Role of thymus-eicosanoids in the immune response

PROSTAfiKAND~SLEUKOTRIE~S AM)mF,NTIAL FATTYACIDS Prostaglandms Leukomenes and E\\mt~al Fatty Acids, ‘0 Longman UK Ltd 1992

Group

1992) 46. 247-255

Review

Role of Thymus-Eicosanoids in the Immune Response M. Juzan, I. Hostein and N. Gualde CNRS URA 1456 Universite’ de Bordeaux II and Fondation Bergonie’. 146. rue Lto-Saignat, 33076 Bordeaux Cedex, France (Reprint requests to MJ) ABSTRACT.

The present review deals with the role(s) of thymus-eicosanoids in the immune response. It reports the production of cyclooxygenase and lipoxygenase metabolites of arachidonic acid by cells of the thymus microenvironment and the role(s) of these eicosanoids in the differentiation and the maturation of immature T-cells. The possibility that these products may be involved in tolerance to self is discussed. Briefly, it is likely that cells from the monocyte-macrophage lineage which constitute a part of the thymus microenvironment could contribute to the education of immature thymocytes by both presenting self-antigens and producing eicosanoids. Tolerance to self might result from PGE,-driven apoptosis and/or LTB,-induced generation of suppressor cells.

INTRODUCTION

one means that T lymphocytes which are the main mechanism responsible for autotolerance are ‘educated’ in the thymus. In other words, they are either positively or negatively selected in the gland. Hence the thymus which is the main central lymphoid organ gives rise to mature, peripheral T lymphocytes which are both circulating cells of the blood and the lymph and resident lymphocytes of the peripheral lymphoid organs. Therefore, most of the process of T-cell education which provides the capability of the immune system to differentiate between self and non-self takes place in the thymus. There is good evidence that eicosanoids are involved in thymocyte education, maturation and/or differentiation (9-13) and that they play a critical role for the future of the tremendous armada of thymusdependent lymphocytes which are the main cells of the whole immune system since they are involved in both the cellular immune response and the regulation of the immune response (14).

In recent years some quite interesting reviews of the scientific literature focused on the modulation of the immune response by eicosanoids. The earliest of these articles dealt mostly with the role of prostaglandins (PGs) as immunosuppressor agents (1). Some of these initial reviews are still frequently cited ( 1, 2) and remain as landmarks of the reports on immunomodulation induced by eicosanoids. The subsequent papers were either more specific for the human immune system (3) or devoted to the role of lipoxygenase metabolites in the immune system (4-6). However, as far as we know, a review concerning the role played by eicosanoids within the thymus has not been published before. Even in his excellent book, which gathers an impressive amount of information on immune response and eicosanoids, Ninnemann does not specifically discuss their role in thymus physiology (7). Before arguing about the role of eicosanoids in thymus physiology one needs to keep in mind that the immune system recognizes most of the molecules which are part of our environment. For that purpose, immune cells express three varieties of receptors which are T-cell receptors for the antigens, surface immunoglobulins of B lymphocytes and molecules encoded by the major histocompatibility complex genes. Hence, with this large panel of receptors the immune system can discriminate between the self and the non-self. This critical capability of immune cells allows them to tolerate the self and to reject the non-self (8). Tolerance to self is both a learned and an ongoing phenomenon during life (9-11). By learned phenomenon

SYNTHESIS OF EICOSANOIDS BY THE THYMUS The question concerning the production of eicosanoids by the thymus is quite puzzling. For instance, it is not yet totally clear whether thymocytes, and by thymocytes one means T lymphocytes, are able or not to produce a significant amount of eicosanoids. The molecular types of arachidonic acid metabolites generated within the thymus is even more open to discussion. In the following pages we would like to briefly summarize the main data 241

248

Prostaglandins

Leukotrienes

Table

and Essential Fatty Acids

Production

of eicosanoids

by cells involved in the immune response and specially thymic cells

Species

Organs

Cells

Eicosanoids

Synthesis modulator

References

Human

Thymus

Epithelium Epithelium Monocytes Moncoytes Monocytes Monocytes Monocytes Monocytes Monocytes Monocytes Monocytes T lymphocytes T lymphocytes Suppressor T-cells T lymphocytes T lymphocytes Cells macrophages

PGs PCS PGE? PGs. LTs PG, PGE, PGE,. TXB? PGE, PGE, PGE>. HETE PGEz PGE,. PGD? LTs PGE? PGs 5-HETE PGI,, TXB,

Steroids Steroids

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Thymocytes Phagocytes

PGS PGs

Lymph node

Macrophages Macrophages ND Thymocytes Phagocytes ND ND Nurse cells Epithelium Epithelium Epithelium Epithelium Macrophages Macrophages Macrophages T lymphocytes T lymphocytes Macrophages Macrophages T lymphocytes Macrophages

PGs PGs, HETEs PGE, PGs PGs 12-HETE PGs PGE,, PGI, PGE,. HETEs PGE, HETEs. HPETEs PGE,, LTB, PGEz HPETE, HETE LTC, PGF,, I2-HETE PGEL PGs PGE, PGs

Rat

Thymus Liver Spleen

Epithelial Kupffer Macrophages

PGE,. HETEs, LTs PGD, PGD?

Bovine

Spleen

ND

PGF?,

Peripheral

blood

Mononuclear

cells

Peritoneum Mouse

Thymus

Peritoneum

Spleen

IL-

1

T lymphocytes FTS anti CD3 anti CD3

Indomethacin Steroids Fetal development Fetal development Alloimmunization Steroids

Kallikrein Inflammation Calcium ionophore

Endotoxin Antigen Autoimmunity Alloimmunization Mitogen Thymosin fraction 5 GVH Thymocytes Glutathion

32333 34.35 36 37 38 39 40 41 42 43 44 45 36 47 48 19 49 50 51 52 53 54 55 56 57 58 59 60

ND: not determined GVH: graft versus host reaction FTS: facteur thymique serique

reported by the scientific duction

of arachidonic

acid

literature derivatives

concerning inside

the prothe thymic

It has been clearly proven that among interacting cells involved in the immune response monocytes and macrophages are important producers of eicosanoids. The Table summarizes published articles demonstrating that most of the arachidonic acid metabolites produced by immune cells and/or lymphoid organs are issued from cells belonging mostly to the monocyte-macrophage lineage. In 1977, Humes et al (46) reported that macrophages were the main source of prostaglandins (PGs) following stimulation by inflammatory products. This former report was confirmed by several authors, such as Bankhurst et al, who actually demonstrated that PGs produced by peripheral blood mononuclear cells gland.

originate from monocytes (17). Since these reports, PGE production by phagocytes was demonstrated among various species such as the mouse (19), guinea-pig (21), human circulating monocytes (18, 20, 23. 48) or peritoneal macrophages (31). In fact, it is very well demonstrated that the monocyte-macrophage lineage does not only produce PGs of the E series but also other metabolites from the cyclooxygenase pathway, such as thromboxane (TX) (3 1, 62) and PGD,. There is also evidence that macrophages express lipoxygenaseactivity, yielding a large variety of arachidonic acid lipoxygenase-derivatives such as hydroxyeicosatetraenoic acids (HETEs) (33), and leukotrienes (LTs) (18, 48, SO). It has even been reported that in terms of eicosanoid production the population of monocyte-macrophages is

Role of Thymus-Eicosanoids

heterogeneous. For instance, some of these phagocytes produce more PGE, than others (22, 59) and the production of eicosanoids depends upon immunostimulation (53), endotoxin treatment (50), drugs (48), presence of interleukins, lipoxygenase metabolites themselves (49), lipopolysaccharide (61), calcium-ionophore, lectins, toxins, indomethacin, steroids (62), and various in vitro conditions (25). To summarize the above reports, one can consider as clearly demonstrated that cells from the monocyte-macrophage lineage produce eicosanoids but in terms of arachidonic acid metabolism these cells are quite heterogeneous. It should be underlined that the in vivo production of eicosanoids by macrophages is probably more complex regarding the regulation of synthesis than what has been observed in vitro since in vitro the cells are kept under the influence of interleukins (ILs) such as IL-l, IL-6, IL-S, various growth factors and even eicosanoids themselves (63). Nevertheless. since it is accepted that the monocyte-macrophage lineage produces eicosanoids everywhere (blood, spleen. peritoneum, etc.) one may consider that it does the same thing within the thymus where it constitutes a part of the microenvironment. Production of eicosanoids by the thymus was reported in 1963 by Bergstrom and Samuelson who demonstrated the presence of a smooth muscle stimulating activity in an ethanol extracted material (39). Using different chromatographic techniques (silicid acid chromatography. reversed phase partition chromatography, thin layer chromatography) before assessing the biological activities of the extracts on duodenal strips of rabbits, they concluded that PGE, was the major product present in the biological extract. This former article does not give any information about the lineage of the PGEproducing cells in the thymus. Similarly, Tomar et al reported that thymus cells produced a small quantity of prostaglandins (43). More information concerning this question was given by the Papiemik group (34) which studied the synthesis of cyclooxygenase metabolites by phagocytic cells of the thymic reticulum in culture. By using both high pressure liquid chromatography and radioimmunoassay Homo-Delarche et al (34) demonstrated the synthesis of TXB,, PGE,. 6-keto-PGF,,, and PGE,, by thymus-phagocytes. Papiemik et al also demonstrated that phagocytic cells of the thymic epithelium which may be issued from thymic-interdigitating cells cultured in vitro did not only produce PGE, but also IL- 1 and were therefore able to modulate thymocyte proliferation (36) via antagonistic influences, PGE, giving the negative signal and IL-1 the positive one. The evidence that prostaglandins could be produced by the so-called ‘non adherent cells’ of the thymus is more controversial and the fact that the prostaglandin-producing cells of the thymus are non-adherent cells in a short-incubation experiment (40) does not mean that these cells belong to the thymocyte lineage.Usually the so-called ‘adherentcells’ are considered as monocytes and/or macrophages which have the very well known capability to stick to the glass- or plastic-dishes. This property is not genuinely

in the Immune Response

249

accurate since, for instance, some adherent-cells do not belong to the monocyte-macrophage system and some monocytes are not adherent. Regarding the generation of prostaglandins, it is likely that production of PGE, is an early event during thymus ontogeny and is necessary for the proliferation and differentiation of thymocytes (38). For example, the production of PGE,,, 6-keto-PGF,, and PGFza by fetal thymic cells was reported as well as the expression of thymocyte markers such as Thy-l and Lyt- 1 surface molecules in relation to prostaglandin production (38). Once again the cell lineage origin of thymus prostaglandins was not clearly demonstrated (38. 37) even if the presence of lipoxygenase metabolites such as HETEs was evidenced during cultures of fetal thymic lobes, and even if cyclooxygenase immunoreactivity appeared to be localized to stromal ie nonlymphoid cells of the fetal thymus (37). There is a good deal of evidence that thymic epithelial cells produce prostaglandins and that this synthesis is stimulated by cell-to-cell contact between epithelial cells and thymocytes (57). The data showing that a thymic hormone, thymulin, increases the production of PGE, and PGD-, by human immature T-cells support the fact that prostaglandins might be synthesized by thymocytes within the thymus (26). In fact, the influence of thymic hormone on prostaglandin synthesis by T-cells remains controversial and might depend upon the experimental conditions since thymosin fraction 5 induces both a release of PGE, and an expression of theta antigen by spleen cells from adult thymectomized mice but not by spleen cells from normal mice (55). These data were not confirmed by Homo-Delarche et al (33). There are fewer reports concerning the production of lipoxygenase metabolites by thymus cells (37, 42). Using both thin layer chromatography and high pressure liquid chromatography of chloroform extracts of culture supematant, Duval demonstrated the production of 12-HETE by thymic cells (42). The interesting fact underlined by this report is that the thymic-12-HETE-producing cells release only small amounts of prostaglandins and leukotrienes and are unaffected by steroid treatment (41). It is likely that leukotrienes as well as HETEs are produced by macrophages of the thymus microenvironment as the above experiments used thymus-macrophage hybridomas (64). Considering the above reports it is likely that most of the eicosanoids produced within the thymus are released from cells of the thymus microenvironment, mostly macrophages, but also epithelial, dendritic cells and nurse cells. The question of eicosanoids production by T lymphocytes remains controversial. For example, according to Goldyne. even mature thymocytes are not able to synthetize LTB, (65) as was reported by other groups (see Table). One cannot exclude a cooperation or more precisely some metabolic interactions between cells of the thymus as it was reported for cells from blood and the vascular system (63). Hence. that could explain the involvement of thymocytes in eicosanoid production and more precisely the

250 Prostaglandins Leukotrienes and Essential Fatty Acids

stimulation of the metabolism of arachidonic acid by thymic epithelial cells (57). Therefore. one can imagine that inside the thymus, one cell (a thymocyte for instance) may influence the metabolic activity of another cell (from the microenvironment) inducing it to produce more or less eicosanoids. In turn, the first cell could be under the influence of the metabolites released by the second one. This type of cell-to-cell interaction may be completed by merely an exchange of metabolites as has been reported between polymorphonuclear and erythrocytes (66) and between leucocytes and platelets (67). This cell-to-cell donation system allows a second cell to metabolize a molecule produced by the first one etc. Hence, regarding the complexity of the potential cellular interactions in the thymus, it is quite difficult to discern what is actually going on in terms of arachidonic acid metabolism. However the production of eicosanoids by cells of the thymus microenvironment may participate to their modulation of the activation of thymocytes (36, 68-71). Among the cells of the microenvironment, macrophages which express an intensive metabolism of arachidonic acid (73), are probably the most efficient in terms of binding to thymocytes of the cortex and the medulla (72).

INTERACTIONS BETWEEN EICOSANOIDS THYMIC HORMONES

AND

The thymus is an endocrine gland which produces hormones involved in the differentiation and the maturation of thymocytes (14). Bach’s group in Paris was the first to report the role of prostaglandins in the expression of membrane molecules and their interaction with the thymic factor, thymulin (previously called FTS (facteur thymique serique)) (74, 75). Bach et al studied three types of thymocyte membrane molecules, respectively the theta antigen, the T-cell receptor, and the receptor of thymocytes for autologous erythrocytes. They observed that the expression of these three membrane-constituents was markedly altered in adult thymectomized mice but corrected soon after treatment by thymulin. The thymulin effect in regard of these three markers is mimicked by both cyclic-AMP and PGE,. On the other hand, the spontaneous expression of theta antigen was inhibited by treatment of the mice with indomethacin (50 pg injected intraperitonealy). In other words, PGE, could be a ‘second messenger’ of thymic hormones. Garaci et al reported similar data since they demonstrated that after adult thymectomy the in vivo expression by splenocytes of the theta marker was induced when animals were treated by an analog of PGE2 (76). This phenomenon was later reported to be linked to the induction of a serum thymic-like activity (77). It is likely that the role of thymic hormones such as thymosin fraction 5 depends upon the immunological status of the animal since the hormone reinduces the expression of membrane theta molecules via the production of PGE,

in thymectomized mice (this phenomenon is inhibited by indomethacin) (55). In contrast, PGE? production by splenocytes from normal mice is inhibited by the hormone (55). Thymic hormones act via the production of cyclic AMP and/or PGE, (78). and it is probable that thymic hormones induce an increase of the intrathymocyte cyclic-AMP either directly or via the production of PGE, (75, 79). The effect of thymic hormones depends upon the level of maturation of thymocytes, for example, thymulin increases the intracellular cyclicAMP/cyclic-GMP ratio of immature but not mature thymocytes, and under some experimental conditions has reverse effects in terms of PGE, production (78). This is in agreement with the fact that peripheral blood human T-cells respond to thymulin by an increase of prostaglandin production. With regard to the effects of thymic hormones on prostaglandins production more controversial data were reported by Homo-Delarche et al (33, 80).

EICOSANOIDS AND THYMOCYTES PROLIFERATIVE RESPONSE, RELATIONS TO APOPTOSIS One cannot discuss the role(s) of eicosanoids on thymocyte proliferative response without arguing about apoptosis or programmed cell death of thymocytes which involves arachidonic acid metabolites. Apoptosis is a mechanism of programmed cell death which is characterized by cleavage of DNA, which appears to be a programmed suicide of the cell, starting with an intranuclear biochemical phenomenon. Endonuclease activation results in the production of oligonucleosomelength DNA fragments giving an electrophoretic aspect of ‘DNA ladder’ (81). It is likely that apoptosis is related with education and/or selection of thymocytes in the thymus (13), i.e. that autoreactive cells are genetically programmed to die in the organ and hence submitted to a negative selection. The question arises as to whether arachidonic acid metabolites produced in the thymocyte-microenvironment play a critical role in physiological apoptosis. It was reported by McConkey et al that agents that elevate cyclic-AMP stimulate DNA fragmentation in rat thymocytes (82), among these agents PGEl is one of the most efficient in terms of programmed cell death. On the contrary PGD?, PGF,, or analogs of cyclic-GMP are inefficient. PGEz induces an endonuclease activation which occurs before cell death. Apoptosis linked to cyclic-AMP-increase is related to an increase of protein kinase A activity (but not protein kinase C activation) and a subsequent protein phosphorylation. Suzuki et al did not observe any effect of PGEl on mouse-thymocyte programmed cell death (83), i.e. the transient increase of CAMP induced by PGE? was not sufficient to induce any DNA fragmentation. However, when PGE, and forskolin are added together to thymocytes, there was an effect mediated

Role of Thymus-Eicosanoids in the Immune Response

via activation of protein kinase C. It was reported that the DNA cleavage is mostly significant in CD4+ CDS+ (double positive) immature thymocytes suggesting that the stage of differentiation and maturation of thymocytes in an important factor in PGE,-induced DNA fragmentation. The discrepancy between the Suzuki data on mouse thymocytes (83) and those on rat thymocytes reported by McConkey (82) is still being elucidated. The report of Harford et al (84) is even more confusing since it suggests that murine thymocyte lysis by corticosteroids may be explained by the inhibition of arachidonic acid metabolism. More specifically, the lytic effect of the 5 lipoxygenase inhibitors, AA 861 and caffeic acid suggests that part of the thymolytic effect of corticosteroids is linked to inhibition of the production of metabolites of arachidonic acid and among them it is likely that LTBa is the more potent anti-apoptosis eicosanoid (84). One can speculate that arachidonic acid metabolites have dualistic effects on programmed cell death, i.e. PGE, induces apoptosis while LTB, prevents it. The question remains if the depression of thymic weight induced by arachidonic acid (85) may be linked to a cyclooxygenase linked apoptosis since indomethacin prevents this effect of arachidonic acid. Hence, when discussing the role played by eicosanoids on thymocyte proliferative response we have to keep in mind that during in vitro experiments programmed cell death of at least a subpopulation of thymocytes could be a factor interfering with the assessment of thymidine uptake which is the most widely used method for measuring DNA synthesis and cell proliferation. Concerning the effect of prostaglandins on thymocyte proliferative response (following binding of prostaglandins to receptors (86)) conflicting data were reported suggesting that either PGE, or PGE, stimulate (87, 88) or inhibit thymidine uptake by thymocytes (71. 89). The early reports of increased thymidine uptake induced by PGE, (87, 88) were attributed to the activity of ‘wound hormones’ expressed by prostaglandins (87, 88). However, the vast majority of the data underlines the inhibitory function of prostaglandins on the whole thymocyte population (71, 89, 90) or on mature and immature thymocytes (89). This is in agreement with the betterestablished role for PGE, as feedback inhibitor of the cellular immune response and it is obvious that most of the PGE, produced in the thymus originates from the thymic microenvironment and mostly from macrophages (37, 38, 71) and epithelial cells (15, 16, 45, 46). The effects of lipoxygenase metabolites in terms of thymocyte proliferation are more subtle since they are closely related to the type (for instance mature or immature) of thymocyte involved in the experiment. We demonstrated that both immature and mature thymocytes proliferate when cultured with concanavalin A plus IL-2 but that PCE, at concentrations of lo-‘) to lo-* M caused significant inhibition of the proliferation of both types of thymocytes in these cultures. In contrast, the lipoxygenase products 15-HETE. LTB,, LTC, and LTD,

25 1

caused marked increase in proliferation of immature thymocytes while having no effect on mature cells (89). Hence, it is likely that the effect of leukotrienes on thymocyte function depends upon the level of cell maturation and mainly affects immature thymocytes. For instance. LTB, which increases the contrasuppressor activity of vicia villosa positive splenocytes has almost no effect on vicia villosa positive thymocytes (91). The effect of lipoxygenase metabolites of arachidonic acid on thymocytes is in agreement with what was reported concerning thymocyte cyclic-AMP and cyclic-GMP response to treatment with metabolites issued from the lipoxygenase pathway (92). Briefly, incubation of mature, immature, and whole thymocytes treated with IO-“’ to 10m7M 15-HETE and lo-“M LTB, showed an approximately 100% increase in cyclic-GMP production. In parallel. it was demonstrated that 15-HETE and LTB,-treated thymocytes inhibited thymidine uptake by fresh allostimulated splenocytes suggesting that the leukotriene-induced generation of suppressor cells follows a rise in lymphocyte cyclic-GMP levels (92).

ROLE OF EICOSANOIDS IN THYMOCYTE DIFFERENTIATION AND MATURATION The thymus plays a critical role in the education of thymocytes by controlling the positive and negative selection of thymocytes, the education of immature thymocytes, and the selection of the repertoire of T-cells (9-13). There is a good deal of evidence that eicosanoids participate in the differentiation and the maturation of thymocytes as they do for bone marrow stem cells during hematopoiesis. Broadly speaking, it was rep&ted that prostaglandins and metabolites of 15-lipoxygenase had an inhibitory effect on myelopoiesis but that 5-lipoxygenase metabolites usually stimulated hematopoiesis (93-98). Therefore. cyclooxygenase and lipoxygenase metabolites of arachidonic acid can modulate the differentiation of committed and uncommitted stem cells. Concerning the thymus. it was reported that prostaglandin production is very low in this organ in the lupus prone NZB/NZW mouse which is characterized by a defect in suppressor cells (99). This may be related to abnormalities of early T-cell development. Recently, by using nothemblots and in situ hybridization to thymus macrophage RNA, we demonstrated the presence of 5 lipoxygenase transcripts within the thymus. The activity of the 5 lipoxygenase enzyme was demonstrated by assessment of leukotrienes produced by thymicmacrophage hybridomas (64). It was reported that LTB, plus IL-2 treatment of immature CD4- CD8- double negative thymocytes induces them to express the CD8 antigen (100) which is the marker of both the suppressor and the cytotoxic T lymphocytes. Later, we demonstrated that the LTB,-induced CD8+ thymocytes are suppressor cells (101) which are mainly involved in tolerance to self ( 102). This is in agreement with the

252

Prostaglandins

Leukotrienes

and Essential Fatty Acids

Cdl-to-Cdl

intetactlons (“cross-talk”)

Microenvironment

Cycloo&eoase pathway (prostaglandins)

Lip&y*enase pathways (leukotrlenes)

eicosanoids play a critical role in tolerance to self; in the first case tolerance to self results from a PGE,-induced apoptosis which is in agreement with the model of the ‘forbidden-clone’ (109-l 11). A forbidden clone is a clone of lymphocytes capable of reaction with selfcomponents of the body but which is regularly eliminated by the normal homeostatic mechanism (111). In the second case auto-tolerance is produced via the generation of suppressor cells which is in agreement with the theory of clonal anergy involved in tolerance to self (108-l 12). Clonal anergy means that the forbidden clone is still present but that some homeostatic mechanism (for example suppressor cells) induces its (clonal) unresponsiveness to self-components.

fancies Microenvironment

cD4+,cD8+

CONCLUSIONS 1 cD8+ s&-essor cell @maI ancxgy) I

Apoptosi! (clmal deletim, forbidden clme)

\

TOLERANCE

TO SELF

J

Figure Predicted pathway for the involvement of thymusmacrophages in tolerance to self (see text). Cells of the microenvironment include macrophages, dendritic cells, epithelial cells. and nurse cells.

already reported role of lipoxygenase metabolites in the generation of suppressor cells (103-106). Hence, we speculated that immature double negative thymocytes in the context of self environment and leukotrienes are induced to differentiate into CD8+ self-specific suppressor T-cells involved in tolerance to self (102). Considering the fact that thymic prostaglandin production is modulated by thymic hormones and that PGE, plays a role in apoptosis which is probably related to the negative selection of immature thymocytes it is likely that eicosanoids are quite important mediators during the early steps of their maturation and selection of thymocytes. Therefore, we speculate that prostaglandins and leukotrienes play a fundamental role in thymus physiology, more precisely that they participate in the thymusdriven tolerance to self either by inducing apoptosis dnd/or generating suppressor cells which shut-off autoreactive lymphocytes. The Figure shows the predicted pathways that we propose for the role of eicosanoids in the education of thymocytes involved in tolerance to self (103-107). On the one hand, anti-self immature double positive (CD4+, CDS+) thymocytes which are both in the context of self antigens (expressed by cells of the thymus microenvironment) and in the presence of PGE, initiate their program of cell death and then are negatively selected. On the other hand immature double negative thymocytes (CD4-, CD8-) which are also in the context of self-antigens are, in the presence of LTB,, induced to give rise to CDS+ suppressor cells involved in tolerance to autologous antigens. Hence, in both cases

Trying to clarify what could be the role of arachidonic acid metabolites in thymus physiology is not an easy challenge. It may be considered relevant that eicosanoids (cyclooxygenase and lipoxygenase metabolites) are produced within the thymus and that they modulate the activity of thymocytes. It is likely that eicosanoid production is, to some extent, modulated by thymic hormones and thymic interleukins. It is possible that most of the eicosanoids are synthetized by cells of the thymus microenvironment and mostly by those of the monocytemacrophage lineage. Hence, eicosanoids may have a major influence on immature T-cells during their education and maturation in the thymus, giving rise to cells which would keep a kind of ‘print’ of that first encounter with arachidonic acid metabolites. For instance, one can imagine that the mature CD8+ suppressor cells involved in the tolerance to self which are generated in the thymus under the influence of LTB, leave the gland for a long journey in the periphery (the rest of the body) but still remain as LTB,-sensitive autologous specific suppressor cells. Acknowledgements The authors are grateful to Maryvonne Birac for her excellent tarial assistance in preparing the manuscript.

References I. Pelus L M. Prostaglandins 2.

3.

4.

5. 6.

and the immune response. Life Sciences 20: 903-914, 1977. Goodwin J S, Webb D R. Regulation of the immune response by prostaglandins. Clinical Immunology Immunopathology 15: 106-122, 1980. Goodwin J S, Ceuppens J L, Gualde N. Control of the immune response in humans by prostaglandins. p 79 in Advances in inflammation research. (I Ottemess, R Capetola, S Wong eds) Raven Press New York, 1984. Rola-Pleszczynski M. Immunoregulation by leukotrienes and other lipoxygenase metabolites. Immunology Today 6: 302-307, 1985. Davies P. Lipoxygenase products in immunity. Immunological Investigations 16: 623-647, 1987-l 988. Vanderhoek J Y. Role of the 15lipoxygenase in the

secre-

Role of Thymus-Eicosanoids

7. 8.

9. 10.

11.

12.

13. 14.

15.

16.

17.

18.

19.

20.

2I

22.

23.

24.

25.

26.

27.

7X.

immune system. Annals of the New York Academy of Sciences 524: 240-251. 1988. Ninnemann J L. Prostaglandins. leukotrienes, and the immune response. Cambridge University Press, 1988. Siskind G W. Immunologic tolerance. p 737 in Fundamental Immunology. (W E Paul ed) Raven, New York, 1984. Bevan MT. Thymic education. Immunology Today 2: 216-219, 1981. Nikolic-Zubic J, Bevan M J. Role of self peptides in positively selecting the T cell repertoire. Nature 344: 65-67. 1990. Von Boehmer H, Teh H S. Kisielow P. The thymus selects the useful, neglects the useless and destroys the harmful. Immunology Today 10: 57-61. 1989. Saito T. Germain R N. The generation and selection of the T cell repertoire: insights from the studies of the molecular basis of T cell recognition. Immunological Reviews 101: 81-l 13, 1988. MacDonald H R. Lees R K. Programmed death of autoreactive thymocytes. Nature 343: 642444. 1990. Sprent J. T lymphocytes and the thymus. p 69 in Fundamental Immunology. (WE Paul ed) Raven. New York, 1984. Russo-Marie F. Homo F. Papiemik M. Effet des hormones steroides sur la s&r&ion de prostaglandines par des cellules Cpitheliales thymiques humaines en culture. Comptes Rendus de 1’ Academic des Sciences S&ie D 290: 767-770. 1980. Homo-Delarche F, Papiemik M. Russo-Marie F. Steroid modulation of in vitro prostaglandin secretion by human thymic epithelium. Journal of Steroid Biochemistry 15: 349-354. 19x1. Bankhurst A D, Hastain E, Goodwin J S. Peake G T. The nature of the prostaglandin-producing mononuclear cell in human peripheral blood. Journal of Laboratory and Clinical Medicine 97: 179-186, 1981. Goldyne M E, Burrish G F, Poubelle P, Borgeat P. Arachidonic acid metabolism among human mononuclear leucocytes. Journal of Biological Chemistry 759: X81.5-8819. 1984. Kurland J I, Bockman R. Prostaglandin E production by human blood monocytes and mouse peritoneal macrophages Journal of Experimental Medicine 137: 952-95s. 1978. Goldyne M E, Stobo J D. Synthesis of prostaglandins by subpopulations of human peripheral blood monocytes. Prostaglandins 18: 6X7-695, 1979. Morley J. Bray M A, Jones R W. Nugteren D H, VanDorp D A. Prostaglandin and thromboxanes production by human and guinea pig macrophages and leucocytes. Prostaglandins 17: 729-736, 1979. Khansari N, Chou Y K, Fudenberg H H. Human monocyte heterogeneity: interleukin 1 and prostaglandin E, production by separate subsets. European Journal of Immunology IS: 4X-5 I. 1985. Kennedy M S. Stobo J D. Goldyne M E. In vitro synthesis of prostaglandins and related lipids by populations of human peripheral blood mononuclear cells. Prostaglandins 20; 135-14.5. 1980. Goldyne M E. Stobo J D. T-cell-derived arachidonic acid and eicosanoid synthesis by macrophages. p 39 in Advance Prostaglandin Thromboxane Leukotriene Research. (B Samuelsson, R Paoletti. P Ramwell eds) Raven Press. New York, 19X6 Bockman R S. Prostaglandin production by human blood monocytea and mouse peritoneal macrophages:synthesis dependent on in vitro culture conditions Proataglandins ?I: 9%3l.1991. Gualde N, Rigaud M. Bach J F. Stimulation of prostaglandin synthesis by the serum factor (FTS). Cellular Immunology 70: 362-366, 1982. Goodwin J S. Behrens T. Role of lipoxygenase metabolites of arachidonic acid in T cell activation. Annals of the New York Academy of Sciences 524: 201-207. 19X8. Garcia-Penarrubia P. Bankhurst A D. Koster FT.

29.

30.

3 1,

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

32.

43.

44.

45.

in the Immune Response

Prostaglandins from human T suppressorlcytotoxic cells modulate natural killer antibacterial activity. Journal of Experimental Medicine 170: 601606. 1989. Aussel C, Mary D, Fehlmann M. Prostaglandin synthesis in human T cells: its partial inhibition by lectins and anti-CD3 antibodies as a possible step in T cell activation. Journal of Immunology 138: 3094-3099. 1987. Goetzl E J. Selective feed-back inhibition of the S-lipoxygenase of arachidonic acid in human T-lymphocytes. Biochimica et Biophysics Acta 101: 344-350. 1981. Foegh M L. Maddox Y T, Ramwell P W. Human peritoneal eosinophils and formation of arachidonate cyclooxygenase products. Scandinavian Journal of Immunology 23: 599-603. 1986. Bauminger S. Differences in prostaglandin formation between thymocyte subpopulations. Prostaglandins 16: 351-355. 197X. Homo-Delarche F, Bach J F, Dardenne M. Thymic hormones and prostaglandins. I. Lack of stimulation of prostaglandin production by thymic hormones. Prostaglandins 38: 183-196, 1989. Homo-Delmche F. Duval D. Papiemik M. Prostaglandin production by phagocytic cells of the mouse thymic reticulum in culture and its modulation by indomethacin and corticosteroids. The Journal of Immunology. 135: 506-512. 1985. Duval D. Huneau J F. Homo-Delarche F. The effects of antiinflammatory steroids on arachidonate metabolism and prostaglandin synthesis vary according to culture conditions. Agents Actions 17: 302-303. 1985. Papiemik M. Homo-Delarche F. Thymic reticulum in mice. III. Phagocytic cells of the thymic reticulum in culture secrete both prostaglandin El and interleukin 1 which regulate thymocyte proliferation. European Journal of Immunology 13: 689-692. 1983. Appasamy P M. Pendino K. Schmidt R R. Chepenik K P. Prystowqky M B, Goldowitr D. Expression of prostaglandin G/H synthase (cyclooxygenase) during murine fetal thymic development. Cellular Immunology 137: 341-357. 1991. Shipman P M, Schmidt RR, Chepenik K P. Relation between arachidonic acid metabolism and development of thymocytes in fetal thymic organ culture\. The Journal of Immunology 140: 2714-2730. 1988. BergstrGm S, Samuelsson B. Isolation of prostaglandin El from calf thymus. Prostaglandins and related faclors 20. Acta Chemica Scandinavia 17: %X2-S287. 1963. Borda E. Genaro A M, Perez Leiros C. Cremaschi G, Peredo H. Sterin-Borda L. Prostaglandins. cyclic AMP production and biological activity of alloimmune rhymocytes. Prostaglandins Leukotrienes Medicine 19: 197-20X. 198s. Duval D. Huneau J F, Homo-Delarche F. Efrects of \erum on the metabolism of exogenous ardchidonic acid by phagocytic cells of the mouse thymic reticulum. Prostaglandins Leukotriene\ and Medicine 73: 67-7 I. 19X6. Duval D. Effect of dexamethasone on arachidonate metabolism in isolated mouse thymocytes. Proata~landins Leukotrienes and EFA. 37: 139-l 56. IYXY. Tomar R H, Darrow T L, John P A. Response to and production of prostaglandin by murine thymus. spleen, bone marron. and lymph node cells. Cellular Immunology 1981: 60: 335-346. 1981. McCormach J E. Kappler J. Marrack P. Wcstcott J Y. Production of prostaglandin E, and prostacyclin by thqmic nurse cells in culture. The Journal of lmmunology 116: 139-243. 199 I Sun L. Piltch A, Hayashi J. Kallikrein stimulates arachidonic acid release and production of prostaglandins from TEA3Al endocrine thymic epithelial cells. Biochemical Journal 258: 351-355. 1039.

254

Prostaelandins

Leukotrienes

and Essential Fattv Acids

46. Humes J L, Bonney R W, Pelus L, Dahlgren M E, Sadowski S S, Kuehl F A, Davies P. Macrophages synthetize and release prostaglandins in response to inflammatory stimuli. Nature 269: 149-151. 1977. 47. Rabinovitch H, Durand J, Gualde N, Rigaud M. Metabolism of polyunsaturated fatty acids by mouse peritoneal macrophages: the lipoxygenase metabolic pathway. Agents Actions 11: 58&583. 1981. 48. Bonney R J, Humes J L. Physiological and pharmacological regulation of prostaglandin and leukotriene production by macrophages. The Journal of Leukocyte and Biology 35: l-10. 1984. 49. Humes J L. Opas E E. Galavage M. Soderman D. Bonney R J. Regulation of macrophage eicosanoid production by hydroperoxy and hydroxy-eicosatetraenoic acids. Biochemical Journal 233: 199-206.1986. 50. Liideritz Th. Rietschele Th, Schade H. Leukotriene C4-release from endotoxinstimulated macrophages. Annals Immunology Hungary 23: 81-91. 1983. 51. Osheroff P L, Webb D R. Paulsrud J. Induction of T-cell dependent splenic prostaglandin Fza by T-cell dependent antigen, Biochimica et Biophysics Acta 66: 425429. 1975. 52. Kato K, Koshihara Y, Fujiwara M, Murota S. Augmentation of 12-lipoxygenase activity of lymph node and spleen T cells in autoimmune mice MRL/l. Prostaglandins Leukotrienes Medicine 12: 273-280, 1983. 53. Dy M, Astoin M. Rigaud M, Hamburger J. Prostaglandin (PG) release in the mixed lymphocyte culture; effect of presensitization by a skin allograft; nature of the PGproducing cell. European Journal of Immunology 10: 121-126. 1980. changes in 54. Webb D R, Nowowiejski I. Mitogen-induced lymphocyte prostaglandin levels: a signal for the induction of suppressor cell activity. Cellular Immunology 41: 72-78. 1978. 55. Garaci R L. Favalli C. Del Gobbo V, Garaci E, Jaffe B M. Is thymosin action mediated by prostaglandin release? Science 220: 1163-l 165, 1983. 56. Lapp W S, Mendes M, Kirchner H. Prostaglandin synthesis by lymphoid tissue of mice experiencing a graft-versus-host reaction: relationship to immunosuppression. Cellular Immunology 50: 271-281. 1980. 57. Sun L, Piltch A S. Liu P-S, Johnson L A. Hayashi J. Thymocytes stimulate metabolism of arachidonic acid in rat thymic epithelial cells. Cellular Immunology 13 1: 86-97. 1990. 58. Urade Y. Ujihara M, Horiguchi Y. Ikai K. Hayaishi 0. The major source of endogenous prostaglandin D? production is likely antigen-presenting cells. Localization of glutathione-requiring prostaglandin D synthetase in histiocytes, dendritic, and Kupffer cells in various rat tissues. The Journal of Immunology 143: 2982-2989. 1989. 59. Ujihara M. Urade Y, Eguchi N. Hayashi H, Ikai K. Hayaishi 0. Prostaglandin D, formation and characterization of its synthetases in various tissues of adult rats. Archives of Biochemistry and Biophysics 260: 521-531, 1988. 60. Urade Y, Watanabe K. Eguchi N, Fujii Y, Hayaishi 0. 9a, 11 B-prostaglandin F? formation in various bovine tissues. Different isozymes of prostaglandin D2 llketoreductase, contribution of prostaglandin F synthetase and its cellular localization. Journal of Biological Chemistry 265: 12029-12035, 1990. 61. Nichols F C, Schenkein H A. Rutherford R B. Prostaglandin E?. prostaglandin E, and thromboxane B? release from human monocytes treated with C3b or bacterial lipopolysaccharide. Biochimica et Biophysics Acta 927: 149-157, 1987. 62. Lewis R A. Austen F K. The biologically active leukotrienes. Biosynthesis. metabolism, receptors. functions, and pharmacology. Journal of Clinical Investigation 73: 889-897. 1984. 63. Lagarde M, Gualde N. Rigaud M. Metabolic interactions

64.

65.

66.

67.

68.

69.

70.

71.

72. 73.

74.

75.

76.

77.

78.

79.

80.

81.

between eicosanoids in blood and vascular cells. Biochemical Journal 257: 921-934. 1989. Delebasste S, Cogny Van Weydevelt F, Gualde N. Effect of arachidonic acid metabolites on thymus tolerance Annals of the New York Academy of Sciences 524: 227-229. 1988. Goldyne M E. Laidler R. Stimulated T cell and natural killer (NK) cell lines fail to synthesize leukotriene B,. Prostaglandins 34: 783-795, 1987. Fitzpatrick F, Liggett W. McGee J. Bunting G, Morton S. Samuelsson B. Metabolism of leukotriene A, by human erythrocytes. A novel cellular source of leukotriene B,. Journal of Biological Chemistry 259: 11403-l 1407, 1984. Maclouf J. Fruteau de Laclos B, Borgeat P. Effects of 12.hydroxy- and 12-hydroperoxy-5, 8. 10, 14eicosatetraenoic acids on the synthesis of 5.hydroxy-6. 8. 11. 14-eicosatetraenoic acid and leukotriene B, in human blood leukocytes. Advances in Prostaglandin and Thromboxane Leukotriene Research 11: 159-162,1983. Papiemik M, Nabbara B,-Savino W, Pontoux C., Barbey S. Thymic reticulum in mice. II. Culture and characterization of nonepithelial phagocytic cells of the thymic reticulum:their role in the syngeneic stimulation of thymic medullary lymphocytes. European Journal of Immunology 13: 147-155. 1983. Papiemik M, Penit C, El Rouby S. Control of prothymocyte proliferation by thymic accessory cells. European Journal of Immunology 17: 1303-1310. 1987. Denning S M, Kurtzberg J. Le P T, Tuck D. Singer K H. Haynes B F. Human thymic epithelial cells directly induce activation of autologous immature thymocytes. Proceedings of the National Academy of Sciences of the United States of America 85: 3125-3129, 1988. Hayari Y. Kukulansky T, Globerson A. Regulation of thymocyte proliferative response by macrophage-derived prostaglandin E, and interleukin I. European Journal of Immunology 15: 4347, 1985. Wood G W. Macrophages in the thymus. Survey of Immunology Research 4: 179-191. 1985. Milicevic N M. Milicevic 2. Colic M. Mujovic S. Ultrastructural study of macrophages in the rat thymus, with special reference to the cortico-medullary zone. Journal de I’Anatomie et de la Physiologie Normales et Pathologiques de I’Homme et des Animaux 150: 89-98, 1987. Bach M A, Bach J F. Effets de I’AMP cyclique sur les cellules formant les rosettes spontanees. Comptes Rendus Academic des Sciences Serie D Paris 275: 2783-2786. 1972. Bach M A. Bach J F Studies on thymus products. VI. The effects of cyclic nucleotides and prostaglandins on rosette-forming cells. Interactions with thymic factor. European Journal of Immunology 3: 778-783. 1973. Garaci E. Rinaldi-Garaci C. Del Gobbo V. Favalli C, Santoro G, et al. A synthetic analog of prostaglandin Ez is able to induce in vivo theta antigen on spleen cells of adult thymectomized mice. Cellular Immunology 62: 8-14, 1981. Rinaldi-Garaci C. Del Gobbo V. Favalli C. Garaci E, Bistoni F. et al Induction of serum thymic-like activity in adult thymectomized mice by a synthetic analog of PGE?. Cellular Immunology 72: 97-101. 1982. Rinaldi-Garaci C. Jezzi T. Baldassare A M. Dardenne M. Bach J F, Garaci E. Effect of thymulin on intracellular cyclic nucleotides and prostaglandins E: in peanut agglutinin- fractionated thymocytes. European Journal of Immunology. 15: 548-552, 1985. Kook A I. Trainin N. The control exerted by thymic hormone (THF) on cellular CAMP levels and immune reactivity of spleen cells in the MLC assay. The Journal of Immunology 115: 8-14.1975. Homo-Delarche F. Gagnerault M C, Bach J F. Dardenne M. Thymic hormones and prostaglandins. II. Synergistic effect on mouse spontaneous rosette forming cells. Prostaglandins 39: 299-3 18.1990. Wyllie A H. Glucocorticoid-induced thymocyte

Role of Thymus-Eicosanoids

82

83

84

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95. 96.

91.

apoptosis is associated with endogenous endonuclease activation. Nature 284: 555-558, 1980. McConkey D J, Orrenius S, Jondal M. Agents that elevate CAMP stimulate DNA fragmentation in thymocytes. The Journal of Immunology 145: 1227-1230, 1990. Suzuki K, Tadakuma T, Harutoshi K. Modulation of thymocyte apoptosis by isoproterenol and prostaglandin Ez Cellular Immunology. 134: 235-240, 199 1. Harford A M, Shopp G M, Goodwin J S, Ebbesen M A. Gibe1 L, Smith A Y, Sterling W A. Murine thymocyte lysis by inhibitors of arachidonic acid metabolism. Advances Prostaglandins Thromboxane Leukotriene Research 21B: 489-492. 199 I. Meade C J. How arachidonic acid depresses thymus weight. International Archives of Allergy and Applied Immunology 59: 432436, 1979. Schaumburg B P. Binding of prostaglandin E, to rat thymocytes. Biochimica et Biophysics Acta 326: 127-133, 1973. Whitfield J F, MacManus J P. Braceland B. Gillan D J. Inhibition by calcium of the cyclic AMP-mediated stimulation of thymic lymphoblast proliferation by prostaglandin E,. Hormone and Metabolic Research 4: 304-308, 1972. Franks D J, MacManus J P, Whitfield J F. The effect of prostaglandins on cyclic AMP production and cell proliferation in thymic lymphocytes. Biochimica et Biophysics Acta 44: 1177-l 183, 1971. Delebassee S, Gualde N. Effect of arachidonic acid metabolites on thymocyte proliferation, Annales de 1’Institut Pasteur/Immunology 139: 383-399, 1988. Toth M, Zakar T, Antoni F. Inhibitory effect of prostaglandin E, on the incorporation of 3H-thymidine into the DNA of thymus ceils. Acta Biochimica Biophysics Academy of Sciences Hungary 10: 161-169, 1975. Gualde N, Cook JM, Guibert F. Effect of PGEz and LTB, on vicia villosa binding lymphocytes. Acta Pathologica et Microbiologica Scandinavica (C) 96: 531-536, 1988. Mexmain S. Cook J M. Aldigier J C, Gualde N, Rigaud M. Thymocyte cyclic AMP and cyclic GMP response to treatment with metabolites issued from the lipoxygenase pathway. The Journal of Immunology 135: 136 I-1 365. 1985. Simantov R, Sachs L. Role of phospholipase Az and prostaglandin E in growth and differentiation of myeloi’d leukemic cells. Biochimica et Biophysics Acta 720: I I l-l 19. 1982. Mahnvaud G, Gaillard S, Chable H. Allegros A. Rigaud M. Effet inhibiteur du 15HPETE sur les cellules souches granulocytaires (CFUGM). Nouvelle Revue Francaise d’HCmatologie (Paris) 25: 319-322, 1983. Patchen M L. Immunomodulators and hemopoiesis. Survey of Immunology Research 2: 237-242. 1983. Claesson H E, Dahlberg N, Gahrton G. Stimulation of human myelopoiesis by leukotriene BJ. Biochimica et Biophysics Acta 131: 579-585. 1985. Snyder D S. Desforges J F. Lipoxygenase metabolites of

98

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111. 112.

in the Immune Response

arachidonic acid modulate hematopoiesis. Blood 67: 1675-1679, 1986. Pelus L M, Gentile P S. In vivo modulation of myelopoiesis by prostaglandin E?. III. Induction of suppressor cells in marrow and spleen capable of mediating inhibition of CFU-GM proliferation. Blood 6: 1633-1640. 1988. Nieburgs A C, Kom J H. Picciano PT. Cohen S. Thymic epithelium in vitro. IV. Regulation of growth and mediator production by epidermal growth factor. Cellular Immunology 108: 396404. 1987. Gualde N. Cook J M. Role of double negative thymocytes (CD4[-]CD8[-1) in the induction of tolerance to self. FASEB Journal 2: A478 (abstract), 198X. Delebassee S. Cogny Van Weydevelt F, Gualde N. Effects of eicosanoids on thymocyte physiology. Immunobiology Supp 3: 227-228. 1987. Gualde N. Cogny Van Weydevelt F, Buffiere F. Jauberteau M 0, Daculsi R. Vaillier D. Influence of LTB, on CD4[-1, CD8[-] thymocytes. Evidence that LTB, plus IL-2 generate CD8[+] suppressor thymocytes involved in tolerance to self. Thymus 18: I 11-I 28, 199 I Gualde N. Rigaud M. Goodwin J S. Induction of suppressor cells from peripheral blood T cells by ISHPETE. Clinical Research 3 I : 490-492, 1983. Gualde N. Goodwin J S. Induction of suppressor T cells in vitro by arachidonic acid metabolites issued from the lipoxygenase pathway. p 565 in Prostaglandins. Leukotrienea and Lipoxins. (M Bailey ed). Plenum Press New York 1985. Atluru D. Goodwin J S. Control of polyclonal immunoglobulin production from human lymphocytes by 1eukotrienes;leukotriene BJ induces an OKT8(+), radiosensitive suppressor cell from resting human OKT8(-)T cells. Journal of Clinical Investigation 74: 1444-1450, 1984. Rola-Pleszczynski M. Leukotriene B4 induces human suppressor lymphocytes. Biochimica et Biophysics Acta 4: 1531-1537, 1985. Swat W, lgnatowicz L, Von Boehmer H. Kisielow P. Clonal deletion of immature CD4+8+ thymocytes in suspension culture by extrathymic antigen-presenting cells. Nature 351: 15&153. 1991. Blackman M A. Gerhard-Burgert H. Woodland D L, Palmer E, Kappler J W, Marrack P. A role for clonal inactivation in T cell tolerance to MIS-l, Nature 345: 540-542. 1990. Ohashi P, Pircher H. Burki 2, Zinkemagel A M, Hengartner H. Distinct sequence of negative or positive selection implied by thymocyte T-cell receptor densities. Nature 346: 861-863, 1990. Fry A M. Jones L A, Kruisbeek A M. Matis L A. Thymic requirement for clonal deletion during T cell development. Science 246: 1044-1046, 1989. Bumet F M. The clonal selection theory of acquired immunity. Cambridge University Press. London, 1959. Cohen I R, Wekerle H. Regulation of autosensitization. The immune activation and specific inhibition of selfrecognizing thymus-derived lymphocytes. Journal of Experimental Medicine 137: 224-238. 1973.

255