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Objective: Regulatory T cells play an important role in the prevention of autoimmunity and have been shown to be effective in the treatment of experimental colitis, a T cell-mediated and organ-specific disease. We previously demonstrated that intrinsic CD25+ regulatory T cells modulate the severity of collagen-induced arthritis (CIA), which, in contrast to colitis, is a systemic antibody-mediated disease and an accepted model of rheumatoid arthritis. We undertook this study to determine whether regulatory T cells have the potential to be used therapeutically in arthritis., Methods: We transferred CD4+,CD25+ T cells into mice exhibiting arthritis symptoms, both immunocompetent mice and mice subjected to lethal irradiation and rescued with syngeneic bone marrow transplantation., Results: A single transfer of regulatory T cells markedly slowed disease progression, which could not be attributed to losses of systemic type II collagen-specific T and B cell responses, since these remained unchanged after adoptive transfer. However, regulatory T cells could be found in the inflamed synovium soon after transfer, indicating that regulation may occur locally in the joint., Conclusion: Our data indicate that CD25+ regulatory T cells can be used for the treatment of systemic, antibody-mediated autoimmune diseases, such as CIA.

Development of a mature T-cell repertoire in the thymus depends on lympho-stromal interaction between thymocytes and stromal cells. To facilitate intercellular contact, the epithelium in the thymus has differentiated into a unique three-dimensionally (3D)-oriented network. Here we analyze factors influencing induction and maintenance of the 3D configuration of the epithelial network in fetal thymic lobes in vitro. We show that the 3D configuration of the thymic stroma depends on (a) the oxygen pressure in vitro and (b) permanent physical contact between stromal cells and developing thymocytes. This latter feature is demonstrated by incubation of fetal thymic lobes with deoxyguanosine (d-Guo), inducing a 2D-organized thymic stroma, with thymic cysts appearing. Reconstitution of d-Guo-treated lobes with a limited number of flow-sorted T-cell progenitors restores the 3D configuration of the thymic epithelium, but only at high oxygen pressure. This study underlines the plasticity of thymic epithelium and shows that the unique organization of the thymic epithelium is dependent on both oxygen and crosstalk signals derived from developing thymocytes.

Type 1 diabetes mellitus is a classical example of a T-cell-mediated autoimmune disease. Several aberrations in immune regulation have been described in both human diabetes patients and animal models of type 1 diabetes. In this review, we summarize how proposed immune defects might be implicated in the loss of T-cell tolerance towards self in autoimmune diabetes in humans, nonobese diabetic (NOD) mice and Biobreeding (BB) rats. For this purpose, we will discuss the tolerance-inducing mechanisms that an autoreactive T cell should encounter from its genesis to its pathogenic role in the pancreas, in order of appearance. These comprise central tolerance mechanisms (i.e. positive and negative selection in the thymus) and those mechanisms operative in the periphery (i.e. activation-induced cell death and regulatory T cells).

This study describes abnormalities of the thymus in mice in which the Stat3 gene has been specifically disrupted behind the keratin 5 promoter. In these mice, virtually all of the thymic epithelial cells (TEC) were deficient for Stat3 activation. Adult mutant mice developed severe thymic hypoplasia, which included alterations in the cortical TEC architecture that coincided with the loss of thymocytes. Even during the asymptomatic period of preadolescence, these mice exhibited a higher susceptibility of the thymus to suboptimal doses of dexamethasone or gamma-irradiation, while their thymocytes per se were no more sensitive than controls. These results indicate that Stat3 in TEC plays an essential role in maintaining thymic architecture and thymocyte survival.

In this study we have tried to detect abnormalities in the immunophenotype and/or function of dendritic cells from the non-obese diabetic mouse (NOD DC), that might be related to islet autoimmunity. The immunophenotype of NOD splenic DC did not show significant abnormalities as compared with the immunophenotype of splenic DC from C57BL/10 mice. Furthermore, NOD splenic and lymph node DC stimulated proliferation of syngeneic T cells as efficiently as DC from C57BL/10 and BALB/c mice. The allogeneic response induced by NOD DC was similar to or only slightly lower than the response induced by C57BL/10 DC. Both a normal immunophenotype of NOD DC and efficient T cell stimulation were observed regardless of the stage of diabetes development. However, the syngeneic T cell proliferation induced by NOD splenic DC, but not by C57BL/10 splenic DC, was significantly prolonged, and it was accompanied by an increased proportion of activated/memory CD4(+)cells. We demonstrated that during the interaction of NOD cells fewer apoptotic cells were generated as compared with the interaction of C57BL/10 cells. Thus, the prolonged T cell response during the syngeneic interaction between NOD DC and T cells might be due to an impaired apoptosis induction. The impaired apoptosis might be of critical importance in the development of islet autoimmunity in the NOD mouse., (Copyright 1999 Academic Press.)

Lympho-stromal interactions in the thymus crucially de- termine the fate of developing T cells. Epithelial cells, inter- digitating reticular cells, macrophages and fibroblasts all play a role in the shaping of the T cell repertoire. Recently published evidence shows that lympho-stromal interaction acts bi-directional. Developing T cell themselves, at different stages of differentiation, control the microarchitecture of thymic microenvironments, a phenomenon designated as 'crosstalk'. This paper reviews experiments showing that developing T cells crosstalk to different thymic epithelial cells in a stepwise fashion. In this way, correctly organized thymic microenvironments guarantee normal thymopoiesis., (Copyright 1999 Academic Press.)

CD27 is a lymphocyte-specific member of the TNF receptor family and has a TNF-related transmembrane ligand, CD70. The CD27/CD70 receptor-ligand pair cooperates with the TCR in the regulation of the peripheral T cell response. The study presented here reveals that CD27 may play a similar role in thymic pre-T cell development. We have previously cloned the cDNA encoding murine CD27, prepared specific mAbs and observed that murine CD27 is expressed on virtually all thymocytes, with the exception of a subpopulation of CD4-8- precursor T cells. It is shown here that induction of murine CD27 expression occurs at the transition from the CD4-8-25+ to the CD4-8-25- precursor T cell stage and is regulated by the pre-TCR. Therefore, we investigated whether CD27 contributes to pre-TCR-mediated thymocyte development. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombination activating gene (RAG)-deficient mice. This in vivo anti-CD3 epsilon mAb treatment induces an about fifty fold numerical expansion of CD4-8-25+ thymocytes and their differentiation to the CD4+8+25- stage. Co-injection of anti-CD27 mAb inhibited the CD3-mediated expansion and differentiation of the CD4-8-25+ precursor population. Also, injection of anti-CD27 mAb in TCR alpha-/- mutant mice led to a reduction in the absolute number of CD4+8+25- thymocytes. We present evidence that in these in vivo systems, anti-CD27 mAb inhibits CD27-ligand interaction. Therefore, we conclude that CD27 may contribute to normal murine T cell development by synergizing with the pre-TCR-mediated signal.

T lymphocytes of the alpha/beta T-cell receptor (TCR) lineage mature in the thymus, where they undergo a series of differentiation, expansion and selection events. For normal T-cell ontogeny to occur, thymocytes must interact physically with cortical and medullary thymic stroma cells. In parallel, interactions of the thymic stromal cells with TCR-positive thymocytes are necessary for the development of the thymic medulla. Comparable requirements for the differentiation of the cortex have not been defined, however. Here we analyse mutant mouse strains to assess the function of early prothymocytes in the induction of the thymic cortex. We find that animals with a developmental block at the earliest stage of T-lineage commitment lack a functional thymic cortex. This abnormality could be corrected in fetal but not adult animals by transplantation of either fetal or adult wild-type haematopoietic stem cells. Thus a developmentally restricted interaction of fetal stromal cells with early prothymocytes is required for the induction of a cortical microenvironment. In addition, a normal thymic architecture is necessary for sustained T-cell ontogeny.

Dividing cells require iron and, therefore, express the transferrin receptor (CD71) on the cell surface to enable internalization of transferrin-bound iron. Since early T cell development is marked by intense proliferation, we questioned whether CD71 might serve as a marker of immature T cells. Therefore, we analyzed the expression of CD71 on fetal, neonatal, and adult thymocytes in correlation with cell size, cell cycle status, and expression of CD3, CD4, CD8, alpha beta TcR, and gamma delta TcR. Phenotypic analysis showed that only the large, immature CD4-8-3-, CD4-8+3-, and CD4+8+3- cells in fetal, neonatal, and adult thymus expressed CD71. In addition, DNA analysis showed that all CD71+ large adult thymocytes were cycling. Downregulation of CD71 occurs when proliferation ceases, i.e., within the CD4+8+3- thymocyte subpopulation. The gradual changes in size and CD71 expression suggest a sequential development within this CD4+8+3- subpopulation from large CD71+ via small CD71+/- to small CD71- cells. As a consequence, CD71 expression is downregulated, in adult T cell development as well as in ontogeny, before the alpha beta TcR appears on the cell surface of the thymocyte. Together, our findings show that CD71 is a marker of immature, proliferating T cells.

Proliferating cells require iron and, therefore, express the transferrin receptor (CD71) that mediates cellular iron uptake. Cycling thymocytes, which have the CD4-8-3-, CD4-8+3-, or CD4+8+3- phenotypes, also express CD71. The importance of CD71-mediated iron uptake for proliferation and maturation of thymocytes was studied using fetal thymus organ cultures at day 14 of gestation and treating them for 7 days with a CD71 monoclonal antibody (mAb). The intracellular iron deficiency caused by this treatment, inhibits both proliferation and maturation of the thymocytes. Cell recovery was reduced by 60%, but cells still expanded tenfold during the culture. Remarkably, the final maturation of alpha beta T cells was completely blocked as no thymocytes with low or high CD3/alpha beta TcR expression developed. Moreover, only few cells reached the CD4+8+3- stage of T cell development. CD4-8-3- thymocytes, however, as well as its CD44-25+ subset developed in normal numbers, suggesting that CD44-25+ CD4-8-3- cells, or their immediate progeny, were most vulnerable to CD71 mAb treatment. The development of gamma delta T cells, which also express CD71, was not affected in these cultures. This suggests that gamma delta T cells are either less iron-dependent or possess alternative iron-uptake mechanisms. Thus, our observations indicate that CD71 treatment, causing decreased intracellular iron levels, severely inhibits the major proliferation phase from the CD44-25+ CD4-8-3- to the CD4+8+3- cells, and completely abrogates the final maturation of CD4+8+3- cells into alpha beta TcR-expressing cells. In contrast, proliferation and differentiation of the earliest thymic precursors into CD44-25+ CD4-8-3- cells is not affected by CD71 treatment.

Unlike medullary thymic epithelial cells (TEC) of normal mice, medullary TEC of TCR- SCID mice are immature and disorganized. In order to assess directly the role of TCR+ cells in the development of medullary TEC, we bred mice which co-expressed the SCID genetic defect and transgenes encoding clonotypic TCR chains. Immunohistologic examination revealed that medullary thymic epithelial cells from TCR beta transgenic SCID mice, whose thymocytes only express TCR beta chains that inefficiently associate with CD3 and zeta components, remained immature and disorganized. In contrast, medullary TEC from TCR alpha beta transgenic SCID mice, whose thymocytes express fully assembled CD3-zeta-TCR alpha beta complexes were mature and organized. Interestingly, the ability of TCR alpha beta(+)-zeta(+)-CD3+ thymocytes to induce maturation of medullary TEC appeared not to be related to the antigen specificity of the TCR as thymi from positively selecting, negatively selecting and non-selecting TCR alpha beta transgenic SCID mice all possessed induced medullary thymic epithelial cells. In addition, we found that induction of medullary TEC cells was associated with the presence of medullary thymocytes, including those of the CD4-CD8- TCR alpha beta+ phenotype. The present findings demonstrate that fully assembled CD3-zeta-TCR complexes are required to induce maturation of medullary thymic epithelial cells and indicate that thymocyte induction of medullary thymic epithelial cells may result from signaling independently of their clonotypic TCR chains.

The thymic stroma is heterogeneous with regard to cellular morphology and cellular function. In this study, we employed the monoclonal antibody ER-TR4 to characterize stromal cells at the ultrastructural level. To identify the labelled cell type, we used two techniques: immunogold labelling on ultrathin frozen sections and immunoperoxidase staining on thick "vibratome" sections. ER-TR4 reacted with thymic Type 1 epithelial cells (according to our classification). A dense labelling appears in the cytoplasm of cortical cells using the two techniques. Immunogold labelling identified small cytoplasmic vesicles whereas the cytoplasm and the cell membrane seem to be labelled with the immunoperoxidase technique. ER-TR4 also identified isolated thymic nurse cells (TNC), and was observed in vitro to inhibit the capacity of some type 1 epithelial cells to establish interactions with immature thymocytes. This finding supports the hypothesis that the factor is involved in the formation of lymphoepithelial interactions within thymic nurse cells, and thus in the relations that immature thymocytes establish with the thymic microenvironment.

The development of mature T cells within the thymus is dependent upon intact cortical and medullary microenvironments. In turn, thymic microenvironments themselves are dependent on lymphoid cells to maintain their integrity. Here, Willem van Ewijk and colleagues discuss experiments that have established the phenomenon of 'crosstalk' within the mouse thymus and suggest a mechanism whereby lymphoid and stromal cells influence each other in a consecutive manner during T-cell development.

The protein Lck (p56lck) has a relative molecular mass of 56,000 and belongs to the Src family of tyrosine kinases. It is expressed exclusively in lymphoid cells, predominantly in thymocytes and peripheral T cells. Lck associates specifically with the cytoplasmic domains of both CD4 and CD8 T-cell surface glycoproteins and interacts with the beta-chain of the interleukin-2 receptor, which implicates Lck activity in signal transduction during thymocyte ontogeny and activation of mature T cells. Here we generate an lck null mutation by homologous recombination in embryonic stem cells to evaluate the role of p56lck in T-cell development and activation. Lck-deficient mice show a pronounced thymic atrophy, with a dramatic reduction in the double-positive (CD4+CD8+) thymocyte population. Mature, single-positive thymocytes are not detectable in these mice and there are only very few peripheral T cells. These results illustrate the crucial role of this T-cell-specific tyrosine kinase in the thymocyte development.

In order to assess the possible role of lymphoid cells in the development of thymic epithelium, we compared the organization and maturation of thymic epithelium in scid mice lacking T cell receptor (TcR)-positive cells with that in scid mice containing TcR+ cells. Immunohistologic examination revealed that thymi from TcR- scid mice were deficient in thymic medullary epithelial cells recognized by the monoclonal antibody ER-TR5, and that the few thymic medullary epithelial cells present were not organized into discrete medullary areas. In contrast, thymi from scid mice containing TcR+ cells possessed ER-TR5+ thymic medullary epithelial cells and these cells were organized normally into discrete medullary regions. Thus, normal organization and maturation of thymic medullary epithelial cells did not occur in the absence of TcR+ cells, but did occur upon introduction of TcR+ cells. We conclude that lympho-stromal cell interactions in the thymus are not unidirectional, and that a symbiotic relationship exists between maturing epithelial cells and developing lymphocytes.

Intrathymic T-cell differentiation requires a symbiotic interaction between thymic microenvironments and developing T cells. This paper attempts to provide insight into lympho-stromal interaction and reviews the architecture of thymic microenvironments, the phenotype of thymic microenvironments, the in vitro culture of thymic microenvironments, and the supportive role of thymic microenvironments in T-cell differentiation. Moreover, we discuss experimental manipulation of thymic microenvironments in vivo and in vitro, using monoclonal antibodies directed to cell surface determinants on stromal cells or their ligands on lymphoid cells. Finally, new types of experimental mouse models are considered with special reference to the role of thymic microenvironments in positive and negative selection of the T-cell repertoire, and the potential influence of T cells on the development of thymic microenvironments.

We examined the immunohistological aspects of the H-Y specific T cell receptor (TcR) alpha/beta transgene expression in the thymus of male and female transgenic (Tg) mice. Virtually all thymocytes expressed the beta transgene in both the male and female thymus. Expression of accessory molecules (co-receptors) in Tg mice deviated from control mice. In the male Tg thymus, CD8 expression was either low or absent on both cortical and medullary thymocytes. In contrast, in the thymus of female mice, CD8+ cells were found both in the cortex and in the medulla. The majority of medullary thymocytes was bright CD8+. This is in clear contrast to the CD8 distribution in control B6 mice, where only a few percent of medullary cells are CD8+. Similarly, the proportion of cells expressing CD4 antigens was reduced in the cortex and medulla of the thymus from male Tg mice, as compared to the thymus of female Tg mice and B6 control mice. Comparative analysis of the stromal cell types of the thymic microenvironments in the three groups of mice revealed that the cortical thymic microenvironment of male Tg mice differed, compared to that of female Tg mice. In particular, the deep cortex showed a closely packed meshwork of epithelial reticular cells. Moreover, H-2Db molecules (which are the restricting elements for the Tg TcR alpha/beta) were abnormally expressed in the thymic cortex of male mice. The cortical microenvironment in female mice, on the other hand, appeared normal. Together, the data indicate that TcR alpha/beta transgene expression in male mice leads to an aberrant co-receptor expression in both cortical and medullary lymphoid cells as well as an abnormal composition of the cortical microenvironment. Both phenomena may be the consequence of "negative selection" of developing H-Y-specific T cells, as it occurs only in the male Tg thymus. The absence of the H-Y antigen, but presence of the restricting element H-2Db in the thymic cortex of female mice, leads to accumulation of CD8+ in the medulla, a phenomenon interpreted as "positive selection".

Recent studies have focused on the potential role of accessory molecules such as CD2, CD28, Thy-1, or TAP in the delivery of activating signals to thymocytes through antigen-independent pathways. To better understand the molecular interactions involved in the expansion of early thymic immigrants, rat mAb were raised against murine thymocyte-surface molecules and screened for their capacity to trigger thymocyte proliferation. One of these mAb (H194-112, IgG2a) was found to recognize a novel heterodimeric thymocyte-activating molecule (THAM) of Mr = 110,000 to 128,000. Flow cytometric analyses and staining patterns on frozen thymus sections subdivided adult thymocytes in three subsets expressing THAM at either low (10%), moderate (80%), or high (5 to 8%) cell-surface density; these cell groups were found to correspond, respectively, to the medullary, the cortical, and the immature CD4-CD8-, J11d+ thymocytes, in which the T cell precursor pool is included. Moreover, most (90%) day 16 fetal thymocytes were also found to upregulate THAM cell-surface expression. The THAMhigh cells were localized in the subcapsular area of the neonatal thymus and scattered throughout the adult organ. Cross-linked mAb H194-112 induced the proliferation of both immature and mature thymocytes in the presence of either PMA or IL-1 and IL-2. The observation that early thymocytes up-regulate THAM along with the IL-2R suggests that this molecule might be involved in an important activation pathway during thymocyte differentiation.

The demonstration of thymic nurse cells (TNC), complexes between stromal cells and thymocytes, in cell suspensions of murine thymuses, prompted us to investigate (1) the relationship of TNC to other thymic stromal cell types defined in situ, and (2) the maturation stage of the enclosed thymocytes. To this purpose we incubated frozen sections of TNC suspensions with various monoclonal antisera directed to T cells and stromal cell types, using immunohistology. This approach enabled us to study antigen expression on the "nursing" cell itself and to analyze the phenotype of the enclosed lymphocytes in cross sections of TNC. The results show that lymphocytes enveloped by TNC express high levels of Thy-1, moderate levels of T200, and variable amounts of Lyt-1. Due to enzymatic degradation Lyt-2 expression could not be studied. The enveloped cells also bear PNA receptors, but no detectable I-A/E antigens. Expression of H-2K antigens on enclosed thymocytes varied from weak to absent. The "nursing" cells react with ER-TR4, a monoclonal antibody which detects cortical epithelial-reticular cells. In addition TNC express I-A/E and H-2K antigens. In contrast, TNC do not react with ER-TR 5 and 7, monoclonal antibodies, which detect medullary epithelial cells and reticular fibroblasts, respectively. TNC do not express the macrophage antigens Mac-1 and Mac-2. We conclude that TNC in vitro represent the in vivo association of epithelial-reticular cells with cortical thymocytes. However, the enclosed thymocytes do not constitute a phenotypically distinct subset of subcapsular or outer cortical cells.

Young adult CBA/H mice were exposed to graded doses of whole-body irradiation with either fast fission neutrons or 300 kVp X rays at center-line dose rates of 0.1 and 0.3 Gy/min, respectively. Dose-response curves were determined at Days 2 and 5 after irradiation for the total thymic cell survival and for the survival of thymocytes defined by monoclonal anti-Thy-1, -Lyt-1, -Lyt-2, and -T-200 antibodies as measured by flow cytofluorometric analysis. Cell dose-response curves of thymocytes show, 2 days after irradiation, a two-component curve with a radiosensitive part and a part refractory to irradiation. The radiosensitive part of the dose survival curve of the Lyt-2+ cells, i.e., mainly cortical cells, has a D0 value of about 0.26 and 0.60 Gy for neutrons and X rays, respectively, whereas that of the other cell types has corresponding D0 values of about 0.30 and 0.70 Gy. The radiorefractory part of the dose-response curves cannot be detected beyond 5 days after irradiation. At that time, the Lyt-2+ cells are again most radiosensitive with a D0 value of 0.37 and 0.99 Gy for neutrons and X rays, respectively. The other measured cell types have corresponding D0 values of about 0.47 Gy. The fission neutron RBE values for the reduction in the thymocyte populations defined by either monoclonal anti-Thy-1, -Lyt-1, -Lyt-2, or -T-200 antibodies to 1.0% vary from 2.6 to 2.8. Furthermore, the estimated D0 values of the Thy-1-, T-200- intrathymic precursor cells which repopulate the thymus during the bone marrow independent phase of the biphasic thymus regeneration after whole-body irradiation are 0.64-0.79 Gy for fission neutrons and 1.32-1.55 Gy for X rays.

The T cell composition of the thymus of sublethal fission neutron-irradiated CBA/H mice was analyzed with cytofluorometry and immunohistology, using monoclonal antibodies directed to the cell surface antigens Thy-1, T-200, MT-4, Lyt-1, Lyt-2, and MEL-14. The results of this investigation show that whole body irradiation with 2.5 Gy fission neutrons results in a severe reduction and degeneration of the cortex, whereas the medulla is affected to a lesser extent. Irradiation selects, within 24 hr, for a population of dull Thy-1+, bright T-200+, bright Lyt-1+ cells localized in the medulla. Phenotype analysis of the regeneration of the thymus, which starts at about 5 days after irradiation, reveals the sequential appearance of: 1) "null" cells, i.e., lymphoblasts negative for all tested antigens, mainly in the subcapsular area but also in the medulla; 2) Thy-1+ "only" and T-200+ "only" cells in the subcapsular area; 3) Thy-1+, T-200+ cells; and 4) Thy-1+, T-200+, MT-4+, Lyt+ cells in the cortex. In addition, an increased MEL-14 expression is observed in correlation with the expression of Thy-1 and T-200 determinants during the regeneration of the thymus. From day 10 on up to at least 150 days after irradiation, no differences can be observed in the thymus of irradiated and age-matched sham-irradiated control mice, as measured by the expression and distribution of Thy-1, T-200, MT-4, Lyt-1, Lyt-2, and MEL-14 antigens. The observed sequence in phenotype shift in the regeneration of the thymus after irradiation is discussed in view of recently published data on the differentiation of the T cell system.

The present paper reports the distribution of lymphoid and non-lymphoid cell types in the thymus of mice. To this purpose, we employed scanning electron microscopy and immunohistology. For immunohistology we used the immunoperoxidase method and incubated frozen sections of the thymus with 1) monoclonal antibodies detecting cell-surface-differentiation antigens on lymphoid cells, such as Thy-1, T-200, Lyt-1, Lyt-2, and MEL-14; 2) monoclonal antibodies detecting the major histocompatibility (MHC) antigens, H-2K, I-A, I-E, and H-2D; and 3) monoclonal antibodies directed against cell-surface antigens associated with cells of the mononuclear phagocyte system, such as Mac-1, Mac-2, and Mac-3. The results of this study indicate that subsets of T lymphocytes are not randomly distributed throughout the thymic parenchyma; rather they are localized in discrete domains. Two major and four minor subpopulations of thymocytes can be detected in frozen sections of the thymus: 1) the majority of cortical thymocytes are strongly Thy-1+ (positive), strongly T-200+, variable in Lyt-1 expression, and strongly Lyt-2+; 2) the majority of medullary thymocytes are weakly Thy-1+, strongly T-200+, strongly Lyt-1+, and Lyt-2- (negative); 3) a minority of medullary cells are weakly Thy-1+, T-200+, strongly Lyt-1+, and strongly Lyt-2+; 4) a small subpopulation of subcapsular lymphoblasts is Thy-1+, T-200+, and negative for the expression of Lyt-1 and Lyt-2 antigens; 5) a small subpopulation of subcapsular lymphoblasts is only Thy-1+ but T-200- and Lyt-; and 6) a small subpopulation of subcapsular lymphoblasts is negative for all antisera tested. Surprisingly, a few individual cells in the thymic cortex, but not in the medulla, react with antibodies directed to MEL-14, a receptor involved in the homing of lymphocytes in peripheral lymphoid organs. MHC antigens (I-A, I-E, H-2K) are mainly expressed on stromal cells in the thymus, as well as on medullary thymocytes. H-2D is also expressed at a low density on cortical thymocytes. In general, anti-MHC antibodies reveal epithelial-reticular cells in the thymic cortex, in a fine dendritic staining pattern. In the medulla, the labeling pattern is more confluent and most probably associated with bone-marrow-derived interdigitating reticular cells and medullary thymocytes. We discuss the distribution of the various lymphoid and non-lymphoid subpopulations within the thymic parenchyma in relation to recently published data on the differentiation of T lymphocytes.

The denaturing effects of various types of fixative solutions on 5 cell surface antigens on mouse T-lymphocytes (Thy-1, T-200, Lyt-1, Lyt-2 and Th-B) were studied. For this purpose, cells were fixed with paraformaldehyde, glutaraldehyde, acrolein and osmium tetroxide at various concentrations. Fixed cells were then incubated with monoclonal antibodies and appropriate second stage antibodies or conjugates. The degree of antibody binding to these cells was determined quantitatively using flow-cytometry with a fluorescence-activated cell sorter or with a semi-automatic micro-ELISA system. The data obtained indicate that paraformaldehyde and glutaraldehyde preserve all five tested antigen molecules, whereas antibody binding to cells fixed in acrolein and osmium tetroxide is rapidly reduced at increasing concentrations of the fixative. The optimal concentration of paraformaldehyde is in the range 0.5-1%, whereas glutaraldehyde should be used at concentrations between 0.05 and 0.1%. Cells fixed with 0.5% paraformaldehyde or with 0.05% glutaraldehyde are stable and can be stored for at least one week prior to incubation with antibodies.

The monoclonal antibody MEL-14 recognizes a lymphocyte surface structure (the MEL-14 antigen) involved in migration of lymphocytes into lymph nodes. Its use as a maturation marker for T cells within the thymus led to the view that a small population (1 to 2%) of MEL-14high thymocytes located in the inner cortex represented fully mature cells about to exit as thymus emigrants. The medulla, in this view, contained only the phenotypically mature but MEL-14low cells, and was not the source of thymus emigrants. The data we present, derived from flow-cytometric analysis of suspension-stained CBA mouse thymocytes, is not in accordance with this view. A high proportion (approximately 20%) of thymocytes express relatively high levels of MEL-14; these include some immature Ly-2- L3T4- and nonmature Ly-2+ L3T4+ thymocytes. Among the 12 to 14% thymocytes of mature phenotype (PNAlow or H-2Khigh or Ly-2+ L3T4- and Ly-2- L3T4+), more than half express relatively high levels of MEL-14. The mature phenotype and MEL-14moderate-to-high cells (8% of thymocytes) appear too numerous to account for the few percent MEL-14high cells seen in the cortex in frozen sections, and the mature phenotype but MEL-14low cells (2 to 3% of thymocytes) too few to fill the medulla; however, both together account numerically for the medullary population. By section staining, the medulla contains Ly-2- L3T4+ and Ly-2+ L3T4- cells in a characteristic 2:1 ratio; by suspension staining this ratio agrees with that of the total mature phenotype population, but not with that of the MEL-14low subset previously claimed to represent medullary cells. Another paradox is apparent when suspension staining and section staining are compared: suspension staining reveals that many mature phenotype cells coexpress high levels of both MEL-14 and H-2K, yet section staining reveals H-2Khigh cells in the medulla but not in the inner cortex, and reveals scattered MEL-14high cells throughout the cortex but not in the medulla. We suggest that section staining for MEL-14 fails to locate the mature cells that stain for MEL-14 in suspension; the few MEL-14high cells localized in both the inner and the outer cortex on section staining are predominantly immature Ly-2- L3T4- and nonmature Ly-2+ L3T4+ thymocytes; the majority of thymocytes of mature phenotype, whether MEL-14high or MEL-14low on suspension staining, are of medullary location; the medulla is the most likely immediate source of thymic emigrants.

Thymuses of CBA/H mice were investigated up to 570 days after whole-body irradiation with 2.5 Gy fast fission neutrons or 6.0 Gy X rays. A number of these thymuses, observed 220-270 days after neutron irradiation, have two equal sized lobes, one of which has an abnormal T cell distribution. The present paper reports on the distribution of lymphoid and stromal cell types in these thymuses. For this purpose, we employed immunohistology using the indirect immunoperoxidase method. We incubated frozen sections of these aberrant thymuses with monoclonal antibodies directed to cell surface differentiation antigens on lymphoid cells, such as Thy-1, T-200, MT-4, Lyt-1, Lyt-2, and MEL-14; monoclonal antibodies directed to major histocompatibility complex (MHC) antigens, such as I-A and H-2K; and monoclonal antibodies directed to determinants in various thymic stromal cell types. The results of this study show a T cell differentiation arrest in only one of the two thymic lobes. T cells in the aberrant lobe express Thy-1, T-200, and MEL-14 antigens but are MT-4- and Lyt-1-. In some lobes, a weak Lyt-2 expression was observed. The observed T cell maturation arrest is mainly restricted to the cortex since in the medulla, in addition to cells with an aberrant cortical phenotype, normal T cell phenotypes are observed. This indicates that cortex and medulla have independent generation kinetics in T cell maturation. The stromal cell composition in these abnormal lobes is not different from that in the normal lobe, but the size of the medulla tends to be smaller. Furthermore, the I-A expression on the cortical epithelial cells does not reveal the characteristic reticular staining pattern that is observed in the normal lobe, since the I-A determinants are not strictly confined to the epithelial cells. In addition, cortical lymphoid and stromal cells in these lobes are slightly H-2K+. These alterations in MHC expression in the cortex are discussed in relation to the observed T cell maturation arrest.

DBA/2 mice which are neonatally infected with Rauscher helper virus (R-MuLV) develop predominantly lymphatic leukemias. From one of these lymphatic leukemias we established a permanent cell line which we named RLD (Rauscher Lymphoid DBA/2). Phenotyping of this cell line with a panel of monoclonal antibodies directed to cell-surface determinants shows that RLD cells have T-cell characteristics: they bind monoclonal antibodies directed to the antigens Thy-1, T-200 and Lyt-1; they do not react with anti-Lyt-2 antibodies, nor do they react with antibodies directed to determinants on B cells or myelomonocytic cells. RLD cells show a high activity of the nuclear enzyme terminal deoxyribonucleotidyl transferase (TdT). RLD cells are able to differentiate after in vitro stimulation with 1% DMSO or with 30 nM tetradecanoylphorbol-1.3-acetate (TPA). This differentiation process is reflected by (1) changes in the 2D gel electrophoresis pattern of metabolically labelled proteins, (2) a decrease in TdT activity and (3) changes in the expression of cell-surface markers. Flow cytometric analysis of stimulated RLD cells shows a strong increase in the Lyt-1 expression. Together these data indicate that RLD cells are immature T lymphocytes which upon appropriate stimulation differentiate along the line of T helper cells.

The spleen of rats and mice was studied with the light and electron microscope. Special attention was paid to the delineation and composition of the white pulp compartments: periarteriolar lymphatic sheath (PALS), follicles and marginal zone. These three compartments each have their specific lymphoid and non-lymphoid cells. Reticulum cells and reticulin fibres, although occurring in all three compartments, from a characteristic pattern in each compartment. In the PALS two areas can be distinguished: a central area, largely devoid of reticulum cells, and a peripheral area where reticulum cells are arranged in cylindrical shells. The central PALS FORMS THE THYMUS DEPENDENT AREA OF THE SPLEEN, THE PERIPHERAL PALS contains both T- and B-lymphocytes. T-B-interactions requiring cell contact could take place in the latter area. Lymph vessels originate from the shells of reticulum cells around the smaller arterioles; these vessels follow the central arteriole to the hilus of the spleen. Circumstantial evidence suggests that the lymph vessels form a recirculation pathway for T-cells and possibly also for B-cells. In two areas of the splenic white pulp characteristic non-lymphoid cells are present. The central PALS contains interdigitating cells (IDC), which show a close contact with surrounding T-lymphocytes. The light zone of the follicle centre exhibits dendritic cells (DC). B-cells are found between the ramifications of the DC. It is conceivable that these cells play a role in the homing of T-cells and B-cells respectively. In addition they might create a microenvironment supporting differentiation and proliferation of T- and B-cells. The marginal zone does not contain a characteristic non-lymphoid cell type. However, in this compartment B-cells are directly exposed to the circulating blood. It is suggested that this factor constitutes one of the essentials of the microenvironment in the marginal zone.

The effect of injection of a range of doses of dexamethasone on the distribution of T-cell subpopulations and stromal cells in the thymus of BALB/c mice was investigated with flowcytometry and immunohistology. To this purpose we used monoclonal antibodies directed to the T-cell differentiation antigens Thy-1, T200, Lyt-1, Lyt-2, T4, MEL-14, and monoclonal antibodies directed to various classes of stromal cells. Injection of dexamethasone in increasing doses of 5-130 mg/kg body weight gradually leads to a depletion of the cortical thymocyte population, i.e., bright Thy-1 + ve, dull T-200 + ve, bright Lyt-2 + ve, and bright T4 + ve cells. These cortical cells are very dull MEL-14 + and express variable numbers of Lyt-1 molecules. Also the medulla is affected by dexamethasone although to a lesser extent. Dexamethasone injection at 130 mg/kg selects for a dull Thy-1 + ve, bright T-200 + ve, and bright Lyt-1 + ve medullary population. These cells are either T4 + ve Lyt-2-ve or T4-ve Lyt-2 + ve. Under these conditions, MEL-14 + ve cells were no longer present in the cortex but accumulated in medullary perivascular spaces. Staining of sequential sections showed that this particular subpopulation has a typical "helper" phenotype. This observation provides strong evidence that perivascular compartments are an exit pathway for emigrating T cells. The medullary population contains a phenotypically distinct, dexamethasone-sensitive subpopulation. This conclusion is based on two findings: 130 mg/kg dexamethasone depletes the thymus of all but 4% of the thymocytes, which form a much smaller subpopulation than the population of dull Thy-1 + ve cells (amounting to 15% of the total thymocytes). The medulla contains a subpopulation of dull Lyt-2 + ve cells, which are resistant to 20 mg/kg dexamethasone, but depleted by 130 mg/kg. Dexamethasone also has a severe effect on thymic nonlymphoid cells. Even at low doses, dexamethasone induces TR4 + ve cortical epithelial-reticular cells to become spherical ("nurse cell-like") structures, depleted of lymphoid cells. These stromal cells no longer express MHC antigens in a membrane-bound fashion. In contrast, the medullary epithelial cells appear morphologically unaffected even at a dexamethasone dose of 130 mg/kg.

The stromal cells of the thymus of sham-irradiated and sublethal fission neutron-irradiated CBA/H mice were analyzed with immunohistology, using monoclonal antibodies directed to I-A and H-2K antigens as well as specific determinants for cortical and medullary stromal elements. In the control thymuses, I-A expression in the thymus shows a reticular staining pattern in the cortex and a confluent staining pattern in the medulla. In contrast, H-2K expression is mainly confluently located in the medulla. Whole body irradiation with 2.5 Gy fission neutrons reduces within 24 hr the cortex to a rim of vacuolized "nurse cell-like" epithelial cells, largely depleted of lymphoid cells. The localization of I-A antigens changes in the cortex and I-A determinants are no longer associated with or localized on epithelial reticular cells. Medullary stromal cells, however, are more or less unaffected. A high rate of phagocytosis is observed during the first 3 days after irradiation. About 5 days after irradiation, the thymus becomes highly vascularized and lymphoid cells repopulate the cortex. The repopulation of the thymic cortex coincides with the appearance of a bright H-2K expression in the cortex which is associated with both stromal cells as well as lymphoid blasts. During the regeneration of the thymus, the thymic stromal architecture is restored before the expression of cell surface-associated reticular MHC staining patterns. The observed sequential changes in the thymic microenvironment are related to the lymphoid repopulation of the thymus.