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Studies on rabbit lymphocytes in vitro
CELLULAR
IMMUNOLOGY
12, 119-126 (1974)
Studies on Rabbit Lymphocytes XIX.
Kinetics
of Reversible Antiallotypic Stimulation of Blast Transformation l STEWART
IIepartmmt
in Vitro
of Pathology,
and Restimulation
SELL
C~nievrsitJ~ of California at San Diego La Jolla. CaliforGa 92037 Rrc&cd
Scptcmbcr
Medical
School,
10, 1973
A study of the kinetics of antiallotypic stimulation of transformation of rabbit peripheral blood lymphocytes demonstrates that : 1. The ability of lymphocytes to respond to stimulation decreases with the time the cells are held in vitro prior to initiation of stimulation (delayed stimulation) ; 2. When stimulated cultures are blocked by the addition of competing immunoglobulin, RNA synthesis is maintanied for 2-3 hr; synthesis then gradually falls to unstimulated levels ; 3. If blocked cultures are restimulated within 3-4 hr after blocking, the amount of RNA synthesis, DNA synthesis, and number of transformed cells will be the same as if blocking had not occurred; after this time these indices will be decreased in comparison to continuously stimulated cultures ; 4. Stimulated and blocked cultures can be restimulated after prestimulation and blocking at times when unprestimulated (delayed stimulated) cells are no longer stimulable. The results confirm and extend earlier findings that stimulated lymphocytes remain in a temporary state of activation following blocking. The relationship of the induction of lymphocyte transformation to the distribution of surface immunoglobulins on rabbit peripheral blood lymphocytes as determined by ultrastructural labeling suggests that the initial step in transformation involves endocytosis of surface receptors.
INTRODUCTION Rabbit peripheral blood lymphocytes may ,be activated in vitro by antiallotypic sera to transf’orm into blast cells (1, 2). This process requires approximately 48 hr with RNA synthesis beginning at 12 hr, DNA synthesis at 36-40 hr and mitotic division at 44-48 hr (3). Stimulation may be reversed by removal of the activating antisera by washing during the first few hours of the culture and much later in the culture period if, in addition to washing, serum containing the allotypic determinant to which the stimulating antiserum is directed is added to the culture (blocking) (4). In fact, substantial inhibition may be affected by blocking as late as 3G40 hr after initiation of the culture (4). If cultures are held in vitro in normal rabbit ,serum prior to addition of stimulating antiallotypic serum (delayed stimulation), the amount of stimulation observed decreases relative to that obtained 1 Supported
by NIH
research
grant
AI-09719-03.
119 Copyright Q 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
120
SELL
if the stimulating antisera is present in the culture from the time of initiation (continuous stimulation) (5). Blocked lymphocyte cultures may be restimulated if an appropriate antiallotypic serum is re-added to the blocked culture (restimulation) (5). The present report describes some of the metabolic events that occur during blocking and restimulation and defines the kinetics of delayed stimulation and stimulation-block-restimulation. bfATERIALS
AND
METHODS
The conditions for lymphocyte culture, blocking, and restimulation have been described previously (5). Each culture contained 3 x 10” lymphocytes, 1.6 ml Eagle’s MEM with glutamine, penicillin, and streptomycin, and 0.4 ml rabbit serum; 0.25 &i[%]U ( sp act > 50 mCi/mmole) and 0.25 &I [1Y]dT (sp act 52 mCi/mmole) were used in labeling experiments carried out as described in the results. The precipitate from 1.5 ml of culture produced by 5% trichloroacetic acid was washed in a Millipore filter disk, dried, and counted (6, 7) ; 0.5 ml of the same culture was used to prepare smears for microscopic examination (7). The antiallotypic sera were prepared ,by the method of Dubiski et al. (8) and tested for specificity by double diffusion in agar and passive hemagglutination (9). The following terminology is used to identify the conditions used in various cultures : Control unstimulated, b4b4 lymphocytes maintained in normal non-b4b4 or b4b4 serum for the entire culture period; Continuous stimulation, b4b4 lymphocytes cultured in the presence of stimulating anti-b4 serum for the entire culture period; Washed cultures, b4b4 lymphocytes stimulated with anti-b4 for a given time period, after which the anti-b4 culture fluid is removed by centrifugation, the cells washed once in medium containing non-b4 serum and resuspended in carrier non-b4 serum until harvesting ; Blocked cultures, b4b4 lymphocytes cultured in anti-b4 serum. washed, and maintained in medium containing normal b4b4 serunl; Delayed stimulation, b4b4 lymphocytes maintained in non-b4b4 serum or b4b4 serum for a given time prior to addition of stimulating anti-b4 serum; those cultures maintained in b4b4 serum must be washed prior to addition of anti-b4 serum; Restimulation, b4b4 lymphocytes stimulated for a given time with anti-b4 serum, washed, blocked for l-3 hr, washed again, and restimulated with anti-b4 serum; Delayed restimulation, b4b4 lymphocytes stimulated for a given time with anti-b4, blocked with b4b4 serum for variable periods of time greater than 1 hr, washed, and restimulated with anti-b4. RESULTS Delayed
Stivnulation
The effect 05 delayed addition of stimulating antiserum is presented in Fig. 1. b4b4 lymphocyte cultures were set up in normal serum and the optimal dose of anti-b4 added at the times indicated. The ability of addition of anti-b4 serum to stimulate transformation falls after 4 hr of culture; after 18 hr, little or significantly reduced stimulation occurs. Since delayed stimulation might shift ‘the peak time of [14C]dT uptake to a later time in the culture period, the later [‘%JdT pulse (45-72 hr) is less affected by delayed stimulation than the 24-48 hr pulse.
ALLOTYPIC
LYMPHOCYTE
TRANSFORMATION
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“cdl 0 0
KINETICS
121
PULSE 24-48 48-72
80
ae
HOURS
OF
CULTURE
FIG. 1. Delayed stimulation with antiallotypic sera. The amount of r(C]dt uptake after delayed stimulation is compared to the effect of continuous stimulation in the same experiment for calculation of the percentage indicated in the ordinate. Lymphocytes were cultured for the time indicated before the addition of the optimal dose of stimulating antiallotypic serum. The closed circles represent [‘VI]dT incorporation from 24 to 48 hr after culture; the opened circles, from 48 to 72 hr after culture. Within 4 hr after culture, the ability to respond to antiallotypic stimulation decreases. By 6 hr, significant decreased stimulation occurs with almost complete inhibition after 12-18 hr.
Blocking of Lymphocyte
Stiwmlation
The effect of removal of stimulating anti-b4 serum by washing and blocking at different times after initial stimulation is given in Fig. 2. The raw data for most of these experiments have been presented previously (4) ; the present figure is provided to emphasize the fact that blocking of lymphocyte transformation is complete up to 16 hr and substantial up to 36-40 hr after stimulation. Pulse periods of different times were used so that a delayed peak of [laC]dT uptake would have been detected. The effect of blocking is essentially the same if thymidine uptake is measured from 24 to 48 hr or from 38 to 72 hr. Effect of Blocking on RNA Synthesis The amount of [“C]U uptake by lymphocyte cultures after blocking was determined by pulsing cultures at different times after blocking with [‘“C]U. The results are presented in Fig. 3. RNA synthesis decreases after blocking, so that by 3 hr it is approaching the control level. Delayed Restimulation To determine the effect of the time of blocking after stimulation prior to restimulation, lymphocyte cultures were stimulated with antiallotypic serum for 4-18 hr, and blocked for the time indicated prior to washing and restimulation. The results are presented in Fig. 4 and illustrate a gradual loss of restimulability following blocking. However, cultures which Ivere stimulated for I-18 hr, blocked for 1-3 hr. and then restimulated responded as well as cultures that had not been blocked. This observation confirms earlier results that demonstrated that continu-
122
SELL
HOURS
OF
CULTURE
FIG. 2. Effect of time of wash or block on lymphocyte response ilz z~itro. Lymphocyte cultures were treated with stimulating antiallotypic serum for the time indicated, and the stimulating antiserum was removed by washing alone or by washing and addition of serum containing the allotype to which the stimulating antiserum was directed (blocking). Each point represents the mean of 2-12 experiments (see Ref. 4). Washing alone was partially effective in reducing transformation up to 18 hr after initiation, while blocking was completely effective up to 18 hr and partially effective up to 45 hr.
ous stimulation and restimulation after short blocking periods gave essentially identical results (5). When compared to the results obtained with delayed stimulation (Fig. l), it should be noted that cultures prestimulated ior 11 hr and blocked for 1 hr will respond maximally when restimulated at 12 hr, whereas cultures held in vitro for 12 hr without prestimulation respond very poorly, if at all. To determine if the time of blocking prior to restimulation might offset the time of maximum DNA synthesis, the effect of 3-6 hr blocking and restimulation on the [1’C]dT uptake between 44 and 56 hr after blocking at 12 hr was compared to continuous stimulation. Cosiderable \.ariation in effect was noted, but there was no consistent difference in the peak time of DNA-synthesis-blocked-restimulated and continuously stimulated cultures, the peak of [14C]dT uptake occurring at the same time in restimulated and continuously stimulated cultures. The effect of the length of blocking of [‘“C]U uptake after restimulation is presented in Fig. 5. If restimulation was carried out within 4 hr after blocking, RNA synthesis after stimulation was essentially the same as in unblocked cultures over the same time period. However, the amount of RNA synthesis induced by restimulation fell steadily from 6 to 24 hr after blocking. The effect of blocking and restimulation after a l-hr block at 12, 24, and 36 hr on [“C]U incorporation was determined. At 12 hr blocking was complete, and restimulation induced, if anything, a greater stimulation of RNA synthesis than continuous stimulation when tested with 12-hr pulses of [‘“C]U. Blocking and restimu-
ALLOTYPTC
LYMI’HOCYTE
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KINETICS
123
CONTROL
“I
I?
zp HOUR
OF
2p
3p
(
CULTURE
FIG. 3. Effect of length of block on rate of RNA synthesis (r’C]U uptake). The amount of [“C]U uptake from initiation of the culture to the time of blocking is given as the starting reference for unstimulated control (0) and stimulated cultures (0). Following blocking r”C]U was added at the times indicated, and the experiment terminated 6 hr after blocking. A continual decrease in [‘“C]U uptake is noted so that by 3 hr RNA synthesis in blocked cultures approaches that of the controls. Each point represents the mean value of three separate experiments from 18 to 24 hr and from 24 to 30 hr. The numbers indicate the time in minutes after blocking that the [“C]U was added to the cultures. NO. indicates no block.
“CdT
PULSE
24
l
048
3
6 HOURS
9
12 AFTER
48 hn. 72 hrs.
18
24
BLOCKING
FIG. 4. Effect of length of block on restimulation (delayed restimulation). Rabbit PBL cultures were stimulated for 4-18 hr, blocked for the times indicated, the blocking serum removed, and the cultures restimulated by addition of the antiallotypic serum. The closed circles represent 2448-hr rC]dT pulses ; the open circle,s, 48-72 hr-pulses. If restimulation was accomplished within 3 hr after blocking, the degree of response was essentially the same as with continuous stimulation. However, after 4 hr the degree of response decreased, so that by 18 hr no stimulation could be obtained.
124
SELL
LENGTH OF BLOCK , 06Ih,S 9 IS 24
4 / P
0.1 HOURS
B AFTER
9
I?
,
RESTIMULATION
FIG. 5. Effect of 6- to 24-hr blocking periods on [“C]U uptake after anti-b4 stimulation. The culturqs were stimulated for 24 hr with anti-b4, blocked with b4 for the times indicated, then restimulated with anti-b4. Separate cultures were pulsed with [‘“C]U for O-3, 3-7, and 6-12 hr after restimulation. The ability to restimulate RNA synthesis decreased with the length of the blocking period from 6 to 24 hr. [“CJU was added at the time of restimulation. Continuously stimulated controls were pulsed at the same time periods for comparison to the restimulated cultures.
lation at 24 hr produced a similar effect, while blocking and/or 36 hr produces no significant effect on RNA synthesis.
restimulation
at
DISCUSSION The results reported herein on the factors affecting antiallotypically induced lymphocyte transformation confirm similar effects noted with concanavalin A (Con A) stimulation and a-methyl-d-mannopyanoside (MAM) or anti-Con A blocking (10, 11). Maximal stimulation of blast transformation occurs when the antiallotypic serum is present during the entire culture period or removed for only short periods of time. The stimulation of lymphocytes may be completely reversed by removal of the stimulating antiserum by washing or blocking. Morphologic observations demonstrate that partially transformed enlarging lymphocytes stimulated by either Con A (11) or antiallotypic sera actually revert to small lymphocytes after blocking (4). However, ‘blocked lymphocytes remain in a state of metabolic activation for approximately 3-6 hr after blocking. This is established by the observations that (i) RNA synthesis in stimulated and blocked cultures does not decrease to that of continuously stimulated cul’tures until 3 hr after blocking, (ii) blocked cultures may be restimulated within 3 hr after blocking, and (iii) the degree and time of maximal DNA synthesis after blocking and restimulation does not differ significantly from that induced by continuous stimulation. The fact that antiallotypically treated cells remain in a state of activation for some time after blocking permits prestimulation with one antiallotypic serum and restimulation with a different allotypic serum (5). This mixed allotype sequential stimulation is only effective if ,both antiallotypic sera react specifically wi’th the allotypes of the lynll~l~o-
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ryte donor (5). Therefore, mixed allotypic sequential stimulation only occurs with lymphocytes which bear Ithe reactive allotypes and is not due to release of some nonspecific “blastogenic” factor. In addition, passive uptake of allotypic determinants has not been demonstrable by lymphocyte transformation in our laboratory either in zivo (12) or in vitro (13) even after treatment with enzymes such as pronase or trypsin (14). The most likely effect of washing and/or blocking of antiallotypically stimulated cells is the removal of antibodies bound to the cells; this explanation has also been used to explain the blocking effect of MAM on Con A stimulation (10, 11). Washing alone removes the unbound antiallotypic antibody or Con A in the medium, thus perimitting a re-equilibration of the bound mitogen so that some of it may come off the cells. Addition of a blocking agent such as b4 Ig for anti-b4 and MAM for Con A provides a competitive ligand that functions to pull mitogen off the cell more effectively than washing alone. This simple explanation requires that the mitogen be bound to the cell during the entire transformation process. However, using the immunoferritin mixed antiglobulin technique (15), we have recently demonstrated that addition of anti-b4 serum to b4b4 lymphocytes at 37°C causes immediate endocytosis of b4 determinants (16). Similar results have been reported using Con A stimulation of human lymphocytes in that immediate endocytosis of Con A is observed (17). The simple binding hypothesis becomes untenable if no surface mitogen is available for unbinding by blocking reagents. An alternate hypothesis is that initiation of blast transformation requires conditions that cause endocytosis of surface components and that these components must be prevented from re-expression on the cell surface if transformation is to proceed to completion. The presence of a mitogen such as anti-b4 not only induces endocytosis of surface immunoglobulin (17)) but also prevents newly synthesized immunoglobulin from establishing itself on the cell surface. If washing or blocking is done, sufficient antiallotypic antibody is removed or neutralized so that cellular immunoglobulin is allowed to reappear on the surface. The fact that the effect of hvashing is much less effective after short periods of time following stimulation than blocking indicates that cells undergoing transformation become less able to re-establish surface receptors as the transformation process continues. This hypothesis is now being tested in our laboratory. ACKNOWLEDGMENTS These experiments were started in England in 1968. I thank Jenny Lowe for the experiments done in Birmingham, England; Stephanie Hughes for those done in Pittsburgh, Pennsylvania; and Haynes B. Sheppard, Jr., for those done in La Jolla, California.
REFERENCES 1. Sell, S., and Gell, P. D. H., J. Ext. Med. 122, 423, 1965. Rev. 5, 19, 1970. 3. Sell, S., Rowe, D. S., and Gell, P. G. H., J. Exp. Med. 122, 823, 1965. 4. Sell, S., Lowe, J. A., and Gell, P. G. H., J. Inz~unol. 108, 674, 1972. 5. Sell, S., Lowe, J. A., and Gell, P. G. H., J. IWWZWIO~.111, 144, 1973. 6. Patterson, M. S., and Green, R. S., Aural. Chcm. 37, 385, 1965. 7. Sell, S., Lowe, J. A., and Gell, P. G. H., IWZVZW~O~O~J~ 16, 285, 1969. 8. Dubiski, S., Dudziak, Z., Skalba, D., and Dubiski, A., Iu~~uut~l~gy 2, 84, 1959. 9. Sell, S., J. E.vp. Med. 128, 341, 1968.
2. Sell, S., Transplalzf.
126 10. 11. 12. 13. 14. 15. 16. 17.
sl%r,r,
Powell, A. E., and Leon, M. E., Exp. Cell Rrs. 62, 315, 1970. Sell, S., and Sheppard, H. W., Jr., Exp. Cell Rcs. (in press). Gell, P. G. H., and Sell, S., J. Exp .ilIrd. 122, 813, 1965. Sell, S., 112 “Cellular Recognition” (R. T. Smith and K. A. Good, Eds.), p. 204. AppletollCentury-Crofts, New York, 1969. Sell, S., unpublished data. An, T., Miyai, K., and Sell, S., J. Inzmunol. 108, 1271, 1972. Linthicum, D. S., Mayr, W., Miyai, K., and Sell, S., Fed. Proc. Fed. Amer. Sot. l&p. Viol. 32, 983, 1973. Barat, N., and Avrameas, S., Exj~. Cell. Rrs. 76, 451, 1973.
IMMUNOLOGY
12, 119-126 (1974)
Studies on Rabbit Lymphocytes XIX.
Kinetics
of Reversible Antiallotypic Stimulation of Blast Transformation l STEWART
IIepartmmt
in Vitro
of Pathology,
and Restimulation
SELL
C~nievrsitJ~ of California at San Diego La Jolla. CaliforGa 92037 Rrc&cd
Scptcmbcr
Medical
School,
10, 1973
A study of the kinetics of antiallotypic stimulation of transformation of rabbit peripheral blood lymphocytes demonstrates that : 1. The ability of lymphocytes to respond to stimulation decreases with the time the cells are held in vitro prior to initiation of stimulation (delayed stimulation) ; 2. When stimulated cultures are blocked by the addition of competing immunoglobulin, RNA synthesis is maintanied for 2-3 hr; synthesis then gradually falls to unstimulated levels ; 3. If blocked cultures are restimulated within 3-4 hr after blocking, the amount of RNA synthesis, DNA synthesis, and number of transformed cells will be the same as if blocking had not occurred; after this time these indices will be decreased in comparison to continuously stimulated cultures ; 4. Stimulated and blocked cultures can be restimulated after prestimulation and blocking at times when unprestimulated (delayed stimulated) cells are no longer stimulable. The results confirm and extend earlier findings that stimulated lymphocytes remain in a temporary state of activation following blocking. The relationship of the induction of lymphocyte transformation to the distribution of surface immunoglobulins on rabbit peripheral blood lymphocytes as determined by ultrastructural labeling suggests that the initial step in transformation involves endocytosis of surface receptors.
INTRODUCTION Rabbit peripheral blood lymphocytes may ,be activated in vitro by antiallotypic sera to transf’orm into blast cells (1, 2). This process requires approximately 48 hr with RNA synthesis beginning at 12 hr, DNA synthesis at 36-40 hr and mitotic division at 44-48 hr (3). Stimulation may be reversed by removal of the activating antisera by washing during the first few hours of the culture and much later in the culture period if, in addition to washing, serum containing the allotypic determinant to which the stimulating antiserum is directed is added to the culture (blocking) (4). In fact, substantial inhibition may be affected by blocking as late as 3G40 hr after initiation of the culture (4). If cultures are held in vitro in normal rabbit ,serum prior to addition of stimulating antiallotypic serum (delayed stimulation), the amount of stimulation observed decreases relative to that obtained 1 Supported
by NIH
research
grant
AI-09719-03.
119 Copyright Q 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
120
SELL
if the stimulating antisera is present in the culture from the time of initiation (continuous stimulation) (5). Blocked lymphocyte cultures may be restimulated if an appropriate antiallotypic serum is re-added to the blocked culture (restimulation) (5). The present report describes some of the metabolic events that occur during blocking and restimulation and defines the kinetics of delayed stimulation and stimulation-block-restimulation. bfATERIALS
AND
METHODS
The conditions for lymphocyte culture, blocking, and restimulation have been described previously (5). Each culture contained 3 x 10” lymphocytes, 1.6 ml Eagle’s MEM with glutamine, penicillin, and streptomycin, and 0.4 ml rabbit serum; 0.25 &i[%]U ( sp act > 50 mCi/mmole) and 0.25 &I [1Y]dT (sp act 52 mCi/mmole) were used in labeling experiments carried out as described in the results. The precipitate from 1.5 ml of culture produced by 5% trichloroacetic acid was washed in a Millipore filter disk, dried, and counted (6, 7) ; 0.5 ml of the same culture was used to prepare smears for microscopic examination (7). The antiallotypic sera were prepared ,by the method of Dubiski et al. (8) and tested for specificity by double diffusion in agar and passive hemagglutination (9). The following terminology is used to identify the conditions used in various cultures : Control unstimulated, b4b4 lymphocytes maintained in normal non-b4b4 or b4b4 serum for the entire culture period; Continuous stimulation, b4b4 lymphocytes cultured in the presence of stimulating anti-b4 serum for the entire culture period; Washed cultures, b4b4 lymphocytes stimulated with anti-b4 for a given time period, after which the anti-b4 culture fluid is removed by centrifugation, the cells washed once in medium containing non-b4 serum and resuspended in carrier non-b4 serum until harvesting ; Blocked cultures, b4b4 lymphocytes cultured in anti-b4 serum. washed, and maintained in medium containing normal b4b4 serunl; Delayed stimulation, b4b4 lymphocytes maintained in non-b4b4 serum or b4b4 serum for a given time prior to addition of stimulating anti-b4 serum; those cultures maintained in b4b4 serum must be washed prior to addition of anti-b4 serum; Restimulation, b4b4 lymphocytes stimulated for a given time with anti-b4 serum, washed, blocked for l-3 hr, washed again, and restimulated with anti-b4 serum; Delayed restimulation, b4b4 lymphocytes stimulated for a given time with anti-b4, blocked with b4b4 serum for variable periods of time greater than 1 hr, washed, and restimulated with anti-b4. RESULTS Delayed
Stivnulation
The effect 05 delayed addition of stimulating antiserum is presented in Fig. 1. b4b4 lymphocyte cultures were set up in normal serum and the optimal dose of anti-b4 added at the times indicated. The ability of addition of anti-b4 serum to stimulate transformation falls after 4 hr of culture; after 18 hr, little or significantly reduced stimulation occurs. Since delayed stimulation might shift ‘the peak time of [14C]dT uptake to a later time in the culture period, the later [‘%JdT pulse (45-72 hr) is less affected by delayed stimulation than the 24-48 hr pulse.
ALLOTYPIC
LYMPHOCYTE
TRANSFORMATION
100
“cdl 0 0
KINETICS
121
PULSE 24-48 48-72
80
ae
HOURS
OF
CULTURE
FIG. 1. Delayed stimulation with antiallotypic sera. The amount of r(C]dt uptake after delayed stimulation is compared to the effect of continuous stimulation in the same experiment for calculation of the percentage indicated in the ordinate. Lymphocytes were cultured for the time indicated before the addition of the optimal dose of stimulating antiallotypic serum. The closed circles represent [‘VI]dT incorporation from 24 to 48 hr after culture; the opened circles, from 48 to 72 hr after culture. Within 4 hr after culture, the ability to respond to antiallotypic stimulation decreases. By 6 hr, significant decreased stimulation occurs with almost complete inhibition after 12-18 hr.
Blocking of Lymphocyte
Stiwmlation
The effect of removal of stimulating anti-b4 serum by washing and blocking at different times after initial stimulation is given in Fig. 2. The raw data for most of these experiments have been presented previously (4) ; the present figure is provided to emphasize the fact that blocking of lymphocyte transformation is complete up to 16 hr and substantial up to 36-40 hr after stimulation. Pulse periods of different times were used so that a delayed peak of [laC]dT uptake would have been detected. The effect of blocking is essentially the same if thymidine uptake is measured from 24 to 48 hr or from 38 to 72 hr. Effect of Blocking on RNA Synthesis The amount of [“C]U uptake by lymphocyte cultures after blocking was determined by pulsing cultures at different times after blocking with [‘“C]U. The results are presented in Fig. 3. RNA synthesis decreases after blocking, so that by 3 hr it is approaching the control level. Delayed Restimulation To determine the effect of the time of blocking after stimulation prior to restimulation, lymphocyte cultures were stimulated with antiallotypic serum for 4-18 hr, and blocked for the time indicated prior to washing and restimulation. The results are presented in Fig. 4 and illustrate a gradual loss of restimulability following blocking. However, cultures which Ivere stimulated for I-18 hr, blocked for 1-3 hr. and then restimulated responded as well as cultures that had not been blocked. This observation confirms earlier results that demonstrated that continu-
122
SELL
HOURS
OF
CULTURE
FIG. 2. Effect of time of wash or block on lymphocyte response ilz z~itro. Lymphocyte cultures were treated with stimulating antiallotypic serum for the time indicated, and the stimulating antiserum was removed by washing alone or by washing and addition of serum containing the allotype to which the stimulating antiserum was directed (blocking). Each point represents the mean of 2-12 experiments (see Ref. 4). Washing alone was partially effective in reducing transformation up to 18 hr after initiation, while blocking was completely effective up to 18 hr and partially effective up to 45 hr.
ous stimulation and restimulation after short blocking periods gave essentially identical results (5). When compared to the results obtained with delayed stimulation (Fig. l), it should be noted that cultures prestimulated ior 11 hr and blocked for 1 hr will respond maximally when restimulated at 12 hr, whereas cultures held in vitro for 12 hr without prestimulation respond very poorly, if at all. To determine if the time of blocking prior to restimulation might offset the time of maximum DNA synthesis, the effect of 3-6 hr blocking and restimulation on the [1’C]dT uptake between 44 and 56 hr after blocking at 12 hr was compared to continuous stimulation. Cosiderable \.ariation in effect was noted, but there was no consistent difference in the peak time of DNA-synthesis-blocked-restimulated and continuously stimulated cultures, the peak of [14C]dT uptake occurring at the same time in restimulated and continuously stimulated cultures. The effect of the length of blocking of [‘“C]U uptake after restimulation is presented in Fig. 5. If restimulation was carried out within 4 hr after blocking, RNA synthesis after stimulation was essentially the same as in unblocked cultures over the same time period. However, the amount of RNA synthesis induced by restimulation fell steadily from 6 to 24 hr after blocking. The effect of blocking and restimulation after a l-hr block at 12, 24, and 36 hr on [“C]U incorporation was determined. At 12 hr blocking was complete, and restimulation induced, if anything, a greater stimulation of RNA synthesis than continuous stimulation when tested with 12-hr pulses of [‘“C]U. Blocking and restimu-
ALLOTYPTC
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CONTROL
“I
I?
zp HOUR
OF
2p
3p
(
CULTURE
FIG. 3. Effect of length of block on rate of RNA synthesis (r’C]U uptake). The amount of [“C]U uptake from initiation of the culture to the time of blocking is given as the starting reference for unstimulated control (0) and stimulated cultures (0). Following blocking r”C]U was added at the times indicated, and the experiment terminated 6 hr after blocking. A continual decrease in [‘“C]U uptake is noted so that by 3 hr RNA synthesis in blocked cultures approaches that of the controls. Each point represents the mean value of three separate experiments from 18 to 24 hr and from 24 to 30 hr. The numbers indicate the time in minutes after blocking that the [“C]U was added to the cultures. NO. indicates no block.
“CdT
PULSE
24
l
048
3
6 HOURS
9
12 AFTER
48 hn. 72 hrs.
18
24
BLOCKING
FIG. 4. Effect of length of block on restimulation (delayed restimulation). Rabbit PBL cultures were stimulated for 4-18 hr, blocked for the times indicated, the blocking serum removed, and the cultures restimulated by addition of the antiallotypic serum. The closed circles represent 2448-hr rC]dT pulses ; the open circle,s, 48-72 hr-pulses. If restimulation was accomplished within 3 hr after blocking, the degree of response was essentially the same as with continuous stimulation. However, after 4 hr the degree of response decreased, so that by 18 hr no stimulation could be obtained.
124
SELL
LENGTH OF BLOCK , 06Ih,S 9 IS 24
4 / P
0.1 HOURS
B AFTER
9
I?
,
RESTIMULATION
FIG. 5. Effect of 6- to 24-hr blocking periods on [“C]U uptake after anti-b4 stimulation. The culturqs were stimulated for 24 hr with anti-b4, blocked with b4 for the times indicated, then restimulated with anti-b4. Separate cultures were pulsed with [‘“C]U for O-3, 3-7, and 6-12 hr after restimulation. The ability to restimulate RNA synthesis decreased with the length of the blocking period from 6 to 24 hr. [“CJU was added at the time of restimulation. Continuously stimulated controls were pulsed at the same time periods for comparison to the restimulated cultures.
lation at 24 hr produced a similar effect, while blocking and/or 36 hr produces no significant effect on RNA synthesis.
restimulation
at
DISCUSSION The results reported herein on the factors affecting antiallotypically induced lymphocyte transformation confirm similar effects noted with concanavalin A (Con A) stimulation and a-methyl-d-mannopyanoside (MAM) or anti-Con A blocking (10, 11). Maximal stimulation of blast transformation occurs when the antiallotypic serum is present during the entire culture period or removed for only short periods of time. The stimulation of lymphocytes may be completely reversed by removal of the stimulating antiserum by washing or blocking. Morphologic observations demonstrate that partially transformed enlarging lymphocytes stimulated by either Con A (11) or antiallotypic sera actually revert to small lymphocytes after blocking (4). However, ‘blocked lymphocytes remain in a state of metabolic activation for approximately 3-6 hr after blocking. This is established by the observations that (i) RNA synthesis in stimulated and blocked cultures does not decrease to that of continuously stimulated cul’tures until 3 hr after blocking, (ii) blocked cultures may be restimulated within 3 hr after blocking, and (iii) the degree and time of maximal DNA synthesis after blocking and restimulation does not differ significantly from that induced by continuous stimulation. The fact that antiallotypically treated cells remain in a state of activation for some time after blocking permits prestimulation with one antiallotypic serum and restimulation with a different allotypic serum (5). This mixed allotype sequential stimulation is only effective if ,both antiallotypic sera react specifically wi’th the allotypes of the lynll~l~o-
ALLOTYPTC
LYMPHOCYTE
TRANSFORMATJOiX
KlNETTC.5
125
ryte donor (5). Therefore, mixed allotypic sequential stimulation only occurs with lymphocytes which bear Ithe reactive allotypes and is not due to release of some nonspecific “blastogenic” factor. In addition, passive uptake of allotypic determinants has not been demonstrable by lymphocyte transformation in our laboratory either in zivo (12) or in vitro (13) even after treatment with enzymes such as pronase or trypsin (14). The most likely effect of washing and/or blocking of antiallotypically stimulated cells is the removal of antibodies bound to the cells; this explanation has also been used to explain the blocking effect of MAM on Con A stimulation (10, 11). Washing alone removes the unbound antiallotypic antibody or Con A in the medium, thus perimitting a re-equilibration of the bound mitogen so that some of it may come off the cells. Addition of a blocking agent such as b4 Ig for anti-b4 and MAM for Con A provides a competitive ligand that functions to pull mitogen off the cell more effectively than washing alone. This simple explanation requires that the mitogen be bound to the cell during the entire transformation process. However, using the immunoferritin mixed antiglobulin technique (15), we have recently demonstrated that addition of anti-b4 serum to b4b4 lymphocytes at 37°C causes immediate endocytosis of b4 determinants (16). Similar results have been reported using Con A stimulation of human lymphocytes in that immediate endocytosis of Con A is observed (17). The simple binding hypothesis becomes untenable if no surface mitogen is available for unbinding by blocking reagents. An alternate hypothesis is that initiation of blast transformation requires conditions that cause endocytosis of surface components and that these components must be prevented from re-expression on the cell surface if transformation is to proceed to completion. The presence of a mitogen such as anti-b4 not only induces endocytosis of surface immunoglobulin (17)) but also prevents newly synthesized immunoglobulin from establishing itself on the cell surface. If washing or blocking is done, sufficient antiallotypic antibody is removed or neutralized so that cellular immunoglobulin is allowed to reappear on the surface. The fact that the effect of hvashing is much less effective after short periods of time following stimulation than blocking indicates that cells undergoing transformation become less able to re-establish surface receptors as the transformation process continues. This hypothesis is now being tested in our laboratory. ACKNOWLEDGMENTS These experiments were started in England in 1968. I thank Jenny Lowe for the experiments done in Birmingham, England; Stephanie Hughes for those done in Pittsburgh, Pennsylvania; and Haynes B. Sheppard, Jr., for those done in La Jolla, California.
REFERENCES 1. Sell, S., and Gell, P. D. H., J. Ext. Med. 122, 423, 1965. Rev. 5, 19, 1970. 3. Sell, S., Rowe, D. S., and Gell, P. G. H., J. Exp. Med. 122, 823, 1965. 4. Sell, S., Lowe, J. A., and Gell, P. G. H., J. Inz~unol. 108, 674, 1972. 5. Sell, S., Lowe, J. A., and Gell, P. G. H., J. IWWZWIO~.111, 144, 1973. 6. Patterson, M. S., and Green, R. S., Aural. Chcm. 37, 385, 1965. 7. Sell, S., Lowe, J. A., and Gell, P. G. H., IWZVZW~O~O~J~ 16, 285, 1969. 8. Dubiski, S., Dudziak, Z., Skalba, D., and Dubiski, A., Iu~~uut~l~gy 2, 84, 1959. 9. Sell, S., J. E.vp. Med. 128, 341, 1968.
2. Sell, S., Transplalzf.
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sl%r,r,
Powell, A. E., and Leon, M. E., Exp. Cell Rrs. 62, 315, 1970. Sell, S., and Sheppard, H. W., Jr., Exp. Cell Rcs. (in press). Gell, P. G. H., and Sell, S., J. Exp .ilIrd. 122, 813, 1965. Sell, S., 112 “Cellular Recognition” (R. T. Smith and K. A. Good, Eds.), p. 204. AppletollCentury-Crofts, New York, 1969. Sell, S., unpublished data. An, T., Miyai, K., and Sell, S., J. Inzmunol. 108, 1271, 1972. Linthicum, D. S., Mayr, W., Miyai, K., and Sell, S., Fed. Proc. Fed. Amer. Sot. l&p. Viol. 32, 983, 1973. Barat, N., and Avrameas, S., Exj~. Cell. Rrs. 76, 451, 1973.