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Extrathymic pathways of T-cell differentiation and immunomodulation
International Immunopharmacology 1 Ž2001. 1261–1273 www.elsevier.comrlocaterintimp
Review
Extrathymic pathways of T-cell differentiation and immunomodulation Toru Abo ) Department of Immunology, Niigata UniÕersity School of Medicine, Niigata 951-8510, Japan Received 11 September 2000; received in revised form 7 February 2001; accepted 9 February 2001
Abstract It is well established that the thymus is an essential organ for the support of T-cell differentiation. However, some T cells, termed extrathymic T cells, have been found to differentiate without such support by the thymus. The major sites of these T cells are the intestine and liver. Subsequent studies have revealed that extrathymic T cells are also present in the uterus and exocrine glands Že.g., the salivary gland.. Depending on the sites, extrathymic T cells have some distinct properties as well as some common properties. For example, all extrathymic T cells have a TCR-CD3 complex similar to thymus-derived T cells. Extrathymic T cells comprise both ab T cells and gd T cells. Although extrathymic T cells are very few in number at any extrathymic sites in youth, they increase in number as a function of age. This phenomenon seems to occur in parallel with thymic involution. Even in youth, extrathymic T cells are activated in number and function by stress, in autoimmune diseases, and during pregnancy. Acute thymic atrophy always accompanies this activation. Therefore, reciprocal regulation between extrathymic T cells and thymus-derived T cells might be present. We hypothesize that extrathymic T cells are intimately associated with innate immunity and that the mechanisms underlying autoimmune diseases and intracellular infection Že.g., malaria. cannot be properly understood without introducing the concept of extrathymic T cells. q 2001 Elsevier Science B.V. All rights reserved. Keywords: T-cell differentiation; Extrathymic T cells; Liver; IL-2R b-chain; Natural killer T cells; Autoimmune disease
1. Properties of extrathymic T cells Although extrathymic T cells have slightly distinct properties depending on the sites, they consistently express IL-2R b-chain ŽIL-2R b . on the surface w1,2x. Similar to the case of NK cells, extrathymic T cells have the IL-2R aybq phenotype Ži.e., an intermediate affinity IL-2R.. In contrast, )
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conventional T cells Ži.e., thymus-derived T cells. have the IL-2R ayby phenotype under resting conditions but have the IL-2R aq bq phenotype Ži.e., a high affinity IL-2R. under activated conditions. In other words, NK cells and extrathymic T cells consistently express intermediate affinity IL-2R under usual conditions w3x. Reflecting this situation, NK cells and extrathymic T cells respond quickly to corresponding antigens w4x. It is speculated that conventional T cells acquired a resting state in phylogenic development but NK cells and extrathymic T
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 0 5 7 - 1
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cells did not yet w5x. Both NK cells and extrathymic T cells are granular lymphocytes in morphology w6x. In the case of extrathymic T cells in the liver, they have an intermediate Žint. density of TCR-CD3 complex on the surface w7x. We call them TCRint cells ŽFig. 1.. On the other hand, conventional T cells are estimated to be TCRhigh cells. Both extrathymic ab T and gd T cells are present in the liver at a ratio of 4:1. This ratio increases with aging w8,9x. In other words, the number and proportion of ab T cells tend to increase in aged mice. Extrathymic T cells, especially ab T cells, in the liver are a mixture of double-negative ŽDN. CD4y 8y, CD4q, and CD8q cells w5x. Extrathymic T cells in the intestine mainly localize at the intraepithelial site ŽIEL. and in the lamina propria ŽLPL. w8,9x. IEL comprise ab T and gd T cells at a ratio of 1:1 and this ratio increases with aging. gd T cells express a higher level of IL-2R b than do ab T cells ŽFig. 2.. LPL are mainly ab T cells and express an intermediate level of IL-2R b between ab T and gd T cells of IEL. Concerning
Fig. 2. IL-2R b expression of ab T and gd T cells in IEL of the small intestine. IEL were isolated from the small intestine and two-color staining of IEL was conducted at indicated combinations.
these results, gd T cells are estimated to be the earliest phenotype of T lymphocytes in phylogeny. gd T cells in the intestine are either DN or CD8q, while ab T cells are CD8q or double-positive ŽDP. CD4q8q. Most, if not all, extrathymic T cells in the intestine are CD8 aa Ža homodimer of CD8. w10x.
Fig. 1. A comparison of phenotype between extrathymic T cells and conventional T cells in young mice Ž8 weeks. and aged mice Ž50 weeks.. Extrathymic T cells are identified as IL-2R bq CD3 int while conventional T cells are IL-2R by CD3 high . The proportion of extrathymic T cells increase with aging Žindicated by arrowheads..
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In contrast, all thymus-derived T cells are CD8 ab Ža heterodimer of CD8.. Although IEL in the small intestine are considered to be mainly of extrathymic origin, it is also true that the generation of these IEL is highly influenced by presence of the thymus. Indeed, athymic nude mice and neonatally thymectomized mice show the decreased number of IEL in the small intestine w11x. With regard to this subject, Lefrancois and Puddington w12x proposed possible pathways of IEL development in the small intestine, in which some precursors of IEL may enter the thymus or IEL may be acted upon by thymic-derived factors Žor cells. for their differentiation in the small intestine. Since I mainly focused attention on extrathymic T cells in the liver and their immunomodulation in this review, I recommend Lefrancois’ reviews to further learn the properties and differentiation of IEL in the small intestine. Extrathymic T cells in the large intestine, uterus, and salivary gland carry different properties, especially in IL-2R b expression. Extrathymic T cells in the large intestine and salivary gland express a high level of IL-2R b , similar to those in the liver w10,13x. On the other hand, extrathymic T cells in the uterus express an intermediate level of IL-2R b , similar to those in LPL of the small intestine. Of course, NK cells, which are the earliest form of lymphocytes in phylogeny, express the highest level of IL-2R b among all lymphocyte subsets. The expression level of IL-2R b might be important for considering their phylogeny and function. The earlier phylogenetic form of extrathymic T cells tends to express a higher level of IL-2R b . Moreover, such extrathymic T cells which express a high level of IL-2R b are able to respond immediately to the corresponding antigens. The expression of IL2R b on NK cells is the highest and therefore these cells respond to antigens most quickly w4x. After infection, the activation of lymphocytes usually occurs in the following order: NK cells ™ extrathymic T cells ™ thymus-derived T cells. Although experimental findings are limited, TCRs used by extrathymic T cells seems to be at the level of oligo-diversity and oligo-clonality w14x. This might be due to their early phylogenic development. For example, Vb 8 is preferentially used for TCR b by extrathymic T cells and there are very few N insertion Že.g., Vb 8..
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2. Relationship between extrathymic T cells and NKT cells We first characterized extrathymic T cells as DN CD4y8y TCRint cells carrying forbidden clones, in the liver around 1990 w7,15,16x. In earlier and subsequent studies w17–19x, T cells with a NK marker, NK1.1, were identified in the thymus, although they are an extremely minor population. Attention has been focused on these T cells, because they carry many properties distinct from conventional T cells which are generated in the mainstream of the thymus. They are CD4q or DN CD4y8y. These T cells were termed natural killer T cells Ži.e., NKT cells.. NKT cells are localized in the medullary region of the thymus and a few of them are also dispersed in the cortex region w20x. It is speculated that NKT cells are generated through an alternative intrathymic pathway. Recent studies have revealed that these NKT cells preferentially use an invariant chain of Va 14Ja 281 for TCR a w21x and recognize glycolipid antigens as well as peptide antigens in the context of MHC class I-like antigen, CD1d w22x. Even in the liver, NKT cells are preferentially present and approximately 50 to 60% of TCRint cells are NK1.1q w5x. In other words, NKT cells belong to TCRint cells and TCRint cells are a mixture of NK1.1q and NK1.1y subsets ŽFig. 3.. The presence of the NK1.1y TCRint subset is obvious, because almost all extrathymic T cells seen in the liver and spleen of athymic nude mice are such cells. They are also IL-2R bq and have the morphology of granular lymphocytes. There has been a controversy as to whether NKT cells in the liver are generated in situ or are all supplied by the thymus. Athymic nude mice w23,24x and neonatally thymectomized mice w25,26x carry extremely few NKT cells in the liver and other immune organs. Moreover, when lethally irradiated, adult-thymectomized mice were subjected to bone marrow cell transplantation or fetal liver cell transplantation, CD4q NKT cells could not be generated in the liver w27,28x. It is therefore conceivable that CD4q NKT cells using Va 14Ja 281 are generated in the thymus and then migrate into the liver. We previously reported that the cell fraction of NKT cells in the liver did not accept the partner cells in parabiotic mice, ever 2 weeks after the operation
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Fig. 3. The NK1.1q and NK1.1y subsets of extrathymic T cells. Intermediate TCR cells Ži.e., TCRint cells. comprise both NK1.1q and NK1.1y subsets, showing the different patterns of CD4q, CD8q, and DN CD4y8y phenotypes.
w29x. On the other hand, up to 50% of conventional T cells and some extrathymic T cells in the liver accepted the partner cells by 2 weeks after the operation. Initially, we considered that NKT cells may be generated in situ in the liver. Concerning the above results of parabiosis, we speculate that NKT cells or their progenitors in the liver are derived from the thymus at the neonatal stage and that these liver NKT cells undergo renewal in situ in the liver. However, if these NKT cells or their progenitors are completely impaired at the adult stage, they have to be supplied by the thymus again.
3. Why the liver and intestine are the major sites for extrathytmic T cell differentiation Extrathymic T cells are abundant in the liver and intestine w8,9x. Since the liver phylogenetically developed from the intestine as an exocrine gland to excrete bile, it and the intestine share similar properties, namely, the continuous renewal of epithelia
Žhepatocytes themselves in the liver. and the presence of extrathymic T cells. The emergence of living beings onto land made possible by the development of pulmonary respiration which occurred 360 million years ago w30x. Until that time, they had carried the gills which then developed into the thymus. Phylogenetically, all these organs in the digestive tract, i.e., the gills, intestine, and liver, developed lymphoid cells in their surroundings Žpossibly from ancient macrophages.. It is therefore speculated that the thymus or gills became the organ which generated conventional T cells, because it continued to be exposed to stimulation by foreign antigens. On the other hand, the intestine and liver remained as sites where extrathymic T cells continued to be generated even thereafter. In the case of the thymus, this organ comprises both tissues of endodermal and epidermal origins. The epidermal tissue originated from the gill holes. In this regard, the medullary region in the thymus still carries the epidermal epithelia and possesses an alternative intrathymic pathway w31x. The main-
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stream of T-cell differentiation in the thymus gives rise to conventional T cells by negative selection of self-reactive forbidden clones. On the other hand, an alternative intrathymic pathway of T-cell differentiation gives rise to the NK1.1q subset of TCRint cells Ži.e., NKT cells. as well as the NK1.1y subset of TCRint cells. This alternative pathway may be a remnant of T-cell differentiation which took place in the ancient skin w32x. With the emergence of living beings onto land, conventional T cells produced by the thymus migrated to the lymph nodes and the spleen Žespecially the white pulp. whereas NKT cells produced by the thymus migrated to the liver and some other organs.
4. Elements supportive of extrathymic T-cell differentiation We have previously reported that c-kitq stem cells exist even in the adult liver w33x. These c-kitq cells isolated from the liver have potential as pluripo-
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tent stem cells; namely, they give rise to erythroid cells in the bone marrow, extrathymic T cells Žand granulocytes. in the liver, and conventional T cells in the thymus when transferred into lethally irradiated mice. When mice are administered with estrogen, many clusters of lymphoid cells appear in the parenchymal space of the liver. c-kitq stem cells are always present in the center of such clusters. In the fetal stage, it is well known that the liver is a hematopoietic organ. It is therefore concluded that the liver retains c-kitq stem cells even after birth and continues to produce extrathymic T cells thereafter. Similar to the case of fetal hematopoiesis, the generation of extrathymic T cells occurs in the parenchymal space. At that time, IL-7 is extremely important as one of the initial growth factors for extrathymic T cells w34x. For functional maturation after extrathymic T cells migrate to the sinusoidal lumen, IL-12, IL-15 and IL-18 might then be important ŽFig. 4.. For extrathymic T cells to maintain their specific localization Že.g., extrathymic sites., the adhesion molecules on their surface might be essentials. In-
Fig. 4. Supportive elements of extrathymic T-cell differentiation. IL-7 which is produced by hepatocytes, is an initial growth factor for extrathymic T cells. IL-12, IL-15, and IL-18 which are produced by Kupffer cells or endothelial cells, are subsequent maturational factors.
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deed, they express the unique pattern of adhesion molecules, CD44q L-selectiny LFA-1high w35,36x. In sharp contrast, conventional T cells have the pattern of adhesion molecules such as CD44y L-selectinqLFA-1low . This inverted expression pattern between extrathymic T cells and conventional T cells may define their localization.
5. Autoreactivity of extrathymic T cells In the case of mice, forbidden clones can be identified by the Mls system. Thus, superantigens which are the products of retrovirus infected in mice, are recognized by some specific Vbq cells. By using this system, we have demonstrated that forbidden clones are confined to IL-2R bq TCRint cells, but are not found in IL-2R by TCRhigh cells at all w31x. In other words, conventional T cells which are generated through the mainstream of T-cell differentiation in the thymus, do not contain forbidden clones ŽFig. 5.. This might be due to the fact that complete
negative selection of forbidden clones occurs in the mainstream. Even under immunosuppressive conditions induced by the administration of immunosuppressive drugs, a leakage of forbidden clones into conventional TCRhigh cells is not seen w37,38x. Namely, forbidden clones are always confined to TCRint cells. As already mentioned, TCRint cells are generated through both an extrathymic pathway in the liver and an alternative intrathymic pathway. Therefore, forbidden clones are generated through both pathways. Incomplete negative selection of forbidden clones might be due to the primitive nature of TCRint cells, for example, an intermediate density of TCR-CD3 complex, the aa homodimer of CD8 antigens, the lack of the expression of CD4 and CD8 antigens in some subsets, etc. In addition to the presence of forbidden clones, TCRint cells eventually mediate autoreactive cytotoxicity w38,39x. Targets of such cytotoxicity include syngeneic thymocytes, regenerating hepatocytes, and others w40x. It is well established that CTL Ži.e.,
Fig. 5. Pathways of T-cell differentiation. The thymus contains an alternative intrathymic pathway as well as the mainstream of T cell differentiation. Extrathymic pathways in the liver and the alternative intrathymic pathway give rise to IL-2R bq TCRint cells which contain forbidden clones.
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cytotoxicic T cells which are TCRhigh cells. could use both the perforin system and the Fas ligandrFas system. In the case of NK cells, the major cytotoxic molecule is perforin. On the other hand, TCRint cells mediate autoreactive cytotoxicity by using mainly the Fas ligandrFas system w38x. However, TCRint cells are able to use perforin Žagainst tumor targets. or TNFa in some situations. Concerning the presence of forbidden clones among TCRint cells and their autoreactive cytotoxicity, TCRint cells may usually mediate their cytotoxicity against some rapidly dividing self-cells. These functions of TCRint cells are beneficial to eliminate abnormal self-cells such as virally infected cells and malignant tumor cells. Since TCRint cells are present in the pregnant uterus, rapidly growing fetal cells might also be the targets for TCRint cells w41x. If such function of TCRint cells in the uterus fails, an invasion of fetal cells into the mother may occur. Examples of this are cases of hydatidiform moles Žbenign. and choriocarcinoma Žmalignant..
6. When are extrathymic T cells activated? 6.1. Aging Extrathymic T cells including NKT cells are very few in number in youth, even in the liver, and expand with aging. In other words, extrathymic T cells tend to expand in parallel with thymic involution. In the case of mice, IL-2R bq TCRint cells become detectable at the age of 4 weeks in the liver w2,8,9x. This is also true of NKT cells w42,43x. Up to the age of 1.5 years, the number and proportion of IL-2R bq TCRint cells and NKT cells increase but the number and proportion of NKT cells then decline. As a result, NK1.1y TCRint cells expand in number and proportion after the age of 1.5 years. In youth, extrathymic T cells and NKT cells are detectable only in the liver, but with aging these T cells become prominent in various immune organs, including the spleen, lymph nodes, bone marrow, and even the thymus. IL-2R bq TCRint cells and NKT cells in the thymus mainly localize in the medullary region. After the age of 50 weeks, the number and proportion of IL-2R bq TCRint cells and NKT cells overcome those of IL-2R by TCRhigh
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cells Ži.e., conventional T cells or thymus-derived T cells. in various immune organs w5x. The increase in the number of extrathymic T cells and NKT cells with aging might be important for the elimination of abnormal self-cells which appear in the body with aging. Even in the exocrine glands such as the salivary glands, infiltration by many extrathymic T cells or NKT cells occurs with aging. These T cells may provide innate immunity for eliminating microbially infected self-cells and malignant tumors. 6.2. Stress When mice are exposed to restraint stress, acute thymic atrophy is induced w44,45x. At this time, the number of DP CD4q8q thymocytes decrease prominently due to their apoptosis. Even the number of TCRhigh cells, which are derived from the thymus and migrate to the peripheral immune organs, tend to decrease in various peripheral immune organs. In sharp contrast, the number of TCRint cells and that of NKT cells hardly decrease in various immune organs due to stress. Therefore, the proportion of TCRint cells and NKT cells becomes prominent in all these organs after exposure to stress. Since TCRint cells and NKT cells mediate autoreactive cytotoxicity, this increased population of T cells might be important for eliminating abnormal self-cells which appear due to stress. Stress-associated factors which induce thymic atrophy and the apoptosis of TCRhigh cells in the peripheral immune organs may include glycocorticoids, catecholamines, and inflammatory cytokines Že.g., IFNg and TNFa . w44,45x. Indeed, any of these factors eventually has the ability to induce acute thymic atrophy and inversely increase the proportion of TCRint cells or NKT cells in the periphery. 6.3. Autoimmune diseases I feel that we cannot understand the mechanisms underlying autoimmune diseases without introducing the concept of extrathymic T cells. When we observe autoimmune prone ŽNZB = NZW. F1 mice ŽNZBrW F1 mice., thymic atrophy is always accompanied by the onset of disease ŽFig. 6.. At that time, the expansion of extrathymic T cells, including NKT
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trathymic T cells. A certain retroviral transposon is inserted into the Fas gene. In the case of conventional T cells, some molecules other than Fas are also involved in the apoptosis of these T cells. On the other hand, only Fas may be associated with the apoptosis of TCRint cells. As a result, only the expansion of TCRint cells is seen in these MRL-lprr lpr mice. A decrease in the number of NKT cells is known to occur in diabetic-prone NOD mice w25,26,47,48x. Therefore, many investigators suspect that NKT cells are immunoregulatory T cells which act against the onset of autoimmune diseases. In these cases, the autoreactivity of NKT cells Ži.e., NK1.1q TCRint cells. functions to suppress some autoreactive T cells Že.g., NK1.1y TCRint cells.. This subject remains to be further investigated. 6.4. Pregnancy Fig. 6. Thymic involution seen in autoimmune prone NZBrW F1 mice. Severe thymic involution is always seen in these autoimmune mice, although the number of lymphocytes increases in other organs.
cells, occurs in the liver and other peripheral immune organs. CD5qB cells which produce autoantibodies, expand in the peritoneal cavity. It is conceivable that the autoreactivity of TCRint cells and NKT cells as well as that of CD5qB cells might be intimately associated with the onset of autoimmune disease. Many investigators have believed that self-reactive forbidden clones are generated by failure of the mainstream of T cell differentiation in the thymus. However, this concept is doubtful. Thus, forbidden clones are always confined to TCRint cells w31,37,38x. Moreover, as mentioned above, thymic atrophy is always accompanied by the onset of autoimmune diseases. We have to consider the possibility that the overactivation of extrathymic T cells, which primarily contain forbidden clones, might be associated with the onset of autoimmune diseases. In the case of MRL-lprr lpr mice, an abnormal expansion of B220q DN CD4y8y TCRint cells is seen in various immune organs w15,46x. This is due to the fact that a defective Fas gene Ži.e., lymphoproliferative, lpr gene. permits the expansion of ex-
Extrathymic T cells are activated in the uterus as well as in the liver in the late phase of pregnancy w41,49x. Similar to the case of stress, thymic atrophy accompanies this phenomenon. We consider that estrogen and sympathetic nerve activation might be associated with this phenomenon w42,43,50,51x. During pregnancy, a large amount of estrogen is secreted from the placenta while sympathetic nerve activation is induced to supply oxygen and nutrients from the mother to the fetus. We have previously reported that the exogenous administration of estrogen or the administration of adrenaline activates extrathymic T cells in the liver of mice in number and function w42,43,50,51x. These administrations also induce acute thymic atrophy. It is therefore conceivable that the major factors which induce the pregnancy-associated phenomenon may be both estrogen and catecholamines. In the case of fetuses, trophoblasts of the placenta, which lack the expression of polymorphic MHC antigens, adhere directly to the mother. This situation enables the mother not to reject the fetus during pregnancy. Instead, there is a danger of fetal tissues invading the mother Že.g., choriocarcinoma.. Activation of NK cells and extrathymic T cells in the uterus might be important for protection of the mother from such invasion w41x. In other words, by using a
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physiologically inevitable response Ži.e., the secretion of estrogen and the activation of the sympathetic nervous system., the mother achieves a successful pregnancy. When the mother experiences over-activation of the sympathetic nerves for some reason, there is a danger that she may fall victim to pregnancy disorders. Such disorders include preeclampsia and hyperemesis gravidarum w41x. Indeed, patients with these disorders suffer from hypertension, tachycardia, palpitation, etc. All these symptoms are induced by the over-activation of the sympathetic nervous system. In more detail, the over-activation of granulocytes, NK cells, and extrathymic T cells might be induced in the uterus due to the over-activation of the sympathetic nerves. Thus, these leukocytes carry adrenergic receptors on the surface and are activated in number and function by sympathetic nerve activation w52x. Causes of the over-activation of sympathetic nerves might include intrinsic factors in the mother Že.g., insufficiency of cardiac function or other organ-functions. and extrinsic factors Že.g., overwork and mental stress.. 6.5. Malarial infection The body seems to be protected against an intracellular protozoa pathogen, malaria, by innate immunity. This notion is supported by the fact that malarial infection induces a prominent expansion of extrathymic T cells Ži.e., IL-2R bq TCRint cells. in the liver. No expansion is seen in conventional T cells Ži.e., IL-2R by TCRhigh cells.. When TCRint cells isolated from the liver of mice which had recovered from malarial infection were injected into irradiated recipient mice, these mice could survive malaria without parasitemia. In the case of the administration of a-galactosylceramide Ž a-GalCer., the major expanding population is the NK1.1q subset of IL-2R bq TCRint cells Ži.e., NKT cells. w53x. In sharp contrast, the major expanding population is the NK1.1y subset of IL-2R bq TCRint cells during malarial infection. Namely, NKT cells, which express CD4 antigen and use an invariant chain of Va 14Ja 281, are not a major population in the protection of the body against malaria.
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Malaria infection is accompanied by severe thymic atrophy and the proportion of TCRhigh cells rather decreases in the periphery. Indeed, there is no protective effect of TCRhigh cells when these T cells Žisolated from mice recovered from malaria. are injected into irradiated mice. In addition to the activation of extrathymic T cells in the liver of mice with malarial infection, we found that the production of autoantibodies such as antiDNA antibody is seen in the sera of these mice. In other words, many signs during malarial infection mimic those seen in autoimmune diseases. These results also support our notion that the body is protected against malaria by innate immunity. 6.6. Administration of nonsteroidal anti-inflammatory drugs (NSAIDs) Many clinicians have believed that the administration of NSAIDs is useful for reducing inflammation. In some cases of inflammation associated with protaglandins Ži.e., inflammation associated with conventional T cells., this may be true. However, when NSAIDs are continuously administered into mice, a prominent activation of granulocytes, NK cells, and extrathymic T cells is induced in the liver and other peripheral organs w54x. This might be due to the fact that NS AIDs suppress the production of prostaglandins via the suppression of cyclooxygenase, and then induce the activation of the sympathetic nervous system. The down-regulation of prostaglandin synthesis always induces subsequent activation of sympathetic nerves, because prostaglandins and catecholamines together comprise an antagonistic system. Since granulocytes, NK cells, and extrathymic T cells carry adrenergic receptors on the surface w52x, such sympathetic nerve activation is responsible for the activation of these leukocyte populations in number and function. In addition, thymic atrophy accompanies this phenomenon. Reflecting these responses, inflammations associated with granulocytes, NK cells, and extrathymic T cells worsen when NSAIDs are administered for a long time. More concretely, purulentive diseases Ži.e., bacterial infections., gastric ulcers, ulcerative colitis, rheumatic arthritis Žespecially the chronic phase., and many autoimmune diseases are granulocyte-as-
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sociated inflammations and are worsened by the continuous administration of NSAIDs. 6.7. Relationship of extrathymic T cells with other immunological responses When allogeneic-combination F1 mice are irradiated and then subjected to bone marrow transplantation of one parent origin, such F1 mice sometimes reject the injected bone marrow cells. This phenomenon is known to be caused hybrid resistance. At this time, not only NK cells but also extrathymic T cells Žincluding NKT cells. are found to be effector cells for this hybrid resistance phenomenon. We previously reported that granulocytes suppress the function of NKT cells in G-CSF transgenic mice w55x. Extrathymic T cells and NKT cells are also associated with chronic graft-versus-host ŽGVH. diseases. It is well known that the target tissues for GVH diseases include mainly the liver, intestine, and skin. All these tissues are sites where extrathymic T cells are present. Therefore, the association of extrathymic T cells with GVH disease is in good agreement with their localization. In our most recent study, we observed that unusual CD8q NKT cells are expanding in the liver of neonatal tolerant mice both in youth and adulthood. Since many allogeneic lymphocytes injected at the neonatal stage Ži.e., within day 3 after birth. remain in the spleen, these allogeneic lymphocytes may stimulate CD8q NKT cells in the liver via the portal vein. At mentioned earlier, NKT cells are primarily autoreactive. In this regard, we speculate that alloactivated CD8q NKT cells then mediate neonatal tolerance phenomenon. Splenectomy results in a decrease in the number of CD8q NKT cells. The neonatal tolerance phenomenon disappears thereafter.
7. Human counterparts of extrathymic T cells or NKT cells Human counterparts of extrathymic T cells have been identified as CD56q T cells and CD57q T cells w56,57x. Similar to the case in mice, these T cells are estimated to be granular lymphocytes in morphology.
When compared with the morphology of NK cells, these extrathymic T cells have a smaller cell size and fewer cytoplasmic granules. This morphological nature indicates that extrathymic T cells are at the intermediate position between NK cells and conventional T cells in phylogeny. CD56q T cells are abundant in the liver, intestine, and uterus in humans while CD57q T cells are abundant in the bone marrow, peripheral blood, and joints Žor joint cavity. w56,57x. This situation shows that CD57q T cells are a major population of T cells when patients with leukemia are subjected to bone marrow transplantation after chemotherapy. Usually, CD56q T cells are extremely few in the intestine of healthy persons. However, such CD56q T cells become abundant in the intestine, especially in the small intestine, of patients with ulcerative colitiss. In the case of natural abortion in early pregnancy, both CD56q T cells and CD57q T cells as well as NK cells and granulocytes appear in the interface between the uterus and placenta w41x. These leukocytes might be associated with the abortion Ži.e., the rejection of fetus.. At this time, many CD56q T cells and CD57q T cells appear even in the peripheral blood. In the case of mice, extrathymic T cells comprise both NK1.1q and NK1.1y subsets among TCRint cells. CD56q T cells correspond to NK1.1q TCRint cells while CD57q T cells correspond to NK1.1y TCRint cells. The majority, if not all, of the NK1.1q TCRint cells use an invariant chain of Va 14Ja 281 for TCR a in mice. In contrast, only a minor population of CD56q T cells use Va 24 Žan equivalent human counterpart of mouse Va 14.. This might be due to the fact that a monomorphic MHC class I-like molecule is the only CD1d in mice. In contrast, monomorphic MHC-class I-like molecules include CD1a, CD1b, CD1c, and CD1d in humans. Moreover, monomorphic MHC class I antigens such as HLA-E, HLA-F, and HLA-G are also present. These monomorphic MHC class I antigens also present some self-antigens to CD56q T cells or CD57q T cells. In a recent study, CD161q T cells were also identified as extrathymic T cells in humans. CD56q T, CD57q T, and CD161q T cells are overlapping populations. Although conventional T cells are extremely important for recognizing and eliminating foreign antigens, these extrathymic T cells in humans are invaluable for interacting with and eliminating
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abnormal self-cells which appear with aging and under conditions of stress, malignancy, pregnancy, autoimmune diseases, chronic GVH disease, intracellular infections, etc.
References w1x Watanabe H, Ohtsuka K, Kimura M, Ikarashi Y, Ohmori K, Kusumi A, et al. Details of an isolation method for hepatic lymphocytes in mice. J Immunol Methods 1992;146:145–54. w2x Iiai T, Watanabe H, Seki S, Sugiura K, Hirokawa K, Utsuyama M, et al. Ontogeny and development of extrathymic T cells in mouse liver. Immunology 1992;77:556–63. w3x Watanabe H, Iiai T, Kimura M, Ohtsuka K, Tanaka T, Miyasaka M, et al. Characterization of intermediate TCR cells in the liver of mice with respect to their unique IL-2R expression. Cell Immunol 1993;149:331–42. w4x Ohtsuka K, Sato K, Watanabe H, Kimura M, Asakura H, Abo T. Unique order of the lymphocyte subset induction in the liver and intestine of mice during Listeria monocytogenes infection. Cell Immunol 1995;161:112–24. w5x Watanabe H, Miyaji C, Kawachi Y, Iiai T, Ohtsuka K, Iwanaga T, et al. Relationships between intermediate TCR cells and NK1.1q T cells in various immune organs. NK1.1q T cells are present within a population of intermediate TCR cells. J Immunol 1995;155:2972–83. w6x Bannai M, Oya H, Kawamura T, Shimizu T, Kawamura H, Miyaji C, Watanabe H, Hatakeyama K, Abo T. Disparate effect of beige mutation on cytotoxic function between NK and NKT cells. Immunology 2000;100:165–9. w7x Seki S, Abo T, Ohteki T, Sugiura K, Kumagai K. Unusual ab-T cells expanded in autoimmune lpr mice are probably a counterpart of normal T cells in the liver. J Immunol 1991;147:1214–21. w8x Ohtsuka K, Hasegawa K, Sato K, Arai K, Watanabe H, Asakura H, et al. A similar expression pattern of adhesion molecules between intermediate TCR cells in the liver and intraepithelial lymphocytes in the intestine. Microbiol Immunol 1994;38:677–83. w9x Ohtsuka K, Iiai T, Watanabe H, Tanaka T, Miyasaka M, Sato K, et al. Similarities and differences between extrathymic T cells residing in mouse liver and intestine. Cell Immunol 1994;153:52–66. w10x Ohtsuka K, Hasegawa K, Yamagiwa S, Sato K, Nakayama M, Watanabe H, et al. Intraepithelial lymphocytes in colon have similar properties to intraepithelial lymphocytes in small intestine and hepatic intermediate TCR cells. Dig Dis Sci 1996;41:902–11. w11x Lefrancois L. Extrathymic differentiation of intraepithelial lymphocytes: generation of a separate and unequal T-cell repertoire? Immunol Today 1991;12:436–8. w12x Lefrancois L, Puddington L. Extrathymic intestinal T-cell development: virtual reality? Immunol Today 1991;16:16–21. w13x Narita J, Kawamura T, Miyaji C, Watanabe H, Honda S,
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Koya T, et al. Abundance of NKT cells in the salivary glands but absence thereof in the liver and thymus of alyr aly mice with Sjogren syndrome. Cell Immunol 1999;192:149–58. ¨ Sugahara S, Kuwano Y, Sato K, Hasegawa K, Yamagiwa S, Kawamura T, et al. Oligoclonality of TCRint cells with a low diversity of TCR complementarity-determining region 3 in mice with graft-versus-host disease. Scand J Immunol 1998; 48:592–604. Ohteki T, Seki S, Abo T, Kumagai K. Liver is a possible site for the proliferation of abnormal CD3q 4y 8y double-negative lymphocytes in autoimmune MRL-lprr lpr mice. J Exp Med 1990;172:7–12. Abo T, Ohteki T, Seki S, Koyamada N, Yoshikai Y, Masuda T, et al. The appearance of T cells bearing self-reactive T cell receptor in the livers of mice injected with bacteria. J Exp Med 1991;174:417–24. Budd RC, Miescher GC, How RC, Lees RK, Bron C, MacDonald HR. Developmentally regulated expression of T cell receptor b chain variable domains in immature thymocytes. J Exp Med 1987;166:577–82. Kikly K, Dennert G. Evidence for extrathymic development of TNK cells. NK1q CD3q cells responsible for acute marrow graft rejection are present in thymus-deficient mice. J Immunol 1992;149:403–12. Bendelac A, Lantz O, Quimby ME, Yewdell JW, Bennink JR, Brutiewicz RR. CD1 recognition by mouse NKq T lymphocytes. Science 1995;168:863–5. Kimura M, Watanabe H, Ohtsuka K, Iiai T, Tsuchida M, Sato S, et al. Radioresistance of intermediate TCR cells and their localization in the body of mice revealed by irradiation. Microbiol Immunol 1993;37:641–52. Makino Y, Yamagata N, Sasho T, Adachi Y, Kanno R, Koseki H, et al. Extrathymic development of Va 14-positive T cells. J Exp Med 1993;177:1399–408. Bendelac A. Positive selection of mouse NKq T cells by CD1-expressing cortical thymocytes. J Exp Med 1995; 182:2091–6. Emoto M, Mittrucker H-W, Schmits R, Mak TW, Kaufmann ¨ SHE. Critical role of leukocyte function-associated antigen-1 in liver accumulation of CD4q NKT cells. J Immunol 1995; 162:5094–8. Emoto M, Emoto Y, Kaufmann SHE. CD8 abq TCR ab intermediate lymphocytes expressing skewed TCRVb repertoire in the liver of aged athymic nu r nu mice. J Immunol 1997;158:1041–50. Hammond K, Cain W, van Driel I, Godfrey D. Three day neonatal thymectomy selectively depletes NK1.1q T cells. Int Immunol 1998;10:1491–9. Hammond KJL, Poulton LD, Palmisano LJ, Silveira PA, Godfrey DI, Baxter AG. a r b-T cell receptor ŽTCR.q CD4y CD8y ŽNKT. thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic ŽNOD.rLt mice by the influence of interleukin ŽIL.-4 andror IL-10. J Exp Med 1998; 187:1047–56. Tilloy F, Di Santo JP, Bendelac A, Lantz O. Thymic dependence of invariant Va 14q natural killer-T cell development. Eur J Immunol 1999;29:3313–8.
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w28x Coles MC, Raulet DH. NK1.1q T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4q CD8q cells. J Immunol 2000;164: 2412–8. w29x Suzuki S, Sugahara S, Shimizu T, Tada T, Minagawa M, Maruyama S, et al. Low level of mixing of partner cells seen in extrathymic T cells in the liver and intestine of parabiotic mice: its biological implication. Eur J Immunol 1998; 28:3719–29. w30x Kawamura T, Toyabe S, Moroda T, Iiai T, TakahashiIwanaga H, Fukuda M, et al. Neonatal granulocytosis is a postpartum event which is seen in the liver as well as in the blood. Hepatology 1997;26:1567–72. w31x Kawachi Y, Watanabe H, Moroda T, Haga M, Iiai T, Hatakeyama K, et al. Self-reactive T cell clones in a restricted population of IL-2 receptor bq cells expressing intermediate levels of the T cell receptor in the liver and other immune organs. Eur J Immunol 1995;25:2272–8. w32x Abo T, Kawamura T, Watanabe H. Physiological responses of extrathymic T cells in the liver. Immunol Rev 2000; 174:135–49. w33x Watanabe H, Miyaji C, Seki S, Abo T. c-kitq stem cells and thymocyte precursors in the livers of adult mice. J Exp Med 1996;184:687–93. w34x Miyaji C, Watanabe H, Osman Y, Kuwano Y, Abo T. A comparison of proliferative response to IL-7 and expression of IL-7 receptors in intermediate TCR cells of the liver, spleen and thymus. Cell Immunol 1996;169:159–65. w35x Arai K, Iiai T, Nakayama M, Hasegawa K, Sato K, Ohtsuka K, et al. Adhesion molecules on intermediate TCR cells: I. Unique expression of adhesion molecules, CD44q L-selectiny, on intermediate TCR cells in the liver and the modulation of their adhesion by hyaluronic acid. Immunology 1995;84:64–71. w36x Nakayama M, Arai K, Hasegawa K, Sato K, Ohtsuka K, Watanabe H, et al. Adhesion molecules on interemdiate TCR cells: II. Hepatoprotective effects of hyaluronic acid on acute liver injury. Cell Immunol 1995;166:275–85. w37x Moroda T, Iiai T, Kawachi Y, Kawamura T, Hatakeyama K, Abo T. Restricted appearance of self-reactive clones into intermediate T cell receptor cells in neonatally thymectomized mice with autoimmune disease. Eur J Immunol 1996;26:3084–91. w38x Moroda T, Kawachi Y, Iiai T, Tsukahara A, Suzuki S, Tada T, et al. Self-reactive forbidden clones are confined to pathways of intermediate T cell receptor cell differentiation even under immunosuppressive conditions. Immunology 1997;91: 88–94. w39x Kawamura T, Kawachi Y, Moroda T, Weerashinghe A, Iiai T, Seki S, et al. Cytotoxic activity against tumour cells mediated by intermediate TCR cells in the liver and spleen. Immunology 1996;89:68–75. w40x Minagawa M, Oya H, Yamamoto S, Shimizu T, Bannai M, Kawamura H, et al. Intensive expansion of natural killer T cells in the early phase of hepatocyte regeneration after partial hepatectomy in mice and its association with sympathetic nerve activation. Hepatology 2000;31:907–15.
w41x Minagawa M, Narita J, Tada T, Maruyama S, Shimizu T, Bannai M, et al. Mechanisms underlying immunologic states during pregnancy: possible association of the sympathetic nervous system. Cell Immunol 1999;196:1–13. w42x Tsukahara A, Seki S, Iiai T, Moroda T, Watanabe H, Suzuki S, et al. Mouse liver T cells: their change with aging and in comparison with peripheral T cells. Hepatology 1997; 26:301–9. w43x Tsukahara A, Tada T, Suzuki S, Iiai T, Moroda T, Maruyama S, et al. Adrenergic stimulation simultaneously induces the expansion of granulocytes and extrathymic T cells in mice. Biomed Res 1997;18:237–46. w44x Maruyama S, Tsukahara A, Suzuki S, Tada T, Minagawa M, Watanabe H, et al. Quick recovery in the generation of self-reactive CD4 low NKT cells by an alternative intrathymic pathway when restored from acute thymic atrophy. Clin Exp Immunol 1999;117:587–95. w45x Shimizu T, Kawamura T, Miyaji T, Oya H, Bannai M, Yamamoto S, et al. Resistance of extrathymic T cells to stress and a role of endogenous glucocorticoids in stressassociated immunosuppression. Scand J Immunol 2000; 51:285–92. w46x Yamagiwa S, Kuwano Y, Hasegawa K, Sato K, Ohtsuka K, Iiai T, et al. Existence of a small population of IL-2R b hi TCRint cells in SCG and MRL-lprr lpr mice which produce normal Fas mRNA and Fas molecules from the lpr gene. Eur J Immunol 1996;26:1409–16. w47x Lehuen A, Lantz O, Beaudoin L, Lalous V, Carnauda C, Bendelac A, et al. Overexpression of natural killer T cells protects Va 14-Ja 281 transgenic nonobese diabetic mice against diabetes. J Exp Med 1998;188:1831–9. w48x Falcone M, Yeung B, Tucker L, Rodriguez E, Sarvetnick N. A defect in interleukin 12-induced activation and interferon g secretion of peripheral natural killer T cells in nonobese diabetic mice suggests new pathogenic mechanisms for insulin-dependent diabetes mellitus. J Exp Med 1999;190:963– 72. w49x Kimura M, Hanawa H, Watanabe H, Ogawa M, Abo T. Synchronous expansion of intermediate TCR cells in the liver and uterus during pregnancy. Cell Immunol 1995; 162:16–25. w50x Okuyama R, Abo T, Seki S, Ohteki T, Sugiura K, Kusumi A, et al. Estrogen administration activates extrathymic T-cell differentiation in the liver. J Exp Med 1992;175:661–9. w51x Narita J, Miyaji C, Watanabe H, Honda S, Koya T, Umezu H, et al. Differentiation of forbidden T cell clones and granulocytes in the parenchymal space of the liver in mice treated with estrogen. Cell Immunol 1998;185:1–13. w52x Suzuki S, Toyabe S, Moroda T, Tada T, Tsukahara A, Iiai T, et al. Circadian rhythm of leukocytes and lymphocyte subsets and its possible correlation with the function of autonomic nervous system. Clin Exp Immunol 1997;110:500–8. w53x Osman Y, Kawamura T, Naito T, Takeda K, van Kaer L, Okumura K, et al. Activation of hepatic NKT cells and subsequent liver injury following administration of agalactosylceramide. Eur J Immunol 2000;30:1919–28. w54x Yamamura S, Arai K, Toyabe S, Takahashi EH, Abo T.
T. Abo r International Immunopharmacology 1 (2001) 1261–1273 Simultaneous activation of granulocytes and extrathymic T cells in number and function by excessive administration of nonsteroidal anti-inflammatory drugs. Cell Immunol 1996; 173:303–11. w55x Kawamura H, Kawamura T, Kokai Y, Mori M, Matsuura A, Oya H, et al. Expansion of extrathymic T cells as well as granulocytes in the liver and other organs of G-CSF transgenic mice: why they lost the ability of hybrid resistance. J Immunol 1999;162:5957–64.
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w56x Takii Y, Hashimoto S, Iiai T, Watanabe H, Hatakeyama K, Abo T. Increase in the proportion of granulated CD56q T cells in patients with malignancy. Clin Exp Immunol 1994; 97:522–7. w57x Okada T, Iiai T, Kawachi Y, Moroda T, Takii Y, Hatakeyama K, et al. Origin of CD57q T cells which increase at tumour sites in patients with colorectal cancer. Clin Exp Immunol 1995;102:159–66.
Review
Extrathymic pathways of T-cell differentiation and immunomodulation Toru Abo ) Department of Immunology, Niigata UniÕersity School of Medicine, Niigata 951-8510, Japan Received 11 September 2000; received in revised form 7 February 2001; accepted 9 February 2001
Abstract It is well established that the thymus is an essential organ for the support of T-cell differentiation. However, some T cells, termed extrathymic T cells, have been found to differentiate without such support by the thymus. The major sites of these T cells are the intestine and liver. Subsequent studies have revealed that extrathymic T cells are also present in the uterus and exocrine glands Že.g., the salivary gland.. Depending on the sites, extrathymic T cells have some distinct properties as well as some common properties. For example, all extrathymic T cells have a TCR-CD3 complex similar to thymus-derived T cells. Extrathymic T cells comprise both ab T cells and gd T cells. Although extrathymic T cells are very few in number at any extrathymic sites in youth, they increase in number as a function of age. This phenomenon seems to occur in parallel with thymic involution. Even in youth, extrathymic T cells are activated in number and function by stress, in autoimmune diseases, and during pregnancy. Acute thymic atrophy always accompanies this activation. Therefore, reciprocal regulation between extrathymic T cells and thymus-derived T cells might be present. We hypothesize that extrathymic T cells are intimately associated with innate immunity and that the mechanisms underlying autoimmune diseases and intracellular infection Že.g., malaria. cannot be properly understood without introducing the concept of extrathymic T cells. q 2001 Elsevier Science B.V. All rights reserved. Keywords: T-cell differentiation; Extrathymic T cells; Liver; IL-2R b-chain; Natural killer T cells; Autoimmune disease
1. Properties of extrathymic T cells Although extrathymic T cells have slightly distinct properties depending on the sites, they consistently express IL-2R b-chain ŽIL-2R b . on the surface w1,2x. Similar to the case of NK cells, extrathymic T cells have the IL-2R aybq phenotype Ži.e., an intermediate affinity IL-2R.. In contrast, )
Tel: q81-25-227-2133; fax: q81-25-227-0766. E-mail address: [email protected] ŽT. Abo..
conventional T cells Ži.e., thymus-derived T cells. have the IL-2R ayby phenotype under resting conditions but have the IL-2R aq bq phenotype Ži.e., a high affinity IL-2R. under activated conditions. In other words, NK cells and extrathymic T cells consistently express intermediate affinity IL-2R under usual conditions w3x. Reflecting this situation, NK cells and extrathymic T cells respond quickly to corresponding antigens w4x. It is speculated that conventional T cells acquired a resting state in phylogenic development but NK cells and extrathymic T
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 0 5 7 - 1
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cells did not yet w5x. Both NK cells and extrathymic T cells are granular lymphocytes in morphology w6x. In the case of extrathymic T cells in the liver, they have an intermediate Žint. density of TCR-CD3 complex on the surface w7x. We call them TCRint cells ŽFig. 1.. On the other hand, conventional T cells are estimated to be TCRhigh cells. Both extrathymic ab T and gd T cells are present in the liver at a ratio of 4:1. This ratio increases with aging w8,9x. In other words, the number and proportion of ab T cells tend to increase in aged mice. Extrathymic T cells, especially ab T cells, in the liver are a mixture of double-negative ŽDN. CD4y 8y, CD4q, and CD8q cells w5x. Extrathymic T cells in the intestine mainly localize at the intraepithelial site ŽIEL. and in the lamina propria ŽLPL. w8,9x. IEL comprise ab T and gd T cells at a ratio of 1:1 and this ratio increases with aging. gd T cells express a higher level of IL-2R b than do ab T cells ŽFig. 2.. LPL are mainly ab T cells and express an intermediate level of IL-2R b between ab T and gd T cells of IEL. Concerning
Fig. 2. IL-2R b expression of ab T and gd T cells in IEL of the small intestine. IEL were isolated from the small intestine and two-color staining of IEL was conducted at indicated combinations.
these results, gd T cells are estimated to be the earliest phenotype of T lymphocytes in phylogeny. gd T cells in the intestine are either DN or CD8q, while ab T cells are CD8q or double-positive ŽDP. CD4q8q. Most, if not all, extrathymic T cells in the intestine are CD8 aa Ža homodimer of CD8. w10x.
Fig. 1. A comparison of phenotype between extrathymic T cells and conventional T cells in young mice Ž8 weeks. and aged mice Ž50 weeks.. Extrathymic T cells are identified as IL-2R bq CD3 int while conventional T cells are IL-2R by CD3 high . The proportion of extrathymic T cells increase with aging Žindicated by arrowheads..
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In contrast, all thymus-derived T cells are CD8 ab Ža heterodimer of CD8.. Although IEL in the small intestine are considered to be mainly of extrathymic origin, it is also true that the generation of these IEL is highly influenced by presence of the thymus. Indeed, athymic nude mice and neonatally thymectomized mice show the decreased number of IEL in the small intestine w11x. With regard to this subject, Lefrancois and Puddington w12x proposed possible pathways of IEL development in the small intestine, in which some precursors of IEL may enter the thymus or IEL may be acted upon by thymic-derived factors Žor cells. for their differentiation in the small intestine. Since I mainly focused attention on extrathymic T cells in the liver and their immunomodulation in this review, I recommend Lefrancois’ reviews to further learn the properties and differentiation of IEL in the small intestine. Extrathymic T cells in the large intestine, uterus, and salivary gland carry different properties, especially in IL-2R b expression. Extrathymic T cells in the large intestine and salivary gland express a high level of IL-2R b , similar to those in the liver w10,13x. On the other hand, extrathymic T cells in the uterus express an intermediate level of IL-2R b , similar to those in LPL of the small intestine. Of course, NK cells, which are the earliest form of lymphocytes in phylogeny, express the highest level of IL-2R b among all lymphocyte subsets. The expression level of IL-2R b might be important for considering their phylogeny and function. The earlier phylogenetic form of extrathymic T cells tends to express a higher level of IL-2R b . Moreover, such extrathymic T cells which express a high level of IL-2R b are able to respond immediately to the corresponding antigens. The expression of IL2R b on NK cells is the highest and therefore these cells respond to antigens most quickly w4x. After infection, the activation of lymphocytes usually occurs in the following order: NK cells ™ extrathymic T cells ™ thymus-derived T cells. Although experimental findings are limited, TCRs used by extrathymic T cells seems to be at the level of oligo-diversity and oligo-clonality w14x. This might be due to their early phylogenic development. For example, Vb 8 is preferentially used for TCR b by extrathymic T cells and there are very few N insertion Že.g., Vb 8..
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2. Relationship between extrathymic T cells and NKT cells We first characterized extrathymic T cells as DN CD4y8y TCRint cells carrying forbidden clones, in the liver around 1990 w7,15,16x. In earlier and subsequent studies w17–19x, T cells with a NK marker, NK1.1, were identified in the thymus, although they are an extremely minor population. Attention has been focused on these T cells, because they carry many properties distinct from conventional T cells which are generated in the mainstream of the thymus. They are CD4q or DN CD4y8y. These T cells were termed natural killer T cells Ži.e., NKT cells.. NKT cells are localized in the medullary region of the thymus and a few of them are also dispersed in the cortex region w20x. It is speculated that NKT cells are generated through an alternative intrathymic pathway. Recent studies have revealed that these NKT cells preferentially use an invariant chain of Va 14Ja 281 for TCR a w21x and recognize glycolipid antigens as well as peptide antigens in the context of MHC class I-like antigen, CD1d w22x. Even in the liver, NKT cells are preferentially present and approximately 50 to 60% of TCRint cells are NK1.1q w5x. In other words, NKT cells belong to TCRint cells and TCRint cells are a mixture of NK1.1q and NK1.1y subsets ŽFig. 3.. The presence of the NK1.1y TCRint subset is obvious, because almost all extrathymic T cells seen in the liver and spleen of athymic nude mice are such cells. They are also IL-2R bq and have the morphology of granular lymphocytes. There has been a controversy as to whether NKT cells in the liver are generated in situ or are all supplied by the thymus. Athymic nude mice w23,24x and neonatally thymectomized mice w25,26x carry extremely few NKT cells in the liver and other immune organs. Moreover, when lethally irradiated, adult-thymectomized mice were subjected to bone marrow cell transplantation or fetal liver cell transplantation, CD4q NKT cells could not be generated in the liver w27,28x. It is therefore conceivable that CD4q NKT cells using Va 14Ja 281 are generated in the thymus and then migrate into the liver. We previously reported that the cell fraction of NKT cells in the liver did not accept the partner cells in parabiotic mice, ever 2 weeks after the operation
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Fig. 3. The NK1.1q and NK1.1y subsets of extrathymic T cells. Intermediate TCR cells Ži.e., TCRint cells. comprise both NK1.1q and NK1.1y subsets, showing the different patterns of CD4q, CD8q, and DN CD4y8y phenotypes.
w29x. On the other hand, up to 50% of conventional T cells and some extrathymic T cells in the liver accepted the partner cells by 2 weeks after the operation. Initially, we considered that NKT cells may be generated in situ in the liver. Concerning the above results of parabiosis, we speculate that NKT cells or their progenitors in the liver are derived from the thymus at the neonatal stage and that these liver NKT cells undergo renewal in situ in the liver. However, if these NKT cells or their progenitors are completely impaired at the adult stage, they have to be supplied by the thymus again.
3. Why the liver and intestine are the major sites for extrathytmic T cell differentiation Extrathymic T cells are abundant in the liver and intestine w8,9x. Since the liver phylogenetically developed from the intestine as an exocrine gland to excrete bile, it and the intestine share similar properties, namely, the continuous renewal of epithelia
Žhepatocytes themselves in the liver. and the presence of extrathymic T cells. The emergence of living beings onto land made possible by the development of pulmonary respiration which occurred 360 million years ago w30x. Until that time, they had carried the gills which then developed into the thymus. Phylogenetically, all these organs in the digestive tract, i.e., the gills, intestine, and liver, developed lymphoid cells in their surroundings Žpossibly from ancient macrophages.. It is therefore speculated that the thymus or gills became the organ which generated conventional T cells, because it continued to be exposed to stimulation by foreign antigens. On the other hand, the intestine and liver remained as sites where extrathymic T cells continued to be generated even thereafter. In the case of the thymus, this organ comprises both tissues of endodermal and epidermal origins. The epidermal tissue originated from the gill holes. In this regard, the medullary region in the thymus still carries the epidermal epithelia and possesses an alternative intrathymic pathway w31x. The main-
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stream of T-cell differentiation in the thymus gives rise to conventional T cells by negative selection of self-reactive forbidden clones. On the other hand, an alternative intrathymic pathway of T-cell differentiation gives rise to the NK1.1q subset of TCRint cells Ži.e., NKT cells. as well as the NK1.1y subset of TCRint cells. This alternative pathway may be a remnant of T-cell differentiation which took place in the ancient skin w32x. With the emergence of living beings onto land, conventional T cells produced by the thymus migrated to the lymph nodes and the spleen Žespecially the white pulp. whereas NKT cells produced by the thymus migrated to the liver and some other organs.
4. Elements supportive of extrathymic T-cell differentiation We have previously reported that c-kitq stem cells exist even in the adult liver w33x. These c-kitq cells isolated from the liver have potential as pluripo-
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tent stem cells; namely, they give rise to erythroid cells in the bone marrow, extrathymic T cells Žand granulocytes. in the liver, and conventional T cells in the thymus when transferred into lethally irradiated mice. When mice are administered with estrogen, many clusters of lymphoid cells appear in the parenchymal space of the liver. c-kitq stem cells are always present in the center of such clusters. In the fetal stage, it is well known that the liver is a hematopoietic organ. It is therefore concluded that the liver retains c-kitq stem cells even after birth and continues to produce extrathymic T cells thereafter. Similar to the case of fetal hematopoiesis, the generation of extrathymic T cells occurs in the parenchymal space. At that time, IL-7 is extremely important as one of the initial growth factors for extrathymic T cells w34x. For functional maturation after extrathymic T cells migrate to the sinusoidal lumen, IL-12, IL-15 and IL-18 might then be important ŽFig. 4.. For extrathymic T cells to maintain their specific localization Že.g., extrathymic sites., the adhesion molecules on their surface might be essentials. In-
Fig. 4. Supportive elements of extrathymic T-cell differentiation. IL-7 which is produced by hepatocytes, is an initial growth factor for extrathymic T cells. IL-12, IL-15, and IL-18 which are produced by Kupffer cells or endothelial cells, are subsequent maturational factors.
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deed, they express the unique pattern of adhesion molecules, CD44q L-selectiny LFA-1high w35,36x. In sharp contrast, conventional T cells have the pattern of adhesion molecules such as CD44y L-selectinqLFA-1low . This inverted expression pattern between extrathymic T cells and conventional T cells may define their localization.
5. Autoreactivity of extrathymic T cells In the case of mice, forbidden clones can be identified by the Mls system. Thus, superantigens which are the products of retrovirus infected in mice, are recognized by some specific Vbq cells. By using this system, we have demonstrated that forbidden clones are confined to IL-2R bq TCRint cells, but are not found in IL-2R by TCRhigh cells at all w31x. In other words, conventional T cells which are generated through the mainstream of T-cell differentiation in the thymus, do not contain forbidden clones ŽFig. 5.. This might be due to the fact that complete
negative selection of forbidden clones occurs in the mainstream. Even under immunosuppressive conditions induced by the administration of immunosuppressive drugs, a leakage of forbidden clones into conventional TCRhigh cells is not seen w37,38x. Namely, forbidden clones are always confined to TCRint cells. As already mentioned, TCRint cells are generated through both an extrathymic pathway in the liver and an alternative intrathymic pathway. Therefore, forbidden clones are generated through both pathways. Incomplete negative selection of forbidden clones might be due to the primitive nature of TCRint cells, for example, an intermediate density of TCR-CD3 complex, the aa homodimer of CD8 antigens, the lack of the expression of CD4 and CD8 antigens in some subsets, etc. In addition to the presence of forbidden clones, TCRint cells eventually mediate autoreactive cytotoxicity w38,39x. Targets of such cytotoxicity include syngeneic thymocytes, regenerating hepatocytes, and others w40x. It is well established that CTL Ži.e.,
Fig. 5. Pathways of T-cell differentiation. The thymus contains an alternative intrathymic pathway as well as the mainstream of T cell differentiation. Extrathymic pathways in the liver and the alternative intrathymic pathway give rise to IL-2R bq TCRint cells which contain forbidden clones.
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cytotoxicic T cells which are TCRhigh cells. could use both the perforin system and the Fas ligandrFas system. In the case of NK cells, the major cytotoxic molecule is perforin. On the other hand, TCRint cells mediate autoreactive cytotoxicity by using mainly the Fas ligandrFas system w38x. However, TCRint cells are able to use perforin Žagainst tumor targets. or TNFa in some situations. Concerning the presence of forbidden clones among TCRint cells and their autoreactive cytotoxicity, TCRint cells may usually mediate their cytotoxicity against some rapidly dividing self-cells. These functions of TCRint cells are beneficial to eliminate abnormal self-cells such as virally infected cells and malignant tumor cells. Since TCRint cells are present in the pregnant uterus, rapidly growing fetal cells might also be the targets for TCRint cells w41x. If such function of TCRint cells in the uterus fails, an invasion of fetal cells into the mother may occur. Examples of this are cases of hydatidiform moles Žbenign. and choriocarcinoma Žmalignant..
6. When are extrathymic T cells activated? 6.1. Aging Extrathymic T cells including NKT cells are very few in number in youth, even in the liver, and expand with aging. In other words, extrathymic T cells tend to expand in parallel with thymic involution. In the case of mice, IL-2R bq TCRint cells become detectable at the age of 4 weeks in the liver w2,8,9x. This is also true of NKT cells w42,43x. Up to the age of 1.5 years, the number and proportion of IL-2R bq TCRint cells and NKT cells increase but the number and proportion of NKT cells then decline. As a result, NK1.1y TCRint cells expand in number and proportion after the age of 1.5 years. In youth, extrathymic T cells and NKT cells are detectable only in the liver, but with aging these T cells become prominent in various immune organs, including the spleen, lymph nodes, bone marrow, and even the thymus. IL-2R bq TCRint cells and NKT cells in the thymus mainly localize in the medullary region. After the age of 50 weeks, the number and proportion of IL-2R bq TCRint cells and NKT cells overcome those of IL-2R by TCRhigh
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cells Ži.e., conventional T cells or thymus-derived T cells. in various immune organs w5x. The increase in the number of extrathymic T cells and NKT cells with aging might be important for the elimination of abnormal self-cells which appear in the body with aging. Even in the exocrine glands such as the salivary glands, infiltration by many extrathymic T cells or NKT cells occurs with aging. These T cells may provide innate immunity for eliminating microbially infected self-cells and malignant tumors. 6.2. Stress When mice are exposed to restraint stress, acute thymic atrophy is induced w44,45x. At this time, the number of DP CD4q8q thymocytes decrease prominently due to their apoptosis. Even the number of TCRhigh cells, which are derived from the thymus and migrate to the peripheral immune organs, tend to decrease in various peripheral immune organs. In sharp contrast, the number of TCRint cells and that of NKT cells hardly decrease in various immune organs due to stress. Therefore, the proportion of TCRint cells and NKT cells becomes prominent in all these organs after exposure to stress. Since TCRint cells and NKT cells mediate autoreactive cytotoxicity, this increased population of T cells might be important for eliminating abnormal self-cells which appear due to stress. Stress-associated factors which induce thymic atrophy and the apoptosis of TCRhigh cells in the peripheral immune organs may include glycocorticoids, catecholamines, and inflammatory cytokines Že.g., IFNg and TNFa . w44,45x. Indeed, any of these factors eventually has the ability to induce acute thymic atrophy and inversely increase the proportion of TCRint cells or NKT cells in the periphery. 6.3. Autoimmune diseases I feel that we cannot understand the mechanisms underlying autoimmune diseases without introducing the concept of extrathymic T cells. When we observe autoimmune prone ŽNZB = NZW. F1 mice ŽNZBrW F1 mice., thymic atrophy is always accompanied by the onset of disease ŽFig. 6.. At that time, the expansion of extrathymic T cells, including NKT
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trathymic T cells. A certain retroviral transposon is inserted into the Fas gene. In the case of conventional T cells, some molecules other than Fas are also involved in the apoptosis of these T cells. On the other hand, only Fas may be associated with the apoptosis of TCRint cells. As a result, only the expansion of TCRint cells is seen in these MRL-lprr lpr mice. A decrease in the number of NKT cells is known to occur in diabetic-prone NOD mice w25,26,47,48x. Therefore, many investigators suspect that NKT cells are immunoregulatory T cells which act against the onset of autoimmune diseases. In these cases, the autoreactivity of NKT cells Ži.e., NK1.1q TCRint cells. functions to suppress some autoreactive T cells Že.g., NK1.1y TCRint cells.. This subject remains to be further investigated. 6.4. Pregnancy Fig. 6. Thymic involution seen in autoimmune prone NZBrW F1 mice. Severe thymic involution is always seen in these autoimmune mice, although the number of lymphocytes increases in other organs.
cells, occurs in the liver and other peripheral immune organs. CD5qB cells which produce autoantibodies, expand in the peritoneal cavity. It is conceivable that the autoreactivity of TCRint cells and NKT cells as well as that of CD5qB cells might be intimately associated with the onset of autoimmune disease. Many investigators have believed that self-reactive forbidden clones are generated by failure of the mainstream of T cell differentiation in the thymus. However, this concept is doubtful. Thus, forbidden clones are always confined to TCRint cells w31,37,38x. Moreover, as mentioned above, thymic atrophy is always accompanied by the onset of autoimmune diseases. We have to consider the possibility that the overactivation of extrathymic T cells, which primarily contain forbidden clones, might be associated with the onset of autoimmune diseases. In the case of MRL-lprr lpr mice, an abnormal expansion of B220q DN CD4y8y TCRint cells is seen in various immune organs w15,46x. This is due to the fact that a defective Fas gene Ži.e., lymphoproliferative, lpr gene. permits the expansion of ex-
Extrathymic T cells are activated in the uterus as well as in the liver in the late phase of pregnancy w41,49x. Similar to the case of stress, thymic atrophy accompanies this phenomenon. We consider that estrogen and sympathetic nerve activation might be associated with this phenomenon w42,43,50,51x. During pregnancy, a large amount of estrogen is secreted from the placenta while sympathetic nerve activation is induced to supply oxygen and nutrients from the mother to the fetus. We have previously reported that the exogenous administration of estrogen or the administration of adrenaline activates extrathymic T cells in the liver of mice in number and function w42,43,50,51x. These administrations also induce acute thymic atrophy. It is therefore conceivable that the major factors which induce the pregnancy-associated phenomenon may be both estrogen and catecholamines. In the case of fetuses, trophoblasts of the placenta, which lack the expression of polymorphic MHC antigens, adhere directly to the mother. This situation enables the mother not to reject the fetus during pregnancy. Instead, there is a danger of fetal tissues invading the mother Že.g., choriocarcinoma.. Activation of NK cells and extrathymic T cells in the uterus might be important for protection of the mother from such invasion w41x. In other words, by using a
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physiologically inevitable response Ži.e., the secretion of estrogen and the activation of the sympathetic nervous system., the mother achieves a successful pregnancy. When the mother experiences over-activation of the sympathetic nerves for some reason, there is a danger that she may fall victim to pregnancy disorders. Such disorders include preeclampsia and hyperemesis gravidarum w41x. Indeed, patients with these disorders suffer from hypertension, tachycardia, palpitation, etc. All these symptoms are induced by the over-activation of the sympathetic nervous system. In more detail, the over-activation of granulocytes, NK cells, and extrathymic T cells might be induced in the uterus due to the over-activation of the sympathetic nerves. Thus, these leukocytes carry adrenergic receptors on the surface and are activated in number and function by sympathetic nerve activation w52x. Causes of the over-activation of sympathetic nerves might include intrinsic factors in the mother Že.g., insufficiency of cardiac function or other organ-functions. and extrinsic factors Že.g., overwork and mental stress.. 6.5. Malarial infection The body seems to be protected against an intracellular protozoa pathogen, malaria, by innate immunity. This notion is supported by the fact that malarial infection induces a prominent expansion of extrathymic T cells Ži.e., IL-2R bq TCRint cells. in the liver. No expansion is seen in conventional T cells Ži.e., IL-2R by TCRhigh cells.. When TCRint cells isolated from the liver of mice which had recovered from malarial infection were injected into irradiated recipient mice, these mice could survive malaria without parasitemia. In the case of the administration of a-galactosylceramide Ž a-GalCer., the major expanding population is the NK1.1q subset of IL-2R bq TCRint cells Ži.e., NKT cells. w53x. In sharp contrast, the major expanding population is the NK1.1y subset of IL-2R bq TCRint cells during malarial infection. Namely, NKT cells, which express CD4 antigen and use an invariant chain of Va 14Ja 281, are not a major population in the protection of the body against malaria.
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Malaria infection is accompanied by severe thymic atrophy and the proportion of TCRhigh cells rather decreases in the periphery. Indeed, there is no protective effect of TCRhigh cells when these T cells Žisolated from mice recovered from malaria. are injected into irradiated mice. In addition to the activation of extrathymic T cells in the liver of mice with malarial infection, we found that the production of autoantibodies such as antiDNA antibody is seen in the sera of these mice. In other words, many signs during malarial infection mimic those seen in autoimmune diseases. These results also support our notion that the body is protected against malaria by innate immunity. 6.6. Administration of nonsteroidal anti-inflammatory drugs (NSAIDs) Many clinicians have believed that the administration of NSAIDs is useful for reducing inflammation. In some cases of inflammation associated with protaglandins Ži.e., inflammation associated with conventional T cells., this may be true. However, when NSAIDs are continuously administered into mice, a prominent activation of granulocytes, NK cells, and extrathymic T cells is induced in the liver and other peripheral organs w54x. This might be due to the fact that NS AIDs suppress the production of prostaglandins via the suppression of cyclooxygenase, and then induce the activation of the sympathetic nervous system. The down-regulation of prostaglandin synthesis always induces subsequent activation of sympathetic nerves, because prostaglandins and catecholamines together comprise an antagonistic system. Since granulocytes, NK cells, and extrathymic T cells carry adrenergic receptors on the surface w52x, such sympathetic nerve activation is responsible for the activation of these leukocyte populations in number and function. In addition, thymic atrophy accompanies this phenomenon. Reflecting these responses, inflammations associated with granulocytes, NK cells, and extrathymic T cells worsen when NSAIDs are administered for a long time. More concretely, purulentive diseases Ži.e., bacterial infections., gastric ulcers, ulcerative colitis, rheumatic arthritis Žespecially the chronic phase., and many autoimmune diseases are granulocyte-as-
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sociated inflammations and are worsened by the continuous administration of NSAIDs. 6.7. Relationship of extrathymic T cells with other immunological responses When allogeneic-combination F1 mice are irradiated and then subjected to bone marrow transplantation of one parent origin, such F1 mice sometimes reject the injected bone marrow cells. This phenomenon is known to be caused hybrid resistance. At this time, not only NK cells but also extrathymic T cells Žincluding NKT cells. are found to be effector cells for this hybrid resistance phenomenon. We previously reported that granulocytes suppress the function of NKT cells in G-CSF transgenic mice w55x. Extrathymic T cells and NKT cells are also associated with chronic graft-versus-host ŽGVH. diseases. It is well known that the target tissues for GVH diseases include mainly the liver, intestine, and skin. All these tissues are sites where extrathymic T cells are present. Therefore, the association of extrathymic T cells with GVH disease is in good agreement with their localization. In our most recent study, we observed that unusual CD8q NKT cells are expanding in the liver of neonatal tolerant mice both in youth and adulthood. Since many allogeneic lymphocytes injected at the neonatal stage Ži.e., within day 3 after birth. remain in the spleen, these allogeneic lymphocytes may stimulate CD8q NKT cells in the liver via the portal vein. At mentioned earlier, NKT cells are primarily autoreactive. In this regard, we speculate that alloactivated CD8q NKT cells then mediate neonatal tolerance phenomenon. Splenectomy results in a decrease in the number of CD8q NKT cells. The neonatal tolerance phenomenon disappears thereafter.
7. Human counterparts of extrathymic T cells or NKT cells Human counterparts of extrathymic T cells have been identified as CD56q T cells and CD57q T cells w56,57x. Similar to the case in mice, these T cells are estimated to be granular lymphocytes in morphology.
When compared with the morphology of NK cells, these extrathymic T cells have a smaller cell size and fewer cytoplasmic granules. This morphological nature indicates that extrathymic T cells are at the intermediate position between NK cells and conventional T cells in phylogeny. CD56q T cells are abundant in the liver, intestine, and uterus in humans while CD57q T cells are abundant in the bone marrow, peripheral blood, and joints Žor joint cavity. w56,57x. This situation shows that CD57q T cells are a major population of T cells when patients with leukemia are subjected to bone marrow transplantation after chemotherapy. Usually, CD56q T cells are extremely few in the intestine of healthy persons. However, such CD56q T cells become abundant in the intestine, especially in the small intestine, of patients with ulcerative colitiss. In the case of natural abortion in early pregnancy, both CD56q T cells and CD57q T cells as well as NK cells and granulocytes appear in the interface between the uterus and placenta w41x. These leukocytes might be associated with the abortion Ži.e., the rejection of fetus.. At this time, many CD56q T cells and CD57q T cells appear even in the peripheral blood. In the case of mice, extrathymic T cells comprise both NK1.1q and NK1.1y subsets among TCRint cells. CD56q T cells correspond to NK1.1q TCRint cells while CD57q T cells correspond to NK1.1y TCRint cells. The majority, if not all, of the NK1.1q TCRint cells use an invariant chain of Va 14Ja 281 for TCR a in mice. In contrast, only a minor population of CD56q T cells use Va 24 Žan equivalent human counterpart of mouse Va 14.. This might be due to the fact that a monomorphic MHC class I-like molecule is the only CD1d in mice. In contrast, monomorphic MHC-class I-like molecules include CD1a, CD1b, CD1c, and CD1d in humans. Moreover, monomorphic MHC class I antigens such as HLA-E, HLA-F, and HLA-G are also present. These monomorphic MHC class I antigens also present some self-antigens to CD56q T cells or CD57q T cells. In a recent study, CD161q T cells were also identified as extrathymic T cells in humans. CD56q T, CD57q T, and CD161q T cells are overlapping populations. Although conventional T cells are extremely important for recognizing and eliminating foreign antigens, these extrathymic T cells in humans are invaluable for interacting with and eliminating
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abnormal self-cells which appear with aging and under conditions of stress, malignancy, pregnancy, autoimmune diseases, chronic GVH disease, intracellular infections, etc.
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