A homing receptor-bearing cortical thymocyte subset: implications for thymus cell migration and the nature of cortisone-resistant thymocytes

The thymus exports a selected subset of virgin T lymphocytes to the peripheral lymphoid organs. The mature phenotype of these thymus emigrants is similar to that of medullary thymocytes and has been cited as supporting a medullary rather than cortical exit site. Using the monoclonal antibody MEL-14, we identify a 1%-3% subpopulation of thymocytes that expresses high levels of a receptor molecule involved in lymphocyte homing to peripheral lymph nodes. We present evidence that these rare MEL-14hi thymocytes are predominantly of mature phenotype and represent the major source of thymus emigrants. Surprisingly, MEL-14hi thymocytes are exclusively cortical in location, although their mature phenotype may allow them to masquerade as medullary cells in conventional studies. We also demonstrate that unlike medullary thymocytes, many cortisone-resistant thymocytes (CRT) are MEL-14hi. Thus, in contrast to current dogma, CRT do not represent a sample of medullary thymocytes as they are found in situ and their level of immunocompetence does not necessarily reflect that of the medullary population. Our findings refute the hypothesis that phenotypically and functionally mature cells are restricted to the medulla, and support our proposition that most thymus emigrants are derived from the MEL-14hi cortical subset.     

Rat T lymphocyte-specific antigens and their cross-reactivity with mouse T cells.

Anti-rat T lymphocyte serum (ATLS)2 was prepared by immunizing rabbits with purified T cells from rat mesenteric nodes and absorbed with rat red cells and syngeneic sarcoma cells. The specificity of ATLS for rat T cells was confirmed by the following reasons: a) ATLS was not toxic for bone marrow cells but lysed most of the thymocytes and a number of spleen and lymph node cells, which were inversely correlated to the percentage of cells with B cell characteristics in respective organs; b) anatomical localization of ATLS-reactive cells in lymphoid organs coincided to the thymus-dependent areas, i.e. the paracortex of lymph node and the periarteriolar region of spleen; c) spleen cells treated with ATLS and complement failed to respond to phytohemagglutinin but normally responded to bacterial lipopolysaccharide; d) those cells treated with ATLS and complement could not induce a graft-vs-host reaction in F1 hosts, whereas the same treatment did not affect direct plaque-forming cells. All of these data confirm the specificity of ATLS and indicate that ATLS recognizes rat T lymphocyte-specific antigens (RTLA). Absorption studies showed that RTLA were present in higher concentration on medullary thymocytes and peripheral T cells than on cortical thymocytes, but absent from bone marrow, liver, and brain tissues. When the cross-reactivity of RTLA with mouse T cells was studied by C-dependent cytotoxicity and immunofluorescence, it was found that mouse T cells shared at least one determinant of RTLA with rat T cells, and that distribution pattern of the cross-reacting antigens in mouse lymphoid tissues was essentially the same as that of RTLA in rat lymphoid organs.     

Antigenic relationship between medullary thymocytes and a subpopulation of peripheral T cells in the rat: description of a masked antigen.

Using differentially absorbed rabbit antisera to rat thoracic duct cells, an antigen is described which normally is expressed on the surface of T cells in thoracic duct lymph and lymph node, but which exists in a masked form on medullary thymocytes and apparently not at all on cortical thymocytes. This antigen is termed the rat masked thymocyte antigen (RMTA). RMTA on medullary thymocytes can be unmasked mechanically by sectioning in a cryostat or enzymatically by treating with neuraminidase. Trypsin destroys or removes RMTA. Nearly all the T cells in thoracic duct lymph and lymph node are RMTA⁺, whereas only 58–66% of T cells in spleen are RMTA⁺. RMTA⁺ T cells, which are cortisone resistant, reside in the paracortex and periarteriolar sheath regions of lymph node and spleen. RMTA^? T cells, which are cortisone sensitive, appear to reside in the red pulp of spleen. The results suggest that (i) two antigenically distinct populations of T cells exist in the rat, RMTA⁺ and RMTA^? T cells, (ii) medullary thymocytes are the immediate precursors of RMTA⁺ T cells, and (iii) cortical thymocytes may be the immediate precursors of RMTA^? cells.     

Isolation of monoclonal antibodies to antigen Lyt-3.2

A hybridoma producing monoclonal antibodies (McAb) NATF9.9 (F9) was obtained from fusion of murine myeloma X63 and splenocytes of AKR mice immunized with a single intravenous injection of 5 X 10(7) thymocytes of CBA mice. F9 McAb were cytotoxic for 80% thymocytes, 10% splenocytes, 20% lymph node cells, 85% cortical and 32% medullary thymocytes of CBA, C57BL/6, BALB/c, DBA/2 and SJL but not for the cells of C58 and AKR mice. F9 McAb reacted only with T cells and did not react with B cells and EL4 thymoma cells (Thy-1.2+, Lyt-1+2-3-). The proportion of F9+ cells accounts for about 40% among T lymphocytes of the lymph nodes and spleen as tested by flow-type cytometry. Lymph node cells treated with F9 McAb plus complement completely lost their reactivity with rat anti-Lyt-2 McAb and only partly (by 30%) with anti-Lyt-1 McAb. The reactivity pattern of F9 McAb attests to their specificity for Lyt-3.2 antigen.     

Ontogeny and regulation of IL-7-expressing thymic epithelial cells

Epithelial cells in the thymus produce IL-7, an essential cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We identified IL-7-expressing thymic epithelial cells (TECs) throughout ontogeny and in the adult mouse thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the early primordium by embryonic day 11.5 and is expressed in a Foxn1-independent pathway. Marked changes occur in the localization and regulation of IL-7-expressing TECs during development. IL-7-expressing TECs are present throughout the early thymic rudiment. In contrast, a major population of IL-7-expressing TECs is localized to the medulla in the adult thymus. Using mouse strains in which thymocyte development is arrested at various stages, we show that fetal and postnatal thymi differ in the frequency and localization of IL-7-expressing TECs. Whereas IL-7 expression is initiated independently of hemopoietic-derived signals during thymic organogenesis, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Moreover, different thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium, suggesting that despite phenotypic similarities, the cortical TEC compartments of wild-type and RAG-1(-/-) mice are developmentally and functionally distinct.     

Pathogenesis of murine enterovirus myocarditis: virus dissemination and immune cell targets.

In order to identify organ and cellular targets of persistent enterovirus infection in vivo, immunocompetent mice (SWR/J, H-2q) were inoculated intraperitoneally with coxsackievirus B3 (CVB3). By use of in situ hybridization for the detection of enteroviral RNA, we show that CVB3 is capable of inducing a multiorgan disease. During acute infection, viral RNA was visualized at high levels in the heart muscle, pancreas, spleen, and lymph nodes and at comparably low levels in the central nervous system, thymus, lung, and liver. At later stages of the disease, the presence of enteroviral RNA was found to be restricted to the myocardium, spleen, and lymph nodes. To characterize infected lymphoid cells during the course of the disease, enteroviral RNA and cell-specific surface antigens were visualized simultaneously in situ in spleen tissue sections. In acute infection, the majority of infected spleen cells, which are located primarily at the periphery of lymph follicles, were found to express the CD45R/B220+ phenotype of pre-B and B cells. Whereas viral RNA was also detected in certain CD4+ helper T cells and Mac-1+ macrophages, no enteroviral genomes were identified in CD8+ cytotoxic/suppressor T cells. Later in disease, the localization of enteroviral RNA revealed a persistent type of infection of B cells within the germinal centers of secondary follicles. In addition, detection of the replicative viral minus-strand RNA intermediate provided evidence for virus replication in lymphoid cells of the spleen during the course of the disease. These data indicate that immune cells are important targets of CVB3 infection, providing a noncardiac reservoir for viral RNA during acute and persistent myocardial enterovirus infection.     

Expression of alpha/beta interferons (IFN-alpha/beta) and their relationship to IFN-alpha/beta-induced genes in lymphocytic choriomeningitis.

Expression of alpha interferon (IFN-alpha)-, IFN-beta-, and IFN-alpha/beta-induced genes was monitored during the development of lymphocytic choriomeningitis (LCM) to assess whether a restricted influence of these antiviral cytokines could be found in the central nervous system (CNS). High levels of IFN-alpha (83 +/- 42 U/ml) were present in the blood of LCM virus-infected mice 3 days postinfection, whereas IFN-beta was not detected (< 1.0 U/ml) at any time point. Spleens contained high levels of IFN-alpha and IFN-beta mRNAs at days 1 and 3 postinfection, whereas no IFN-alpha mRNA and only low levels of IFN-beta mRNA were detected in brains. In situ hybridization showed IFN-alpha mRNA-expressing cells in the marginal zones of the spleen and in the subcapsular sinus and outer cortex of cervical lymph nodes. The expression of 2',5'-oligoadenylate synthetase (2',5'-OAS) mRNA followed the expression of IFN-beta mRNA in the brain, whereas 2',5'-OAS mRNA in the periphery was associated with systemic IFN-alpha. The localization of IFN-alpha-expressing cells in the spleen and lymph nodes in proximity to T- and B-cell compartments is consistent with a role for these cytokines in immune regulation. Furthermore, the absence of IFN-alpha and the relatively low level and delayed expression of IFN-beta in the brain suggest that the CNS is an especially vulnerable organ for virus replication. With certain strains of LCM virus, the absence of early antiviral IFN-alpha/beta activity and preferential virus growth in the brain might lead to targeted T-cell inflammation of the CNS, resulting in death of the animal.     

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.     

T cell maturation: thymocyte and thymus migrant subpopulations defined with monoclonal antibodies to MHC region antigens

The maturation sequences of thymocytes is known to some extent: A generative layer of subcapsular large lymphoblasts gives rise to a major population of small cortical thymocytes and a minor population of midsize medullary thymocytes. The relative contribution of these three populations to the peripheral T cell populations is not yet known. In this study, subcapsular lymphoblasts, cortical small cells, medullary cells, and thymic emigrant cells have all been analyzed by immunofluorescence for expression of the antigens H-2D, I-A, H-2K, and TL. H-2D is expressed brightly on all subcapsular large cells, dimly on cortical small cells, and brightly on all migrants, cortisone-resistant thymocytes (CRT), and peripheral T cells. I-A can be detected at low levels on 30 to 50% of cells in all the thymic subpopulations, and on 30 to 50% of migrants and peripheral T cells. Fifty to 80% of small cortical cells do not express detectable H-2K, but all the other subpopulations, both inside and outside the thymus, stain uniformly quite brightly. TL3 is expressed on 70 to 80% of subcapsular and cortical thymocytes, 30 to 40% of CRT, is undetectable on migrants but can be seen at low levels on 10 to 20% of spleen and lymph node T cells. The possibility that some or all of these antigens represent stable markers of separate lineages rather than unstable, stage-specific markers is discussed.     

Spectrum of reactivity of monoclonal antibodies against specific T-suppressors

Monoclonal antibodies (MoAb) against specific T-suppressors CI and C4 are characterized by their reactivity with normal lymphoid cells and some tumour cell lines cultivated in vitro. MoAb CI and C4 react with T and B cells from spleen and lymph nodes. The amount of CI and C4 T and B subsets are equal in the spleen (25-29%), while lymph node T-lymphocytes contain twice as much CI and C4 cells than B-lymphocytes (40 and 20%, respectively). In the thymus CI is expressed mostly on immature (cortical) thymocytes and C4--on the mature (medullary) thymocytes. CI is expressed on some T-lymphoma cell lines (BW 5147, EL4, LBRM33), but not on thymoma RDM4 and mastocytoma P815. C4 is not found on the above cell lines but is expressed on the intermediate filament of mouse and quail fibroblasts and in lymphoid cells. This cross-reactivity may result from the existance of similar determinants in cytoskeleton proteins and lymphocyte membranes or from the intermediate filament expression on T-suppressor cellular membranes, but not on other functional T subsets.     

Are any functionally mature cells of medullary phenotype located in the thymus cortex?

Experiments were undertaken to test if thymocytes of "mature" or "medullary" phenotype were restricted to the medullary area of the thymus. A calculation based on direct cell counts on serial sections indicated that 11.5% of adult male CBA thymic lymphoid cells were within the medullary zone. Since only 3-4% of thymocytes were cortisone resistant, the majority of thymocytes within the medulla were, like cortical thymocytes, cortisone sensitive. A series of cell surface antigenic markers, used alone or in pairs, suggested that 13-15% of thymocytes were of medullary phenotype, somewhat more than the number of thymocytes actually present in the medulla. However, much of this discrepancy could be explained by differential death of cortical cells during isolation and staining, and by the existence in the cortex of a subpopulation of early blast cells which shared some, but not all markers with medullary thymocytes. A direct test for mature or medullary phenotype cells in the cortex involved selective transcapsular labeling of outer-cortical cells with fluorescent dyes, followed by multiparameter immunofluorescent analysis of the 10% labeled population. Outer-cortical thymocytes included some cells (mainly early blasts) sharing some markers with medullary thymocytes, but very few (less than 1%) of these cells expressed all the characteristic "mature" markers. Limit-dilution precursor frequency studies showed the level of functional cells in the outer cortex was extremely low. The overall conclusion was that the vast majority of cells of complete "mature" phenotype are confined to the thymic medulla. These findings favor the view that thymus migrants originate from the thymic medulla, but do not exclude a cortical origin. The results also illustrate the need for multiparameter analysis to distinguish medullary thymocytes from early blast cells.     

Fate of thymocytes: studies with 125I-iododeoxyuridine and 3H-thymidine in mice.

Cortical thymocytes of young adult mice were labeled in situ with radioactive DNA precursors. As a result of cell emigration and cell death, total thymic radioactivity decreased within 8 days to 10% or less of that present on day 1. Accumulation of thymic migrants in peripheral lymphoid organs was estimated by computing the net thymus-derived radioactivity in these tissues. Thymic cell death was assessed by comparing values obtained with 125I-UdR to those acquired with 3H-TdR; The results indicate that cortical thymocytes migrate to the spleen, mesenteric lymph node, femurs and intestine; nevertheless, only a small fraction of the activity originally present in the thymus was recovered in these organs; the vast majority of newly formed cortical thymocytes apparently die after a relatively short life span. Exclusive of the fraction which dies in situ, evidence for thymocyte death is seen in bone marrow; however, most migrants appear to terminate in the intestine.     

The distribution of adenosine deaminase among lymphocyte populations in the rat.

Adenosine deaminase (ADA) activity was determined in young rat lymphocyte populations. The ADA-specific activity (per 10(8) cells and per milligram protein) was 3- to 10-fold higher in thymocytes than in lymphocytes from thoracic duct, lymph node, spleen, and bone marrow. The high ADA activity in thymocytes appeared to be preferentially associated with cortical thymocytes. Enrichment or depletion of cortical thymocytes by density gradient centrifugation, cortisone treatment, or selective lysis with anti-Thy-1 plus complement resulted in parallel increases or decreases in ADA levles. These results also suggested that medullary thymocytes have ADA levels similar to those of peripheral lymphocytes. "Immature" cortical thymocytes and thymocyte progenitors appeared to have low ADA activity; low enzyme levels were found in fetal thymus at 16 days of embryonic life, in the early phases of thymus regeneration, and in a "null" cell population isolated from bone marrow. This study demonstrates that ADA activity varies markedly during T lymphocyte differentiation and suggests that fundamental differences in nucleotide metabolism may exist in T cells at different stages of development.     

Antigenic relationship between bone marrow lymphocytes cortical thymocytes and a subpopulation of peripheral T cells in the rat: description of a bone marrow lymphocyte antigen.

Populations of rat bone marrow lymphocytes (BML) consisting of approximately 90 percent, “tnull” cells were prepared by density gradient centrifugation, passage through a column of fine glass beads, and treatment with anti-T cell and anti-B cell serum plus complement. Antisera to these bone marrow lymphocytes were raised in rabbits. After absorption with RBC and peritoneal exudate cells, the anti-BML sera were found by immunofluorescence to react selectively with “null” cells in bone marrow, with cortical thymocytes, and with a cortisone-sensitive subset of T cells in blood and in spleen, possibly in red pulp. The antigen that is common to these cell types is designated the rat bone marrow lymphocyte antigen (RBMLA). Lymphocytes that are positive fur KBMLA are negative for another lymphocyte-specific heteroantigen, rat musked thymocyte antigen (RMTA). As shown previously, RMTA is present on medullary thymocytes and ou cortisone-resistant T cells in white pulp of spleen, paracortex of lymph node and thoracic duct lymph. It is postulated that two developmentally and functionally distinct lines of T cells exist in peripheral lymphoid tissues of the rat, one derived from cortical thymocytes and one derived from medullary thymocytes. It is further postulated that the “null” population of bone marrow lymphocytes contains the lymphopoietic stem cells from which these two lines of T cells originate.     

Regulation of RAG-2 protein expression in avian thymocytes.

The recombinase-activating genes, RAG-1 and RAG-2, have been shown to be necessary to initiate the process of V(D)J recombination during the ontogeny of lymphocytes. While much is known about the end products of this rearrangement process, little is known about the function or regulation of the components of the recombinase system. To this end, we have generated a monoclonal antibody to the chicken RAG-2 protein. Chicken thymocytes were found to express high levels of RAG-2, part of which is phosphorylated. Within thymocytes, RAG-2 is expressed primarily within the nucleus. RAG-2 protein levels are high in the CD4- CD8- and CD4+ CD8+ immature thymocytes but absent at the single-positive CD4+ CD8- or CD4- CD8+ stage of thymocyte development. Mitogenic stimulation of thymocytes with phorbol myristate acetate and ionomycin results in down-regulation of RAG-2 expression. Consistent with these data, in vivo levels of RAG-2 are markedly lower in proliferating thymocytes than in smaller, G0/G1 cells. Down-regulation of RAG-2 expression appears to occur before cells enter S phase, suggesting that RAG-2 function may be limited to noncycling cells.     

Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen

A monoclonal antibody (MAb), BLT-1, with specificity for bovine mature T cells was prepared by somatic cell hybridization of myeloma NS-1 and spleen cells from BALB/c mice hyperimmunized with bovine T lymphocytes. The MAb reacted with over 92% of nylon wool-nonadherent lymphocytes (T cells) but not with nylon wool-adherent EAC-positive lymphocytes (B cells) in the indirect immunofluorescence assay. It is an IgM, with kappa-light chains, which fixed complement well and killed over 95% of mature T cells in complement-mediated cytotoxicity assays. It reacted with the same proportions of peripheral lymphoid cells (peripheral blood, lymph nodes, and spleen) as the polyclonal goat anti-bovine thymocyte serum (GABTS), but only with 25% of GABTS-positive thymocytes. Immunoperoxidase staining of frozen tissue sections showed that the BLT-1-positive cells were located in the medulla of the thymus and in the T lymphocyte areas of lymph nodes. Western immunoblotting assays showed that the BLT-1-reactive membrane antigen is a 22,000 m.w. protein which was inducible in bovine thymocytes with bovine thymic hormones, thymosin fraction 5, thymosin alpha 1, and thymopentin ORF-18150, indicating that it is a mature T lymphocyte differentiation antigen. The thymosin alpha 1 and thymopentin were found to show additive effects on mature T cell antigen expression by bovine thymocytes.