Phenotypic analysis of thymocytes that express homing receptors for peripheral lymph nodes

Thymocytes that express high levels of homing receptors for peripheral lymph nodes can be detected with the monoclonal antibody MEL-14. We have shown that in adult mice these rare MEL-14hi thymocytes a) are cortical in location and typically constitute 1 to 3% of the total thymocyte population, b) may be a major source of thymus emigrants, and c) contain a high frequency of precursors of alloreactive cytotoxic T lymphocytes. In this study we have analyzed the phenotype of the MEL-14hi thymocyte subset. Most normal adult MEL-14hi thymocytes are midsize and express the mature phenotype typical of thymus emigrants, medullary thymocytes, and peripheral T cells: they are predominantly PNAlo, H-2K+, Thy-1+, Ly-1hi, and either Lyt-2-/L3T4+ or Lyt-2+/L3T4-. These findings argue strongly for the presence of rare MEL-14hi immunocompetent cortical thymocytes that, aside from their homing receptor expression, are phenotypically indistinguishable from medullary thymocytes. However, a minority (20 to 30%) of MEL-14hi thymocytes are large and phenotypically nonmature: they express intermediate to high levels of PNA binding sites, and are H-2K- to H-2Klo, Thy-1hi, Ly-1+, and either Lyt-2+/L3T4+ or Lyt-2-/L3T4-. Through a technique that selectively labels outer cortical cells, phenotypically nonmature MEL-14hi thymocytes have been shown to be concentrated in the subcapsular blast region of the outer cortex. Although we have no direct evidence of a precursor-product relationship, we consider it likely that the phenotypically nonmature outer cortical MEL-14hi lymphoblasts give rise to phenotypically mature MEL-14hi cells located deeper in the cortex. These results are consistent with our previous proposal that MEL-14hi thymocytes are a major source of thymus emigrants, and indicate that expression of high levels of MEL-14-defined homing receptors may be closely linked to the intrathymic selection process.     

Phenotype and localization of thymocytes expressing the homing receptor-associated antigen MEL-14: arguments for the view that most mature thymocytes are located in the medulla

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.     

Dual immunofluorescence studies of cortisone-induced thymic involution: evidence for a major cortical component to cortisone-resistant thymocytes

Cortisone-resistant thymocytes (CRT) have been used as the experimental equivalent of medullary thymocytes for the past 15 yr. Studies with CRT have provided evidence that the medullary population is similar to mature T cells in phenotype and function and may therefore be the major source of thymus emigrants. However, we have recently demonstrated that CRT differ from medullary thymocytes in their expression of the homing receptor molecule recognized by the monoclonal antibody MEL-14. Thus, many CRT express high levels of the MEL-14-defined homing receptor, whereas medullary thymocytes are MEL-14- to MEL-14lo. In normal adult mice, only 1 to 3% of thymocytes are MEL-14hi; these cells are located exclusively in the cortex and many are phenotypically and functionally mature. In this study we have used dual immunofluorescence techniques to further characterize those thymocytes resistant to cortisone treatment. Aside from being of mature phenotype with respect to expression of peanut agglutinin binding sites and the cell surface molecules H-2K, Ly-1, Lyt-2, and L3T4, CRT can be divided into MEL-14lo and MEL-14hi subpopulations, suggesting that they may actually be derived from both the medullary and the MEL-14hi cortical thymocyte subsets.     

Identification of early stages of T lymphocyte development in the thymus cortex and medulla

Thymocyte subpopulations with a phenotype suggesting they are early stages of T cell development in the adult mouse thymus were characterized and isolated by using multiparameter flow cytometry and sorting, in conjunction with selective killing with antibody and complement (C). The intrathymic localization of these subpopulations was assessed by dipping the thymus in fluorescent dyes to selectively label outer-cortical cells. The main phenotypic markers used were sensitivity to C-mediated lysis by the monoclonal antibody B2A2 (which spares most prothymocytes but kills most thymocytes), the expression of the T cell lineage specific markers Ly-2 and L3T4, and the levels of the common T cell antigens Ly-1 and Thy-1. A preliminary selection for cells lacking Ly-2 and L3T4, or resistant to B2A2 and C, produced a population of large cells, only 5% of all thymocytes and distinct from the typical cortical blast cells. This population of putative early thymocytes was itself heterogeneous, consisting of eight subpopulations separable by phenotype and intrathymic localization. One group of two subpopulations (B2A2-, Ly-1++, Thy-1+ and either Ly-2+ L3T4- or Ly-2- L3T4+) appeared to be of medullary location, and their phenotype suggested they could have been early members of the medullary lineages. Another group of two subpopulations (B2A2-, Ly-1++, Thy-1-, Ly-2-, L3T4- and B2A2-, Ly-1++, Thy-1+, Ly-2- L3T4-) did not show a clear localization pattern and may have represented cells in an earlier stage of transition to medullary phenotype and location. A quite different group of three subpopulations (B2A2++, Ly-1-, Thy-1-, Ly-2- L3T4-; B2A2++, Ly-1-, Thy-1+, Ly-2-, L3T4-; and B2A2++, Ly-1+, Thy-1++, Ly-2- L3T4-) was concentrated in the outer cortex and seemed to represent a series of stages of a cortical pathway, before the typical cortical blast cells. Finally, a very minor subset (0.2% of thymocytes), lacking all these markers, was concentrated in the outer cortex; this fifth group had the phenotype expected of the earliest intrathymic precursor cells. The results suggest that the separate developmental streams of cortical and medullary thymocytes may be traced back, via these minor early blast subpopulations, to common precursor cells in the outer cortex.     

Cultured Lyt-2- L3T4- T lymphocytes from normal thymus or lpr mice express a broad spectrum of cytolytic activity

T lymphocytes expressing the surface phenotype Lyt-2- L3T4- represent a minor population of immature thymocytes that appear to be the precursors of mature T cells. Cells with the same apparent surface phenotype also accumulate in vast numbers in the lymphoid tissues of the autoimmune lpr mouse. Lyt-2- L3T4- T lymphocytes from lpr lymph node (LN) or normal thymus express low to undetectable levels, respectively, of surface antigen receptor. In addition, they produce reduced amounts of lymphokines compared with normal T cells and lack precursors of alloantigen-specific cytolytic T lymphocytes. We previously showed that after culture with phorbol esters and interleukin 2, lpr Lyt-2- L3T4- T lymphocytes proliferate and differentiate, acquiring increased levels of surface antigen receptor by most cells, as well as Lyt-2 by a portion. We now show that cultured Lyt-2- L3T4- T cells from lpr LN or normal thymus are very efficiently cytolytic toward not only allogeneic tumor targets, but also natural killer (NK)-susceptible targets and syngeneic targets. Such killing was not inhibited by antibodies to H-2 or Lyt-2. In contrast, cultured mature Lyt-2+ L3T4- T cells from normal LN, thymus, or lpr LN were cytolytic only toward allogeneic targets and were dependent on Lyt-2 expression and H-2 recognition. The similarities of cultured Lyt-2- L3T4- T cells to NK and lymphokine-activated killer cells are discussed.     

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.     

Distinction of virgin and memory T lymphocytes. Stable acquisition of the Pgp-1 glycoprotein concomitant with antigenic stimulation

The Pgp-1 glycoprotein was identified on a minor (27%) subset of peripheral Lyt-2+ or L3T4+ T cells. In contrast, mature medullary-type thymocytes (Lyt-2+ L3T4-, Lyt-2- L3T4+) were nearly devoid of cells expressing detectable surface Pgp-1. The appearance of peripheral Pgp-1- T cells was found to be thymus dependent, as demonstrated by the diminished proportion of Pgp-1- T cells after thymectomy and their virtual absence in athymic nude mice. The subsequent acquisition of surface Pgp-1 was found to be a stable differentiation event occurring concomitantly with primary antigenic stimulation; selected Pgp-1- mature T cells from thymus or periphery acquired constitutive expression of Pgp-1 after stimulation in vitro with alloantigen or mitogens. These observations were extended by studies in vivo showing that immunization with various antigens augmented the percentage of Pgp-1+ spleen cells within the Lyt-2+ subset. Furthermore, the frequencies of antigen-specific CTLp, after immunization by any of three different antigens tested, were greatly enriched in the Pgp-1+ compared with the Pgp-1- subpopulations. Peritoneal exudate Lyt-2+ cells, after a localized allograft rejection, demonstrated a particularly prominent Pgp-1+ subpopulation (78%) that contained virtually all the allospecific cytolytic activity. A model consistent with all of these data proposes that mature thymocytes lacking surface Pgp-1 upon emigration to the periphery acquire its expression at the time of primary antigenic stimulation. Hence, expression of Pgp-1 among peripheral T cells is an important differentiation marker for identifying antigen-stimulated memory T cells.     

Expression and function of a 90-kilodalton homodimeric molecule (10D1 antigen) on human thymocytes

The 10D1 Ag is a 90-kDa homodimeric molecule specifically expressed on a subpopulation of human T cells, and is involved in an alternative pathway of T cell activation. In the present study, we have examined the expression and function of the 10D1 Ag on human thymocytes. Three-color FMF analysis showed that the 10D1 Ag was highly expressed on minor but distinct subpopulations of double-negative and CD4 single-positive thymocytes, and weakly on a part of double-positive thymocytes, but not on CD8 single-positive thymocytes. In double-negative thymocytes, the vast majority of 10D1+ cells were immature thymocytes of CD7+2+3- phenotype. Interestingly, 10D1 mAb could induce the proliferation of CD4 single-positive thymocytes in the presence of goat anti-mouse Ig to cross-link the 10D1 Ag. The treatment of thymocytes with OKT4 mAb plus C but not with OKT8 mAb plus C totally abrogated the proliferative response induced by 10D1 mAb, indicating that the 10D1-responsible thymocytes were of CD4+8- phenotype. This 10D1 mAb-induced thymocyte proliferation was perfectly dependent on the endogenous IL-2/IL-2R system since a complete inhibition was observed with anti-IL-2 and anti-IL-2R mAb. The proliferating CD4 single positive thymocytes predominantly expressed the IL-2R alpha (p55) but not a detectable level of the IL-2R beta (p75). These results indicate that, although the 10D1 Ag can be detected on the CD7+2+3-4-8- thymocytes, its functional expression is restricted to a minor more mature CD4+ thymocyte population as well as in peripheral blood T cells, and the implications of these findings are discussed.     

Expression and functional significance of the J11d marker on mouse thymocytes

Subpopulations of thymocytes have been characterized phenotypically and functionally in relation to their expression of the marker defined by the monoclonal antibody J11d. Cortical-type L3T4+, Lyt-2+ thymocytes are all J11d+. Thymocytes that share the phenotype L3T4-, Lyt-2+ with peripheral Lyt-2+ T cells contain a J11d+ and a J11d- subset. These J11d- cells behave like peripheral Lyt-2+ T cells in two functional assays: they form clonal growth bursts in response to immobilized antibody against the T cell antigen receptor, and they act as precursors of alloreactive cytotoxic T cells. The J11d+ cells are inert in both of these assays. In contrast, L3T4+, Lyt-2- thymocytes do not contain a J11d+ subset.     

Thymic stroma-derived T cell growth factor (TSTGF). IV. Capacity of TSTGF to promote the growth of L3T4- Lyt-2- thymocytes by synergy with phorbol myristate acetate or various IL

The present study investigates the role of thymic stroma-derived T cell growth factor (TSTGF) in promoting the growth of L3T4- Lyt2- (double-negative) thymocytes. Partially purified TSTGF samples were prepared from the culture supernatant of a newly established thymic stromal cell line, MRL104.8a. The TSTGF alone induced only marginal proliferation of double-negative thymocytes, whereas this factor exerted a potent growth-promoting effect on these cells in combination with PMA. Because such an enhanced proliferation was not inhibited by anti-IL-4 or anti-IL-2R antibody, this was not due to the stimulation of an autocrine mechanism involving the production and utilization of IL-4 or IL-2. In scrutinizing PMA-equivalent physiologic substance(s), IL-1 was revealed to be capable of replacing the role of PMA in the above co-stimulation cultures and including enhanced proliferation of double-negative thymocytes in combination with TSTGF. Although TSTGF plus IL-2 or IL-4 also exhibited an appreciable or moderate synergistic effect on the growth of double-negative thymocytes, its magnitude was weaker compared with that obtained by TSTGF plus IL-1. More important, the strikingly enhanced proliferation was induced in the combinations of TSTGF, IL-1, and IL-2 or IL-4 under conditions in which the proliferation induced by IL-1 plus IL-4 or IL-1 plus IL-2 was marginal or slight. Furthermore, such strongly enhanced proliferation was also observed in the double-negative thymocyte population which was additionally depleted of T3+ cells (namely, the L3T4- Lyt-2- T3- or dull population). These results indicate the crucial role of TSTGF in the proliferation of immature thymocytes by synergy with various cytokines.     

Thymus cell migration: cells migrating from thymus to peripheral lymphoid organs have a "mature" phenotype

To gain information on the lineage relationship of cells leaving the thymus, we studied the phenotype of thymus emigrants within hours of their exit. The migrants were identified in the peripheral lymphoid organs by their fluorescence, 3 to 4 hr after intrathymic injection of a solution of fluorescein isothiocyanate, a technique that initially only labels thymocytes. Migrants identified in this way were analyzed with rhodamine-anti-Thy-1 or rhodamine peanut agglutinin (PNA). They were found to express Thy-1 antigen and PNA binding sites at levels very similar to those found on the majority of peripheral T cells or medullary thymocytes and quite different from cortical thymocytes. Taken together with our previous experiments on Lyt-1, Lyt-2, and H-2 levels, the data show that cells leaving the thymus are quite mature in phenotype and are indistinguishable from peripheral T cells by all the criteria examined.     

Developmental sequence of T200 antigen modifications in murine T cells

The T200 glycoproteins of T cells were analyzed at different stages of T cell development. Immunoprecipitation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that Lyt-2-L3T4-, and Lyt-2+L3T4+ thymocytes had similar T200 proteins, whereas Lyt-2+L3T4- and Lyt-2-L3T4+ thymocytes expressed a distinct set of T200 molecules. This result indicated a molecular switch in regulation of T200 protein expression upon differentiation of thymocytes to mature phenotype T cells. Further modifications were evident when the T200 proteins of peripheral T cell subsets were examined. In particular L3T4+ T cells expressed T200 proteins of m.w. 220,000, 200,000, and 175,000, whereas Lyt-2+ lymph node T cells expressed an additional T200 protein of m.w. 235,000. Antigenic differences in the T200 glyco-proteins of peripheral L3T4+ and Lyt-2+ T cells were also detected. The anti-B220 monoclonal antibody, 14.8, reacted with lymph node Lyt-2+ T cells but did not react with lymph node L3T4+T cells or with Lyt-2+L3T4- thymocytes. This finding demonstrated a lineage-specific modification of the T200 protein of Lyt-2+ T cells that occurred after exit of these cells from the thymus into peripheral lymphoid organs. This modification apparently occurred on the m.w. 235,000 and 220,000 proteins since these species were precipitated by 14.8, whereas the others were not. In vitro growth and activation also resulted in further T200 antigen alterations. The monoclonal antibody, RA3, which reacts with the B220 antigen of B cells but, unlike 14.8, does not react with any peripheral T cells, showed significant reactivity with Lyt-2+ cytotoxic T cell (CTL) clones but not with L3T4+ T helper cell clones. CTL clones were also 14.8+ but T helper cell clones were not. Immunoprecipitation by 14.8 and RA3 of T200 proteins from CTL clones yielded a single protein of m.w. 240,000 that co-migrated with the B cell form of T200. Overall, the results indicate the presence of developmentally regulated mechanisms that control T200 glycoprotein expression during T cell differentiation in the thymus and in peripheral lymphoid organs.     

Distinct BMI-1 and EZH2 expression patterns in thymocytes and mature T cells suggest a role for Polycomb genes in human T cell differentiation

BMI-1 and EZH2 Polycomb-group (PcG) proteins belong to two distinct protein complexes involved in the regulation of hematopoiesis. Using unique PcG-specific antisera and triple immunofluorescence, we found that mature resting peripheral T cells expressed BMI-1, whereas dividing blasts were EZH2(+). By contrast, subcapsular immature double-negative (DN) (CD4(-)/CD8(-)) T cells in the thymus coexpressed BMI-1 and EZH2 or were BMI-1 single positive. Their descendants, double-positive (DP; CD4(+)/CD8(+)) cortical thymocytes, expressed EZH2 without BMI-1. Most EZH2(+) DN and DP thymocytes were dividing, while DN BMI-1(+)/EZH2(-) thymocytes were resting and proliferation was occasionally noted in DN BMI-1(+)/EZH2(+) cells. Maturation of DP cortical thymocytes to single-positive (CD4(+)/CD8(-) or CD8(+)/CD4(-)) medullar thymocytes correlated with decreased detectability of EZH2 and continued relative absence of BMI-1. Our data show that BMI-1 and EZH2 expression in mature peripheral T cells is mutually exclusive and linked to proliferation status, and that this pattern is not yet established in thymocytes of the cortex and medulla. T cell stage-specific PcG expression profiles suggest that PcG genes contribute to regulation of T cell differentiation. They probably reflect stabilization of cell type-specific gene expression and irreversibility of lineage choice. The difference in PcG expression between medullar thymocytes and mature interfollicular T cells indicates that additional maturation processes occur after thymocyte transportation from the thymus.     

Selective binding of Dolichos biflorus agglutinin to L3T4-, Lyt-2- thymocytes. Expression of terminal alpha-linked N-acetyl-D-galactosamine residues defines a subpopulation of fetal and adult murine thymocytes

Using histochemical and immunocytochemical techniques, a lectin with nominal specificity for alpha-linked N-acetyl-D-galactosamine, Dolichos biflorus agglutinin (DBA), was found to preferentially label thymocytes with an L3T4-, Lyt-2- phenotype from fetal/newborn and adult mice. Through days 14 to 16 of gestation, virtually all thymocytes bound DBA, followed by a dramatic reduction of DBA labeling during the last 4 days of gestation, reaching adult levels of about 2 to 4% of total thymocytes. At later stages of fetal development, the DBA+ cells were confined to the subcapsular area of the thymus. This apparent loss of DBA+ cells was caused by an expansion of the thymocyte population not labeled with this lectin. Affinity purification of thymocyte cell surface components with insolubilized DBA indicated that virtually all of the lectin binding to fetal thymocytes was mediated by a 120-kDa glycoprotein. In addition to thymocytes, DBA also labeled about 5% of bone marrow cells from both normal or nude mice and a small population of spleen cells as well. These results suggest that this lectin may be useful to positively select for LT34-, Lyt-2- thymocytes, and, possibly, other immature populations within the T cell lineage.     

Functional status of interleukin 2 receptors expressed by immature (Lyt-2-/L3T4-) thymocytes

A subpopulation of phenotypically immature (Lyt-2-/L3T4-) thymocytes express receptors for the polypeptide hormone interleukin 2 (IL 2); however, these cells do not proliferate in vitro in response to IL 2. In investigating this phenomenon in greater detail, we observed that the IL 2 receptors (IL 2-R) on freshly isolated immature thymocytes bound IL 2 with about fivefold lower affinity (Kd approximately 100 pM) than IL 2-R on activated mature T cells and T cell lines (Kd approximately 20 pM). Furthermore, in contrast to activated T cells, Lyt-2-/L3T4- thymocytes did not endocytose bound IL 2. When stimulated in short-term culture with a combination of phorbol ester (PMA) and calcium ionophore, Lyt-2-/L3T4- thymocytes proliferated in a largely IL 2-dependent fashion. IL 2-R expression on these activated cells initially disappeared (at 24 hr) and subsequently reappeared (at 48 to 72 hr). Reexpressed IL 2-R on activated thymocytes resembled those on mature T lymphocytes in that they bound IL 2 with high affinity (Kd = 15 to 25 pM) and were capable of endocytosing IL 2. Taken together, these data place certain constraints on the putative physiologic role of IL 2 in intrathymic growth regulation.     

Transgenic expression of RasGRP1 induces the maturation of double-negative thymocytes and enhances the production of CD8 single-positive thymocytes

RasGRP1 is a guanine nucleotide exchange factor for Ras that is required for the efficient production of both CD4 and CD8 single-positive thymocytes. We found that RasGRP1 expression is rapidly up-regulated in double-negative thymocytes following pre-TCR ligation. Transgenic overexpression of RasGRP1 compensated for deficient pre-TCR signaling in vivo, enabling recombinase-activating gene 2(-/-) double-negative thymocytes to mature to the double-positive stage. RasGRP1 transgenic mice had a 4-fold increase in CD8 single-positive thymocytes, most of which had atypically low levels of CD3. The RasGRP1 transgene lowered the threshold of TCR signaling needed to initiate proliferation of single-positive thymocytes, with this effect being particularly evident among CD8 single-positive cells. In 3-day cultures, TCR stimulation via anti-CD3 caused a 10-fold increase in the ratio of CD8 to CD4 thymocytes among RasGRP1 transgenic vs nontransgenic thymocytes. These results demonstrate that in addition to driving the double-negative to double-positive transition, increased expression of RasGRP1 selectively increases CD8 single-positive thymocyte numbers and enhances their responsiveness to TCR signaling.     

Thymocytes express the golli products of the myelin basic protein gene and levels of expression are stage dependent

The golli products of the myelin basic protein gene have been shown to be expressed in mouse thymus and brain. The full repertoire of thymic cell types expressing golli products has not yet been determined, although immunoreactivity has been found in some macrophages. We have analyzed the cellular expression of golli mRNAs and proteins in the thymus. The results showed that MTS5(+) cortical/MTS10(+) medullary epithelial cells and NLDC145(+) dendritic cells did not express golli, while some macrophages did exhibit strong immunoreactivity. GOLLI: mRNAs were not detected in macrophages by in situ hybridization. Thymocytes expressed significant levels of golli mRNAs and proteins by in situ hybridization and immunohistochemistry. Interestingly, golli immunoreactivity varied with thymocyte stage of differentiation. For example, CD4(-)CD8(-) (double-negative) thymocytes expressed relatively high levels of golli. Upon further differentiation into CD4(-)CD8(-) (double-positive) thymocytes, golli protein expression declined dramatically. When thymocytes developed into CD8(-) or CD4(+) (single-positive) thymocytes, golli protein expression increased again, but it never achieved the levels found in double-negative thymocytes. Thus, the altered levels of expression of golli proteins in developing thymocytes correlated with the transitions from double-negative to double-positive and double-positive to single-positive stages. The lack of significant golli expression in thymic stromal cells may offer an alternative explanation for the mechanism of inefficient negative selection of those autoreactive thymocytes with specificity for myelin basic proteins.     

Premature expression of chemokine receptor CCR9 impairs T cell development

During thymocyte development, CCR9 is expressed on late CD4-CD8- (double-negative (DN)) and CD4+CD8+ (double-positive) cells, but is subsequently down-regulated as cells transition to the mature CD4+ or CD8+ (single-positive (SP)) stage. This pattern of expression has led to speculation that CCR9 may regulate thymocyte trafficking and/or export. In this study, we generated transgenic mice in which CCR9 surface expression was maintained throughout T cell development. Significantly, forced expression of CCR9 on mature SP thymocytes did not inhibit their export from the thymus, indicating that CCR9 down-regulation is not essential for thymocyte emigration. CCR9 was also expressed prematurely on immature DN thymocytes in CCR9 transgenic mice. Early expression of CCR9 resulted in a partial block of development at the DN stage and a marked reduction in the numbers of double-positive and SP thymocytes. Moreover, in CCR9-transgenic mice, CD25high DN cells were scattered throughout the cortex rather than confined to the subcapsular region of the thymus. Together, these results suggest that regulated expression of CCR9 is critical for normal development of immature thymocytes, but that down-regulation of CCR9 is not a prerequisite for thymocyte emigration.     

Viral infection of the thymus.

We have examined infection of the thymus during congenitally acquired chronic lymphocytic choriomeningitis virus (LCMV) infection of mice, a classic model of antigen-specific T-cell tolerance. Our results show that (i) infection starts at the fetal stage and is maintained throughout adulthood, and (ii) this chronic infection of the thymus can be eliminated by transfer of virus-specific cytotoxic T lymphocytes (CTL) that infiltrate the thymus and clear all viral products from both medullary and cortical regions. Elimination of virus from the thymus results in abrogation of tolerance. During the fetal stage, the predominant cell type infected is the earliest precursor of T cells with a surface phenotype of Thy1+ CD4- CD8- J11d+. In the adult thymus, infection is confined primarily to the cortisone-resistant thymocytes present in the medullary region. The infected cells are CD4+ and J11d+. The presence of J11d, a marker usually associated with immature thymocytes, on infected single positive CD4+ "mature" thymocytes is intriguing and suggests that infection by this noncytolytic virus may affect development of T cells. There is minimal infection of the CD8+ medullary thymocytes or of the double positive (CD4+ CD8+) cells present in the cortex. Infection within the cortex is confined to the stromal cells. Interestingly, there is infection of the double negative (CD4- CD8-) thymocytes in the adult thymus, showing that even during adulthood the newly developing T cells are susceptible to infection by LCMV. Virus can be eliminated from the thymuses of these carrier mice by adoptive transfer of medullary region first and then from the thymic cortex. This result clearly shows the need to reevaluate the widely held notion that mature T cells are unable to reenter the thymus. In fact, in our experiments the donor T cells made up to 20 to 30% of the total cells in the thymus at 5 to 7 days after the transfer. The number of donor T cells declined as virus was eliminated from the thymus, and at 1 month posttransfer, the donor T cells were hardly detectable. The results of this study examining the dynamics of viral infection and clearance from the thymus, the primary site of T-cell development, have implications for understanding tolerance induction in chronic viral infections.     

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.