Thymic shared antigen-1. A novel thymocyte marker discriminating immature from mature thymocyte subsets.

In a previous study, we raised a mAb (MTS 35) reacting with a plasma membrane Ag expressed on both cortical thymocytes and a subset of thymic medullary epithelial cells. In view of the shared expression of this molecule, we have defined it as thymic shared Ag-1 (TSA-1). Considering its selective reactivity with cortical, but not medullary thymocytes, the relevance of TSA-1 as a marker of immature T cells was investigated in detail in this study, using multicolor flow cytometric analysis. TSA-1 was found on all immature thymocyte subsets (CD3-4-8-, CD3-4+8-, CD3-4-8+, CD3-4+8+, CD3low4+8+). Conversely, CD3high4+8- and CD3high4-8+ thymocytes, early thymic migrants and peripheral T cells were TSA-1-. More refined gating and analysis of the transitional CD3intermediate/high4+8+ thymocytes, proposed candidates for negative selection, demonstrated that approximately one half were TSA-1-. In fact, there was a directly inverse relationship between TSA-1 and CD3 expression on thymocytes. In the periphery, TSA-1 was detected on B lymphocytes. TSA-1 is PI-linked and has a molecular mass of 17 kDa nonreduced, or 12 to 13 kDa reduced. Through cross-correlation analysis, this molecule was distinct from H-2K, PNA-R, CD5, CD11a/18, Thy-1, HSA, Ly6A/E, Ly6C, ThB, CD25, CD44. Hence TSA-1 appears to be a unique marker which exquisitely separates mature from immature thymocytes.     

Pertussis toxin-induced lymphocytosis is associated with alterations in thymocyte subpopulations.

Pertussis toxin (Ptx), an important adjuvant for inducing certain organ-specific autoimmune diseases in mice, exerts multiple effects upon the immune system. In addition to its adjuvant effects, which include enhancement of delayed-type hypersensitivity and increased antibody production. Ptx elicits a marked lymphocytosis with a concomitant decrease in thymic weight. In vitro studies indicate that Ptx acts directly on thymocytes and that both susceptible and resistant populations exist. It is believed that these susceptible cells are released into the circulation and account, in part, for the T cell component of the lymphocytosis. We have used flow cytometry to analyze the CD4, CD8, and Thy-1 phenotypes of thymic and peripheral T cells from Ptx-treated mice. In the thymus, there is a dramatic decrease in the number of CD4+CD8+ (double positive) cells at all doses tested (0.25, 0.50, and 1.0 microgram) by day 4 after Ptx treatment. The double negative and single positive populations remain relatively constant. Analysis of Thy-1 expression reveals a significant reduction in Thy-1hi thymocytes, with little change in the Thy-1lo population. Thus Ptx primarily affects and depletes, in a dose-dependent fashion, thymic T cells with an immature phenotype. These results mimic those of corticosteroids, although neither prior adrenalectomy nor treatment with the antiglucocorticoid RU486 are able to prevent the effects of Ptx. In the periphery of Ptx-treated animals, the relative increase in the number of CD4+ T cells is more than that of CD8+ T cells. Double positive and Thy-1hi cells cannot be detected in appreciable numbers. These results are consistent with the concept that Ptx may drive immature thymocytes through accelerated maturation for release into the periphery as single positive, predominantly CD4+, Thy-1lo cells. Increased numbers of such cells may in part account for the immunopotentiating effects of Ptx, particularly as they relate to the induction of organ-specific autoimmune disease. Treatment with purified Ptx beta-oligomer fails to elicit any of the responses described above, indicating that the holotoxin is required for such activities.     

Thymic involution and thymocyte phenotypic alterations induced by murine mammary adenocarcinomas

A profound thymic atrophy has been observed in mice bearing large adenocarcinomas of the mammary gland. Only 2 to 5% of thymocytes remained 4 wk after tumor implantation. Although there is a slight decrease in the overall percentages of Thy-1+ cells in tumor bearers, the majority of the remaining cells are of a Thy-1 low phenotype. There was a lower percentage of double positive (CD4+, CD8+) cells, an increase of CD4+ CD8- thymocytes, similar percentages of CD4- CD8+ cells and double negative (CD4- CD8-) thymocytes in tumor-bearing mice. In addition, an increased percentage of CD3 cells could be detected in these animals. These results indicate that proportionally less immature thymocytes are present in the atrophic thymuses of mammary tumor bearers. Enhanced levels of glucocorticoids are known to produce similar effects on the thymus. However, adrenalectomy of mice followed by tumor implantation did not result in reversal of the thymic atrophy. Furthermore, a study of serum corticosterone levels in tumor bearers indicated no significant changes during tumorigenesis. A study of several parameters of bone marrow (BM) populations indicate that there is an increase in cells of the granulocyte-macrophage lineage and a decrease in lymphocytes induced by tumor-derived granulocyte macrophage-CSF. An alteration of prothymocytes in the BM is not the main cause of the thymic atrophy because BM cells from normal and tumor-bearing mice reconstituted irradiated normal mice equally well. There was no preferential recruitment of double positive cells to the spleen as indicated by no significant differences in the levels of T cells of immature phenotype including the CD4+ CD8+ population in the spleens of tumor bearers. Because no major changes were observed in tumor bearers, either at their capacity to repopulate the thymus or at the patterns of subsequent redistribution of thymocytes, it was postulated that the thymic atrophy may be caused by a direct or indirect effect of the tumor or tumor-associated factor(s). Intrathymic injections of tumor cells into young normal recipient mice resulted in a significant reduction of the thymus weight and cellularity. These data suggest that mammary tumors can secrete factor(s) that are capable of severely impairing the normal development of cells of the T cell lineage.     

Human T cell antigen expression during the early stages of fetal thymic maturation

Human thymus tissue was examined from 7 wk of gestation through birth for the expression of antigens reacting with a panel of anti-T cell monoclonal antibodies. Additionally, the reactivities of reagents against the transferrin receptor, against leukocytes, against low m. w. keratins, and against major histocompatibility complex antigens were studied on human fetal thymic tissue. Frozen tissue sections were evaluated by using indirect immunofluorescence assays. At 7 wk of gestation, no lymphoid cells were identified within the epithelial thymic rudiment; however, lymphoid cells reacting with both antibody 3A1, a pan T cell marker, and antibody T200, a pan leukocyte reagent, were identified in perithymic mesenchyme. After lymphoid colonization of the thymic rudiment at 10 wk of fetal gestation, fetal thymic tissue reacted with antibodies T1, T4, and T8. At 12 wk of gestation, antibodies T3, T6, A1G3 (anti-p80, a marker of mature thymocytes), and 35.1 (anti-E rosette receptor) all reacted with thymic tissue. Our findings indicate that T cell antigens were acquired sequentially on thymocytes at discrete stages during the first trimester of human fetal development. The 3A1 antigen was present on fetal lymphocytes before lymphoid cell colonization of thymic epithelium, suggesting that passage through the thymus was not required for the expression of the 3A1 antigen by T cell precursors. The appearance of mature T cell antigens, T3 and p80, on thymocytes by 12 wk of gestation implies that the T cell antigen repertoire may be established in the thymus during the first trimester. Thus, a critical period of T cell maturation appears to occur between 7 and 12 wk of human fetal gestation.     

Helper cells active in the generation of cytotoxicity to a syngeneic tumor

We have examined a population of thymic lymphocytes that augment the generation in vitro of specific cytotoxic T cells against P815.X2, a syngeneic tumor in DBA/2 mice. These helper cells are evident in the thymus of DBA/2 mice 5 to 7 days after subcutaneous injection of live P815 cells. They are Thy-1 positive, resistant to gamma radiation, antigen specific, and appear to exert their influence most effectively when added at the beginning of the culture.     

Immunohistology of lymphoid and non-lymphoid cells in the thymus in relation to T lymphocyte differentiation

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.     

Lymphocyte lineages at mucosal effector sites: rat salivary glands

Development of T cell lineages and the role of the thymus as a source of immature T cells in parotid (PG) and submandibular salivary glands (SMG) were studied in Fischer 344 rats using the Thy-1/CD45RC/RT6 expression model. In addition, the phenotypes of salivary gland lymphocytes were compared with other conventional and extrathymic populations. PG mononuclear cells consisted of T cells (38%), B cells (29%), and NK cells (4%). SMG had 19% T cells, 7% B cells, 37% NK cells, and an unusual population of CD3(-)/RT6(+) cells. In comparison with lymph node (LN), both PG and SMG were enriched in immature (Thy-1(+)) and activated (Thy-1(-)/CD45RC(-)/RT6(-)) T cells. Unchanged percentages of Thy-1(+) T cells in PG and SMG following short-term adult thymectomy indicated that immature salivary gland T cells had an extrathymic source. In contrast, thymectomy eliminated LN recent thymic emigrants. SMG had T cells with characteristics of extrathymic populations, expressing TCRgammadelta(+) (28%), the CD8alphaalpha homodimer (11%), and NKR-P1A (66%). Many SMG T cells expressed integrin alpha(E)beta(7). PG T cells resembled those isolated from LN in respect to TCR and CD8 isoform usage, but were enriched in alpha(E)beta(7)(+) T cells and in NKT cells. Thus, salivary gland mononuclear cells are composed of a variety of subpopulations whose distributions differ between SMG and PG and are distinct from LN. These studies provide a basis for further investigation of regionalization in the mucosal immune network and are relevant to the design of vaccine regimens and intervention during pathological immune processes.     

The stem cell antigens Sca-1 and Sca-2 subdivide thymic and peripheral T lymphocytes into unique subsets

Stem cell Ag 1 and 2 (Sca-1 and Sca-2), so named due to their expression by mouse bone marrow stem cells, were evaluated for expression by populations of cells within the thymus. Immunohistochemical analysis demonstrated that Sca-1 was expressed by cells in the thymic medulla and by some subcapsular blast cells, as well as by the thymic blood vessels and capsule. Sca-2 expression, which was limited to the thymic cortex, could be associated with large cycling thymic blast cells. Both Sca-1 and Sca-2 were expressed on a sub-population of CD4-CD8- thymocytes, and this subpopulation was entirely contained within the Ly-1lo progenitor fraction of cells. Sca-1 expression by a phenotypically mature subset of CD4+CD8- thymocytes was also noted. Conversely, Sca-2 expression was observed on a phenotypically immature or nonmature subpopulation of CD4-CD8- thymocytes. MEL-14, an antibody that defines functional expression of a lymphocyte homing molecule, identified a small population of thymocytes that contained all four major thymic subsets. Sca-2 split the MEL-14hi thymocyte subset into two Sca-2+ non-mature/immature phenotype fractions and two Sca-2- mature phenotype fractions. In peripheral lymphoid organs, Sca-1 identified a sub-population of mature T lymphocytes that is predominantly CD4+CD8-, in agreement with the thymic distribution of Sca-1. Peripheral T cells of the CD4-CD8+ phenotype were predominantly Sca-1-. In contrast, Sca-2 did not appear to stain peripheral T lymphocytes, but recognized only a subset of B lymphocytes which could be localized by immunohistochemistry to germinal centers. Thus, expression of Sca-1 is observed throughout T cell ontogeny, whereas Sca-2 is expressed by some subsets of thymocytes, including at least one half of thymic blasts, but not by mature peripheral T lymphocytes.     

Induction of high level IL-2 production in CD4+8- T helper lymphocytes requires post-thymic development.

Triggering of the CD3:TCR complex by optimal concentrations of anti-CD3, anti-TCR beta-chain, and allogeneic stimulator cells induced dramatically higher levels (fivefold for anti-CD3, greater than 10-fold for anti-TCR beta-chain, 84-fold for alloantigen) of IL-2 production in spleen CD4+8- T cells than their thymic counterparts, despite comparable levels of CD3 and TCR beta-chain expression. The nature of the reduced IL-2 production was examined by analysis of anti-CD3-induced IL-2 production at the single cell level. The frequency of IL-2-producing cells in spleen CD4+8- T cells (40.0%) was approximately threefold that of thymus CD4+8- T cells (14.5%). Furthermore, the average IL-2 levels among positive IL-2 producers was also approximately threefold higher in spleen CD4+8- T cells than their thymic counterparts. Adoptive transfer of purified Thy-1.2+ CD4+8- T cells into Thy-1.1-congenic hosts provided a physiologic and histocompatible system that enabled identification of transferred donor (Thy-1.2+) among a sea of host (Thy-1.2-) CD4+ T cells, whose immune function with respect to IL-2 inducibility was examined after isolation by electronic cell sorting. Donor CD4+ T cells thus isolated from host spleen shortly (1 day) after i.v. transfer of thymus CD4+8- T cells were similar to freshly isolated thymus CD4+8- T cells in that they both produced little IL-2 in response to anti-CD3. However, by day 3 post-transfer, IL-2 production by donor CD4+8- T cells had more than doubled and by day 8, they produced IL-2 levels comparable to those of host spleen CD4+8- T cells. A similar acquisition of high level IL-2 inducibility in thymus CD4+8- T cells upon i.v. transfer into Thy-1.1-congenic hosts was also observed using allogeneic cells as the stimulus of IL-2 production. When thymus CD4+8- T cells were intra-thymically transferred into Thy-1.1-congenic hosts, those donor cells that emigrated to the periphery became high IL-2 producers in a time-dependent manner, whereas those that remained inside the thymus showed no signs of up-regulation in IL-2 inducibility. Intrathymic transfer of CD4-8- thymocytes revealed that the most recent thymic emigrant CD4+8- T cells contained few IL-2-producing cells and were not functionally mature with respect to high level IL-2 inducibility.     

Evidence for the existence of two parallel differentiation pathways in the thymus of MRL lpr/lpr mice.

MRL mice homozygous for the lpr/lpr gene develop a massive lymphadenopathy caused by the accumulation of CD4-CD8-, Thy-1-positive T cells that express B220. This phenotypically unusual T cell population coexists with normal, B220- T cells in lpr/lpr animals. To investigate the origin and differentiation pathway of B220+ T cells, the expression of a panel of developmentally regulated cell surface markers including TCR, CD4, CD8, Thy-1, and B220 was examined. Thymocytes and peripheral T lymphocytes from lpr/lpr mice were analyzed by four-color flow cytometry. The results showed that both B220+ and B220- thymocytes contained all of CD4-CD8-, CD4+CD8+, and CD4 or CD8 single positive T cell subpopulation in the lpr thymus. Expression of the V beta 11 TCR, measured by flow cytometry and reverse polymerase chain reaction, was demonstrated in lpr thymus. However, the number of T cells expressing V beta 11 was greatly reduced in both the B220+ and B220- T cell populations in lymph node, spleen, and liver. Taken together, the data provide evidence for maturation and selection of a distinct population of B220+ T cells in the thymus of MRL lpr/lpr mice.     

Differentiation of an immature T cell line: a model of thymic positive selection.

Thymocyte differentiation is dependent upon recognition of major histocompatibility complex (MHC) molecules on thymic stroma, a process called positive selection. Here we describe an immature CD4+8+ T cell line derived from a TCR transgenic mouse that differentiates into CD4+8- cells in response to antigen and nonthymic antigen-presenting cells. When injected intrathymically, these cells differentiate in the absence of antigen. The ability of immature T cells to recognize MHC molecules in the absence of foreign antigen in the thymus can thus be attributed to a unique property of thymic antigen-presenting cells. These studies also demonstrate the phenotypic and functional changes associated with TCR-mediated T cell maturation and establish an in vitro model system of positive selection.     

Mink cell focus-forming murine leukemia virus infection induces apoptosis of thymic lymphocytes

In a previous study we identified the subpopulations of thymus cells that were infected by the lymphomagenic MCF13 murine leukemia virus (MLV) (F. K. Yoshimura, T. Wang, and M. Cankovic, J. Virol. 73:4890-4898, 1999) and observed an effect on thymus size by virus infection. In this report we describe our results which demonstrate that MCF13 MLV infection of thymuses reduced the number of T lymphocytes in this organ. Histological examination showed diffuse lymphocyte depletion, which was most striking in the CD4(+) CD8(+) lymphocyte-enriched cortical zone. Consistent with this, flow cytometric analysis showed that the lymphocytes which were depleted were predominantly the immature CD3(-) CD4(+) CD8(+) and CD3(+) CD4(+) CD8(+) cells. A comparison of the percentages of live, apoptotic, and dead cells of the gp70(+) and gp70(-) thymic lymphocytes suggested that this effect on thymus cellularity is a result of virus infection. Studies of the survival of thymic T lymphocytes in culture showed that cells from MCF13 MLV-inoculated mice underwent greater apoptosis and death than cells from control animals. Assays for apoptosis included 7-amino-actinomycin D staining, DNA fragmentation, and cleavage of caspase-3 and poly(ADP-ribose) polymerase proenzymes. Our results suggest that apoptosis of thymic lymphocytes by virus infection is an important step in the early stages of MCF13 MLV tumorigenesis.     

Ontogenesis of rat immune system: Proteasome expression in different cell populations of the developing thymus

Immune proteasomes in thymus are involved in processing of self-antigens, which are presented by MHC class I molecules for rejection of autoreactive thymocytes in adults and probably in perinatal rats. The distribution of immune proteasome subunits LMP7 and LMP2 in thymic cells have been investigated during rat perinatal ontogenesis. Double immunofluorescent labeling revealed LMP7 and LMP2 in thymic epithelial and dendritic cells, as well as in CD68 positive cells - macrophages, monocytes - at all developmental stages. LMP2 and LMP7 were also detected by flow cytometry in almost all thymic CD90 lymphocytes through pre- and postnatal ontogenesis. Our results demonstrate that the immune proteasomes are expressed in all types of thymic antigen presenting cells during perinatal ontogenesis, suggesting the establishment of the negative selection in the thymus at the end of fetal life. The observation of the immune proteasome expression in T lymphocytes suggests their role in thymocyte differentiation besides antigen processing in thymus.     

Subpopulations of early thymocytes. A cross-correlation flow cytometric analysis of adult mouse Ly-2-L3T4-(CD8-CD4-) thymocytes using eight different surface markers

Putative early thymocytes, the Ly-2-L3T4-(CD8-CD4-) cells representing 3 to 4% of adult CBA mouse thymic lymphocytes, were isolated in high purity (99.5%). They were then stained by using mAb and analyzed by flow cytometry for the expression of six additional surface antigenic markers. Cross-correlation of the data obtained from a complete series of successive two-parameter analyses revealed the existence of about 11 discrete subsets, falling into four-main groups, within the Ly-2-L3T4- population. All subsets consisted of relatively large lymphoid cells. The most numerous group of Ly-2-L3T4- cells was Ly-1 low B2A2-M1/69 high Thy-1 high Pgp-1 low and by these markers resembled Ly-2+L3T4+ cortical blasts. Many of the cells in this group were positive for the IL-2R and/or for MEL-14. A second major group of Ly-2-L3T4- cells was Ly-1 high B2A2-M1/69 low Pgp-1 high, and resembled in some respects activated mature T cells. This group had previously been shown to be absent from the embryonic thymus. The group could be divided into Thy-1 high and Thy-1 low subsets. None of the cells in this group were positive for the IL-2R and very few expressed MEL-14. A third group, 13% of the Ly-2-L3T4- population, was Ly-1 low B2A2-M1/69 low Pgp-1 high, and could also be divided into Thy-1 high and Thy-1 low subsets. A final minor group, 9% of the Ly-2-L3T4- population, was Ly-1 high B2A2-M1/69 high Pgp-1 low Thy-1 high. The particular pattern of markers on these subsets, combined with subsequent information on their properties, makes it unlikely that they all represent sequential steps in one continuous developmental stream, and indicates that complex developmental steps have occurred, even at this supposedly early stage of T cell differentiation.     

Acquisition of non-MHC restricted cytotoxic function by IL 2 activated thymocytes with an "immature" antigenic phenotype

Culture of human thymocytes in interleukin 2 (IL 2) results in the generation of cytotoxic T lymphocytes (CTL) that kill tumor cell targets without major histocompatibility complex (MHC) restriction. Thymic non-MHC restricted CTL expressed Leu-19 antigen, but were generated from thymic precursor cells that lacked expression of Leu-19. In contrast, short term culture in Il 2 of peripheral blood lymphocytes depleted of Leu-19+ lymphocytes did not result in the generation of cytotoxic activity. IL 2 was necessary and sufficient for the generation of cytotoxic thymocytes and induction of Leu-19 antigen expression. Thymic non-MHC restricted CTL were generated from precursor cells expressing CD1, an antigen present on the majority of thymocytes. Furthermore, cytotoxic activity was detected in IL 2 cultured thymocyte populations with an "immature" antigenic phenotype, i.e. CD1+ and CD4+, CD8+. Upon subsequent culture, thymic non-MHC restricted CTL lost expression of CD1, and developed an antigenic phenotype similar to peripheral blood non-MHC-restricted CTL, suggesting that peripheral non-MHC-restricted CTL may originate from these thymic precursors.     

Analysis of Ly-6.2-bearing murine lymphocyte subpopulations in relation to the T-lymphocyte markers,Thy-1, Lyt-1, and Lyt-2

Using immunofluorescence with a monoclonal anti-Ly-6.2 antibody and FACS analysis we have confirmed that the Ly-6.2 antigen is present on approximately 70% of mature T cells and B cells but on few immature lymphocytes. There is a wide range of antigen density among the Ly-6.2⁺ populations, with the mean density higher on T cells than B cells. Following Con A activation of splenocytes there was a sixfold increase in Ly-6.2 antigen density though approximately 20% of the activated lymphocytes were Ly-6.2^?. The increase in Ly-6.2 density was specific since similar density increases did not occur for the closely linked antigens ThB and H $\text{9}\text{25}$. By panning a predominantly T-cell population for Lyt-2-bearing cells, it was found that Lyt-2⁺ lymphocytes were either negative or dully staining for Ly-6.2. However, activated cells bearing the Lyt-2 antigen were all Ly-6.2 positive. Double-staining experiments showed that T cells which had high Ly-6.2 antigen densities also had high Thy-1 antigen densities. Corticosteroid-resistant thymocytes were highly enriched for Ly-6.2-bearing cells compared to untreated thymocytes and had staining profiles for Ly-6.2 which were similar to peripheral T cells, supporting the idea that steroid treatment selects for a phenotypically mature thymic population.     

Thymus involution induced by mouse hepatitis virus A59 in BALB/c mice.

Mouse hepatitis virus A59 (MHV-A59) infection of adult BALB/c mice induced a severe, transient atrophy of the thymus. The effect was maximal at 1 week after infection, and thymuses returned to normal size by 2 weeks after infection. There was no effect of glucocorticoids, since thymus atrophy was also found in adrenalectomized, infected mice. In infected thymus, immature CD4+ CD8+ lymphocytes were selectively depleted, and apoptosis of lymphocytes was increased. The MHV receptor glycoprotein MHVR was detected on thymus epithelial cells but not on T lymphocytes. In a small number of stromal epithelial cells, but in very few lymphocytes, the viral genome was detectable by in situ hybridization. These observations suggested that MHV-A59-induced thymic atrophy results not from a generalized lytic infection of T lymphocytes but rather from apoptosis of immature double-positive T cells that might be caused by infection of a small proportion of thymus epithelial cells or from inappropriate secretion of some factor, such as a cytokine.     

Differentiation and functional maturation of bone marrow-derived intestinal epithelial T cells expressing membrane T cell receptor in athymic radiation chimeras

The thymus dependency of murine intestinal intraepithelial lymphocytes (IEL) was studied in an athymic F1----parent radiation chimera model. IEL, although not splenic or lymph node lymphocytes, from athymic chimeras displayed normal levels of cells bearing the class-specific T cell Ag, CD4 and CD8; the TCR-associated molecule, CD3; and the Thy-1 Ag. Moreover, two-color flow cytometric analyses of IEL from athymic mice demonstrated regulated expression of T cell Ag characteristic of IEL subset populations from thymus-bearing mice. In immunoprecipitation experiments, surface TCR-alpha beta or TCR-gamma delta were expressed on IEL, although not on splenic lymphocytes, from athymic chimeras. That IEL from athymic chimeras constituted a population of functionally mature effector cells activated in situ, similar to IEL from thymus-bearing mice, was demonstrated by the presence of CD3-mediated lytic activity of athymic lethally irradiated bone marrow reconstituted IEL. These data provide compelling evidence that intestinal T cells do not require thymic influence for maturation and development, and demonstrate that the microenvironment of the intestinal epithelium is uniquely adapted to regulate IEL differentiation.     

Ontogeny of thymic B cells in normal mice.

Ontogeny of thymic B cells and their surface characteristics were analyzed using monoclonal antibodies (mAbs) against B220 molecules (CD45, CD45R). A small number of B cells were detected in fetal thymus on Gestation Day 14 (approximately 3.5% of the low-density fraction). Similarly, the percentage of B cells in the low-density fraction was 3.2% on Gestation Day 18, and 3.5% on Day 1 after birth. These were the same level as that of adult mice. CD5+ B cells, which form the major population of thymic B cells, were also found in the fetal life (0.5% on Day 14 and 2.2% on Day 16 in the low-density cells). The percentage of CD5+ B cells in B cell-enriched fraction was about 65% on Day 1 after birth, which is the same level as that in adult mice. These results indicate that a small number of B cells or cells in the B-cell lineage are present in the fetal thymus and also suggest the importance of these thymic B cells in the negative selection of T cells during early developmental stages.     

A novel cofactor produced by a thymic epithelial cell line: promotion of proliferation of immature thymic lymphocytes by the presence of interleukin-1 and various mitogens

Three thymic epithelial cell lines (TEC1C5, TEC1-4, and TEC2-3) were established from the thymus of newborn C57BL/6 mice. TEC1C5 was revealed to be an interleukin (IL)-1 producing cell line. TEC1-4 produced a cofactor to promote proliferation of double negative (CD4-8-) thymic lymphocytes by the presence of IL-1. Production of the same cofactor was also seen in TEC2-3, but only when it was cultured by the presence of indomethacin. The chemical analysis of the TEC1-4 culture supernatant by ion-exchange column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the factor was approximately 35 kDa in molecular weight. The present study revealed that a factor produced by TEC1-4 acted as a cofactor to promote the proliferation of immature T cells stimulated by IL-1 and various mitogens and was considered to be a new one in terms of molecular weight.