I. INTRODUCTION Research on the Thymus Gland Has Revealed an Organ of Unexpected Com- Plexity and Diversity of Function

Japan. J. Med. Sci. Biol., 29, 289-321, 1976

REVIEW

SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND

Louis V. CASO

Department o f Anatomic Sciences, Temple University, Health Sciences Center, School of Dentistry, Philadelphia, Pennsylvania

(Received: May 8, 1976)

CONTENTS

I. Introduction ...... 289 II. Active Factors Present in the Thymus Gland ...... 290 Lymphopoietin Lymphocyte Stimulating Hormone Other Stimulators of Lymphatic Tissue III. Thymectomy and the Immune Response...... 293 Endocrine Effects Immunological Effects IV. Action of Thymic Extracts on the Immune Response...... 295 Humoral Antibodies Cell-mediated Antibodies V. The B and T Cell Systems of Immunocompetence ...... 298 Action of Thymic Extracts on the Maturation of T Lymphocytes VI. Inhibitory Action of the Thymus Gland ...... 301 Growth Inhibitory Factor from Thymus Gland VII. Cellular Origin of Thymic Hormones...... 303 VIII. Role of the Thymus in Neoplastic Disease...... 304 Thymus in Relation to Immunity in Aging Studies of Aging Mice The Thymus in Relation to the Neoplasms of the Immune System The Thymus in Relation to Tumor Growth in Experimental Animals Immunosuppression and Immune Surveillance Thymic Humoral Factor Alpha Globulins Other Immunosuppressive Thymic Fractions IX. Summary ...... 311

I. INTRODUCTION Research on the thymus gland has revealed an organ of unexpected com- plexity and diversity of function. The mechanisms of the immune response have been associated with the thymus gland, and the intricacies of the differentiation of immunocompetent cells and their activation by antigens are at present the subject of elaborate investigation. Much of this research has been carried out in the mouse, and Metcalf (1964) has described the structure of the mouse thymus, which consists of 90% by weight

289 290 CASO Vol. 29 of lymphocytes, which in turn account for 99.9% of observed mitoses in the gland. These lymphocytesare not unique to the thymus and are interchangeable with those from other organs. Thymic grafts have been found to be infiltrated by lymphocytes from spleen and bone marrow, but not from thymus or lymph nodes. Thymic lymphocytes in the mouse have been shown to be replaced normally and continually by stem cells from the circulation. The lymphocytes are embedded in a meshwork of reticular epithelial cells, derived from endoderm, and are closely packed in the outer region to form a cortex, while the inner region or medulla has fewer lymphocytes in a meshwork of reticular epithelial cells and mesenchymal reticular cells. Radiating from the medulla are arteries which end in capillaries in the subcapsular region of the cortex. These radial arteries are surrounded by reticular epithelial cells which enclose a space around the arteries, forming a double-wall barrier about the blood vesselsin the cortex. This barrier is incomplete in the subcapsular region. Lymphocytes form a cull around the radial arteries, and in thymic grafts many primitive lymphocytes are found along the arteries. The thymus of the intact mouse, however, contains primitive cells not along blood vessels but in clusters scattered through the subcapsular zone and the middle and inner regions of the cortex (Metcalf, 1964). There is evidence that mesenchymal reticular cells, scattered among the epithelial cells in the outer cortex or along the double wall of the radial vessels, stimulate mitosis in adjacent lymphocytes. These mesenchymal reticular cells are PAS-positiveand phagocytic in contrast to the reticular epithelial cells of the vascular sheath and medulla, which are PAS-negative and non-phagocytic and do not show direct association with mitosis in adjacent lymphocytes (Clark, 1963; Hoshino, 1963). Metcalf (1964) postulates that the PAS-positive mesenchymal cells trigger off mitoses in adjacent lymphocytes after initial stimulation of mitosis by the reticular epithelial cells. In addition, he notes the presence of PAS-positive granules in the cytoplasm of some of the reticular epithelial cells of the medulla, a fact which suggests that they may be secretory even though mitoses are infrequent in this region.

II. ACTIVEFACTORS PRESENT IN THE THYMUSGLAND

Lymphopoietin: Lymphoid cells exhibit intense proliferation on entering the thymus, the primitive lymphocytes and mitoses being concentrated in the cortex. The stimulus appears to be intrinsic to the thymus. It is notable that antigenic stimulation is without effect in stimulating thymic lymphopoiesis, the blood-thymic barrier of reticular epithelial cells, though incomplete, perhaps being the major obstacle. Evidence for the production of a lymphocyte-stimulating factor within the thymus, active on thymic lymphocytes, comes from the behavior of thymic tissue in organ grafts. The grafts are only successful if a minimal proportion of medullary tissue is present in the graft. Only the epithelial and mesenchymal 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND 291 reticular cells survive the graft, while lymphoid cells are replaced by lymphocytes from the host (Metcalf and Wakonig-Vaartaja, 1964). Thymic graft growth is determined by the age, strain and mitotic pattern of the donor mouse and not the recipient, despite genetic differences between the donor and recipient animals and migration of lymphocytes into the thymic graft (Metcalf et al., 1961). The grafts are not affected by the host's thymus, by thymectomy in the host, or other thymic grafts in the host. In turn the thymus of the host is not affected by a thymic graft. Metcalf (1964) presents the hypothesis that lympho-

poietin is produced by the epithelial reticular cells of the cortex and medulla. This substance would be active in the context of PAS-positive mesenchymal

reticular cells or epithelial reticular cells in the cortex. In addition, the medulla may secrete a separate substance which is necessary for the maintenance of thymic

epithelial cells, lymphoid cells or phagocytic (mesenchymal) cells. Recent work by Goldstein (1975) has produced two purified polypeptides from calf thymus. These have been found to be closely related to each other

in activity and have been named thymopoietin I and II by Goldstein. One effect is the in vivo induction of neuromuscular block similar to that found in myasthenia gravis (Goldstein, 1968; Goldstein and Manganaro, 1971). The other effects indicate that this substance can induce the appearance of antigens found on thymic lymphocytes, TL and Thy-1 (ƒÆ), when the extract is incubated with bone marrow hematopoietic cells (Back and Goldstein, 1975). It fails, however,

to induce lymphopoiesis in thymic lymphocytes with or without mitogens such as Concanavalin A, and therefore would not appear to be the substance respon-

sible for the in vivo proliferation of lymphocytes in the thymus. These experiments were carried out on thymus cell suspensions, whereas Metcalf envisaged the hormone acting in the structural context of the thymic cortex. Thymo-

poietin does, however, induce lymphopoiesis in suspensions of spleen cells by enhancing the proliferation caused by Concanavalin A. lymphocyte Stimulating Hormone: One effect of thymectomy in neonatal

animals is a depletion of lymphoid cells in the spleen and lymph nodes. This is accompanied by marked lymphopenia. Metcalf (1964) prepared an extract from mouse thymus gland, heat labile and non-diffusible, which when injected into neonatally thymectomized mice produced temporary lymphocytosis and an elevated lymphocyte/polymorphonuclear leukocyte ratio (L/P). Extracts from spleen, lymph node, liver and other organs were ineffective. He called this thymic extract lymphocytosis stimulating factor (LSF), and further studies

revealed its presence in supernatants from thymus gland growing in tissue culture, and from serum of patients with chronic lymphoid leukemia and lymphosarcoma. It was also found in supernatants from serum of high leukemic

strains of mice (AKR and C58). The sera from normal persons, or from patients with acute leukemia or chronic myeloid leukemia, were negative for LSF (Met- calf, 1966). Thymic extracts from calf, rat or isologous mouse caused increased lymph node weight, increased uptake of tritiated thymidine by DNA of lymph nodes 292 CASO Vol. 29

and increased uptake of 14C-labeled glycine into protein of lymph nodes (Klein , Goldstein and White, 1965).

While thymic extracts gave only partial replacement in thymectomy so that lymphopenia and lymphoid organ atrophy were not corrected completely , grafts of isogeneic or allogeneic thymus in neonatally thymectomized mice prevented these effects if the grafts were done prior to extensive deterioration of the lym-

phatic system (Leuchars et al., 1964; Miller, 1965). Moreover, these authors found that some dividing lymphocytes in the host spleen and lymph nodes , after antigenic stimulation, can be demonstrated to be derived from the thymus

graft; while in thymectomized animals receiving thymus graft but no antigenic stimulation, most cells from the host are found in both the graft and the host's lymphoid organs (Miller, 1962). That a humoral factor from the thymus is responsible for the maintenance of lymphoid tissue in the spleen and lymph nodes was demonstrated by the use of millipore diffusion chambers (0.45 to 0.1ƒÊm pore size) which contain thymus grafts. These preparations prevented the development of severe lympho-

penia in neonatally thymectomized animals, although the blood lymphocyte count and lymphoid cellularity of the peripheral lymphoid organs were not completely normal. Although there is some criticism that millipore chambers have been known to leak (Davies, 1975), corroborative findings from different experiments and laboratories seem to support the conclusion that there is indeed a diffusible, cell-free substance (called by Miller competence-inducing factor , CIF) which maintains lymphatic tissue (Osoba and Miller , 1963, 1964; Levy, Trainin and Law, 1963). A factor which restores the formation of humoral antibody has been observed for diffusion-chamber preparations of bursa of Fabricius in bursectomized chicks, and extracts from the bursa identical to thymus extracts

have been obtained (St. Pierre and Ackerman , 1965; Luckey, 1973). Hand, Caster and Luckey (1967) succeeded in purifying a thymus extract which had properties similar to Metcalf's LSF and Miller's CIF. This substance is a heat-labile, water-soluble protein of 1,700 molecular weight, showing a single

band on electrophoresis. It is active in the neonatal mouse at a dose of 0 .1 ƒÊg, increasing the L/P ratio, spleen size and probably lymph node size . It does not

increase the total leukocyte count. It is designated by Luckey , Robey and Campbell (1973) as lymphocyte stimulating hormone (LSH h), and is probably a purified variety of LSF and CFI.

A second purified compound, isolated by Robey, Campbell and Luckey

(1972) from calf thymus and named LSHr (Luckey et al., 1973) is a heat-stable protein showing a single band on electrophoresis and having a molecular weight of 79,800. It differs chemically, therefore, from LSHh, but is active at the 0.1 ƒÊg/2 g dose in the mouse, and is seen to increase the L/P ratio and spleen weight in the neonatal mouse. Unlike LSHh, it increases the total leukocyte count. Both LSHh and LSHr may stimulate lymphopoiesis in spleen and lymph

node and have effects on the immune response which will be considered later . Metcalf assumed that LSF in producing lymphopoiesis in the spleen and 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND 293 lymph nodes needed the additional stimulus of heterologous antigens. While animals reared in a germ-free environment have underdeveloped spleen and lymph nodes (Luckey, 1963), increased lymphoid organ size and lymphocytosis can be induced by injection of bacteria or soluble antigen (Caster et al., 1966). However, injection of LSH into germ-free mice, without addition of antigen, was found to cause a three-fold increase in spleen and lymph node size and an increased L/P ratio (Luckey et al., 1973). Other Stimulators o f Lymphatic Tissue: Thymosin is another highly purified extract from calf thymus obtained by Goldstein, Slater and White (1966). The purified active substance is a non-dializable protein of molecular weight of 12,600 (Luckey, 1973; Goldstein, Asanuma and White, 1970; Robey, 1973). Much of the biological work has been done on thymosin Fr. 3, a somewhat less purified fraction. In thymectomized mice this substance slightly alleviated leukopenia and provided some improvement in lymphoid histology. In adult mice following sublethal irradiation, thymosin stimulated lymphoid tissue regeneration. Injection prior to irradiation retarded lymphoid involution and attenuated the deleterious effects. Histological studies of peripheral lymphoid organs showed that thymosin stimulated proliferation of primitive lymphocytes in thymus- dependent areas (Goldstein et al., 1970b). Results on sublethally and lethally irradiated rats, however, failed to show restoration of thymus-dependent areas in peripheral lymphoid tissue by thymosin (Kruger, Goldstein and Waksman, 1970). Less purified active thymic extracts are thymic humoral factor (THF) (Trainin, Small and Kimhi, 1973; Trainin et al., 1975) and homeostatic thymic hormone (HTH) (Bernardi and Cosma, 1965). THE is a small, dializable molecule (mol. wt.<1,000) which is probably not a protein. It has been shown to enhance lymphoid cell proliferation in neonatally thymectomized mice in 3H- thymidine studies (Trainin, Small and Kimhi, 1973). HTH is a substance containing nucleic acid and having a molecular weight of 2,000. It has been shown in guinea pigs to maintain normal levels of lymphocyte counts in blood, spleen and bone marrow up to 45 days after thymectomy (Cosma, 1973a). All of these substances have positive effects on the immune responses which will be considered in a later section.

III. THYMECTOMYAND THE IMMUNERESPONSE Endocrine Effects: While it is well known that thymectomy produces lymphopenia and atrophy of the spleen and lymph nodes, there are much more extensive effects which suggest additional endocrine involvement. Wasting disease and other effects of thymectomy develop after a variable period of time depending on the maturity of the animal at thymectomy. These effects depend also on the condition of the animal and the species, wasting disease occurring in the mouse, rat and guinea pig, but not in the rabbit or lamb. Wasting disease is the result of deficiency of the immune response mecha- 294 CASO Vol. 29 nism with subsequent generalized infection. It can be prevented in thymec-

tomized mice by germ-free conditions (Wilson , Sjopin and Bealmear, 1964), and pathogen-free mice show no evidence of wasting disease (Hess and Stoner, 1966). Antibiotics reduce the incidence of the disease (Azar, 1964), and onset of wasting is accelerated by injection of endotoxin (Salvin, Peterson and Good , 1965). Wasting disease is of course prevented or reduced in incidence or severity by thymus extracts such as thymosin, THE and HTH. To what extent endocrine changes are due to the wasting syndrome and other effects, such as stress, is not clear, but Cosma and Hook (1973) cite numerous organs which are affected by thymectomy. Bone shows increased fragility in the mouse, and cartilage proliferation in the epiphyseal plate ceases in the rat .

Skeletal muscle shows atrophy in the guinea pig. In the rat , shortly after thymectomy, in the anterior hypophysis there is an increase in the number and size of ƒ¿, ƒÀ, and o cells and the appearance of big chromophobes. The posterior hypophysis shows evidence of neurosecretion. In the guinea pig after thymectomy, the epithelium of the thyroid gland increases in height with resorption of colloid from most follicles. The adrenals are increased in size and the gonads show transitory signs of stimulation. Later during the terminal stages of wasting disease many endocrine glands show signs of exhaustion. Immunological Effects: The results observed after neonatal thymectomy indicate that the thymus is unique in its influence on the immunological competence of the lymphoid system. The thymic hormones which control lymphopoiesis also affect the immune response mechanisms. The cell-mediated immune response is impaired concomitantly with the impaired development and reduction in numbers of small lymphocytes in blood , lymph nodes and spleen. The peripheral lymphoid organs eventually degenerate after thymectomy. The ability of neonatally thymectomized mice to reject allogeneic skin grafts has been demonstrated to be reduced significantly (Miller, 1961; Miller, Marshall and White, 1962). This effect was also observed in the chicken. Other cellular mediated responses which have been observed to decrease in intensity after thymectomy are the delayed hypersensitivity reaction, auto- allergic encephalitis, allergic thyroiditis and lethal hypersensitivity reactions . The ability of transplanted lymphoid cells to mount a graft versus host reaction in the allogeneic host has been reduced after thymectomy of the donor animal

(Cosma and Hook, 1973). The humoral antibody responses are variably affected after thymectomy depending on the species of animal and especially on the type of antigen. Moreover, in all these reactions, the longer after birth that thymectomy is per- formed, the less severe and more delayed are the effects. In the neonatally thymectomized mouse, hamster, rat or rabbit the primary immune response has been reduced when the antigen injected was bovine serum albumin, influenza A virus, T2 coliphage or Salmonella typhi H-O or Vi. Con- versely, normal levels of antibody were found when thymectomized mice or rats were immunized with tetanus toxoid, Pneumococcus III capsule polysaccharide, 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND 295 ferritin and others (Cosma and Hook, 1973). The secondary response can be shown to be depressed after thymectomy, but in the case of sheep erythrocytes the primary but not the secondary response is depressed (Cosma and Hook, 1973).

IV. ACTIONOF THYMIC EXTRACTSON THE IMMUNERESPONSE

Humoral Antibodies: Plaque-forming cells (PFC) have been used to quanti- tate the number of cells from a spleen suspension which have been stimulated by antigen injected into the intact animal. The number of colonies to which these cells give rise in vitro in agar is an index of the number of potential antibody-forming cells which have been stimulated. Ceylowski and co-workers used this method to demonstrate the effect of antigen injected into neonatal ICR mice, in which the immune response mechanisms had not yet reached functional maturity. They found that the administration of LSHr to these animals increased the number of PFC to four times that of control animals. Adult animals showed no effect (Ceylowski et al., 1972). Inoculation of neonatal mice with sheep erythrocytes, after treatment with LSHh, also stimulated the formation of antibody-forming cells (PFC) (Luckey, Robey and Campbell, 1973). In two strains of 11-day-old mice, after inoculation with sheep erythrocytes and injection of LSHr, direct lysis of sheep erythrocytes by immune antiserum could be demonstrated (Luckey et al., 1973). Here relatively high doses of LSHr were found to be less effective than lower doses (Robey, 1973). Goldstein and colleagues (1970a), using injections of thymosin Fr. 3, were unable to show PFC or agglutinating antibody after inoculation with sheep erythrocytes. This was true whether the mice used were thymectomized after birth or as adults, or were adults which had been irradiated followed by syngeneic bone marrow transplants. A series of three injections of S. typhi H antigen into 5-day-old thymectomized guinea pigs, together with repeated injections of HTH, produced higher antibody titers than in guinea pigs given only antigen and saline. Moreover, the toxic allergic reaction in the thymectomized guinea pig, inoculated with egg albumin, was restored after HTH administration (Cosma, 1973a). Thymic humoral factor (THF), the extract prepared by Trainin and associates (Trainin et al., 1973), was shown to stimulate PFC from neonatally thymectomized mice after inoculation with sheep erythrocytes. Repeated injection of calf thymus extract gave the limited restoration. Cell-mediated Antibodies: Trainin and co-workers conducted extensive tests with THE and reported that extracts from calf, sheep, rabbit or mouse thymus are all active. The extract from syngeneic or allogeneic mice restored the ability of thymectomized mice to reject skin grafts (Trainin et al., 1973). Trainin et al. (1969) demonstrated that the graft versus host reaction of spleen lymphocytes could be restored by THF. They used an in vitro method 296 CASO Vol. 29

to show increased spleen size in allogeneic grafts (Auerbach and Globerson , 1966). When fragments of (C3H/eb •~C57 B1/6) F1 mouse spleen were grown in culture medium containing C57 B1/6 spleen cells explanted from intact mice , the F1 spleen fragments showed characteristic enlargement after 4 days in culture. If the C57 Bl/6 mice had been thymectomized before the spleen cells were ex-

planted, they were found to have no effect on the F1 spleen fragments and these did not reach the critical increase in growth measurement. When, however, the allogeneic spleen cells (C57 B1/6) from thymectomized mice were incubated for one hour with THF, washed and grown in the F1 culture, the F1 spleen fragments reacted by increased growth. Therefore, THF restored the immunocompetence of the C57 B1/6 spleen cells. Additional graft versus host in vitro experiments were reported by Globerson, Umiel and Friedman (1975) and Trainin et al. (1975).

Essentially the same type of experiment was done in vivo, but allogeneic lymphoid cells from a normal intact mouse which was still immunologically incompetent, when injected into the F1 mouse could cause the graft versus host reaction provided that thymic extract was also injected (Trainin and Small , 1970).

Trainin et al. (1973) reported findings that thymic hormone influences both bone marrow cells and peripheral lymphoid tissue (spleen and lymph nodes) in producing immunocompetent cells which would cause the graft versus host reaction. When bone marrow from a C57BL mouse was incubated with thymic extract, then injected into the (C3H•~C57BL) F1 , allogeneic mouse, spleen suspensions made from the second mouse, injected into another F1 mouse, produced the graft versus host reaction. If the bone marrow was incubated in lymph node extract or culture medium instead of THF, the bone marrow cells were not matured to immunocompetence and the graft versus host reaction did not occur. A variation of this experiment also produced the graft versus host reaction and consisted of incubating immature bone marrow cells from C57BL/6 mice in thymic extract, followed by 24 hr of culture with spleen or lymph node cells from irradiated (C3H•~C57BL/6) F1 mice. This combination of cells and thymic extract, assayed in vitro for splenic enlargement (Auerbach and Glober- son, 1966), produced the graft versus host reaction. However , if the spleen cells or lymph node cells were omitted from the incubation in thymic extract , the reaction did not occur. In this instance, in the maturation of bone marrow stem cells to fully immunocompetent lymphocytes, in addition to thymic extract , peripheral lymphoid cells were required. Thymosin has also been found to influence cell-mediated antibodies . Gold- stein and co-workers (1970a) reported that thymosin, injected three times weekly in the mouse for 9 weeks after thymectomy , brought about rejection of allogeneic skin grafts nearly as rapidly as in intact mice. Results of experiments with thymosin Fr. 3 have paralleled those obtained with THF. Spleen cells from neonatally thymectomized mice , which had been injected with thymosin, produced the graft versus host reaction when implanted 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND 297

into allogeneic mice. This restoration of immunocompetence was partial, 60% of the thymosin-treated animals showing the reaction as compared to 890 of normal intact control mice. None of the thymectomized, untreated mice showed the reaction (Robey, 1973). Thymosin was found to accelerate the development of immunocompetence in spleen cells in normal, neonatal mice after injection of the extract on days 1 and 2 after birth. Spleen cell suspensions from these animals on day 4, when injected into irradiated allogeneic mice intraperitoneally, produced the graft versus host reaction as measured by spleen size of the host. Normally the spleen cells would not have been active until 6 to 8 days after birth (Goldstein et al., 1971). The graft versus host reaction could also be produced when spleen cells, from 3-day-old CBA/wh mice, were incubated with thymosin and injected intraperitoneally into irradiated B6AF1/J hosts. Bone marrow from mature CBA/wh mice, incubated with thymosin in vitro, caused the graft versus host reaction in irradiated B6AF1/J mice after intravenous injection. Normally this reaction does not occur in these mice (Goldstein et al., 1971). Antiserum made to calf thymosin, when injected into normal intact mice, prolongs the time for rejection of allogeneic skin grafts. However, anti-thymus or anti-lymphocyte antiserum (mouse) when injected into normal mice produces a longer delay in graft rejection (Hardy, Quint and State, 1969; Hardy et al., 1968). Therefore, other factors in addition to thymosin must be involved in graft rejection. When thymosin and rabbit anti-mouse lymphocyte antiserum are injected into mice 7 days prior to grafting, there is increased delay in graft rejection (35 days compared to 25 days with anti-lymphocyte antiserum alone). Perhaps thymosin mobilized more target cells which were then more efficiently attacked by the anti-lymphocyte antiserum (Quint, Hardy and Monaco, 1969; Hardy et al., 1969). If thymosin is given after grafting, the effects of anti-lymphocyte antiserum are reduced, the graft lasting 21.2 days. Thymosin partially reverses the antiserum action. The antigenicity of thymosin prompted Kruger, Goldstein and Waksman (1970) to question whether thymosin injections were eliciting an immunological rather than hormonal response, especially if bacteria or endotoxin were present as adjuvants. It would seem that adequate controls, such as lymph node or spleen extracts, would eliminate this possibility, as would care in using only sterile extracts. Especially vulnerable to extraneous antigen is the one-way mixed lymphocyte reaction (MLR), reported by Cohen, Hooper and Goldstein (1975) to have been activated by spleen and liver extracts, and endotoxin (lipopolysaccharide), as well as by thymosin Fr. 8. A further objection is that the thymus gland itself has antigens different from spleen and lymph node, and could therefore be acting immunologically. In the case of cytotoxicity, thymosin antiserum is specific for thymic cells and probably shows no cross-reactivity with spleen or lymph node lymphocytes (Hardy et al., 1968). However, recent findings 298 CASO Vol. 29

that thymosin stimulates synthesis of T cell antigens on the surface of non- immunocompetent cells argues against this objection (Komuro and Boyse, 1973b).

V. THE B- AND T-CELL SYSTEMS OF IMMUNOCOMPETENCE

Glick and others found in the chick that removal of the thymus in early life brought about impairment of cell-mediated immunity (CMI), whereas removal of the bursa of Fabricius was followed by impairment of the humoral antibody response (Glick, Chang and Jaap, 1956; Warner, Szenberg and Burnet, 1962). Miller and Osaba (1967) found that removal of the thymus in the mouse also caused impairment of CMI as well as the impairment of the humoral antibody response. Claman, Chaperon and Triplett (1966) postulated that antibody is produced by bone marrow cells, while thymus lymphocytes act as necessary auxiliaries. Nossal et al. (1968) demonstrated in tissue culture the distinction between thymus-derived lymphocytes (T cells), which control CIM responses, and bone marrow-derived lymphocytes (B cells), which control humoral antibody responses, with the co-operation of T cells. Macrophages are necessary at some stage in both types of reaction. The precise number of steps between the uncommitted stem cell and the development of the mature, immunocompetent T cell is unknown. The first step involves the modification of the stem cell to the T-cell pathway (prethymic cell), which can develop under the influence of thymic factors into •gimmature•h thymus cells. These have high ƒÆ density and TL+ markers on the cell surface. •g Mature•h thymus cells, which have low ƒÆ density and TL- markers, can derive either from the •gimmature•h thymus cells or directly from the committed prethymic cells. The •gmature•h cells proceed to post-thymic stages to become antigen-

sensitive cells. B cells also arise from stem cells in the hematopoietic tissue

(in the bird in the burst of Fabricius, in the mammal perhaps in the bone marrow or the lymphatic tissue of the large intestine). Lymphocytes in the bursa of Fabricius first acquire IgM on the cell surface, followed by the ap-

pearance of IgG. Some lymphocytes bear both IgM and IgG simultaneously. Both incompletely matured B cells and T cells are •gseeded•h to the peripheral lymphoid organs. In the peripheral lymphoid tissue further maturation occurs on exposure of the lymphocytes to antigens at their specific receptors, i.e., the cells divide and differentiate to produce antibody in the case of B cells, or factors which cause CMI and •ghelper•h activity in the case of T lymphocytes. The mechanisms governing this evolution are complex and at present controversial

(see Miller, 1975; Claman, 1975; Raff, 1974). In the mouse, the stem cells from which B and T lymphocytes develop are believed to arise from large basophilic cells (haemocytoblasts) in the yolk sac

or embryonic liver. Lymphopoiesis in the thymus gland depends on population of the epithelial anlage by the migrating stem cells. Owen (1972) places the time of entry of stem cells at 11 days of gestation approximately, the 12-day mouse thymus being fully lymphoid and the 10-day mouse thymus having no 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND 299

differentiating lymphocytes. At this time the large basophilic cells are seen, having large nuclei and an electron-dense cytoplasm due to large numbers of polyribosomes. After proliferation, during the transition from large basophilic cells to small lymphocytes, the small cells modify their plasma membrane to produce new antigens. These are demonstrated by inoculating the lymphocytes into the allogeneic mouse, producing antiserum, and demonstrating its cyto-

toxicity to the thymic lymphocytes. In this way it has been found that the small lymphocytes gain Thy-1 (ƒÆ) and TL antigens (Reif and Allen, 1966; Aoki et al., 1969). In tissue culture, thymus from the 14-day embryo has been shown to contain the large basophilic cells which are negative for Thy-1 (ƒÆ) and TL antigens. After two days in culture a proportion of the medium and small lymphocytes are seen to possess Thy-1 (ƒÆ) and TL antigens, while after four

days' culture the majority of the lymphocytes are Thy-1 (ƒÆ) and TL positive

(Owen and Raff, 1970). This is the same sequence seen in vivo, but tissue culture eliminates the possibility of new cells coming into the thymus from the circulation. Marker studies show that these thymus-derived or T lymphocytes migrate to certain areas of peripheral lymphoid organs, where they may undergo further maturation, because they are less reactive for Thy-1 (ƒÆ) antigen. Aoki et al. (1969) have demonstrated that these T lymphocytes quantitatively have less Thy-1 (ƒÆ) antigen on their cell surfaces. These more mature T lymphocytes have lost reactivity for TL antigen. The same kind of maturation of T cells can be seen within the thymus itself. In the thymus the majority of the lymphocytes are cells with high density Thy-1 (ƒÆ) antigens and which possess TL antigen (in TL+ mice). These cells

are sensitive to corticosteroids and are not immunocompetent. However, depending on the mouse strain, from 3 to 10% of the thymic lymphocytes are less sensitive or resistant to corticosteroids, are immunocompetent, have much lower surface density of Thy-1 (ƒÆ) antigens and have lost TL+ antigens (Blomgren and Andersson, 1970). These are probably located in the medulla (Oven, 1972). It is at present unclear whether the •ghigh ƒÆ•h TL+ cells are thee direct precursors of the •glow ƒÆ•h TL- cells (Miller, 1975). In addition, T cells undergo further maturation to fully competent, antigen-sensitive lymphocytes as the cells pass through •gpost-thymic•h stages and populate the peripheral lymphoid tissue (Mil- ler, 1975). A thymic humoral factor has been implicated in this final maturation

(Stutman, 1975). The highly immature •gprethymic•h cells are also present in the peripheral lymphoid organs. B lymphocytes generally have been assumed to arise in bone marrow in mammals, in analogy to the bursa of Fabricius of birds. Even less is known about their maturation than is known of the maturation of competent T cells. Both B and T immunocompetent lymphocytes can recognize and respond to antigens in a highly specific manner. When fully differentiated, bath B and T lymphocytes express specific receptors on their cell surfaces, each cell being committed to a single antigen, or possibly a small group of antigens. In the case of B lymphocytes there is evidence that the receptor is an immunoglobulin 300 CASO Vol. 29

molecule (Singhal and Wigzell, 1971), and the specific antigen stimulates the cell to secrete this specific immunoglobulin (antibody). The actual signal for activation may be a structure other than the Ig receptor (Moller and Coutinho, 1975). The receptor on the T lymphocyte cell surface is uncertain, but it reacts specifically to recognized antigen, producing non-specific factors (lymphokines), which mobilize macrophages and other elements seen in the cell-mediated responses (Raff, 1974). While B and T lymphocytes can be distinguished immunologically, morphologically they have been found to be indistinguishable by the light microscope, transmission E/M and probably scanning E/M (Alexan- der and Wetzel, 1975). The two cell systems, however, are not exclusive of each other but rather in part are complementary. That is why thymectomy in the mouse impairs the humoral response to sheep erythrocytes. Antigens of this type are thymus- dependent and need the intervention of T lymphocytes to produce antibodies. Other antigens are able to produce the normal antibody response in thymectomized animals and are said to be thymus-independent. These include pneu- mococcal polysaccharide, dextran, levan, lipopolysaccharide (endotoxin), etc. They are polymeric antigens which elicit IgM antibody almost entirely. In contrast, the highly T-cell-dependent antibodies consist of high affinity antibody (IgG or IgE), compared to T-cell-independent (or marginally dependent) antibody which is low affinity antibody (IgM) (Miller, 1975). There is evidence, however, that some thymus-independent antigens can elicit IgG antibody without T-cell intervention. In the mouse, there are surface antigenic differences in lymphocytes which migrate from the thymus to lymph nodes or spleen, and it is possible to speak of •gspleen-seeking•h or •glymph node-seeking•h lymphocytes. Among various factors, antiserum to Thy-1 (ƒÆ) and TL produces more inhibition to cells migrating to the spleen than to those migrating to lymph nodes, while anti-H-2 locus antiserum inhibits the cells migrating to lymph nodes more strongly than to spleen. All of the five factors tested show considerable overlapping in the peripheral distribution of lymphocytes (Schlesinger, Schlomai-Korzash and Israel, 1973). Action o f Thymic Extracts on the Maturation o f T Lymphocytes: The research of Reif and Allen (1963, 1966), Boyse et al. (1968) and Aoki et al. (1969) have established that the T lymphocytes of the thymus have the characteristic surface antigens Thy-1 (ƒÆ), TL and Ly, obtained by cytotoxicity tests with appro- priately absorbed antisera to the mouse genotype. Komuro and Boyse (1973a, 1973b) showed that spleen cells and bone marrow cells, as well as cells from liver of 14-day embryos, contain a proportion of cells which are low in reactivity for these antigens. After incubation of these cells in thymosin (Goldstein et al., 1972) for 3 hr they gave a higher expression of the antigens, showing increased maturity from prethymic cells to T cells. Cell division is not involved because the time interval is short. Extracts of spleen, liver and kidney were without effect. That thymosin action is an active process and not simply a passive altera- tion of the cell surface is shown by the effect of cyclohexamide added to the 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND 301 thymosin-prethymic cell mixture. An inhibitor of protein synthesis, cyclohexamide also inhibited the expression of T antigens on the surface of these cells. Therefore, it is indicated that the antigens must be synthesized by the prethymic cells after thymosin treatment (Komuro and Boyse, 1973b). Thymic extracts are, however, regarded as signaling the development of cells which had already been genetically determined to the T cell-line. This is shown by the action of other agents, such as cyclic-AMP or polyadenylic uridylic acid, in activating expression of Thy-1 (ƒÆ) or TL antigens on prethymic lymphocytes (Scheid et al., 1973). Cyclic-AMP may be acting as an intermediary in thymic extract stimulation of T antigens (Scheid et al., 1975). The findings of Komuro and Boyse (1973a) have been confirmed by Miller and Esselman (1975), who treated bone marrow cells, separated by density diffusion gradients, with thymosin Fr. 3. One-hour incubation was sufficient to convert these prethymic cells to those expressing Thy-1 (ƒÆ) antigen in two strains of mice. These T-cell precursors were found to be in the lower density region of the density gradients. This cytotoxicity test has been used as a bioassay for thymosin (Fr. 8) (Hooper et al., 1975). Bach and Goldstein (1975) used thymopoietin to stimulate expression of TL and Thy-1 (ƒÆ) antigens in prethymic cells. Bone marrow from A/J mice was incubated 6 hr with either thymopoietin I or II, or 18 hr with thymopoietin I, after which the treated prethymic cells were used to absorb anti-TL antibodies and anti-Thy-1 (ƒÆ) antibodies from the respective antiserum. When bone marrow from A•TL- mice, which are genetically negative for TL but positive for Thy-1 (ƒÆ) antigens, was treated with thymopoietin, the prethymic cells gave expression to the Thy-1 (ƒÆ) antigen only. A specific antigen for human thymic lymphocytes has been detected by anti-human T-cell serum (ATCS) (Touraine et al., 1974), an antiserum made to peripheral blood lymphocytes from a patient with agammaglobulinemia and absorbed with human erythrocytes, B cells from lymphocytes of chronic lymphatic leukemia and from a lymphoblast cell-line with high density surface Ig. The T-cell antigen detected in this way was called human specific T lymphocyte antigen, HTLA. Touraine and associates (1975), using human bone marrow cells separated by density diffusion gradients, treated prethymic cells with human and calf thymic extracts as prepared by Goldstein et al. (1972). After 2-hr incubation in thymosin, the HTLA marker was expressed by the prethymic lymphocytes. It should be noted that cells treated with the less purified fraction from spleen control extract also showed some activity in expressing the HTLA marker.

VI. INHIBITORYACTION OF THE THYMUSGLAND Evidence from experiments with rodents indicates that the thymus acts to suppress tumors of viral carcinogen origin (Defendi, Roosa and Koprowski, 1964; Law, 1965; Allison and Taylor, 1967). Thymectomy has been found to inhibit the immunization of mice against polyoma virus-induced tumor formation, while 302 CASO Vol. 29 antibody titers against the virus remain at normal levels. Law and Ting (1965) concluded that a thymic humoral factor must therefore be active against a cellular antigen of polyoma virus. Natural resistance to tumor virus (adenovirus 12) was overcome by neonatal thymectomy (Kirschstein, Rabson and Peters, 1964), and natural resistance to polyoma virus was overcome by thymectomy followed by irradiation (Defendi and Roosa, 1964). The results with chemical carcinogens have been variable. Neonatal thymectomy has been reported to enhance the carcinogenic effects of 20-methylcholanthrene (Grant and Miller, 1962; Nishizuka, Nakakuki and Usui, 1965), and 3,4-benzopyrene (Miller, Grant and Roe, 1963). In contrast, after thymectomy no differences were found in the effects of 3-methylcholanthrene (Law, 1965) or 7,12-dimethylbenz(a) anthracene (Allison and Taylor, 1967). Thymectomy has been reported to have positive effects in promoting resistance to chemical carcinogens (Prehn, cited by Metcalf, 1966), and to mammary tumor virus (Martinez, 1964). The effects of thymectomy in suppression of mammary tumor virus have been found to be influenced by the strain and breeding of the test animals (Squartini, Olivi and Bolis, 1970). Thymectomy prevents lymphoid leukemia in the mouse, and in thymectomized animals a thymus graft can restore susceptibility to leukemia, whether spontaneously re-occurring or induced by virus, chemical carcinogen or irradiation (Metcalf, 1966). It is possible that the virus may interfere with the controls of epithelial and mesenchymal reticular cells over the differentiation and proliferation of lymphocytes (Furth et al., 1966). Szent-Gyorgyi, Hegyeli and McLoughlin (1962) separated two fractions from an unpurified thymus extract and found one to be stimulative, the other inhibitory, to tumor growth. A purified fraction of thymus lipid which is stimulative to tumor growth was described by Potop and Milcu (1973). In all of these effects attributed to the thymus gland, two functional aspects of thymic T cells should be kept in mind. Nossal (1974) demonstrated in tissue culture that T cells, activated by allogeneic cells or by cells containing a tumor- associated transplantation antigen (TATA), became cytotoxic to the specific allogeneic cells or the TATA cells. On the other hand, recent evidence indicates that in addition to the •ghelper•h effect of T cells in the humoral immune response of B cells, subpopulations of T cells have been shown to be immunosuppressive, i.e., to inhibit lymphocyte stimulation by mitogens or allogeneic cells (MLR) (Bash, Durkin and Waksman, 1975; Folch, Yoshinaga and Waks- man, 1973). Moreover, extracts from antigen-primed T cells (suppressive T-cell component) have also been shown to suppress the humoral antibody reaction

(Tada, 1975). These diverse actions could account for the various effects of the thymus in experimental systems designed to evaluate thymic regulation of cell growth. Growth Inhibitory Factor from Thymus Gland: Potop and Milcu (1973) found that total lipid extract from the thymus gland had strong antitumorigenic activity against methylcholanthrene-induced tumors in rats. Fractionating the 303 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND extract from calf thymus, these workers isolated several components, some of which were antineoplastic in varying degrees and one of which was stimulative to tumor growth (Fr. IIIB). The inhibitory antitumor fractions were found to reduce the incidence of tumors with the Walker tumor in thymectomized rats; to reduce the weight increase in Ehrlich ascites tumor in rats; to increase survival time of mice with Ehrlich tumor transplants. A highly purified fraction of the thymic lipid extract was obtained by thin layer chromatography and called by Potop and Milcu fraction S or thymosterin. Tested on KB tumor cells in tissue culture, thymosterin decreased proliferation of the cells and decreased cell population and protein concentration. A less purified fraction was found in the mouse to stimulate humoral antibody formation, to induce a rise in hemoglobin and a rise in total leukocyte count in peripheral blood. Thymosterin is known to be a steroid, but its relationship to the other thymic extracts is at present unknown. Its possible action on the maturation of T cells in its participation in the immune response would seem to be a vital area for future research.

VII. CELLULARORIGIN OF THYMIC HORMONES

The data from experiments on the thymus gland leave no doubt that this organ has vital and extensive effects on the lymphoid system. More recent studies of purified thymic extracts support the hypothesis that an important measure of thymic humoral control is exercised over the functioning elements of the immune response and the maturation of functional lymphocytes. Other, non-lymphoid effects of thymic extracts, such as that of Mizutani (Mizutani, 1973; Mizutani et al., 1975) which lowers serum calcium, and the action of thymopoietin on neuromuscular transmission (Goldstein and Manganaro, 1971), suggest a most widespread influence of the thymus gland. This extends even to effects on other endocrine glands. It is known that the thymus antagonizes the action of ACTH on the adrenal cortex, and acts synergistically with growth h nrm nn e in enhancing the proliferation of lymphoid tissue (Cosma, 197 3b) . Still unknown is the cellular origin of the thymic humoral factors. An experiment of Goldstein (1975), as yet in the tentative stage, utilized antiserum to thymopoietin to show its localization in the thymus of man, guinea pig, calf and other species. Indirect immunofiuorescence localized thymopoietin in the epithelial cells of the cortex with one antiserum, and in the epithelial cells of the medulla with another. There is good evidence that thymic lymphopoiesis is correlated with the uptake of 35S in the epithelial cells of the mouse thymus. Radioautography showed rapid incorporation of 35S into mucoid vacuoles and colloid follicles of the epithelial cells of the medulla. Moreover, gel filtration chromatography demonstrated that 35S was incorporated into a macromolecule which was pre- sumed to be a mucoid epithelial secretory product (Clark, 1968). 304 CASO Vol. 29

More indirect is the evidence presented by Dardenne et al. (1974) who studied a serum thymic factor present in the circulation of the normal mouse and which confers immunocompetence on lymphocytes (Back and Dardenne , 1972; Bach and Dardenne, 1973). Thymectomy causes its disappearance in a few hours. Using a pure population of thymic epithelial cells in millipore diffusion chambers, this thymic factor could be restored by transplants into thymectomized mice. The histological appearance of the thymic graft had the configuration characteristic of epithelial cells. These results were repeated with a transplant of non-lymphoid epithelial thymoma. Later work , however (Back et al., 1975), indicated that the thymic serum factor could be reintroduced into thymectomized mice by injection of large numbers of dissociated thymocytes , which would contain both epithelial and lymphoid cells, the contribution of each being impossible to separate. The activity of thymus reticular epithelial cells has been studied by Wekerle , Cohen and Feldman (1973). These workers cultured lymphocyte-free thymus reticular cells in tissue culture with non-immunocompetent spleen lymphocytes . The immature cells became sensitive to Concanavalin A, as shown by increased DNA synthesis monitored by thymidine uptake. Waksal et al. (1975) again grew thymic epithelial cells, devoid of lymphoid elements, in tissue culture with immunodeficient spleen cell suspensions. The lymphocytes were shown to mature by proliferation with Concanavalin A and 3H-thymidine uptake and the ability to mount the graft versus host reaction. These results were independent of difference in species between the reticular epithelial cells and spleen cells, the former being either from the syngeneic mouse or the xenogeneic (Lewis rat) thymus. Epithelial cells were also implicated in T-cell maturation by the studies of Potworowski et al. (1975), who used an absorbed antiserum made to reticuloepithelial cells of chicken thymus. Injected into embryonated eggs, this substance severely depleted thymus cortex of small lymphocytes , which were replaced by large, undifferentiated cells. The percentage of thymus lymphocytes bearing "T" antige n was reduced markedly. ECM sections showed cytoplasmic disorgani - zation of the reticuloepithelial cells of the thymus medulla . The stromal elements of the thymus would appear to be implicated directly in the maturation of T cells and perhaps in the secretion of thymic hormone . The role of the thymic epithelial reticular cells and the mesenchymal reticular cells has not been defined, although Wekerle , Cohen and Feldman (1973) observed both PAS-positive and PAS-negative cells in their reticulum cultures . The E/M micrograph studies of the reticulum cultures show highly active, secretory type cells (Waksal et al., 1975).

VIII. ROLE OF THE THYMUS IN NEOPLASTIC DISEASE

The thymus gland has often been assumed to be related to the development of malignancy, but until recently the experimental methods for testing this 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND 305

hypothesis have not been available. In particular the involution of the thymus from the size attained at puberty to the decline seen in middle life and old age has been observed to coincide with the increased occurrence of neoplasms in later life. In addition, congenital, hypoimmune diseases have been associated with significantly increased incidence of neoplasms, some of which have been out- lined by Good (1970). Data from immunosuppression therapy for organ transplantation have shown a similar significantly increased occurrence of malignant tumors (Good, 1970; Nossal, 1974). The theory of immune surveillance has attempted to account for these phenomena and, simply stated, proposes that all cells of the body are monitored for changes in antigenicity (tumor-associated transplantation antigens, TATA) and destroyed by the body's immune mechanisms when discovered, thereby preventing clones of abnormal cells from developing into malignant cells and finally into a tumor. Impairment of this mechanism would result in the development of neoplasms. Since the thymus is the focal gland for the development and functioning of the immune responses, with a direct role in cell-mediated immunity, there has been renewed interest in discovering its part in the origin or prevention of neoplastic disease. With the identification of lymphocyte cell membrane markers, which facili- tate the separation of human T and B lymphocytes, new information has been accumulating concerning the problems relating to tumor immunology. In the application of these methods the number of experiments and human subjects studied has necessarily been limited, and therefore caution should be observed in generalizing from the results. The methods themselves bear examination, but their refinement and wider application should throw additional light on both the theory of immune surveillance and the part the thymus plays in neo- plasia. The detection of B lymphocytes has been accomplished by the methods of Froland et al. (1971), Unanue et al. (1971), Dickler and Kunel (1972), and Holm and Wigzell (1972). T lymphocytes have been detected by the methods of Lay et al. (1971), Froland (1972), and Aiuti and Wigzell (1973). Thymus in Relation to Immunity in Aging: The human thymus gland is largest in relation to total body size during foetal life and this continues till the end of the second year of postnatal life. Thereafter the thymus continues to grow at a slower rate till puberty, at which time it weighs 30 to 40 g. After puberty involution begins, and then it slowly declines in size through old age. It is a misconception that the thymus completely atrophies in later years, because it remains a substantial organ through middle and old age (Boyd, 1932). Severe disease causes atrophy in 24 hr, and therefore estimates of size based on post mortem or cadaver material have fostered the belief in complete thymic atrophy as a simple result of aging. Nevertheless it is true that the incidence of malignant tumors increases with age, and there is some evidence that at least part of the immune response mechanism operates at lower levels in later years. Waldorf, Willkens and Decker (1968) studied delayed hypersensitivity in 116 individuals aged 59 to 98. The challenge dose of 2,4-dinitrochlorobenzene (DNCB) showed sensitization in only 306 CASO Vol. 29

84 subjects (72%), significantly lower than in younger individuals. In those over

70 years of age sensitization was lower still (69%). Sixteen of the non-reactive subjects could still react to the tuberculin skin test, perhaps showing that pre- vious delayed hypersensitivity persists despite anergy to the new antigen (DNCB). Similar results were obtained by Gross (1965), who used the antigen with fewer

subjects. The influence of the thymus during aging can be seen from a study by Augener et al. (1974) who measured the absolute number of T lymphocytes detected by the rosette-forming technique. Peripheral blood at ages 3 to 12 contained a mean of 2,490 T lymphocytes per mm3 while at ages 67-83 the count was 1,250 per mm3. There was no significant change in absolute num- hers of B lymphocytes. It is of interest that Bach et al. (1972) could detect

a thymic serum factor which was higher in children than adults and absent in normal subjects over 50 years of age. These workers mention the need for more information and a test system with a better rationale before drawing definite conclusions. Studies of Aging Mice: The incidence of neoplasms in mice reared in the laboratory has shown that most of the tumors occur after 18 months of age, at which time the normal mortality for females is 50%. Normal 50% mortality for males occurs at 16 months. Tumor incidence in this mouse strain was 54% for the males and 75% for the females (Homburger et al., 1975). In wild house mice, spontaneous tumors are infrequent till over 2 years of age, tumors occurring in 28 of 293 mice, murine Gtype virus being found in most of the lymphomas (Gardner et al., 1973). Studies of long-lived inbred mice showed that the F-1 hybrids lived longer and had more tumors than the isogenous

parental strain, and that tumors tended to occur at a later age (Smith, Walford and Mickey, 1973). Spleen cells from aging mice, as seen by the number of

plaque-forming cells in vitro, have been found to be less immunocompetent than those from young mice. Moreover, when spleen cells from older mice are mixed with those from young mice, the normal lymphocytes undergo immunosuppression and show a limited response to antigenic stimuli in vitro (Jaroslow et al., 1975). In the mouse mutant (nu/nu) which is athymic and hairless (nude), longe- vity does not seem to be related to increased tumor incidence. Of 1,100 nude mice, no malignant neoplasms were observed up to 3 months of age, the normal life expectancy (Rygaard and Povlsen, 1974). The relatively short life span

would seem to complicate comparisons with normal mice, making conclusions about the thymus in immune surveillance uncertain. The Thymus in Relation to the Neoplasms of the Immune System: Some evidence has been presented in recent years that neoplasms associated with the lymphatic system can be divided into those of B-cell and T-cell origin. Lukes and Collins (1975) list three lymphomas as possibly belonging to the T-cell type: Hodgkin's disease, mycosis fungoides and Sð¥zary's syndrome. These authors note several pertinent facts concerning Hodgkin's disease. 307 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND

There is frequent delayed responsiveness to hypersensitivity, delayed graft rejection and depletion of lymphocytes as the disease progresses. The initial foci of involvement in lymph nodes are in the paracortical region, normally occu- pied by T-cells, showing the selectivity of Hodgkin's disease for thymic-dependent lymphoid tissue. B-cell neoplasms would derive from the centers of follicles of lymphoid organs, the region where B-cells are concentrated. Order and Hellman (1972) theorize that in Hodgkin's disease T-cells are altered antigenically by virus, while other unaffected, immunocompetent T-cells within the same lymphoid organ react against the altered cells, setting up a chronic immune reaction resembling the graft versus host reaction. However, Denton (1973) studied immune responsiveness in Hodgkin's disease and found mostly IgG immunoglobulin on the surface of lymphocytes, depending on the stage of the disease. Of 17 patients investigated for cell-mediated immunity (by allogeneic cell stimulation in vitro), none showed impairment of cell-mediated immunity. Brouet, Flandrin and Seligman (1973) studied six patients with Sezary's syndrome using specific antiserum of T lymphocytes and the rosette-forming test for T lymphocytes. No membrane-bound immunoglobulin was detected on abnormal cells. Lymphocytes showing membrane-bound immunoglobulin were counted as low as 1 to 4%. Specific anti-T cell antiserum was cytotoxic to 54 to 88% of the mononucleated cells in three patients, while rosette formation was seen in 65 to 90% of the mononucleated cells in five patients. Cooper and colleagues (1975) studied 22 patients with lymphoproliferative disease using standard methods for separating and identifying B and T cells (Special Technical Report, 1974). They identified T cell lymphomas in 5 patients. Three other patients were unclassifiable and the remainder showed lymphomas and chronic lymphatic leukemias of B cell origin. These results were from cells from lymph nodes, while lymphocytes from peripheral blood gave inconclusive results. Leech et al. (1975) studied follicular center cells in lymphomas and found B lymphocytes in the region. The authors concluded that their findings sup- ported the theory that many lymphomas are derived from follicular center cells. They believe these immunological techniques to be valuable methods for identifying lymphomas when used in conjunction with histochemical, ultrastruc- tural and histological examination. The Thymus in Relation to Tumor Growth in Experimental Animals: While depression of the immune response in man has been observed in association with the growth of neoplasms, as in the observations of Gross (1965), Papatestas and Kark (1974) and Sharma and Terasaki (1974), there is in addition much evidence to suggest that normal immune surveillance by T cells is impaired by neoplasms in experimental animals. Athymic or nude mice normally lack the thymus and as a result do not possess mature T cells, although normal thymic precursor cells are present in their bone marrow. Predictably, they are more susceptible to transplanted tumors than normal mice, and several studies have shown nude mice to be 308 CASO Vol. 29

fitting subjects for the growth of human malignant cell lines (Giovanella , Stehlin and Williams, 1974). T cells have been implicated in many of these studies, such as the experiments with Moloney sarcoma virus-induced tumor (Davis, 1975) in which the tumors were found to grow progressively in nude mice while they regressed in 53% of their hairy (thymic) litter mates. IgG antibody was detected in all the normal mice which showed tumor regression but in only 2 of the athymic mice at very low titer. The antibody in this case is judged to be T-cell dependent. Another study (Schmidt and Good, 1975) with a human tumor cell line (HUTU-80), which invariably grows in athymic mice, demonstrated that thymus grafts in these mice caused regression of the tumor , and if implanted before challenge with the tumor cells could effectively cause resistance to tumor growth. It should be borne in mind that the state of the tissue affects the success of the transplant in nude mice as in other strains . Surgical explants from human tumors give rise to slow-growing, non-invasive and non-metastasizing tumors in certain experiments, while human cultured cell lines which show normal growth patterns do not produce tumors (Giovanella , Stehlin and Wil- liams, 1974). This is perhaps an indication that there are other mechanisms operative besides those mediated by T-cell immunity . When an anti-thymocyte antiserum was injected into thymic mice, it was found that 13 human tumor cell lines (carcinomas and sarcomas) could be transplanted successfully into the mice to form neoplasms (Arnstein et al., 1974). Here again primary tumor explants were relatively unsuccessful in producing tumors when transplanted into these mice. T-cell association with tumor growth can be seen in immunosuppression caused by growth of mammary tumor in C3H/He mice. Those immune mechanisms which have greatest T-cell dependence were found to be depressed the most, i.e., the antibody (7S) response to horse erythrocytes was found to fall below control levels in 2 weeks. The thymic-independent response to lipopolysaccharide was not affected. These results are confirmed by the histologic findings that the cortical areas of lymph nodes and the thymus gland showed atrophy in the tumor-bearing mice (Rowland et al., 1973). The thymus has been implicated in suppressing metastasis in the 3LL Lewis tumor (a line derived from spontaneous lung tumor in the C57BL mouse). If tumor-bearing mice are thymectomized, the number of metastases increases significantly. Conversely, in thymectomized mice, thymus grafts or injection of thymic cells is found to reduce the number of metastases (Carnaud, Hoch and Trainin, 1974). Other studies have linked thymus-derived cells to the establishment of immunity to tumors, but there have been exceptions. Whitney, Levy and Smith (1975) found that T lymphocytes and not B lymphocytes were responsible for anti-tumor effects against a methylcholanthrene-induced rhabdomyosarcoma, because lymphocyte activity could be abolished by anti-BOO antiserum . Leclerc and coworkers (1973), also by use of anti-O antiserum, concluded that T-cells were the effector cells against the murine sarcoma virus-induced tumor . The 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND 309

antiserum was cytotoxic in vitro against the lymphocytes immunized by the tumor. Gorczynski (1974), using anti-e antiserum against the immunized lymphocytes, found that T lymphocytes were responsible for protection in vivo against the tumor induced by murine sarcoma virus. At variance with these findings are those of Lamon et al. (1972) who concluded that non-thymus-derived lymphocytes were responsible for the immune reaction against the Moloney sarcoma

virus-determined tumor, and Owen and Seeger (1973) who tentatively implicated macrophages in a non-specific reaction against the same type of tumor. Immunosupibression and Immune Surveillance: The theory of immune surveillance is difficult to prove or disprove directly because of the many vari- ables and unknown processes which it presupposes. One experimental approach is to attempt to simulate the beginnings of a neoplastic growth by administering small injections of tumor cells which have low antigenicity, or by injecting very low concentrations of chemical car-

cinogen. If immune surveillance exists, it would be expected to detect minute changes in antigenicity of cells developing in response to these procedures. The incipient neoplasms would then be eliminated by the immune reactions of the host. This is essentially the experiment devised by Andrews (1974). This worker found that the primary immune response did not prevent tumor growth, and hence immune surveillance failed to be demonstrated. The assumption which must be made in such experiments is that the

physiological conditions under which neoplasms develop have been duplicated. Certainty concerning these conditions is impossible at present. Moreover, as Kripke and Borsos (1974) indicate in their consideration of the problem, unknown immune processes may exist, which our present methods, based on

standard laboratory procedures, would not detect. Therefore immune surveillance remains an elusive concept in the normal control of cell growth. It would seem, assuming that the protection against tumor formation is of

the immune type (and it need not be exclusively so), that there is an essential conflict encompassed in the body's immune reactions. We tend to assume that these reactions normally do not operate against the isogeneic cells of the organism, yet this very possibility always exists, as attested by the ample evidence for autoimmune reactions and autoimmune diseases. If this is the case, there must be an area in which there is a critical balance in withholding or applying

the immune response against the body's own cells when there is antigenic change. It is possible that there is another control, an immune reaction •gover- ride,•h that is capable of suppressing the immune reaction in order to protect the integrity of the body's own organs. Errors in the suppression of immunity would allow escape and growth of malignant cells. There is some evidence that the thymus gland is associated with suppression

of the response of lymphocytes at some point after their stimulation by antigen. This evidence comes from the action of suppressor T cells in inhibiting certain

phases of the immune reaction (Bash, Durkin and Waksman, 1975; Tada, 1975) and also from the action of certain extracts from the thymus gland and from 310 CASO Vol. 29

the plasma of normal individuals. Thymic Humoral Factor: The action of THE in eliciting the immunocompetence of lymphocytes in both humoral and cell-mediated immune reactions has been discussed in an earlier section. Carnaud et al. (1974) investigated the ability of certain lymphocytes to become autosensitized in vitro by syngeneic cell monolayers. These sensitized lymphocytes then exhibited a dual function: they were cytotoxic to syngeneic tumor cells in vitro, but injected in viva they caused the enhancement of growth of syngeneic tumorcells. There is some evidence that the lymphocytes are T cells. When the lymphocytes were incubated with syngeneic fibroblasts in the presence of THF, and subsequently grown in vitro with syngeneic tumor cells (3LL tumor from C57BL mouse), they showed reduced cytotoxicity toward the tumor cells. Hence there is an experimental model which indicates that THE prevented early recognition of syngeneic antigens by lymphocytes with subsequent lymphocyte triggering when grown with syngeneic cells. Alpha Globulins: Kamrin (1959) found that a crude extract of rat blood when injected into mature rats prolonged allogeneic skin grafts. Mowbray (1963), using a partially purified fraction from ox blood, and Mannick and Schmid (1967), using human a globulin, confirmed that these substances can prolong skin allografts. Mowbray (1967) found that ribonuclease conjugated to protein would cause immunosuppression of the primary and secondary immune responses. Ribonuclease alone was without effect. Milton (1971) found that a-2 glycoprotein, which had ribonuclease activity, would inhibit human lymphocyte proliferation in response to phytohemagglutinin (PHA), PPD and allogeneic cells. Glaser, Cohen and Nelken (1972) and Glaser, Ofek and Nelken (1972) demonstrated that a globulin from human plasma would prolong skin allograf t survival, depress antibody production to sheep erythrocytes, lessen the ability of mouse spleen cells to confer immunity on irradiated isologous recipients, and reduce phagocytic activity in peritoneal leukocytes. Alpha globulin was found not to be cytotoxic and since it could be washed from lymphocytes was concluded to be loosely bound. These activities were accomplished by concentrations of a globulin 20 to 120 times the normal serum concentration, and were seen as possibly operative in diseases in which its serum concentration is known to rise. Cooperband et al. (1969) concluded that the a globulin from serum acted in suppressing PHA stimulation of lymphocytes by competitive blockade of recognition sites at the earliest events of immune responsiveness. Inhibition of the macrophage migration-inhibition factor was reported. These conclusions were not confirmed by later work on thymus a globulin extract (Carpenter, Phillips and Merrill, 1971). Alpha-2 globulin has been extracted from bovine thymus and has been found to have actions similar to the serum extract: suppression of the hemagglutinin response to sheep erythrocytes and suppression of lymphocyte proliferation and DNA synthesis induced by PHA in vitro (Carpenter, Boylston and 311 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND

Merrill, 1971). The thymic extract was obtained by the same method which was used by Cooperband et al. (1969) in preparing Fraction C from serum. Thymus a globulin has been found to prevent DNA synthesis in PHA-stimulated lymphocytes but does not prevent blast formation and it allows synthesis of

RNA at the time of DNA suppression. There is no blockade of the triggering device for lymphocyte stimulation or competition with PHA for antigenic sites on the cell surface (Carpenter, Phillips and Merrill, 1971). The globulin extract from the thymus has been found to be the same

functionally as that from serum (Fraction C). An important dose relationship exists for a globulin suppression of DNA synthesis in PHA stimulated lymphocytes. Larger doses in vitro (150 ƒÊg/ml) were found to suppress DNA synthesis, but smaller doses (50 ƒÊg/ml) were found to augment DNA synthesis after PHA

stimulation. The same relationship holds for the mixed lymphocyte reaction,

greater proliferation of cells resulting from lower a globulin concentrations, depressed proliferation at higher concentrations. Rosettes with sheep erythrocytes were found to be unaffected by a globulins (Phillips, Carpenter and Lane, 1975). The response of lymphocytes to PHA and the mixed lymphocyte reaction is believed to be due to T lymphocytes exclusively. Hence there is a possible mechanism for augmenting or suppressing cell-mediated immunity depending on the concentration of a globulins in the area. Other Immunosuppressive Thymic Fractions: Another thymic extract has

been isolated, by the method used for extracting chalones, by Kiger, Florentin and Mathe (1973) and appears to be an acidic protein rather than a-2 glyco-

protein. This fraction is suppressive toward: lymphocyte transformation by PHA, the graft versus host reaction, skin allograft rejection and the formation of hemolysins against sheep erythrocytes. The first three reactions involve T cells, while the last reaction is accomplished by B cells with co-operating T cells. The existence of this factor in blood plasma has not been explored. Histones extracted from the thymus are also found to be immunosuppressive (Pelletier and Delaunay, 1970).

IX. SUMMARY*

In studies of the mouse thymus, lymphocyte mitoses are seen to be most

frequent in the thymus cortex. There is evidence from thymic grafts that a hypothetical factor, thymopoietin, may stimulate mitosis of thymic lymphocytes. It is a factor which is postulated to act in conjunction with the PAS-positive mesenchymal reticular cells and epithelial reticular cells of the cortex. The thymus medulla is necessary for the integrity of thymic grafts, and may also elaborate a secretion for maintaining the cellular functions of the gland. Thymectomy has been used as a gauge for judging normal thymic function and results, in the mouse, in lymphopenia, degeneration of spleen and lymph

* Thymic factors are summarized in Table I. 312 CASO Vol. 29

TABLE I Comparisons of Thymic Factors Discussed in Text. Actions in Appropriate Test Systems

1 Some indication.

2 Slight alleviation of leukopenia. 3 In higher doses PHA and MLR effects augmented at the 50 ƒÊg/ml dose . 4 Autosensitization in vitro.

nodes, delayed rejection of skin allografts, reduced ability of spleen cells to mount the graft versus host reaction, and reduced primary immune response to certain antigens. Correction of these deficiencies offers a means of evaluating various thymic extracts and grafts. Lymphocytosis-stimulating hormone (LSH) is known to maintain the peripheral lymphoid organs and cause lymphocytosis in the thymectomized animal. Diffusion chamber studies of thymic grafts also show restored lymphoid tissue by a cell-free factor (CIF). These two factors may be the same and probably represent the basis of the highly purified lymphocyte-stimulating proteins , LSHr and LSHh, which restore the L/P ratio in thymectomized animals and may stimulate lymphopoiesis in spleen and lymph nodes . LSHr, unlike LSHh, increases the total lymphocyte count . LSHr has been found to increase the humoral antibody response in neonatal mice both by the PFC technique and by direct hemolysis of sheep erythrocytes. Homeostatic thymic hormone (HTH) is a thymic extract of small mole- 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND 313

cular weight and contains nucleic acid. In the thymectomized guinea pig it has been found to maintain normal levels of lymphocytes in the blood, spleen and lymph nodes, to restore antibody titers to typhoid H antigen and to restore

the toxic allergic reaction. Thymic humoral factor (THF) is of smaller molecular weight (<1,000) and

probably is not a protein. It also enhances lymphoid proliferation in neonatally thymectomized mice. There is evidence that THF participates in humoral antibody formation because it stimulates PFC formation from neonatally thymectomized mice after inoculation with sheep erythrocytes. Its effects on cell- mediated immunity are seen from findings that injection of THF restores the ability of thymectomized mice to reject skin allograf ts. THF enables spleen cells from thymectomized or neonatal animals to mount the graft versus host reaction, and causes maturation of bone marrow cells and spleen or lymph node cells so that they can participate in the graft versus host reaction. It has been

reported to stimulate lymphocytes to kill isogeneic tumor cells in vitro. Thymosin is protein extracted from the thymus. It has been found to

alleviate leukopenia slightly and provide some improvement in lymphoid histology in thymectomized mice. Injected into thymectomized mice it brings about rejection of skin allografts nearly as rapidly as in intact mice. It can give partial restoration to spleen cells from thymectomized mice for exerting the graft versus host reaction, and has been reported to accelerate the development of immunocompetence in spleen cells of normal, neonatal mice for producing the graft versus host reaction. Thymosin has been found capable of partially reversing the effects of antilymphocyte antiserum, which prolongs the time of allograf t skin rejection, when injected after the time of grafting. Evidence is available that thymosin accelerates resistance to virus-induced tumor in neonatal mice. Within the mouse thymus gland the majority of thymocytes express high density Thy-1 (ƒÆ) antigen and the TL antigen in TL+ mice, but in very immature cells these antigens are lacking. These cells are non-immunocompetent and sensitive to corticosteroids. A small percentage of thymic lymphocytes are im-

munocompetent, have a lower density of Thy-1 (ƒÆ), have lost TL antigen and are probably located in the medulla. Further maturation of these immunocompetent cells occurs as they populate peripheral lymphoid tissue. The transition from prethymic cells to immature T cells to post-thymic cells has yet to be demonstrated, but thymosin has been shown to cause •gmaturation•h of cells, low in Thy-1 (ƒÆ), TL and Ly antigens in bone marrow and spleen, to cells fully expressing these antigens. Thymopoietin I and II, two purified polypeptides extracted from thymus, have also been reported to cause expression of Thy-1 (ƒÆ) and TL antigens in immature lymphocytes in hematopoietic tissue. The thymus is known to suppress tumor growth in experimental animals infected with polyoma virus, SV 40 and adenovirus 12. The inhibitory phase of thymic action is exemplified by thymosterin, purified from the lipid fraction of thymic extract and known to be a steroid. Thymosterin was found to be inhibitory to the proliferation of KB tumor cells in tissue culture. The total lipid 314 CASO Vol. 29 fraction and Fr. B were found to reduce the incidence of experimental tumor growth and increase the survival time of tumor-bearing animals. Fr. B has been reported to stimulate humoral antibody formation in mice and to increase blood hemoglobin and leukocyte counts. Fr. III B of the lipid fraction of thymus is stimulative to tumor growth. Additional research is indicated on the thymic lipid extracts. Other functions of the thymus have been indicated. Thymopoietin has been found to cause neuromuscular block, and the extract of Mizutani causes a lowering of serum calcium. Obviously these findings constitute a major area for future research, as does the inter-relationship of all the thymic factors. Another area for future research is the cellular origin of the thymic factors, which are believed to be secreted by the epithelial and mesenchymal reticular cells of the cortex and medulla. The thymus has long been associated with the development of malignancy because of the increased incidence of neoplasms in immune deficiency diseases, therapeutic immunosuppression and the decline of immune responses in aging. With the advent of methods for identifying B and T lymphocytes in human subjects, recent studies have associated the thymus, with its role as chief organ moderator of the immune response, with various aspects of the development of neoplasms. Efforts are being made to classify, and identify clinically, by B- and T-cell analysis the various forms of lymphomas. T lymphocytes have been identified in several patients with Sezary's syndrome. Athymic or nude mice have been shown to be more susceptible to tumor transplantation, and the thymus has been shown to repress tumor-cell transplantation and tumor-cell metastasis. Thymus-derived cells have been linked to the establishment of immunity to experimental tumors of chemical and viral carcinogen origin, although here some experiments are contradictory. A well known feature of tumor growth is the suppression of various aspects of the immune responses. It appears possible that immunosuppression may also be a property of thymic extracts, and some recent work has demonstrated five types of extracts to be immunosuppressive: THF, a globulins from human serum, a globulins from bovine thymus, chalone-like extract from calf thymus, and histones extracted from calf thymus. The a globulins of serum and thymus are functionally similar in that they both suppress the hemagglutinin response to sheep erythrocytes and suppress DNA synthesis and lymphocyte proliferation induced by PHA. The PHA effect is dependent on the dose of a globulins, higher doses causing suppression and lower doses stimulation. Suppressor T cells are also inhibitory to certain immune responses.

ACKNOWLEDGMENT The authorexpresses his thanksand appreciationto Mrs.Margaret Pickard for her valuable assistancewith the manuscript. 1976 SOME ENDOCRINE ASPECTS OF THE THYMUS GLAND 315

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