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Effect of glass-adherent cells on the blastogenic response of ‘purified’ lymphocytes to phytohemagglutinin
Experimental Cell Research61 (1970) 153-158
EFFECT OF GLASS-ADHERENT OF ‘PURIFIED’
CELLS ON THE BLASTOGENIC
LYMPHOCYTES
RESPONSE
TO PHYTOHEMAGGLUTININ
W. R. LEVIS and J. H. ROBBINS Dermatology
Branch, National
Cancer Institute, National Institutes of Health, Bldg IO, Room 12-N-238, Bethesda, Md 20014, USA
SUMMARY When human, peripheral blood leukocytes are cultured with phytohemagglutinin, (PHA), the lymphocytes respond by undergoing blastogenesis, DNA synthesis, and mitotic division. However, this response is markedly decreased in cultures of lymphocytes which have been separated from those leukocytes which adhere to nylon fiber and glass. The response of these ‘purified’ lymphocyte cultures can be restored by adding either autologous, or homologous, glass-adherent, phagocytic leukocytes. The responding cells in these reconstituted cultures are derived from the ‘purified’ lymphocytes. Our results indicate an obligatory role for glass-adherent cells in the response of ‘purified’ lymphocytes to phytohemagglutinin.
It has been known for some time that the small lymphocyte undergoes blastogenesis, DNA synthesis, and mitotic division in cultures of human peripheral blood leukocytes in response to phytohemagglutinin (PHA), an extract of the red kidney bean [5, 91. It has not previously been determined, however, if the other leukocytes present in these cultures serve an essential function in this response of the lymphocytes. One approach in resolving this question is to study the response to PHA of lymphocytes which have been ‘purified’ by varying degrees of separation from those periperal blood leukocytes, most of which are phagocytic, which adhere to glass, cotton, or nylon fiber. The response of such ‘purified’ lymphocytes to PHA has been studied, but the reported results have been conflicting. Walker & Fowler [12] reported that ‘purified’ lymphocytes give an increased response to PHA, while Wilson [13] found a decreased response. Other in-
vestigators [3, 81 stated that ‘purified’ lymphocytes respond normally to PHA, while Oppenheim et al. [7] claimed that such cultures have an impaired response to “suboptimal” but not to “optimal” concentrations of PHA. We have investigated the response to PHA of highly purified lymphocyte cultures and have utilized a very sensitive and rapid assay for DNA synthesis which enabled accurate determinations to be performed as early as the second and third days of the culture period before any of the cultures had passed the time of maximal DNA synthesis. In this report we present the results of our studies which indicate that the blastogenic response to all concentrations of PHA is markedly impaired in lymphocyte cultures which have been highly ‘purified’ and that addition of either autologous or homologous glass-adherent cells to such cultures increases the response of the lymphocytes. Exptl Cell Res 61
154
W. R. Levis & J. H. Robbins MATERIALS
AND METHODS
Obtaining glass-adherent cells and ‘purified’ lymphocytes All cells were obtained from heparinized, human venous blood. The culture fluid was composed of one part of autologous plasma and of 4 parts of Medium 199 (Difco, Detroit, Mich.) containing bicarbonate, penicillin, and streptomycin. The glass-adherent (g-a) cells were obtained by collecting the plasma and the upper portion of the buffy coat from 10 ml aliquots of heparinized blood subjected to a room temperature centrifugation for 5 min at 300 g. The mononuclear-rich plasma aliquots so obtained were then recentrifuged at 150 g for 6 min, the supernates were discarded, and the cell buttons were pooled and then dispersed to a concentration of 24 x lo8 cells/ml of the culture fluid. 0.4 ml aliquots of this mononuclearrich culture fluid were incubated at 38°C with room air as the gas phase in stoppered, 16 x 85 mm Leighton culture tubes (Bellco Glass, Vineland, N.J.), each of which contained a 9 x 9 mm glass coverslip to which many cells adhered. The coverslips were removed after 1 to 5 days’ incubation and, in our early experiments, washed free of most of the non-g-a cells by being dipped into three washes of 38°C Medium 199. However, since this procedure often still left a considerable number of lymphocytes on the coverslips, the dipping process was supplemented by subjecting each side of a dipped coverslip to a flow of 2 ml of the Medium 199 dispensed from a pipette. Coverslips so washed usually contained either very few or no morphologically identifiable lymphocytes and, when placed in culture fluid with PHA (Burroughs-Wellcome, Tuckahoe, N.Y.), gave relatively little or no evidence of DNA synthesis. The number of g-a cells appeared to be reasonably constant on each of the coverslips prepared for a given experiment from any batch of mononuclearrich culture fluid; however, the number of g-a cells per coverslip obtained from different batches varied over at least the twenty-fold range of 2 x lo3 to 4 x lo4 cells per coverslip. Most of the mononuclear cells on the coverslips had the morphology of macrophages and were capable of phagocytosing polystyrene latex particles of 1 pm diameter (Dow Chemical, Midland, Mich.). Such phagocytic cells have been demonstrated to be derived predominantly, if not solely, from the monocytes [ll]. ‘Purified’ lymphocytes were obtained from the leukocyte-rich plasma resulting after 1 to 14 h of gravity sedimentation of heparinized blood at 38°C. This leukocyte-rich ulasma was diluted with an eaual vol of Medium 199 and passed onto a nylon fiber column 121(Fenwal, Morton Grove, Ill.). The column was then washed with 2 vol of Medium 199, and the effluent, which contained predominantly small lymphocytes, erythrocytes, and platelets, was collected in a glass bottle. Most of the few, contaminating, g-a leukocytes were then removed bv uermitting them to adhere to the bottle’s inner surfaces usually until more than 99 % of the remaining leukocytes had the morphology of lymphocytes. The resulting cell suspension was decanted and centrifuged at 150 Exptl
Cell Res 61
g, the supemate was removed, and the centrifuged cells were diluted to a concentration of 0.5 to 3 x lo6 lymphocytes/ml of the culture fluid. 0.4 ml aliquots of these lymphocyte suspensions, cultured in stoppered Leighton tubes with air as the gas phase, are hereinafter referred to as ‘purified’ lymphocyte cultures. When required, such cultures were ‘reconstituted with g-a cells by placing a coverslip containing g-a cells in the Leighton tube’s window. In contrast to the terms ‘purified’ lymphocyte culture and ‘reconstituted’ culture, the term ‘leukocyte’ culture will refer to the widely used culture containing all the formed elements of peripheral blood obtained without any separatory technique other than gravity sedimentation or gentle centrifugation of the heparinized whole blood to remove most of the erythrocytes.
Assessing DNA synthesis and blastogenesis DNA synthesis was evaluated after addition to each culture of 4 &i cf 8HTdR (spec. act. 17-22 Ci/mM) folllwed by a 3 h incubation period at 38°C. The cells were then collected by passing the culture fluid through a 0.45 pm poresize Millipore filter (Millipore, Bedford, Mass.) which was then washed with saline, 5 % trichloroacetic acid, 95 % and 100 % ethanol. The filter was then dried in vacua at 80°C wet with a ‘Liquifluor’-toluene scintillation fluid (New England Nuclear. Boston. Mass.). and its radioactivity was determined on a”Tri-Carl;’ spectrometer (Packard. Downers Grove. Ill.). This simule and rapid filter technique has been shown [IO]-to be a very sensitive and specific assay for determining the incorporation of radioactivity into the DNA of lymphocytes undergoing blastogenesis. Blastogenesis was assessed morphologically by Giemsa staining of the Leighton tubes’ coverslips or the smears made of the cell pellets obtained from gentle centrifugation of the culture fluid.
RESULTS Effect of g-a cells on the response of ‘purified lymphocytes to PHA When ‘purified’ lymphocytes were cultured with either a high or a low concentration of PHA in the absence of g-a cells, either very little or no significant levels of DNA synthesis were detected (broken curves of fig. la, b). However, when these ‘purified’ lymphocytes were cultured with PHA in the presence of g-a cells, markedly increased DNA synthesis occurred (solid curves of fig. la, b). It did not seem likely that the increased synthesis was due to synthesis by the g-a cells, since PHA-containing cultures of
‘Purified’ lymphocytes and phytohemagglutinin
155
Fig. I. Abscissa: culture time (hours); cpm. O-O, lymphocytes with added g-a cells; O---O, lymphocytes without addedg-a cells.(a) 5 ,ugof PHA/ml of culture fluid; (b) 0.33 pg of PHA/ml
ordinate:
.
-
the g-a cells alone showed either no detectable DNA synthesis or a level of synthesis far too low simply to account for the massive increases in DNA synthesis obtained after the g-a cells had been added to the PHA-containing, ‘purified’ lymphocyte cultures. For example, when a coverslip containing the g-a cells used in the experiment of fig. 1 a was cultured with PHA at a concentration of 5 ,ug/ml of culture fluid, only 250 cpm were obtained at the 48th hour of culture. However, as shown in fig. la, addition of similar coverslips to the ‘purified’ lymphocyte cultures containing PHA led to increased DNA synthesis represented by approx. 45000 cpm at the 48th h of culture. Nor did it seem likely that the increased synthesis caused by addition of the g-a cells was due simply to a non-specific increase by the g-a cells in the number of cells in the culture, for increasing the number of lymphocytes by 40 % gave no more than a 25 % increase in DNA synthesis at the 48th hour of culture in contrast to approximately a 1000 % increase given by adding the g-a cells.
lymphocytes/ml of culture fluid.
The amount of DNA synthesized in our ‘purified’ lymphocyte cultures in response to a given concentration of PHA is correlated with the number of g-a cells in the culture. Thus, there was considerable DNA synthesis in PHA-containing cultures comprised 99 % or less of lymphocytes and 1 % or more of other leukocytes. In most of our ‘purified’ lymphocyte cultures which, as previously stated, had a cell population in which more than 99 % of the cells had the morphology of small lymphocytes, it was not practicably possible to obtain a precise count of the very small number of contaminating g-a cells from morphological examination of stained smears. However, the relative degrees of the very low contamination in such cultures could be reasonably assessed by placing in such cultures a clean coverslip to which contaminating g-a cells would adhere. After several days in culture the coverslips were removed and, after being air-dried and fixed in methanol, were stained with Giemsa stain. In this manner we observed that batches of ‘purified’ lymphocytes giving the greatest response to Exptl Cell Res 61
156
W. R. Levis & J. H. Robbins
Fig. 2. Effect of homologous g-a cells on the blastogenic response of ‘purified’ lymphocytes to PHA. Lymphocytes, 106/ml of culture fluid; PHA, 2.3 @g/ml of culture fluid. Giemsa stained, x 400, 48th hour of culture. (a) Smear from a culture of lymphocytes alone; (b) coverslip from a culture of lymphocytes with added g-a cells; (c) coverslip from a culture of g-a cells with PHA; (d) smear from a culture of lymphocytes with PHA; (e) coverslip from a culture of lymphocytes with PHA and added g-a cells. Further details in text.
PHA also deposited the most g-a cells on their coverslips. We observed also that our purest lymphocyte preparations, which gave no evidence of having deposited any cells on such coverslips, synthesized the least amount of DNA in response to PHA. It should be noted, however, that it is nevertheless possible that the response to PHA given by these very pure preparations of lymphocytes may be due to the presence of a small number of contaminating g-a cells which we are unable to detect morphologically. Further evidence that a very small number of g-a cells may lead to considerable DNA synthesis in ‘purified’ lymphocyte cultures containing PHA was shown by the fact that we have obtained increases of many thousands of cpm in cultures containing 0.5 to 1 X lo6 lymphocytes by the addition to such cultures of a coverslip containing less than 4 x lo3 g-a cells. We have observed an increase in DNA synthesis in the presence of g-a cells in each of the 20 experiments conducted with ‘purified’ lymphocytes and PHA. The effect could Exptl Cell Res 61
be detected with all the concentrations of PHA tested, including very high and even toxic concentrations, if the DNA assayswere conducted before the reconstituted cultures had passed the time of their maximal DNA synthesis. In this regard it is important to note that we have performed an experiment in which highly stimulated, reconstituted cultures had a peak of DNA synthesis prior to the 84th hour of culture, while the ‘purified’ lymphocyte cultures peaked at a later time. Obviously, in such experiments valid comparisons between the two types of cultures. can be obtained only if the DNA determinations are made early in the culture period and, thus, prior to the time of peak DNA synthesis in either of the cultures. In PHA-stimulated leukocyte cultures the amount of DNA synthesized is related to the transformation of small lymphocytes into blastoid cells which undergo mitotic division [5]. In our ‘purified’ lymphocyte and reconstituted cultures a similar correlation was observed between DNA synthesis and the number of blastoid cells. Thus, in cultures
‘Purified’ lymphocytes and phytohemagglutinin with no detectable DNA synthesis, no blastoid cells were found in stained smears. In cultures synthesizing DNA, on the other hand, blastoid cells were regularly observed, their numbers increasing with increases in the amounts of DNA synthesized. This correlation is illustrated by the results at the 48th hour of culture in an experiment in which homologous g-a cells increased the response of ‘purified’ lymphocyte cultures to PHA (fig. 2). In cultures of lymphocytes alone (fig. 2a), of lymphocytes with g-a cells (fig. 2b), or of g-a cells with PHA (fig. 2c), no blastoid cells were apparent and none of the cultures gave more than 160 cpm. Lymphocyte cultures containing PHA (fig. 2d) showed a very rare blastoid cell (arrow) and gave up to 450 cpm. However, cultures containing lymphocytes with g-a cells and PHA gave up to 50000 cpm and had large numbers of blastoid cells many of which were found in the typical rosette formation surrounding a centrally located g-a cell (fig. 2e). Origin of responding cells in the reconstituted cultures The possibility has already been briefly considered that the DNA-synthesizing and dividing blastoid cells in the reconstituted cultures might have been derived from the g-a cells or from lymphocytes unintentionally transferred with them. Despite the fact that this possibility seemed unlikely, it was, nevertheless, considered essential to determine definitively the population of origin of the responding cells in the reconstituted cultures. A definitive determination was made possible when we found that homologous, as well as autologous, g-a cells could increase the response to PHA of ‘purified’ lymphocyte cultures, for we were then able to identify a cell line by the sex chromosomes of the dividing cells when the lymphocytes and the g-a
157
cells were obtained from donors of the opposite sex. One of these determinations was performed in the experiment already presented in fig. 2 in which the lymphocytes were obtained from a female and the g-a cells from a male donor. At the 48th hour of culture all 25 of the dividing cells examined in the reconstituted cultures had a female chromosome complement, indicating that most, if not all, of the responding cells had been derived from the female’s lymphocyte population. DISCUSSION Our results show that lymphocytes which have been adequately separated from other leukocytes have a markedly diminished blastogenic response to PHA. The lymphocytes’ response is markedly increased if either autologous or homologous g-a cells are added to the cultures. The way in which the g-a cells affect the response of the lymphocytes is not known. The mechanism may be due to effects of the g-a cells on the cultural conditions, for example, through changes in the nutrients, pH, or oxygen levels of the medium. Or the mechanism may be more specific involving, for example, the production by the g-a cells of specific substances or physical conditions which affect the lymphocytes either through the medium or via direct cell-to-cell contact between the lymphocytes which respond and the g-a cells. Of course, the possibility must also be considered that the lymphocytes were injured during or after their separation from the other leukocytes; however, if this were the case, the interesting question still would remain as to how the g-a cells are able to repair, or to compensate for, such an injury. We have found also that the ability of g-a cells to increase the blastogenic response of ‘purified’ lymphocyte cultures is not limited Exptl Cell Res 61
158 W. R. Levis & J. H. Robbins only to PHA as the blastogenic stimulus but applies also to homologous cells and to antigens such as histoplasmin, tuberculin purified protein derivative, and tetanus and diphtheria toxoids. Thus, with our technique we have confirmed the observation originally reported by other investigators [3,6,7] that cultures of ‘purified’ lymphocytes, obtained from a person whose leukocyte cultures normally responded to a specific antigen, did not undergo significant blastogenesis in response to that antigen unless autologous g-a cells were added to the lymphocyte cultures. In addition, we have shown that the immunologic specificity of the response of such reconstituted cultures is determined by the lymphocyte population [4], that not only autologous, but also homologous, g-a cells can restore responses to such cultures, and that sex chromosome analysis reveals the dividing cells in such cultures to be derived from the lymphocyte and not from the g-a cell donor. We have confirmed also the reports by McFarland [6] and by Gordon [l] that mixtures of ‘purified’ lymphocytes from two unrelated individuals give increased DNA synthesis if they contain g-a cells from at least one of the individuals. However, since we have found that coverslips containing g-a cells generally serve as a very potent blastogenic stimulus when placed in an unrelated individual’s leukocyte or ‘purified’ lymphocyte cultures, analysis of the sex chromosome complement was undertaken in order to show that cells from both populations of ‘purified’ lymphocytes underwent cell division in a mixed culture when a coverslip containing only one of the individual’s g-a cells was included in that culture. Thus, our results show that g-a cells are required in order for ‘purified’ lymphocytes
Exptl Cell Res 61
to respond with a maximal blastogenic response in cultures containing PHA, antigens, or homologous cells. These findings suggest that the g-a cells which are present in all, typical, unpurified leukocyte cultures may serve essential functions in the blastogenic response in such cultures. If these functions are not related only to the in vitro conditions of culture, it is possible that the g-a cells or their progenitors may function in a similar manner in immunologic processes occurring in vivo in which lymphocytes are stimulated to synthesize DNA and to undergo mitotic division, for example, in delayed hypersensitivity and homograft reactions. The authors thank Mrs Lisa C. van de Water for performing the chromosome analyses and Professor Leonard Ornstein for encouragement and a critical reading of this manuscript.
REFERENCES 1. Gordon, J, Proc sot exptl biol med 127 (1968) 30. 2. Greenwalt, T J, Gajewski, M & McKenna, J C, Transfusion 2 (1961) 221. 3. Hersh, E M & Harris, J E, J immunol 100 (1968) 1184. 4. Levis, W R & Robbins, J H, Fed proc 28 (1969) 566. 5. Ling, N R, Lymphocyte stimulation. NorthHolland Publishing Co., Amsterdam, (1968). 6. McFarland, W, Proceedings of the third annual leukocyte culture conference (ed W 0 Rieke) p. 77. Aonleton-Centurv-Crofts. New York (1969). 7. Oppenheim, J J, Leienthal, B G & Hers& E M, J immunol 101 (1968) 262. 8. Rabinowitz, Y, Blood 23 (1964) 811. Robbins. J H. Science 146 (1964) 1648. 1;: Robbins; J H, Burk, P G‘ & Levis, W R, Fed proc 28 (1969) 363. 11. Schrek, R & Rabinowitz, Y, Proc sot exptl biol med 113 (1963) 191. 12. Walker, R I & Fowler, I, Exptl cell res 38 (1965) 379. 13. Wilson, D B, J exptl zoo1 162 (1966) 161. Received December 16, 1969.
EFFECT OF GLASS-ADHERENT OF ‘PURIFIED’
CELLS ON THE BLASTOGENIC
LYMPHOCYTES
RESPONSE
TO PHYTOHEMAGGLUTININ
W. R. LEVIS and J. H. ROBBINS Dermatology
Branch, National
Cancer Institute, National Institutes of Health, Bldg IO, Room 12-N-238, Bethesda, Md 20014, USA
SUMMARY When human, peripheral blood leukocytes are cultured with phytohemagglutinin, (PHA), the lymphocytes respond by undergoing blastogenesis, DNA synthesis, and mitotic division. However, this response is markedly decreased in cultures of lymphocytes which have been separated from those leukocytes which adhere to nylon fiber and glass. The response of these ‘purified’ lymphocyte cultures can be restored by adding either autologous, or homologous, glass-adherent, phagocytic leukocytes. The responding cells in these reconstituted cultures are derived from the ‘purified’ lymphocytes. Our results indicate an obligatory role for glass-adherent cells in the response of ‘purified’ lymphocytes to phytohemagglutinin.
It has been known for some time that the small lymphocyte undergoes blastogenesis, DNA synthesis, and mitotic division in cultures of human peripheral blood leukocytes in response to phytohemagglutinin (PHA), an extract of the red kidney bean [5, 91. It has not previously been determined, however, if the other leukocytes present in these cultures serve an essential function in this response of the lymphocytes. One approach in resolving this question is to study the response to PHA of lymphocytes which have been ‘purified’ by varying degrees of separation from those periperal blood leukocytes, most of which are phagocytic, which adhere to glass, cotton, or nylon fiber. The response of such ‘purified’ lymphocytes to PHA has been studied, but the reported results have been conflicting. Walker & Fowler [12] reported that ‘purified’ lymphocytes give an increased response to PHA, while Wilson [13] found a decreased response. Other in-
vestigators [3, 81 stated that ‘purified’ lymphocytes respond normally to PHA, while Oppenheim et al. [7] claimed that such cultures have an impaired response to “suboptimal” but not to “optimal” concentrations of PHA. We have investigated the response to PHA of highly purified lymphocyte cultures and have utilized a very sensitive and rapid assay for DNA synthesis which enabled accurate determinations to be performed as early as the second and third days of the culture period before any of the cultures had passed the time of maximal DNA synthesis. In this report we present the results of our studies which indicate that the blastogenic response to all concentrations of PHA is markedly impaired in lymphocyte cultures which have been highly ‘purified’ and that addition of either autologous or homologous glass-adherent cells to such cultures increases the response of the lymphocytes. Exptl Cell Res 61
154
W. R. Levis & J. H. Robbins MATERIALS
AND METHODS
Obtaining glass-adherent cells and ‘purified’ lymphocytes All cells were obtained from heparinized, human venous blood. The culture fluid was composed of one part of autologous plasma and of 4 parts of Medium 199 (Difco, Detroit, Mich.) containing bicarbonate, penicillin, and streptomycin. The glass-adherent (g-a) cells were obtained by collecting the plasma and the upper portion of the buffy coat from 10 ml aliquots of heparinized blood subjected to a room temperature centrifugation for 5 min at 300 g. The mononuclear-rich plasma aliquots so obtained were then recentrifuged at 150 g for 6 min, the supernates were discarded, and the cell buttons were pooled and then dispersed to a concentration of 24 x lo8 cells/ml of the culture fluid. 0.4 ml aliquots of this mononuclearrich culture fluid were incubated at 38°C with room air as the gas phase in stoppered, 16 x 85 mm Leighton culture tubes (Bellco Glass, Vineland, N.J.), each of which contained a 9 x 9 mm glass coverslip to which many cells adhered. The coverslips were removed after 1 to 5 days’ incubation and, in our early experiments, washed free of most of the non-g-a cells by being dipped into three washes of 38°C Medium 199. However, since this procedure often still left a considerable number of lymphocytes on the coverslips, the dipping process was supplemented by subjecting each side of a dipped coverslip to a flow of 2 ml of the Medium 199 dispensed from a pipette. Coverslips so washed usually contained either very few or no morphologically identifiable lymphocytes and, when placed in culture fluid with PHA (Burroughs-Wellcome, Tuckahoe, N.Y.), gave relatively little or no evidence of DNA synthesis. The number of g-a cells appeared to be reasonably constant on each of the coverslips prepared for a given experiment from any batch of mononuclearrich culture fluid; however, the number of g-a cells per coverslip obtained from different batches varied over at least the twenty-fold range of 2 x lo3 to 4 x lo4 cells per coverslip. Most of the mononuclear cells on the coverslips had the morphology of macrophages and were capable of phagocytosing polystyrene latex particles of 1 pm diameter (Dow Chemical, Midland, Mich.). Such phagocytic cells have been demonstrated to be derived predominantly, if not solely, from the monocytes [ll]. ‘Purified’ lymphocytes were obtained from the leukocyte-rich plasma resulting after 1 to 14 h of gravity sedimentation of heparinized blood at 38°C. This leukocyte-rich ulasma was diluted with an eaual vol of Medium 199 and passed onto a nylon fiber column 121(Fenwal, Morton Grove, Ill.). The column was then washed with 2 vol of Medium 199, and the effluent, which contained predominantly small lymphocytes, erythrocytes, and platelets, was collected in a glass bottle. Most of the few, contaminating, g-a leukocytes were then removed bv uermitting them to adhere to the bottle’s inner surfaces usually until more than 99 % of the remaining leukocytes had the morphology of lymphocytes. The resulting cell suspension was decanted and centrifuged at 150 Exptl
Cell Res 61
g, the supemate was removed, and the centrifuged cells were diluted to a concentration of 0.5 to 3 x lo6 lymphocytes/ml of the culture fluid. 0.4 ml aliquots of these lymphocyte suspensions, cultured in stoppered Leighton tubes with air as the gas phase, are hereinafter referred to as ‘purified’ lymphocyte cultures. When required, such cultures were ‘reconstituted with g-a cells by placing a coverslip containing g-a cells in the Leighton tube’s window. In contrast to the terms ‘purified’ lymphocyte culture and ‘reconstituted’ culture, the term ‘leukocyte’ culture will refer to the widely used culture containing all the formed elements of peripheral blood obtained without any separatory technique other than gravity sedimentation or gentle centrifugation of the heparinized whole blood to remove most of the erythrocytes.
Assessing DNA synthesis and blastogenesis DNA synthesis was evaluated after addition to each culture of 4 &i cf 8HTdR (spec. act. 17-22 Ci/mM) folllwed by a 3 h incubation period at 38°C. The cells were then collected by passing the culture fluid through a 0.45 pm poresize Millipore filter (Millipore, Bedford, Mass.) which was then washed with saline, 5 % trichloroacetic acid, 95 % and 100 % ethanol. The filter was then dried in vacua at 80°C wet with a ‘Liquifluor’-toluene scintillation fluid (New England Nuclear. Boston. Mass.). and its radioactivity was determined on a”Tri-Carl;’ spectrometer (Packard. Downers Grove. Ill.). This simule and rapid filter technique has been shown [IO]-to be a very sensitive and specific assay for determining the incorporation of radioactivity into the DNA of lymphocytes undergoing blastogenesis. Blastogenesis was assessed morphologically by Giemsa staining of the Leighton tubes’ coverslips or the smears made of the cell pellets obtained from gentle centrifugation of the culture fluid.
RESULTS Effect of g-a cells on the response of ‘purified lymphocytes to PHA When ‘purified’ lymphocytes were cultured with either a high or a low concentration of PHA in the absence of g-a cells, either very little or no significant levels of DNA synthesis were detected (broken curves of fig. la, b). However, when these ‘purified’ lymphocytes were cultured with PHA in the presence of g-a cells, markedly increased DNA synthesis occurred (solid curves of fig. la, b). It did not seem likely that the increased synthesis was due to synthesis by the g-a cells, since PHA-containing cultures of
‘Purified’ lymphocytes and phytohemagglutinin
155
Fig. I. Abscissa: culture time (hours); cpm. O-O, lymphocytes with added g-a cells; O---O, lymphocytes without addedg-a cells.(a) 5 ,ugof PHA/ml of culture fluid; (b) 0.33 pg of PHA/ml
ordinate:
.
-
the g-a cells alone showed either no detectable DNA synthesis or a level of synthesis far too low simply to account for the massive increases in DNA synthesis obtained after the g-a cells had been added to the PHA-containing, ‘purified’ lymphocyte cultures. For example, when a coverslip containing the g-a cells used in the experiment of fig. 1 a was cultured with PHA at a concentration of 5 ,ug/ml of culture fluid, only 250 cpm were obtained at the 48th hour of culture. However, as shown in fig. la, addition of similar coverslips to the ‘purified’ lymphocyte cultures containing PHA led to increased DNA synthesis represented by approx. 45000 cpm at the 48th h of culture. Nor did it seem likely that the increased synthesis caused by addition of the g-a cells was due simply to a non-specific increase by the g-a cells in the number of cells in the culture, for increasing the number of lymphocytes by 40 % gave no more than a 25 % increase in DNA synthesis at the 48th hour of culture in contrast to approximately a 1000 % increase given by adding the g-a cells.
lymphocytes/ml of culture fluid.
The amount of DNA synthesized in our ‘purified’ lymphocyte cultures in response to a given concentration of PHA is correlated with the number of g-a cells in the culture. Thus, there was considerable DNA synthesis in PHA-containing cultures comprised 99 % or less of lymphocytes and 1 % or more of other leukocytes. In most of our ‘purified’ lymphocyte cultures which, as previously stated, had a cell population in which more than 99 % of the cells had the morphology of small lymphocytes, it was not practicably possible to obtain a precise count of the very small number of contaminating g-a cells from morphological examination of stained smears. However, the relative degrees of the very low contamination in such cultures could be reasonably assessed by placing in such cultures a clean coverslip to which contaminating g-a cells would adhere. After several days in culture the coverslips were removed and, after being air-dried and fixed in methanol, were stained with Giemsa stain. In this manner we observed that batches of ‘purified’ lymphocytes giving the greatest response to Exptl Cell Res 61
156
W. R. Levis & J. H. Robbins
Fig. 2. Effect of homologous g-a cells on the blastogenic response of ‘purified’ lymphocytes to PHA. Lymphocytes, 106/ml of culture fluid; PHA, 2.3 @g/ml of culture fluid. Giemsa stained, x 400, 48th hour of culture. (a) Smear from a culture of lymphocytes alone; (b) coverslip from a culture of lymphocytes with added g-a cells; (c) coverslip from a culture of g-a cells with PHA; (d) smear from a culture of lymphocytes with PHA; (e) coverslip from a culture of lymphocytes with PHA and added g-a cells. Further details in text.
PHA also deposited the most g-a cells on their coverslips. We observed also that our purest lymphocyte preparations, which gave no evidence of having deposited any cells on such coverslips, synthesized the least amount of DNA in response to PHA. It should be noted, however, that it is nevertheless possible that the response to PHA given by these very pure preparations of lymphocytes may be due to the presence of a small number of contaminating g-a cells which we are unable to detect morphologically. Further evidence that a very small number of g-a cells may lead to considerable DNA synthesis in ‘purified’ lymphocyte cultures containing PHA was shown by the fact that we have obtained increases of many thousands of cpm in cultures containing 0.5 to 1 X lo6 lymphocytes by the addition to such cultures of a coverslip containing less than 4 x lo3 g-a cells. We have observed an increase in DNA synthesis in the presence of g-a cells in each of the 20 experiments conducted with ‘purified’ lymphocytes and PHA. The effect could Exptl Cell Res 61
be detected with all the concentrations of PHA tested, including very high and even toxic concentrations, if the DNA assayswere conducted before the reconstituted cultures had passed the time of their maximal DNA synthesis. In this regard it is important to note that we have performed an experiment in which highly stimulated, reconstituted cultures had a peak of DNA synthesis prior to the 84th hour of culture, while the ‘purified’ lymphocyte cultures peaked at a later time. Obviously, in such experiments valid comparisons between the two types of cultures. can be obtained only if the DNA determinations are made early in the culture period and, thus, prior to the time of peak DNA synthesis in either of the cultures. In PHA-stimulated leukocyte cultures the amount of DNA synthesized is related to the transformation of small lymphocytes into blastoid cells which undergo mitotic division [5]. In our ‘purified’ lymphocyte and reconstituted cultures a similar correlation was observed between DNA synthesis and the number of blastoid cells. Thus, in cultures
‘Purified’ lymphocytes and phytohemagglutinin with no detectable DNA synthesis, no blastoid cells were found in stained smears. In cultures synthesizing DNA, on the other hand, blastoid cells were regularly observed, their numbers increasing with increases in the amounts of DNA synthesized. This correlation is illustrated by the results at the 48th hour of culture in an experiment in which homologous g-a cells increased the response of ‘purified’ lymphocyte cultures to PHA (fig. 2). In cultures of lymphocytes alone (fig. 2a), of lymphocytes with g-a cells (fig. 2b), or of g-a cells with PHA (fig. 2c), no blastoid cells were apparent and none of the cultures gave more than 160 cpm. Lymphocyte cultures containing PHA (fig. 2d) showed a very rare blastoid cell (arrow) and gave up to 450 cpm. However, cultures containing lymphocytes with g-a cells and PHA gave up to 50000 cpm and had large numbers of blastoid cells many of which were found in the typical rosette formation surrounding a centrally located g-a cell (fig. 2e). Origin of responding cells in the reconstituted cultures The possibility has already been briefly considered that the DNA-synthesizing and dividing blastoid cells in the reconstituted cultures might have been derived from the g-a cells or from lymphocytes unintentionally transferred with them. Despite the fact that this possibility seemed unlikely, it was, nevertheless, considered essential to determine definitively the population of origin of the responding cells in the reconstituted cultures. A definitive determination was made possible when we found that homologous, as well as autologous, g-a cells could increase the response to PHA of ‘purified’ lymphocyte cultures, for we were then able to identify a cell line by the sex chromosomes of the dividing cells when the lymphocytes and the g-a
157
cells were obtained from donors of the opposite sex. One of these determinations was performed in the experiment already presented in fig. 2 in which the lymphocytes were obtained from a female and the g-a cells from a male donor. At the 48th hour of culture all 25 of the dividing cells examined in the reconstituted cultures had a female chromosome complement, indicating that most, if not all, of the responding cells had been derived from the female’s lymphocyte population. DISCUSSION Our results show that lymphocytes which have been adequately separated from other leukocytes have a markedly diminished blastogenic response to PHA. The lymphocytes’ response is markedly increased if either autologous or homologous g-a cells are added to the cultures. The way in which the g-a cells affect the response of the lymphocytes is not known. The mechanism may be due to effects of the g-a cells on the cultural conditions, for example, through changes in the nutrients, pH, or oxygen levels of the medium. Or the mechanism may be more specific involving, for example, the production by the g-a cells of specific substances or physical conditions which affect the lymphocytes either through the medium or via direct cell-to-cell contact between the lymphocytes which respond and the g-a cells. Of course, the possibility must also be considered that the lymphocytes were injured during or after their separation from the other leukocytes; however, if this were the case, the interesting question still would remain as to how the g-a cells are able to repair, or to compensate for, such an injury. We have found also that the ability of g-a cells to increase the blastogenic response of ‘purified’ lymphocyte cultures is not limited Exptl Cell Res 61
158 W. R. Levis & J. H. Robbins only to PHA as the blastogenic stimulus but applies also to homologous cells and to antigens such as histoplasmin, tuberculin purified protein derivative, and tetanus and diphtheria toxoids. Thus, with our technique we have confirmed the observation originally reported by other investigators [3,6,7] that cultures of ‘purified’ lymphocytes, obtained from a person whose leukocyte cultures normally responded to a specific antigen, did not undergo significant blastogenesis in response to that antigen unless autologous g-a cells were added to the lymphocyte cultures. In addition, we have shown that the immunologic specificity of the response of such reconstituted cultures is determined by the lymphocyte population [4], that not only autologous, but also homologous, g-a cells can restore responses to such cultures, and that sex chromosome analysis reveals the dividing cells in such cultures to be derived from the lymphocyte and not from the g-a cell donor. We have confirmed also the reports by McFarland [6] and by Gordon [l] that mixtures of ‘purified’ lymphocytes from two unrelated individuals give increased DNA synthesis if they contain g-a cells from at least one of the individuals. However, since we have found that coverslips containing g-a cells generally serve as a very potent blastogenic stimulus when placed in an unrelated individual’s leukocyte or ‘purified’ lymphocyte cultures, analysis of the sex chromosome complement was undertaken in order to show that cells from both populations of ‘purified’ lymphocytes underwent cell division in a mixed culture when a coverslip containing only one of the individual’s g-a cells was included in that culture. Thus, our results show that g-a cells are required in order for ‘purified’ lymphocytes
Exptl Cell Res 61
to respond with a maximal blastogenic response in cultures containing PHA, antigens, or homologous cells. These findings suggest that the g-a cells which are present in all, typical, unpurified leukocyte cultures may serve essential functions in the blastogenic response in such cultures. If these functions are not related only to the in vitro conditions of culture, it is possible that the g-a cells or their progenitors may function in a similar manner in immunologic processes occurring in vivo in which lymphocytes are stimulated to synthesize DNA and to undergo mitotic division, for example, in delayed hypersensitivity and homograft reactions. The authors thank Mrs Lisa C. van de Water for performing the chromosome analyses and Professor Leonard Ornstein for encouragement and a critical reading of this manuscript.
REFERENCES 1. Gordon, J, Proc sot exptl biol med 127 (1968) 30. 2. Greenwalt, T J, Gajewski, M & McKenna, J C, Transfusion 2 (1961) 221. 3. Hersh, E M & Harris, J E, J immunol 100 (1968) 1184. 4. Levis, W R & Robbins, J H, Fed proc 28 (1969) 566. 5. Ling, N R, Lymphocyte stimulation. NorthHolland Publishing Co., Amsterdam, (1968). 6. McFarland, W, Proceedings of the third annual leukocyte culture conference (ed W 0 Rieke) p. 77. Aonleton-Centurv-Crofts. New York (1969). 7. Oppenheim, J J, Leienthal, B G & Hers& E M, J immunol 101 (1968) 262. 8. Rabinowitz, Y, Blood 23 (1964) 811. Robbins. J H. Science 146 (1964) 1648. 1;: Robbins; J H, Burk, P G‘ & Levis, W R, Fed proc 28 (1969) 363. 11. Schrek, R & Rabinowitz, Y, Proc sot exptl biol med 113 (1963) 191. 12. Walker, R I & Fowler, I, Exptl cell res 38 (1965) 379. 13. Wilson, D B, J exptl zoo1 162 (1966) 161. Received December 16, 1969.