Selective
Responses of Mouse T Lymphocytes
T Mitogens
and in Mixed Lymphocyte Cytolysis Reaction
RrcriTvd
March
to Different
Culture
Induced
16, 1976
CBA spleen T lymphocytes were stimulated by the T mitogens concanavalin-.4 (Con-A), phytohemagglutinin (PHA), and leukoagglutinin ( LA). On the 2nd to 3rd culture day the activated cells (blasts) were separated from the nonactivated cells The lymphocytes which were not acti(lymphocytes) by lg velocity sedimentation. vated during the primary culture (lymphocyte fraction from the velocity sedimcntation) were then stimulated by the same mitogens or in one-way MLC to DRA/-’ m. and tested for relevant target lysis after MLC stimulation. Primary stimulation with Con-A abolished the responses to Con-A, to PHA, and to L.4, whereas primaq stimulation with PHA or with LA abolished the responses to these mitogens but left behind a considerable Con-A response. Stimulation Lvith any one of the listed T mitogens did not significantly affect the RILC responses. While primary stimulation \vith Con-A abolished the relevant target cell lysis after MI-C stimulation, primary stimulation with PHA or with LA reduced it only slightly. Assuming that the various mitogens stimulate separate subpopulations of T cells, the results seem to indicate that the Con-A-responsive population includes the PHAand LA-responsive populations but not the MLC-responsive population. It also appears that the T cell\ generated to killer cells during hILC are mainly confined to the concanavalillresponsive population.
INTRODUCTIOX Mixed lymphocyte culture (fiKC> primed T blasts have the capacity of reverting “back” to “secondary” lymphocytes (1). The secondary lymphoqtes respond promptly to the original stimulator cells (but to a lesser extent to nonrelated stimulator cells) in secondary RILC, and they are promptly reinduced to maximal cytotoxicity to relevant allogeneic target cells (2, 3). However, although these secondary lymphocytes are T cells, they are nonresponsive or I-espcmd only marginally to the T mitogens concanavalin-A (Con-A) and phytohemagglutinin (PHA) (2). Presently it is not known whether this is due to the MLC-responding cells losing their capacity to respond to these mitogens, or clue to the existence of two possibly nonoverlapping subpop~llatio~~s, the resting MLC-responsive T cells and the resting mitogen-responsive T cells. Evidence exists that there are at least two, possibly three, subpopulations of mature
T
cells
in the
mouse
(4,
5).
These 121
Copyright 0 1976 by Academic Press, All rights of reproductiun in any form
sul)l)ol’ulations
seem
to
I,eh;lvr
tlif-
122
SHORT
COMMUNICATIONS
ferently in immune responses. Using the Ly-alloantigens as markers for T lymphocyte subpopulations, Cantor and Boyse (4) demonstrated that helper activity in anti-SRBC response is generated by the Ly-1 carrying cells, whereas lysis of relevat target cells by in vitro sensitized lymphocytes was performed by the T subclass carrying Ly-2.3. On the other hand, the maturation of Ly-2.3carrying T cells to killer cells was amplified by the Ly-1 cells (6). Mitogen responsiveness has also been used as a probe for T cell subpopulations in the mouse. Stobo and Paul (7) treated mouse spleen cells with decreasingly strong concentrations of anti-0 serum plus complement. They found that after subtotal killing of T cells, the PHA-response disappeared but left behind a population of T cells that was still capable of responding to Con-A. Stout and Herzenberg (8, 9) separated mouse spleen cells carrying receptors to the Fc part of IgG (FcR) from FcR negative T cells, and compared their functions. While both FcR positive and negative cells responded in vitro to PHA, only the FcR positive cells responded to Con-A. Our study was undertaken to clarify whether and to what extent the PHA and Con-A responsive T cell populations overlap, and to examine which one of these populations, if any, is generated to killer cells in one-way MLC. The results demonstrate that the PHA-responsive cell population is included within the Con-A responsive population and that while removal of the PHA-responsive population has little effect on the generation of killer cells, removal of the Con-A responsive population abolishes the cytolytic effect. Indirectly, these results also explain why MLC-primed secondary T cells do not respond to the T mitogens PHA and Con-A. MATERIALS
AND METHODS
Mice The CBA (CBA/H-TGTG), from the Jackson Laboratory, Preparation
A and DBA/2 breeding stocks were obtained Bar Harbor, Maine and bred in our colony.
of Cells
Spleen cells were used both as stimulators and as responders in the MLC. Spleen fragments were teased apart, single spleen cells were recovered by sedimentation of the clumps, and the erythrocytes were lysed with 0.83% NH4CI (10). The MLC-stimulator cells were incubated with Mitomycin-C (Calbiothem, Los Angeles, California) as previously described (10). A lower case “m” is used here in DBA/2m to denote the mitomycin-blocked counterpart. The responder cells were further purified by attachment to a glass surface and/or by iron powder plus magnetic treatment, resulting in a population containing more than 85% lymphocytes (10). These “further purified” cells will be referred to as “spleen lymphocytes.” Spleen T lymphocyfes were fractionated by passing 50 x lo6 spleen lymphocytes through a Leukopak nylon wool column (11). The passed cells were contaminated with less than 2% surface Ig-containing cells, as shown by staining with fluorescein isothyocyanate (FITC)-conjugated anti-mouse Ig (a gift from Professor A. Fagraeus, State Bacteriological Laboratory, Stockholm).
Hydroxyurea (HU) was a gift from Dr. J. Anderssoii (Department of Immunology, University of Uppsala) and was used in DNA synthesis inhibiting nontoxic concentration of 5 x 1O-3 M (12). Phytohemagglutinin (PHA-M) was purchased from Difco Pharmaceuticals, Detroit, Michigan and was used in the optimal final dilution of 1: 150. Concanavalin-A (Con-A) was a gift from Professor H. Wigzell, Department of Immunology, IIniversity of Uppsala, and was used in the optima1 concentration of 5 pg/ml. Leukoagglutining (T,A) was purified from I-‘haseoZz~svulgaris as described (13) and was used in the optima1 stimulatory concentration of 1 pg/ml. All chemicals were directly dissolved into the culture medium (without serum). The induction of lymphocyte activation by Con-A needed 15 min incubation in serum-free medium prior to adding of the serum to the culture. The use of the indicated chemicals in in sdfro cu1turc.s of mouse lymphocytes has been described in detail elsewhere ( 10). Clfzflfrcs
Production of large numbers of MLC-activated cells ; “mui” calturc conditions : The cultures used were large tube cultures in 00 X 130 mm round bottom glass tubes containing 25 x 10F responder lymphocytes and the mitogens at indicated concentrations in 50 ml of medium. The culture medium was Eagle’s Minimum Essential Medium supplemented with 5% fetal calf serum of a selected batch and the cultures were carried out in a humidified atmosphere of 5% co2 in air. Details of the “maxi” culture conditions have been described earlier ( 10 j . Quantitation of lymphocyte activation ; tube culf~we conditions : The cultures were ordinary tube cultures in 11.25 X 100 mm disposable glass tubes (Miinnerstadt Glasswahrenfabrik, Miinnerstadt, Germany) with 1.5 x 10” responder lymphocytes plus mitogen at indicated concentrations or 3.0 x 10” stimulator cells in 2 ml of medium. Details of the tube culture conditions have been described previously (10). The MLC responses were quantitated (1) by differential blast cell counts from May-Gruenwald-Giemsa stained cytocentrifuged (Shandon Scientific Co., London) cell smears and/or (2) by quantitated mitosis counts after 3 hr Colcemitl-Ciba arrest (10). Quantitation of the cell counts was assessed 1~~ adding a known number of glutaraldehyde-fixed chicken erythrocytes to the cultures prior to making the cell smears as previously reported (10). Since these two methods of assessing the MLC response did not significantly differ from each other, we have given the results as quantitatetl blast cell counts only. Separation
of Activated
Cells from Non-Activated
Cells
For separation of activated cells (blasts) and from nonactivated cells (small lymphocytes) we used the lg velocity sedimentation method of hliller and Phillips (14). This method fractionates cells according to their size. The fractionation of cells was performed in a linear 15 to 30% newborn calf serum/ phosphate-buffered saline gradient as described ( 15). Mitogen-activated cells were recovered from the “maxi” cultures on the 2nd to 3rd culture day. The cells were washed three times with Dulbecco’s BSS and
124
SHORT
COMMUNICATIONS
placed on the gradient. Since the blasts were larger than the lymphocytes they tended to sediment more quickly. Thus three fractions were recovered: Fraction I containing predominantly blasts, Fraction II containing blasts and small lymphocytes, and Fraction III containing only small lymphocytes. Fractions I and II were discarded and the lymphocyte fraction-consisting of approximately 60-70s of cultured cells-was used for subsequent stimulations with the mitogens or in the MLC. Cell-Mediated
Lysis
(CML)
Assay
The %r (sodium 51chromate ; Radiochemical Centre, Amersham) method for quantitation of target cell damage by in vitro-activated lymphocytes has been described in detail (10). In CML experiments the P-815-X2 mastocytoma cells (DBA/2m ; obtained from Dr. K. T. Brunner, Swiss Institute for experimental Cancer Research, Lausanne) were used as targets. Standardized conditions and 6 hr exposure time were used (10). Specific 51Cr release was calculated according to the formula: y0 specific release = 100 X
exp. release-background release maximum release-background release’
Under these conditions the background release never maximum (Triton X-100 detergent) release.
exceeded 10% of the
RESULTS Number
of MLC
and Mitoyen
Responsive
T Cells
The first experiment was designed to estimate the percentage of CBA spleen T cells reactive in our culture conditions to the mitogens, PHA, La, and Con-A, and in one-way MLC to DBA/2m. To estimate the number of responding cells we used hydroxyurea (HU). HU is a drug that in proper concentration ( 1O-2-1O-3 M) allows blast transformation but reversibly inhibits the cells entering DNA synthesis. When it is used to estimate the number of responding cells, the response cells are “arrested” at the blast stage (when the cells were easily identified), and the peak number of blasts in cultures containing HU (usually observed on the 2nd to 3rd culture day) is used to indicate the number of responding cells (2). We are aware that the estimate in the MLC is probably slightly too high, since the mitomycin-blocked stimulator cells contribute to some extent to the blast response before they die (unpublished observations). CBA spleen T lymphocytes obtained via iron powder and nylon wool purification were stimulated in the presence of 5 X 10m3M HU by the various mitogens and in the MLC. When 1.5 x 10” responder cells were used per culture, the number of blasts in HU-arrested mitogen cultures was (Table 1) : PHA 0.12-0.18 x 106, LA 0.10-0.16 x 106, and Con-A 0.35-0.41 X 106. The percentage of responding T cells, as calculated from these figures, was : PHA S-12$%, LA 7-11%, and Con-A 23-26s. When similar amounts of CBA spleen T cells were stimulated in one-way mixed lymphocyte culture by DBA/2m, the peak number of blasts in
Number of responder cells (X lo’),’
I’eak blast response with HI: (X 10”)”
Input (‘; I
3.000 1.500 1.500 750
700 180 lh(l 60
6.7 12.0 10.6 8.0
IA L:I
1.500 1.500
100 1ho
6.6 10.6
Cell-A
Con-A
3.000 1.500 1..WO 750
5.50 .1.50 108 200
18.3 23.3 26.6 26.6
DBA/2m DBA/Zm DBA/2m DBA/2m DBH/2m DB4/2m
3.000 1.soo 1.500 1 .soo 7.50 375
80 1.5 80 110 75 3s
2.6 3.0 5.3 7.3 10.0 10.0
Mitogen or stimulus ccl1
Con-A Con-r\
n Number of responder CBA spleen T cells per culture. * Number of blasts per culture in the presence of D.Y.4 synthesis rentration of hydrosyurea (HIT).
inhibiting
(5 X 1OP U) con-
HU arrested cultures was 0.04-0.08 x 10”. The number of T cells resl)onding to Dl3A/Zm was calculated from these figures and was J-SC/r. It should also be noted that the number of T cells transforming to blasts upon mitogen or allogeneic stimulation seems to be influenced by the culture condtions employed : Somewhat higher numbers of blasts are usually observe
Responses of Mouse T Lymphocytes
T Mitogens
and in Mixed Lymphocyte Cytolysis Reaction
RrcriTvd
March
to Different
Culture
Induced
16, 1976
CBA spleen T lymphocytes were stimulated by the T mitogens concanavalin-.4 (Con-A), phytohemagglutinin (PHA), and leukoagglutinin ( LA). On the 2nd to 3rd culture day the activated cells (blasts) were separated from the nonactivated cells The lymphocytes which were not acti(lymphocytes) by lg velocity sedimentation. vated during the primary culture (lymphocyte fraction from the velocity sedimcntation) were then stimulated by the same mitogens or in one-way MLC to DRA/-’ m. and tested for relevant target lysis after MLC stimulation. Primary stimulation with Con-A abolished the responses to Con-A, to PHA, and to L.4, whereas primaq stimulation with PHA or with LA abolished the responses to these mitogens but left behind a considerable Con-A response. Stimulation Lvith any one of the listed T mitogens did not significantly affect the RILC responses. While primary stimulation \vith Con-A abolished the relevant target cell lysis after MI-C stimulation, primary stimulation with PHA or with LA reduced it only slightly. Assuming that the various mitogens stimulate separate subpopulations of T cells, the results seem to indicate that the Con-A-responsive population includes the PHAand LA-responsive populations but not the MLC-responsive population. It also appears that the T cell\ generated to killer cells during hILC are mainly confined to the concanavalillresponsive population.
INTRODUCTIOX Mixed lymphocyte culture (fiKC> primed T blasts have the capacity of reverting “back” to “secondary” lymphocytes (1). The secondary lymphoqtes respond promptly to the original stimulator cells (but to a lesser extent to nonrelated stimulator cells) in secondary RILC, and they are promptly reinduced to maximal cytotoxicity to relevant allogeneic target cells (2, 3). However, although these secondary lymphocytes are T cells, they are nonresponsive or I-espcmd only marginally to the T mitogens concanavalin-A (Con-A) and phytohemagglutinin (PHA) (2). Presently it is not known whether this is due to the MLC-responding cells losing their capacity to respond to these mitogens, or clue to the existence of two possibly nonoverlapping subpop~llatio~~s, the resting MLC-responsive T cells and the resting mitogen-responsive T cells. Evidence exists that there are at least two, possibly three, subpopulations of mature
T
cells
in the
mouse
(4,
5).
These 121
Copyright 0 1976 by Academic Press, All rights of reproductiun in any form
sul)l)ol’ulations
seem
to
I,eh;lvr
tlif-
122
SHORT
COMMUNICATIONS
ferently in immune responses. Using the Ly-alloantigens as markers for T lymphocyte subpopulations, Cantor and Boyse (4) demonstrated that helper activity in anti-SRBC response is generated by the Ly-1 carrying cells, whereas lysis of relevat target cells by in vitro sensitized lymphocytes was performed by the T subclass carrying Ly-2.3. On the other hand, the maturation of Ly-2.3carrying T cells to killer cells was amplified by the Ly-1 cells (6). Mitogen responsiveness has also been used as a probe for T cell subpopulations in the mouse. Stobo and Paul (7) treated mouse spleen cells with decreasingly strong concentrations of anti-0 serum plus complement. They found that after subtotal killing of T cells, the PHA-response disappeared but left behind a population of T cells that was still capable of responding to Con-A. Stout and Herzenberg (8, 9) separated mouse spleen cells carrying receptors to the Fc part of IgG (FcR) from FcR negative T cells, and compared their functions. While both FcR positive and negative cells responded in vitro to PHA, only the FcR positive cells responded to Con-A. Our study was undertaken to clarify whether and to what extent the PHA and Con-A responsive T cell populations overlap, and to examine which one of these populations, if any, is generated to killer cells in one-way MLC. The results demonstrate that the PHA-responsive cell population is included within the Con-A responsive population and that while removal of the PHA-responsive population has little effect on the generation of killer cells, removal of the Con-A responsive population abolishes the cytolytic effect. Indirectly, these results also explain why MLC-primed secondary T cells do not respond to the T mitogens PHA and Con-A. MATERIALS
AND METHODS
Mice The CBA (CBA/H-TGTG), from the Jackson Laboratory, Preparation
A and DBA/2 breeding stocks were obtained Bar Harbor, Maine and bred in our colony.
of Cells
Spleen cells were used both as stimulators and as responders in the MLC. Spleen fragments were teased apart, single spleen cells were recovered by sedimentation of the clumps, and the erythrocytes were lysed with 0.83% NH4CI (10). The MLC-stimulator cells were incubated with Mitomycin-C (Calbiothem, Los Angeles, California) as previously described (10). A lower case “m” is used here in DBA/2m to denote the mitomycin-blocked counterpart. The responder cells were further purified by attachment to a glass surface and/or by iron powder plus magnetic treatment, resulting in a population containing more than 85% lymphocytes (10). These “further purified” cells will be referred to as “spleen lymphocytes.” Spleen T lymphocyfes were fractionated by passing 50 x lo6 spleen lymphocytes through a Leukopak nylon wool column (11). The passed cells were contaminated with less than 2% surface Ig-containing cells, as shown by staining with fluorescein isothyocyanate (FITC)-conjugated anti-mouse Ig (a gift from Professor A. Fagraeus, State Bacteriological Laboratory, Stockholm).
Hydroxyurea (HU) was a gift from Dr. J. Anderssoii (Department of Immunology, University of Uppsala) and was used in DNA synthesis inhibiting nontoxic concentration of 5 x 1O-3 M (12). Phytohemagglutinin (PHA-M) was purchased from Difco Pharmaceuticals, Detroit, Michigan and was used in the optimal final dilution of 1: 150. Concanavalin-A (Con-A) was a gift from Professor H. Wigzell, Department of Immunology, IIniversity of Uppsala, and was used in the optima1 concentration of 5 pg/ml. Leukoagglutining (T,A) was purified from I-‘haseoZz~svulgaris as described (13) and was used in the optima1 stimulatory concentration of 1 pg/ml. All chemicals were directly dissolved into the culture medium (without serum). The induction of lymphocyte activation by Con-A needed 15 min incubation in serum-free medium prior to adding of the serum to the culture. The use of the indicated chemicals in in sdfro cu1turc.s of mouse lymphocytes has been described in detail elsewhere ( 10). Clfzflfrcs
Production of large numbers of MLC-activated cells ; “mui” calturc conditions : The cultures used were large tube cultures in 00 X 130 mm round bottom glass tubes containing 25 x 10F responder lymphocytes and the mitogens at indicated concentrations in 50 ml of medium. The culture medium was Eagle’s Minimum Essential Medium supplemented with 5% fetal calf serum of a selected batch and the cultures were carried out in a humidified atmosphere of 5% co2 in air. Details of the “maxi” culture conditions have been described earlier ( 10 j . Quantitation of lymphocyte activation ; tube culf~we conditions : The cultures were ordinary tube cultures in 11.25 X 100 mm disposable glass tubes (Miinnerstadt Glasswahrenfabrik, Miinnerstadt, Germany) with 1.5 x 10” responder lymphocytes plus mitogen at indicated concentrations or 3.0 x 10” stimulator cells in 2 ml of medium. Details of the tube culture conditions have been described previously (10). The MLC responses were quantitated (1) by differential blast cell counts from May-Gruenwald-Giemsa stained cytocentrifuged (Shandon Scientific Co., London) cell smears and/or (2) by quantitated mitosis counts after 3 hr Colcemitl-Ciba arrest (10). Quantitation of the cell counts was assessed 1~~ adding a known number of glutaraldehyde-fixed chicken erythrocytes to the cultures prior to making the cell smears as previously reported (10). Since these two methods of assessing the MLC response did not significantly differ from each other, we have given the results as quantitatetl blast cell counts only. Separation
of Activated
Cells from Non-Activated
Cells
For separation of activated cells (blasts) and from nonactivated cells (small lymphocytes) we used the lg velocity sedimentation method of hliller and Phillips (14). This method fractionates cells according to their size. The fractionation of cells was performed in a linear 15 to 30% newborn calf serum/ phosphate-buffered saline gradient as described ( 15). Mitogen-activated cells were recovered from the “maxi” cultures on the 2nd to 3rd culture day. The cells were washed three times with Dulbecco’s BSS and
124
SHORT
COMMUNICATIONS
placed on the gradient. Since the blasts were larger than the lymphocytes they tended to sediment more quickly. Thus three fractions were recovered: Fraction I containing predominantly blasts, Fraction II containing blasts and small lymphocytes, and Fraction III containing only small lymphocytes. Fractions I and II were discarded and the lymphocyte fraction-consisting of approximately 60-70s of cultured cells-was used for subsequent stimulations with the mitogens or in the MLC. Cell-Mediated
Lysis
(CML)
Assay
The %r (sodium 51chromate ; Radiochemical Centre, Amersham) method for quantitation of target cell damage by in vitro-activated lymphocytes has been described in detail (10). In CML experiments the P-815-X2 mastocytoma cells (DBA/2m ; obtained from Dr. K. T. Brunner, Swiss Institute for experimental Cancer Research, Lausanne) were used as targets. Standardized conditions and 6 hr exposure time were used (10). Specific 51Cr release was calculated according to the formula: y0 specific release = 100 X
exp. release-background release maximum release-background release’
Under these conditions the background release never maximum (Triton X-100 detergent) release.
exceeded 10% of the
RESULTS Number
of MLC
and Mitoyen
Responsive
T Cells
The first experiment was designed to estimate the percentage of CBA spleen T cells reactive in our culture conditions to the mitogens, PHA, La, and Con-A, and in one-way MLC to DBA/2m. To estimate the number of responding cells we used hydroxyurea (HU). HU is a drug that in proper concentration ( 1O-2-1O-3 M) allows blast transformation but reversibly inhibits the cells entering DNA synthesis. When it is used to estimate the number of responding cells, the response cells are “arrested” at the blast stage (when the cells were easily identified), and the peak number of blasts in cultures containing HU (usually observed on the 2nd to 3rd culture day) is used to indicate the number of responding cells (2). We are aware that the estimate in the MLC is probably slightly too high, since the mitomycin-blocked stimulator cells contribute to some extent to the blast response before they die (unpublished observations). CBA spleen T lymphocytes obtained via iron powder and nylon wool purification were stimulated in the presence of 5 X 10m3M HU by the various mitogens and in the MLC. When 1.5 x 10” responder cells were used per culture, the number of blasts in HU-arrested mitogen cultures was (Table 1) : PHA 0.12-0.18 x 106, LA 0.10-0.16 x 106, and Con-A 0.35-0.41 X 106. The percentage of responding T cells, as calculated from these figures, was : PHA S-12$%, LA 7-11%, and Con-A 23-26s. When similar amounts of CBA spleen T cells were stimulated in one-way mixed lymphocyte culture by DBA/2m, the peak number of blasts in
Number of responder cells (X lo’),’
I’eak blast response with HI: (X 10”)”
Input (‘; I
3.000 1.500 1.500 750
700 180 lh(l 60
6.7 12.0 10.6 8.0
IA L:I
1.500 1.500
100 1ho
6.6 10.6
Cell-A
Con-A
3.000 1.500 1..WO 750
5.50 .1.50 108 200
18.3 23.3 26.6 26.6
DBA/2m DBA/Zm DBA/2m DBA/2m DBH/2m DB4/2m
3.000 1.soo 1.500 1 .soo 7.50 375
80 1.5 80 110 75 3s
2.6 3.0 5.3 7.3 10.0 10.0
Mitogen or stimulus ccl1
Con-A Con-r\
n Number of responder CBA spleen T cells per culture. * Number of blasts per culture in the presence of D.Y.4 synthesis rentration of hydrosyurea (HIT).
inhibiting
(5 X 1OP U) con-
HU arrested cultures was 0.04-0.08 x 10”. The number of T cells resl)onding to Dl3A/Zm was calculated from these figures and was J-SC/r. It should also be noted that the number of T cells transforming to blasts upon mitogen or allogeneic stimulation seems to be influenced by the culture condtions employed : Somewhat higher numbers of blasts are usually observe
