CELLULARIMMUNOLOGY23,l-10 (1976)
The Lymphocyte Inhibiting Factor Extracted from the Thymus Inhibition of the DNA Synthesis in Vitro1 IRENE FLORENTIN, Institut
(Lift):
NICOLE KIGER AND GEORGES MATHS
de CancCrologie et d’lmmztnogine’tique, Ho*pital Paul-Browse, Received
(I.N.S.E.R.M. 94800-Villejuif, August
et Association France
Claude Bernard)
25, 1975
The action of a lymphocyte inhibiting factor extracted from the thymus (LIFT) has been investigated by studying its capacity to inhibit in vitro, independently of any cytotoxic effect, DNA synthesis in different types of mouse lymphoid cells. [3H]TdR incorporation was strongly depressed under the influence of the thymic extract in short term cultures of thymus, bone marrow cells as well as of spleen cells coming from: 1) normal CBA mice; 2) mice treated with PHA. Similarly in vitro PHA stimulation of spleen cells coming from normal mice was depressed when thymic extract was added to the culture. By contrast, the thymic extract does not significantly decrease [3H]TdR uptake in spleen cells coming from: 1) normal CBA mice injected with LPS; 2) T lym’phocyte deprived mice, whether or not their B lymphocytes were activated by in viva LPS administration. These results suggest that the LIFT acts on T lymphocytes at different stages of their maturation and differentiation. The inhibiting effect of the thymic extract upon [aH]TdR incorporation in bone marrow cells could be the result of an action on #erythrocytic and leukocytic precursors.
INTRODUCTION We have previously described a thymic extract designated a lymphocyte inhibiting factor (LIFT) due to its ability specifically to inhibit lymphocyte proliferation, (1, 2). From its immunosuppressive properties “in Z&JO” it was concluded that this factor affected only thymus-dependent (T) lymphocytes. Indeed, immune reactions involving T lymphocytes alone such as allogeneic skin graft rejection (1) and graft versus host reaction (1, 3, 4) or in collaboration with thymus independent (B) lymphocytes such as antibody formation against sheep red blood cells (SRBC) and DNP coupled to human y globulin (5) could be efficiently depressed. However, immune responses not involving T lymphocyte participation such as antibody formation to DNP-polymerized flagellin was unaffected. Preliminary observations had shown that the uptake of tritiated thymidine into thymocytes in short term culture was inhibited in the presence of LIFT (1). The question was raised whether the specificity of action observed in vivo could be demonstrated more directly upon the in vitro proliferation of lymphoid cells. For this purpose we have compared the effects of this factor upon the DNA synthesis in different lymphoid cell populations enriched in or deprived of one lymphocyte cell type, namely spleen cells coming from normal mice, stimulated or 1 Supported by Grant N.74.5.015.01, I.N.S.E.R.M. 1 Copyright 1976by AcademicPress,Inc. All rights o9 repmductfonin any form reserved.
and by Grant ATP
1-73-16, I.N.S.E.R.M.
2
FLORENTIN,
KIGER
AND
MATHfi
not by phytohemagglutinin (PHA) in vi&o and spleen cells from mice injected either with phytohemagglutinin (PHA) or with lipopolysaccharide (LPS) in order to stimulate specifically proliferation and differentiation of T (6) or B (7) lymphocyte populations. In addition, LIFT effect on DNA synthesis in bone marrow cells was studied. In the course of this study we have investigated the kinetics and dose dependence of the inhibition by LIFT of tritiated thymidine uptake in thymocytes and spleen cells in short term cultures. To exclude the possibility that cytotoxicity of the thymic extract might be the basis of the inhibition of DNA synthesis, its reversibility of action was demonstrated. MATERIAL Preparation
AND
METHODS
of LIFT
The extraction and partial purification of the thymic extract have been described in detail elsewhere (1). Briefly, lamb thymus was homogenized in distilled water. After centrifugation at ZO,OOOgfor 1 hr the supernatant was fractionated by alcohol precipitation. The inhibitory activity was recovered in the fraction soluble in a final ethanol concentration of 42% ; this is referred to as the T4 fraction. A similar procedure was applied to lamb kidney to obtain a control extract called the K4 fraction. Preparation
of Lymphocyte
Subpopulations
Various treatments were given to &week-old CBA mice (Centre d’Elevage, Orleans, CNRS) in order to enrich or deplete the thymus or spleen of one lymphocyte population or subpopulation. Preparation of T-Lymphocyte
Deprived Spleen Cells
Adult CBA mice were thymectomized, lethally irradiated 1 wk later and immediately reconstituted by an intravenous injection of lo7 isogeneic bone marrow cells. These animals referred to as “B mice”, were killed 4 wk later and their spleens were removed and processed as described below. Spleen cells coming from (nu/nu) mice are used as a source of T-lymphocyte deprived cell population. Control spleen cell populations are provided by (nu/+) sibs. Preparation of Spleens with Activated T or B-Lymphocytes In order to enrich spleen cell populations with activated T-lymphocytes, 100 pg of purified phytohemagglutinin (PHA, MR58, Wellcome) were injected intravenously into normal CBA mice. Similarly, B-lymphocytes were stimulated by one injection of 100 pg of lipopolysaccharide B from E. coli (LPS, Difco) given to normal or to “B” CBA mice. The spleens were removed 4 days after the injection of PHA or of LPS.
LYMPHOCYTE
In Vitro
DNA
or RNA
INHIBITORY
Synthesis Inhibition
THYMIC
FACTOR
3
Tests
Single cell suspensions were prepared from thymus, spleen, and femoral bone marrow in RPM1 1640 culture medium (Gibco) supplemented with antibiotics and adjusted to 3.3 X 10” cells per ml; 3 ml aliquots were distributed into glass culture tubes. In the kinetic studies, 250 pg in protein of the Tq fraction or the K4 control extract were added to each 3 ml aliquot of the thymocyte suspension at time zero. Cells were incubated at 37°C for 20, 30, 45, 60 or 90 min in a humidified atmosphere of air-CO2 (95 : 5) and 5 &i of methyl [SH] thymidine ( [SH]TdR, specific activity 7 Ci/mmole) were added 10 min before the end of incubation. When the cells were incubated for longer periods : 34, 5, 24, or 48 hr, [3H]TdR or [3H] uridine (specific activity 10 Ci/mM) was added 30 min before the end of each incubation. After the acid insoluble fraction had been precipitated by addition of trichloracetic acid to a final concentration of 7%, it was prepared for radioactivity counting as previously described (1). The cultures were made in triplicate and the results shown are examples from repeated experiments. For the dose effect studies 500, 250, 125, 75 or 30 pg in protein of the T4 fraction were added to 3 ml of thymic cell suspension. The cells were incubated for 34 hours as described above. In the comparative studies of the thymic extract’s action on different lymphoid cell populations, the cells were incubated for 39 hours at 37°C in the presence of 250 pg of the T4 fraction per culture tube and then processed as described above. In Vitro
PHA
Stiwdation
Normal spleen cell suspensions were prepared in RPM1 1640 culture medium supplemented with heat inactivated mule serum (Gibco Laboratories) and antibiotics. 5 X lo5 cells in 0.2 ml were cultured in each well of tissue culture treated U plates (Falcon Microtest II) for 54 hr in an air-CO2 atmosphere. 20 ~1 of l/50 dilution of stock solution of PHA and 16 pg in protein in 10 ~1 of the Td fraction or K4 fraction were added to some wells at the beginning of the culture. One microcurie of i3H]TdR per well was added for 4 hr at the end of the culture. The cells were harvested on glass fibre filters using a multiple automated sample harvester (MASH II, Microbiological Associates). Cell Viability At the end of the incubation period, cell counts were made using a hemocytometer and viability was assessed by the trypan blue dye exclusion test. Reversibility
Assay
Thymocytes were incubated for 1 hr in the presence or absence of the T, fraction under the same conditions as described above. The cells were spun down and resuspended either in LIFT-free fresh medium or in their first incubation medium. Cultures were then incubated for 1, 2 or 3 hr, after which [3H]TdR was added and the incubation continued for further 30 min.
FLORENTIN,
KIGER
AND
MATHfi
RESULTS Kinetics of the Inhibition the InfEuence of LIFT
of DNA
and RNA
Synthesis in Thymocytes
under
Mouse thymocytes were incubated from 20 min to 48 hr in the presence of the thymic extract, and inhibition of DNA or RNA precursor incorporation was estimated at different times during incubation. Results recorded in Table 1 show that [3H] TdR incorporation was strongly depressed after the cells had been in contact with the T4 fraction for 20 min. This inhibitory effect increased slightly with time, reaching its maximal value after 60 min to 5 hr of incubation, and then decreased to become nonsignificant after 48 hr of incubation. A slight but significant inhibition of [3H]uridine incorporation was observed after 3 hr of incubation with the thymic extract, this inhibition lasted for 24 hr but was no longer significant at 48 hr. In all these experiments the Kb control fraction was ineffective in decreasing thymidine or uridine incorporation in thymocytes. Dose Efect of the T4 Fraction on DNA
Synthesis in Lymphoid
Cell Suspensions
Results recorded in Table 2 show that the T4 fraction inhibitory effect on DNA synthesis in thymus or spleen cells is dose dependent. Moreover, spleen cells appear to be less sensitive than thymic cells to the action of LIFT. The amount of T4 fraction required to produce a definite percentage of inhibition of DNA synthesis in spleen cells is twice that required to produce an identical inhibition in thymic cells. In all further experiments a dose of 250 pg of the T4 fraction in 3 ml of culture was chosen as it generally gives a 50% inhibition of the DNA synthesis in normal spleen cells, although this inhibition can fluctuate through a range of 15% from one experiment to another. Cell Viability In cultures exposed to the T4 or Kq fractions there was no loss of cells compared to control cultures incubated for the same period in medium alone. Cell viability was in all cases greater than 90% by the trypan blue test, indicating that these fractions have no toxic effect. TABLE Kinetics
Time
of cultivation
[aH]TdR uptake Mean c.p.m. no fraction T4 fraction ‘%$Sb;Kb~“, by the 4
[“gz:
of DNA and RNA Synthesis Inhibition in Thymocytes Under the Influence of the Tc Fraction
20 min
30 mina
45 miw
60 mina
75 miw
90 mine
3$ hrb
5 hrb
24 hrb
48 bra
12,096 5.382
13,872 4,813
14,254 4,533
20,794 5,073
23,340 5,601
28,742 5,662
138,080 27,717
124,092 40,146
15,950 8,103
58,995 51,612
74%
79%
49%
13%
14,398 14,275
17,106 16,982
24,838 17,809
62,872 57.776
56%
65%
8,346 8.452
8,978 8,905
0%
0%
358%
,“;‘$e . .
no fr&ion Tc fraction % Inhibition TI fraction 0 [‘H]TdR b [‘H]TdR
1
13,076 13,217
80% 18,395 17,942
by the
is added is added
0%
0%
0%
10 min before the end of the cultivationqperiod. for 30 min before the endlof the cultivation~period.
0%
78%
107,937 85,183
21%
68% 139,778 91,415 35%
28%
8%
LYMPHOCYTE
INHIBITORY
TABLE
THYMIC
5
FACTOR
2
Dose Effect of the Tc Fraction on DNA Synthesis in Lymphoid Cell Suspensions Dose of T, fraction (pg of protein) added to 3 ml of culture
500pg
25opg
125rg
60 Irg
30 rg
1.5Pg
7spg
0
[3H]TdR uptake in thymocytes, Mean c.p.m. % inhibition
16,340 32,785 68,303 105,328 118,985 125,006 123,957 123,280 34% 0% 0% 73% 45% W% 95%
[3H]TdR uptake in spleen cells, mean c.p.m. y0 inhibition
3,919 72%
Reversibility
6,400 10,695 18% 51%
12,162 7%
12,768 2%
13,153 0%
12,975 0%
13,065 -
of Action
The 85% inhibition in [3H]TdR incorporation tion of thymocytes with the T4 fraction remained suspended in the same medium after centrifugtion. resuspended in chalone-free fresh medium the increased progressively with time reaching nearly recuperation (Fig. 1). LIFT Efect on DNA Bone Marrow Cells
Synthesis in Different
observed after a 1 hr incubaconstant when cells were reHowever, when the cells were [3H]TdR incorporation rate normal values after 3$ hr of
Spleen Cell Populations
and in
Histological studies of the spleens from animals stimulated by PHA or LPS have been performed in order to verify their specific mitogenic activity. In normal animals injected with PHA the periarteriolar T dependent areas of the splenic white pulp were larger and more cellular than those of the normal controls, the periarteriolar zones accumulatin, (+small and medium sized lymphocytes. In “B mice” injected with LPS the follicular zones showed marked hyperplasia with numerous small and medium sized lymphocytes. The periarteriolar zones seemed to be compressed by the proliferating lymphocytes of the follicle.
FIG. 1. Reversibility
of the inhibition of [‘H]TdR
uptake in thymocytes.
6
FLORENTIN,
KIGER
AND
TABLE Effect
Fraction added in vitro
None
None l-4 K4 None l-4 K4 None T4 K,
LPS
D In comparison
3
of the Thymic Extract on C3H]TdR Uptake in Spleen Cells Obtained from Normal Mice, PHA or LPS Treated Mice
Treatment of spleen donors in vi00
PHA
MATHI?
with [3H]TdR
uptake
[3H]TdR
uptake &SD
17,450 6,857 20,588 101,324 33,708 105,964 111,845 91,744 128,621 in culture
i f f f f f f f f
incubated
(c.p.m.)
1,230 262 375 12,253 5,191 19,090 10,878 10,870 10,953 without
% of inhibition0
61 -18 69 -4 17 -15 any fraction.
We have compared the inhibitory effect of LIFT upon tritiated thymidine incorporation in different lymphoid cell populations, and all the results presented here were confirmed in several experiments. We first compared LIFT inhibitory activity on the DNA synthesis of spleen cells coming from normal mice, PHA treated mice and LPS treated mice. Results are shown in Table 3. When spleen cells from normal CBA mice are incubated for 39 hr in the presence of T* fraction, the [3H]TdR uptake is 61% depressed in comparison with that of control cells cultured without any fraction. In spleen cells selectively enriched with PHA activated T-lymphocytes, we first observed that the spontaneous DNA synthesis is increased sixfold compared to that in nonactivated spleen cells. Nevertheless, the addition of the T4 fraction to the cultures reduced the [3H]TdR incorporation by 69%. Similarly LPS administration induces cell proliferation which results in a 6.5 fold increase in the spontaneous DNA synthesis in vitro. In contrast, with the results observed in PHA stimulated spleen cells, the addition of Td fraction induces only a 17% decrease of [3H] TdR incorporation in in tivo LPS activated spleen cells. In the second type of experiment the action of the thymic extract on DNA synthesis in spleen cell populations coming from normal CBA mice, “B mice”, and LPS treated “B mice” were compared. The results are shown in Table 4. In spleen cell populations depleted in T-lymphocytes the [SH] TdR incorporation in the cultures exposed to the Tq fraction is reduced by 34%, whereas the percentage of inhibition in normal spleen cell cultures reaches 58%. Furthermore, when B lymphocytes have been stimulated by LPS injected into “B” mice”, the spontaneous DNA synthesis is increased threefold when compared to nonstimulated “B mice” spleen cells. In this case, we have observed that the thymic extract exerts only a slight inhibitory effect on DNA synthesis, as the inhibition of [8H]TdR uptake is only 17%. In spleen cells coming from “nude” mice we have observed an inhibition of 40% of the [3H]TdR uptake under the influence of the Tq fraction; at the same
LYMPHOCYTE
INHIBITORY
THYMIC
TABLE Effect
7
FACTOR
4
of the Thymic Extract on C3H]TdR Uptake in Spleen Cells Obtained Normal Mice or “B Mice” with or without LPS Treatment
Origin of spleen cells
Normal
mice
“B mice”
Treatment in viva
Fraction added in vitro
None
None T4 K4 None l-4 K4 None T4 K4
None
“B mice”
LPS
a In comparison
with C3H]TdR
CH]TdR uptake (c.p.m.) f SD 16,552 7,030 15,960 34,514 24,159 34,610 106,673 88,474 109,980
uptake in culture
incubated
f f f f f f f i f
without
from
% of inhibition0
2,371 201 640 340 2,664 1,615 12,664 6,899 7,875
68 3 34 1 17 -3
any fraction.
time the inhibition was of 73% in the spleen cell population taken from normal littermates (Table 5). In this experiment we observed that when LIFT was added to cultures of bone marrow cells it, induced a strong inhibition of t3H]TdR uptake. This inhibitory effect was more striking when bone marrow cells came from normal mice than from nude mice. In all these experiments the K4 fraction did not significantly effect [3H]TdR incorporation by spleen cell populations. TABLE Effect
Origin
of the Thymic Extract on C3H]TdR Uptake in Spleen and Bone Marrow Cells from “Nude” Mice and Normal SIBS
of cells
Nude mice
Nude mice
Normal
5
mice
Fraction added
Spleen cells
Bone marrow
None ‘I.4 K4 cells
Spleen cells
40 1
None l-4 K4
548,952 f 26,325 120,333 f 14,126 327,330 rt 8,699
78 17
None
15,310 f- 1,664 4,195 f 1,045 17,265 h-r 634
73 -12
470,244 f 42,587 56,540 f 6,375 537,755 f 39,538
89 -13
K4 mice
Bone marrow
cells
None Ta
K* a In comparison
with C3H]TdR
uptake in culture
incubated
13,295 f 8,026 i 13,404 rt
% of inhibitiona
610 1,832 1,775
T4
Normal
C3H]TdR uptake (c.p.m.) f SD
without
any fraction.
8 Efect
FLORENTIN,
of the Thymic Extract
KIGER
on in Vitro
AND
PHA
MATHI?
Transformation
of Spleen Cells
The T4 fraction used in the same final contraction as in all other experiments (250 &3 ml) m ’ d uces a 61% inhibition in the nonstimulated spleen cells. On the other hand, PHA induced a 17-fold stimulation of DNA synthesis compared to that seen in unstimulated cultures. The T4 fraction reduced this PHA induced stimulation at least 68% ; in all cases the Kd fraction did not significantly affect the [ 3H] TdR incorporation in unstimulated and PHA stimulated spleen cells. DISCUSSION In order to determine if the LIFT immunosuppressive action on thymodependent reactions observed in viva (5) can be the result of a specific inhibition of T lymphocyte proliferation, we have investigated its effect on in vitro DNA synthesis in cell populations containing different proportions of T or B lymphocytes. We have first shown that the DNA synthesis suppression by LIFT is dose dependent and is greater in thymocytes than in spleen cells. In all further studies LIFT has been used at a concentration of 80 pg/ml which was shown to depress DNA synthesis by about 50% in normal spleen cells and 70% in thymocytes. Under these conditions, we have compared the LIFT effect on [ 3H] TdR uptake in short term culture of thymus cells, containing a great proportion of immature T-lymphocytes with a high proliferative activity and of spleen cell in which T-lymphocytes have been selectively activated by in viva administration of PHA. In both cases we have noticed a strong inhibition of DNA synthesis under the influence of LIFT. A similar inhibition of PHA-induced DNA synthesis was observed when LIFT was added at the same time as the mitogen to normal spleen cell cultures. In contrast, in T-lymphocyte-deprived spleen cell populations, coming either from “B mice” or from “nude” mice, the inhibition of DNA synthesis after incubation with LIFT is half as much as that in normal spleen cells. After LPS administration in normal mice or in “B mice” in order to selectively activated B-lymphocytes, DNA synthesis in their spleen cells was not significantly depressed under the influence of LIFT. From these data, it can be concluded that LIFT exerts a strong inhibitory effect on DNA synthesis in thymus-dependent lymphocytes which proliferate either during their maturation process in the thymus or after mitogenic stimulation. In contrast, it only slightly inhibits DNA synthesis in T-lymphocyte-depleted spleen cell populations and does not act on B-lymphocytes activated by LPS. Thus the specific immunosuppressive properties of LIFT may be the result of an inhibition of T-lymphocyte proliferation. However we cannot conclude an exclusive specificity of action of LIFT for T-lymphocytes. Indeed, we have noticed a strong inhibitory effect of this substance on bone marrow cells. This cannot be explained only by an action on lymphocyte precursors; it is most likely also due to an inhibition of erythroi’d or myeloi’d precursor proliferation. Experiments are actually in progress to further investigate this possibility. According to the kinetic studies and the finding that LIFT produced an inhibition of [3H]TdR uptake within the first 20 min of incubation, we can propose that binding of LIFT to membrane surface constituents of cells triggers
LYMPHOCYTE
INHIBITORY
THYMIC
FACTOR
9
the events leading to the inhibition of the proliferation. This early inhibition indicates that the substance acts by inhibiting S phase cells in their progression through the S phase to Gs, and perhaps by decreasing the flux of late G1 cells to S phase. However, by the method used in this study we cannot exclude an action of LIFT on other phases of the mitotic cell cycle. It cannot be argued that the inhibitory effect is due to a cytotoxic action of the thymic extract since, at the end of the incubation period, cell viability was identical in all samples. Moreover, the observation that in thymocytes DNA synthesis is inhibited in a totally reversible way rules out any selective cytotoxic effect of the extract. We can also argue against the hypothesis that our observations are the result of technical artefacts, such as the presence of cold nucleotide or thymidine binding protein in the the thymic extract or of enzymes which can disturb labelled thymidine incorporation, for it would be difficult to explain how such substances could interfere with T and not with B-lymphocyte cellular metabolism. Other immunosuppressive substances have been recently isolated from thymus (8, 9) and identified as alpha globulins similar to the immuno-regulatory alpha globulin (IRA) extracted from the serum (10, 11). IRA possesses physiological properties nearly identical to that found for LIFT, and in addition exhibits in V&O and in vitro (12) the same type of lymphocyte specificity. But the physicochemical properties of the two substances seems to be completely different from one to another and, at the present time, we have to suppose that these extracts are not identical substances (13, 14). A model for the regulation of cell production in all tissues which is now supported by a growing body of experimental data implies that differentiated cells secrete substances (“chalones”) that regulate the rate of cell renewal by a negative feedback mechanism (15). Some authors now refer to DNA synthesis inhibitors extractable from lymphoid tissue as the “lymphocyte chalone” (16-18). The thymic inhibitory factor could also be identified with the lymphocytes chalone implicated in the mechanisms controlling lymphopoiesis in the thymus. However, this extract does not seem to exhibit the close specificity of action postulated in the “chalone” concept : indeed it may inhibit erthyro’id and/or myelo’id precursors as well as lymphocyte precursors. On the other hand, the existence of a thymic stimulating factor seems now well established. This substance mediates at least one step of the maturation of Tlymphocyte (19, 20) and may trigger off colony-forming-unit proliferation (2123). The lymphocyte inhibitory factor described here, which inhibits T lymphoblast proliferation, seems to be at the same time capable of inhibiting erythrocyte and leukocyte precursor proliferation. Thus it is not impossible that these two substances exert antagonistic effects in order to maintain not only homeostasis of T-cell production, as suggested by Olsson et al. (24), but also of erythrocyte and leukocyte precursors. ACKNOWLEDGMENTS The authors are grateful to Dr. A. Khalil, I.C.I.G., HBpital Paul-Brousse, Villejuif, France, who performed histological studies. They also thank Miss Martine Davigny and Miss Nicole Saou for their excellent technical assistance, Dr. J. C. Salomon, Institut de Recherches
10
FLORENTIN,
KIGER
AND
MATHfi
Scientifiques sur le Cancer, Villejuif, France, for the supply of “nud’e mice” and Dr. A. J. S. Davies, Chester Beatty Research Institute, London, for his helpful criticism.
REFERENCES 1. Kiger, N., Florentin, I., and Math& G., Transplantation, 14, 448, 1972. 2. Kiger, N., Florentin, I., and Math& G., Nat. Cancer. Inst. Monogr., 38, 135, 1973. 3. Math& G., Kiger, N., Florentin, I., Garcia-Giralt, E., Martyre, M. C., Halle-Pannenko, O., and Schwarzenberg, L., Transplant. Proc. 5, 933, 1973. 4. Kiger, N., Florentin, I., and Math& G., Transplantation 16, 393, 1973. 5. Florentin, I., Kiger, N., and Math&, G., Eur. J. ZwwnunoZ.3, 624, 1973. 6. Dukor, P., and Dietrich, F. M., Intern. Arch. All. Appl. Zmmwnol. 32, 34, 1967. 7. Manning, J. K., Reed, N. D., and Jutila, J. M., J. Zmmzmol. 108, 1470, 1972. 8. Carpenter, C. B., Boylston, A. W., and Merrill, J. P., Cell Zmmunol. 2, 425, 1971. 9. Phillips, S. M., Carpenter, C. A., and Lane, P., N.Y. Acad. SC. 249,236, 1975. 10. Davis, R. C., Cooperband, S. R., and Mannick, J. A., J. Zmmzmol. 106, 755, 1970. 11. Cooperband, S. R., Badger, A. M., Davis, R. C., Schmid, K., and Mannick, J. A., J. Zmmunol. 109,154, 1972. 12. Menzoian, J. O., Glasgow, A. H., Nimberg, R. D., Cooperband, S. R., Schmid, K., Sanoroschets, T., and Mannick, J. A., J. Zmmzlnol. 113, 266, 1974. 13. Occhino, J. C., Glasgow, A. H., Cooperband, S. R., Mannick, J. A., and Schmid, K., J. Zmmunol. 110,685, 1973. 14. Kiger, N., Florentin, I., and Math;, G., Boll Zst. Sieroter Milanese 54, 244, 1975. 15. Bullough, W. S., Biol. Rev. 37, 307, 1962. 16. Garcia-Giralt, E., Lasalvia, E., Florentin, I., and Math& G., Europ. J. Cl&. Biol. Res. 15, 1012, 1970. 17. Houck, J. C., and Irausquin, H., Nut. Cancer Inst. Monog. 38, 117, 1973. 18. Tibbetts, L. M., Glade, P. R., and Papagcorgiou, P. S., Cell Zmmunol. 18, 384, 1975. 19. Trainin, N., Small, M., and Globerson, A., J. Exp. Med. 130, 765, 1969. 20. Goldstein, A. L., and White, A., In “Contemporary Topics in Immunology.” A. J. S. Davies and R. L. Carter, Eds.), Plenum Publ. Co., New York, 2, 339, 1973. 21. Resnitsky, P., Zipori, D., and Trainin, N., Blood 37, 634, 1971. 22. Frindel, E., and Croizat, H., N.Y. Acad. of Sci. 249, 468, 1975. 23. Trainin, N., Zipori, D., and Umiel, T., Boll. Zst. Sieroter. Milanese 54, 211, 1975. 24. Olsson, L., and Glacsson, M. H., Cell. Tissue Kinet. 8, 491, 1975.
The Lymphocyte Inhibiting Factor Extracted from the Thymus Inhibition of the DNA Synthesis in Vitro1 IRENE FLORENTIN, Institut
(Lift):
NICOLE KIGER AND GEORGES MATHS
de CancCrologie et d’lmmztnogine’tique, Ho*pital Paul-Browse, Received
(I.N.S.E.R.M. 94800-Villejuif, August
et Association France
Claude Bernard)
25, 1975
The action of a lymphocyte inhibiting factor extracted from the thymus (LIFT) has been investigated by studying its capacity to inhibit in vitro, independently of any cytotoxic effect, DNA synthesis in different types of mouse lymphoid cells. [3H]TdR incorporation was strongly depressed under the influence of the thymic extract in short term cultures of thymus, bone marrow cells as well as of spleen cells coming from: 1) normal CBA mice; 2) mice treated with PHA. Similarly in vitro PHA stimulation of spleen cells coming from normal mice was depressed when thymic extract was added to the culture. By contrast, the thymic extract does not significantly decrease [3H]TdR uptake in spleen cells coming from: 1) normal CBA mice injected with LPS; 2) T lym’phocyte deprived mice, whether or not their B lymphocytes were activated by in viva LPS administration. These results suggest that the LIFT acts on T lymphocytes at different stages of their maturation and differentiation. The inhibiting effect of the thymic extract upon [aH]TdR incorporation in bone marrow cells could be the result of an action on #erythrocytic and leukocytic precursors.
INTRODUCTION We have previously described a thymic extract designated a lymphocyte inhibiting factor (LIFT) due to its ability specifically to inhibit lymphocyte proliferation, (1, 2). From its immunosuppressive properties “in Z&JO” it was concluded that this factor affected only thymus-dependent (T) lymphocytes. Indeed, immune reactions involving T lymphocytes alone such as allogeneic skin graft rejection (1) and graft versus host reaction (1, 3, 4) or in collaboration with thymus independent (B) lymphocytes such as antibody formation against sheep red blood cells (SRBC) and DNP coupled to human y globulin (5) could be efficiently depressed. However, immune responses not involving T lymphocyte participation such as antibody formation to DNP-polymerized flagellin was unaffected. Preliminary observations had shown that the uptake of tritiated thymidine into thymocytes in short term culture was inhibited in the presence of LIFT (1). The question was raised whether the specificity of action observed in vivo could be demonstrated more directly upon the in vitro proliferation of lymphoid cells. For this purpose we have compared the effects of this factor upon the DNA synthesis in different lymphoid cell populations enriched in or deprived of one lymphocyte cell type, namely spleen cells coming from normal mice, stimulated or 1 Supported by Grant N.74.5.015.01, I.N.S.E.R.M. 1 Copyright 1976by AcademicPress,Inc. All rights o9 repmductfonin any form reserved.
and by Grant ATP
1-73-16, I.N.S.E.R.M.
2
FLORENTIN,
KIGER
AND
MATHfi
not by phytohemagglutinin (PHA) in vi&o and spleen cells from mice injected either with phytohemagglutinin (PHA) or with lipopolysaccharide (LPS) in order to stimulate specifically proliferation and differentiation of T (6) or B (7) lymphocyte populations. In addition, LIFT effect on DNA synthesis in bone marrow cells was studied. In the course of this study we have investigated the kinetics and dose dependence of the inhibition by LIFT of tritiated thymidine uptake in thymocytes and spleen cells in short term cultures. To exclude the possibility that cytotoxicity of the thymic extract might be the basis of the inhibition of DNA synthesis, its reversibility of action was demonstrated. MATERIAL Preparation
AND
METHODS
of LIFT
The extraction and partial purification of the thymic extract have been described in detail elsewhere (1). Briefly, lamb thymus was homogenized in distilled water. After centrifugation at ZO,OOOgfor 1 hr the supernatant was fractionated by alcohol precipitation. The inhibitory activity was recovered in the fraction soluble in a final ethanol concentration of 42% ; this is referred to as the T4 fraction. A similar procedure was applied to lamb kidney to obtain a control extract called the K4 fraction. Preparation
of Lymphocyte
Subpopulations
Various treatments were given to &week-old CBA mice (Centre d’Elevage, Orleans, CNRS) in order to enrich or deplete the thymus or spleen of one lymphocyte population or subpopulation. Preparation of T-Lymphocyte
Deprived Spleen Cells
Adult CBA mice were thymectomized, lethally irradiated 1 wk later and immediately reconstituted by an intravenous injection of lo7 isogeneic bone marrow cells. These animals referred to as “B mice”, were killed 4 wk later and their spleens were removed and processed as described below. Spleen cells coming from (nu/nu) mice are used as a source of T-lymphocyte deprived cell population. Control spleen cell populations are provided by (nu/+) sibs. Preparation of Spleens with Activated T or B-Lymphocytes In order to enrich spleen cell populations with activated T-lymphocytes, 100 pg of purified phytohemagglutinin (PHA, MR58, Wellcome) were injected intravenously into normal CBA mice. Similarly, B-lymphocytes were stimulated by one injection of 100 pg of lipopolysaccharide B from E. coli (LPS, Difco) given to normal or to “B” CBA mice. The spleens were removed 4 days after the injection of PHA or of LPS.
LYMPHOCYTE
In Vitro
DNA
or RNA
INHIBITORY
Synthesis Inhibition
THYMIC
FACTOR
3
Tests
Single cell suspensions were prepared from thymus, spleen, and femoral bone marrow in RPM1 1640 culture medium (Gibco) supplemented with antibiotics and adjusted to 3.3 X 10” cells per ml; 3 ml aliquots were distributed into glass culture tubes. In the kinetic studies, 250 pg in protein of the Tq fraction or the K4 control extract were added to each 3 ml aliquot of the thymocyte suspension at time zero. Cells were incubated at 37°C for 20, 30, 45, 60 or 90 min in a humidified atmosphere of air-CO2 (95 : 5) and 5 &i of methyl [SH] thymidine ( [SH]TdR, specific activity 7 Ci/mmole) were added 10 min before the end of incubation. When the cells were incubated for longer periods : 34, 5, 24, or 48 hr, [3H]TdR or [3H] uridine (specific activity 10 Ci/mM) was added 30 min before the end of each incubation. After the acid insoluble fraction had been precipitated by addition of trichloracetic acid to a final concentration of 7%, it was prepared for radioactivity counting as previously described (1). The cultures were made in triplicate and the results shown are examples from repeated experiments. For the dose effect studies 500, 250, 125, 75 or 30 pg in protein of the T4 fraction were added to 3 ml of thymic cell suspension. The cells were incubated for 34 hours as described above. In the comparative studies of the thymic extract’s action on different lymphoid cell populations, the cells were incubated for 39 hours at 37°C in the presence of 250 pg of the T4 fraction per culture tube and then processed as described above. In Vitro
PHA
Stiwdation
Normal spleen cell suspensions were prepared in RPM1 1640 culture medium supplemented with heat inactivated mule serum (Gibco Laboratories) and antibiotics. 5 X lo5 cells in 0.2 ml were cultured in each well of tissue culture treated U plates (Falcon Microtest II) for 54 hr in an air-CO2 atmosphere. 20 ~1 of l/50 dilution of stock solution of PHA and 16 pg in protein in 10 ~1 of the Td fraction or K4 fraction were added to some wells at the beginning of the culture. One microcurie of i3H]TdR per well was added for 4 hr at the end of the culture. The cells were harvested on glass fibre filters using a multiple automated sample harvester (MASH II, Microbiological Associates). Cell Viability At the end of the incubation period, cell counts were made using a hemocytometer and viability was assessed by the trypan blue dye exclusion test. Reversibility
Assay
Thymocytes were incubated for 1 hr in the presence or absence of the T, fraction under the same conditions as described above. The cells were spun down and resuspended either in LIFT-free fresh medium or in their first incubation medium. Cultures were then incubated for 1, 2 or 3 hr, after which [3H]TdR was added and the incubation continued for further 30 min.
FLORENTIN,
KIGER
AND
MATHfi
RESULTS Kinetics of the Inhibition the InfEuence of LIFT
of DNA
and RNA
Synthesis in Thymocytes
under
Mouse thymocytes were incubated from 20 min to 48 hr in the presence of the thymic extract, and inhibition of DNA or RNA precursor incorporation was estimated at different times during incubation. Results recorded in Table 1 show that [3H] TdR incorporation was strongly depressed after the cells had been in contact with the T4 fraction for 20 min. This inhibitory effect increased slightly with time, reaching its maximal value after 60 min to 5 hr of incubation, and then decreased to become nonsignificant after 48 hr of incubation. A slight but significant inhibition of [3H]uridine incorporation was observed after 3 hr of incubation with the thymic extract, this inhibition lasted for 24 hr but was no longer significant at 48 hr. In all these experiments the Kb control fraction was ineffective in decreasing thymidine or uridine incorporation in thymocytes. Dose Efect of the T4 Fraction on DNA
Synthesis in Lymphoid
Cell Suspensions
Results recorded in Table 2 show that the T4 fraction inhibitory effect on DNA synthesis in thymus or spleen cells is dose dependent. Moreover, spleen cells appear to be less sensitive than thymic cells to the action of LIFT. The amount of T4 fraction required to produce a definite percentage of inhibition of DNA synthesis in spleen cells is twice that required to produce an identical inhibition in thymic cells. In all further experiments a dose of 250 pg of the T4 fraction in 3 ml of culture was chosen as it generally gives a 50% inhibition of the DNA synthesis in normal spleen cells, although this inhibition can fluctuate through a range of 15% from one experiment to another. Cell Viability In cultures exposed to the T4 or Kq fractions there was no loss of cells compared to control cultures incubated for the same period in medium alone. Cell viability was in all cases greater than 90% by the trypan blue test, indicating that these fractions have no toxic effect. TABLE Kinetics
Time
of cultivation
[aH]TdR uptake Mean c.p.m. no fraction T4 fraction ‘%$Sb;Kb~“, by the 4
[“gz:
of DNA and RNA Synthesis Inhibition in Thymocytes Under the Influence of the Tc Fraction
20 min
30 mina
45 miw
60 mina
75 miw
90 mine
3$ hrb
5 hrb
24 hrb
48 bra
12,096 5.382
13,872 4,813
14,254 4,533
20,794 5,073
23,340 5,601
28,742 5,662
138,080 27,717
124,092 40,146
15,950 8,103
58,995 51,612
74%
79%
49%
13%
14,398 14,275
17,106 16,982
24,838 17,809
62,872 57.776
56%
65%
8,346 8.452
8,978 8,905
0%
0%
358%
,“;‘$e . .
no fr&ion Tc fraction % Inhibition TI fraction 0 [‘H]TdR b [‘H]TdR
1
13,076 13,217
80% 18,395 17,942
by the
is added is added
0%
0%
0%
10 min before the end of the cultivationqperiod. for 30 min before the endlof the cultivation~period.
0%
78%
107,937 85,183
21%
68% 139,778 91,415 35%
28%
8%
LYMPHOCYTE
INHIBITORY
TABLE
THYMIC
5
FACTOR
2
Dose Effect of the Tc Fraction on DNA Synthesis in Lymphoid Cell Suspensions Dose of T, fraction (pg of protein) added to 3 ml of culture
500pg
25opg
125rg
60 Irg
30 rg
1.5Pg
7spg
0
[3H]TdR uptake in thymocytes, Mean c.p.m. % inhibition
16,340 32,785 68,303 105,328 118,985 125,006 123,957 123,280 34% 0% 0% 73% 45% W% 95%
[3H]TdR uptake in spleen cells, mean c.p.m. y0 inhibition
3,919 72%
Reversibility
6,400 10,695 18% 51%
12,162 7%
12,768 2%
13,153 0%
12,975 0%
13,065 -
of Action
The 85% inhibition in [3H]TdR incorporation tion of thymocytes with the T4 fraction remained suspended in the same medium after centrifugtion. resuspended in chalone-free fresh medium the increased progressively with time reaching nearly recuperation (Fig. 1). LIFT Efect on DNA Bone Marrow Cells
Synthesis in Different
observed after a 1 hr incubaconstant when cells were reHowever, when the cells were [3H]TdR incorporation rate normal values after 3$ hr of
Spleen Cell Populations
and in
Histological studies of the spleens from animals stimulated by PHA or LPS have been performed in order to verify their specific mitogenic activity. In normal animals injected with PHA the periarteriolar T dependent areas of the splenic white pulp were larger and more cellular than those of the normal controls, the periarteriolar zones accumulatin, (+small and medium sized lymphocytes. In “B mice” injected with LPS the follicular zones showed marked hyperplasia with numerous small and medium sized lymphocytes. The periarteriolar zones seemed to be compressed by the proliferating lymphocytes of the follicle.
FIG. 1. Reversibility
of the inhibition of [‘H]TdR
uptake in thymocytes.
6
FLORENTIN,
KIGER
AND
TABLE Effect
Fraction added in vitro
None
None l-4 K4 None l-4 K4 None T4 K,
LPS
D In comparison
3
of the Thymic Extract on C3H]TdR Uptake in Spleen Cells Obtained from Normal Mice, PHA or LPS Treated Mice
Treatment of spleen donors in vi00
PHA
MATHI?
with [3H]TdR
uptake
[3H]TdR
uptake &SD
17,450 6,857 20,588 101,324 33,708 105,964 111,845 91,744 128,621 in culture
i f f f f f f f f
incubated
(c.p.m.)
1,230 262 375 12,253 5,191 19,090 10,878 10,870 10,953 without
% of inhibition0
61 -18 69 -4 17 -15 any fraction.
We have compared the inhibitory effect of LIFT upon tritiated thymidine incorporation in different lymphoid cell populations, and all the results presented here were confirmed in several experiments. We first compared LIFT inhibitory activity on the DNA synthesis of spleen cells coming from normal mice, PHA treated mice and LPS treated mice. Results are shown in Table 3. When spleen cells from normal CBA mice are incubated for 39 hr in the presence of T* fraction, the [3H]TdR uptake is 61% depressed in comparison with that of control cells cultured without any fraction. In spleen cells selectively enriched with PHA activated T-lymphocytes, we first observed that the spontaneous DNA synthesis is increased sixfold compared to that in nonactivated spleen cells. Nevertheless, the addition of the T4 fraction to the cultures reduced the [3H]TdR incorporation by 69%. Similarly LPS administration induces cell proliferation which results in a 6.5 fold increase in the spontaneous DNA synthesis in vitro. In contrast, with the results observed in PHA stimulated spleen cells, the addition of Td fraction induces only a 17% decrease of [3H] TdR incorporation in in tivo LPS activated spleen cells. In the second type of experiment the action of the thymic extract on DNA synthesis in spleen cell populations coming from normal CBA mice, “B mice”, and LPS treated “B mice” were compared. The results are shown in Table 4. In spleen cell populations depleted in T-lymphocytes the [SH] TdR incorporation in the cultures exposed to the Tq fraction is reduced by 34%, whereas the percentage of inhibition in normal spleen cell cultures reaches 58%. Furthermore, when B lymphocytes have been stimulated by LPS injected into “B” mice”, the spontaneous DNA synthesis is increased threefold when compared to nonstimulated “B mice” spleen cells. In this case, we have observed that the thymic extract exerts only a slight inhibitory effect on DNA synthesis, as the inhibition of [8H]TdR uptake is only 17%. In spleen cells coming from “nude” mice we have observed an inhibition of 40% of the [3H]TdR uptake under the influence of the Tq fraction; at the same
LYMPHOCYTE
INHIBITORY
THYMIC
TABLE Effect
7
FACTOR
4
of the Thymic Extract on C3H]TdR Uptake in Spleen Cells Obtained Normal Mice or “B Mice” with or without LPS Treatment
Origin of spleen cells
Normal
mice
“B mice”
Treatment in viva
Fraction added in vitro
None
None T4 K4 None l-4 K4 None T4 K4
None
“B mice”
LPS
a In comparison
with C3H]TdR
CH]TdR uptake (c.p.m.) f SD 16,552 7,030 15,960 34,514 24,159 34,610 106,673 88,474 109,980
uptake in culture
incubated
f f f f f f f i f
without
from
% of inhibition0
2,371 201 640 340 2,664 1,615 12,664 6,899 7,875
68 3 34 1 17 -3
any fraction.
time the inhibition was of 73% in the spleen cell population taken from normal littermates (Table 5). In this experiment we observed that when LIFT was added to cultures of bone marrow cells it, induced a strong inhibition of t3H]TdR uptake. This inhibitory effect was more striking when bone marrow cells came from normal mice than from nude mice. In all these experiments the K4 fraction did not significantly effect [3H]TdR incorporation by spleen cell populations. TABLE Effect
Origin
of the Thymic Extract on C3H]TdR Uptake in Spleen and Bone Marrow Cells from “Nude” Mice and Normal SIBS
of cells
Nude mice
Nude mice
Normal
5
mice
Fraction added
Spleen cells
Bone marrow
None ‘I.4 K4 cells
Spleen cells
40 1
None l-4 K4
548,952 f 26,325 120,333 f 14,126 327,330 rt 8,699
78 17
None
15,310 f- 1,664 4,195 f 1,045 17,265 h-r 634
73 -12
470,244 f 42,587 56,540 f 6,375 537,755 f 39,538
89 -13
K4 mice
Bone marrow
cells
None Ta
K* a In comparison
with C3H]TdR
uptake in culture
incubated
13,295 f 8,026 i 13,404 rt
% of inhibitiona
610 1,832 1,775
T4
Normal
C3H]TdR uptake (c.p.m.) f SD
without
any fraction.
8 Efect
FLORENTIN,
of the Thymic Extract
KIGER
on in Vitro
AND
PHA
MATHI?
Transformation
of Spleen Cells
The T4 fraction used in the same final contraction as in all other experiments (250 &3 ml) m ’ d uces a 61% inhibition in the nonstimulated spleen cells. On the other hand, PHA induced a 17-fold stimulation of DNA synthesis compared to that seen in unstimulated cultures. The T4 fraction reduced this PHA induced stimulation at least 68% ; in all cases the Kd fraction did not significantly affect the [ 3H] TdR incorporation in unstimulated and PHA stimulated spleen cells. DISCUSSION In order to determine if the LIFT immunosuppressive action on thymodependent reactions observed in viva (5) can be the result of a specific inhibition of T lymphocyte proliferation, we have investigated its effect on in vitro DNA synthesis in cell populations containing different proportions of T or B lymphocytes. We have first shown that the DNA synthesis suppression by LIFT is dose dependent and is greater in thymocytes than in spleen cells. In all further studies LIFT has been used at a concentration of 80 pg/ml which was shown to depress DNA synthesis by about 50% in normal spleen cells and 70% in thymocytes. Under these conditions, we have compared the LIFT effect on [ 3H] TdR uptake in short term culture of thymus cells, containing a great proportion of immature T-lymphocytes with a high proliferative activity and of spleen cell in which T-lymphocytes have been selectively activated by in viva administration of PHA. In both cases we have noticed a strong inhibition of DNA synthesis under the influence of LIFT. A similar inhibition of PHA-induced DNA synthesis was observed when LIFT was added at the same time as the mitogen to normal spleen cell cultures. In contrast, in T-lymphocyte-deprived spleen cell populations, coming either from “B mice” or from “nude” mice, the inhibition of DNA synthesis after incubation with LIFT is half as much as that in normal spleen cells. After LPS administration in normal mice or in “B mice” in order to selectively activated B-lymphocytes, DNA synthesis in their spleen cells was not significantly depressed under the influence of LIFT. From these data, it can be concluded that LIFT exerts a strong inhibitory effect on DNA synthesis in thymus-dependent lymphocytes which proliferate either during their maturation process in the thymus or after mitogenic stimulation. In contrast, it only slightly inhibits DNA synthesis in T-lymphocyte-depleted spleen cell populations and does not act on B-lymphocytes activated by LPS. Thus the specific immunosuppressive properties of LIFT may be the result of an inhibition of T-lymphocyte proliferation. However we cannot conclude an exclusive specificity of action of LIFT for T-lymphocytes. Indeed, we have noticed a strong inhibitory effect of this substance on bone marrow cells. This cannot be explained only by an action on lymphocyte precursors; it is most likely also due to an inhibition of erythroi’d or myeloi’d precursor proliferation. Experiments are actually in progress to further investigate this possibility. According to the kinetic studies and the finding that LIFT produced an inhibition of [3H]TdR uptake within the first 20 min of incubation, we can propose that binding of LIFT to membrane surface constituents of cells triggers
LYMPHOCYTE
INHIBITORY
THYMIC
FACTOR
9
the events leading to the inhibition of the proliferation. This early inhibition indicates that the substance acts by inhibiting S phase cells in their progression through the S phase to Gs, and perhaps by decreasing the flux of late G1 cells to S phase. However, by the method used in this study we cannot exclude an action of LIFT on other phases of the mitotic cell cycle. It cannot be argued that the inhibitory effect is due to a cytotoxic action of the thymic extract since, at the end of the incubation period, cell viability was identical in all samples. Moreover, the observation that in thymocytes DNA synthesis is inhibited in a totally reversible way rules out any selective cytotoxic effect of the extract. We can also argue against the hypothesis that our observations are the result of technical artefacts, such as the presence of cold nucleotide or thymidine binding protein in the the thymic extract or of enzymes which can disturb labelled thymidine incorporation, for it would be difficult to explain how such substances could interfere with T and not with B-lymphocyte cellular metabolism. Other immunosuppressive substances have been recently isolated from thymus (8, 9) and identified as alpha globulins similar to the immuno-regulatory alpha globulin (IRA) extracted from the serum (10, 11). IRA possesses physiological properties nearly identical to that found for LIFT, and in addition exhibits in V&O and in vitro (12) the same type of lymphocyte specificity. But the physicochemical properties of the two substances seems to be completely different from one to another and, at the present time, we have to suppose that these extracts are not identical substances (13, 14). A model for the regulation of cell production in all tissues which is now supported by a growing body of experimental data implies that differentiated cells secrete substances (“chalones”) that regulate the rate of cell renewal by a negative feedback mechanism (15). Some authors now refer to DNA synthesis inhibitors extractable from lymphoid tissue as the “lymphocyte chalone” (16-18). The thymic inhibitory factor could also be identified with the lymphocytes chalone implicated in the mechanisms controlling lymphopoiesis in the thymus. However, this extract does not seem to exhibit the close specificity of action postulated in the “chalone” concept : indeed it may inhibit erthyro’id and/or myelo’id precursors as well as lymphocyte precursors. On the other hand, the existence of a thymic stimulating factor seems now well established. This substance mediates at least one step of the maturation of Tlymphocyte (19, 20) and may trigger off colony-forming-unit proliferation (2123). The lymphocyte inhibitory factor described here, which inhibits T lymphoblast proliferation, seems to be at the same time capable of inhibiting erythrocyte and leukocyte precursor proliferation. Thus it is not impossible that these two substances exert antagonistic effects in order to maintain not only homeostasis of T-cell production, as suggested by Olsson et al. (24), but also of erythrocyte and leukocyte precursors. ACKNOWLEDGMENTS The authors are grateful to Dr. A. Khalil, I.C.I.G., HBpital Paul-Brousse, Villejuif, France, who performed histological studies. They also thank Miss Martine Davigny and Miss Nicole Saou for their excellent technical assistance, Dr. J. C. Salomon, Institut de Recherches
10
FLORENTIN,
KIGER
AND
MATHfi
Scientifiques sur le Cancer, Villejuif, France, for the supply of “nud’e mice” and Dr. A. J. S. Davies, Chester Beatty Research Institute, London, for his helpful criticism.
REFERENCES 1. Kiger, N., Florentin, I., and Math& G., Transplantation, 14, 448, 1972. 2. Kiger, N., Florentin, I., and Math& G., Nat. Cancer. Inst. Monogr., 38, 135, 1973. 3. Math& G., Kiger, N., Florentin, I., Garcia-Giralt, E., Martyre, M. C., Halle-Pannenko, O., and Schwarzenberg, L., Transplant. Proc. 5, 933, 1973. 4. Kiger, N., Florentin, I., and Math& G., Transplantation 16, 393, 1973. 5. Florentin, I., Kiger, N., and Math&, G., Eur. J. ZwwnunoZ.3, 624, 1973. 6. Dukor, P., and Dietrich, F. M., Intern. Arch. All. Appl. Zmmwnol. 32, 34, 1967. 7. Manning, J. K., Reed, N. D., and Jutila, J. M., J. Zmmzmol. 108, 1470, 1972. 8. Carpenter, C. B., Boylston, A. W., and Merrill, J. P., Cell Zmmunol. 2, 425, 1971. 9. Phillips, S. M., Carpenter, C. A., and Lane, P., N.Y. Acad. SC. 249,236, 1975. 10. Davis, R. C., Cooperband, S. R., and Mannick, J. A., J. Zmmzmol. 106, 755, 1970. 11. Cooperband, S. R., Badger, A. M., Davis, R. C., Schmid, K., and Mannick, J. A., J. Zmmunol. 109,154, 1972. 12. Menzoian, J. O., Glasgow, A. H., Nimberg, R. D., Cooperband, S. R., Schmid, K., Sanoroschets, T., and Mannick, J. A., J. Zmmzlnol. 113, 266, 1974. 13. Occhino, J. C., Glasgow, A. H., Cooperband, S. R., Mannick, J. A., and Schmid, K., J. Zmmunol. 110,685, 1973. 14. Kiger, N., Florentin, I., and Math;, G., Boll Zst. Sieroter Milanese 54, 244, 1975. 15. Bullough, W. S., Biol. Rev. 37, 307, 1962. 16. Garcia-Giralt, E., Lasalvia, E., Florentin, I., and Math& G., Europ. J. Cl&. Biol. Res. 15, 1012, 1970. 17. Houck, J. C., and Irausquin, H., Nut. Cancer Inst. Monog. 38, 117, 1973. 18. Tibbetts, L. M., Glade, P. R., and Papagcorgiou, P. S., Cell Zmmunol. 18, 384, 1975. 19. Trainin, N., Small, M., and Globerson, A., J. Exp. Med. 130, 765, 1969. 20. Goldstein, A. L., and White, A., In “Contemporary Topics in Immunology.” A. J. S. Davies and R. L. Carter, Eds.), Plenum Publ. Co., New York, 2, 339, 1973. 21. Resnitsky, P., Zipori, D., and Trainin, N., Blood 37, 634, 1971. 22. Frindel, E., and Croizat, H., N.Y. Acad. of Sci. 249, 468, 1975. 23. Trainin, N., Zipori, D., and Umiel, T., Boll. Zst. Sieroter. Milanese 54, 211, 1975. 24. Olsson, L., and Glacsson, M. H., Cell. Tissue Kinet. 8, 491, 1975.
