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Ontogeny of cellular immunity: Size and turnover of rat thymocytes responsive to in vitro stimulation
CELLULAR
IMMUNOLOGY
Ontogeny
9,273-281
(1973)
of Cellular Immunity: Size and Turnover Responsive to in Vitro Stimulation STELLAC. KNIGHT,
lktkion
of Surgical
Sciences,
BARBARA
Clinical
Research
NEWEY, AND Centre,
Harrow,
of Rat Thymocytes
hT.
R.
LIKc
Middlcscw
H.41
3UJ,
and lepartment of Experimental
Pathologljl, Received
The Medical Janwary
School,
Birminghum
B15 27‘J
22, 1973
Velocity sedimentation of thymus cell suspensions prepared from Wistar rats of different ageshasbeenusedto separatecellsmainly on the basisof size. Large cells, many in division, sediment faster than the major population of small thymocytes and can be separated from them on this basis. In vitro responsiveness to mitogens has been found to be associated with the minor populations of larger cells, particularly the medium-large cells (230-270 pms). The mitogen-responsive populations also contain cells which show a high autonomous DNA synthesis. The size distribution of thymus cells in neonatal animals is more varied than in adults and there is frequently a higher proportion of large cells. The mitogen-responsive cells were again found among the fast sedimenting large cells and the highest responses were seen with the very largest cells (approximately 450 pms). The cells in both adult and neonatal animals which have the capacity to respond to mitogens in vitro are probably larger in size than the normal peripheral recirculating thoracic duct cells. The major population of normal small thymocytes showed a much lower spontaneous uptake of sH-thgmidine and did not respond to mitogens.
INTRODUCTION The thymus is known to be the source of a substantial part of the recirculating pool of lymphocytes (1) and clearly plays a vital role in the development and maintenance of the immune system (2). The thymus-derived lymphocyte populations are involved in various forms of cellular immunity (3-10) and may also participate in some collaborative way with other cell types in the induction of humoral immunity (11-13). The capacity to give mixed lymphocyte reactions (MLR ; 6-S) and to respond in vitro to some nonspecific mitogens such as phytohaemagglutinin (PHA; 9) and Concanavalin A (Con A; 8, 10) are among these properties of the peripheral thymus-derived cells. The thymus itself contains some cells which have the characteristics of the peripheral thymus-derived cells. They are few in number, possibly constituting only 2-5s of thymus cells and can be distinguished from the major population of thymocytes by differences in their antigenic markers (14, 15) and sensitivities to corticosteroids (10, 16, 17) or to x-irradiation (18) and by their different density (28). The mitogen-responsive subpopulation of thymocytes is believed to reside in the medullary areas of the thymus (19) and the nonresponsive cells in the cortex (20). 273 Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserwd
274
KNIGHT,
NEWICY
CONTROL
x
.4ND
LING
2%
%
31234501234
‘5012345
DAY 66lKl
o
12345012345
DAY O-0 x-x
Spleen Thymus
FIG. 1. Uptake of 3H-thymidine into l-ml cultures containing 2 X 10G adult thymus cells. Cultures containing different amounts of Con A were incubated thymidine for 24 hr before harvesting on successive days of culture as shown.
spleen or with SW-
Human fetal cells vary in their responsiveness to in z&o stimulation (21, 22), but the higher PHA responsesof thymus cells taken before 18 weeks of gestation are probably correlated with the preponderance of medullary cells at that time (20, 31). With increasing postnatal age an increase of both MLR (23) and PHA activity (24, 25) has been reported. We have separated subpopulations of thymocytes taken from rats of different ages on the basis of cell size using a velocity sedimentation technique (26). An analysis has been undertaken of the autonomous DNA synthesis and responsiveness to mitogens of the separatedthymus cells. MATERIALS
AND METHODS
Animals. Inbred Wistar rats (ASH) were used throughout the experiments. Cell szfspensions.The thymus was removed from rats of different ages and teased gently apart with forceps in RPM1 1640 medium containing 10% fetal calf serum (FCS) (Bio-Cult Lab.) and then filtered through gauze. When cells from neonatal animals were used two or three thymuses from littermates were pooled. Immediately before loading onto the sedimentation apparatus between 1 and 3 X 10’ viable (trypan blue dye-excluding) thymus cells were suspended in 20 ml of 0.2% bovine serum albumin (BSA Armour Fraction v) in sterile phosphate buffered saline (PBS). Cell fractionation. A velocity sedimentation apparatus similar to that described by Miller and Phillips (26) was used with some modifications. A discontinuous albumin gradient was used and both albulnin ScJhitiollS and the cell suspensionwere loaded from a single loading vessel into a sedimentation vessel 12 cm in diameter (apparatus manufactured by G. K. Scientific, Jacey Road, Shirley, Warwickshire.
SIZE
AND
IN
VITRO
ACTIVITY
OF
RAT
THYMOCYTES
12
14 16
18
275
;iA 2
4
6 8 10 FRACTION
20
FIG. 2. Trypan blue dye-excluding cells recovered in each fraction after velocity sedimentation of adult rat thymus cells in four experiments.
England). The incorporation of a small amount of fetal calf serum into the albumin solutions greatly improved cell survival. The stock solution of 2% BSA was prepared by adding BSA (Sg) to RPM1 1640 medium (100 ml) buffered with Hepes (20 mM) and adding PBS (140 ml) and fetal calf serum ( 10 ml). Dilutions of this solution were prepared by the addition of PBS. The following solutions were run consecutively into the sedimentation chamber via the loading vessel: PBS (20 ml), Cell suspension (20 ml), 0.5% BSA (50 ml), 1% BSA (50 ml), 1.59% BSA (100 ml), 2% BSA (130 ml). The total loading time was 30 minutes. The apparatus was left for 24 hr at room temperature and the cells were then collected in 10 ml fractions from the bottom of the vessel. Cuhres. Fractions were centrifuged and the cells resuspended in 1 ml of Hepes buffered RPM1 1640 containing 10% FCS. Trypan blue dye-excluding cells were counted and cell suspensions made up at 2 X loo per ml. Some fractions were pooled to obtain sufficient cells for culture. Duplicate or triplicate cultures were set up in volumes of 0.5 ml in 7.5 x 1.25 cm Pyrex tubes or 0.2 ml in U-shaped microtiter plate wells (Cooke Engineering). Cultures were stimulated with Con A (Miles-Yeda, made up in solution and stored in aliquots at -2O”C, PHA or in a one-way mixed lymphocyte reaction Burroughs Wellcome, MRIO), (MLR) with Fisher rat spleen cells which had been exposed to 25 pg mitomycin C (Nutritional Biochemicals Corp.) for 20 minutes at 37°C and washed three
276
KNIGHT,
NEWEY
AND
LING
a
0 12 dpm x10-3 viable cells x 10-6
0 . a
4
FRACTION FIG. 3. Viable cells recovered in each fraction after velocity sedimentation of adult rat thymus cells. Dpm= increase in uptake of sH-thymidine on the second day of culture of separated cells due to the presence of Con A, PHA, or mitomycin-treated allogeneic cells (MLR). Brackets show where cells from different fractions were pooled. Cultures were 0.2 ml in microplates.
times. 3H.-Thymidine (methyl-T, Radiochemical Centre, Amersham) diluted to a specific activity of 150 mCi/mmole was added to macro (0.5 &i) or micro (0.2 &i) cultures 24 hr before harvesting. The uptake of 3H-thymidine into the acid-insoluble fraction was assessedby liquid scintillation counting as previously described (27,32). RESULTS Responses of whole thynm cell populations to dogens. Marked responses to Con A and PHA were regularly obtained. The dose-responsecurves and kinetics of the responseto Con A are shown in Fig. 1. The response of spleen cells is also shown for comparison. A concentration of Con A of 20 pg/ml was consistently found to be optimal for stimulating thymocytes under the conditions used whereas the concentration for spleen cells was less critical, similar levels of stimulation being recorded in the 20-100 pgg/ml range. Incorporation of 3H-thymidine was usually maximal over the 2-3 day period but in many experiments a l-2 day maximum was found. The optimal dose of PHA for stimulation of thymus cells was found to be 2 pg/ml and the optimal time of culture 2 days. In the light of these findings the thymus subpopulations from the sedimentation experiments were routinely cultured with Con A at 20 pg/ml for 2 and 3 days and with PHA for 2 days.
SIZE
AND
dpm x 10s3 viable cells
IN
VITRO
0
ACTIVITY
OF
RAT
277
THYMOCYTES
a
x 10-6 .6
FRACTION
4. Viable cells recovered in each fraction after velocity sedimentation of neonatal rat (< l-day-old) thymus cells. Dpm = increment in uptake of sH-thymidine on the second day of culture due to the presence of Con A. Pooled fractions are indicated by brackets. Cultures were 0.2 ml in microplates. FIG.
Morphological observations. There were marked differences in the cellular composition of the fractions. The lowest (quickly sedimenting) fractions contained many blast cells and many cells in division. In the higher fractions the cells were smaller and the major peak of cells consisted of typical small lymphocytes with very little cytoplasm. No dividing cells were seen. The lower fractions were slightly more heterogeneous containing a few cells which appeared in stained smears to be smaller in size. Dead cells were found in varying numbers at the top of the gradient, peaking at around fractions 18 to 20. After sedimentation and collection of cells between 40 and 7070 of the cells loaded were recovered in a viable state. Size-distribution of viable thymus cells of adltlt and neonatal rats. The sedimentation pattern of adult rat thymocytes always showed a major peak of slowly sedimenting small lymphocytes and a less prominent zone corresponding to smaller numbers of faster sedimenting cells. The numbers of viable (dye-excluding) cells recovered in each fraction after sedimentation of adult thymus cell suspensionsis shown in Figs. 2 and 3. Half the animals less than 1 week of age gave a thymus cell distribution pattern similar to that seen with adult cells. However, in many young animals the distribution of cells showed a shift to larger cells, the actively DNA/synthesizing fractions of larger cells constituting a higher proportion of the total cell recovery (Figs. 4 and 5). Autonomous DNA synthesis. The uptake of 3H-thymidine over the first 24 hr of culture was high in the fractions containing the faster sedimenting (larger) cells whereas
a much lower
level of incorporation
was obtained
with
cells from
the major
peak consisting mainly of small cells (Table 1). The uptake of 3H-thymidine declined over the 3-day culture period in both 0.5 and 0.2 ml cultures but continued to be associatedwith the faster sedmenting fractions. Responses to Ynztogen: adult cells. The cells responding most markedly to stimulation with Con A and PHA (Fig. 3) were regularly found in fractions S-10. A response was also obtained with the largest cells in the first few fractions and this tendency was always most marked with Con A stimulation (Fig. 3). Cells responding in a MLR were present in fractions 8-12 (expressed as increments above background 3H-thymidine incorporation of unstimulated cultures in Fig. 3).
278
KNIGHT,
4r
NEWEY
AND
LING
3 DAYS
12 1 4 DAYS
FRACTION
5. Viable cells recovered in each fraction after velocity sedimentation of 3-, 4-, and 7day-old rat thymus cells. Dpm = increment in uptake of 3H-thymidine on the second day of culture due to the presence of Con A. Pooled fractions are indicated by brackets. Cultures were 0.2 ml in microplates. FIG.
In all experiments, without exception, responding cells were in the more rapidly sedimenting fractions and not present in the major population of slowly sedimenting small lymphocytes. Taking the mean volume of the major population of rat thymocytes to be 122 pm3 (28) the volume of rat thymus cells in the most responsive fractions [calculated from the sedimentation rate using the formula S in millimeters per hour = ir2 where r is the mean cell radius (26) ] was shown to lie between 230 and 270 pm3. Responsesto mitogens: neonatal cells. In rats under 1 week of age the Con A responsive cells were detected most strikingly in the bottom 8 fractions recovered (Figs. 4 and 5). This was true whether the cell distribution was similar to that of adult cells or not. In some animals no response to mitogens was observed in the S-10 fraction region where the adult response was most marked (Fig. 5). The highest response to these mitogens was thus seen in very large cells of approximately 450 pm3 in size. The reactivity in a MLR was more difficult to detect but in a single 3-day-old animal positive MLRs were obtained using cells from fractions S-10. DISCUSSION Repeated tests on rat thymus cells fractionated according to size hare shown that the small resting lymphocytes from the major population are not reactive to in vitro
SIZE
AND
IN
VITRO
ACTIVITY
OF
TABLE UPTAKE
OF
“H-THYMIDINE L’ELOCITY
INTO
Fraction 110.
Dl’1l
CELLS
SEPARATIOK
in cultures
incubated
13 14 15 16
17 18 19
299 299 299 299 82 166 1055 10.55
1055 146 61 59 170 122 113 113 113 113 113
a Some fractions were pooled value for successive fractions. 2 X lo6 cells per ml.
FHOM
with
DIFFERENT SIX
“H-thymidine
FRACTIONS
ADULT
8725 8725 8725 8725 3681 3681 3030 3030 2451 3527 3511 1248 138 150 150 1.50 150 150 150 to provide Cultures
ml
2360 2360 3458 3458 2041 2041 1310 297 466 169 275 52 36 83 85 111
13 30 39 adequate contained
AFTER
RATS~
on day:
l-2 0.2
2 3 4 5 6 7 8 9 10 11 12
FKOM
OF CELLS
Ok 1
1
279
THYMOCYTES
1
UNSTIMULATED
SEDIMENTATION
R.4T
2-3 0.5
ml
6425 6425 6125 6425 6425 509 2883 3827 5664 3393 4941 678 948 691 112
46 62 44
0.2 1111 904 901 904 66 66 126 253 286 1116 646 198 107 37 32 47 38 41 44 44
cell numbers and this is indicated 0.2 or 0.5 ml of cell suspension
0.5
ml
1134 1134 1134 1134 113-l
150 150
3308 4302 4548 2762 2619 704 549 72 80 280
by a single containing
stimulation with mitogen, whereas the minor population containing larger cells with a high rate of spontaneous DNA synthesis is responsive to a variety of mitogens. The cells within the thymus which have the properties of the peripheral thymusderived cells are significantly larger than the small lymphocytes present in the thoracic duct of rats whereas the cells of the major unresponsive population are smaller. This result, although unexpected on the grounds that the mitogenresponsive cells of the thymus have been generally thought to be mature, nondividing cells, is in agreement with reports by Waksman and co-workers (36, 37) who fractionated rat thymus cells by a density-gradient technique and similarly found mitogen responses to be associated almost exclusively with the large cell fraction. The results are in general agreement with many studies in the mouse indicating that only a small proportion of thymocytes have “mature” characteristics (10, I I18). Potentially cytotoxic lymphocytes in the mouse thymus are recovered in a medium density fraction distinct from the typical dense small lymphocyte (30)) and the GVH activity of a minor, TL negative population of mouse thymus is much greater than that of a major TL positive population (38). The “mature” population in the mouse thymus is a cortisone resistent minor population which behaves like a recirculating lymphocyte population when injected (39). Many of the cells emigrating from the thymus are known to have been recently in DNA
280
KNIGHT,
NEWEY
AND
LING
synthesis (29) and the larger cells separated from rat thymus in our experiments could be mitotically active prior to emigrating to the periphery. The “spontaneous” DNA synthesis that we have measured refers to the uptake of 3H-thymidine in vitro over early periods of culture and probably reflects the mitotic activity those cells would have had in the thymic environment rather than any in vitro stimulation due to the fetal calf serum present in the medium. Labeled cells were found in the same fractions after intravenous injection of 3Hthymidine. The unresponsiveness of the major peak of small thymocytes in all the systems tested requires more careful investigation. It is conceivable that a comprehensive kinetic study using different doses of mitogens might yield positive stimulation. The survey experiments on dose requirements for Con A (Fig. 1) show that only a very narrow dose range can provide good thymus cell stimulation and this dose could differ slightly with separated populations. A possibilty also exists that culture conditions are less favorable when only resting thymocyte cells are present, or that these cells are more fragile in vitro. Support for this may be indicated by the comparatively poor survival of thymus cells compared with other lymphoid cells in vitro (33). The complete failure to detect responsesof these cells in every experiment, including those in which allogeneic spleen cells were present, make this unlikely. However, PHA responsesof small dense thymocytes of the rat are reported to occur in the presence of supernates from activated human or mouse lymphoid cells. The varied size distribution patterns of the preparations of neonatal rat thymus cells indicate that there may be considerable individual variation in the time during ontogeny when the proportion of large cells decreases.Our results also demonstrate that the very large blast cells present in young animals are the ones which show sensitivity to in vitro stimulation with PHA and ConA. This suggests, in agreement with other workers (20, 31), that the early blast cells present in the medulla of fetal thymus could have some of the characteristics associated with the so-called “mature” or immunocompetent thymus-derived cells, although they are larger in size. Our preliminary MLR data suggests that possibly only the medium-large lymphocytes give an MLR response. The appearance of an MLR response later in ontogeny than PHA responsiveness, (34, 35) could thus correspond to the gradual development from a population of very large PHA-responsive blast cells to slightly smaller cells responding to PHA and in the MLRs. The early disappearance of MLR reactive cells after adult thymectomy in rats (8) would, therefore, suggest that cells from this population of medium-large thymocytes are continuously entering the peripheral lymphocyte pool. In the adult rat thymus the cells giving optimal mitogen responses are slightly smaller than those in the neonatal animals but are still larger than the peripheral thymus-derived cells. An important problem emphasized by this work is the function of the major population of small thymocytes. This question may be tackled more easily with the use of the technique of velocity sedimentation which permits a clear separation of the cells and the possibility of the preparation of specific antisera as well as a comparison of the biochemical and antigenic characteristics of the two populations. REFERENCES 1. Davies, 2. Miller,
A. J. S., Tmn.rplantotio?z Rev. 1, 43, 1969. J. F. A. P., and Osoba, D., Physiol. Rcrj. 47,
437, 1967.
SIZE
AND
IN
VITRO
ACTIVITY
OF
RAT
THYMOCYTES
281
3. Cooper, M. D., Peterson, R. D. A., South, M. A., and Good, R. A., J. Exp. Med. 123, 75, 1966. 4. Warner, N. L., Szenberg, A., and But-net, F. M., Amt. J. E.rp. Biol. 40, 373, 1962. 5. Nisbet, N. W., Simonsen, M., and Zaleski, M., J. Exp. Med. 129, 459, 1969. 6. Wilson, D. B., Silvers, W. K., and Nowell, P. C., J. Exp. Med. 126, 655, 1967. 7. Kisken, W. A., and Swenson, N. A., Nature (London), 224,76, 1969. 8. Knight, S. C., Newey, B., and Ling, N. R., Cytobios, 7, 35, 1973. 9. Janossy, G., and Greaves, M. F., C/ix Exp. ZmnznwnoZ.9, 483, 1971. 10. Janossy, G., and Greaves, M. F., Clm. E.rp. Zamzunol. 10, 525, 1972. 11. Claman, H. N., Chaperon, E. A., and Triplett, R. F., Proc. Sot. Exp. Biol. 122, 1167, 1966. 12. Mitchell, G. F, and Miller, J. F. A. P., 1. Exp. Med. 128, 821, 1968. 13. Miller, J. F. A. P., and Mitchell, G. F., 128, 801, 1968. 14. Raff, M. C., and Cantor, H., 11%“Progress in Immunology” Academic Press, New York, p. 83, 1971. 15. Owen, J. J. T., Zn “Ontogeny of Acquired Immunity” p. 35, North Holland, 1972. 16 Blomgren, H., and Anderson, B., Cell. Immunol. 1, 545, 1971. 17. Blomgren, H., and Svedmyr, E., Cell. Znzmunol. 2, 285, 1971. 18. Jacobsson, H., and Blomgren, H., Cell. Znzmunol. 4, 93, 1972. 19. Weber, W. T., J. Cell. Physiol. 68, 117, 1966. 20. Papiernik, M., Blood, 36, 470, 1970. 21. Jones, W. R., Amer. J. Obstet. Gynecol. 104, 586,1%9. 22. Haywood, A. R., and Soothill, J. F., In “Ontogeny of Acquired Immunity” p. 261, North Holland 1972. 23. Knight, S. C., and Thorbecke, G. J., Cell. Zmmunol. 2, 91, 1971. 24. Schwarz, M. R., and Rieke, W. D., Anat. Rec. 155, 493, 1966. 25. Claman, H. N., and Brunstetter, F. H., J. Immtnol. 100, 1127, 1968. 26. Miller, R. G., and Phillips, R. A., J. Cell. Physiol. 7,3, 191, 1969. 27. Knight, S. C., Farrant, J., and Morris, G. J., Nature (Londosl) New Biol. 239, 88, 1972. 28. Aisenberg, A. A., and Murray, C., J. Znzmztnol. 107, 284, 1971. 29. Ernstrom, V., and Larsson, B., Nature (Londox) 222, 279, 1969. 30. Shortman, K., Brunner, K. T., and Cerottini, J. C., J. E-2-p. Med. 135, 1375, 1972. 31. Papiernik, M., In “Proceedings of the Symposium,” Phylogenic and Ontogenic study of the immune response and its contribution to the immunological theory. p. 341, INSERM, 1972. 32. Knight, S. C., Newey, B., and Ling, N. R., Cytobios, in press. 33. Knight, S. C., and Ling, N. R., Clin. Exp. Immunol. 4, 667, 1969. 34. Carr, M. C., Stites, D. P., and Fudenberg, H. H., In “Proceedings of the Symposium,” Phylogenic and Ontogenic study of the immune response and its contribution to the immunological theory. p. 333, INSERM, 1972. 35. Meo, T., Pospisil, M., Massobrio, G., and Miggiano, V. Zn “Proceedings of the Symposium,” Phylogenic and Ontogenic study of the immune response and its contribution to the immunological theory. p. 315, INSERM, 1972. 36. Colley, D. G., Shih, A. Y., and Waksman B. H., J. Exp. Med. 132, 1107, 1970. 37. Wu, Hu Ya S., and Waksman, B. H., Cell. Znznmunol. 3, 516, 1972. 38. Leckland, E., and Boyse, A. E., Science 172, 1258, 1971. 39. Blomgren, H., and Anderson, B., Clin. Exp. Zmmzmol. 10, 297, 1972. 40. Gery, I., Gershon, R. K., and Waksman, B. H., J. Exp. Med. 136, 128, 1972.
IMMUNOLOGY
Ontogeny
9,273-281
(1973)
of Cellular Immunity: Size and Turnover Responsive to in Vitro Stimulation STELLAC. KNIGHT,
lktkion
of Surgical
Sciences,
BARBARA
Clinical
Research
NEWEY, AND Centre,
Harrow,
of Rat Thymocytes
hT.
R.
LIKc
Middlcscw
H.41
3UJ,
and lepartment of Experimental
Pathologljl, Received
The Medical Janwary
School,
Birminghum
B15 27‘J
22, 1973
Velocity sedimentation of thymus cell suspensions prepared from Wistar rats of different ageshasbeenusedto separatecellsmainly on the basisof size. Large cells, many in division, sediment faster than the major population of small thymocytes and can be separated from them on this basis. In vitro responsiveness to mitogens has been found to be associated with the minor populations of larger cells, particularly the medium-large cells (230-270 pms). The mitogen-responsive populations also contain cells which show a high autonomous DNA synthesis. The size distribution of thymus cells in neonatal animals is more varied than in adults and there is frequently a higher proportion of large cells. The mitogen-responsive cells were again found among the fast sedimenting large cells and the highest responses were seen with the very largest cells (approximately 450 pms). The cells in both adult and neonatal animals which have the capacity to respond to mitogens in vitro are probably larger in size than the normal peripheral recirculating thoracic duct cells. The major population of normal small thymocytes showed a much lower spontaneous uptake of sH-thgmidine and did not respond to mitogens.
INTRODUCTION The thymus is known to be the source of a substantial part of the recirculating pool of lymphocytes (1) and clearly plays a vital role in the development and maintenance of the immune system (2). The thymus-derived lymphocyte populations are involved in various forms of cellular immunity (3-10) and may also participate in some collaborative way with other cell types in the induction of humoral immunity (11-13). The capacity to give mixed lymphocyte reactions (MLR ; 6-S) and to respond in vitro to some nonspecific mitogens such as phytohaemagglutinin (PHA; 9) and Concanavalin A (Con A; 8, 10) are among these properties of the peripheral thymus-derived cells. The thymus itself contains some cells which have the characteristics of the peripheral thymus-derived cells. They are few in number, possibly constituting only 2-5s of thymus cells and can be distinguished from the major population of thymocytes by differences in their antigenic markers (14, 15) and sensitivities to corticosteroids (10, 16, 17) or to x-irradiation (18) and by their different density (28). The mitogen-responsive subpopulation of thymocytes is believed to reside in the medullary areas of the thymus (19) and the nonresponsive cells in the cortex (20). 273 Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserwd
274
KNIGHT,
NEWICY
CONTROL
x
.4ND
LING
2%
%
31234501234
‘5012345
DAY 66lKl
o
12345012345
DAY O-0 x-x
Spleen Thymus
FIG. 1. Uptake of 3H-thymidine into l-ml cultures containing 2 X 10G adult thymus cells. Cultures containing different amounts of Con A were incubated thymidine for 24 hr before harvesting on successive days of culture as shown.
spleen or with SW-
Human fetal cells vary in their responsiveness to in z&o stimulation (21, 22), but the higher PHA responsesof thymus cells taken before 18 weeks of gestation are probably correlated with the preponderance of medullary cells at that time (20, 31). With increasing postnatal age an increase of both MLR (23) and PHA activity (24, 25) has been reported. We have separated subpopulations of thymocytes taken from rats of different ages on the basis of cell size using a velocity sedimentation technique (26). An analysis has been undertaken of the autonomous DNA synthesis and responsiveness to mitogens of the separatedthymus cells. MATERIALS
AND METHODS
Animals. Inbred Wistar rats (ASH) were used throughout the experiments. Cell szfspensions.The thymus was removed from rats of different ages and teased gently apart with forceps in RPM1 1640 medium containing 10% fetal calf serum (FCS) (Bio-Cult Lab.) and then filtered through gauze. When cells from neonatal animals were used two or three thymuses from littermates were pooled. Immediately before loading onto the sedimentation apparatus between 1 and 3 X 10’ viable (trypan blue dye-excluding) thymus cells were suspended in 20 ml of 0.2% bovine serum albumin (BSA Armour Fraction v) in sterile phosphate buffered saline (PBS). Cell fractionation. A velocity sedimentation apparatus similar to that described by Miller and Phillips (26) was used with some modifications. A discontinuous albumin gradient was used and both albulnin ScJhitiollS and the cell suspensionwere loaded from a single loading vessel into a sedimentation vessel 12 cm in diameter (apparatus manufactured by G. K. Scientific, Jacey Road, Shirley, Warwickshire.
SIZE
AND
IN
VITRO
ACTIVITY
OF
RAT
THYMOCYTES
12
14 16
18
275
;iA 2
4
6 8 10 FRACTION
20
FIG. 2. Trypan blue dye-excluding cells recovered in each fraction after velocity sedimentation of adult rat thymus cells in four experiments.
England). The incorporation of a small amount of fetal calf serum into the albumin solutions greatly improved cell survival. The stock solution of 2% BSA was prepared by adding BSA (Sg) to RPM1 1640 medium (100 ml) buffered with Hepes (20 mM) and adding PBS (140 ml) and fetal calf serum ( 10 ml). Dilutions of this solution were prepared by the addition of PBS. The following solutions were run consecutively into the sedimentation chamber via the loading vessel: PBS (20 ml), Cell suspension (20 ml), 0.5% BSA (50 ml), 1% BSA (50 ml), 1.59% BSA (100 ml), 2% BSA (130 ml). The total loading time was 30 minutes. The apparatus was left for 24 hr at room temperature and the cells were then collected in 10 ml fractions from the bottom of the vessel. Cuhres. Fractions were centrifuged and the cells resuspended in 1 ml of Hepes buffered RPM1 1640 containing 10% FCS. Trypan blue dye-excluding cells were counted and cell suspensions made up at 2 X loo per ml. Some fractions were pooled to obtain sufficient cells for culture. Duplicate or triplicate cultures were set up in volumes of 0.5 ml in 7.5 x 1.25 cm Pyrex tubes or 0.2 ml in U-shaped microtiter plate wells (Cooke Engineering). Cultures were stimulated with Con A (Miles-Yeda, made up in solution and stored in aliquots at -2O”C, PHA or in a one-way mixed lymphocyte reaction Burroughs Wellcome, MRIO), (MLR) with Fisher rat spleen cells which had been exposed to 25 pg mitomycin C (Nutritional Biochemicals Corp.) for 20 minutes at 37°C and washed three
276
KNIGHT,
NEWEY
AND
LING
a
0 12 dpm x10-3 viable cells x 10-6
0 . a
4
FRACTION FIG. 3. Viable cells recovered in each fraction after velocity sedimentation of adult rat thymus cells. Dpm= increase in uptake of sH-thymidine on the second day of culture of separated cells due to the presence of Con A, PHA, or mitomycin-treated allogeneic cells (MLR). Brackets show where cells from different fractions were pooled. Cultures were 0.2 ml in microplates.
times. 3H.-Thymidine (methyl-T, Radiochemical Centre, Amersham) diluted to a specific activity of 150 mCi/mmole was added to macro (0.5 &i) or micro (0.2 &i) cultures 24 hr before harvesting. The uptake of 3H-thymidine into the acid-insoluble fraction was assessedby liquid scintillation counting as previously described (27,32). RESULTS Responses of whole thynm cell populations to dogens. Marked responses to Con A and PHA were regularly obtained. The dose-responsecurves and kinetics of the responseto Con A are shown in Fig. 1. The response of spleen cells is also shown for comparison. A concentration of Con A of 20 pg/ml was consistently found to be optimal for stimulating thymocytes under the conditions used whereas the concentration for spleen cells was less critical, similar levels of stimulation being recorded in the 20-100 pgg/ml range. Incorporation of 3H-thymidine was usually maximal over the 2-3 day period but in many experiments a l-2 day maximum was found. The optimal dose of PHA for stimulation of thymus cells was found to be 2 pg/ml and the optimal time of culture 2 days. In the light of these findings the thymus subpopulations from the sedimentation experiments were routinely cultured with Con A at 20 pg/ml for 2 and 3 days and with PHA for 2 days.
SIZE
AND
dpm x 10s3 viable cells
IN
VITRO
0
ACTIVITY
OF
RAT
277
THYMOCYTES
a
x 10-6 .6
FRACTION
4. Viable cells recovered in each fraction after velocity sedimentation of neonatal rat (< l-day-old) thymus cells. Dpm = increment in uptake of sH-thymidine on the second day of culture due to the presence of Con A. Pooled fractions are indicated by brackets. Cultures were 0.2 ml in microplates. FIG.
Morphological observations. There were marked differences in the cellular composition of the fractions. The lowest (quickly sedimenting) fractions contained many blast cells and many cells in division. In the higher fractions the cells were smaller and the major peak of cells consisted of typical small lymphocytes with very little cytoplasm. No dividing cells were seen. The lower fractions were slightly more heterogeneous containing a few cells which appeared in stained smears to be smaller in size. Dead cells were found in varying numbers at the top of the gradient, peaking at around fractions 18 to 20. After sedimentation and collection of cells between 40 and 7070 of the cells loaded were recovered in a viable state. Size-distribution of viable thymus cells of adltlt and neonatal rats. The sedimentation pattern of adult rat thymocytes always showed a major peak of slowly sedimenting small lymphocytes and a less prominent zone corresponding to smaller numbers of faster sedimenting cells. The numbers of viable (dye-excluding) cells recovered in each fraction after sedimentation of adult thymus cell suspensionsis shown in Figs. 2 and 3. Half the animals less than 1 week of age gave a thymus cell distribution pattern similar to that seen with adult cells. However, in many young animals the distribution of cells showed a shift to larger cells, the actively DNA/synthesizing fractions of larger cells constituting a higher proportion of the total cell recovery (Figs. 4 and 5). Autonomous DNA synthesis. The uptake of 3H-thymidine over the first 24 hr of culture was high in the fractions containing the faster sedimenting (larger) cells whereas
a much lower
level of incorporation
was obtained
with
cells from
the major
peak consisting mainly of small cells (Table 1). The uptake of 3H-thymidine declined over the 3-day culture period in both 0.5 and 0.2 ml cultures but continued to be associatedwith the faster sedmenting fractions. Responses to Ynztogen: adult cells. The cells responding most markedly to stimulation with Con A and PHA (Fig. 3) were regularly found in fractions S-10. A response was also obtained with the largest cells in the first few fractions and this tendency was always most marked with Con A stimulation (Fig. 3). Cells responding in a MLR were present in fractions 8-12 (expressed as increments above background 3H-thymidine incorporation of unstimulated cultures in Fig. 3).
278
KNIGHT,
4r
NEWEY
AND
LING
3 DAYS
12 1 4 DAYS
FRACTION
5. Viable cells recovered in each fraction after velocity sedimentation of 3-, 4-, and 7day-old rat thymus cells. Dpm = increment in uptake of 3H-thymidine on the second day of culture due to the presence of Con A. Pooled fractions are indicated by brackets. Cultures were 0.2 ml in microplates. FIG.
In all experiments, without exception, responding cells were in the more rapidly sedimenting fractions and not present in the major population of slowly sedimenting small lymphocytes. Taking the mean volume of the major population of rat thymocytes to be 122 pm3 (28) the volume of rat thymus cells in the most responsive fractions [calculated from the sedimentation rate using the formula S in millimeters per hour = ir2 where r is the mean cell radius (26) ] was shown to lie between 230 and 270 pm3. Responsesto mitogens: neonatal cells. In rats under 1 week of age the Con A responsive cells were detected most strikingly in the bottom 8 fractions recovered (Figs. 4 and 5). This was true whether the cell distribution was similar to that of adult cells or not. In some animals no response to mitogens was observed in the S-10 fraction region where the adult response was most marked (Fig. 5). The highest response to these mitogens was thus seen in very large cells of approximately 450 pm3 in size. The reactivity in a MLR was more difficult to detect but in a single 3-day-old animal positive MLRs were obtained using cells from fractions S-10. DISCUSSION Repeated tests on rat thymus cells fractionated according to size hare shown that the small resting lymphocytes from the major population are not reactive to in vitro
SIZE
AND
IN
VITRO
ACTIVITY
OF
TABLE UPTAKE
OF
“H-THYMIDINE L’ELOCITY
INTO
Fraction 110.
Dl’1l
CELLS
SEPARATIOK
in cultures
incubated
13 14 15 16
17 18 19
299 299 299 299 82 166 1055 10.55
1055 146 61 59 170 122 113 113 113 113 113
a Some fractions were pooled value for successive fractions. 2 X lo6 cells per ml.
FHOM
with
DIFFERENT SIX
“H-thymidine
FRACTIONS
ADULT
8725 8725 8725 8725 3681 3681 3030 3030 2451 3527 3511 1248 138 150 150 1.50 150 150 150 to provide Cultures
ml
2360 2360 3458 3458 2041 2041 1310 297 466 169 275 52 36 83 85 111
13 30 39 adequate contained
AFTER
RATS~
on day:
l-2 0.2
2 3 4 5 6 7 8 9 10 11 12
FKOM
OF CELLS
Ok 1
1
279
THYMOCYTES
1
UNSTIMULATED
SEDIMENTATION
R.4T
2-3 0.5
ml
6425 6425 6125 6425 6425 509 2883 3827 5664 3393 4941 678 948 691 112
46 62 44
0.2 1111 904 901 904 66 66 126 253 286 1116 646 198 107 37 32 47 38 41 44 44
cell numbers and this is indicated 0.2 or 0.5 ml of cell suspension
0.5
ml
1134 1134 1134 1134 113-l
150 150
3308 4302 4548 2762 2619 704 549 72 80 280
by a single containing
stimulation with mitogen, whereas the minor population containing larger cells with a high rate of spontaneous DNA synthesis is responsive to a variety of mitogens. The cells within the thymus which have the properties of the peripheral thymusderived cells are significantly larger than the small lymphocytes present in the thoracic duct of rats whereas the cells of the major unresponsive population are smaller. This result, although unexpected on the grounds that the mitogenresponsive cells of the thymus have been generally thought to be mature, nondividing cells, is in agreement with reports by Waksman and co-workers (36, 37) who fractionated rat thymus cells by a density-gradient technique and similarly found mitogen responses to be associated almost exclusively with the large cell fraction. The results are in general agreement with many studies in the mouse indicating that only a small proportion of thymocytes have “mature” characteristics (10, I I18). Potentially cytotoxic lymphocytes in the mouse thymus are recovered in a medium density fraction distinct from the typical dense small lymphocyte (30)) and the GVH activity of a minor, TL negative population of mouse thymus is much greater than that of a major TL positive population (38). The “mature” population in the mouse thymus is a cortisone resistent minor population which behaves like a recirculating lymphocyte population when injected (39). Many of the cells emigrating from the thymus are known to have been recently in DNA
280
KNIGHT,
NEWEY
AND
LING
synthesis (29) and the larger cells separated from rat thymus in our experiments could be mitotically active prior to emigrating to the periphery. The “spontaneous” DNA synthesis that we have measured refers to the uptake of 3H-thymidine in vitro over early periods of culture and probably reflects the mitotic activity those cells would have had in the thymic environment rather than any in vitro stimulation due to the fetal calf serum present in the medium. Labeled cells were found in the same fractions after intravenous injection of 3Hthymidine. The unresponsiveness of the major peak of small thymocytes in all the systems tested requires more careful investigation. It is conceivable that a comprehensive kinetic study using different doses of mitogens might yield positive stimulation. The survey experiments on dose requirements for Con A (Fig. 1) show that only a very narrow dose range can provide good thymus cell stimulation and this dose could differ slightly with separated populations. A possibilty also exists that culture conditions are less favorable when only resting thymocyte cells are present, or that these cells are more fragile in vitro. Support for this may be indicated by the comparatively poor survival of thymus cells compared with other lymphoid cells in vitro (33). The complete failure to detect responsesof these cells in every experiment, including those in which allogeneic spleen cells were present, make this unlikely. However, PHA responsesof small dense thymocytes of the rat are reported to occur in the presence of supernates from activated human or mouse lymphoid cells. The varied size distribution patterns of the preparations of neonatal rat thymus cells indicate that there may be considerable individual variation in the time during ontogeny when the proportion of large cells decreases.Our results also demonstrate that the very large blast cells present in young animals are the ones which show sensitivity to in vitro stimulation with PHA and ConA. This suggests, in agreement with other workers (20, 31), that the early blast cells present in the medulla of fetal thymus could have some of the characteristics associated with the so-called “mature” or immunocompetent thymus-derived cells, although they are larger in size. Our preliminary MLR data suggests that possibly only the medium-large lymphocytes give an MLR response. The appearance of an MLR response later in ontogeny than PHA responsiveness, (34, 35) could thus correspond to the gradual development from a population of very large PHA-responsive blast cells to slightly smaller cells responding to PHA and in the MLRs. The early disappearance of MLR reactive cells after adult thymectomy in rats (8) would, therefore, suggest that cells from this population of medium-large thymocytes are continuously entering the peripheral lymphocyte pool. In the adult rat thymus the cells giving optimal mitogen responses are slightly smaller than those in the neonatal animals but are still larger than the peripheral thymus-derived cells. An important problem emphasized by this work is the function of the major population of small thymocytes. This question may be tackled more easily with the use of the technique of velocity sedimentation which permits a clear separation of the cells and the possibility of the preparation of specific antisera as well as a comparison of the biochemical and antigenic characteristics of the two populations. REFERENCES 1. Davies, 2. Miller,
A. J. S., Tmn.rplantotio?z Rev. 1, 43, 1969. J. F. A. P., and Osoba, D., Physiol. Rcrj. 47,
437, 1967.
SIZE
AND
IN
VITRO
ACTIVITY
OF
RAT
THYMOCYTES
281
3. Cooper, M. D., Peterson, R. D. A., South, M. A., and Good, R. A., J. Exp. Med. 123, 75, 1966. 4. Warner, N. L., Szenberg, A., and But-net, F. M., Amt. J. E.rp. Biol. 40, 373, 1962. 5. Nisbet, N. W., Simonsen, M., and Zaleski, M., J. Exp. Med. 129, 459, 1969. 6. Wilson, D. B., Silvers, W. K., and Nowell, P. C., J. Exp. Med. 126, 655, 1967. 7. Kisken, W. A., and Swenson, N. A., Nature (London), 224,76, 1969. 8. Knight, S. C., Newey, B., and Ling, N. R., Cytobios, 7, 35, 1973. 9. Janossy, G., and Greaves, M. F., C/ix Exp. ZmnznwnoZ.9, 483, 1971. 10. Janossy, G., and Greaves, M. F., Clm. E.rp. Zamzunol. 10, 525, 1972. 11. Claman, H. N., Chaperon, E. A., and Triplett, R. F., Proc. Sot. Exp. Biol. 122, 1167, 1966. 12. Mitchell, G. F, and Miller, J. F. A. P., 1. Exp. Med. 128, 821, 1968. 13. Miller, J. F. A. P., and Mitchell, G. F., 128, 801, 1968. 14. Raff, M. C., and Cantor, H., 11%“Progress in Immunology” Academic Press, New York, p. 83, 1971. 15. Owen, J. J. T., Zn “Ontogeny of Acquired Immunity” p. 35, North Holland, 1972. 16 Blomgren, H., and Anderson, B., Cell. Immunol. 1, 545, 1971. 17. Blomgren, H., and Svedmyr, E., Cell. Znzmunol. 2, 285, 1971. 18. Jacobsson, H., and Blomgren, H., Cell. Znzmunol. 4, 93, 1972. 19. Weber, W. T., J. Cell. Physiol. 68, 117, 1966. 20. Papiernik, M., Blood, 36, 470, 1970. 21. Jones, W. R., Amer. J. Obstet. Gynecol. 104, 586,1%9. 22. Haywood, A. R., and Soothill, J. F., In “Ontogeny of Acquired Immunity” p. 261, North Holland 1972. 23. Knight, S. C., and Thorbecke, G. J., Cell. Zmmunol. 2, 91, 1971. 24. Schwarz, M. R., and Rieke, W. D., Anat. Rec. 155, 493, 1966. 25. Claman, H. N., and Brunstetter, F. H., J. Immtnol. 100, 1127, 1968. 26. Miller, R. G., and Phillips, R. A., J. Cell. Physiol. 7,3, 191, 1969. 27. Knight, S. C., Farrant, J., and Morris, G. J., Nature (Londosl) New Biol. 239, 88, 1972. 28. Aisenberg, A. A., and Murray, C., J. Znzmztnol. 107, 284, 1971. 29. Ernstrom, V., and Larsson, B., Nature (Londox) 222, 279, 1969. 30. Shortman, K., Brunner, K. T., and Cerottini, J. C., J. E-2-p. Med. 135, 1375, 1972. 31. Papiernik, M., In “Proceedings of the Symposium,” Phylogenic and Ontogenic study of the immune response and its contribution to the immunological theory. p. 341, INSERM, 1972. 32. Knight, S. C., Newey, B., and Ling, N. R., Cytobios, in press. 33. Knight, S. C., and Ling, N. R., Clin. Exp. Immunol. 4, 667, 1969. 34. Carr, M. C., Stites, D. P., and Fudenberg, H. H., In “Proceedings of the Symposium,” Phylogenic and Ontogenic study of the immune response and its contribution to the immunological theory. p. 333, INSERM, 1972. 35. Meo, T., Pospisil, M., Massobrio, G., and Miggiano, V. Zn “Proceedings of the Symposium,” Phylogenic and Ontogenic study of the immune response and its contribution to the immunological theory. p. 315, INSERM, 1972. 36. Colley, D. G., Shih, A. Y., and Waksman B. H., J. Exp. Med. 132, 1107, 1970. 37. Wu, Hu Ya S., and Waksman, B. H., Cell. Znznmunol. 3, 516, 1972. 38. Leckland, E., and Boyse, A. E., Science 172, 1258, 1971. 39. Blomgren, H., and Anderson, B., Clin. Exp. Zmmzmol. 10, 297, 1972. 40. Gery, I., Gershon, R. K., and Waksman, B. H., J. Exp. Med. 136, 128, 1972.