Studies on the Thymus in Chagas'' Disease II. Thymocyte Subset Fluctuations in Trypanosoma cruzi-infected mice: Relationship to Stress

Changes in thymic T-cell subsets in mice acutely infected with Trypanosoma cruzi have been studied in both C3H/HeJ and C57BL/6 mice. The significant decrease in thymocyte number, observed in both mouse strains on day 14 post-infection correlated with a drastic decrease in CD4+CD8+ cell number, whereas the number of CD4-CD8-, CD4+CD8- and CD4-CD8+ cells remained essentially unchanged. The important increase in CD3hi cell frequency confirmed that resistant thymocytes during Chagas' disease development were mostly medullary thymocytes, whereas the thymic cortex was largely depleted, as previously observed on thymus sections. This involution of the thymus could have been due to the increase of circulating glucocorticoid levels observed after infection. However, similar cell modifications were found in infected adrenalectomized mice whose serum corticosterone levels were only slightly augmented. Thus, the thymic alterations appear not to be linked to stress responses, at least those dependent on high levels of circulating glucocorticoids.     

Estrogen Blocks Early T Cell Development in the Thymus

PROBLEM: Pregnancy and estrogen are known to suppress B lymphopoiesis as well as lead to thymic involution in the mouse. Additionally, estrogen deficiency by oophorectomy reportedly causes a selective increase in the B220+ B cells in the murine bone marrow. The purpose of this study was to determine if estrogens played a regulatory role in T cell development. METHODS: The first experimental group consisted of 5–6-week-old Balb/c mice that received subcutaneous pellets of placebo, estriol, estradiol, or progesterone. The thymus glands were examined 2–4 weeks after treatment. The second group consisted of 6-week-old Balb/c mice who underwent either bilateral oophorectomy or a sham procedure. Two weeks after the surgery, extensive phenotypic characterization of the thymus and spleen cells was performed by flow cytometry using monoclonal antibodies to surface markers of T cell subsets. RESULTS: Estrogen treatment causes a dramatic reduction of thymic size and cellularity. All defined T cell subsets of CD4 and CD8 were reduced, with a disproportionate loss of CD4+CD8+ double positive cells. Examination of the triple negative (CD3-CD4-CD8-) subset revealed a striking loss of TN developmental progression of the early precursor cells. Based on the expression of CD44 (pgp-1) and CD25 (IL-2Rα) markers, the TN thymic compartment was composed almost entirely of the earliest population (CD44+, CD25-), with the remaining maturational stages (CD44+, CD25+; CD44-, CD25+; CD44-, CD25-) depleted. In contrast, all T cell developmental stages in the thymus were found to be in normal proportions in the oophorectomized mice, with no differences in the splenic T and B cell subsets. CONCLUSIONS: The study demonstrates that estrogen but not progesterone blocks T cell development in the thymus. However, contrary to our expectation, estrogen deprivation by oophorectomy does not enhance T cell development.     

Characterization of immature CD4+CD8-CD3- thymocytes.

Previously we have described (Hugo, P. et al., Int. Immunol. 1990. 2: 209) an immature CD4+CD8-CD3- thymocyte subset which is thought to be the counterpart of the CD4-CD8+CD3- subset. In this study we show that the ontogeny of these two subsets is parallel in fetal thymic organ culture. Extensive phenotypic characterization of CD4+CD8-CD3- cells reveals that they closely resemble CD4-CD8+CD3- thymocytes being: HSAhigh, Thy-1high, interleukin 2 receptor alpha chain negative, CD44-, H-2K+/-, CD5low, MEL-14low/intermediate, CD2+, LFA-1+ and MTS 35+. Finally, we show that the proportion of CD4+CD8-CD3- thymocytes is highly variable between mouse strains.     

Organotin-induced thymus atrophy concerns the OX-44+ immature thymocytes. Relation to the interaction between early thymocytes and thymic epithelial cells?

After single oral application of the organotin compound di-n-butyltindichloride (DBTC) to rats, a reversible dose-dependent thymus weight reduction is observed. This is maximal at day 4 and recovers to the control value approximately at day 9 after administration. In this study the changes in thymocyte subpopulations after a single oral dose of 15 mg DBTC/kg body weight were analysed by immunohistology. Thymus glands of exposed rats were collected at day 1,2,3,4,5,7 and 9 after DBTC dosing and frozen sections were screened for various thymocyte differentiation antigens. Staining by mAb HIS-44 that labels a subset of cortical thymocytes showed that the thymus atrophy was restricted to the cortex. Here a time-dependent decrease of labelling by CD2 (OX-34), CD8 (OX-8), CD4 (ER-2), and CD5 (OX-19) was observed. In contrast, the number of cortical OX-44+ cells increased from day 2 to day 5. This increase can reflect an increase of CD4-CD8- double-negative thymocytes or of macrophages. However, most of these OX-44+ cells were negative for acid phosphatase, which is present in most macrophages. We concluded that these OX-44+ cells were mainly CD4-CD8- thymocytes and that the thymocyte subpopulation of this phenotype, i.e. CD4-CD8-OX-44+, may be the target cell for DBTC. It is discussed whether DBTC might disturb the interaction of early thymocytes and thymic epithelium, probably by an interaction with the CD2 antigen.     

3, 3', 4, 4'-Tetrachlorobiphenyl Inhibits Proliferation of Immature Thymocytes in Fetal Thymus Organ Culture

The environmental pollutant 3, 3', 4, 4'-tetrachlorobiphenyl (TCB) leads to thymic atrophy and immuno-suppression, the former possibly causing the latter. TCB binds lo the cytosolic aryl-hydrocarbon receptor (AhR) and transforms it into a DNA-binding state. The development of fetal thymocyles is severely affected by TCB and other AhR-binding xenobiotics, leading to a skewed pattern of thymocyte maturation stages. Murine thymocyte proliferation after exposure to TCB was studied in fetal thymus organ culture (FTOC). C57BL/6 fetus thymic lobes from day 15 of gestation were explanted and grown for 2, 4, 6. and 8 days in organ culture in the presence or absence of 3.3 μM TCB. Subsets of thymocytes were defined by CD4 and CD8 surface markers, and their cell cycle was analysed by DNA staining with 7-amino-actinomycin D (7-AAD). Exposure of fetal thymi in vitro to 3.3 μM TCB significantly reduced the total number of thymocytes. and fewer thymocytes were in S/G₂M phase. The inhibition of cell proliferation induced by TCB treatment affected mainly the CD4⁻ CD8⁻ (double-negative, DN) and CD4⁻ CD8⁺ (single-positive, SP) subsets, and these inhibition appeared mainly in more immature thymocytes, i. e. DNCD3⁻ and CD8⁺CD3⁻ subpopulations, whereas no effect of TCB on CD4⁺CD8⁺ (double-positive, DP) cell proliferative activity was observed. Analysis of the relation of cell proliferation and development of subsets in differentiating fetal ihymocytes suggests that TCB enhanced thymocyte differentiation into mature CD8⁺ cells.     

Delineation of chicken thymocytes by CD3-TCR complex, CD4 and CD8 antigen expression reveals phylogenically conserved and novel thymocyte subsets.

To further define the relationship between thymocyte subsets and their developmental sequence, multi-parameter flow cytometry was used to determine the distribution of the CD3-TCR complex and the accessory molecules CD4 and CD8 on chicken thymocytes. As in mammals, adult thymocytes could be subdivided into CD3-, CD3lo, and CD3hi staining populations. CD4 and CD8 distribution on such populations revealed the presence of CD3-CD4+CD8- and CD3-CD4-CD8+ thymocytes, putative precursors to CD4+CD8+ cells, detectable in the adult and at high frequency during ontogeny. Of particular interest was the existence of CD3lo expression on CD4+CD8- and CD4-CD8+, and in some instances, on CD4-CD8- thymocytes. Such phenotypes are not easily detectable in the mammalian thymus but were readily observed in both adult and embryonic chicken thymus from 16 days of embryogenesis. Further analysis of the TCR lineage of these CD3lo cells revealed that they were essentially all of the alpha beta TCR type. Mature CD3hi thymocytes were found within the CD4+CD8+ and CD4+CD8- and CD4-CD8+ subsets. Both alpha beta and gamma delta TCR lineage thymocytes were detected within all CD4- and CD8-defined subsets, thus identifying novel thymocyte subsets in the chicken thymus, namely alpha beta TCR+CD4-CD8- and gamma delta TCR+ CD4+CD8- cells. Hence, this analysis of chicken thymocytes, while confirming the phylogenically conserved nature of the thymus, has revealed novel T cell subsets, providing further insight into the complexity of mainstream thymocyte maturation pathways.     

Two differential pathways from double-negative to double-positive thymocytes.

Murine thymocytes are divided into four major populations on the basis of expression of CD4 and CD8 antigens. The bulk of evidence favours the view that CD4-CD8- cells can develop into CD4-CD8+ and CD4+CD8- cells via the CD4+CD8+ stage in the thymus. However, CD4-CD8+ and CD4+CD8- thymocyte subsets contain not only CD3+ mature cells but also CD3- immature cells, which seem to be intermediate cells between CD4-CD8- and CD4+CD8+ cells. Here we demonstrate mouse strain differences in the proportion of immature single-positive thymocyte subsets in thymus at the steady or developing state. In C3H mice, immature CD4+CD8- is dominant in proportion over CD4-CD8+ in foetal thymus and in donor-derived thymocytes at an early stage of bone marrow transplantation. On the other hand, immature CD4-CD8+ is dominant over CD4+CD8- during T-cell development in the case of B10.BR mice. An intermediate pattern was shown in the case of F1 mice. Both of these immature single-positive subsets gave rise to double-positive cells after 24 hr culture. These results suggest that there exist two distinct differential pathways; one is from CD4-CD8- cells to CD4+CD8+ cells via CD4-CD8+ cells, and another is via CD4+CD8- cells, and that an application of the 'CD8 pathway' or 'CD4 pathway' seems to be genetically destined by BM-derived cells but not by thymic stromal cells.     

Kinetics of chicken embryonic thymocyte development in ovo and in organ culture.

Thymocyte development was monitored in an embryonic thymus organ culture system to establish a model in the chicken in which the functional nature of the thymic microenvironment could be assessed. Thymus lobes were removed from 10-day-old embryos and cultured for 2-10 days. Cell yield increased to a maximum in 4-8 days of culture with a corresponding decrease in average cell size. An initial thymocyte population of predominantly CD3-CD4-CD8- cells gave rise to all CD3/CD4/CD8-defined subpopulations in vitro, maintaining high levels of CD3-CD4-CD8+ and CD3+CD4-CD8+ cells and a low representation of CD3-CD4+CD8-, CD3+CD4+CD8-, CD3-CD4+CD8+ and CD3+CD4+CD8+ thymocytes. This is the first observation of a CD3-CD4+CD8- population in the chicken. Developmental kinetics of CD3+ cells were similar to that in the embryo, suggesting that the in vitro environment is sufficient to promote and maintain thymocyte maturation. Thymocytes of both the gamma delta and alpha beta T cell receptor (TcR) lineages developed in that order, confirming in ovo data and the lineage potential of the first wave of thymocyte precursors. One unusual finding was a relative accumulation of gamma delta TcR+ thymocytes in culture, incorporating all CD4/CD8 subsets, including a previously undetected population, CD4+CD8-. This may indicate a favorable developmental environment or simply a lack of normal cellular emigration. A detailed comparison with T cell development in the embryo demonstrated that the chicken thymus organ culture system reflects thymic events in ovo during a limited time period and thus should prove useful in the identification of functionally relevant thymic molecules.     

Variant isoforms of CD44 are required in early thymocyte development

The earliest T cells homing to the thymus (CD3-CD4loCD8-) express CD117 (c-kit), CD43 (leukosialin), and the integrins CD11a (alphaL), CD11b (alphaM), CD29 (beta1), CD49f (alpha6), and CD44. Using reagents specific for CD44 variant isoforms (CD44v), we demonstrated that CD44v were expressed on virtually all early thymocytes,whereas cells carrying only the standard molecule (CD44s, not containing any variant domains), which is ubiquitously found on mature lymphocytes later, are very sparse. The expression of CD44v was closely correlated with CD43 and CD117 and was restricted to the CD3-CD4loCD8- stage. CD44v were detected on lymphocyte progenitor populations in the fetal blood, liver, thymus and spleen, as well as in the adult bone marrow. Functional studies demonstrated that only cells expressing CD44v from fetal liver and adult bone marrow could efficiently populate fetal thymic stroma and develop into mature T cells. In fetal thymic organ cultures anti-CD44v antibodies specifically blocked thymocyte development. We also present evidence that CD44v were required for the initial interaction of hematopoietic progenitor cells with the thymic stroma. Our data imply that CD44v are not only a useful marker for hematopoietic progenitors, but also play a functional role in the initiation of thymocyte development.     

Developmental status of CD4-CD8+ and CD4+CD8- thymocytes with medium expression of CD3

In normal mice, more than 10% of thymocytes in the CD4+CD8- and CD4-CD8+ single-positive (SP) subsets express a medium level of CD3 on the cell surface. However, the fate of CD3medium cells is unclear. The CD3medium SP subpopulations might contain (i) cells in an immature stage of the pathways leading to CD3high cells, (ii) cells in developmental pathways that do not lead to CD3high cells, or (iii) cells that have been negatively selected. We found that sorted CD3medium CD4+CD8- thymocytes from adult mice up-regulated CD3 to high levels in reaggregation thymus organ culture. Unlike their CD3high counterparts, CD3medium CD4+CD8- thymocytes were unable to undergo chemotaxis towards the chemokines CCL19 and CCL21. CD3medium thymocytes of both CD4+CD8- and CD4-CD8+ subsets were also considerably more responsive than CD3high SP cells to apoptotic signals induced in vitro by ligation of CD95 (Fas/APO-1) or by dexamethasone. In both SP subsets, a higher frequency of thymocytes expressing forbidden Vbeta+ T cell receptors reactive with endogenous mammary tumor virus superantigens was found in CD3medium subpopulations than in CD3high subpopulations. These findings argue that the CD3medium SP thymocyte subpopulations contain apoptosis-susceptible precursor cells of CD3high SP cells and are subject to negatively selecting pressures.     

Modulation of T-cell differentiation in murine fetal thymus organ cultures

The effects of IL-1, IL-2, and a panel of monoclonal antibodies to thymic stroma and/or thymocytes, on T-cell differentiation in murine fetal thymus organ cultures were followed. Day-14 fetal thymic lobes were cultured for up to 12 days in the presence of IL-1 and/or IL-2 at concentrations of 100 U/ml. Development of all the major subpopulations defined by CD4 and CD8 expression was inhibited by IL-2, however the degree of inhibition was greatest for CD4+CD8- and CD4+CD8+ subsets. IL-1 alone caused only minor shifts in the subpopulations, but when added together with IL-2 the inhibitory effects of IL-2 were markedly enhanced. Analyses of subsets of CD4-CD8- cells demonstrated that the inhibition was most dramatic at the IL-2R positive and subsequent stages of CD4-CD8- differentiation. Interestingly, the putative precursors of IL-2R+CD4-CD8- increased in the presence of IL-2. In preliminary studies the organ culture system was used to examine the effects of a panel of antibodies to thymocytes and/or thymic stromal cells. Out of 14 antibodies tested, MTS 35 and MTS 37 have caused relative increases in the CD8 and CD4 single-positives, respectively. Both antibodies also induced increases in the percentages of CD4-CD8- cells and decreases in the percentages of CD4+CD8+ cells.     

Cell death and thymic export during fetal life

In the fetus the peripheral T cell pool expands as the fetus grows, but the mechanisms that regulate T cell homeostasis during fetal life are unknown. Here, we show that the peripheral T cell pool in the sheep fetus is established by the export from the fetal thymus of twice as many CD8+ as CD4+ thymic emigrants every day. Clonal deletion of CD4+ thymocytes in the fetal thymus appeared to be more stringent than was the case for CD8+ thymocytes because only 1 in 35 single-positive CD4 (SPCD4) thymocytes was exported from the thymus whereas the majority (2/3) of the single-positive CD8 (SPCD8) thymocytes were exported from the fetal thymus each day. Furthermore, within the thymus, the number of apoptotic SPCD4 thymocytes was 40 times greater than the number of apoptotic SPCD8 thymocytes. A tissue-specific migration of CD8+ emigrants localizing in the spleen was also established in the fetus in contrast to CD4+ emigrants, which migrated randomly to spleen and LN.     

Different subpopulations of developing thymocytes are associated with adherent (macrophage) or nonadherent (dendritic) thymic rosettes.

Thymic rosettes (ROS), structures consisting of thymic lymphoid cells attached to a central stromal cell, were isolated from mouse thymus by collagenase digestion and unit-gravity elutriation. The ROS were then separated into those where the stromal cells were either macrophage-like (M-ROS) or dendritic cell-like (D-ROS), on the basis of the differences in adherence properties or in the level of MAC-1 surface antigen. The ROS were then dissociated and the thymocyte content analyzed by immunofluorescent staining and flow cytometry. M-ROS and D-ROS differed in thymocyte composition, although the major component of both was the CD4+CD8+ cortical thymocyte. D-ROS were enriched in thymocytes expressing high levels of surface T-cell antigen receptor (TcR) and the associated CD3 complex, and these included both CD4+CD8-CD3++ and CD4-CD8+CD3++ mature thymocytes. M-ROS were enriched in CD4-CD8- thymocytes and had a reduced content of thymocytes expressing high TcR-CD3 levels; they nevertheless contained some mature thymocytes, but only of the CD4+CD8-CD3++ category. Several lines of evidence indicated that the mature thymocytes in ROS were cells recently formed in the cortex, and were not from the medullary pool. ROS-associated mature thymocytes expressed lower levels of H-2K than free, mature thymocytes. The CD4+CD8+CD3++ subpopulation, believed to be a developmental intermediate between cortical thymocytes and mature T cells, was present in both ROS populations. Further, late intermediates leading to both mature T-cell categories were evident in D-ROS, but only those leading to CD4+CD8-CD3++ T cells were evident in M-ROS. The results are compatible with a role for ROS in TcR-specificity selection and in the final maturation steps in the thymic cortex.     

T cell receptor-negative thymocytes from SCID mice can be induced to enter the CD4/CD8 differentiation pathway

In order to investigate the role of T cell receptor (TcR) expression in thymocyte maturation, we have analyzed thymocytes from C.B-17/SCID mice, which are unable to productively rearrange their antigen receptor genes and fail to express TcR. Despite this defect, SCID thymocytes are functional as they produce lymphokines and proliferate in response to a variety of stimuli. Phenotypic analysis revealed that thymocyte populations from young adult SCID mice resemble thymocyte populations from normal embryonic mice in that they are large, Thy-1.2+, CD4-, CD8-, TcR- and enriched in CD5lo, IL2R+ and Pgp1+ cells. However, other TcR- populations normally present in adult mice (i.e., CD4-CD8+ cells and CD4+CD8+ cells) are absent from the thymus of TcR- adult SCID mice. To understand the basis of the developmental arrest of TcR- SCID thymocytes at the CD4-CD8- stage of differentiation, we analyzed thymi from the occasional "leaky" SCID mouse which possesses small numbers of TcR+ thymocytes. We found that the presence of TcR+ cells within a SCID thymus was invariably associated with the presence of CD4+ and/or CD8+ SCID thymocytes. Interestingly, however, the CD4+/CD8+ SCID thymocytes were not themselves necessarily TcR+. That is, emergence of SCID thymocytes expressing CD4/CD8 was tightly linked to the presence of TcR+ cells within that SCID thymus, but the SCID thymocytes that expressed CD4/CD8 were not necessarily the same cells that expressed TcR. Finally, we found that the introduction into TcR- SCID mice of normal bone marrow cells that give rise to TcR+ cells within the SCID thymus promoted the differentiation of SCID thymocytes into CD4-CD8+ and CD4+CD8+ TcR- cells. These data indicate that TcR+ cells within the thymic milieu provide critical signals which promote entry of CD4-CD8-TcR- precursor T cells into the CD4/CD8 differentiation pathway. When applied to differentiation of normal thymocytes, these findings may imply a critical role for early appearing CD4-CD8- TcR (gamma/delta)+ cells in initiating normal thymic ontogeny.     

Human intrathymic T cell differentiation

The human thymus develops early on in fetal gestation with morphologic maturity reached by the beginning of the second trimester. Endodermal epithelial tissue from the third pharyngeal pouch gives rise to TE3+ cortical thymic epithelium while ectodermal epithelial tissue from the third pharyngeal cleft invaginates and splits during development to give rise to A2B5/TE4+ medullary and subcapsular cortical thymic epithelium. Fetal liver CD7+ T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression on thymocytes of other T lineage surface molecules. Human thymic epithelial cells grown in vitro bind to mature and immature thymocytes via CD2 and CD11a/CD18 (LFA-1) molecules on thymocytes and by CD58 (LFA-3) and CD54 (ICAM-1) molecules on thymic epithelial cells. Thymic epithelial cells produce numerous cytokines including IL1, IL6, G-CSF, M-CSF, and GM-CSF--molecules that likely are important in various stages of thymocyte activation and differentiation. Thymocytes can be activated via several cell surface molecules including CD2, CD3/TCR, and CD28 molecules. Finally, CD7+ CD4-CD8- CD3- thymocytes give rise to T cells of both the TCRab+ and TCR gd+ lineages.     

CD45RA antibodies split the CD3bright T cell subset.

Thymocyte subsets have been well characterized on the basis of CD4 and CD8 antigen expression. Recently, the use of anti-CD3 antibodies has allowed more precise phenotyping of these subsets. The most immature T cell precursors are largely CD3-CD4-CD8-, while the most mature are CD3brightCD4+CD8- or CD3brightCD4-CD8+. Moreover, the expression of CD45RA on thymocytes appears to define a progenitor population and may define a continuous lineage of cells. Using a panel of CD45RA antibodies, we have further characterized the CD45RA+ thymocyte population in the murine system. The size of this subset is greatly enhanced in cortisone-treated mice and in sublethally irradiated mice. Moreover, the CD45RA+ population is present early in foetal life and is maintained thereafter. Using three-colour immunofluorescence, we show that (i) while most CD45RA+ cells are present amongst the CD4-CD8- thymocyte subset in the normal thymus, after cortisone treatment or irradiation, all four thymocyte subsets co-express significant amounts of CD45RA. This suggests that not only progenitor cells but also the mature population which can survive such manipulation are CD45RA+; and (ii) a large proportion of CD45RA+ cells are CD3bright and this subset is represented in the thymus at all stages of maturation tested. These data suggest that a proportion of TCR-gamma delta + CD3+ cells in the fetus as well as of TCR-alpha beta+ CD3+ cells in the adult co-express CD45RA.     

CD4+CD8+CD3high thymocytes appear transiently during ontogeny: evidence from phenotypic and functional studies.

During T cell development thymocyte subsets emerge in a defined order, reflective of their maturational stage. In this study we determined the timing of appearance of CD4+CD8+CD3high thymocytes during in vivo and in vitro embryonic development, and thymic reconstitution after cortisone treatment. In these models, CD4+CD8+CD3high cells followed CD4+CD8+CD3low and preceded mature CD4+CD8-CD3high/CD4-CD8+CD3high thymocytes, while cortisone resistance was first seen among CD4+CD8+CD3high cells. CD4+CD8+CD3high thymocytes were also shown to display a pattern of antigen receptor-mediated calcium influx intermediate between that induced in other CD4+CD8+ cells and mature thymocytes. These results are consistent with a precursor-progeny relationship between CD4+CD8+CD3low and CD4+CD8+CD3high thymocytes, the latter developing to mature thymocytes (Hugo, P. et al., Int. Immunol. 1991. 3: 265).     

Alterations in the thymocyte phenotype of EphB-deficient mice largely affect the double negative cell compartment

In the present study, we have analysed the phenotype of EphB2 and/or EphB3 deficient thymocytes confirming and extending previous studies on the role of this family of molecules in T-cell differentiation. In all mutant thymuses statistically significant reduced cell contents were observed. This reduction of thymic cellularity correlated with increased proportions of apoptotic cells, largely both double negative (DN; CD4- CD8-) and double positive (CD4+ CD8+) cells, and decreased proportions of DN cycling cells. Adult deficient thymuses also showed increased proportions of DN cells but not significant variations in the percentages of other thymocyte subsets. In absolute terms, the thymocyte number decreased significantly in all thymocyte compartments from the DN3 (CD44- CD25+) cell stage onward, without variations in the numbers of both DN1 (CD44+ CD25-) and DN2 (CD44+ CD25+) cells. Remarkably, all these changes also occurred from the 15-day fetal EphB2 and/or EphB3 deficient mice, suggesting that adult phenotype results from the gradual accumulations of defects appearing early in the thymus ontogeny. As a reflection of thymus condition, a reduction in the number of T lymphocytes occurred in the peripheral blood and mesenteric lymph nodes, but not in spleen, maintaining the proportions of T-cell subsets defined by CD4/CD8 marker expression, in all cases.     

Involvement of CCR9 at multiple stages of adult T lymphopoiesis

The chemokine CCL25 is constitutively expressed in the thymus, and its receptor CCR9 is expressed on subsets of developing thymocytes. Nevertheless, the function of CCL25/CCR9 in adult thymopoiesis remains unclear. Here, we demonstrate that purified CCR9(-/-) hematopoietic stem cells are deficient in their ability to generate all major thymocyte subsets including double-negative 1 (DN1) cells in competitive transfers. CCR9(-/-) bone marrow contained normal numbers of lineage(-) Sca-1+c-kit+, common lymphoid progenitors, and lymphoid-primed multipotent progenitors (LMPP), and CCR9(-/-) LMPP showed similar T cell potential as their wild-type (WT) counterparts when cultured on OP9-delta-like 1 stromal cells. In contrast, early thymic progenitor and DN2 thymocyte numbers were reduced in the thymus of adult CCR9(-/-) mice. In fetal thymic organ cultures (FTOC), CCR9(-/-) DN1 cells were as efficient as WT DN1 cells in generating double-positive (DP) thymocytes; however, under competitive FTOC, CCR9(-/-) DP cell numbers were reduced significantly. Similarly, following intrathymic injection into sublethally irradiated recipients, CCR9(-/-) DN cells were out-competed by WT DN cells in generating DP thymocytes. Finally, in competitive reaggregation thymic organ cultures, CCR9(-/-) preselection DP thymocytes were disadvantaged significantly in their ability to generate CD4 single-positive (SP) thymocytes, a finding that correlated with a reduced ability to form TCR-MHC-dependent conjugates with thymic epithelial cells. Together, these results highlight a role for CCR9 at several stages of adult thymopoiesis: in hematopoietic progenitor seeding of the thymus, in the DN-DP thymocyte transition, and in the generation of CD4 SP thymocytes.