ONTOGENY OF THYMUS CELL FUNCTION * A. Chakravarty, L. Kubai, Y. Sidky, and R. Auerbach Department of Zoology University of Wisconsin Madison, Wisconsin 53706
In our laboratory we have been studying the differentiation of the mouse embryonic thymus both in terms of the ontogeny of thymic lymphocytes and in terms of the functional capacity of these cells in both humoral and cellular immune systems. For our studies we have primarily employed in vitro procedures and have used tissue culture both to obtain differentiation of thymic lymphocytes from early embryonic thymus rudiments, and, subsequently, to assess immunocompetence of such in vitro-derived cells. Our experiments have used two test systems: one for assessment of cellular immunity, the other for detection of humoral immune function. For cellular immunity we have used the in vitro graft-versus-host (GvH) reaction, described originally by Auerbach and Globerson and recently modified to permit quantitative assessment of immunocompetence.3 In this reaction, cells to be assessed for immunocompetence are added to neonatal semiallogeneic spleen explants where they can induce splenomegaly; this splenomegaly is detected by the increase in incorporation of isotope-labeled amino acids into the test explants. Both the onset of spleen-fragment enlargement and the degree of enlargement can be used to characterize the relative immunocompetence of various cell suspension^.^ The in vitro GvH reaction has, for example, been used to show that thymus cells from adult mice are only about 10% as active as spleen cells, but t h q a 10-20-fold increase in activity occurs when thymus cells from coFticosteroid-treated animals are used. Similarly, we have been able to show that aliquots of thymus cell suspension treated with anti-TL serum are as effective as control serum treated aliquots in spite of the 90% cytotoxicity of the anti-T1 In our studies of humoral immunity we have restricted our investigations to the study of the response to sheep red blood cells (SRBC). For most of these studies we have employed the Click modification of the Mishell-Dutton cell suspension culture system; the organ culture methods developed by Globerson and Auerbach were employed for some ancillary studies. For both procedures assessment of immunological response was measured by enumeration of direct ( 19s) plaque-forming cells (PFC) , using the Cunningham-Szenberg liquid monolayer assay.7 THE PRIMITIVETHYMUSSTEM CELL
As background information for the studies of thymus stem-cell differentiation we determined the onset of immunocompetence as measured by the in vitro
* Supported by Grant GB 36767 from the National Science Foundation and Grant CA 13548 from the National Cancer Institute. 34
Chakravarty et al.: Ontogeny of Thymus Cell Function
35
gvh reaction. In confirmation of earlier in vivo studies,8. we found that neonatal thymus cells were as effective as were adult thymus cells.l0 (TABLE 1). We were unable to obtain positive responses with any embryonic thymus cells, however, suggesting that a considerable time period was required following the appearance of thymic small lymphocytes at day 16 of embryonic life before immunocompetence could be exerted as measured by our assay.
TABLE1 ABILITYOF SEMIALLOGENEIC EMBRYONIC, NEWBORNOR ADULTTHYMUS CELLS TO INDUCE SPLENOMEGALY (GRAFT-VERSUS-HOST REACTION)In Vitro Source of Thymus Cells adult
newborn 19-day embryo 18-day embryo 17-day embryo 16-day embryo I3-day embryo, after 7 days in organ culture adult
newborn 13-day embryo, after 7 days in organ culture 13-day embryo, 850 R x-ray, then 7 days in vitro I3-day nu/nu embryo, 7 days in vitro 13-day embryo, 1-3 days in hydrocortisone, 4-6 days further culture
Number of Cells
Number of Ex- Number of periments Cultures
1.ox loo 1.ox loo 1.ox lo"
24 28 4
1.ox loa
10
Spleen Index 1.25 * 1.25 * 1.01 0.99 1.12 0.99
1.ox lo6
5
145 212 41 107 47 38
1.ox loo
1
4
1.45 * (1.00) t (0.97) t
0.2 x loa
5
20
1.22 *
0.2x loa
I
21
1.33 *
0.2 x loa
3
4
1.29 t
0.2x loa
5
18
1.18 t
1.ox 10"
5
0.2x lo0 0.2x loo
'>Highly significant; see Auerbach and Shalaby (1973) for validation and experimental detail. .t From previous experiments. $ .05 > p < .lo.
In Vitro Maturation Some time ago work from our laboratory showed that the thymus rudiment isolated from 12-13 day embryos, is capable of becoming lymphoid when grown in vitro for 6-7 days 1'. 1* demonstrating that the thymus rudiment at the time of explanation must have contained a stem-cell population capable of differentiating into morphologically normal thymus lymphocytes. The yet earlier history of that stem-cell population (i.e. yolk-sac-derived) suggested by the work of Moore and Owen13 and Owen and Richter,14 need not concern us within the context of the studies we are reporting. To examine the func-
36
Annals New York Academy of Sciences
tional capacity of tissue-culture-derived thymus lymphocytes, thymus rudiments from 13-day C57BL/6 embryos were cultured for one week, and lymphocytes from such cultures were then assayed for their ability to induce spleen enlargement in BDF, neonatal spleen explants. The experiments showed that not only are in vitro-derived thymus lymphocytes immunocompetent, but their effectiveness is equivalent to that of the cortisone-resistant thymus cell; i.e. splenomegaly could be induced by as few as 5 X lo4 cells (TABLE1 ) . This finding suggests that the primitive thymus stem cells present in 13-day rudiments are capable of exhaustive differentiation in the absence of bone-marrow-derived precursor cells to yield a population of lymphocytes enriched for effector activity for cell-mediated immunity. Radiation Sensitivity
To examine the radiation sensitivity of the thymus stem cell, thymus rudiments from 13-day embryos were irradiated with x-rays ranging from 250 R to 1000 R and then cultured for a 6-7 day period.15 Irradiation had a profound effect on growth of the rudiment, as seen by the fact that the overall size of the explants as well as the total number of lymphocytes was drastically reduced at the higher doses. For example, whereas thymus rudiments in vitro yield about 5 X lo4 to lo5 lymphocytes,lG rudiments irradiated with 1000 R give rise to only about loJ cells. Interestingly, however, histological examination showed that the differentiation of lymphocytes appeared normal; i.e. although the explants were smaller, their histiotypic development was not severely altered. Indeed, it appeared that the most pronounced radiation effect was seen in the mesenchymal tissue suggesting that stem-cell proliferation, rather than differentiation, was dependent on the induction effects known to be operative in thymus deve1opment.l' To assess the immunocompetence of thymic lymphocytes obtained following x-irradiation, 13- or 14-day rudiments were irradiated with 850 R and subsequently cultured for 6-7 days. After this time cell suspensions were prepared and assayed in the in vitro GvH reaction assay. As can be seen from TABLE1, irradiated thymus explants yielded lymphocytes capable of causing a gvh reaction. Sensitivity to Corticosteroids
Relatively low concentrations of some corticosteroids can inhibit the in vitro lymphoid differentiation of the 13-day embryonic mouse thymus.ls Corticosterone was the most effective of the steroids studied, and thymus lymphoid differentiation remained arrested as long as the culture medium contained as little as 0.6 pg/ml. After careful washing and further culturing for 4-6 days, thymus rudiments developed lymphocytes that were normal in appearance and the cultures were essentially indistinguishable from untreated control cultures.ls Thymus lymphoid cells obtained from corticosterone treated rudiments have now been tested in the in vitro GvH reaction assay. As can be seen from TABLE1, these lymphocytes are capable of inducing splenomegaly in vitro, indicating that they are capable of acting as effector cells in cell-mediated immunity.
Chakravarty et al.: Ontogeny of Thymus Cell Function
37
It had previously been noticed that corticosterone treatment causes a disappearance of basophilic cells in the thymus rudiment, yet it is the basophilic cells that have been considered to be the stem cells for thymus 1ymph0cytes.l~ We must therefore entertain the suggestion that the basophilic cells are not the sole stem cells within the thymus rudiment. It may well be, however, that corticosterone treatment leads to cytochemical alterations within the stem cells resulting in their inability to absorb basophilic dyes. Thymocytes jrom Nude Embryos
Although mice homozygous for the nude gene are thymusless from birth, it had previously been reported that thymus rudiments do indeed appear during embryogeny, but that these fail to complete their migration into the thoracic cavity.19 To examine the nature of thymus stem cells in nu/nu mice, 13-day embryos from crosses of nu/+ x nu/+ (Balb/C) were dissected to obtain thymus rudiments and skin fragments. Since at the time of dissection it was not possible to distinguish nu/nu embryos from normal ones (nu/+ or +/+) each embryo was separately handled. Thymus rudiments from each embryo were grown in organ culture, while several fragments of skin from each embryo were grafted into the anterior eye chamber of adult Balb/C mice. After one week in culture, thymuses were either fixed for histological examination or prepared as a cell suspension to be tested for G v H competence. Eye-chamber grafts were recovered after two weeks and scored for the presence of hair. When skin fragments from normal embryos are grown in the anterior eye chamber, they readily produce hair; 2n nude skin grafts, however, fail to do so. Thus eye-graft assessment permitted the distinction of nu/nu embryonic thymus from normal thymus. Admittedly, this distinction could only be made after the experimental studies of thymus cultures was completed, and for this reason each pair of thymuses had to be handled separately throughout, limiting the number of lymphocytes that could be tested in any single GvH assay. Histological examination of nu/ nu thymus cultures revealed that lymphopoiesis in such cultures was normal; there was no visible distinction between cultures derived from nu/nu embryos and those from their littermates. Furthermore, nu/nu lymphocytes gave positive spleen indexes and the mean spleen index of nu/nu lymphocytes was not distinguishable from the index of littermate controls (TABLE 1). Kindred has recently reported that thymuses grafted into nu/ nu animals become populated with cells from the nu/nu host.?' Similarly, studies carried out with thymus extracts indicate that bone marrow cells from nu/nu mice can become &positive T-cells.?' Our work suggests that not only are there presumptive T-cells in nu/nu mice, but there are embryonic thymus stem cells, and that these cells can be detected in the thymus itself.
THYMUS HELPERCELL FUNCTION Although many studies have described interactions between T- and B-cells in the immune response to SRBC, thymus cells themselves have not been able to act synergistically when assayed directly. Thus virtually all studies involving T-B collaboration have employed the use of an intermediate irradiated host,
38
Annals New York Academy of Sciences
previously injected with thymus cells and usually with antigen. This thymus “education” or “activation” has been an obligatory event in yielding functional thymus-derived cells. To obtain thymus-cell helper function without cell passage, thymus cells were incubated for 24 hours with 5 pg c0ncanavalin-A,1~as suggested by studies of Anderson et aLZ4and Sjoberg et d Z 6 To test for helper-cell activity, thymus cells were then washed and 3 x 106 cells were combined with 5 X 108 spleen cells obtained from adult, irradiated, thymectomized animals previously injected with bone marrow cells (B spleen cells). After 4 days of culture in the presence of SRBC the cultures were examined for presence of PFC. The results (FIGURE 1 ) clearly indicate that the pretreatment of thymus cells with concanavalin A permits them to act as helper cells in the immune response to SRBC. The specificity of action of concanavalin A was suggested by the fact that con-Atreated bone marrow cells failed to enhance the immune response. Against this background information about adult thymus cells, we next examined the ontogeny of thymus helper-cell a~tivity.2~9 z6, 27 We found that even thymus lymphocytes obtained from 16-day embryos were capable of collaborating in the response to SRBC following 24 hours of incubation in medium containing con-A. Moreover, although the activity of 16-day embryonic thymus cells appeared lower on a per cell basis, the capacity of embryonic thymus cells obtained from 18-day embryos was virtually identical with that of adult thymus cells (FIGURE 2). Since, moreover, the 16-day embryonic thymus has a lymphocyte population in which only a small proportion of cells
FIGURE1. Functional ontogeny of T-cells.
Chakravarty et al.: Ontogeny of Thymus Cell Function
39
TREATED WITH CON-A WITHOUT CON-A "B" SPLEEN CELLS (background)
FIGURE
2. I n
vilro collaboration.
are small lymphocytes,1G* 2s our findings suggest that as soon as thymic small lymphocytes appear they have the ability to collaborate in the response to SRBC provided they are activated by con-A in vitro. In an effort to determine the mechanism of con-A action on thymus cell activity, the 0 and H-2 antigenicity of thymus cells before and after con-A treatment was determined.2:'- 26. 2; The results indicate that con-A treatment is accompanied by a decrease in 0 and an increase in H-2 surface antigens, a change generally considered indicative of thymus cell maturation; ?0-31 moreover, this shift in antigen levels of thymocytes by con-A treatment is achieved equally in both embryonic and adult thymus cell populations. In contrast to our findings of con-A-induced maturation of helper cell function, we found that con-A treatment of embryonic thymus cells did not alter the ontogeny of immunocompetence as measured in the gvh reaction. Thus, there appears here to be a real dissociation between the maturation of helper cell activity and of GvH reactivity of thymus cells. Such a dissociation had previously been suggested by Feldman et ~ 1 1 . 3 ~ GENERAL COMMENTS
Our in vitro studies of embryonic thymus rudiments seem to define a thymus stem cell that is somewhat different from the thymus precursor cell residing in the bone marrow of adult animals. Clearly, it is radiation resistant; it need not be Giemsa-positive; and it is not destroyed by corticosteroid treatment. Moreover, the presence of a thymus stem cell in nude embryos needs to be recognized.
40
Annals New York Academy of Sciences
We have found that concanavalin-A activation of thymus cells can lead to functional helper cells without influencing the maturation of the effector cells for cell-mediated immunity as measured by the gvh reaction. This should serve as a caution that any single scheme for thymus cell maturation may not accurately reflect the complex series of differentiative events leading to the production of immunocompetent cells. At the same time, since concanavalin-A can cause detectable maturational shifts in H-2 and 0 antigenicity of embryonic cells without concomitantly causing them to become immunocompetent in the gvh assay, it is clear that the shift in surface antigenicity associated with maturation is not the only signal of functionality of developing thymus lymphoid cell populations. The ontogeny of immune competence appears to coincide with the morphological appearance of lymphocytes, at least to the extent that such lymphocytes can be activated to function as helper cells in the immune response to SRBC. Moreover, the demonstration that even before thymus cells make their appearance, there may be immunocompetence of liver cells 33-55 and even of yolk-sac cells 36 indicates that the differentiation of immunocompetence is achieved early in development, concomitant for cellular immunity with the appearance of blood-borne cells, and for humoral immunity no later than with the appearance of thymus lymphocytes. One of us has argued elsewhere 37, 38 that this finding places new constraints on current immunological theories of tolerance and on the machinery by which an embryo can learn epigenetically to recognize self as different from non-self; for many of the antigens against which the embryo must not respond do not arise until long after the immune system itself has achieved competence. The concept of allosteric stem-cell tolerance S T , 38 attempts to explain tolerance as the result of an immune-type response leading to the production of blocking factors. Finally, an earlier analogy drawn between the ontogeny of thymus cells and the ontogeny of germ cells may be recalled in the light of several newer findings about the thymus stem cell.4o The fact that the thymus stem cell is alkaline phosphatase positive 4 1 brings to mind the studies on presumptive germ cells which have relied so heavily on just that property of these cells.42 The radiation-resistance of the primordial germ cells has long been in sharp contrast to the radiation sensitivity of later stages in the development of the functional germ cells; a similar pattern is now seen for the thymus, where the stem cell, in contrast to the mature thymocytes, is also highly radiation-resistant. Perhaps the most striking parallel, however, resides in the complex cell surface changes that accompany activation, as measured by altered antigenicity and functionality following mitogenic stimulation.
REFERENCES 1. GLOBERSON, A. & R. AUERBACH. 1965. Primary immune reactions in organ cultures. Science 1 4 9 991-993. 2. AUERBACH, R. & A. GLOBERSON. 1966. In vitro induction of the graft versus host reaction. Exp. Cell Res. 42: 3 1 - 4 1 . 3. AUERBACH, R. & M.-R. SHALABY. 1973. Graft-versus-host reaction in tissue culture. J. Exp. Med. 138 1506-1520. 4. DOELL, R. & R. AUERBACH. Unpublished observations.
Chakravarty et al.: Ontogeny of Thymus Cell Function
41
5. CLICK,R. E., L. BENCK& B. J. ALTER. 1972. Immune response in vitro. I. Culture conditions for antibody synthesis. Cell. Immunol. 3: 264-276. A. & R. AUERBACH. 1966. Primary antibody response in organ cul6. GLOBERSON, tures. J. Exp. Med. 124: 1001-1016. J. A. & A. SZENBERG.1968. Further improvement in the plaque 7. CUNNINGHAM, technique for detecting single antibody forming cells. J. Immunol. 14: 599600. G. M. HOCHWALD & E. B. JACOBSON.1963. 8. COHEN,M. W., G. J. THORBECKE, Induction of a graft-versus-host reaction in newborn mice by injection of newborn or adult homologous thymus cells. Proc. SOC. Exp. Biol. Med. 114: 242-244. 1973. Studies on the development of immunity: 9. UMIEL,T. & R. AUERBACH. The graft-versus-host reaction. Pathobiol. Ann. 7: 27-45. 10. CHAKRAVARTY, A., L. KUBAI,C. LANDAHL, J. ROETHLE,M.-R. SHALABY & R. 1973. Studies on the development of immunity in the mouse. AUERBACH. Phylogenic and Ontogenic Study of the Immune Response and its Contribution to the Immunological Theory. : 269-278. INSERM Coll. Soc. Franc. d’Immunol. 1960. 112 vitro formation of lymphocytes from 1 1 . BALL,W. D. & R. AUERBACH. embryonic thymus. Exp. Cell Res. 20: 245-247. R. 1961. Experimental analysis of the origin of cell types in the de12. AUERBACH, velopment of the mouse thymus. Develop. Bid. 3: 336-354. 13. MOORE,M. A. S. & J. J. T. OWEN. 1967. Experimental studies on the development of the thymus. J. Exp. Med. 126: 715-725. 14. OWEN,J. J. T. & M. A. RITTER. 1969. Tissue interaction in the development of thymus lymphocytes. J. Exp. Med. 129: 431-437. 1973. Radiation resistant thymic stem cells. Proc. 15. KUBAI,L. & R. AUERBACH. Soc.Exp. Biol. Med. 142: 554-559. R. 1964. Experimental analysis of mouse thymus and spleen mor16. AUERBACH, phogenesis. In The Thymus in Immunobiology. R. A. Good & A. Gabrielsen, Eds. :95-1 13. Harper and Row. 17. AUERBACH, R. 1960. Morphogenetic interactions in the development of the mouse thymus gland. Develop. Biol. 2: 271-284. 18. SIDKY,Y. 1968. Effect of steroids on thymus lymphoid development in vitro. Anat. Rec. 161: 187-191. 19. WORTIS,H. H., S. NEHLSEN & .I. J. OWEN. 1971. Abnormal development of the thymus in “nude” mice. 3. Exp. Med. 134: 681-692. 20. AUERBACH, R. 1954. Analysis of the developmental effects of a lethal mutation in the house mouse. J. Exp. Zool. 127: 305-330. 21. KINDRED, B. 1975. Am. Zool. In press. 22. BOYSE,E. A., G. H. COHEN,J. A. HOOPER,R. S. SCHULOF & A. L. GOLDSTEIN. 1973. Differentiation of T cells induced by preparations from thymus and by non thymic agents. The determined state of the precursor cell. J. Exp. Med. 138: 1027-1032. 23. CHAKRAVARTY, A. 1974. Functional ontogeny of thymus cells. (Abstr.) Fed. Proc. 33: 735. 24. ANDERSON,J., G. MOLLER& 0. SJOBERG. 1972. Selective induction of DNA synthesis in T and B lymphocytes. Cell. Immunol. 4: 381-393. 25. BARTH,R. F. & 0. SINGLA.1973. Differential effects of concanavalin-A on Thelper dependent and independent antibody responses. Cell. Immunol. 9: 96-103. 26. CHAKRAVARTY, A. 1975. Ph.D. Dissertation. University of Wisconsin. Madison, Wisc. 27. CHAKRAVARTY, A. 1975. In preparation. 28. BALL,W. D. 1963. A quantitative assessment of mouse thymus differentiation. Exp. Cell Res. 31: 82-88.
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Annals New York Academy of Sciences
29. OWEN,J. J. T. & M. C. RAFF. 1970. Studies on the differentiation of thymusderived lymphocytes. J. Exp. Med. 132: 1216-1232. 30. OWEN,J. J. T. 1972. The origin and development of lymphocytes populations. I n Ontogeny of acquired Immunity. Ciba Found. Symp. R. Porter & J. Knight, Eds. : 35-64. Elsevier. Amsterdam, The Netherlands. 31. RAFF, M. C. 1971. T and B lymphocytes in mice studied by using antisera against surface antigenic markers. Am. J. Pathol. 65: 467. 32. SEGAL,S., I. R. COHEN& M. FELDMAN. 1972. Thymus-derived lymphocytes:
Humoral and cellular reactions distinguished by hydrocortisone. Science 175: 1126-1128. 33. UMIEL,T., A. GLOBERSON & R. AUERBACH.1968. Role of the thymus in the development of immunocompetence of embryonic liver cells in vitro. Proc. SOC.Exp. Biol. Med. 129 598-600. 34. UMIEL, T. 197 1. Thymus-influenced maturation of embryonic liver cells. Transplantation 11: 531-535. 35. UMIEL,T. 1973. Requirements for development of immunocompetence of embryonic liver cells: The graft-versus-host response. Differentiation 1: 295. 36. HOFMAN,F. & A. GLOBERSON.1973. Graft-versus-host response induced in v i m by mouse yolk sac cells. Eur. J. Immunol. 3: 179-181. 37. AUERBACH, R. 1974. Development of immunity and the concept of stem cell
tolerance. Am. Zool. In press. 38. AUERBACH, R. 1974. Towards a developmental theory of immunity: Ontogeny of immunocompetence and the concept of allosteric tolerance. 111 Cellular Selection and Regulation in the Immune Response. G. Edelman, Ed. : 59-70.
Raven Press. New York, N.Y. In press. 39. AUERBACH, R. & J. ROETHLE. 1974. Tolerance to heterologous erythrocytes. Science 183: 332-334. 40. AUERBACH, R. 1970. Toward a developmental theory of antibody formation:
The Germinal Theory of immunity. In Developmental Aspects of Antibody Formation and Structure. J. Sterzl & I. Riha, Eds. : 23-33. Academic Press. New York, N.Y. 41. RUUSKANEN, 0. & K. KOUVALAINEN. 1974. Differentiation of thymus and thymocytes. A study in foetal guinea-pigs using alkaline phosphatase as a label of thymocytes. Immunology. 26: 187-195.
In our laboratory we have been studying the differentiation of the mouse embryonic thymus both in terms of the ontogeny of thymic lymphocytes and in terms of the functional capacity of these cells in both humoral and cellular immune systems. For our studies we have primarily employed in vitro procedures and have used tissue culture both to obtain differentiation of thymic lymphocytes from early embryonic thymus rudiments, and, subsequently, to assess immunocompetence of such in vitro-derived cells. Our experiments have used two test systems: one for assessment of cellular immunity, the other for detection of humoral immune function. For cellular immunity we have used the in vitro graft-versus-host (GvH) reaction, described originally by Auerbach and Globerson and recently modified to permit quantitative assessment of immunocompetence.3 In this reaction, cells to be assessed for immunocompetence are added to neonatal semiallogeneic spleen explants where they can induce splenomegaly; this splenomegaly is detected by the increase in incorporation of isotope-labeled amino acids into the test explants. Both the onset of spleen-fragment enlargement and the degree of enlargement can be used to characterize the relative immunocompetence of various cell suspension^.^ The in vitro GvH reaction has, for example, been used to show that thymus cells from adult mice are only about 10% as active as spleen cells, but t h q a 10-20-fold increase in activity occurs when thymus cells from coFticosteroid-treated animals are used. Similarly, we have been able to show that aliquots of thymus cell suspension treated with anti-TL serum are as effective as control serum treated aliquots in spite of the 90% cytotoxicity of the anti-T1 In our studies of humoral immunity we have restricted our investigations to the study of the response to sheep red blood cells (SRBC). For most of these studies we have employed the Click modification of the Mishell-Dutton cell suspension culture system; the organ culture methods developed by Globerson and Auerbach were employed for some ancillary studies. For both procedures assessment of immunological response was measured by enumeration of direct ( 19s) plaque-forming cells (PFC) , using the Cunningham-Szenberg liquid monolayer assay.7 THE PRIMITIVETHYMUSSTEM CELL
As background information for the studies of thymus stem-cell differentiation we determined the onset of immunocompetence as measured by the in vitro
* Supported by Grant GB 36767 from the National Science Foundation and Grant CA 13548 from the National Cancer Institute. 34
Chakravarty et al.: Ontogeny of Thymus Cell Function
35
gvh reaction. In confirmation of earlier in vivo studies,8. we found that neonatal thymus cells were as effective as were adult thymus cells.l0 (TABLE 1). We were unable to obtain positive responses with any embryonic thymus cells, however, suggesting that a considerable time period was required following the appearance of thymic small lymphocytes at day 16 of embryonic life before immunocompetence could be exerted as measured by our assay.
TABLE1 ABILITYOF SEMIALLOGENEIC EMBRYONIC, NEWBORNOR ADULTTHYMUS CELLS TO INDUCE SPLENOMEGALY (GRAFT-VERSUS-HOST REACTION)In Vitro Source of Thymus Cells adult
newborn 19-day embryo 18-day embryo 17-day embryo 16-day embryo I3-day embryo, after 7 days in organ culture adult
newborn 13-day embryo, after 7 days in organ culture 13-day embryo, 850 R x-ray, then 7 days in vitro I3-day nu/nu embryo, 7 days in vitro 13-day embryo, 1-3 days in hydrocortisone, 4-6 days further culture
Number of Cells
Number of Ex- Number of periments Cultures
1.ox loo 1.ox loo 1.ox lo"
24 28 4
1.ox loa
10
Spleen Index 1.25 * 1.25 * 1.01 0.99 1.12 0.99
1.ox lo6
5
145 212 41 107 47 38
1.ox loo
1
4
1.45 * (1.00) t (0.97) t
0.2 x loa
5
20
1.22 *
0.2x loa
I
21
1.33 *
0.2 x loa
3
4
1.29 t
0.2x loa
5
18
1.18 t
1.ox 10"
5
0.2x lo0 0.2x loo
'>Highly significant; see Auerbach and Shalaby (1973) for validation and experimental detail. .t From previous experiments. $ .05 > p < .lo.
In Vitro Maturation Some time ago work from our laboratory showed that the thymus rudiment isolated from 12-13 day embryos, is capable of becoming lymphoid when grown in vitro for 6-7 days 1'. 1* demonstrating that the thymus rudiment at the time of explanation must have contained a stem-cell population capable of differentiating into morphologically normal thymus lymphocytes. The yet earlier history of that stem-cell population (i.e. yolk-sac-derived) suggested by the work of Moore and Owen13 and Owen and Richter,14 need not concern us within the context of the studies we are reporting. To examine the func-
36
Annals New York Academy of Sciences
tional capacity of tissue-culture-derived thymus lymphocytes, thymus rudiments from 13-day C57BL/6 embryos were cultured for one week, and lymphocytes from such cultures were then assayed for their ability to induce spleen enlargement in BDF, neonatal spleen explants. The experiments showed that not only are in vitro-derived thymus lymphocytes immunocompetent, but their effectiveness is equivalent to that of the cortisone-resistant thymus cell; i.e. splenomegaly could be induced by as few as 5 X lo4 cells (TABLE1 ) . This finding suggests that the primitive thymus stem cells present in 13-day rudiments are capable of exhaustive differentiation in the absence of bone-marrow-derived precursor cells to yield a population of lymphocytes enriched for effector activity for cell-mediated immunity. Radiation Sensitivity
To examine the radiation sensitivity of the thymus stem cell, thymus rudiments from 13-day embryos were irradiated with x-rays ranging from 250 R to 1000 R and then cultured for a 6-7 day period.15 Irradiation had a profound effect on growth of the rudiment, as seen by the fact that the overall size of the explants as well as the total number of lymphocytes was drastically reduced at the higher doses. For example, whereas thymus rudiments in vitro yield about 5 X lo4 to lo5 lymphocytes,lG rudiments irradiated with 1000 R give rise to only about loJ cells. Interestingly, however, histological examination showed that the differentiation of lymphocytes appeared normal; i.e. although the explants were smaller, their histiotypic development was not severely altered. Indeed, it appeared that the most pronounced radiation effect was seen in the mesenchymal tissue suggesting that stem-cell proliferation, rather than differentiation, was dependent on the induction effects known to be operative in thymus deve1opment.l' To assess the immunocompetence of thymic lymphocytes obtained following x-irradiation, 13- or 14-day rudiments were irradiated with 850 R and subsequently cultured for 6-7 days. After this time cell suspensions were prepared and assayed in the in vitro GvH reaction assay. As can be seen from TABLE1, irradiated thymus explants yielded lymphocytes capable of causing a gvh reaction. Sensitivity to Corticosteroids
Relatively low concentrations of some corticosteroids can inhibit the in vitro lymphoid differentiation of the 13-day embryonic mouse thymus.ls Corticosterone was the most effective of the steroids studied, and thymus lymphoid differentiation remained arrested as long as the culture medium contained as little as 0.6 pg/ml. After careful washing and further culturing for 4-6 days, thymus rudiments developed lymphocytes that were normal in appearance and the cultures were essentially indistinguishable from untreated control cultures.ls Thymus lymphoid cells obtained from corticosterone treated rudiments have now been tested in the in vitro GvH reaction assay. As can be seen from TABLE1, these lymphocytes are capable of inducing splenomegaly in vitro, indicating that they are capable of acting as effector cells in cell-mediated immunity.
Chakravarty et al.: Ontogeny of Thymus Cell Function
37
It had previously been noticed that corticosterone treatment causes a disappearance of basophilic cells in the thymus rudiment, yet it is the basophilic cells that have been considered to be the stem cells for thymus 1ymph0cytes.l~ We must therefore entertain the suggestion that the basophilic cells are not the sole stem cells within the thymus rudiment. It may well be, however, that corticosterone treatment leads to cytochemical alterations within the stem cells resulting in their inability to absorb basophilic dyes. Thymocytes jrom Nude Embryos
Although mice homozygous for the nude gene are thymusless from birth, it had previously been reported that thymus rudiments do indeed appear during embryogeny, but that these fail to complete their migration into the thoracic cavity.19 To examine the nature of thymus stem cells in nu/nu mice, 13-day embryos from crosses of nu/+ x nu/+ (Balb/C) were dissected to obtain thymus rudiments and skin fragments. Since at the time of dissection it was not possible to distinguish nu/nu embryos from normal ones (nu/+ or +/+) each embryo was separately handled. Thymus rudiments from each embryo were grown in organ culture, while several fragments of skin from each embryo were grafted into the anterior eye chamber of adult Balb/C mice. After one week in culture, thymuses were either fixed for histological examination or prepared as a cell suspension to be tested for G v H competence. Eye-chamber grafts were recovered after two weeks and scored for the presence of hair. When skin fragments from normal embryos are grown in the anterior eye chamber, they readily produce hair; 2n nude skin grafts, however, fail to do so. Thus eye-graft assessment permitted the distinction of nu/nu embryonic thymus from normal thymus. Admittedly, this distinction could only be made after the experimental studies of thymus cultures was completed, and for this reason each pair of thymuses had to be handled separately throughout, limiting the number of lymphocytes that could be tested in any single GvH assay. Histological examination of nu/ nu thymus cultures revealed that lymphopoiesis in such cultures was normal; there was no visible distinction between cultures derived from nu/nu embryos and those from their littermates. Furthermore, nu/nu lymphocytes gave positive spleen indexes and the mean spleen index of nu/nu lymphocytes was not distinguishable from the index of littermate controls (TABLE 1). Kindred has recently reported that thymuses grafted into nu/ nu animals become populated with cells from the nu/nu host.?' Similarly, studies carried out with thymus extracts indicate that bone marrow cells from nu/nu mice can become &positive T-cells.?' Our work suggests that not only are there presumptive T-cells in nu/nu mice, but there are embryonic thymus stem cells, and that these cells can be detected in the thymus itself.
THYMUS HELPERCELL FUNCTION Although many studies have described interactions between T- and B-cells in the immune response to SRBC, thymus cells themselves have not been able to act synergistically when assayed directly. Thus virtually all studies involving T-B collaboration have employed the use of an intermediate irradiated host,
38
Annals New York Academy of Sciences
previously injected with thymus cells and usually with antigen. This thymus “education” or “activation” has been an obligatory event in yielding functional thymus-derived cells. To obtain thymus-cell helper function without cell passage, thymus cells were incubated for 24 hours with 5 pg c0ncanavalin-A,1~as suggested by studies of Anderson et aLZ4and Sjoberg et d Z 6 To test for helper-cell activity, thymus cells were then washed and 3 x 106 cells were combined with 5 X 108 spleen cells obtained from adult, irradiated, thymectomized animals previously injected with bone marrow cells (B spleen cells). After 4 days of culture in the presence of SRBC the cultures were examined for presence of PFC. The results (FIGURE 1 ) clearly indicate that the pretreatment of thymus cells with concanavalin A permits them to act as helper cells in the immune response to SRBC. The specificity of action of concanavalin A was suggested by the fact that con-Atreated bone marrow cells failed to enhance the immune response. Against this background information about adult thymus cells, we next examined the ontogeny of thymus helper-cell a~tivity.2~9 z6, 27 We found that even thymus lymphocytes obtained from 16-day embryos were capable of collaborating in the response to SRBC following 24 hours of incubation in medium containing con-A. Moreover, although the activity of 16-day embryonic thymus cells appeared lower on a per cell basis, the capacity of embryonic thymus cells obtained from 18-day embryos was virtually identical with that of adult thymus cells (FIGURE 2). Since, moreover, the 16-day embryonic thymus has a lymphocyte population in which only a small proportion of cells
FIGURE1. Functional ontogeny of T-cells.
Chakravarty et al.: Ontogeny of Thymus Cell Function
39
TREATED WITH CON-A WITHOUT CON-A "B" SPLEEN CELLS (background)
FIGURE
2. I n
vilro collaboration.
are small lymphocytes,1G* 2s our findings suggest that as soon as thymic small lymphocytes appear they have the ability to collaborate in the response to SRBC provided they are activated by con-A in vitro. In an effort to determine the mechanism of con-A action on thymus cell activity, the 0 and H-2 antigenicity of thymus cells before and after con-A treatment was determined.2:'- 26. 2; The results indicate that con-A treatment is accompanied by a decrease in 0 and an increase in H-2 surface antigens, a change generally considered indicative of thymus cell maturation; ?0-31 moreover, this shift in antigen levels of thymocytes by con-A treatment is achieved equally in both embryonic and adult thymus cell populations. In contrast to our findings of con-A-induced maturation of helper cell function, we found that con-A treatment of embryonic thymus cells did not alter the ontogeny of immunocompetence as measured in the gvh reaction. Thus, there appears here to be a real dissociation between the maturation of helper cell activity and of GvH reactivity of thymus cells. Such a dissociation had previously been suggested by Feldman et ~ 1 1 . 3 ~ GENERAL COMMENTS
Our in vitro studies of embryonic thymus rudiments seem to define a thymus stem cell that is somewhat different from the thymus precursor cell residing in the bone marrow of adult animals. Clearly, it is radiation resistant; it need not be Giemsa-positive; and it is not destroyed by corticosteroid treatment. Moreover, the presence of a thymus stem cell in nude embryos needs to be recognized.
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Annals New York Academy of Sciences
We have found that concanavalin-A activation of thymus cells can lead to functional helper cells without influencing the maturation of the effector cells for cell-mediated immunity as measured by the gvh reaction. This should serve as a caution that any single scheme for thymus cell maturation may not accurately reflect the complex series of differentiative events leading to the production of immunocompetent cells. At the same time, since concanavalin-A can cause detectable maturational shifts in H-2 and 0 antigenicity of embryonic cells without concomitantly causing them to become immunocompetent in the gvh assay, it is clear that the shift in surface antigenicity associated with maturation is not the only signal of functionality of developing thymus lymphoid cell populations. The ontogeny of immune competence appears to coincide with the morphological appearance of lymphocytes, at least to the extent that such lymphocytes can be activated to function as helper cells in the immune response to SRBC. Moreover, the demonstration that even before thymus cells make their appearance, there may be immunocompetence of liver cells 33-55 and even of yolk-sac cells 36 indicates that the differentiation of immunocompetence is achieved early in development, concomitant for cellular immunity with the appearance of blood-borne cells, and for humoral immunity no later than with the appearance of thymus lymphocytes. One of us has argued elsewhere 37, 38 that this finding places new constraints on current immunological theories of tolerance and on the machinery by which an embryo can learn epigenetically to recognize self as different from non-self; for many of the antigens against which the embryo must not respond do not arise until long after the immune system itself has achieved competence. The concept of allosteric stem-cell tolerance S T , 38 attempts to explain tolerance as the result of an immune-type response leading to the production of blocking factors. Finally, an earlier analogy drawn between the ontogeny of thymus cells and the ontogeny of germ cells may be recalled in the light of several newer findings about the thymus stem cell.4o The fact that the thymus stem cell is alkaline phosphatase positive 4 1 brings to mind the studies on presumptive germ cells which have relied so heavily on just that property of these cells.42 The radiation-resistance of the primordial germ cells has long been in sharp contrast to the radiation sensitivity of later stages in the development of the functional germ cells; a similar pattern is now seen for the thymus, where the stem cell, in contrast to the mature thymocytes, is also highly radiation-resistant. Perhaps the most striking parallel, however, resides in the complex cell surface changes that accompany activation, as measured by altered antigenicity and functionality following mitogenic stimulation.
REFERENCES 1. GLOBERSON, A. & R. AUERBACH. 1965. Primary immune reactions in organ cultures. Science 1 4 9 991-993. 2. AUERBACH, R. & A. GLOBERSON. 1966. In vitro induction of the graft versus host reaction. Exp. Cell Res. 42: 3 1 - 4 1 . 3. AUERBACH, R. & M.-R. SHALABY. 1973. Graft-versus-host reaction in tissue culture. J. Exp. Med. 138 1506-1520. 4. DOELL, R. & R. AUERBACH. Unpublished observations.
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5. CLICK,R. E., L. BENCK& B. J. ALTER. 1972. Immune response in vitro. I. Culture conditions for antibody synthesis. Cell. Immunol. 3: 264-276. A. & R. AUERBACH. 1966. Primary antibody response in organ cul6. GLOBERSON, tures. J. Exp. Med. 124: 1001-1016. J. A. & A. SZENBERG.1968. Further improvement in the plaque 7. CUNNINGHAM, technique for detecting single antibody forming cells. J. Immunol. 14: 599600. G. M. HOCHWALD & E. B. JACOBSON.1963. 8. COHEN,M. W., G. J. THORBECKE, Induction of a graft-versus-host reaction in newborn mice by injection of newborn or adult homologous thymus cells. Proc. SOC. Exp. Biol. Med. 114: 242-244. 1973. Studies on the development of immunity: 9. UMIEL,T. & R. AUERBACH. The graft-versus-host reaction. Pathobiol. Ann. 7: 27-45. 10. CHAKRAVARTY, A., L. KUBAI,C. LANDAHL, J. ROETHLE,M.-R. SHALABY & R. 1973. Studies on the development of immunity in the mouse. AUERBACH. Phylogenic and Ontogenic Study of the Immune Response and its Contribution to the Immunological Theory. : 269-278. INSERM Coll. Soc. Franc. d’Immunol. 1960. 112 vitro formation of lymphocytes from 1 1 . BALL,W. D. & R. AUERBACH. embryonic thymus. Exp. Cell Res. 20: 245-247. R. 1961. Experimental analysis of the origin of cell types in the de12. AUERBACH, velopment of the mouse thymus. Develop. Bid. 3: 336-354. 13. MOORE,M. A. S. & J. J. T. OWEN. 1967. Experimental studies on the development of the thymus. J. Exp. Med. 126: 715-725. 14. OWEN,J. J. T. & M. A. RITTER. 1969. Tissue interaction in the development of thymus lymphocytes. J. Exp. Med. 129: 431-437. 1973. Radiation resistant thymic stem cells. Proc. 15. KUBAI,L. & R. AUERBACH. Soc.Exp. Biol. Med. 142: 554-559. R. 1964. Experimental analysis of mouse thymus and spleen mor16. AUERBACH, phogenesis. In The Thymus in Immunobiology. R. A. Good & A. Gabrielsen, Eds. :95-1 13. Harper and Row. 17. AUERBACH, R. 1960. Morphogenetic interactions in the development of the mouse thymus gland. Develop. Biol. 2: 271-284. 18. SIDKY,Y. 1968. Effect of steroids on thymus lymphoid development in vitro. Anat. Rec. 161: 187-191. 19. WORTIS,H. H., S. NEHLSEN & .I. J. OWEN. 1971. Abnormal development of the thymus in “nude” mice. 3. Exp. Med. 134: 681-692. 20. AUERBACH, R. 1954. Analysis of the developmental effects of a lethal mutation in the house mouse. J. Exp. Zool. 127: 305-330. 21. KINDRED, B. 1975. Am. Zool. In press. 22. BOYSE,E. A., G. H. COHEN,J. A. HOOPER,R. S. SCHULOF & A. L. GOLDSTEIN. 1973. Differentiation of T cells induced by preparations from thymus and by non thymic agents. The determined state of the precursor cell. J. Exp. Med. 138: 1027-1032. 23. CHAKRAVARTY, A. 1974. Functional ontogeny of thymus cells. (Abstr.) Fed. Proc. 33: 735. 24. ANDERSON,J., G. MOLLER& 0. SJOBERG. 1972. Selective induction of DNA synthesis in T and B lymphocytes. Cell. Immunol. 4: 381-393. 25. BARTH,R. F. & 0. SINGLA.1973. Differential effects of concanavalin-A on Thelper dependent and independent antibody responses. Cell. Immunol. 9: 96-103. 26. CHAKRAVARTY, A. 1975. Ph.D. Dissertation. University of Wisconsin. Madison, Wisc. 27. CHAKRAVARTY, A. 1975. In preparation. 28. BALL,W. D. 1963. A quantitative assessment of mouse thymus differentiation. Exp. Cell Res. 31: 82-88.
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29. OWEN,J. J. T. & M. C. RAFF. 1970. Studies on the differentiation of thymusderived lymphocytes. J. Exp. Med. 132: 1216-1232. 30. OWEN,J. J. T. 1972. The origin and development of lymphocytes populations. I n Ontogeny of acquired Immunity. Ciba Found. Symp. R. Porter & J. Knight, Eds. : 35-64. Elsevier. Amsterdam, The Netherlands. 31. RAFF, M. C. 1971. T and B lymphocytes in mice studied by using antisera against surface antigenic markers. Am. J. Pathol. 65: 467. 32. SEGAL,S., I. R. COHEN& M. FELDMAN. 1972. Thymus-derived lymphocytes:
Humoral and cellular reactions distinguished by hydrocortisone. Science 175: 1126-1128. 33. UMIEL,T., A. GLOBERSON & R. AUERBACH.1968. Role of the thymus in the development of immunocompetence of embryonic liver cells in vitro. Proc. SOC.Exp. Biol. Med. 129 598-600. 34. UMIEL, T. 197 1. Thymus-influenced maturation of embryonic liver cells. Transplantation 11: 531-535. 35. UMIEL,T. 1973. Requirements for development of immunocompetence of embryonic liver cells: The graft-versus-host response. Differentiation 1: 295. 36. HOFMAN,F. & A. GLOBERSON.1973. Graft-versus-host response induced in v i m by mouse yolk sac cells. Eur. J. Immunol. 3: 179-181. 37. AUERBACH, R. 1974. Development of immunity and the concept of stem cell
tolerance. Am. Zool. In press. 38. AUERBACH, R. 1974. Towards a developmental theory of immunity: Ontogeny of immunocompetence and the concept of allosteric tolerance. 111 Cellular Selection and Regulation in the Immune Response. G. Edelman, Ed. : 59-70.
Raven Press. New York, N.Y. In press. 39. AUERBACH, R. & J. ROETHLE. 1974. Tolerance to heterologous erythrocytes. Science 183: 332-334. 40. AUERBACH, R. 1970. Toward a developmental theory of antibody formation:
The Germinal Theory of immunity. In Developmental Aspects of Antibody Formation and Structure. J. Sterzl & I. Riha, Eds. : 23-33. Academic Press. New York, N.Y. 41. RUUSKANEN, 0. & K. KOUVALAINEN. 1974. Differentiation of thymus and thymocytes. A study in foetal guinea-pigs using alkaline phosphatase as a label of thymocytes. Immunology. 26: 187-195.
