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Graft-versus-host reactivity of human lymphoid cells from established cell lines
CELLULAR
2, 508-512
IMMUNOLOGY
Graft-Versus-Host
(1971)
Reactivity Established
from
of
Human Lymphoid Cell Lines 1
Cells
JULIUS GORDON,’ KENNETH TAGUCHI,~ RODRIQUE I. CONSTANTINE, AND LLOYD D. MACLEAN Department
of
Experimental
Szlrgery, Received
McGill May
University,
Montreal,
Canada
13, 1971
Cells from three out of five established human lyrnphoid cell lines gave reactions when injected under the kidney capsule of rats pretreated with cyclophosphamide. These reactions were characterized by mononuclear cell infiltration and destruction of the kidney cortex. No reactions were obtained when the cells were incubated with anti-human lymphocyte serum or were treated with mitomycin. Reactions could not be elicited in irradiated or i\LS-treated recipients. The reactions obtained were indistinguishable from the graft-versus-host reactions given by fresh human buffy coat leukocytes.
Recently Moore and his associates (l), Broder et al. (2) and Gerber and Monroe (3) described methods to establish lymphoid cells in permanent long-term culture. The cells are derived from the blood of patients with metabolic disorders or from normal individuals. Whether these cells are normal, or how they are related to the normal lymphocyte is not quite clear. Morphologically they appear as “blast cells” seen in short-term mixed leukocyte cultures, or as cells stimulated to divide with phytohemagglutinin. However, unlike the latter these divide with a generation time of 12 hr, and cells of some lines have been in culture for several years. Some lines have been shown to synthesize immunoglobulins (4), interferon (5), migration inhibition factor, lymphotoxin (6), and blastogenic factor (7). These criteria, however, do not necessarily characterize cultured cells as normal. The purpose of the experiments to be described was to find out whether these cells are capable of generating cell-mediated immune reactions. The method of assay used was the graft-versus-host (GVH) reaction we recently described for human lymphoid cell populations (8). MATERIALS
AND
METHODS
The GVH assay was carried out as previously described for mouse (9) and human (8) lymphoid cells. Briefly, 30-50 X lo6 lymphoid cells, suspended in 0.1 ml of Medium 199, containing 1.5% fetal calf serum was injected under the left i This investigation was supported by grants from and from the John A. Hartford Foundation, Inc. * Research 3 Fellow
Associate of the Medical
of the
Medical
Research
Research
Council
Council
of Canada. 508
the Medical
Research
of Canada.
Council
of Canada
SHORT
509
COMMUNICATIONS
kidney capsule of rats, injected with 100 mg/kg body weight cyclophosphamide (CY) 1 day earlier. The reaction was evaluated on the 6th day after inoculation of cells : both kidneys were excised, weighed, and sections were taken for histology. The reaction was expressed as a ratio of the weights of the injected to the uninjetted kidney and was confirmed by examination of the histological sections. In some experiments the rats were pretreated with 6 ml of a horse anti-rat lymphocyte serum (ARLS) or were given 1200 R total-body x-irradiation. Outbred (hooded) or (Lewis X BN) Fl female rats were used weighing 180-200 g. The cells inoculated were normal, human, buffy coat leukocytes, cells from a murine lymphatic leukemia (BW5147 from the Jackson Laboratories, Bar Harbor, Maine), and cells from 5 established human lymphoid cell lines. The designations and some characteristics of these cells are shown in Table 1. Cells from 4 of these lines were used in one experiment in Dr. G. E. Moore’s laboratory in Buffalo. Most of the experiments reported were carried out with the cell line No. 806s established in Buffalo and maintained in our laboratory in Montreal. In some experiments the cells were treated prior to injection with mitomycin according to the method of Bach and Voynow (10) or were exposed to a rabbit anti-human lymphoblast serum (AHLS). Treatment with AHLS was either in viva or in vitro. In vitro 40 X lo7 cells were incubated at 37°C for 30 min in Z-ml aliquots of AHLS diluted with Medium 199. In viva the AHLS was administered to the recipient rats in 3 daily injections beginning 1 day before inoculation of the cells. RESULTS
Normal peripheral blood lymphocytes or cells from 5 established cell lines were injected under the kidney capsule of rats given 100 mg/kg body weight cyclophosphamide 1 day earlier. As can be seen in Table 2, cells from 3 of the lines gave reactions comparable to that elicited by an equal number of peripheral blood lymphocytes. Histologically, the reactions obtained were indistinguishable from those given by fresh lymphoid cells and were characterized by round cell infiltration of the cortex, numerous mitoses, and destruction of the tubules. Cells from two of the cell lines gave no reactions. TABLE Sour;:
Designation
CHARACTERISTICS
source
1
OF THE
Established
CELL
LINES
IgM
_______RPM1 RPM1
6237 8068
RPM1 RPM1 RPM1
1788
Normal ?vlalignant melanoma Normal
1120 5287
Normal Normal
u All the cell lines and the information tory. * Lymphotosin. c Not tested.
1967 1969
NTc -
1968 1966 1967
+ -
in this table
were obtained
USED
a
Synthesis of -___ Interferon IgA LT h _~~ .~~. NT + + f NT + +
IgG
from
NT + +
+ + +
Dr. G. E. Moore’s
+ -I f labora-
510
SHORT
COMMUNICATIONS
TABLE REACTIVITY
2
OF CULTURED
CELLS
Cells No. Designation RPM1 RPM1 RPM1 RPM1 RPM1 RPM1 Fresh
a
1788 5287 1120 6237 8068 8068 blood
of recipients
Reaction
b Ki/Kc
No. injected 50 50 50 50 40 25 2.5
x x x x x x x
106 106 106 10” 106 106 10”
8 9 5 9 10 8 7
1.01 1.02 1.18 1.36 1.39 1.18 1.19
a The numbers designating each line are those of Dr. G. E. Moore of the Roswell Institute. b Ratio of the weight of the injected to that of the uninjected kidney. Mean f limits.
f f f f f f f
0.07 0.06 0.04 0.16 0.24 0.12 0.03
Park
Memorial
95’+J0 confidence
To rule out the possibility that the reactions obtained were that of a “host-versus-graft” variety, the donor cells injected were pretreated with mitomycin or were exposed to a rabbit anti-human lymphoblast serum (AHLS). The results shown in Tables 3 and 4 indicate that both treatments abrogated the reaction. To ascertain that the reaction does not represent a tissue culture in viva in which cells that proliferate in vitro merely carry on this process in viva, cell-line cells were injected into x-irradiated or ALS-treated recipients. Whereas it may be expected that such recipients would provide a milieux conducive for continued proliferation of cells, we have shown previously that x-irradiated or ALS-treated animals do not support GVH reactions (11). Indeed in 5 out of 6 experiments, cellline cells injected into x-irradiated recipients gave no reactions. Similarly in 2 out of 2 experiments no reactions were obtained in ARLS pretreated animals. Leukemic cells on the other hand did proliferate extensively in x-irradiated rats. One such experiment is illustrated in Table 5. DISCUSSION
A reaction has been defined by two criteria. Firstly by cell proliferation as judged and measured by an increase in the weight of the injected kidney and secondly, by TABLE INHIBITION Cells
injected
40 x 40 x
106 106
OF THE REACTION
3
BY TREATMENT WITH MITOMYCIN
Treatment Nil Mitomycin
No. b
OF THE
CULTURED
of recipients 7 7
CELLS
Reaction 1.20 1.00
Ki/Kc * f
0
0.10 0.06
a Ratio of the weight of the injected to the uninjected kidney Mean f 9.5’% confidence limits. b Cells were incubated at 37°C for 30 min with 25 pg/ml mitomycin (Nutritional Biochemicals) They were washed 3 times before injection. The cells used in the control group, incubated without mitomycin were handled in an identical manner.
SHORT
TABLE IYHIRITIO~
Cells -__ 40 40 40
OF THE REACTION
injected
h
x
106
40
x x x
10” 106 106
40
x
106
40 x 40 x 40 x
100 106 106
511
COMMUNICATIONS
BY TIIEATMENT
4 OF THE CULTURED No. of recipients
AHLS
CELLS
Reaction
?\Til In oii~~, 0.5 ml In rim, 0.2 ml Nil Iti oitra, 1 :1 In @ifro, 1:4 ITZ z~itro, 1 :16 In vitro, 1:64
CLAHIS, or anti-human lvmphoblast serum Treatment with AHLS was performed either as recipients in ciao. In the latter instance AHLS ginning 1 day before injection of the cells. b The cells injected were from cell line RPM1 c Ratio of the weight of the injected to that of limits.
TYITH ;\HLS
a
c Ki/Kc
1.27
zt
0.05
1.10 1.13 1.21
f f f
(I.05 0.05 0.06
1.01 1.06 1.09
i i i
0.05 0.07 0.05
1.11
i
0.06
was raised in rabbits, a preincubation in vitro was given intraperitoneally
with cell line No. 8068. or administration to the in 3 dail!- doses be-
8068. the uninjected
?iIean
kidney.
& 957,
confidence
invasion and destruction of the kidney cortex as judged from histological sections. These two criteria coincide and overlap in all instances but one. Proliferation is always accompanied by destruction except in the case of the injection of tumor cells which proliferate and infiltrate the cortex but do not cause destruction. The criteria for a positive reaction were met following the injection of cells from 3 of the 5 established cell lines studied. Since cells from some of these lines were used in only a single experiment, no conclusion can be derived regarding the possible significance of the negative results obtaind. The positive reactions may be viewed either as one given by the host or as a proliferative reaction an
TABLE REACTIONS
IN X-IRRADIATED
5 OR ALS-TREATED
RECIPIENTS
Recipient Cells RPM1 RPM1 RPM1 RPM1 Murine
injected 8068 25 8068 25 8068 40 8068 40 leukemic
Reaction X x X X
10” 106 lo6 lo6 cells c 30 X lo6
No.
Treatment
8 6 4 12 6
CY ARLS CY X-irradiation X-irradiation
t Ki/Kc
a 1.24 0.97 1.24
* f f
0.11 0.04 0.09
1.02
f
0.05
3.24
f
0.24
(i The concentration of cyclophosphamide (CY) injected was 100 mg/kg body wt. The ARLS used was a horse anti-rat lymphocyte serum; 6 ml was administered in 15 daily doses. Irradiation was from a WZo source; the dose administered was 1200 R at a rate of 58 R/mm. The cells were injected one day after pretreatment. b Ratio of the weight of the .njected to that of the uninjected kidney; mean & 95% Con& dence limits. c Lymphatic leukemia cells (BW 5147 from the Jackson Laboratories, Bar Harbor, Maine) maintained in AKR mice.
512
SHORT
COMMUNICATIONS
of the donor cells of an immune or nonimmune nature. The former may be excluded on the grounds that donor cells whose proliferation was inhibited by mitomycin treatment, or cells exposed to AHLS failed to induce a reaction. It is more difficult to discriminate between an immune and nonimmune proliferation of donor cells, Arguments which would tend to favor the former alternative are as follows: we have shown previously that x-irradiated as well as ARLS-treated recipients cannot support a GVH reaction ( 11) . Animals so treated, however, would be expected to sustain the nonspecific proliferation of cells. This was indeed shown to be the case when leukemic cells from AKR mice were injected. Cell-line cells, however, did not give a reaction in these recipients. Furthermore, malignant cells which proliferate in the kidney cortex will infiltrate between the tubules, causing no cell destruction (12)) whereas the reaction produced by the cells from the established lines give reactions indistinguishable from that given by fresh lymphoid cells. Accordingly, the simplest interpretation of the results described would be that cells from at least some established lymphoid cell lines are capable of mounting a GVH reaction and are thus immunocompetent. However, because of the importance of this issue, and the indirect nature of the evidence, this conclusion must remain tentative until more direct proof becomes available. ACKNOWLEDGMENT We wish to thank Dr. G. E. Moore for the supply of cells and for his kind hospitality allowing us to carry out an experiment in his laboratory.
in
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Moore, G. E., Gerner, R. E., and Franklin, H. A., J. Anzer. Med. Ass. 199, 519, 1967. Broder, S. W., Glade, P. R., and Hirshhorn, K., Blood 35, 539, 1970. Gerber, P., and Monroe, J. H. J. Nat. Cancer Ilzst. 40, 855, 1968. Tanigaki, N., Yagi, Y., Moore, G. E., and Pressman, D., J. ImmunoZ. 95, 634, 1966. Kasel, J. A., Haase, A. T., Glade, P. R., and Chessin, L. N., Proc. Sot. Exp. Biol. Med. 128, 351, 1968. Granger, G. A., Moore, G. E., White, J. G., Matzinger, P., Sundsmo, J. S., Shupe, S., Kolb, W. P., Kramer, J., and Glade, P. R., J. Immunol. 104, 1476, 1970. Smith, R. T., Bausher, J. A. C., and Adler, W. H. Amer. J. Pathol. 60, 495, 1970. Gordon, J., Saleh, W. S., and MacLean, L. D., Excerpta Med. Int. Congr. Ser. No. 197, 185, 1970. Saleh, W. S., MacLean, L. D., and Gordon, J., J. Immuml. 100, 114, 1969. Bach, F. H., and Voynow, N. K., Scietice 153, 545,1%6. Constantine, R. I., Nakouz, J., MacLean, L. D. and Gordon, J., J. Im~m~nol. (in press). Elkins. W. I... 1. Ezp. Med. 120, 329, 1964.
2, 508-512
IMMUNOLOGY
Graft-Versus-Host
(1971)
Reactivity Established
from
of
Human Lymphoid Cell Lines 1
Cells
JULIUS GORDON,’ KENNETH TAGUCHI,~ RODRIQUE I. CONSTANTINE, AND LLOYD D. MACLEAN Department
of
Experimental
Szlrgery, Received
McGill May
University,
Montreal,
Canada
13, 1971
Cells from three out of five established human lyrnphoid cell lines gave reactions when injected under the kidney capsule of rats pretreated with cyclophosphamide. These reactions were characterized by mononuclear cell infiltration and destruction of the kidney cortex. No reactions were obtained when the cells were incubated with anti-human lymphocyte serum or were treated with mitomycin. Reactions could not be elicited in irradiated or i\LS-treated recipients. The reactions obtained were indistinguishable from the graft-versus-host reactions given by fresh human buffy coat leukocytes.
Recently Moore and his associates (l), Broder et al. (2) and Gerber and Monroe (3) described methods to establish lymphoid cells in permanent long-term culture. The cells are derived from the blood of patients with metabolic disorders or from normal individuals. Whether these cells are normal, or how they are related to the normal lymphocyte is not quite clear. Morphologically they appear as “blast cells” seen in short-term mixed leukocyte cultures, or as cells stimulated to divide with phytohemagglutinin. However, unlike the latter these divide with a generation time of 12 hr, and cells of some lines have been in culture for several years. Some lines have been shown to synthesize immunoglobulins (4), interferon (5), migration inhibition factor, lymphotoxin (6), and blastogenic factor (7). These criteria, however, do not necessarily characterize cultured cells as normal. The purpose of the experiments to be described was to find out whether these cells are capable of generating cell-mediated immune reactions. The method of assay used was the graft-versus-host (GVH) reaction we recently described for human lymphoid cell populations (8). MATERIALS
AND
METHODS
The GVH assay was carried out as previously described for mouse (9) and human (8) lymphoid cells. Briefly, 30-50 X lo6 lymphoid cells, suspended in 0.1 ml of Medium 199, containing 1.5% fetal calf serum was injected under the left i This investigation was supported by grants from and from the John A. Hartford Foundation, Inc. * Research 3 Fellow
Associate of the Medical
of the
Medical
Research
Research
Council
Council
of Canada. 508
the Medical
Research
of Canada.
Council
of Canada
SHORT
509
COMMUNICATIONS
kidney capsule of rats, injected with 100 mg/kg body weight cyclophosphamide (CY) 1 day earlier. The reaction was evaluated on the 6th day after inoculation of cells : both kidneys were excised, weighed, and sections were taken for histology. The reaction was expressed as a ratio of the weights of the injected to the uninjetted kidney and was confirmed by examination of the histological sections. In some experiments the rats were pretreated with 6 ml of a horse anti-rat lymphocyte serum (ARLS) or were given 1200 R total-body x-irradiation. Outbred (hooded) or (Lewis X BN) Fl female rats were used weighing 180-200 g. The cells inoculated were normal, human, buffy coat leukocytes, cells from a murine lymphatic leukemia (BW5147 from the Jackson Laboratories, Bar Harbor, Maine), and cells from 5 established human lymphoid cell lines. The designations and some characteristics of these cells are shown in Table 1. Cells from 4 of these lines were used in one experiment in Dr. G. E. Moore’s laboratory in Buffalo. Most of the experiments reported were carried out with the cell line No. 806s established in Buffalo and maintained in our laboratory in Montreal. In some experiments the cells were treated prior to injection with mitomycin according to the method of Bach and Voynow (10) or were exposed to a rabbit anti-human lymphoblast serum (AHLS). Treatment with AHLS was either in viva or in vitro. In vitro 40 X lo7 cells were incubated at 37°C for 30 min in Z-ml aliquots of AHLS diluted with Medium 199. In viva the AHLS was administered to the recipient rats in 3 daily injections beginning 1 day before inoculation of the cells. RESULTS
Normal peripheral blood lymphocytes or cells from 5 established cell lines were injected under the kidney capsule of rats given 100 mg/kg body weight cyclophosphamide 1 day earlier. As can be seen in Table 2, cells from 3 of the lines gave reactions comparable to that elicited by an equal number of peripheral blood lymphocytes. Histologically, the reactions obtained were indistinguishable from those given by fresh lymphoid cells and were characterized by round cell infiltration of the cortex, numerous mitoses, and destruction of the tubules. Cells from two of the cell lines gave no reactions. TABLE Sour;:
Designation
CHARACTERISTICS
source
1
OF THE
Established
CELL
LINES
IgM
_______RPM1 RPM1
6237 8068
RPM1 RPM1 RPM1
1788
Normal ?vlalignant melanoma Normal
1120 5287
Normal Normal
u All the cell lines and the information tory. * Lymphotosin. c Not tested.
1967 1969
NTc -
1968 1966 1967
+ -
in this table
were obtained
USED
a
Synthesis of -___ Interferon IgA LT h _~~ .~~. NT + + f NT + +
IgG
from
NT + +
+ + +
Dr. G. E. Moore’s
+ -I f labora-
510
SHORT
COMMUNICATIONS
TABLE REACTIVITY
2
OF CULTURED
CELLS
Cells No. Designation RPM1 RPM1 RPM1 RPM1 RPM1 RPM1 Fresh
a
1788 5287 1120 6237 8068 8068 blood
of recipients
Reaction
b Ki/Kc
No. injected 50 50 50 50 40 25 2.5
x x x x x x x
106 106 106 10” 106 106 10”
8 9 5 9 10 8 7
1.01 1.02 1.18 1.36 1.39 1.18 1.19
a The numbers designating each line are those of Dr. G. E. Moore of the Roswell Institute. b Ratio of the weight of the injected to that of the uninjected kidney. Mean f limits.
f f f f f f f
0.07 0.06 0.04 0.16 0.24 0.12 0.03
Park
Memorial
95’+J0 confidence
To rule out the possibility that the reactions obtained were that of a “host-versus-graft” variety, the donor cells injected were pretreated with mitomycin or were exposed to a rabbit anti-human lymphoblast serum (AHLS). The results shown in Tables 3 and 4 indicate that both treatments abrogated the reaction. To ascertain that the reaction does not represent a tissue culture in viva in which cells that proliferate in vitro merely carry on this process in viva, cell-line cells were injected into x-irradiated or ALS-treated recipients. Whereas it may be expected that such recipients would provide a milieux conducive for continued proliferation of cells, we have shown previously that x-irradiated or ALS-treated animals do not support GVH reactions (11). Indeed in 5 out of 6 experiments, cellline cells injected into x-irradiated recipients gave no reactions. Similarly in 2 out of 2 experiments no reactions were obtained in ARLS pretreated animals. Leukemic cells on the other hand did proliferate extensively in x-irradiated rats. One such experiment is illustrated in Table 5. DISCUSSION
A reaction has been defined by two criteria. Firstly by cell proliferation as judged and measured by an increase in the weight of the injected kidney and secondly, by TABLE INHIBITION Cells
injected
40 x 40 x
106 106
OF THE REACTION
3
BY TREATMENT WITH MITOMYCIN
Treatment Nil Mitomycin
No. b
OF THE
CULTURED
of recipients 7 7
CELLS
Reaction 1.20 1.00
Ki/Kc * f
0
0.10 0.06
a Ratio of the weight of the injected to the uninjected kidney Mean f 9.5’% confidence limits. b Cells were incubated at 37°C for 30 min with 25 pg/ml mitomycin (Nutritional Biochemicals) They were washed 3 times before injection. The cells used in the control group, incubated without mitomycin were handled in an identical manner.
SHORT
TABLE IYHIRITIO~
Cells -__ 40 40 40
OF THE REACTION
injected
h
x
106
40
x x x
10” 106 106
40
x
106
40 x 40 x 40 x
100 106 106
511
COMMUNICATIONS
BY TIIEATMENT
4 OF THE CULTURED No. of recipients
AHLS
CELLS
Reaction
?\Til In oii~~, 0.5 ml In rim, 0.2 ml Nil Iti oitra, 1 :1 In @ifro, 1:4 ITZ z~itro, 1 :16 In vitro, 1:64
CLAHIS, or anti-human lvmphoblast serum Treatment with AHLS was performed either as recipients in ciao. In the latter instance AHLS ginning 1 day before injection of the cells. b The cells injected were from cell line RPM1 c Ratio of the weight of the injected to that of limits.
TYITH ;\HLS
a
c Ki/Kc
1.27
zt
0.05
1.10 1.13 1.21
f f f
(I.05 0.05 0.06
1.01 1.06 1.09
i i i
0.05 0.07 0.05
1.11
i
0.06
was raised in rabbits, a preincubation in vitro was given intraperitoneally
with cell line No. 8068. or administration to the in 3 dail!- doses be-
8068. the uninjected
?iIean
kidney.
& 957,
confidence
invasion and destruction of the kidney cortex as judged from histological sections. These two criteria coincide and overlap in all instances but one. Proliferation is always accompanied by destruction except in the case of the injection of tumor cells which proliferate and infiltrate the cortex but do not cause destruction. The criteria for a positive reaction were met following the injection of cells from 3 of the 5 established cell lines studied. Since cells from some of these lines were used in only a single experiment, no conclusion can be derived regarding the possible significance of the negative results obtaind. The positive reactions may be viewed either as one given by the host or as a proliferative reaction an
TABLE REACTIONS
IN X-IRRADIATED
5 OR ALS-TREATED
RECIPIENTS
Recipient Cells RPM1 RPM1 RPM1 RPM1 Murine
injected 8068 25 8068 25 8068 40 8068 40 leukemic
Reaction X x X X
10” 106 lo6 lo6 cells c 30 X lo6
No.
Treatment
8 6 4 12 6
CY ARLS CY X-irradiation X-irradiation
t Ki/Kc
a 1.24 0.97 1.24
* f f
0.11 0.04 0.09
1.02
f
0.05
3.24
f
0.24
(i The concentration of cyclophosphamide (CY) injected was 100 mg/kg body wt. The ARLS used was a horse anti-rat lymphocyte serum; 6 ml was administered in 15 daily doses. Irradiation was from a WZo source; the dose administered was 1200 R at a rate of 58 R/mm. The cells were injected one day after pretreatment. b Ratio of the weight of the .njected to that of the uninjected kidney; mean & 95% Con& dence limits. c Lymphatic leukemia cells (BW 5147 from the Jackson Laboratories, Bar Harbor, Maine) maintained in AKR mice.
512
SHORT
COMMUNICATIONS
of the donor cells of an immune or nonimmune nature. The former may be excluded on the grounds that donor cells whose proliferation was inhibited by mitomycin treatment, or cells exposed to AHLS failed to induce a reaction. It is more difficult to discriminate between an immune and nonimmune proliferation of donor cells, Arguments which would tend to favor the former alternative are as follows: we have shown previously that x-irradiated as well as ARLS-treated recipients cannot support a GVH reaction ( 11) . Animals so treated, however, would be expected to sustain the nonspecific proliferation of cells. This was indeed shown to be the case when leukemic cells from AKR mice were injected. Cell-line cells, however, did not give a reaction in these recipients. Furthermore, malignant cells which proliferate in the kidney cortex will infiltrate between the tubules, causing no cell destruction (12)) whereas the reaction produced by the cells from the established lines give reactions indistinguishable from that given by fresh lymphoid cells. Accordingly, the simplest interpretation of the results described would be that cells from at least some established lymphoid cell lines are capable of mounting a GVH reaction and are thus immunocompetent. However, because of the importance of this issue, and the indirect nature of the evidence, this conclusion must remain tentative until more direct proof becomes available. ACKNOWLEDGMENT We wish to thank Dr. G. E. Moore for the supply of cells and for his kind hospitality allowing us to carry out an experiment in his laboratory.
in
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Moore, G. E., Gerner, R. E., and Franklin, H. A., J. Anzer. Med. Ass. 199, 519, 1967. Broder, S. W., Glade, P. R., and Hirshhorn, K., Blood 35, 539, 1970. Gerber, P., and Monroe, J. H. J. Nat. Cancer Ilzst. 40, 855, 1968. Tanigaki, N., Yagi, Y., Moore, G. E., and Pressman, D., J. ImmunoZ. 95, 634, 1966. Kasel, J. A., Haase, A. T., Glade, P. R., and Chessin, L. N., Proc. Sot. Exp. Biol. Med. 128, 351, 1968. Granger, G. A., Moore, G. E., White, J. G., Matzinger, P., Sundsmo, J. S., Shupe, S., Kolb, W. P., Kramer, J., and Glade, P. R., J. Immunol. 104, 1476, 1970. Smith, R. T., Bausher, J. A. C., and Adler, W. H. Amer. J. Pathol. 60, 495, 1970. Gordon, J., Saleh, W. S., and MacLean, L. D., Excerpta Med. Int. Congr. Ser. No. 197, 185, 1970. Saleh, W. S., MacLean, L. D., and Gordon, J., J. Immuml. 100, 114, 1969. Bach, F. H., and Voynow, N. K., Scietice 153, 545,1%6. Constantine, R. I., Nakouz, J., MacLean, L. D. and Gordon, J., J. Im~m~nol. (in press). Elkins. W. I... 1. Ezp. Med. 120, 329, 1964.