Thymic lymphocytes and haemopoiesis

12

Ier FORUM D'IMMUNOLOGIE

selective disappearance of cycling cells resulting in an observed low proliferative activity of the remaining cell population. The properties of the remaining population are disclosed following t h y m e c t o m y and this may explain the observed changes in the E / G ratios and proliferative properties of bone marrow CFU-S that follow thymus removal. The subpopulation of CFU-S which remains in the bone marrow may then continue to function and repopulate the haemopoietic tissues of the animal, even under stress conditions such as those caused by phenylhydrazine. The observed normal numbers of CFU-S in the bone marrow of C57BL/6 mice may be a result of compensatory proliferation of the (~thymus-independent ), population. Furthermore, the thymus dependent CFU-S population may be derived from the other slowly cycling CFU-S population. Exposure of bone marrow from neonatally thymectomized mice to t h y m u s humoral activity induces CFU-S cycling which may be a first step in the re-establishment of the thymus dependent population. Production of specific markers for stem cell subpopulations, such as monoclonal antibodies, may help to further examine the above propositions.

Be[erences. [1] [2] [3] [4] [5] [6]

TRAININ, N. & RESNITZKY, P., Nature (Lond.), 1969, 221, 1154. I:{ESNITZKu P., ZIPORI, D. & TRAI~IN, N., Blood, 1971, 37, 634. ZIPORI, D. & TRAININ, N., Exp. Hemalol., 1975, 3, 1. ZiPol~i, D. & TRAININ, N., Blood, 1973, 42, 671. ZIPORI, D. & TRAININ, N., Exp. Hematol., 1975, 3, 389. TRAININ, N., PECHT, M. & HANDZEL, Z. T., Immunol. Today, 1983, 31, 16.

THYMIC LYMPHOCYTES AND HAEMOPOIESIS by J. W. Goodman and S. G. Shinpock

The Laurence Berkeley Laboralory, 1, Cyclolron Road, Berkeley, CA 94720 (USA) The discovery of a role for thymocytes in myelopoiesis in our laboratory [I] occurred in the mid 60's at about the same time t h a t Claman and his coworkers' experiments documenting a synergism between thymus and marrow on the immune response [2, 3] pointed the way to today's elaborate interactions among different kinds of lymphocytes. Our finding, in all candor, was serendipitous. We were seeking an explanation for studies in which poor growth of parental (P) marrow (BM) in irradiated F1 hybrids (later called genetic resistance) could be substantially augmented by pretreating hosts with viable spleen or lymph node cells of the future donor t y p e [4]. Cell dose and time course considerations, along with the ineffectiveness of comparable numbers of thymocytes or liver cells, indicated t h a t induction of tolerance to a still hypothetical parent-specific antigen was not a plausible explanation. We wanted to test the hypothesis t h a t cells which were effective in the pretreatment of hybrids (F1) persisted, even after lethal irradiation of the host, in providing whatever was lacking before in the F1 for successful growth of transplanted parental cells. For this, we needed a cell source t h a t would (a) provide a relatively large quantity, (b) be easy to prepare with reasonable viability and (c) not itself be myelopoietic. Obviously, spleen and marrow don't meet requirement (c), and lymph nodes are clearly inferior to thymus with respect to (a)

T CELLS AS R E G U L A T O R S OF HAEMATOPOIESIS

13

and (b). Thus we first used parental thymocytes injected together with (actually 1-4 h before) bone marrow to reconstitute irradiated FI hybrid mice. The augmentation achieved was dramatic and, under the circumstances, unexpected. The criterion used for marrow growth in the first published studies [1, 5] was 5gFe uptake as a measure of erythropoiesis, but histologic examination [6, 7] showed t h a t all myelopoietic elements were augmented when parental thymocytes were transplanted to P --> F1 chimeras. One possibility, that the augmentation was somehow a by-product of graft-versus-host (GVH) activity, was thoroughly investigated [8] with the use of tolerant thymocytes and also in experiments designed to make use of immunogenetics (i. e., three strains or hybrids were chosen for host, BM donor, and thymus donor such that the various possibilities of GVH or GVG would or would not be expressed). The conclusion was reached that GVH was not a necessary component of augmentation. Throughout these early studies we were, of course, very much interested in the physiological implications of what was being measured. The P --> F1 chimera, after all, is not really a good model for the normally functioning intact animal. Aware of clinical observations as well as experimental data from the late 50's and early 60's which suggested a ~cnormal ~ function for the thymus in myelopoiesis in general, and in erythropoiesis in particular, we certainly attempted but failed to show an effect in isogemc chimeras. Lord and Schofield's [9] finding thus came as a pleasant surprise because, even though radiation-damaged marrow isn't physiological, an effect of thymocytes was seen in an isogenic situation. As documented in a recent review [10], however, these studies cannot be regularly reproduced. Again with a not completely ~ normal ~ model, the W / W v mouse, Wiktor-Jedrzejczak and his colleagues [11] obtained evidence that a theta-sensitive cell is involved in erythropoiesis. This encouraged us to renew our efforts in the isogenic chimera, and we obtained a few positive data in a painstaking histologic study [12] which corroborated Zipori and his colleagues much earlier finding that BM lacking thymusdependent cells is defective. By the end of the 70's many short-term culture methods had been devised for assaying BM and peripheral blood for stem cells committed to specific differentiation pathways, and several investigators [13, 14, 15] used such methods to investigate thymic effects on mouse bone marrow in vitro. In general, it can be said t h a t isogenic thymocytes augment CFU-C and B F U - E and, under certain culture conditions, CFU-E. The lack of correspondence between CFU-E results from the plasma clot, and methylcellulose methods shake one's faith in the ability to extrapolate from in vitro data to the intact animal, to say the least. Nonetheless, whatever the explanation for these apparent contradictions (see [15] for discussion), in vitro assays continue to be valuable tools for experimental haematologists because of the advantages they offer for controlling at least some of the growth conditions. The use of monoclonal and/or monospecific antibodies has been especially helpful in the investigation of human lymphocyte subpopulation in vitro. In fact there are so many positive data from studies of human BM and peripheral blood lymphocytes (see [15] for citations) added to earlier experimental work that it is possible to say with little fear of contradiction that it has been firmly established that T lymphocytes play an important role in regulation of haemopoiesis. The mechanism(s) whereby T cells effect this regulation remains to be elucidated. It seems highly likely, however, that production of lymphokines is involuted. The recently reported finding [16] that activated T cells were much more effective than resting cells in stimulating CFU-C growth in vitro is a good indication of this, as lymphocytes are known to produce interleukins only when activated. Studies in our own laboratory also strongly point to involvement of interleukins in regulation, as shown in figure 1, which presents data from one of three similar experiments. B6D2F1 bone marrow (BM) cells were grown for three days in the presence of 10% horse serum in medium RPMI 1640 with no additional factor (CBM), crude supernatant from 7-day pokeweed-mitogen-stimulated spleen cells (PWM-CM), II-1, II-2 (both purified mouse interleukins prepared and supplied by R. I. Mishell) or

14

1 er F O R U M

D'IMMUNOLOGIE

,,

I--

0~1 _1

1"

0 O-

0

~ ii~!8ii4~i!i~i i~~ii! 0

iiiiiiiiiiiii:ii: , E i,i

OC

I

D

0

._J

--

I

I

~

0

I

Z

::~. i~!i _o

.~

o w r~

I i,I

w > 0 m

~ ',~,.,~ Z ~ . ~ ~

LL 0

z

,,-,•

zu~ '

O.

~.

~, .~

.~

Z w

hZ

D 0

0

~' ~~'::~i~ :~,iI i !ii!~:: :~

.-~.~

r~ 0

83 0

i

0 u

Z w

~,~i:i:i:i:i:i:i:i:i:i:l Is_ ~ ~

0

N

O_

Fw

I

I

I

I

0 ~0

uO c~J

0 ~j

if~ --

~

I

~ --

ic)

-2

iiiiiiiiiii -L)

I

!

o

&

o

&,

C~

r'--

i#~

o,J

n 0 W "1w

6 m

I

0

I

0

I

0

!

I

I

0

0

0

T CELLS AS R E G U L A T O R S OF H A E M A T O P O I E S I S

15

I1-3, purified to homogeneity from W E H I - 3 supernatant (shown to be lacking CSF and endotoxin) and kindly supplied by J. N. Ihle. The number of (( colonies )~ (clusters of more t h a n 100 cells of uniform appearance) produced at 3 days is shown in figure 1 a. Culture contents (clusters together with individual cells) were harvested and assayed in vivo (CFU-S) as depicted in figure 1 c and in vitro for B F U - E (fig. 1 b) and CFU-C (fig. 1 d). Both L929 supernatant, which results in a very high percentage of macrophage colonies, and mouse endotoxin serum (ET), which stimulates a larger proportion of granulocytic colonies, were used as sources of CSF. I1-3 supported growth of all three stem cell types and was about 2 times as effective at the dose recommended by J. N. Ihle as the crude supernatant, PWM-CM. II-1 and 21-2 were less effective than PWM-CM at maintaining CFU-S and slightly less effective at maintaining CFU-C; none of the three factors was effective in promoting B F U - E . Cells from I1-2-stimulated primary cultures maintained or promoted growth of CFU-S, but because of the paucity of cells harvested initially, B F U - E and CFU-C assays could not always be carried out as they were in this particular experiment. The results with I1-3 stimulated cells are very interesting, as this glycoprotein, thought to be produced by activated T lymphocytes, has been thought primarily to maintain in culture progenitors of T lymphocytes (pre-T's) and mast-like cells [17]. The preliminary data presented here suggest t h a t II-3 acts either on an even earlier, less committed progenitor or alternatively on m a n y different precursors. Goldwasser e t a l . [18] have similar findings, although their methods did not employ an initial 3-day culture. Clearly these approaches will be fruitful in the eventual understanding of how thymic lymphocytes are involved in haemopoiesis. Re/erences.

[1] GOODMAN,J. W. 85 SHINPOCK, S. G., Influence of thymus cells on erythropoiesis of parental marrow in irradiated hybrid mice. Proc. Soc. exp. Biol. (N. Y.), 1968, 129, 417-422. [2] CLAMAN, H. N., CHAPERON, E. A. & THIPLETT, R. W., Thymus-marrow cell combinations. Synergism in antibody production. Proc. Soc. exp. Biol. (N. Y.), 1966, 122, 1167-1171. [3] CLAMAN, H. N., CHAPERON, E. A. & TRIPLETT, R. F., Immunocompetence of transferred thymus-marrow cell combinations. J. Immunol., 1966, 97, 828-832. [4] GOODMAN, J. W. & WHEELER, U. B., Factors influencing growth of parental marrow grafts in irradiated F1 hybrid mice. Transplant., 1968, 6, 173-186. [5] GOODMAN, J. W. & SmNPOCK, S. G., Further studies on the relationship of the thymus to hemopoiesis. Transplant., 1972, 13, 203-211. [6] GOODMAN,J. W. & GRUBBS, C. G., The relationship of the thymus to erythropoiesis, in (( Hemopoietic cellular proliferation )) (F. Stohlman, Jr) (p. 2635). Grune and Stratton, New York, 1970. [7] BASFORD, N. L. & GOODMAN, J. W., Effects of lymphocytes from the thymus and lymph nodes on differentiation of hemopoietic spleen colonies in irradiated mice. J. Cell. Physiol., 1974, 84, 37-48. [8] GOODMAN, J. W., BURCH, K. T. & BASFORD, N. L., Graft-vs.-host activity of thymocytes: relationship to the role of thymocytes in hemopoiesis. Blood, 1972, 39, 850-861. [9] LORD, B. I. & SCHOFIELD, R., The influence of thymus cells in hemopoiesis: stimulation of hemopoietic stem cells in a syngeneic, in vivo, situatTon. Blood, 1973, 42, 395-404. [1O] GOODMAN, J. W. & SHINPOCK, S. G., Interaction between T lymphocytes and hemopoietic stem cells. A critical mini-review, in (~ Biology of bone marrow transplantation, ICN-UCLA Symposia on Molecular and Cellular Biology ~) (R. P. Gale & C. F. Fox), vol. XVII (p. 461-476). Academic Press, London, New York, 1980. [11] WIKTOH-JEDRZEJCZAK, W., SHARKIS, S., AHMED, A. & SELL, K. W., Thetasensitive cell and erythropoiesis: identification of a defect in W/Wv anemic mice. Science, 1977, 196, 313-315.

16

1er F O R U M D ' I M M U N O L O G I E

[I2] GOODMAN, J. W., BASFORD, N. L. & SHINPOCK, S. G., On the role of thymus in hemopoietic differentiation. Blood Cells, 1978, 4, 53-64. [13] SHARKIS, S. J., SPIVAK, J. L., AHMED, A., MISITI, J., STUART, R. K., •IKTORJEDRZEJCZAK, W., SELL, K. W. & SENSENBRENNER, L. L., Regulation of hematopoiesis: helper and suppressor influences of the thymus. Blood, 1980, 55, 524-527. [14] SAWADA,U. & ADLER, S. S., In vitro hemopoiesis of W/Wv and + / + marrow cells cultured alone and with thymocytes. Exp. Hemat., 1980, 8, 702-708. [15] GOODMAN,J. W. & GOODMAN, D. R., Involvement of cells of the immune system in regulation of erythropoiesis, in Current concepts in erythropoiesis (C. D. R. Dunn) (p. 59-79), 1983. [16] BACON, E. R., SING, A. P. & REINISH, C. L., Amplification of granulopoiesis by T cell subpopulations. Exp. Hemalol., 1983, 11, 747-756. [17] IHLE, J. N., Biochemical and biological properties of interleukin 3: a lymphokine mediating the differentiation of a lineage of cells which includes prothymocytes and mast-like cells. Cont. Top. Mol. Immunology, 1983 (in press). [18] GOLDWASSER,E., IHLE, J. N., PRYSTOWSKY, M. D., RICH, I. & VAN ZANDT, G., Effects of interleukin-3 on hemopoietic precursor cells. Syrup. Mol. Cell Biol., 1983 (in press). This work was supported by NIH Grant AM28430-02 and by the Officeof Health and Environmental Research, Officeof Energy Research, US Department of Energy, under Contract No. DEAC03-76SF00098.

THYMIC R E G U L A T I O N OF HAEMOPOIETIC STEM CELL K I N E T I C S D U R I N G IMMUNE RESPONSES by F. Lepault, M. P. Fache and E. Frindel (*)

Laboraloire de Cin~lique Cellulaire, I N S E R M U250, Inslilul Guslave-Roussy, rue Camille-Desmoulins, 91800 Villejui[ (France) The pluripotential haemopoietic stem cell (CFU-S) is capable of self-renewal and of differentiation into all blood cell lineages including the T and B lymphocytes. The CFU-S is detected by its capacity to form colonies in the spleen of lethally irradiated recipients [24] containing cells of either one of the three lineages of myelopoiesis (erythrocytic, granulocytic and megakaryocytic cells) or are mixed [26]. L y m p h o c y t e colonies are not observed. In normal mice, bred under germ-free or specific pathogen-free conditions, the CFU-S are quiescent. Under various circonstances, the CFU-S are triggered into cell cycle, as assessed by the suicide technique of Becker el al. [1]. One of the events which leads to CFU-S proliferation is when mice are taken out of sterile conditions or contract infectious diseases. This recruitment into cycle also occurs after antigenic stimulation [2]. At the time we made these observations, clinical and experimental evidence had suggested a possible role for the neonatal thymus or thymus-processed cells in the (*) To whom correspondence should be addressed.