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Direct evidence for the response of B and T cells to pokeweed mitogen
CELLULAR
Direct
IMMUKOLOGY
Evidence
482-487
9,
(1973)
for the Response of B and T Cells Mitogen ’ W.
Department
of Pathobiology,
T. WEBER
School of Veterinary Medicine, Philadelphia, Penmylvania 19104 Received
to Pokeweed
July
University
of
Pennsylvania,
30, 1973
Chicken spleen cells containing chromosomally marked thymus derived (T) and bursa-derived (Bz) cells were evaluated for their ability to respond to pokeweed mitogen (PWM), concanavalin A (Con A), and to anti-immunoglobulin serum during a 4-day culture period. The results indicate that soluble PWM induces a proliferative response of B2 cells in addition to a predominant T cell response. The PWM-induced Bf cell proliferative response was clearly detected only at 4 days after culture initiation. Soluble Con A did not induce detectable proliferation of Bz cells and stimulated T cells exclusively. In contrast, anti-immunoglobulin serum was a specific stimulant for B2 cells under the culture conditions used.
INTRODUCTION The type of lymphocyte stimulated by pokeweed mitogen (PWM) continues to be a subject of considerable controversy. Based to a large extent on experiments in mice, three conflicting sets of experimental data are found in the literature. Some reports suggested that only bone marrow derived, thymus-independent B cells were activated (1,2) whereas in others evidence only for the responseof T cells is found (3). The conclusion that PWM can activate both T and B cells is represented in several more recent reports (4-6). In vitro studies on lymphocyte populations obtained from B cell depleted chickens clearly indicated that T cells could respond to PWM, and evaluation of PWM-induced proliferative responses by lymphocytes from normal and B cell depleted chickens suggested furthermore that bursa-derived (B,) cells did not respond to this phytomitogen (7). We have attempted to develop experimental models in the chicken that would allow identification of thymus-derived (T) and bursa-derived ( B2) cells through the use of chromosome markers. The use of experimental chicks containing identified T and Bz cell populations have already proven useful for the detection of antigen-induced proliferation of B2 cells in viva (8) and for determining the ability of Bz and T cells to respond to lipopolysaccharide (9). In this report, direct evidence for the PWM-induced response of B2 cells, in addition to a predominant T cell responseto this stimulant will be presented. 1 This work was supported by United States Public Health Service Grant AM 11693. 482 1973 by Academic Press, reproduction in any form
Inc. reserved.
483
SHORT COMMUNICATIONS TABLE
1
DONOR AND HOST CELL PROLIFERATIVE RESPONSES TO SOLUBLE PWM, CON A AND TO ANTI-IMMUNOGLOBULIN SERUM IN SPLEEN CELL CULTURES OF A CYCLOPHOSPHAMIDE-TREATED CHICKEN FOLLOWING EMBRYONIC BURSAL CELL TRANSFER Culture
Stimulant % Donor mitoses
M.1.a
PWM Con A Control Anti-CIg
7 1.5 40 85
11 50 1 16
NRS
33
period-48
hr -
No. donor mitoses/lOl cellsb
No. host mitoses/lOJ cellsb
7.7 7.5 4 136
102.3 492.5 6 24
ye Donor mitoses at culture initiation
Weeks after bursal cell transfer
-__ (1.9) (1.8)
~1Mitotic Index (M.I.) = b Number of donor (host) mitoses X mitotic index X Figures in (parenthesis) stimulated cultures divided
9.9
34
10
(1.2)
(13.7) 3
(17.1) (82.1)
20.1
No. mitoses/103 cells, based on no. mitoses/2000 nucleated cells. mitoses/l04 cells in culture calculated by multiplying ‘% donor (host) 10. represent stimulatory indices (S.I.) = no. of mitoses/104 cells in by no. mitoses/104 cells in control cultures.
MATERIALS
AND
METHODS
The experimental animal model containing chromosomally marked host T and donor B, cells has recently been described (9). Briefly, embryonic bursal cells of known chromosome composition were transferred to histocompatible (blood type BsB2) S-day-old chicks carrying the opposite sex chromosome marker. Bursal cell recipients were first treated with cyclophosphamide to induce B cell depletion as described by Linna et al. (10). At lo-16 wk after bursal cell transfer, spleen cell suspensionswere obtained from the recipients and cultures initiated in serum-free medium (11). Cultures were stimulated with the following optimal concentrations of stimulants : Pokeweed Mitogen (Grand Island Biological Company, Grand Island, NY) 2 pi/ml ; concanavalin A, 3 X crystallized and carbohydrate-free (Miles Laboratories, Kankakee, Illinois) 2.5 rg/ml. Anti-immunoglobulin serum (AntiCIg) and normal rabbit serum (NRS) were prepared as described in detail elsewhere (13) and used at a concentration of 25 pi/ml. Chromosome preparations were prepared from 2 to 4 day stimulated and unstimulated cultures according to the method of Moorhead et al. (12) with minor modifications found especially suitable for avian cells (11). At least 100 mitotic figures were examined from each culture to determine the proportion of proliferating host T and donor Bz cells. RESULTS When spleen cell cultures of a bursal cell recipient were established 10 wk after cell transfer and evaluated 48 hr after culture initiation, significant proliferation of host T cells was detected in PWM and in Con A-stimulated cultures (Table 1). The responseof host cells to Con A (S.I. 82.1) was almost five times greater than their responseto PWM (S.I. 17.1). In contrast, the number of donor mitoses (B2) per 10’ cells was not significantly elevated in cultures stimulated by PWM (S. I.
28 91 17
Control Anti-CIg NRS
(I Cultures b Cultures r Mitotic d Number Figures in
13 1.3
PWM Con A
IIb
1 6 2
6 25
7 25 1 9 0.5
M.1.C
2.8 54.6 3.4 (16.6)
7.8 (2.8) 3.3 (1.2)
4.2 (
No. donor mitoses/ 10’ cellsd
48 hr
7.8 5.4 16.6
52.2 246.7 (
(6.7) (31.6)
65.8 (11.9) 245.0 (44.5) 5.5 5.4 (1.3) 3.95
No. host mitoses/ lo4 cells”
Culture
period
12 74 11
29 1
11 0 5 40 2
% Donor mitoses
1.5 4.5 2.5
7 12
8 27 2.5 8 3.5
M.I.
SERUM
1.8 33.3 2.8
20.3 1.2
(11.9)
(11.3) (
8.8 (7.0) 0 (0) 1.25 32.0 (45.7) 0.7 96 hr
No. donor mitoses/ 10’ cells
96 hr
TRANSFER
index X 10. by no. mitoses/l@i
CON A AND TO ANTI-IMMUNOGLOBULIN FOLLOWING EMBRYONIC BURSAL CELL
2
initiated 12 wk after bursal cell transfer; 60% donor mitoses at time of culture initiation. initiated 16 wk after bursal cell transfer; 27% donor mitoses at time of culture initiation. Index (M.I.) = no. mitoses/lOs cells, based on no. mitoses/2000 nucleated cells. of donor (host) mitoses/104 cells in culture calculated by multiplying y0 donor (host) mitoses X mitotic (parentheses) represent stimulatory indices (S.I.) = no. of mitoses/104 cells in stimulated cultures divided
6 2 45 94 21
PWM Con A Control Anti-CIg NRS
% Donor mitoses
IQ
CULTURES
TABLE
RESPONSES TO SOLUBLE PWM, OF CYCLOPHOSPHAMIDE-TREATED CHICKENS
HOST CELL PROLIFERATIVE
Stimulant
AND
Expt
DONOR
cells in control
13.2 11.7 22.2
49.7 118.8
cultures.
(
(3.8) (9.0)
(1.4)
(3.0) (11.4)
No. host mitoses/ lo4 cells
CELL
71.2 270.0 23.75 48.0 34.3
IN SPLEEN
E 2, ;: 5 5 z rn
z
z ;; T:
SHORT
COMMUXICATIOiYS
485
1.9) and by Con A (S.I. 1.S). At the 4S-hr culture period the percentage of spontaneously dividing donor mitoses in control cultures was remarkably high (40%) and the 1.5% and 7% donor mitoses encountered in Con A and PWM-stimulated cultures can be attributed to this spontaneously dividing donor cell population. In cultures stimulated by Anti-CIg a nearly 14-fold increase in the number of donor Bz mitoses was detected as compared to NRS-stimulated cultures, whereas dividing host T cells were not significantly elevated when compared to values obtained in control cultures (S.I. 1.2). Upon extension of the culture period to 4 days, a strikingly different pattern emerged in PWM-stimulated cultures. As shown by two experiments represented in Table 2, the percentage of donor B, mitoses in PWM-stimulated cultures was now more than twice that found in control cultures and a convincing increase in the number of donor mitoses/104 cells in culture was detected in the stimulated as compared to unstimulated cultures (S.I. 7.0-11.3). Although the absolute increase in, the number of PWM-stimulated donor mitoses/104 cells in culture (no. of donor mitoses/104 cells in stimulated minus the number of donor mitoses in control cultures) was 7.5 and 18.5 in Expts I and II, respectively, PWM-activated dividing host T cells still predominated, however, as shown by an increase of 47.4 and 36.5 host mitoses/104 cells in PWM-stimulated as compared to control cultures. The values obtained in 4S-hr cultures in Expt I, Table 2, confirmed that no significant response of donor Bz cells to PWM (S.I.
486
SHORT
COMMUNICATIONS
The failure to detect a significant response of I32 cells to PWM in 48-hr cultures and only a questionable response at 72 hr after culture initiation is in agreement with observations presented previously (14). It is apparent from these additional studies, however, that a contiincing response of Bz cells to PWM was detectable with this system after a 96-hr culture period. The relatively small and apparent delayed response of Bz cells to this phytomitogen has at least two possible explanations. One possibility is that the number of dividing Bs cells detected in 4 day PW~~-stimulated cultures resulted from the repeated division of an initially small population of PWM-responsive B2 cells. Their proliferation during the early phases of the culture period may be partially masked and difficult to detect because of the predomir~ant T cell response and the still active spontaneous proliferation of Bz cells. A second possibility is that Bz cells may not be activated by PWM as rapidly as T cells apparently are, and their late detection could have resulted from a slower activation, coupled perhaps with a slower turnover time, of a considerably larger Bz cell population. Additional experiments with T cell depleted, clearly identified B, cell populations should allow determination of the size of the Bz cell pool which is capable of responding to PWM as well as to other stimulants. Our experiments, although in agreement with those reports in the literature that have suggested a PW~-induced proliferative response of both T and B cells (4-6), did not establish whether the presence of T cells or T cell products, that may have been liberated into the culture medium, were essential for the B:, cell response to PWM. Recent experiments in mice have suggested, however, that in this species thymus-independent B celis can respond to PWM in the apparent absence of T cells (5). In view of several reports which suggest that other soluble phytomitogens including PN[A and Con A can also stimulate B cells when these are cultured in the presence of T cells (15--17), the failure of Con A to induce detectable proliferation of chromosomally marked I32 cells throughout a 4-day culture period in our experiments is noteworthy. Clearly, the presence and proliferation of T cells in the same culture vessel did not significantly alter the unresponsiveness of Bz cells to Con A under the culture conditions employed. At present it is unclear whether this discrepancy is due to species differences, to differences in culture conditions, or is the result of different methods of evaluation with varyin g degrees of sensitivity and specificity. It is still conceivable that avian B2 cells may respond to Con ,A if the stimulant were presented to the cells in an insoluble form, but to date we have not been able to obtain convincing evidence for this view (14). In other experiments we have shown that viable bursal (B,) cells within bursal follicles fail to reSponr1 to soluble and insoluble, sepharose-coupled forms of phytomitogens, even when ctxltured in the presence of supernatants of phytomitogen-activated T cells (18). Additional studies with chromosomally marked B, cell subpopulations should now make it possible to determine the functional potential of these cells. ACKNOWLEDGMENT The
valuable
technical
assistance of Mrs. anne Sciascia is
gratefully
acknowledged.
REFERENCES t.. Janossy,
G., and Greaves,
hi.
F., Clin.
Exp.
Immunol.
9, 483,
2. Stockman, G. O., Gallagher, M. T., Heim. L. R., South, Sot.
Exp.
Biol.
Med.
136,
980,
1971.
1971.
7~4. A.,
and
Trentin,
J. J. Pmt.
SHORT
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
COMMUNICATIONS
487
Watson, J., Epstein, R., Nakoinz, I., and Ralph, P. J. Immzmol. 110, 43, 1973. Janossy, G., and Greaves, M. F., Cl&. Exp. Immunol. 10, 525, 1972. Shortman, K., Byrd, W. J., Cerottini, J. C., and Brunner, K. T., Cell. Immulzol. 6, 25, 1973. Peavy, D. L., Adler, W. H., Shands, J. W., and Smith, R. T., Cell. Immunol. (in press). Kirchner, H., Oppenheim, J. J., and Blaese, R. M., In “Proceedings of the Seventh Leucocyte Culture Conference (F. Daguillard, Ed.), pp. 501-511. Academic Press, New York, 1973. Weber, W. T., Cell. Immunol. 4, 51, 1972. Weber, W. T., J. Immunol. 111,127i’,l973. Linna, T. J., Frommel, D., and Good, R. A., Int. Arch. Allergy Appl. Immunol. 42, 20, 1972. Weber, W. T. J Reticuloendothelial. Sot. 8, 37, 1970. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M., and Hungerford, D. A., Exp. Cell. Res. 20, 613, 1960. Weber, W. T., In “Proceedings of the Seventh Leucocyte Culture Conference (F. Daguillard, Ed.), pp. 535-546, Academic Press, New York, 1973. Weber, W. T., Fed. Proc. 32, 956, 1973. Piquet, P. F., and Vassalli, P., J. Exp. Med. 136, 962, 1972. Elfenbein, G. J., Harrison, M. R., and Green, I., .I. Immunol. 110, 1334, 1973. Vischer, T. L., Clint. Exp. Immunol. 11, 253, 1972. Weber, W. T., J. Reticuloendotheliol. Sot. (in press).
Direct
IMMUKOLOGY
Evidence
482-487
9,
(1973)
for the Response of B and T Cells Mitogen ’ W.
Department
of Pathobiology,
T. WEBER
School of Veterinary Medicine, Philadelphia, Penmylvania 19104 Received
to Pokeweed
July
University
of
Pennsylvania,
30, 1973
Chicken spleen cells containing chromosomally marked thymus derived (T) and bursa-derived (Bz) cells were evaluated for their ability to respond to pokeweed mitogen (PWM), concanavalin A (Con A), and to anti-immunoglobulin serum during a 4-day culture period. The results indicate that soluble PWM induces a proliferative response of B2 cells in addition to a predominant T cell response. The PWM-induced Bf cell proliferative response was clearly detected only at 4 days after culture initiation. Soluble Con A did not induce detectable proliferation of Bz cells and stimulated T cells exclusively. In contrast, anti-immunoglobulin serum was a specific stimulant for B2 cells under the culture conditions used.
INTRODUCTION The type of lymphocyte stimulated by pokeweed mitogen (PWM) continues to be a subject of considerable controversy. Based to a large extent on experiments in mice, three conflicting sets of experimental data are found in the literature. Some reports suggested that only bone marrow derived, thymus-independent B cells were activated (1,2) whereas in others evidence only for the responseof T cells is found (3). The conclusion that PWM can activate both T and B cells is represented in several more recent reports (4-6). In vitro studies on lymphocyte populations obtained from B cell depleted chickens clearly indicated that T cells could respond to PWM, and evaluation of PWM-induced proliferative responses by lymphocytes from normal and B cell depleted chickens suggested furthermore that bursa-derived (B,) cells did not respond to this phytomitogen (7). We have attempted to develop experimental models in the chicken that would allow identification of thymus-derived (T) and bursa-derived ( B2) cells through the use of chromosome markers. The use of experimental chicks containing identified T and Bz cell populations have already proven useful for the detection of antigen-induced proliferation of B2 cells in viva (8) and for determining the ability of Bz and T cells to respond to lipopolysaccharide (9). In this report, direct evidence for the PWM-induced response of B2 cells, in addition to a predominant T cell responseto this stimulant will be presented. 1 This work was supported by United States Public Health Service Grant AM 11693. 482 1973 by Academic Press, reproduction in any form
Inc. reserved.
483
SHORT COMMUNICATIONS TABLE
1
DONOR AND HOST CELL PROLIFERATIVE RESPONSES TO SOLUBLE PWM, CON A AND TO ANTI-IMMUNOGLOBULIN SERUM IN SPLEEN CELL CULTURES OF A CYCLOPHOSPHAMIDE-TREATED CHICKEN FOLLOWING EMBRYONIC BURSAL CELL TRANSFER Culture
Stimulant % Donor mitoses
M.1.a
PWM Con A Control Anti-CIg
7 1.5 40 85
11 50 1 16
NRS
33
period-48
hr -
No. donor mitoses/lOl cellsb
No. host mitoses/lOJ cellsb
7.7 7.5 4 136
102.3 492.5 6 24
ye Donor mitoses at culture initiation
Weeks after bursal cell transfer
-__ (1.9) (1.8)
~1Mitotic Index (M.I.) = b Number of donor (host) mitoses X mitotic index X Figures in (parenthesis) stimulated cultures divided
9.9
34
10
(1.2)
(13.7) 3
(17.1) (82.1)
20.1
No. mitoses/103 cells, based on no. mitoses/2000 nucleated cells. mitoses/l04 cells in culture calculated by multiplying ‘% donor (host) 10. represent stimulatory indices (S.I.) = no. of mitoses/104 cells in by no. mitoses/104 cells in control cultures.
MATERIALS
AND
METHODS
The experimental animal model containing chromosomally marked host T and donor B, cells has recently been described (9). Briefly, embryonic bursal cells of known chromosome composition were transferred to histocompatible (blood type BsB2) S-day-old chicks carrying the opposite sex chromosome marker. Bursal cell recipients were first treated with cyclophosphamide to induce B cell depletion as described by Linna et al. (10). At lo-16 wk after bursal cell transfer, spleen cell suspensionswere obtained from the recipients and cultures initiated in serum-free medium (11). Cultures were stimulated with the following optimal concentrations of stimulants : Pokeweed Mitogen (Grand Island Biological Company, Grand Island, NY) 2 pi/ml ; concanavalin A, 3 X crystallized and carbohydrate-free (Miles Laboratories, Kankakee, Illinois) 2.5 rg/ml. Anti-immunoglobulin serum (AntiCIg) and normal rabbit serum (NRS) were prepared as described in detail elsewhere (13) and used at a concentration of 25 pi/ml. Chromosome preparations were prepared from 2 to 4 day stimulated and unstimulated cultures according to the method of Moorhead et al. (12) with minor modifications found especially suitable for avian cells (11). At least 100 mitotic figures were examined from each culture to determine the proportion of proliferating host T and donor Bz cells. RESULTS When spleen cell cultures of a bursal cell recipient were established 10 wk after cell transfer and evaluated 48 hr after culture initiation, significant proliferation of host T cells was detected in PWM and in Con A-stimulated cultures (Table 1). The responseof host cells to Con A (S.I. 82.1) was almost five times greater than their responseto PWM (S.I. 17.1). In contrast, the number of donor mitoses (B2) per 10’ cells was not significantly elevated in cultures stimulated by PWM (S. I.
28 91 17
Control Anti-CIg NRS
(I Cultures b Cultures r Mitotic d Number Figures in
13 1.3
PWM Con A
IIb
1 6 2
6 25
7 25 1 9 0.5
M.1.C
2.8 54.6 3.4 (16.6)
7.8 (2.8) 3.3 (1.2)
4.2 (
No. donor mitoses/ 10’ cellsd
48 hr
7.8 5.4 16.6
52.2 246.7 (
(6.7) (31.6)
65.8 (11.9) 245.0 (44.5) 5.5 5.4 (1.3) 3.95
No. host mitoses/ lo4 cells”
Culture
period
12 74 11
29 1
11 0 5 40 2
% Donor mitoses
1.5 4.5 2.5
7 12
8 27 2.5 8 3.5
M.I.
SERUM
1.8 33.3 2.8
20.3 1.2
(11.9)
(11.3) (
8.8 (7.0) 0 (0) 1.25 32.0 (45.7) 0.7 96 hr
No. donor mitoses/ 10’ cells
96 hr
TRANSFER
index X 10. by no. mitoses/l@i
CON A AND TO ANTI-IMMUNOGLOBULIN FOLLOWING EMBRYONIC BURSAL CELL
2
initiated 12 wk after bursal cell transfer; 60% donor mitoses at time of culture initiation. initiated 16 wk after bursal cell transfer; 27% donor mitoses at time of culture initiation. Index (M.I.) = no. mitoses/lOs cells, based on no. mitoses/2000 nucleated cells. of donor (host) mitoses/104 cells in culture calculated by multiplying y0 donor (host) mitoses X mitotic (parentheses) represent stimulatory indices (S.I.) = no. of mitoses/104 cells in stimulated cultures divided
6 2 45 94 21
PWM Con A Control Anti-CIg NRS
% Donor mitoses
IQ
CULTURES
TABLE
RESPONSES TO SOLUBLE PWM, OF CYCLOPHOSPHAMIDE-TREATED CHICKENS
HOST CELL PROLIFERATIVE
Stimulant
AND
Expt
DONOR
cells in control
13.2 11.7 22.2
49.7 118.8
cultures.
(
(3.8) (9.0)
(1.4)
(3.0) (11.4)
No. host mitoses/ lo4 cells
CELL
71.2 270.0 23.75 48.0 34.3
IN SPLEEN
E 2, ;: 5 5 z rn
z
z ;; T:
SHORT
COMMUXICATIOiYS
485
1.9) and by Con A (S.I. 1.S). At the 4S-hr culture period the percentage of spontaneously dividing donor mitoses in control cultures was remarkably high (40%) and the 1.5% and 7% donor mitoses encountered in Con A and PWM-stimulated cultures can be attributed to this spontaneously dividing donor cell population. In cultures stimulated by Anti-CIg a nearly 14-fold increase in the number of donor Bz mitoses was detected as compared to NRS-stimulated cultures, whereas dividing host T cells were not significantly elevated when compared to values obtained in control cultures (S.I. 1.2). Upon extension of the culture period to 4 days, a strikingly different pattern emerged in PWM-stimulated cultures. As shown by two experiments represented in Table 2, the percentage of donor B, mitoses in PWM-stimulated cultures was now more than twice that found in control cultures and a convincing increase in the number of donor mitoses/104 cells in culture was detected in the stimulated as compared to unstimulated cultures (S.I. 7.0-11.3). Although the absolute increase in, the number of PWM-stimulated donor mitoses/104 cells in culture (no. of donor mitoses/104 cells in stimulated minus the number of donor mitoses in control cultures) was 7.5 and 18.5 in Expts I and II, respectively, PWM-activated dividing host T cells still predominated, however, as shown by an increase of 47.4 and 36.5 host mitoses/104 cells in PWM-stimulated as compared to control cultures. The values obtained in 4S-hr cultures in Expt I, Table 2, confirmed that no significant response of donor Bz cells to PWM (S.I.
486
SHORT
COMMUNICATIONS
The failure to detect a significant response of I32 cells to PWM in 48-hr cultures and only a questionable response at 72 hr after culture initiation is in agreement with observations presented previously (14). It is apparent from these additional studies, however, that a contiincing response of Bz cells to PWM was detectable with this system after a 96-hr culture period. The relatively small and apparent delayed response of Bz cells to this phytomitogen has at least two possible explanations. One possibility is that the number of dividing Bs cells detected in 4 day PW~~-stimulated cultures resulted from the repeated division of an initially small population of PWM-responsive B2 cells. Their proliferation during the early phases of the culture period may be partially masked and difficult to detect because of the predomir~ant T cell response and the still active spontaneous proliferation of Bz cells. A second possibility is that Bz cells may not be activated by PWM as rapidly as T cells apparently are, and their late detection could have resulted from a slower activation, coupled perhaps with a slower turnover time, of a considerably larger Bz cell population. Additional experiments with T cell depleted, clearly identified B, cell populations should allow determination of the size of the Bz cell pool which is capable of responding to PWM as well as to other stimulants. Our experiments, although in agreement with those reports in the literature that have suggested a PW~-induced proliferative response of both T and B cells (4-6), did not establish whether the presence of T cells or T cell products, that may have been liberated into the culture medium, were essential for the B:, cell response to PWM. Recent experiments in mice have suggested, however, that in this species thymus-independent B celis can respond to PWM in the apparent absence of T cells (5). In view of several reports which suggest that other soluble phytomitogens including PN[A and Con A can also stimulate B cells when these are cultured in the presence of T cells (15--17), the failure of Con A to induce detectable proliferation of chromosomally marked I32 cells throughout a 4-day culture period in our experiments is noteworthy. Clearly, the presence and proliferation of T cells in the same culture vessel did not significantly alter the unresponsiveness of Bz cells to Con A under the culture conditions employed. At present it is unclear whether this discrepancy is due to species differences, to differences in culture conditions, or is the result of different methods of evaluation with varyin g degrees of sensitivity and specificity. It is still conceivable that avian B2 cells may respond to Con ,A if the stimulant were presented to the cells in an insoluble form, but to date we have not been able to obtain convincing evidence for this view (14). In other experiments we have shown that viable bursal (B,) cells within bursal follicles fail to reSponr1 to soluble and insoluble, sepharose-coupled forms of phytomitogens, even when ctxltured in the presence of supernatants of phytomitogen-activated T cells (18). Additional studies with chromosomally marked B, cell subpopulations should now make it possible to determine the functional potential of these cells. ACKNOWLEDGMENT The
valuable
technical
assistance of Mrs. anne Sciascia is
gratefully
acknowledged.
REFERENCES t.. Janossy,
G., and Greaves,
hi.
F., Clin.
Exp.
Immunol.
9, 483,
2. Stockman, G. O., Gallagher, M. T., Heim. L. R., South, Sot.
Exp.
Biol.
Med.
136,
980,
1971.
1971.
7~4. A.,
and
Trentin,
J. J. Pmt.
SHORT
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
COMMUNICATIONS
487
Watson, J., Epstein, R., Nakoinz, I., and Ralph, P. J. Immzmol. 110, 43, 1973. Janossy, G., and Greaves, M. F., Cl&. Exp. Immunol. 10, 525, 1972. Shortman, K., Byrd, W. J., Cerottini, J. C., and Brunner, K. T., Cell. Immulzol. 6, 25, 1973. Peavy, D. L., Adler, W. H., Shands, J. W., and Smith, R. T., Cell. Immunol. (in press). Kirchner, H., Oppenheim, J. J., and Blaese, R. M., In “Proceedings of the Seventh Leucocyte Culture Conference (F. Daguillard, Ed.), pp. 501-511. Academic Press, New York, 1973. Weber, W. T., Cell. Immunol. 4, 51, 1972. Weber, W. T., J. Immunol. 111,127i’,l973. Linna, T. J., Frommel, D., and Good, R. A., Int. Arch. Allergy Appl. Immunol. 42, 20, 1972. Weber, W. T. J Reticuloendothelial. Sot. 8, 37, 1970. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M., and Hungerford, D. A., Exp. Cell. Res. 20, 613, 1960. Weber, W. T., In “Proceedings of the Seventh Leucocyte Culture Conference (F. Daguillard, Ed.), pp. 535-546, Academic Press, New York, 1973. Weber, W. T., Fed. Proc. 32, 956, 1973. Piquet, P. F., and Vassalli, P., J. Exp. Med. 136, 962, 1972. Elfenbein, G. J., Harrison, M. R., and Green, I., .I. Immunol. 110, 1334, 1973. Vischer, T. L., Clint. Exp. Immunol. 11, 253, 1972. Weber, W. T., J. Reticuloendotheliol. Sot. (in press).