Use of monoclonal antibodies in a study of the development of T lymphocytes in the human fetus.

A panel of monoclonal antibodies (OKT3, 4, 6, 8, 10, 11) was used for the identification of T lymphocyte subpopulations in cell suspensions of human fetal liver, thymus, bone marrow and spleen. In liver suspensions of 8-16 week old fetuses and in bone marrow suspensions (12-20 weeks) less than 5% of lymphocytes reacted with either OKT3, 11, 4, 8 or 6, whereas the OKT10 antibody bound to, respectively, 35 and 86% of lymphocytes in these tissues. In liver suspensions of 17-20 week old fetuses, about 20% of lymphocytes carried either the T3, 11, 4 or 8 antigen and more than 60% of lymphocytes were OKT10+. The maturation stages in fetal thymus (11-20 weeks) are comparable to those in the post-natal thymus, with the exception that a substantial proportion of fetal thymocytes expresses the T3 and T6 antigen simultaneously. In the fetal spleen (12-20 weeks), 40% of lymphocytes reacts with OKT3. These OKT3+ spleen cells may be divided into two subsets expressing either the T4 antigen or the T8 antigen. These OKT3+/OKT4+ and OKT3+/OKT8+ lymphoid cells of the fetal spleen can be further subdivided into a T10+ and T10- subpopulation. These data suggest that T lymphoid precursor cells, reacting with either none of the monoclonal antibodies or only with OKT10, are generated in fetal liver (up till 16 weeks gestational age) and bone marrow. Further maturation takes place in the fetal thymus, but also to a certain extent in peripheral lymphoid organs such as the fetal spleen, as evidenced by the coexistence of a T3+/T10+ and T3+/T10- subpopulation in this organ.     

A bone marrow-derived stroma cell line, ST2, can support the differentiation of fetal thymocytes from the CD4−CD8−double negative to the CD4+CD8+ double positive differentiation stage in vitro

T-cell precursors differentiate into mature T cells predominantly in the thymus. However, it has also been reported that T-cell precursors mature in extrathymic organs such as the liver, bone marrow, or intestines. In order to investigate the nature of the extrathymic microenvironment that supports T-cell maturation, we examined the effect of a bone marrow-derived stroma cell line, ST2, on T-cell precursors by using a reaggregate thymic organ culture (RTOC) system. We found that ST2 cells supported the differentiation of fetal thymocytes at day 14.5 of gestation from a CD4- CD8- double negative (DN) to a CD4+ CD8+ double positive (DP) differentiation stage in a manner similar to that observed in thymus. Anti-interleukin-7 receptor (IL-7R) and anti-c-kit antibodies blocked the growth of thymocytes in RTOC with ST2 cells, but did not inhibit the generation of DP thymocytes. These data indicate that a bone marrow-derived stroma cell, ST2, which supports B-cell differentiation, is also able to support T-cell development and may constitute one of the microenvironmental components for extrathymic T-cell development.     

Characterization of early lymphoid precursor cells in the human fetus using monoclonal antibodies and anti-terminal deoxynucleotidyl transferase.

Monoclonal antibodies (MoAbs) directed primarily against immature lymphoid cells (VIL-A1, BA-2, OKT10) or recognizing antigens associated with the B cell lineage (VIB-C5, OKI1) were used for the identification of lymphoid cells in liver, bone marrow, spleen and thymus of human fetuses between 8 and 20 weeks of gestational age. Many lymphocytes in liver, bone marrow and spleen reacted with the MoAbs used. In the fetal thymus, however, cells did not bind to the VIL-A1 and VIB-C5 MoAbs and only a few cells were BA-2+ or OKI1+. In the liver and bone marrow the VIL-A1, VIB-C5 and BA-2 MoAbs reacted almost exclusively with terminal deoxynucleotidyl transferase (TdT) containing cells, pre-B and B cells. TdT+ cells were present in liver, bone marrow and thymus, but not in the spleen. In liver and bone marrow the relative numbers of TdT+ cells decreased during gestation, in the thymus they increased. The antigenic make-up of the TdT+ cells in liver and bone marrow was comparable to that of pre-B and B cells in these organs: most of them reacted with VIL-A1, VIB-C5 and OKT10 MoAbs and many were BA-2+ and OKI1+. TdT+ cells in liver and bone marrow did not bind to T-cell-markers, i.e. OKT6 and WT-1. A few lymphoid cells in these organs contained TdT and mu heavy chains. TdT+ cells in the thymus had a completely different phenotype: most of them were OKT6+ and they did not react with the VIL-A1 and VIB-C5 MoAbs. These findings suggest that TdT+ cells in fetal liver and bone marrow are precursors of the B cell lineage, whereas those in the thymus probably belong to the T cell lineage. In the fetal spleen almost all B cells displayed the VIB-C5 and OKI1 antigens. At 12 weeks of gestation greater than 80% of splenic B cells were also VIL-A1+ and BA-2+; with ongoing gestation far less B cells in spleen expressed these antigens, however, indicating that these B cells are more mature than those in fetal liver and bone marrow, but still less mature than the B cells in postnatal blood and bone marrow, which do not display the VIL-A1 and BA-2 markers. These findings suggest that some further maturation of B cell stages takes place in the spleen during human fetal life.     

The development of lymphocytes with T- or B-membrane determinants in the human foetus

The development of T- or B-membrane determinants on human foetal lymphoid cells was studied by the direct immunofluorescence technique, using a tetramethyl rhodamine isothiocyanate (TRITC) labelled horse antihuman T-cell conjugate (ATC) for the detection of T lymphocytes and a fluorescein isothiocyanate (FITC) labelled goat antihuman Fab conjugate for the demonstration of Ig-bearing B lymphocytes. Human foetal lymphocytes were also tested for spontaneous rosette formation with sheep red blood cells (SRBC).

Cell suspensions of liver, spleen, thymus, bone marrow and blood of twenty-five human foetuses of 5·5–26 weeks of gestational age have been investigated. ATC-positive lymphoid cells were first seen in the liver at 5·5 weeks; E rosette-forming cells (ERFC) and Ig-bearing lymphoid cells were first found at 9 weeks. ERFC were also present in the thymus at 9 weeks. By 12 weeks, fluorescent B and T lymphocytes were found in bone marrow and spleen. ERFC were also found in bone marrow at this age, but not in spleen. At 15 weeks, more than 80% of blood lymphoid cells had T or B determinants.

A difference in the reactivity of lymphoid cells with the ATC and their capacity to form E rosettes was observed. In liver and spleen, the ATC determinant was detectable before the SRBC receptor. In bone marrow, blood and thymus the ATC determinant was found on a higher percentage of lymphoid cells than was the SRBC receptor when those organs were first investigated. During the entire investigated period of gestation, the majority of lymphoid cells in liver and bone marrow did not react with either of the conjugates, nor did they form E rosettes. In all organs investigated, except in the thymus, lymphoid cells were occasionally seen which reacted with both conjugates. By the 16th week of foetal age, more than 90% of lymphoid cells in thymus, spleen and blood had acquired T- or B-membrane determinants.

     

Peripheral tolerance through clonal deletion of mature CD4-CD8+ T cells.

Transgenic mice bearing the alpha beta transgenes encoding a defined T cell receptor specific for the male (H-Y) antigen presented by the H-2Db class I MHC molecule were used to study mechanisms of peripheral tolerance. Female transgenic mice produce large numbers of functionally homogeneous CD8+ male antigen-reactive T cells in the thymus that subsequently accumulate in the peripheral lymphoid organs. We have used three experimental approaches to show that male reactive CD8+ T cells can be eliminated from peripheral lymphoid organs after exposure to male antigen. (i) In female transgenic mice that were neonatally tolerized with male spleen cells, male reactive CD8+ T cells continued to be produced in large numbers in the thymus but were virtually absent in the lymph nodes. (ii) Injection of thymocytes from female transgenic mice into female mice neonatally tolerized with the male antigen, or into normal male mice, led to the specific elimination of male-reactive CD8+ T cells in the lymph nodes. (iii) Four days after male lymphoid cells were injected intravenously into female transgenic mice, male antigen-reactive CD8+ T cells recovered from the lymph nodes of recipient mice were highly apoptotic when compared to CD4+ (non-male reactive) T cells. These data indicate that tolerance to extrathymic antigen can be achieved through elimination of mature T cells in the peripheral lymphoid organs.     

免疫组化技术分析人淋巴组织的多种分化抗原

用间接免疫过氧化物酶和PAP技术检测本室制备的31种抗人分化抗原单抗与淋巴组织的反应性.结果表明,CD3、CD5单抗与扁桃腺和淋巴结的T细胞、大部分成熟髓质胸腺细胞和脾白髓的中央动脉周围淋巴鞘呈非常强的反应.CD4~+细胞在扁桃腺的分布与CD3~+细胞类似,但数量稍少.只有少部扁桃腺和淋巴结T细胞与CD8单抗反应,CD8单抗主要染大部分胸腺皮质细胞,但抗CD8单抗与脾窦的内皮细胞呈强阳性交叉反应.Wu59单抗同时与扁桃腺、淋巴结和脾脏的T、B细胞呈非常强的膜染色,并与胸腺皮质和髓质细胞呈阳性反应,该单抗可能识别白细胞共同抗原或LFA-1.Wu 26.145单抗除与扁桃腺生发中心呈弱阳性反应外,还与脾红髓窦状结构内的血小板呈强阳性反应.此外,抗B细胞及其亚群单抗与扁桃腺、淋巴结、脾白髓生发中心呈强阳性反应.抗IL-2受体单抗与上述组织基本上呈阴性反应。     

Both activated and nonactivated leukocytes from the periphery continuously enter the thymic medulla of adult rats: phenotypes,sources and magnitude of traffic

Although the thymus is primarily noted for the export of T cells to the periphery, a small influx of cells has also been observed. It is still a matter of debate whether entry into the thymus depends on prior activation. The phenotypes, sources and degree of immigration are largely unknown. We monitored by quantitative immunohistochemistry the entry of cells from the periphery into the rat thymus in three experimental models. We injected i.v. recirculating, small, nonactivated CD4⁺ T cell subsets, often referred to as naive (CD45RC⁺) and memory or antigen-experienced (CD45RC⁻) cells, purified from thoracic duct lymph of allotype-marked donors, allotype-marked leukocytes released from spleen or lung transplants, or leukocytes labeled in the periphery for 12 weeks during the S-phase of the cell cycle by oral application of 5-bromo-2-deoxyuridine (BrdUrd). Early after i.v. injection (0.5 h), significantly more antigen-experienced (CD45RC⁻) CD4⁺ T cells entered the thymus, and by 24 h four times as many cells from the CD45RC⁻ subset as from the CD45RC⁺ subset had entered the thymus and localized to the medulla. None of the thymic entrants expressed the interleukin (IL)-2 receptor. Following spleen transplantation ∼ 40% of donor cells entering the thymic medulla were T cells and ∼ 55% were B cells. In contrast, from a lung transplant, ∼ 85% of peripheral immigrants were T cells and ∼ 10% were B cells. After both procedures, a small number of NK cells and monocytes/macrophages were found among the immigrants (< 5%). Rats were fed BrdUrd continuously for 12 weeks, a procedure which labeled ∼ 30% of peripheral lymphocytes but not cortical thymocytes. BrdUrd-labeled cells were localized almost exclusively to the thymic medulla and represented ∼ 10% of medullary cells. Of the thymic immigrants ∼ 50% were T cells, ∼ 30% were B cells (including ∼ 15% IgD⁺ cells), ∼ 15% were NK cells and the remainder (∼ 5%) were monocytes/macrophages. Only a quarter of BrdUrd-labeled cells expressed the IL-2 receptor. The thymus is continuously infiltrated by both activated and nonactivated leukocytes from the periphery, including T cells, B cells, NK cells and monocytes. These immigrants are supplied by lymphoid and nonlymphoid organs in a characteristic subset composition. Their entry is facilitated by prior antigen experience or activation. Thus, the participation of the thymic medulla in general leukocyte traffic suggests a mechanism by which the T cell repertoire could potentially be modulated by the peripheral tissues.     

Characterization of intermediate T-cell receptor cells expanding in the liver, thymus and other organs in autoimmune lpr mice: parallel analysis with their normal counterparts.

Autoimmune MRL-lpr/lpr (lpr) mice were previously demonstrated to have an abnormal proliferation of intermediate T-cell receptor (TCR) cells of extrathymic origin in the liver. Despite this situation, thymectomy in lpr mice resulted in amelioration of autoimmune disease. To understand the underlying mechanism, we investigated associated T-cell differentiation in the thymus and other organs of these mice. When the disease was evoked, T cells with extrathymic properties, i.e. intermediate TCR-alpha beta cells expressing double-negative (DN) CD4-8- phenotype and interleukin-2 (IL-2) receptor beta-chain, became prominent not only in the liver, but also in the thymus. Such thymic T cells mainly resided in the medulla. A small-scale localization of such T cells was seen in the thymic medulla even in normal control mice. There was a heterogeneity among intermediate TCR cells in terms of the composition of DN cells and the expression of CD2 and B220 antigens, depending on the organs and the sites in the same organ. Intermediate TCR cells in the liver, thymus and autoimmune target organs (e.g. kidney) contained a high proportion of the active form (CD2+B220-), while intermediate TCR cells accumulating in peripheral organs, the spleen and lymph nodes, were mainly of the inactive form (CD2-B220+). The active form had an ability to proliferate in response to IL-2 and SEB, whereas the inactive form did not. The present results suggest that the proliferation of intermediate TCR cells occur at multiple sites; this may explain the effect of thymectomy, namely, the retarded onset of disease, in lpr mice.     

Developmentally regulated expression of the neural cell adhesion molecule (NCAM) by mouse thymocytes

The expression of the neural cell adhesion molecule (NCAM) has been investigated during thymus ontogeny. NCAM mRNA was readily detectable at day 19 of gestation, the youngest age studied. Its level declined after birth to become undetectable at 3 weeks of age. Cell surface expression of NCAM protein was detected on 14% of day 15 fetal thymocytes and peaked during the perinatal period, when around 40% of the thymocytes expressed low to medium levels of NCAM. At postnatal day 2, the vast majority of the NCAM+ cells were also CD4+ and CD8+. At embryonic day 15, NCAM appeared also to be expressed by CD4- thymocytes since 14% of the cells were already NCAM+ whereas CD4 was virtually undetectable. In frozen section of the newborn thymus, surface staining for NCAM was present on a subpopulation of cells in the cortex, rare in the medulla and absent from the sub-capsular area. In conjunction with other cell adhesion molecules, NCAM could play a role in cell interactions during thymic development.     

Mouse hepatitis viral infection induces an extrathymic differentiation of the specific intrahepatic alpha beta-TCRintermediate LFA-1high T-cell population.

Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent model for the study of thymic and extrathymic T-cell subpopulation disorders induced during viral hepatitis. It was recently reported that, in addition to the intrathymic T-cell differentiation pathway, an extrathymic differentiation pathway of alpha beta-T-cell receptor (TCR) T lymphocytes exists in the liver, and becomes important under pathological situations such as autoimmune diseases, malignancies or hepatic bacterial infections. In the present study, we compared the phenotypes of resident hepatic, splenic or thymic T-cell subpopulations during the acute viral hepatitis induced by HMV3 in susceptible C57BL/6 mice. The number of liver-resident mononuclear cells (MNC) increased during the viral infection, while cellularity decreased. Single positive (SP) CD4+ cells strongly increased in both the liver and thymus, while double positive (DP) (CD4+ CD8+) cells, present in the liver and thymus of mock-infected mice, decreased in C57BL/6 mice during the viral infection. A shift of alpha beta-TCRintermediate T cells toward alpha beta-TCRhigh was evidenced in the liver and thymus of infected mice, but not in the spleen. The few alpha beta-TCRint double negative (DN) (CD4-CD8-) cells also decreased following viral infection. alpha beta-TCRint or high lymphocytes expressing high levels of leucocyte function antigen-1 (LFA-1) increased in the liver of MHV3-infected mice. In addition, liver-resident T cells expressed strongly the CD44 (Pgp-1) activation marker, suggesting that they were either activated or antigen experienced during the viral infection. No significant change in T-cell subpopulations was detected in the spleen, suggesting that MHV3 infection could induce an early in situ differentiation of resident hepatic T cells rather than a recruitment of lymphocytes from peripheral lymphoid organs.     

Intra- and extrathymic B cells in physiologic and pathologic conditions

Summary Normal thymuses and thymuses with lymphofollicular hyperplasia have been examined immunohistologically using immunoenzymatic single and double labelling methods and a panel of monoclonal antibodies against B lymphocyte differentiation antigens (CD19-, CD20-, CD21-, CD22-, CD23- and CD37ag) and human immunoglobulins (IgM, IgD) for the presence and localisation of B lymphocytes and cells expressing B cell differentiation antigens. The numerous hyperplastic lymph follicles which occur in the pathological condition of lymphofollicular hyperplasia of the thymus were found to originate in the extrathymic compartment of the interlobular septal space. This area was found to be blown up by the growing lymph follicles with exactly the same cellular composition as their counterparts in the peripheral lymphatic tissue. Some of the B lymphocytes expressing the immunophenotype of follicular mantle zone lymphocytes which were detected in the thymic medulla probably infiltrated through discontinuities of the border between the perivascular space and the thymic medulla. Apart from this primarily extrathymic B cell compartment, B lymphocytes and cells expressing B cell antigens were found within the thymus medulla of normal control thymuses of different ages from fetal to adult life. These cells were detected as a small subpopulation in normal fetal, juvenile and adult thymuses. Morphologically they could be subdivided into small, round lymphoid cells accounting for less than 1% of medullary lymphoid cells, and into a larger variant, asteroidally shaped because of short cytoplasmic processes. These asteroid cells were even more infrequent than the lymphoid variant. Immunophenotype (CD19ag+, CD20ag+, CD22ag+, CD37ag+, IgM+, IgD+) and morphology of the first cell type led to the conclusion that the lymphoid cells were in fact B lymphocytes. They were scattered throughout the medulla of fetal and juvenile and adult thymuses alike. The second, the asteroid cell type, constantly expressed CD20ag and inconstantly IgM, CD22ag and CD37ag; furthermore, CD23ag was detected in a subset of the asteroid cells either restricted to the perinuclear zone or expressed in the entire cytoplasma and on the plasma membrane. The asteroid cells were located in the corticomedullary region of the fetal thymuses but were randomly distributed with a tendency to Hassall's corpuscles in juvenile and adult thymuses. They often formed rosettes with non-B lymphocytes. It can be concluded that a small number of B cells and asteroid cells of still uncertain origin, but expressing B cell antigens, are constitutive elements of the fetal and adult thymic medulla. It can be assumed that the asteroid cell might represent a novel type of thymic accessory cell and that the rosetting of non-B lymphocytes around this asteroid cell might simulate or in fact be the earliest B cell interaction of maturing T cells.Abbreviation mAb(s) monoclonal antibody(-ies) - CDxxag antigen defined by the mAb cluster xx - CDxx(mAb) mAb of the cluster xx This study was supported by the Land Baden-Württemberg (Förderung der AIDS Forschung)Dedicated to Prof. Dr. V. Becker on the occasion of his 65th birthday     

Influence of age on the proliferation and peripheralization of thymic T cells

Bone marrow cells obtained from B10.Thy-1.1 mice (H-2b, Thy-1.1) were injected directly into the thymus of C57BL/6 mice (H-2b,Thy 1.2) of various ages. Thymocyte precursors in the injected donor-bone marrow cells could proliferate in the thymic microenvironment in the following manner: first, preferentially proliferating into the subcapsular cortex; and second, spreading to the whole layer of the cortex, a portion of them gradually moving into the medulla. The proliferation of donor-type thymocytes was most pronounced when intrathymic injection of bone marrow cells (ITB) was performed in newborn mice and especially prominent in week-old mice; it took approximately ten weeks for donor-type thymocytes to finish the whole course of proliferation, differentiation, and emigration to the periphery. When ITB was performed in mice 4 weeks of age and older, the proliferation of donor-type thymocytes was retarded at onset, less pronounced in magnitude, and disappeared earlier. Emigration of donor-type T cells from the thymus to the peripheral lymphoid tissues occurred most rapidly when ITB was performed in newborn mice, and these T cells continued to reside thereafter in the peripheral lymphoid tissues. However, when ITB was performed in mice 4 weeks of age and older, the number of emigrated T cells in the spleen decreased (about a tenth of that in newborn mice) and, moreover, these T cells resided only transiently in the spleen. It was suggested that T cells emigrating from the thymus of mice from newborn to 2 weeks of age are long-lived, whereas those from the thymus in mice 4 weeks of age and older are short-lived. However, when 4-week-old young adult mice were treated by irradiation or hydrocortisone, the thymic capacity was enhanced in terms of proliferation and peripheralization of thymocytes, and emigrated T cells became long-lived.     

Cytoplasmic CD3+ surface CD8+ lymphocytes develop as a thymus-independent lineage in chick-quail chimeras

We have analyzed the embryonic development of a population of lymphoid cells that express a CD3 antigenic determinant in the cytoplasm but not on the cell surface. Since these cells lack T cell receptor (TcR) molecules, we have provisionally named them TCRO cells. Their development, expansion and distribution was investigated following transplantation of splenic and bursal fragments from chicken embryos into quail embryos. Since quail cells are not recognized by our panel of monoclonal antibodies against chicken TcR1, TcR2, TcR3, CD3, CD4 and CD8 molecules, these antibodies provided reliable markers for donor chick lymphocytes in the tissues of the quail recipients. Transplanted spleen and bursa both generated CD3+ cells, the number of which increased as a function of age. Notably, approximately half of these CD3+ cells expressed surface CD8, but none acquired TcR1 (gamma/delta), TcR2 (alpha/beta) or TcR3 expression. Since TCRO cells normally appear first in the spleen of 8-day chick embryos (E8), their generation in E6 splenic transplants indicated an extrathymic origin. The TCRO cells of chick splenic origin migrated to the spleen, bursa and thymus of the quail recipients. In six of seven chimeras acquiring CT3+ cells in the recipient thymus, these cells were restricted to the medulla and displayed the typical TCRO phenotype: CD3+CD8+TcR1-TcR2-TcR3-. These intrathymic TCRO cells also lacked the CT1 thymocyte antigen. We conclude that the TCRO cells represent a thymus-independent lineage of lymphoid cells that can migrate into a receptive thymus by rarely, if ever, differentiate into conventional T cells.     

Functional maturation of recent thymic emigrants in the periphery: development of alloreactivity correlates with the cyclic expression of CD45RC isoforms.

The transition from fully developed CD4+CD8- single-positive (SP) thymocytes into fully mature recirculating peripheral T cells is both poorly understood with regard to the expression of restricted isoforms (CD45R) of the leukocyte common antigen and in terms of T cell function. The present investigation monitored the extrathymic development of CD4+CD8- SP thymocytes in euthymic recipients using allotype-marked donor cells and monoclonal antibody OX22 which recognizes an epitope on the C exon of rat CD45R. We established that donor-derived cells in the blood 1 day later bore the phenotype of the injected SP thymocytes (CD4+ Thy-1+ CD45RC-). T cells with the identical phenotype were also present in the thoracic duct lymph of uninjected rats, suggesting that the Thy-1+ CD45RC- T cells represent recent thymic emigrants (RTE) which have migrated to the periphery of their own accord. During extrathymic maturation donor-derived peripheral RTE lost Thy-1 within 3 days and expressed the CD45RC+ high molecular weight isoform by day 7; between days 8 and 14 a proportion (25%-30%) of the donor cells once again lost the high molecular weight isoform (CD45RC-). The transition of SP (CD45RC-) thymocytes to fully mature CD45RC+ CD4 T cells via intermediate peripheral RTE was accompanied at each stage by an increased ability of the maturing T cells to induce skin allograft rejection. Unexpectedly, the subsequent loss of the high molecular weight isoform, following presumed antigen encounter, was associated with a significant reduction in the ability of this Thy-1-CD45RC- subpopulation to effect graft rejection. The cyclic expression of CD45RC isoforms on both immature and mature CD4 T cells and the fact that the low molecular weight isoform was found in the periphery on both RTE (unquestionably naive) and antigen-experienced CD4 T cells, makes it unlikely that this isoform uniquely identifies memory T cells, at least in the rat.     

Development and activation of regulatory T cells in the human fetus

There is an increasing amount of knowledge on the functional properties of regulatory T cells (Treg) in the adult immune system, but data on the generation and function of these cells during human embryonic development are scarce. In this study, we show that in the fetal thymus, double-positive cells initiate expression of CD25, GITR, CTLA4 and CD122 during their transition from the CD27- to the CD27+ stage. Moreover, CD4+CD25+ fetal thymocytes already have the potential to suppress proliferation of CD25- cells. After leaving the thymus, FoxP3+CD4+CD25+ Treg enter the fetal lymph nodes and spleen, where they acquire a primed/memory phenotype. A model is proposed for the development of human fetal Treg that encompasses two sequential maturation steps: initiation of a regulatory phenotype and suppressive activity in the thymus; and subsequent activation within the peripheral lymphoid organs. Upon activation, FoxP3+CD4+CD25+ Treg suppress potentially deleterious responses by autoreactive lymphocytes and maintain homeostasis within the developing fetus.     

Expression and role of interleukin-2 receptor beta chain on CD4-CD8- T cell receptor alpha beta+ cells [corrected]

Using anti-murine interleukin-2 receptor beta chain (IL-2R beta) monoclonal antibody (mAb), we have examined the expression of IL-2R beta on murine thymocyte subpopulations. We found that it was constitutively expressed on 1%-4% of thymocytes in an almost mutually exclusive fashion with IL-2R alpha. The expression of IL-2R beta is developmentally regulated. While it is expressed mainly on T cell receptor gamma delta+ (TcR gamma delta+) cells during fetal age, the major subpopulation expressing IL-2R beta in adult mouse shifts to CD4-CD8-TcR alpha beta+ thymocytes. A considerable portion of CD4-CD8- TcR alpha beta+ cells in other organs, including spleen, bone marrow and liver, was also found to express IL-2R beta. In fetal thymus organ culture, the above thymocyte subset was induced to expand in response to exogeneous IL-2, and the expansion was inhibited by addition of anti-IL-2R beta mAb, suggesting that IL-2R beta is functional in this subpopulation. However, in vivo blockade of the IL-2/IL-2R pathway with the mAb did not exert any effects on the appearance of CD4-CD8- TcR alpha beta+ cells both in the thymus and the periphery. This indicates that the development of CD4-CD8- TcR alpha beta+ cells is not solely controlled by IL-2 but also by other complex elements.     

Developmentally regulated interactions of human thymocytes with different laminin isoforms

The gene family of heterotrimeric laminin molecules consists of at least 15 naturally occurring isoforms which are formed by five different alpha, three beta and three gamma subunits. The expression pattern of the individual laminin chains in the human thymus was comprehensively analysed in the present study. Whereas laminin isoforms containing the laminin alpha1 chain (e.g. LN-1) were not present in the human thymus, laminin isoforms containing the alpha2 chain (LN-2/4) or the alpha5 chain (LN-10/11) were expressed in the subcapsular epithelium and in thymic blood vessels. Expression of the laminin alpha4 chain seemed to be restricted to endothelial cells of the thymus, whereas the LN-5 isoform containing the alpha3 chain could be detected on medullary thymic epithelial cells and weakly in the subcapsular epithelium. As revealed by cell attachment assays, early CD4- CD8- thymocytes which are localized in the thymus beneath the subcapsular epithelium adhered strongly to LN-10/11, but not to LN-1, LN-2/4 or LN-5. Adhesion of these thymocytes to LN-10/11 was mediated by the integrin alpha6beta1. During further development, the cortically localized CD4+ CD8+ thymocytes have lost the capacity to adhere to laminin-10/11. Neither do these cells adhere to any other laminin isoform tested. However, the more differentiated single positive CD8+ thymocytes which were mainly found in the medulla were able to bind to LN-5 which is expressed by medullary epithelial cells. Interactions of CD8+ thymocytes with LN-5 were integrin alpha6beta4-dependent. These results show that interactions of developing human thymocytes with different laminin isoforms are spatially and developmentally regulated.     

Characterization of human lymphocytes separated from fetal liver and spleen at different stages of ontogeny

Membrane markers on human lymphocytes separated from fetal liver and spleen were studied. Depending on the period of intrauterine development, a growing percentage of T- and B-lymphocytes (up to 16% and 45%, respectively) among spleen cells was seen, but in liver the number was low independent of the gestational age (T cells less than 10% and B cells less than 15%). The majority of early CD3+ spleen cells (21st-28th week) expressed TCR alpha beta but not TCR gamma delta, although a significant proportion of these cells was still lacking CD4, CD8, and CD5 differentiation antigens, suggesting their immaturity. Later spleen T cells (29th-36th week) expressed the phenotype as mature adult-type T cells (CD3+TCR alpha beta +CD4/8+CD5+). During ontogeny in fetal spleen, a growing number of B cells could be estimated without any changes in the proportion of subsets, expressing the different light and heavy chains. However, the proportion of CD5+ B cells decreased with gestational age. The results suggest that the functional immaturity of antenatal splenocytes could not be caused by dramatic phenotypical differences in comparison with adult-type splenic lymphocytes.     

Duration dependent effect of chronic stress on primary and secondary lymphoid organs and their reversibility in rats

The present study was undertaken to investigate whether or not chronic stress effect and its reversibility on lymphoid organs is duration dependent. Male rats were exposed to restraint (1?h) followed by a gap of 4?h to forced swimming exercise (15?min) daily for 2, 4 and 8 weeks. After each exposure period, rats were allowed to recover for 6 weeks. Stress exposure resulted in duration dependent decreases in weight of thymus and axillary lymph nodes, lymphocyte counts of spleen, thymus and axillary lymph nodes and number of islets of white pulp of spleen and increases in apoptotic index of splenocytes, thymocytes and lymphocytes of axillary lymph nodes. All the parameters of lymphoid organs studied showed significant alterations in 2 weeks of stress exposure indicated their sensitivity to stress effects in short term exposure and thymus was the most sensitive organ among all. The alterations in all the parameters of spleen and majority of parameters of thymus and axillary lymph nodes returned to control level in recovery group rats of 2 and 4 weeks exposure but not in that of 8 weeks exposure. The present study for the first time reveal that severity of stress effects on lymphoid organs increases with increasing duration of exposure and shorter the exposure period faster the recovery. In addition, an in vitro study showed that corticosterone caused apoptosis of thymocytes, splenocytes and lymphocytes of axillary lymph nodes in dose dependent manner. Thus corticosterone induced death of cells of lymphoid organs under stress is the major cause of involution of lymphoid organs.     

Characterization of CCR9 expression and thymus-expressed chemokine responsiveness of the murine thymus, spleen and mesenteric lymph node

CC chemokine receptor 9 (CCR9) is a receptor expressed at high levels in immature thymocytes, small intestine trafficking T cells and IgA-producing plasma cells. CCR9 mediates chemotaxis in response to thymus-expressed chemokine (TECK) selectively expressed in the thymus and small intestine. CCR9 expression in different subpopulations of thymus, spleen and mesenteric lymph node (MLN) cells was analyzed by flow cytometry and TECK responsiveness of those lymphoid cells was assessed by a Transwell migration assay. CCR9 surface expression level did not completely correlate with cellular chemotaxis to its cognate ligand TECK. The active chemotaxis to TECK was observed in CD4 single positive thymocytes and CD4(-)B220(hi) splenocyte and MLN cells, which poorly expressed CCR9 on their surface. TECK responsiveness of CCR9-abundant subpopulations in the thymus and MLN was unremarkable except for CD4(+)B220(hi) subset of the MLN, and was evident in the CCR3(+) subsets of the thymus and spleen. Exposure to TECK did not affect CCR9 expression in the thymus, spleen and MLN, except for the CD4(+)CD8(+) thymocyte. CCR9 was exuberantly expressed in the cytoplasm of lymphoid cells. CCR9 may act in concert with CCR3 for in terms of TECK responsiveness. Its cytoplasmic location may allow precise regulation of leukocyte responsiveness to TECK.