High resistance of immunocompetent spleen cells to repeated exposure of γ-radiation and its abolition by syngeneic lymphocytes

CELLULAR

3, 326-332 (1972)

IMMUNOLOGY

SHORT COMMUNICATIONS High

Resistance

Repeated

of

lmmunocompetent

Exposure

of Y-Radiation

by Syngeneic

R. V. Imtitate

PETRW

of Biophysics,

and

Cells

to

Its Abolition

Lymphocytes

AND Ministry

Spleen

A.

S.

CHEREDEEV

of Public Henlth,

Moscow,

USSR

Receipted June 23, 1971

Spleen cells from mice, subjected to y-radiation at dose 500 R and taken 14 days later, possessed an increased radioresistance by their capacity to accumulate antibody-producing cells in culture in &JO. The addition of syngeneic lymphocytes to radioresistant spleen cells abolished the effect of radioresistance. It is suggested that high radioresistance of immunocompetent cell population may be a consequence of interference with normal cell-to-cell cooperation processes under conditions of decreased level of lymphocytes and of relatively elevated content of stem elements in postirradiated spleens. It seems likely that the change of lymphocyte: stem cell ratio on Day 14 after irradiation of mice decreases the occurrence of transmission of immunological information from lymphocyte to stem cell, and this situation may be estimated phenomenologically as high radioresistance of antibody-producing cells. INTRODUCTION

In our previous work (1, 2) it has been shown that a population of lymphoid cells from sublethally irradiated mice possessed an increased radioresistance when their capacity was tested to produce antibody-forming cells after transplantation into lethally irradiated recipients (Gz viva culture). The dose-effect curve for population of spleen cells taken from mice 14 days after irradiation at 500 R is characterized for D, = 220 R, n = 10.2 (for normal spleen cell population Do = 188.3 R and n = 0.8). However, no increased radioresistance of haematopoietic stem cells was demonstrated in the spleens of such previously irradiated donors (2). A prediction was made that the increased radioresistance of immunocompetent spleen cells in the previously irradiated mice depends upon the population imbalance of cell types observed after exposure to ionizing radiation. Earlier, it was shown that 2-3 weeks after sublethal irradiation an overshoot of stem cells occurred in the spleen of mice (300-7007’ 0 as compared with normal level) with a simultaneous sharp depletion of lymphoid elements (3, 4). The purpose of this investigation

was to evaluate

the above

supposition

by adding

normal

syngeneic

lymphocytes to “radioresistant” spleen cells and by following the estimation of radioresistance of antibody-forming cell accumulation in culture in viva. 326 0

1972 by Academic

Press, Inc.

SHORT

COMlRl

MATERIALS AND

CKICATIONS

327

METHODS

(CBA X C57BL)F,, male and female mice, 3-4 months old, weighing 20-22 g were used throughout these experiments. Mouse donors of spleen cells were irradiated at 500 R. Fourteen days after irradiation the donors were killed, their spleens were removed, and pooled spleen cell suspensions were prepared in tissue culture medium 199 by the generally accepted technique (5). Lymph node cells were isolated from nonirradiated mice of the same genotype. Spleen cell suspensions obtainmed from previously irradiated or normal donors were subjected to gamma radiation in vitro (100-800 R range) either alone or mixed with normal lymph node cells. The cell suspensions together with 2 X 108 sheep red blood cells (SRBC) were injected into lethally irradiated syngeneic recipients. Each recipient received a certain cell dose in the volume of 0.5 ml. Recipients were irradiated at lethal dose (850 R) 24 hr before cell injection. Six days :after cell transfer (maximal response) the recipients were killed, and the number of plaque-forming cells was determined by the technique described by Jerne et al. (6). The dose-effect curves were depicted by the method of least squares (7). The estimation of these curves can be completely accomplished with two parameters D, and n (8). Mice and cell suspensions were irradiated with @‘COy-rays in apparatus EGO-2 at a dose rate of 517-431 R/min. RESULTS

In the firs,t set of experiments the dose-effect curves were obtained for unmixed single spleen and lymph node cell suspensions. Results of these experiments are presented in Table 1 and Fig. 1. As seen from the table, transfer of 25 X lo6 normal spleen cells together with 2 X lo8 SRBC resulted in the accumulation in the spleens of lethally irradiated recipients of approximately 3000/108 plaque-forming cells. The exposure of cells to y-radiation in vitro before cell transfer inhibited plaque formation. The parameters of radioresistance were D, = 188.3 R and n = O.S. The transfer of the same amount of spleen cells from previously irradiated donors was accompanied by accumulation of significantly fewer antibody producers (+ lOO/lO*). Nevertheless, irradiation in vitro had very little effect. Up to 400 R, the survival of plaque-forming capacity remained at 100% level. The parameters of dose-effect curve were D, = 2’20 R and n = 10.2. An analogous result after radiation inactivation of antibody-producing cells was observed when 10 X 10” spleen cells from irradiated donors were lodged in culture in viva. Antibody--producing cells, revealed after transfer of normal lymph node cells at dose 1 X lo6 or 10 X lo”, were subjected to the same radiation inactivation in vitro that followed transfer of normal spleen cells (D, = 147 R, n = 0.98). Results of the second set of experiments, consisting in the establishment of the dose-effect dependencies for various cell mixtures, are presented in Table 2. The addition of 10 X 10Gnormal lymphocytes to 10 X 1OFspleen cells from 14-day irradiated donors and transfer of this mixture together with SRBC into lethally irradiated syngeneic recipients resulted in accumulation of 20,000/10s plaque-forming cells. If both components taken at the same dosage were derived from tissues of normal mice, the number of antibody-producing cells \vas equal to 4600/10s. In

328

SHORT

COMMUXICATIONS

PFC (No./108 Amount and t?;pe of transferred cells 0 25 x 106 “PI. c.u

25 x 10” 14-day spl. c.”

10 x lo6 l&day

10 x 10” 1.n.c.d

1 x 106 1.n.c.

spl. c.

100

recipient Ijose (R)

200

spleen cells)

400

600

800

52*13

2910f288 (l()O%Y

1455z!zl61 (50%)

803+82 (27.6%)

249f31 (8.5%~

(1.8%~

4lf8 (1.4%)

115fll (100%)

182zk27 (lj7%)

102115

126+17 (109%j

86fl4 (74.5%)

29&6 (25.5%)

97+15 (100%)

12119

79z!z21

(81%)

41zk26 (43%)

16&S

(125%)

(16%)

914 (9%)

(88%)

1461zt532 (100%)

697+94 (47.7%)

286~1x32 (19.5%)

102+14 (6.9%)

(5.2%!

6zk3 (0.4%)

410f92 (100%)

134f12 (33.5%)

79&13 (19.7%)

93136 (23.2%)

42flL (10.5%)

13+2 (3.2%)

a spl. c. = spleen cells from normal mice. * The sur\-ival of PFC (%) is given in parentheses. c 14-day spl. c. = spleen cells from 14day irradiated d 1.n.c. = lymph node ceils from normal mice.

7614

donors (500 Ii).

other words, a spleen population of 14-day irradiated donors contains approsimately 3-7 times more stem elements (CFU) than in normal spleen (3, -t) ; and after interaction with an equivalent dose of lymph node cells, gave a 5-fold greater accumulation of plaque-forming cells than normal spleen cel1s.l The use in the misture of spleen cells from 21-day irradiated donors led to an accumulation of 9000/108 plaque-forming cells. Spleen CFU level at this time after sublethal irradiation usually exceeds the normal one by 1.5-2 times (3, 4). It is interesting to note that the use of 1 X lo6 lymph node cells in the mixture with 10 X 10Gspleen cells from 14-day irradiated donors, i.e., in ratio 1 :lO, has not resulted in an increase in the numbers of antibody-producing cells. However, all of the mixtures studied were subjected to the same radiation inactivation that normal spleen cells were. The radioresistance of the mixtures by the criterion of accumulation of antibody-producing cells did not differ significantly from normal. The doseeffect curves for various cell mixtures are depicted in Fig. 1. In all cases D, and +z had approximately the same values. Thus, the addition of normal syngeneic lymphocytes to radioresistant spleen cell populations, obtained from mice on Day 14 after sublethal irradiation, abolished the effect of increased radioresistance whether lymphocytes were taken in 1 :l or 1 :lO ratio. 1 The question whether CFU can carry a function of antibody-forming lymphocytes) remains to be open; we don’t exclude such a possibility.

precursors

(B-

SHORT

COMMUNICATIONS

329

FIG. 1. The dose-effect curves for antibody-producing cells, derived from : I (0-O) normal lymph node cells; 2 (A-A) mixture of normal lymph node and spleen cells; 3 (0-O) normal spleen cells ; 4 (&--a) mixture of normal lymph node cells and spleen cells from 14-day irradiated donors ; and 5 (A---A) spleen cells from 14-day irradated donors.

DISCUSSION Xt present the data obtained about the increased radioresistance of iinmunoconlpetent precursors in spleen population of irradiated donors (14 days after 500 K) can be well understood and adequately explained by modern concepts of cellular immunology. During the last years many studies have been published in which the great role of cooperative processes between different types taking part in the antibody response is stressed (9-11). Mechanisms involving a collaboration of two (10, 11) or even three (9, 12) different types of cells have been postulated for different models of immunopoiesis. It is likely that, in addition to macrophages, a cooperation, of at least, two different cell types is required for the development of the immune response. The first type is thymus-derived (antigen-reactive) lymphocytes which can react to antigen, but do not produce antibody (13-15). The second type is bone marrow stem precursor cells which, under the influence of the former. undergo proliferation and differentiation to a great number of antibody-producers (11, 14-15). Cell-to-cell cooperation seems to be a necessary prerequisite for realization of the immune response, at least, to SREC. If this is so, then after sublethal irradiation of mice the spleen cell population is defective for realization of normal cell-to-cell interaction processes. The number of stem elements which can serve as precursors

330

SHORT

COMMUNICATIONS

TABLE

2

RADIATION INACTIVATION OF ANTIBODY-PRODUCER ACCUMULATION AFTER TRANSFER OF \.ARIOI'S MIXTURES TOGETHER WITH 2 x 108 SRBC TO SYNGENEIC LETHALLY IRRADIATED (CBA X C57BL)F1 RECIPIENTS PFC (No./lOB

recipient Dose (R)

100

200

Amount and type of transferred cells 0

spleen cells)

400

10 x 106 spl.c.= + 10 x lo~I.n.c.*

467OztSSl (loo%o)c

2700f248 (57.8%)

1454zk206 (31.1%)

4971100

97+24

(10.6%)

(2.1%)

10 X 10’14 d.spkd + 10 x 106I.n.c.

21123~~3405 (100%)

11465f1257 (54.3%)

5531zk657

1708f263

(26.2%)

(8.0%)

10 X lo6 21 d.spl.ce + 10 x 106I.n.c.

8954+1032 (100%)

4793~626 (53.2%)

1862zk204 (20.7%)

10 x lo6 14 d.sp1.c. + 1 X 106I.n.c.

348f.53 (100%)

159zk22 (45.4%)

68f1.5 (19.4%)

p sp1.c. = spleen cells b 1.n.c. = lymph node c The survival of PFC d 14 d.sp1.c. = spleen 6 21 d.sp1.c. = spleen

from cells (%) cells cells

800

600

66~1~26 (1.4%)

308flOl (1.5%)

945x10 (0.5%)

287zt91

259zt.68

(8.2%)

(3.2%)

(2.8%)

28zfz6 (8.0%)

38f9 (10.8%)

6+3 (1.6%)

741f242

normal mice. from normal mice. is given in parentheses. from 14-day irradiated donors (500 R). from 21day irradiated donors (500 R).

for antibody-producing cells is elevated 3-7 times with IO-fold reduction of the normal lymphocyte numbers (3, 4). In spite of the increased number of stem elements, the spleen cells from 14-day irradiated donors after their transfer to syngeneic recipients gave a poor immune response. However, the decreased level of antibody-producer accumulation remained constant under the influence of irradiation of cell suspensions in vitro in the range of 100-100 R. Such an accumulation of radioresistant immunocytes may be explained if the above facts about the key role of cellular cooperative processes in the immune response are kept in mind. As a schematic example, let us take into consideration the cooperative events which may emerge in normal mice and after radiation exposure. In normal mice, when the number of antigen-stimulated lymphocytes (N) much exceeds the level of stem precursor cells (n) , i.e., when we have N > n, then the all-potential stem cells may be involved in the immune response to SRBC and the terminal number of antibody producers will be determined by the original value n. Irradiation of cell suspension in vitro at continuously increasing doses D,, Dz, D3, * * , DI, would result in accumulation of n,, n2, vz3, . , nk antibody-producing cells, and n > n, > n2 >, * , > Sk. In other words, the number of antibody-producing cells is decreased in proportion to the magnitude of the radiation dose. In sublethally irradiated animals the ratio of lymphocytes : stem cells in the spleen is changed in favor of the latter (3,4). Suppose the number of lymphocytes is decreased in w times, and the number of stem cells is increased in f times in such a way, that an inequality N > n under normal conditions is converted into K/W < fn in irradiated animals, Then, of the fn stem cells, only a small part of l

l

l

l

l

SHORT

331

COMMUNICATIONS

them equal to L\:/Iu, will be recruited in the immune response. The use of the in, Di, , Dk will result in the depletion of creasing doses of radiation D1, D2, stem cells so that fn > fn, > fn, > * * > fni > > fnk. However, the number of stem elements to be involved in immunopoiesis remains at the same N/m level, until the number of antigen-stimulated lymphocytes at Di would be compared with the level of stem precursor cells (i.e., N/W = fni). In other words, since JV/UL < fn, the number of antibody-producing cells remains constant and equal to :V/WL, simulating a 100% survival of cells. And when at a certain radiation dose Di we have N/W = f?zi, then after this moment the inactivation of antibodyproducing capacity will accept a typical character. This prediction was confirmed experimentally. The great shoulder of dose-effect curve for antibody producers, dervied from 14day spleen of irradiated donors clisappeared after normal syngeneic lymphocytes were added to these spleen cells. It is possible that the addition of L lymphocytes to N/M caused a conversion of inequalacquired ity N/M < fn into N/W + L > fn and, therefore, the radiosensitivity the normal character. There are grounds to believe that the suppression of antibody production caused by ionizing radiation may be not only a result of the quantitative decrease of immunocompetent precursors, but also due to the disturbances of the quantitative cooperative interrelations between stem precursor cells and antigen-reactive lymphocytes. As a consequence of such disturbances, the spleen tissue at certain intervals after irradia.tion of animals acquires a new property which is manifested by an imaginary high radioresistance of immunocompetent cells. l

l

l

l

l

l

l

l

l

l

REFERENCES 1. Petrov, 1~. V., and Cheredeev, resistance of immunologically

A. N., Effect of preliminary irradiation of mice on radiocompetent spleen cells. Natwe Londo~z 220, 1349, 1968.

2. Cheredeev, A. N., Resistentnost’ k povtornomu oblucheniju kletok immunokompetentnoj i krovotvornoj tkani u mysheij (Resistance of cells from immunocompetent and hemopoietic tissues to repeated y-ray exposure). Radiobiologija (Radiobiology) 10, 633 [Russian], 1970. 3. Koslov, V. A., and Seslavina, L. S., Kolichestvo immunokompetentnykh i kolonieobrazujuschikh kletok v selezenke myshej v raznye sroki posle obluchenija (The quantity of imrnunocompetent and colony-forming cells in spleen of mice in various intervals after whole body radiation). Radiobiologija (Radiobiology) 8, 72 [Russian]. 1968. 4. Cheredeev, A. N., Radiorezistentnost’ immunocompetentnykh i hemopoeticheskikh predshestvennikov v razlichnye sroki posle luchevogo vozdejstvija (Radioresistance of immunocompetent and haemopoietic precursors at various intervals after radiation exposure). Thesis, 1970 [Russian]. 5. Petrov, :R. V., and Zaretzkaja, Y. M. In “Transplantatzionnyj immunitet i radiatzionnye khimery (Transplantation Immunity and Radiation Chimerae”), p. 127. Atomizdat, 1965. [Russian]. 6. Jerne, N., Nordin, H., and Henry, C., The agar plaque technique producing cells. In “Conference on Cell-Bound Antibodies,” Press, Philadelphia, Pennsylvania, 1963. 7. Kudrin, A. N., Ponomareva, cheskoj meditzine” (“The p. 102. Meditzina, Moscow,

for recognizing p. 109. Wistar

antibodyInstitute

G. T., In “Primenenie matematiki v eksperimental’noj i kliniUse of Mathematics in Experimental and Clinical Medicine”), 1967.

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8. Alper, T., Fowler, 9.

10. 11.

12. 13. 14. 15. 16.

17.

18.

CO1\IMUNICATIONS

J. F., Morgan, R. I,., von Berg, D. D., Ellis, F., and Oliver, R., The characterization of “Type C” survival curve. Brit. J. Radio]. 35, 722, 1962. Petrov, R. V., Formy vzaimodejstvija geneticheski razlichajuschikhsja kletok limfoidnykh tkanej (trekhkletochnaja sistema immunogcneza) /The kinds of interaction of genetically differing cells of lymphoid tissues (Three-cell system of immunogenesis)/. Uspcklri ~o~lr~nzewzoj biologii (Advaw. Mod. Biol.) 69, 261 (Russian), 1970. Claman, H. N., Chaperon, E. A., and Triplett, R. F., Thymus-marrow combinations. Synergism in antibody production. E’r-oc. Sot. Exfi. B&l. Med. 122, 1165, 1966. Miller, J. F. A. P., and Mitchell, G. F.. Cell to cell interaction in the immune response. I. Hemolysin-forming cells in neonatally thymectomized mice reconstituted with thymus or thoracic duct lymphocytes. J. IZrp. Jfcd. 128, 801, 1968. Mosier, D. E., and Coppleson, L. W., A three-cell interaction required for the induction of the primary immune response i,c zsitvn. Proc. Nat. Acad. Sci. U.S.A. 61, 542, 1968. Claman, H. N., Chaperon, E. A., Triplett, R. F., Immunocompetence of transferred thymusmarrow cell combinations. J. 1~~~~m~~zo/. 87, 828, 1968. Tyan, M. L., and Herzenberg, L. A., Studies on the ontogeny of the mouse immune system. II. Immunoglobulin-producing cells. J. Iw~r~unoZ. 101, 446, 1968. Davies, A. J. S., Leuchars, E., Wallis, V., Marchant, R., and Elliott, E. V., The failure of thymus-derived cells to produce antibody. 7’l.cllrsp~alftation 5, 222, 1967. Mitchell, G. F., and Miller, J. F. A. P., Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes. J. Exp. ~2lrtl. 128. 821, 1968. Nossal, G. J. V., Cunningham, A., Mitchell, G. F.. and Miller, J. F. A. P., Cell to cell interaction in the immune response. III. Chromosomal analysis of single antibody-forming cells in reconstituted, irradiated, or thymectomized mice. J. Exp. Med. 128, 839. 1968. Fontalin, L. N., Pevnitzkij, I.. A., and Kraskina. S. A., Eksperimental’nyj analiz proiskhozhdenija antiteloobrazujuschikh kletok v selezenke intaktnogo retzipienta posle transplantatzii emu kletok immunizirovannogo donora (;\n experimental analysis of the origin of antibody-forming cells in the spleen of intact recipients after transplantation of immune donor cells). Bzc11. Exp. Biol. Med. U.S.S.R. 32, 108 (Russian), 1967.