in RA the colony formation was significantly lower than in the normal controls. No increase of colony formation was observed at low concentrations (less than 10(-7) M). Impromidine was less effective than histamine, and pyridylethylamine (PEA) was inactive. Cimetidine counteracted the effect of histamine while chlorpheniramine did not. The results show that colony formation may be inhibited through H2-receptors. This action may be of importance in cellular interactions in tissues with high local histamine concentrations.

Autoreactive T cells in rheumatic disease (1) analysis of growth frequencies and autoreactivity of T cells in patients with rheumatoid arthritis and lyme disease

A limiting dilution system was established in order to estimate frequencies of interleukin-2 (IL-2)-responsive, autoreactive and alloreactive T cells in samples of peripheral blood (PBL) and synovial fluid lymphocytes (SFL), from patients with rheumatoid arthritis (RA) and lyme disease, as well as from healthy donors and a patient with osteoarthrosis. The frequencies of IL-2-dependent T-cell colony formation were significantly higher in patients with RA and lyme disease (median: 1 287 ) as compared to controls (median: 1 1,313 ) indicating a preactivation of T cells in these patients in vivo. Autoreactivity was measured by the proliferative response of T-cell lines to autologous irradiated PBL as stimulating cells. The frequencies of autoreactive T cells in blood were significantly higher in patients (median: 1 2,615 ) as compared to controls (median: 1 19,607 ). There was no significant difference in autoreactive T-cell frequencies between the patients' SFL (median: 1 3,185 ) and PBL (median: 1 2,615 ). In every case the frequency of alloreactive T cells exceeded the frequency of autoreactive T cells. Most autoreactive T-cell lines were also alloreactive and were shown to be MHC Class II-restricted. There is evidence of a down regulation of autoreactive T cells by suppressor cells in peripheral blood in two cases with elevated autoreactive T-cell frequencies (one RA patient and one control patient suffering from a viral infection). In contrast, no suppression of autoreactive T cells was observed in the RA patients' SFL or in PBL and SFL from patients with lyme disease. These results suggest that the chronic inflammation observed in RA and lyme disease may be supported by an elevated number of autoreactive T cells in the absence of suppressive mechanisms.

T-Cell Colony Formation in Healthy Homosexual Men without Anti-Human Immunodeficiency Virus Antibody

In order to study the abnormal differentiation and proliferation of precursor T cells in homosexual men, a study was made of T-cell colony formation in 15 healthy homosexual men without anti-human immunodeficiency virus (HIV) antibody. The colony forming method used was the one-step monolayer technique using phytohemagglutinin as an inducer. Our results revealed that colony formation was extremely reduced and 3 cases showed no colony growth. However, with the addition of recombinant interleukin 2 to the culture system, the number of colonies increased up to the same level as in heterosexual controls. In addition, the analysis of CD3+, 4+, and 8+ cells of colony component cells revealed a decrease in CD3+ and 4+ cells. However, with the addition of recombinant interleukin 2, the numbers of CD3+ cells distinctly increased. The above results suggest that a disorder in the differentiation and proliferation of precursor T cells in homosexual men might be one of the causes of immunodeficiency.

Inhibition of the colony-forming capacity of human T-lymphocytes exerted by theophylline

The effects of theophylline on T-lymphocyte colony formation were investigated both by in vitro studies (adding theophylline in cultures at three different concentrations—5, 15 and 30 μg/ml) and by in vivo experiments (after oral ingestion of 10 mg/kg time-released theophylline) in a group of young healthy volunteers. In vitro the drug showed a significant dose-dependent inhibitory effect on both peripheral mononuclear cells (PMNC) and purified T-lymphocyte colony formation. No significant difference in PMNC colony formation before and 12 h after the oral ingestion of theophylline was detected in the in vivo study, even if at the same time the plasma concentration of the drug remained at therapeutic levels in every subject. In addition, in vitro experiments mimicking the in vivo conditions suggested that the apparent disagreement between the in vitro and in vivo results was due to experimental procedures (i.e. the inhibitory effect requires the constant presence of the theophylline in the culture medium). The experimental data provided in this study confirm, using different experimental procedures, the results of our recent investigations showing that theophylline exerts an inhibitory effect on T-cell proliferation.

T-lymphocyte colony formation of murine thymocytes in agar contained in glass capillaries.

Optimal growth conditions are presented for a new colony test with mouse thymocytes in agar contained in glass capillaries. The kinetics of colony growth and the dependence from the PHA-, I1-2-, agar- and 2-mercaptoethanol concentration are shown. The colony forming cells are identified as T-lymphocytes by usual morphology and by an indirect immunoperoxidase method using mouse anti-Thy 1.2 antibodies.

T-Lymphocyte Colony Formation by Peripheral Lymphocytes of Patients with Non-Hodgkin's Lymphoma

T-Lymphocyte Colony Formation by Peripheral Lymphocytes of Patients with Non-Hodgkin's Lymphoma

Peripheral blood adherent cells from AIDS patients inhibit normal T-colony growth through decreased expression of interleukin 2-receptors and production of interleukin 2

Adherent cells display an important accessory role on normal T-cell colony formation. Since the in-vitro proliferation of T colony-forming cells (T-CFC) from AIDS patients is extremely impaired we studied the effect of patients' adherent cells on T-CFC growth. Patients' peripheral blood mononuclear cells (PBMC) were fractionated on the basis of rosette formation with sheep red blood cells and complement-mediated cytotoxicity with OKT3 monoclonal antibody (E −T3 −). Both mature (E +) T cells and E −OKT3 − cell fractions failed to generate T-cell colonies although colony growth could be obtained from unfractionated PBMC. In five out of 12 AIDS patients, adherent cell-depletion of PBMC enhanced the plating efficiency. Moreover, patients' but not normal adherent cells could inhibit normal T-cell colony growth in a dose-dependent manner. Media conditioned by patients' unstimulated adherent cells (LCM-A + p) also inhibit normal T-cell colony formation. In addition, LCM-A +p were capable of inhibiting interleukin 2-receptor (IL2-R) expression and interleukin 2 (IL2) production by normal mitogen-stimulated T cells. These LCM-A +p did not contain detectable reverse transcriptase activity nor could they infect the CEM T-cell line which is permissive to human immunodeficiency virus (HIV). Conversely, this adherent cell-derived inhibitory activity could be abrogated by heating or treatment with proteolytic enzymes. These findings indicate that the low T-cell colony formation in some AIDS patients could be due to adherent cell-derived inhibitory activity(ies).

Refinement of a T-lymphocyte cloning assay to quantify the in vivo thioguanine-resistant mutant frequency in humans.

Cell cloning by limiting dilution in 96-well microtiter plates has been employed to isolate colonies of human T-lymphocytes resistant to the purine analogue, 6-thioguanine (TG). These colonies show stability of the TG-resistant (TG1) phenotype, lack hypoxanthine guanine phosphoribosyl transferase (HPRT) activity and thus appear to be the result of in vivo somatic cell mutation events. In order to employ this T-lymphocyte cloning assay for quantitative determination of the in vivo TGr mutant frequency in humans, we have defined the optimal conditions for T-cell colony formation with both nonselected and TG-selected cells. The parameters investigated include medium, serum, amount of the mitogen phytohaemagglutinin, amount of T-cell growth factor (TCGF) and the number of irradiated feeder or accessory cells. Under the optimal conditions, the fraction of positive wells is proportional to the number of cells plated per well with both nonselection and TG selection conditions. T-cell cloning efficiencies therefore are independent of inoculum size. There was some evidence for a decline in TGr mutant cell cloning at densities greater than 2 x 10(4) cells per round-bottom well, possibly due to metabolic cooperation between wild-type and mutant cells. The conditions defined in this study appear to provide a quantitative measurement of the in vivo TGr mutant frequency in human T-lymphocytes.

T-cell colony formation in patients with T-cell malignancies: Growth factor requirements for in-vitro proliferation of peripheral blood T-cell colony-forming cells

Peripheral blood T colony-forming cells (T-CFC) from patients with T-cell malignancies are capable of proliferation in methylcellulose, in the absence of added growth factors or mitogenic stimulation. We show here that based on the spontaneous plating efficiencies of their T-CFC, two groups of patients can be established: Group A (10 patients) with a high colony number (more than 100 colonies/10 5 seeded cells), and group B (12 patients) with less than 100 colonies/10 5 cells. The addition of interleukin 2-(IL2)containing PHA-leukocyte conditioned medium enhanced colony growth from group B but not group A patients. Moreover, both biochemically purified and recombinant IL2 induced the colony growth from group B but not group A patients without any other stimulation. In addition, a monoclonal antibody (moAb) against the IL2 receptor (IL2-R; anti-Tac) inhibited the spontaneous colony formation from T-CFC of both groups of patients. These observations strongly suggest that IL2-R are involved in the spontaneous colony growth of T-CFC. To determine whether IL2 is also involved in the spontaneous colony formation, media conditioned (LCM) by unstimulated leukemic cells were tested for IL2 activity. A constitutive release of IL2 was detected in LCM from only 2 out of 10 patients tested, and some of them were capable to inhibit thymidine incorporation by 1L2-dependent cells cultured in the presence of highly purified IL2. However, most of these LCM contained a T-cell colony-promoting activity (TCPA) inducing colony formation from both normal resting and PHA-stimulated E' clonogenic cells without added IL2 or mitogenic stimulation. TCPA-containing LCM induced the expression of functional IL2-R and IL2 release by normal resting lymphocytes. TCPA was constitutively released by blast-enriched cell fractions suggesting that it is released by the leukemic cells.

Response to hydrocortisone of blast progenitors in acute myeloblastic leukemia: An aspect of lineage infidelity

The blast population in acute myeloblastic leukemia (AML) contains cells capable of forming blast-cell colonies in culture. The purpose of this study was to measure the effects of hydrocortisone on this process, using two end-points. First, we measured the effects of increasing concentrations of hydrocortisone on the primary plating efficiency of T-lymphocyte-depleted blast cell preparations from AML peripheral blood. Second, colonies forming in the presence or absence of the hormone were pooled and replated; changes in the plating efficiencies (secondary plating efficiency or PE2) of these suspensions reflected the effect of the hormone on blast progenitor self-renewal. For comparison, we measured the hydrocortisone dose response curves for normal granulopoietic and T-lymphocyte colony-formation. The latter showed little individual variation; T-lymphocyte colony-formation was regularly sensitive to the hormone while granulopoietic colony-formation was resistant. In contrast, wide variations were found in the hydrocortisone dose response curve for blast from 24 patients with AML (FAB 1–6). A significant association was found between successful remission induction and resistance to hydrocortisone in 24 treated patients. The associated was maintained when the data was stratified by other risk factors, including PE2 and the presence of blasts bearing immunologically-defined markers of more than one differentiation lineage (lineage infidelity). We propose that sensitivity to hydrocortisone may reflect the passage of blast cells through lymphopoiesis-associated components of differentiation programs. From this point of view, the poor prognosis associated with sensitivity of blast progenitors to hyrocortisone may be similar to the response-failure of patients whose blasts exhibit lineaged infidelity when tested with immunological procedures.

HLA-DR-antigen-associated restriction of EBV-specific cytotoxic T-cell colonies

EBV-specific cytotoxic T cells can be generated in vitro in a secondary response. Several previous studies with bulk cultures provided evidence that cytotoxicity was restricted by HLA-A,B-related antigens. In the present family study, the EBV-specific cytotoxic T-cell response of a normal EBV-seropositive donor was analysed in detail by T-cell colony formation. Peripheral blood mononuclear cells were stimulated by the gamma-irradiated autologous lymphoblastoid cell line (LCL) and 3 days later seeded into agarose. Colonies were harvested, amplified by addition of interleukin-2 (IL-2), and analysed for T-cell markers and specificity in 51Cr-release assays. Twenty-two colonies were studied: all colonies were OKT3+, five were predominantly OKT4+, 9 were OKT8+ and 8 were mixtures. As expected from previous work, the OKT8+ colonies were cytotoxic for the autologous LCL target and cytotoxicity was blocked by monoclonal antibody (W6/32) to the nonpolymorphic determinants of HLA-A,B,C antigens. Significantly, the OKT4+ colonies tested also showed specific cytotoxicity, but lysis of the autologous LCL was blocked by the monoclonal antibody (OKlal) to the non-polymorphic determinants of HLA-DR antigens. Two interesting patterns of specificity were seen in cytotoxicity tests on sibling LCL targets. In one pattern, targets bearing the A11, B5, DR7 haplotype were lysed, while those bearing the A1, B8, DR3 were not, indicating haplotype preference. In the other pattern, there was lysis of the autologous cell line but not of the sibling targets. These results including HLA-DR-associated restriction, haplotype preference and strict self-preference, further illustrate the complexity of the EBV-cytotoxic T-cell response.

Opposing effects of 12‐o‐tetradecanoylphorbol 13‐acetate on human myeloid and lymphoid cell proliferation

The effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on human hematopoietic cells has been investigated. It was found that 1-10 ng/ml of TPA totally abrogated erythroid and granulocytic colony growth and, simultaneously in the presence of PHA, stimulated T-lymphocyte colony formation. TPA concentrations insufficient to inhibit myeloid colony growth also failed to stimulate lymphocyte colony formation. Optimal culture conditions for the growth of these colonies required the presence of TPA, PHA, and leukocyte-conditioned medium in the cultures. Cells within the colonies were 80-90% E-rosette positive and by monoclonal antibody characterization contained 45-66% OKT3-positive cells. Colony-forming cells were found in both E-rosette-positive and-negative fractions. Although by cell surface marker characterization the cells within the colonies had properties of T-cells, the exact relationship of cells forming colonies under these conditions to those detected in other T-cell colony assays remain to be determined.

Depressed functions of T cells and the presence of suppressor macrophages in patients with sarcoidosis

T-cell functions of patients with sarcoidosis were studied by employing PWM-induced immunoglobulin (Ig) synthesis and PHA-induced T-cell colony formation. Con A-induced suppressor function as well as helper function in PWM-induced Ig production was markedly depressed in T cells from patients with sarcoidosis. PHA-induced T colony formation was also found to be markedly depressed in patients with sarcoidosis. However, the depressed colony formation was recovered by depletion of macrophages or by addition of indomethacin. The addition of the soluble factors derived from patients' macrophages suppressed the colony formation of T cells from healthy adult volunteers. Taken together, the results suggest that the presence of suppressor macrophages may induce the depressed state of several T-cell functions in patients with sarcoidosis.

Effect of 2’-Deoxycoformycin on Erythroid, Granulocytic, and T-Lymphocyte Colony Growth

The finding of elevated intracellular levels of adenosine deaminase (ADA) in some patients with acute lymphoblastic leukemia has led to attempts to control this disease with the adenosine deaminase inhibitor 2'-deoxycoformycin (dCF). Because of clinical reports indicating its relative freedom from myelotoxicity, we have tested the effects of this drug on erythroid, granulocytic, and T-lymphocyte colony formation by normal marrow and peripheral blood cells. While clinically the drug has been found to be active at serum concentrations of approximately 10 microM, we have tested it at concentrations up to and including 1 mM. It was found that both erythroid and granulocytic colony growth was completely unaffected by 1 mM dCF, a concentration at least 2 magnitudes higher than that necessary to totally ablate intracellular ADA levels. T-lymphocyte colony growth was unaffected by 100 microM dCF, but at 1 mM some inhibition was observed. These findings therefore indicate that dCF, while able to cause leukemic cell lysis in vivo, has no inhibitory effect on the proliferative capacity of normal hematopoietic cells.

Effect of 2'-deoxycoformycin on erythroid, granulocytic, and T- lymphocyte colony growth

The finding of elevated intracellular levels of adenosine deaminase (ADA) in some patients with acute lymphoblastic leukemia has led to attempts to control this disease with the adenosine deaminase inhibitor 2′-deoxycoformycin (dCF). Because of clinical reports indicating its relative freedom from myelotoxicity, we have tested the effects of this drug on erythroid, granulocytic, and T-lymphocyte colony formation by normal marrow and peripheral blood cells. While clinically the drug has been found to be active at serum concentrations of approximately 10 microM, we have tested it at concentrations up to and including 1 mM. It was found that both erythroid and granulocytic colony growth was completely unaffected by 1 mM dCF, a concentration at least 2 magnitudes higher than that necessary to totally ablate intracellular ADA levels. T-lymphocyte colony growth was unaffected by 100 microM dCF, but at 1 mM some inhibition was observed. These findings therefore indicate that dCF, while able to cause leukemic cell lysis in vivo, has no inhibitory effect on the proliferative capacity of normal hematopoietic cells.

Pharmacologic and biochemical modulation of human T-lymphocyte colony formation: Hormonal influences

The generation of T-cell colonies from human peripheral blood lymphocytes is a sensitive in vitro measure of cell-mediated immunity, considered to be under different and/or additional regulatory controls than short-term liquid cultures. The influences of steroids (aldosterone, estradiol, diethylstilbesterol, hydrocortisone, prednisolone, progesterone, testosterone), prostaglandins (PGA1, PGA2, PGB1, PGB2, PGE1, PGE2, PGF1α), bradykinin, cyclic adenosine monophosphate (AMP), cyclic guanosine monophosphate (GMP), epinephrine, glucagon, histamine, insulin, luteninizing hormone, luteotropic hormone, serotonin, and thyroxin on the generation of both T-cell colonies in semisolid phase and induction of transformation in liquid culture was assessed in parallel assays. Steroids uniformly suppressed both types of culture systems, although colony formation appeared more sensitive by several hours of magnitude. In contrast, significant differences in the response of lymphocytes in colony formation assay, compared to liquid transformation, was noted for the other agents. Prostaglandins significantly inhibited colony formation even in the presence of as little as 10−12 M PGE2; however, liquid culture responses were suppressed only by higher concentrations (10−15 M) and enhanced transformation was found at lower concentrations (10−9 M). Bradykinin, glucagon, and luteninizing hormone did not significantly influence either colony formation or liquid transformation. In contrast, cyclic AMP inhibited and cyclic GMP stimulated colony formation and liquid transformation. Histamine, insulin, epinephrine, and serotonin all had significant positive or negative influences on colony growth formation in concentrations that produced no detectable effects using conventional liquid transformation assays. Finally, correlation analysis of drug effects for each system extends the thesis that these assays quantitate different parameters of T-cell function. T-lymphocyte colony formation is a promising diagnostic tool for rapid screening of immune modulating agents.