A Micro-Culture System for Cloning Human T Lymphocytes in Agar

Immunobiol., vol. 160, pp. 145-158 (1981)

Original Papers Department of Internal Medicine, University of Innsbruck, Austria

A Micro-Culture System for Cloning Human T Lymphocytes in Agar G. KONWALINKA, CH. PESCHEL,

D. GEISSLER, B. TOMASCHEK,

C. GATIRINGER, G. SCHULERl,R. ODAVIC, and H. BRAUNSTEINER Received May 13, 1981 . Accepted June 30, 1981

Abstract A simple and reproducible single-layer micro-agar culture system for cloning of human T lymphocytes has been described. The system consists of an agar layer, in which mononuc­ lear cells from peripheral blood were suspended, and a liquid overlayer containing the mitogenic substance. The advantages of the described method are a low incubation volume (0.5 ml) and the liquid overlayer. The addition of different test substances to the liquid overlayer is simple and easily controllable. Depending on the agar concentration a different number of formed colonies can be found floating in the liquid phase. The morphological, cytochemical and immunological characteristics of the cells from those aggregates could be easily studied. The T-cell characteristics of formed clusters and colonies was confirmed by immunofluorescence and E rosette formation. The effects of agar, serum and cell concentra­ tions, as well as the mitogenic activation caused by three lectins on the development and number of colonies were studied on day 7, 10 and 14 of incubation.

Introduction Cloning of hematopoietic progenitor cells in vitro has been widely used to study the proliferation and differentiation of these cells under experi­ mental and clinical conditions. Several authors have recently described methods for the induction of growth of normal human T lymphocytes in semi-solid media (agar and methylcellulose) under continuous stimulation of lectins such as phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) (1-12). The colonies formed by proliferat­ ing lymphocytes can be obtained either by preincubation of cells in liquid Department of Dermatology, University of Innsbruck, Austria Abbreviations: FCS: Fetal calf serum; PHA: Phytohaemagglutinin; PWM: Pokeweed mitogen; ConA: Concanavalin A; 2-ME: 2-mercaptoethanol; SRBC: Sheep red blood cells; ATS: Anti human T-cell serum; AcP: Acid phosphatase; ANAE: Alpha naphthylacetate acid esterase; BG: Beta-glucuronidase; E-RFC: Sheep erythrocyte rosette forming cells; TEM: Translucent electron microscopy. This work was supported in part by the «Fonds zur F6rderung der wissenschaftlichen Forschung», Projekt-Nr. 4177 1

146 . G. KONWALINKA et al.

suspension with mitogen followed by seeding in agar (1-3, 7, 9), or by a simpler procedure of cloning the cells with lectin directly in agar (4-6,8, 10) or agarose (11). Most of these methods use double layer system requiring a PHA-containing underlayer and are carried out in petri dishes with a minimum volume of 1 ml. Furthermore, the cell growth in these systems was enhanced by the addition of different factors such as red blood cells, autologous plasma, fetal calf serum (FCS) supplemented with bovine serum albumin, transferrin, L-glutamine, L-cystine, FeCl3 and 2-mercaptoethanol (2-ME) (13). A one-stage microculture assay in glass capillary tubes was recently described (8, 14). The purpose of this work was to develop a simple, single-layer micro agar culture system which enables a study of T­ lymphocyte proliferation in vitro. This system consists of an agar layer, containing the mononuclear cells and a liquid phase overlayer with the mitogenic substance. The effects of sera, cell and agar concentrations and different lectins (PHA, Con A and PWM) on the development of colonies were studied. Depending on the agar concentration a different percentage of colonies was found in the liquid phase. The cells from the colonies were studied by immunofluorescence, cytochemical stainings and electron mi­ croscopy. Materials and Methods Materials The lectins used were: Phytohaemagglutinin (PHA-P, Difco, Detroit, Mich.); Concanavalin A (Pharmacia, Uppsala); Pokeweed Mitogen (Gibco, Glasgow). 2-mercaptoethanol was purchased from Merck-Schuchardt (Darmstadt). Lymphoprep used for the separation of cells was obtained from Nyegaard & Co.; Oslo. Fetal calf serum (FCS) was a selected batch purchased from Gibco (Glasgow). It was heat inactivated at 56°C for 30 minutes. Human pool serum was obtained from 20 healthy male donors. After heat inactiva­ tion, the red cell antibodies were adsorbed by incubation of serum with erythrocytes of blood group 0 for 12 hours. Autologous serum was also heat inactivated. Multiwell tissue culture plates, obtained from Falcon (Cat. No. 3008, Oxnard, California), were used for incubation of cells.

Cell separation Peripheral blood from healthy donors was obtained by venepuncture and anticoagulated by heparin (10 units/ml). Mononuclear cells were separated by centrifugation over Lymphoprep as previously described (15). The cells were washed three times in Hank's balanced salt solution without calcium and magnesium ions and resuspended in McCoy SA modified medium containing 20 per cent FCS.

Culture method McCoys SA medium containing 20 per cent FCS and the desired final cell concentration per ml was mixed with 2 per cent boiled agar (Difco-Bacto-Agar) to give a final agar concentration of 0.2 per cent. 0.25 ml of this mixture (containing 7.5 X 104 cells) were repeatedly pipetted into six units of Multiwell Tissue culture plates. Agar was allowed to solidify at room temperature and was overlayered with 0,25 ml of medium containing 20 per cent serum, the lectin and 0.02 ml 1 X 10-2 M 2-ME. Culture plates were incubated at 3rC in a fully humidified atmosphere containing 5 per cent CO 2 for 7,10 and 14 days. Aggregates containing more than 50 cells were defined as colonies and counted through an inverted microscope. For

Microagar Culture System for Lymphocytes· 147 each experiment six units of the Multiwell tissue culture plates were used. The results are expressed as mean per well ± 1 SEM. The reproducibility test has been carried out on a single cell suspension obtained from one healthy donor. The cells were plated into six wells of 5 different Multiwell tissue culture plates. The seeded cell concentration was 10 X 104 cells per well stimulated by 5 !!g PHA per millilitre in the presence of 2-ME. The concentration of agar was 0.2 per cent. After 7 days of incubation colonies were counted and the number (mean ± 1 SEM) of colonies per Multiwell plate and for all 30 samples calculated. The statistical evaluation of the results was performed by analysis of variance according to FISHER, because a normal distribution of the values was found.

Determination of T antigens and rosette formation on lymphocytes from colonies Rosette formation and immunofluorescence tests were performed as previously described (16). PHA-induced colonies were harvested by a Pasteur pipette on day 7 and 14 and one cell suspension prepared by repeated aspiration through a narrow gauge (No. 20) needle. Larger colonies could be disrupted by this procedure, while most of the smaller aggregates remained undissociated. Neuraminidase treated sheep red blood cells (SRBC) were used as indicator cells for rosette formation, while untreated human red blood cells served as controls. B cells were identified by a direct membrane immunofluorescence test (conjugate F-I009, Antihuman Ig Dakopatts, Copenhagen). T -cells were identified by an indirect test using specific horse anti human T-cell serum (ATS) (16) and anti-horse immunoglobulin conjugate (B 0401, Behring­ werke, Marburg). This latter conjugate alone served as the control for direct test (identifying B-cells). Normal horse serum was used as a control for ATS.

Morphological and cytochemical studies Under direct inspection through an inverted microscope PHA-induced colonies and clusters growing in the liquid phase were harvested with a Pasteur pipette on day 5,7,10,14,16 and 18 of incubation. These were placed on microscopic slides by cytocentrifugation (1500 rqm) and stained by May-Grunwald Giemsa. For the evaluation of the colonies grown in agar layer, the liquid was carefully aspirated under the inverted microscope. The plates were then placed on a warm plate (40°). The dried agar layer containing the lymphocytic colonies was stained as previously described (17). In our cytochemical studies the activity of acid phosphatase (AcP) (18), alpha naphthyl acetate acid esterase (ANAE) (19), and II-glucuronidase (BG) (20) were determined. T lymphocytes that showed an intense reaction product of AcP, ANAE, and BG in the paranuclear zone were considered positive. The percentage of positive cells in 20 single colonies and clusters was estimated on day 5, 7, 10 and 14. The mitotic index was also determined in clusters and colonies on day 5 and 7.

Electron microscopy Colonies, aspirated with a Pasteur pipette from the liquid phase on day 7, 10 and 14, were fixed for 1 hour at room temperature in Karnovsky's 1:1 paraformaldehyde-glutaraldehyde fixative, and washed three times with 0.1 M cacodylate buffer (pH 7.4) at +4°C by centrifugation. After postfixation with a 3 percent aqueous solution of OS04 for 1 hour at 0 °C and staining with veronal-acetate buffered uranyl acetate for 1 hour at room temperature, the specimens were rapidly dehydrated in a graded series of alcohol and embedded in Epon 812. Ultrathin sections of silver interference colour were cut with a Reichert Ultracut ultrami­ crotome, stained with lead citrate and examined with a Philips EM 400 electron microscope operating at 80 KV.

Results

Development and morphology of colonies By incubation of 5-20 X 104 mononuclear cells per well in the presence of a mitogen and under the described conditions, the first clusters can be

148 . G. KONWALINKA et

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Fig. 1. Two colonies of the compact, round type after 14 days of incubation, growing in the liquid overlayer (Orig. magnif. 400 X).

found after 2-3 days of incubation. Two days later some large aggregates were observed on the surface of the agar layer. Their subsequent detach­ ment resulted in the transition of the clusters into the liquid phase where the cells continued to proliferate. The other aggregates finished their develop­ ment in agar layer, remained there, or detached later. Some colonies increased markedly in size from days 5 to 14. Two types of colonies can be identified: a compact, round type, which can mainly be seen in the liquid phase (Fig. 1), and a diffuse type, which remains in the agar layer. The same two types of lymphocytic colonies have been previously described (1, 13). More than 90 per cent of all colonies developed under the mitogenic activity of PHA or Con A were of the compact type. The colonies formed in the presence of PWM were not as compact, and degenerated after 7 days. In order to study the optimal growth conditions for in vitro culture of human T lymphocytes and the influence of various factors on the development of colonies the following experiments were carried out.

Effect of different agar concentrations In this experiment the influence of different agar concentrations (0.10; 0.15; 0.20; 0.25; and 0.30 per cent) on the number of colonies after 7,10 and 14 days of incubation was studied (Fig. 2). The results indicate, that no significant difference in colony numbers was obtained on days 10 and 14, when the agar concentration was kept between 0.15 and 0.25 per cent. A very high response on day 7 was found with 0.10 per cent agar, followed by a rapid decrease of colony numbers on day 10 and 14. Therefore all subsequent experiments were carried out with the agar concentration of 0.20 per cent.

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In this experiment the distribution of colonies between liquid and agar layer was also studied (Fig. 3). At the agar concentration of 0.30 per cent almost all cell aggregates were found in the agar layer. By lowering the agar concentration to 0.25-0.15 per cent the percentage of colonies in agar decreased to 20-30 per cent, while in 0.10 per cent agar nearly all colonies could be found in the liquid phase.

Effect of varying concentrations of different lectins PHA, Con A, or PWM in the indicated concentrations (Fig. 4) with 5 X 10-4 M 2-ME were added to the liquid phase. The cell concentration was 2 X 105 per ml. After incubation for 7, 10 and 14 days the formed colonies were counted. The highest mitogenic effect on colony number during the

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observed period of incubation was obtained with PHA. The optimal concentration of this lectin was 3 to 5 micrograms per ml. The highest colony number was found after 7 days of incubation. An optimal mitogenic effect of Con A was observed with the concentration of 10 micrograms per ml. The lowest stimulation of all three lectins was obtained by PWM. The colonies induced by this mitogen could be counted only on day 7, because they quickly degenerated during further incubation.

Relation between the seeded cell concentration and the number of colonies Increasing cell numbers between 2.5 and 20 X 104 cells per well were incubated in the presence of 3 and 5 micrograms PHA per ml with and without 2-ME. In the whole range of the tested cell concentrations and in the presence of 2-ME the linearity was obtained on day 7 with 3 and 5 micrograms PHA per ml (Fig. 5). On day 10 a linearity existed only up to 15 X 104 cells per well with 5 micrograms PHA per ml, while after 14 days of incubation a linear relationship could be observed only up to 10 X 104 cells per well. Without 2-ME a linearity was retained up to a cell concentration of 15 X 104 cells per well with 3 or 5 micrograms PHA per ml on all days of incubation. The addition of 2-ME to the cultures resulted in a longer linearity on day 7, and a significantly increased colony number.

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Effect of serum

The comparison between FSC, human pool and autologous serum was carried out with lymphocytes obtained from four healthy donors. In these experiments the same serum was always used in agar and liquid layer. The tested concentrations of serum were 10, 20 and 30 per cent. The colonies were counted on days 7, 10 and 14 (Fig. 6). The optimal concentration for autologous serum and FCS was 20 per cent on all days of incubation, E ~

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152 . G.

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whereas the human pooled serum did not reach a plateau even at the concentration of 30 per cent.

Plating efficiency By using mononuclear peripheral blood cells from 10 healthy persons under the described conditions in the presence of FCS, the mean colony number was 82±37 per lOX 104 seeded cells on day 7; 69±37 on day 10; and 57.7±36.4 on day 14.

Reproducib11ity test The analysis of variance according to Fisher showed no significant difference in the growth of colonies between the 5 Multiwell tissue culture plates (p > 0.05).

Immunofluorescence and rosette formation In order to investigate the proportion of T-cells in the PHA-induced colonies and clusters, two methods for identification of T -cells were used: E-rosette formation and specific anti T-cell sera. On day 7, 90 per cent rosette-fonning cells (E-RFC) (= 3 erythrocytes per lymphocyte) were counted. To exclude non-specific adherence of red blood cells due to residual membrane-bound agar, native human red blood cells were used as controls. In accordance with other authors (24) 15 per cent of lymphocytes fonned rosettes with human red blood cells. On day 14 identical results were obtained. The T-cell identification was confirmed by membrane­ immunofluorescence using ATS on days 7 and 14. About 90-95 per cent of cells showed a characteristic diffuse, patchy, spot-like or cap-like fluores­ cence. No B-cells could be detected by using anti-immunoglobulin conju­ gate.

Morphological and cytochemical studies The colonies and clusters that form in the liquid phase and agar layer stained by May Grunwald Giemsa had morphological characteristics of PHA-transformed blast-like cells. In order to study the T-cell differentiation of cells in clusters and colonies, the cytoplasmic enzyme activities of AcP, ANAE, and BG were detennined on days 5, 7, 10, 14, 16 and 18. Strong activity was present in all cells during the observed period. Two patterns of ANAE activity were found from day 5 to 14: a dot-like reaction product, or a distinct granular cytoplasmic staining. The analysis of colonies showed that up to 25 per cent of cells had these staining characteristics. In clusters the percentage of ANAE-positive cells varied from 5 to 40 per cent. A slightly lower percentage of BG positive cells was found in the colonies (15 per cent) and clusters from day 5 to day 14. On day 16 and 18 of incubation most of the lymphocytes had lost their enzymatic activity for ANEA and BG.

Microagar Culture System for Lymphocytes· 153

Fig. 7. A cluster in the liquid phase after 6 days of incubation stained by acid phosphatase showing a mitotic figure.

As described by other authors (23) some colonies and clusters also contained large cells with a typical macrophage-like, diffuse cytoplasmatic ANAE and AcP activity. While single, central-located macrophages could be observed up to day 10 of incubation, their number markedly increased in some colonies up to 5 per cent after a longer incubation period. The mitotic index determined in clusters and colonies (Fig. 7) was 2-3 per cent on day 5, this percentage decreased to 0.1 per cent on day 7 of incubation. Electron microscopic studies Translucent electron microscopy (TEM) of single colonies on day 7, 10 and 14 showed that the majoritiy of cells had ultrastructural characteristics of lymphoblast-like cells, while some cells morphologically resembled small, resting lymphocytes (Fig. 8). The lymphoblast-like cells had large, round or markedly indented lobular nuclei often with prominent nucleoli, but little condensed chromatin. The cytoplasm contained numerous mitochondria, rough endoplasmatic reticulum and polyribosomes. There were also variable numbers of electron-dense, lysosomal-like organelles and some translucent lipid droplets. Discussion The purpose of our investigation was to develop a simple and reproduc­ ible micro-agar culture method, which can be easily used for studying the regulation of colony-forming lymphocytes. The described procedure has

154 . G . KONWALINKA et al.

Fig. 8. The ultrastructural presentation of cells from a colony after 14 days of incubation, showing the heterogeneous structure of the lymphocytes. Small resting lymphocyte (SL) (arrow) with a high nuclear to cytoplasmic ratio and no evidence of cytoplasmic organelles. Orig. magnif. 6200 X •

several advantages compared to the existing agar systems. In this culture method no preincubation of cells in liquid phase, or addition of red cells, bovine serum albumin, transferrin, L-glutamine, L-cystine and FeCl3 are necessary. According to obtained results, the addition of 2-ME was not essential for the growth of T lymphocytes, but enhanced colony formation and improved the linearity.

Microagar Culture System for Lymphocytes· 155

In recently described methods for cloning T-Iymphocytes the mononuc­ lear cells were suspended in a liquid overlayer and stimulated by an underlayer containing irradiated mononuclear cells and PHA (21). Since it is well known that lymphocytes agglutinate in suspensions containing lectins, the mononuclear cells were immobilized in agar and the lectins added to the liquid phase. The proliferation of cells takes place in agar during the first 5 days of incubation. Some of the formed aggregates detach from the surface and continue their growth in liquid phase. The maximum proliferation occurs around day 5 of incubation as can be seen from the high mitotic index. The low incubation volume (0.5 ml) makes possible the use of Multiwell tissue culture plates and consequently a low consumption of material. The liquid phase of our system maintains the cells during a prolonged period of incubation (up to three weeks). Furthermore, the addition of different test substances to this phase is simple and can be easily controlled. The presence of clusters and colonies in liquid phase makes morphological, cytochemical, immunofluorescence and electronmicro­ scopic studies of the cells from these aggregates extremely convenient. It is not yet established whether the mitogen-stimulated colony growth results from the clonal proliferation of PHA-transformed mature lympho­ cytes or from T cell progenitors expressing PHA receptors (21). As high density peripheral mononuclear cells were cloned in our experiments, it is likely that the blastic transformation was the main cause of colony forma­ tion on day 7 of incubation. Since the liquid phase of our system improves the culture conditions during a prolonged period of incubation, it is possible that some immature progenitors were involved in colony forma­ tion on days 10 and 14 of incubation. This hypothesis is also supported by the fact that some aggregates picked up and stained after 10 to 14 days of incubation contained as a sign of immaturity only a low percentage of BG and ANAE positive cells. Further experiments using discontinous albumin gradient centrifugation and immunological methods are necessary to estab­ lish the nature of the cells responsible for the colony growth after 7,10 and 14 days of incubation. The agar concentration plays an important role in the process of colony formation. Not only the colony number (21), but also the colony size depend on the agar concentration. We found that the most constant number of colonies during 7, 10 and 14 days of incubation can be obtained between 0.15 and 0.25 per cent agar. In the higher agar concentration of 0.3 per cent the colony formation was decreased on day 7. Moreover, in this agar concentration most of the colonies remained in the agar layer and thereby by morphological and other studies became more difficult. In our test of mitogenic activity all three lectins induced colony forma­ tion, as was found by other authors (5,11). PHA showed the best mitogenic stimulation with all cells and on all days of observation. We also studied the diffusibility of PHA by adding this lectin either to the liquid or to agar layer. After 7, 10 and 14 days of incubation no

156 . G. KONWALINKA et al.

significant change was found in the total number and size of induced colonies. A linear correlation was demonstrated between the number of seeded cells and the number of colonies. In the presence of 2-ME a significantly higher number of colonies was obtained with all cell concentrations and during the whole observation period. On the other hand, a «breaking point» of linearity was observed at a lower number of seeded cells with a longer incubation. Without 2-ME this tendency was found at a higher cell concentration. Both can be interpreted as the result of the consumption of nutritional factors in the medium because of an increased cell proliferation. The absence of growth at the concentrations below 5 X 104 seeded cells could be explained by the insufficient cell-cell contact and low concentra­ tion of diffusible mediators (22). The source and quality of serum are essentially important for the cell maintenance and proliferation in vitro. Some authors (10, 13, 22) have found, that the addition of autologous plasma significantly improves the growth of lymphocytic colonies. FCS was also used in a system in which special nutritional supplements were necessary for optimal growth (13). Therefore we tested the effects of the addition of FCS, human pool and autologous serum in our system. The best growth was obtained in the presence of autologous serum at the concentra­ tion of 20 and 30 per cent. The addition of 30 per cent of human pool serum resulted in a slightly lower number of colonies compared to the autologous serum. The poorest growth was obtained in the presence of FCS. Since it is not always convenient for routine examinations to prepare autologous serum, we suggest the use of human pool serum as an alternative. Spontaneous rosette formation with sheep red blood cells (E-rosetting) and specific A TS were used for the identification of T -cells. We selected these two methods for characterisation of lymphocytes within the colonies as the earlier embryonic appearance of T antigens compared to E receptors has been reported (25). However, in our system no differences in the percentage of E-rosetting and ATS positive cells (90 per cent) could be found on day 7 and 14. Moreover, the finding of 90 to 95 per cent E-RFC on day 14 confirmed the viability and metabolic activity of these cells, since only viable T cells bind SRBC (24). No B-cells could be identified using anti-immunoglobulin antisera. The morphological and cytochemical studies of PHA-induced colonies and clusters grown in the liquid phase could also be easily performed from day 5 to day 18. ANAE and BG, which are considered as markers for T cell maturation, were clearly positive in 15 to 25 per cent of cells in colonies from day 5 to 14, whereas AcP showed a high percentage of positive cells regardless of their maturity grade. A variable number of ANAE and BG positive cells was found in clusters from day 5 to 14. It seems that the clusters with a high number of ANAE and BG-positive cells remain in this proliferation stage, whereas those with a low percentage of positive cells proliferate further and form colonies. Pure macrophagic colonies were not

Microagar Culture System for Lymphocytes· 157

found even after 18 days of incubation, unlike the results of other authors (23). It is possible that the liquid overlayer of our system enables a longer survival of T-cell colonies and does not favor the formation of pure macrophagic colonies. TEM of the colonies confirmed the lymphoblastic nature of the cells and their ultrastructural characteristics closely resembled those described by other authors for PHA-transformed human T-cell blasts (1, 23) and lym­ phoblastoid cell lines (26, 27). In accordance with the cytochemical findings the variable number of mitochondria and lysosomes revealed by TEM provides further evidence for the different stages of maturation of cells within individual colonies. The described single-layer micro-agar system for in vitro culture of human T-Iymphocytes has the advantage of low incubation volume (0.5 ml). This enables a better cell-cell interaction at a lower concentration of seeded cells, and a higher concentration of lymphocyte colony enhancing factor which stimulates the development of T-cell colonies (22). Further­ more, the described method is an easy and reproducible system for the routine study of the regulating mechanisms of human lymphocyte function. Acknowledgment We wish to thank Dr. E. CRONKITE for fruitful discussions and reading the manuscript. The excellent technical assistance of A. MANESCHG, E. BOGUSCH, F. OBERWASSERLECHNER, E. ORQUE, ist gratefully appreciated.

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