Human T Lymphocyte Colonies in Agar: A Comparison With Other T Cell Assays in Healthy Subjects and Cancer Patients

Wilson, J. D.; Dalton, Gael

doi:10.1038/icb.1976.3

Cited by 23 publications

(4 citation statements)

References 7 publications

(10 reference statements)

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“…The number of colonies which were obtained from blood agreed with previously published studies with similar methods (26)(27)(28). Greater numbers of colonies arose from nontumor-involved human lymph nodes than had previously been reported for the mouse (28), or for the human spleen (23 (7).…”

Section: Discussionsupporting

confidence: 79%

Prostaglandin E inhibition of T-lymphocyte colony formation: a possible mechanism of monocyte modulation of clonal expansion.

Bockman¹,

Rothschild²

1979

J. Clin. Invest.

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A B S T R A C T Prostaglandin and monocyte modulation ofa T-lymphocyte cell capable ofundergoing clonal expansion was studied. Circulating human mononuclear cells were isolated by density centrifugation. After 24 h in culture with phytohemagglutinin present, the cells were mixed with 0.3% agar and overlayed onto a 0.5% agar layer that contained media and phytohemagglutinin. At day 6, colonies that contained >50 cells were counted. These colonies represented clonal proliferation ofa phytohemagglutinin-responsive T-lymphocyte precursor. This responder cell accounted for <0.3% of the starting cell population. Colonies were comprised ofcells which, when isolated, formed E rosettes. These colony cells could be shown to have helper or suppressor function as measured by their ability to promote or inhibit immunoglobulin synthesis. By these latter criteria the colony cells were considered to be mature T lymphocytes. The addition of prostaglandin E to the cultures demonstrated a linear, r = 0.82, dose-dependent inhibition of colony formation with a 50% point of inhibition (150) = 0.18 uM. Low numbers of normal monocytes when added to the cultures mimicked the effect of synthetic prostaglandin E2. A highly significant correlation could be shown for endogenous prostaglandin E levels and colony counts. It appears that monocytes through their synthesis of prostaglandin E2 can restrict the clonal expansion of a circulating T-lymphocyte precursor.

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Section: Discussionsupporting

confidence: 79%

Prostaglandin E inhibition of T-lymphocyte colony formation: a possible mechanism of monocyte modulation of clonal expansion.

Bockman¹,

Rothschild²

1979

J. Clin. Invest.

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“…Mitogen-dependent colony formation by T-cells in semi-solid agar has been previously reported (Fibach et al 1976, Riou et al 1976, Sredni et al 1976, Wilson & Dalton 1976. This approach appeared very interesting for providing direct measurements of reactive cell frequencies, as well as clones which couid possibly be important in the analysis of effector T-cell speciH-cities.…”

Section: T-cell Responses To Con Amentioning

confidence: 89%

Clonal Growth of T Cells in vitro: Preliminary Attempts to a Quantitative Approach

Watanabe¹,

Fathman²,

Coutinho³

1977

Immunological Reviews

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“…It is interesting in this regard that PHA-induced colony formation has been reported to be severely depressed in unselected patients with malignant disease (8,18). Such studies will be facilitated by the ease with which growth of lymphoid cell lines and preparation of large pools of CM can be accomplished.…”

Section: Discussionmentioning

confidence: 99%

“…In the mouse, B lymphoid colonies are formed in the presence of 2-mercaptoethanol (5), and T lymphoid colonies can be induced with the plant lectins phytohemagglutinin (PHA) and concanavalin A (6). T lymphoid colonies can also be established from human peripheral blood lymphocytes in the presence of PHA (7)(8)(9), whereas with pokeweed mitogen mixed T and B lymphoid colonies are formed (9). Established human lymphoid cell lines multiply spontaneously in the absence of plant lectins or mercaptoethanol, and it seemed possible that such cells might release growth-stimulating substances into the culture medium.…”

mentioning

confidence: 99%

Lymphocyte culture: induction of colonies by conditioned medium from human lymphoid cell lines.

Galbraith¹,

Goust²,

Fudenberg³

1977

The Journal of Experimental Medicine

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It has been shown that macrophage and granulocyte colonies can be induced m semisolid agar (1-3) in the presence of substances termed colony-stimulating factors (CSF), which are released predominantly by monocytes (4). However, attempts to induce formation of lymphoid colonies with CSF have so far proved unsuccessful. In the mouse, B lymphoid colonies are formed in the presence of 2-mercaptoethanol (5), and T lymphoid colonies can be induced with the plant lectins phytohemagglutinin (PHA) and concanavalin A (6). T lymphoid colonies can also be established from human peripheral blood lymphocytes in the presence of PHA (7-9), whereas with pokeweed mitogen mixed T and B lymphoid colonies are formed (9). Established human lymphoid cell lines multiply spontaneously in the absence of plant lectins or mercaptoethanol, and it seemed possible that such cells might release growth-stimulating substances into the culture medium. We have therefore investigated the ability of conditioned medium (CM) obtained from lymphoid cell lines to induce normal human peripheral blood lymphocytes (PBL) to form lymphoid colonies in agar. Materials and MethodsThree B-cell lines-4061 (provided by Dr. J. Sinkovics, Houston, Tex.), PGLC 33H, and RPMI 1788-were used as sources of CM. After 3-4 days of culture in serum-free medium at 37°C, cellfree supernates were obtained by centrifugation at 800 g for 10 rain, filtration through 0.22-~m filters (Millipore Corp., Bedford, Mass.), and ultracentrifugation at 47,500 g for 1 h.Peripheral blood was collected from adult healthy volunteers in pyrogen-and preservativefree heparin. Lymphocytes were obtained by centrifugation over Ficoll Isopaque gradients and washed three times in RPMI 1640 medium (Grand Island Biological Co., Grand Island, N.Y.). Contamination with monocytes varied between 3 and 20% as assessed by latex ingestion and staining for peroxidase (10). Cells were seeded at various concentrations in 2 ml 0.3% agar made by the addition of I vol of double-strength RPMI 1640 (containing 200 gg/ml streptomycin, 200 U/ ml benzyl penicillin, and 20% fetal calf serum [Gibco]) to 1 vol of 0.6% agar (Difco Laboratories, Detroit, Mich.) made up in pyrogen-free distilled water. Various amounts of CM were added directly to the 0.3% agar mixture. After culture in 35-ram plastic Petri dishes at 37°C in 5% CO2 in air for various times, the plates were counted under an inverted microscope. Colonies were defined as clumps containing more than 50 cells. Colony cells were tested for their ability to form E rosettes, by using the technique of Wybran et al. (11), and for the presence of surface * Publication no. 145 from the

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Human T Lymphocyte Colonies in Agar: A Comparison With Other T Cell Assays in Healthy Subjects and Cancer Patients

Cited by 23 publications

References 7 publications

Prostaglandin E inhibition of T-lymphocyte colony formation: a possible mechanism of monocyte modulation of clonal expansion.

Prostaglandin E inhibition of T-lymphocyte colony formation: a possible mechanism of monocyte modulation of clonal expansion.

Clonal Growth of T Cells in vitro: Preliminary Attempts to a Quantitative Approach

Lymphocyte culture: induction of colonies by conditioned medium from human lymphoid cell lines.

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