Kinetics of thymus repopulation by intrathymic progenitors after intravenous injection: evidence for successive repopulation by an IL-2R+, Pgp-1- and by an IL-2R-, Pgp-1+ progenitor

A kinetic study of thymus repopulation after intravenous injection of L3T4-, Lyt-2- thymocytes further depleted of IL-2R+ or Pgp-1+ cells indicates that donor cells within the thymus at Day 8 after injection descend primarily from an IL-2R+, Pgp-1- progenitor, while at Day 17 after injection most progeny of donor phenotype descend from an IL-2R-, Pgp-1+ progenitor. Repopulation studies in organ culture demonstrate that IL-2R+, Pgp-1+ cells also have progenitor activity. All three progenitors give rise to differentiated cell types normally present in the thymus. These results are consistent with the interpretation that the L3T4-, Lyt-2- population is composed of a heterogeneous collection of progenitors which repopulate the thymus with differing kinetics and imply that it will be difficult to establish lineage relationships within this population in the absence of a clonal assay for thymocyte progenitors.     

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.     

Thymocyte subpopulations during early fetal development in the BALB/c mouse

Phenotypic analysis of thymocytes during murine fetal development may be of use in determining the pathways of thymocyte differentiation. The expression of the functionally significant molecules Lyt-2 (CD8), L3T4 (CD4), and the TCR has already been described. However, mAb specific for several other murine lymphocyte surface markers are now available and, although these have been used to characterize adult thymocytes, a detailed analysis of fetal thymocytes with these antibodies has not previously been undertaken. In this study, we have used mAb specific for Thy-1, J11d, Pgp-1, and the IL-2R, in addition to those for Lyt-2 and L3T4, to identify subpopulations of early fetal thymocytes. By using two-color flow cytometric analysis of cells obtained from fetal thymuses on sequential days of gestation, we have been able to follow the development of various subpopulations through early fetal ontogeny. Our data indicate that the earlier thymocytes are found in the J11d+/Pgp-1+ subset which is abundant at fetal day 14 but constitute a numerical minority by day 16.     

T cell antigen receptor expression by subsets of Ly-2-L3T4- (CD8-CD4-) thymocytes

The V beta 8-specific mAb F23.1 and KJ16 were used as fluorescent stains to test for TCR expression on the surface of subpopulations of early, CD4-CD8- (L3T4-Ly-2-) thymocytes from adult CBA mice. A surprisingly high proportion (27%) of Ly-2-L3T4- thymocytes were strongly F23.1 and KJ16 positive. No positive cells were detected among Ly-2-L3T4- thymocytes from V beta 8-negative SJL mice. In contrast to the adult thymus, Ly-2-L3T4- cells from embryonic CBA thymus lacked F23.1-positive cells. Subsets of adult CBA Ly-2-L3T4- thymocytes were separated to determine which expressed V beta 8. The major subset, Ly-1 low B2A2-M1/69+Thy-1+Pgp-1-, representing a phenotype similar to embryonic Ly-2-L3T4- thymocytes and the phenotype commonly isolated from adult thymocytes as Ly-1 "dull," lacked cells strongly positive for F23.1. In contrast, a series of subsets of adult CBA Ly-2-L3T4- thymocytes which were B2A2-M1/69- and Pgp-1+ all included strongly F23.1-positive cells. A minor subset, negative for most markers except Pgp-1 and presumed on the basis of this phenotype and some reconstitution studies to include the earliest intrathymic precursors, contained 28% F23.1-positive cells. However, no F.23.1-positive cells were detected in equivalent "prethymic" populations from bone marrow or from athymic mouse spleen. The subsets of Ly-2-L3T4- thymocytes which were Ly-1 high, B2A2-M1/69-, and Pgp-1+ all contained about 70% F23.1-positive cells, indicating a V beta 8 usage much higher than the mature T cell average. These results indicate that a series of distinct developmental events have occurred within these CD4-CD8- thymocytes previously considered as a single group of early precursor cells, and that some aspects of repertoire selection may be occurring amongst thymocytes which lack CD4 or CD8.     

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.     

Expansion of large granular lymphocytes in IL-2-driven 14-day-old fetal thymocytes in organ culture

These experiments were designed to evaluate the role of cytokines in early T cell development within the thymus. By using a thymic organ culture model, we have studied the influence of high dose of IL-2 (10 to 1000 IU/ml) on the cell populations that are generated during 12 days starting from a thymic rudiment of 14-day-old mouse embryo. The IL-2 treatment resulted in the expansion of Thy-1+/-, CD4-, CD8-, CD3-, Fc gamma RII+, CD5 (Lyt-1)-, HSA-, Pgp- 1+, Mel-14- population. These cells had the morphology of large granular lymphocytes and displayed broad cytotoxic activity. In addition, IL-2-treated organ cultures had a dramatic decrease in CD4+CD8+ thymocytes, a marked reduction in TCR-alpha beta+ thymocytes--even more pronounced in the TCR-V beta 6+ and TCR-V beta 8+ thymocytes--and no significant changes in the number of TCR-gamma delta+ as compared to control organ cultures.     

Progenitor cells in the thymus: most thymus-homing progenitor cells in the adult mouse thymus bear Pgp-1 glycoprotein but not interleukin-2 receptor on their cell surface

Pgp-1-positive and interleukin-2-receptor (IL-2R)-positive cells are both minor (less than 5%) subpopulations within the adult thymus. A thymocyte population enriched for these two cell types, obtained by killing the bulk of thymocytes with anti-Thy-1 antibody and complement, contains thymus-homing progenitor cells which can transiently repopulate the thymus of irradiated recipients. Using two-color immunofluorescence, we demonstrate that the Pgp-1+ and IL-2R+ cells present in this enriched population represent largely nonoverlapping subsets, although some cells do express both markers. We also show by depletion of these two cell types that the bulk of the thymus-homing progenitors present in this enriched population are found in the Pgp-1+ population, and not in the IL-2R+ population. We discuss the relationship between the thymus-homing progenitors in this depleted thymus subpopulation and the thymus-homing progenitors present in the thymus as a whole.     

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.     

Natural killer cells in the thymus. Studies in mice with severe combined immune deficiency

The relationship between NK cell and T cell progenitors was investigated by using mice with severe combined immune deficiency (scid). Scid mice are devoid of mature T and B cells because they cannot rearrange their Ig and TCR genes. However, they have normal splenic NK cells. Thymus of scid mice, although markedly hypocellular, contains cells that lyse YAC-1, an NK-sensitive tumor cell. By flow cytometry, two populations of cells were identified in the scid thymus. Eighty percent of the cells were Thy-1+, IL-2R(7D4)+, J11d+, CD3-, CD4-, CD8- whereas the remaining were IL-2R-, J11d-, CD3-, CD4-, and CD8-. By cell sorting, all NK activity was found in the latter population, which is phenotypically similar to splenic NK cells. To determine if the thymus contains a bipotential NK/T progenitor cell, J11d+, IL-2R+ cells were cultured and analyzed for the generation of NK cells in vitro. These cells were used because they resemble 15-day fetal and adult CD4- CD8- thymocytes that are capable of giving rise to mature T cells. Cultured J11d+ thymocytes acquired non-MHC-restricted cytotoxicity, but in contrast to mature NK cells, the resulting cells contained mRNA for the gamma, delta, and epsilon-chains of CD3. This suggests that J11d+ cells are early T cells that can acquire the ability to kill in a non-MHC-restricted manner, but which do not give rise to NK cells in vitro. The differentiative potential of scid thymocytes was also tested in vivo. Unlike bone marrow cells, scid thymocytes containing 80% J11d+ cells failed to give rise to NK cells when transferred into irradiated recipients. Together these results suggest that mature NK cells reside in the thymus of scid mice but are not derived from a common NK/T progenitor.     

T cell lineages in the thymus of lpr/lpr mice. Evidence for parallel pathways of normal and abnormal T cell development

Autoimmune mice homozygous for the lpr/lpr (lpr) gene develop a profound lymphadenopathy resulting from the accumulation of immature Thy-1+ Lyt-2- L3T4- cells in peripheral lymphoid tissues. The source of these cells is not known although the presence of a thymus is necessary to manifest both the lymph node enlargement and the autoimmunity. For this reason and the fact that the abnormal lpr cell phenotypically resembles immature thymocytes, we studied the thymus in lpr mice. Adult lpr thymuses were found to contain an immature population phenotypically identical to the peripherally accumulating cells, including the expression of B220 and Pgp-1 antigens as well as the presence of surface T cell receptor molecules as defined by the antibody KJ16-133. Evidence is presented that some of these lpr precursor T cells are capable of intrathymic differentiation, whereas the vast majority are exported unchanged to the lymph nodes where a portion differentiate further into mature T cells. This lpr-specific lineage could be distinguished from a normal component of the lpr thymus by surface phenotype and immunohistology. The results suggest that the massive accumulation of cells in lpr lymph nodes is not so much the result of abnormal proliferation of T cells as abnormal intrathymic differentiation. In addition, a minor subpopulation of normal Lyt-2- L3T4- thymocytes was identified that resembles the phenotype of the lpr cell and similarly expresses surface T cell receptor molecules. The presence of two parallel lineages in the lpr thymus thus also provides insight into normal T cell development.     

Up-regulation of interleukin 4 receptor expression on immature (Lyt-2-/L3T4-) thymocytes

In this report, the effect of interleukin 4 (IL-4) on the growth and differentiation of Lyt-2-/L3T4-(2-4-) thymocytes was investigated. It was found that these thymocytes proliferated extensively when cultured in the presence of IL-4 + phorbol myristate acetate without apparent differentiation to Lyt-2+ or L3T4+ cells. We also demonstrated that 2-4- thymocytes constitutively express a high affinity (dissociation constant of 20 to 40 pM) receptor for IL-4. Freshly isolated 2-4- thymocytes expressed on average about 100 IL-4 receptors per cell, but the number of receptors increased approximately 8-fold within 3 days after activation by IL-4 + phorbol myristate acetate. These findings suggest that IL-4 may play an important role in T cell ontogeny by promoting self-renewal of stem cells within the thymus.     

Heterogeneity of immature (Lyt-2-/L3T4-) thymocytes. Identification of four major phenotypically distinct subsets differing in cell cycle status and in vitro activation requirements

Thymocytes that bear neither Lyt-2 nor L3T4 differentiation Ag (2-4- thymocytes) contain the precursors of mature Lyt-2+ and L3T4+ T cells. In the present study, we have identified four major subpopulations of 2-4- cells in adult C57BL/6 mice that differ in surface phenotype and in situ proliferative status. Two-color immunofluorescence analysis with RL-73 (a mAb recognizing an as yet unidentified activation Ag) and PC-61 (an anti-IL-2R mAb) revealed three distinct subsets of 2-4-thymocytes: RL-73+ IL-2R- (30%), RL-73+/-IL-2R+ (45%), and RL-73- IL-2R- (25%). The RL-73+ IL-2R- subset had the highest percentage of large blasts and cycling cells, whereas the RL-73+/- IL-2R+ and RL-73- IL-2R- subsets had intermediate and low percentages, respectively, indicating that in situ proliferation correlated better with RL-73 intensity than with IL-2R expression. An additional marker, heat-stable Ag (HSA), was found to further subdivide the RL-73- population into RL-73- HSA- (10% of total 2-4-) and RL-73- HSA+ (15%) fractions. The two latter (RL-73-) subsets appeared to be more "mature" than the former since they expressed high levels of Lyt-1 and appeared later during fetal thymus ontogeny. In parallel with the phenotypic analysis, we compared the in vitro activation requirements of each of the four purified 2-4- subsets. All four populations proliferated well to the combination of phorbol ester (PMA), ionomycin, and IL-2. In response to PMA and ionomycin (without added IL-2), only RL-73- HSA-cells proliferated and this proliferation was correlated with IL-2 production. However, if IL-1 was included with PMA and ionomycin then all four populations responded. Finally, a proliferative response to Con A or mitogenic anti-Thy-1 mAb was observed only for RL-73- HSA+ and (to a lesser extent) RL-73- HSA-cells. These data indicate that each of the four phenotypically distinct subpopulations of immature thymocytes can also be distinguished on the basis of their in vitro activation requirements.     

Cell proliferation and differentiation in the fetal and early postnatal mouse thymus

The relationships between cell proliferation and cell differentiation during thymus ontogeny were studied by labeling DNA-synthesizing thymocytes with bromodeoxyuridine and staining with antibodies against CD4, CD8, J11d, phagocytic glycoprotein 1, TCR V beta 8 chain, Thy-1, and IL-2R surface proteins. The development of the thymus was discontinuous, with two well defined growth periods from 13 days to 18 days of fetal life and from 3 days to 6 days after birth, and more progressive growth from day 8 to 2 wk. Cell proliferation started on fetal day 12, 1 day after the arrival of hemopoietic stem cells in the third branchial pouch. These cells were phagocytic glycoprotein 1-positive but IL-2R and Thy-1 negative. Thus, cell proliferation preceded IL-2R expression. Until day 15, CD4-8- thymocytes expanded without differentiation. Then CD4-8+ and CD4+8+ cells appeared; this induction was proliferation dependent and occurred on cells which had already lost IL-2R, but just after maximum expression of this receptor. During several days, the thymus remained of constant size (around 10(7) cells) and behaved like the steady state thymus. On day 3 after birth, expansion started again and was correlated with an increase in CD4-8- proliferation index and IL-2R expression. At the same time, the thymic subset capable of expansion without differentiation was again, transiently, detectable. These results suggest that the inflow of precursor cells into the thymus is permanent but transiently increased at several times during ontogeny. Moreover, the behavior of fetal CD4-8- cells does not appear radically different from that of adult precursors, but the actual difference resides in the variation of the relative proportion of CD4-8- cells at different maturation stages, as revealed by striking variations of IL-2R expression by cycling cells.     

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.     

Evidence that the Pgp-1 glycoprotein is expressed on thymus-homing progenitor cells of the thymus

The Pgp-1 glycoprotein is found on the bone marrow prothymocyte; however, only a few percent of cells within the normal thymus express significant quantities of Pgp-1 glycoprotein. One hypothesis is that some or all of these Pgp-1+ thymocytes represent thymocyte progenitors or the immediate descendents of the bone marrow-derived prothymocyte. A cell present in the thymus which is able to home back to the thymus and to transiently repopulate it represents one class of thymocyte progenitor. Thymocyte populations enriched in this thymus-homing progenitor are enriched in Pgp-1+ cells. Treatment of these enriched populations with anti-Pgp-1 antibody inhibits activity of the thymus-homing progenitor. These results are consistent with the hypothesis that the thymus-homing progenitor bears Pgp-1 on its surface.     

Interferon-gamma is produced by activated immature mouse thymocytes and inhibits the interleukin 4-induced proliferation of immature thymocytes

We have recently shown that interleukin 4 (IL-4) (formerly called BSF-1) is a potent stimulator of fetal and adult immature thymocyte proliferation and that adult L3T4-/Lyt-2-thymocytes can be stimulated by calcium ionophore (A23187) and phorbol ester to secrete IL-4 (Zlotnik, A., J. Ransom, G. Frank, M. Fischer, and M. Howard. 1987. Proc. Natl. Acad. Sci. (USA) 84:3856). This report shows that fetal thymocytes (day 15 of gestation) can also be activated to produce IL-4 suggesting that IL-4 may be a mediator of fetal as well as adult immature thymocyte proliferation. Furthermore, we demonstrate that interferon-gamma (IFN-gamma) inhibits the IL-4-mediated proliferation of both fetal and adult L3T4-/Lyt-2-thymocytes. The inhibition of proliferation is blocked by anti-IFN-gamma antibody and is unaffected by indomethacin suggesting that IFN-gamma directly inhibits immature thymocyte proliferation. IFN-gamma does not block the IL-4/phorbol myristate acetate-mediated proliferation of an adult thymocyte population, which is enriched for L3T4-/Lyt-2+ and L3T4+/Lyt-2- cells, suggesting that the inhibitory effect of IFN-gamma is limited to the immature thymocyte population. Both fetal (day 15) and adult L3T4-/Lyt-2--thymocytes can be activated to secrete an IFN-gamma like activity. This activity is neutralized by a monoclonal anti-IFN-gamma antibody indicating that the activity is due to IFN-gamma. mRNA analysis of adult L3T4-/Lyt-2- thymocytes stimulated with A23187 and phorbol myristate acetate confirms that mRNA for both IL-4 and IFN-gamma is induced in adult L3T4-/Lyt-2- thymocytes. These results indicate that IL-4 and IFN-gamma can regulate immature thymocyte proliferation.     

Role of a thymic stromal cell clone in inducing the stage-specific differentiation of various subpopulations of double negative thymocytes

The monolayer of a thymic stromal cell clone termed MRL104.8a induced the differentiation of adult double negative (DN) thymocytes (CD3-4-8-) through a CD3-4-8+ intermediate into CD3- (or dull) 4+8+ stages. DN thymocytes were separated into three subpopulations depending on their cell-surface expression of Pgp-1 and IL-2R, namely, Pgp-1+IL-2R-, Pgp-1-IL-2R+, and Pgp-1-IL-2R-. The present study investigated the requirements of the MRL104.8a monolayer for inducing the differentiation of these DN thymocyte subpopulations. The following were revealed: i) the MRL104.8a monolayer failed to induce the differentiation of a Pgp-1+IL-2R- subpopulation; ii) whereas a Pgp-1-IL-2R+ subpopulation did not express either CD4 or CD8 Ag when cultured in medium, culturing this subpopulation on the thymic stromal cell monolayers resulted in the expression of CD8 but not CD4 Ag; and iii) a Pgp-1-IL-2R- DN subpopulation obtained through less extensive treatments with anti-CD4 and anti-CD8 antibodies in the presence of C before sorting procedures spontaneously differentiated into double positive cells in medium. In contrast, most of DN cells with the same phenotype obtained through extensive anti-CD4 and -CD8 treatments before sorting failed to express CD4 and/or CD8 Ag in medium but could differentiate through a CD3-4-8+ into more mature stages only when they were cultured on the thymic stromal monolayer. These results indicate differential requirements of thymic stromal cells for the differentiation of various DN subpopulations with qualitatively distinct phenotypes and different magnitudes (very low vs almost zero levels) of CD4/CD8 expression.     

The Pgp-1 antigen is expressed on early fetal thymocytes

The Pgp-1 glycoprotein is expressed on the bone marrow prothymocyte and on a class of intrathymic progenitor cells in the adult animal. Only 5% of adult thymocytes are strongly Pgp-1⁺. When fetal thymocytes of day 13–14 of gestation are examined by flow cytometric analysis, 80–90% of thymocytes are Pgp-1⁺, while the bulk of thymocytes are Thy-1^–. By day 15–16, the percentage of Pgp-1⁺ cells begins to fall while nearly all cells become Thy-1⁺. Two-color immunofluorescence indicates that many Pgp-1⁺ cells are Thy-1⁺. The percentage of Pgp-1⁺ cells continues to fall over the next several days, reaching adult levels by day 19. These observations are consistent with the interpretation that at least some classes of thymocyte progenitors are Pgp-1⁺.