Abnormal thymocyte subset distribution and differential reduction of CD4+ and CD8+ T cell subsets during peripheral maturation in diabetes-prone BioBreeding rats

In this study we quantified CD8+ and CD4+ T cells in T lymphocytopenic BB rats as compared with control rats at given stages along the maturational pathway from immature thymocytes to mature peripheral T cells. Our results show that BB rats exhibit abnormal thymocyte subset distribution. Numbers of mature TCRhigh/CD4-8+ thymocytes, and also their TCRhigh/CD4+8+ precursors were decreased, as were levels of CD8 expression on all thymocyte subsets investigated. By analogy with mouse thymocyte development, these findings suggest a decreased efficiency for positive selection of CD8 precursors in BB rats. Furthermore, as related to the number of available mature TCRhigh single positive thymocytes, numbers of CD4+ and CD8+ T cells most recently migrated from the thymus were severely decreased in BB blood, indicating either reduced thymic output or rapid cell death after migration. Subsequently, in peripheral blood and cervical lymph nodes, a 95% decrease of CD8+ and a 50 to 80% decrease of CD4+ T cells were demonstrated upon maturation from recent thymic migrants to mature peripheral T cells, leaving the BB rat with a severely reduced T cell population, consisting of CD4+ T cells and a minute population of CD8+ T cells. The vast majority of the latter was found to have an immature peripheral phenotype. Possible consequences of our findings for the generation of autoreactive CD8+ T cells are discussed.     

Human natural killer cell committed thymocytes and their relation to the T cell lineage

Recent studies have demonstrated that mature natural killer (NK) cells can be grown from human triple negative (TN; CD3-, CD4-, CD8-) thymocytes, suggesting that a common NK/T cell precursor exists within the thymus that can give rise to both NK cells and T cells under appropriate conditions. In the present study, we have investigated human fetal and postnatal thymus to determine whether NK cells and their precursors exist within this tissue and whether NK cells can be distinguished from T cell progenitors. Based on the surface expression of CD56 (an NK cell-associated antigen) and CD5 (a T cell-associated antigen), three phenotypically distinctive populations of TN thymocytes were identified. CD56+, CD5-; CD56-, CD5-, and CD56-, CD5+. The CD56+, CD5- population of TN thymocytes, although displaying a low cytolytic function against NK sensitive tumor cell targets, were similar in antigenic phenotype to fetal liver NK cells, gave rise to NK cell clones, and were unable to generate T cells in mouse fetal thymic organ cultures (mFTOC). This population of thymocytes represents a relatively mature population of lineage-committed NK cells. The CD56-, CD5- population of TN thymocytes were similar to thymic NK cells in antigenic phenotype and NK cell clonogenic potential. Clones derived from this population of TN thymocytes acquired CD56 surface expression and NK cell cytolytic function. CD56-, CD5- TN thymocytes thus contain a novel population of NK cell-committed precursors. The CD56-, CD5- population of TN thymocytes also contains a small percentage of CD34+ cells, which demonstrate no in vitro clonogenic potential, but possess T cell reconstituting capabilities in mFTOC. The majority of TN thymocytes do not express CD56, but coexpress CD34 and CD5. These CD56-, CD5+, CD34+ cells demonstrate no NK or T cell clonogenic potential, but are extremely efficient in repopulating mFTOC and differentiating into CD3+, CD4+, CD8+ T cells. The results of this investigation have identified NK cells and NK cell precursors in the human thymus and have shown that these cell types are unable to differentiate along the T cell lineage pathway. Thus, while a common NK/T cell progenitor likely exists, once committed to the NK cell lineage these cells no longer have the capacity to develop along the T cell developmental pathway.     

CD4+ CD8+ thymocytes are preferentially induced to die following CD45 cross-linking, through a novel apoptotic pathway

Ligation of the protein tyrosine phosphatase CD45 on both mature and immature T cells modulates the amplitude of TCR-mediated signals. In this work, we have evaluated the consequences of CD45 ligation on immature T cells, in the absence of TCR engagement. Cross-linking of CD45 on thymocytes by mAbs led to the induction of cellular death, characterized by a reduction in mitochondrial membrane potential (delta psi(m)), production of reactive oxygen species, loss in membrane asymmetry, exposure of phosphatidylserine residues, and incorporation of vital dyes. In sharp contrast to most stimuli causing thymocyte death, CD45 cross-linking did not lead to DNA degradation. Cell death was not blocked by Bcl-2 overexpression or treatment with caspase inhibitor. However, death was inhibited by the addition of scavengers of reactive oxygen species. We also established that susceptibility to CD45-mediated death is acquired during the transition of early CD4- CD8- TCR- T cell precursors into CD4+ CD8+ TCR- thymocytes and is increased with further acquisition of surface TCR on these cells. Moreover, mature thymocytes were much less sensitive to CD45 cross-linking than CD4+ CD8+ cells. We propose that during T cell development, CD45 ligation could induce the death of those immature thymocytes that do not fulfill the requirements for positive selection.     

Comparisons of endothelial cell G- and F-actin distribution in situ and in vitro

Precursor of T lymphocytes undergo proliferation and maturation under the influence of the thymic microenvironment. In our study, we have attempted to determine the distribution of human postnatal thymocytes in division according to their stage of differentiation. Our data show that about 11.5% of all thymic cells are in S/G2/M phases, and that a subset of the cortical and precortical subpopulations contains most of the dividing cells. Rate of cell division is maintained at high levels from the prethymocyte precursor along the successive stages of differentiation represented by CD1+CD3-CD4-CD8- and CD1+CD3-CD4+CD8- cells. The percentage of dividing cells is maximal in an intermediate subset of CD1+CD3-CD4+CD8-CD45RO+ cells defined by the distinct expression of class I HLAdim/high molecules, which could contain cells in transit from prethymocytes to double-positive cortical cells. The CD3- fraction of the double-positive cortical cells contains most of the dividing thymocytes, although the rate of division within this subset is much less than that of the precursor CD1+CD3-CD4+CD8- cells. In a linear scheme of differentiation, cell division stops at or near the point of initiation of CD3 expression. These results suggest that in human thymus cell expansion takes place before the initiation of the positive selection process. According to this view the stringency of the selection process would require the previous generation of a large number of precursors to permit the production of sufficient numbers of mature T cells.     

Cyclosporin A inhibits the decrease of CD4/CD8 expression induced by protein kinase C activation

Cyclosporin A (CsA) is a powerful immunosuppressive drug widely used in transplantation medicine. A major effect of CsA is inhibition of the differentiation of immature double-positive (DP) CD4+ CD8+ thymocytes into mature single-positive (SP) CD4+ CD8- or CD4- CD8+ thymocytes. The mechanisms underlying the changes in CD4/CD8 expression during normal differentiation of thymocytes and the way CsA interferes with this differentiation process are still unknown. Here we show that protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) causes a decrease of both CD4 and CD8 expression at the cell surface level and at the mRNA level in a CD4+ CD8+ T cell line and in freshly isolated thymocytes. A PKC inhibitor, staurosporin, interferes with the differentiation from DP to SP in fetal thymus organ culture system. These data suggest that the alternation of CD4/CD8 expression from DP to SP is dependent on PKC activation. CsA blocks this decrease of CD4/CD8 expression by PMA in vitro. Moreover, this PMA effect is also blocked by treatment with cycloheximide. These results suggest that the reduction of CD4/CD8 expression requires de novo synthesis of a protein(s) induced in response to a signal conveyed by activated PKC. CsA may block the transition from DP to SP by inhibition of CD4/CD8 down-regulation induced by PKC activation.     

CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes

Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti-CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death.     

Linomide enhances apoptosis in CD4+CD8+ thymocytes

Linomide, a quinoline-3-carboxamide, has a pleiotropic immune modulating capacity and inhibits development as well as progression of disease in animal models of autoimmunity. Linomide treatment of mice resulted in a dramatic, dose-dependent decrease of the thymic cell number shortly after the start of administration. Flow cytometric analysis revealed that the major thymocyte subset, the early immature type CD4+CD8+ thymocytes, were reduced in number by 75%, mature CD4+CD8- or CD4-CD8+ thymocytes were less sensitive to treatment. The polyclonal T cell activator Con A (Concanavalin A) was used together with IL-2 to evaluate the potential proliferative responsiveness of ex vivo thymocytes. Thymocytes from mice treated with Linomide exhibited a more vigorous proliferation than control cultures. An effect shown to not only be due to the enrichment of mature thymocytes in the cultures from Linomide treated animals, but also when purified, mature thymocytes (CD4+CD8- and CD4-CD8+) were cultured with Con A and IL-2, these cells responded with a significantly enhanced proliferation. In vivo Linomide treatment did not result in increased plasma concentrations of corticosterone and treatment of adrenalectomized mice resulted in a reduction of thymocytes which was comparable to the effect in intact mice, indicating that glucocorticoids (GC) are not major mediators of Linomide-induced thymocyte deletion. In addition to this, and supporting a glucocorticoid independent mode of action, Linomide treatment of thymocytes in vitro resulted in a significant increase in the number of apoptotic cells, specifically in the CD4+CD8+ subset, implicating apopotosis as one component in the course of thymocyte reduction. In addition to this, in vivo treatment with Linomide resulted in an identical pattern to that seen in vitro in that there was significantly increased apoptosis only in the CD4+CD8+. These data indicate that Linomide modifies thymocyte development using a glucocorticoid independent pathway and results in the increased apoptosis of the CD4+CD8+ subset.     

Fibroblast dependency during early thymocyte development maps to the CD25+ CD44+ stage and involves interactions with fibroblast matrix molecules

We have investigated the role of specific components of the thymic stroma during development of CD4-8-T cell precursors by separating and reaggregating precursor subsets with individual or combinations of stromal cells. We show that while the development of CD25+ 44+ precursors is dependent upon a combination of major histocompatibility complex (MHC) class II+ thymic epithelial cells and fibroblasts, their direct descendants, CD25+ 44- precursors, develop to the CD4+ 8+ stage in the presence of MHC class II+ thymic epithelial cells alone. Thus, CD25+ 44+ precursors are the last developmental stage to be dependent upon fibroblast support. In addition, while metabolically inactive, 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide (ECDI)-treated fibroblasts retain the ability to promote T cell development, prior treatment with hyaluronidase abrogates this effect, suggesting that fibroblast-associated extracellular matrix components are the key elements involved. In support of this, we show that fibroblasts are located in cortical regions of the thymus where T cell precursors are known to reside, and that these fibroblasts are associated with an extensive extracellular matrix not found on thymic epithelial cells. Finally, antibodies to alpha 4 integrin and CD44 interfere with the efficiency with which CD4+ 8+ cells are generated from CD25+ 44+ precursors in reaggregate cultures and also reduce the binding of the latter to 3T3 fibroblasts, suggesting these molecules play a role in bringing T cell precursors into contact with fibroblast-associated extracellular matrix.     

Calnexin association is not sufficient to protect T cell receptor alpha proteins from rapid degradation in CD4+CD8+ thymocytes

During T cell development, assembly of the mutisubunit T cell receptor (TCR) complex is regulated by the differential stability of newly synthesized TCRalpha molecules, having a half-life of approximately 20 min in immature CD4+CD8+ thymocytes compared with >75 min in mature T cells. The molecular basis for TCRalpha instability in CD4+CD8+ thymocytes is unknown but has been postulated to involve abnormalities in N-glycan processing and calnexin assembly as perturbation of these pathways markedly destabilizes TCRalpha proteins in all other T cell types examined. Here, we compared the processing of TCRalpha glycoproteins and their assembly with calnexin and calreticulin chaperones in CD4+CD8+ thymocytes and splenic T cells. These studies show that TCRalpha glycoproteins synthesized in CD4+CD8+ thymocytes were processed in a similar manner as those made in splenic T cells and that TCRalpha proteins stably associated with calnexin in both cell types. Interestingly, however, TCRalpha association with the calnexin-related molecule calreticulin was decreased in CD4+CD8+ thymocytes compared with splenic T cells. Finally, TCRalpha degradation in CD4+CD8+ thymocytes was impaired by inhibitors of proteasome activity, which was correlated with stabilization of calnexin.TCRalpha complexes. These data demonstrate that calnexin association is not sufficient to protect TCRalpha proteins from rapid degradation in CD4+CD8+ thymocytes, suggesting that additional components of the quality control system of the endoplasmic reticulum operate to ensure the proper folding of nascent TCRalpha glycoproteins.     

The cytoplasmic domain of CD4 promotes the development of CD4 lineage T cells

Thymocytes must bind major histocompatibility complex (MHC) proteins on thymic epithelial cells in order to mature into either CD8+ cytotoxic T cells or CD4+ helper T cells. Thymic precursors express both CD8 and CD4, and it has been suggested that the intracellular signals generated by CD8 or CD4 binding to class I or II MHC, respectively, might influence the fate of uncommitted cells. Here we test the notion that intracellular signaling by CD4 directs the development of thymocytes to a CD4 lineage. A hybrid protein consisting of the CD8 extracellular and transmembrane domains and the cytoplasmic domain of CD4 (CD884) should bind class I MHC but deliver a CD4 intracellular signal. We find that expression of a hybrid CD884 protein in thymocytes of transgenic mice leads to the development of large numbers of class I MHC-specific, CD4 lineage T cells. We discuss these results in terms of current models for CD4 and CD8 lineage commitment.     

Thymic alterations in feline GM1 gangliosidosis

GM1 gangliosidosis is an inherited metabolic disease characterized by progressive neurological deterioration with premature death seen in children and numerous animals, including cats. We have observed that thymuses from affected cats greater than seven months of age (GM1 mutant cats) show marked thymic reduction compared to age-matched normal cats. The studies reported here were done to describe alterations in the thymus prior to (less then 90 days of age) and during the development of mild (90 to 210 days of age) to severe (greater than 210 days of age) progressive neurologic disease and to explore the pathogenesis of the thymic abnormality. Although histologic examination of the thymus from GM1 affected cats less than 210 days of age showed no significant differences from age-matched control cats, thymuses from GM1 mutant cats greater than 210 days of age were significantly reduced in size (approximately 3-fold). Histologic sections of lymph nodes, adrenal glands, and spleens from GM1 gangliosidosis-affected cats showed no significant differences. Flow cytometric analyses showed a marked decrease in the percentage of immature CD4+CD8+ thymocytes (p < 0.001) and significantly increased CD4-CD8+ cells (p < 0.01) in GM1 mutant cats greater than 210 days of age when compared to normal age matched cats. Co-labelling with CD4, CD8, and CD5 indicated an increase in the percentage of GM1 mutant cat thymocytes at this age which were CD5high, suggesting the presence of more mature cells. Cytometric analyses of subpopulations of peripheral lymphocytes indicated an increase in CD4-CD8+ cells (p < 0.05) with concurrent decreases in CD4+CD8- and CD4-CD8- cells (which were not significant). Similar analyses of thymocyte and lymphocyte subpopulations from cats < 210 days of age showed no significant differences between GM1 mutant and normal cells. GM1 mutant cats at all ages had increased surface binding of Cholera toxin B on thymocytes, indicating increased surface GM1 ganglioside expression. Increases were highly significant in GM1 mutant cats greater than 210 days of age. In situ labelling for apoptosis was increased in GM1 mutant cats between 90 to 200 days of age when thymic masses were within normal limits. In GM1 mutant cats over 200 days of age, decreased labelling was observed when thymic mass was reduced and the CD4+CD8+ subpopulation, known to be very susceptible to apoptosis, was significantly decreased. These data describe premature thymic involution in feline GM1 gangliosidosis and suggest that increased surface GM1 gangliosides alters thymocyte development in these cats.     

Interactive effects of cocaine and gender on thymocytes: a study of in vivo repeated cocaine exposure

This study used a mouse model including both sexes to assess the impact of repeat cocaine exposure on the differentiation and function of T cell in thymus. Cocaine hydrochloride in 0.9% saline, 5 mg or 40 mg/kg, was administrated by i.p. injection to C57BL/6 mice for 10 days. Thymocytes were obtained 24 h after the 10th injection. Repeat in vivo cocaine exposure inhibited the proliferation of T lymphocytes in response to Con-A and Con-A plus anti-CD28. The proliferation induced by IL-2 in the Con-A stimulated T blasts was attenuated in cocaine treated mice. These effects were seen at a lower cocaine dose in female mice. The total number of thymocytes was reduced. Although the percentage of mature thymocytes (CD4+ CD8- and CD4- CD8+ cells) was not altered, the absolute cell numbers were attenuated. Both percentage and absolute cell number of immature thymocytes (CD4+ CD8+) decreased and the pre-mature (CD4- CD8-) cells increased. CD28 and CD25 expression were attenuated in Con-A stimulated thymocytes of mice treated with cocaine at 40 mg/kg. Interleukin 2 production was not significantly altered, however, gamma-IFN production was decreased by cocaine exposure at 40 mg/kg. In conclusion, cocaine exerts inhibitory effects on the function of mature thymocytes, and on the differentiation of thymocytes. A gender difference in response to cocaine was noted in that female mice were more sensitive to lower dose of cocaine exposure.     

Natural killer cell development and function precede alpha beta T cell differentiation in mouse fetal thymic ontogeny

Natural killer (NK) cells mediate MHC-unrestricted cytolysis of virus-infected cells and tumor cells. In the adult mouse, NK cells are bone marrow-derived lymphocytes that mature predominantly in extrathymic locations but have also been suggested to share a common intrathymic progenitor with T lymphocytes. However, mature NK cells are thought to be absent in mouse fetal ontogeny. We report the existence of thymocytes with a mature NK cell phenotype (NK1.1+/CD117-) as early as day 13 of gestation, approximately 3 days before the appearance of CD4+/CD8+ cells in T lymphocyte development. These mature fetal thymic NK cells express genes associated with NK cell effector function and, when freshly isolated, display MHC-unrestricted cytolytic activity in vitro. Moreover, the capacity of fetal thymic NK cells for sustained growth both in vitro and in vivo, in addition to their close phenotypic resemblance to early precursor thymocytes, confounds previous assessments of NK lineage precursor function. Thus, mature NK cells may have been inadvertently included in previous attempts to identify multipotent and bipotent precursor thymocytes. These results provide the first evidence of functional NK lymphocytes in mouse fetal ontogeny and demonstrate that NK cell maturation precedes alpha beta T cell development in the fetal thymus.     

Central T cell tolerance in lupus-prone mice: influence of autoimmune background and the lpr mutation

Lupus-prone mice develop a systemic autoimmune disease that is dependent upon the B cell help provided by autoreactive alphabeta CD4+ T cells. Since autoreactive T cells with high affinity for self peptides are normally deleted in the thymus, their presence in these mice suggests the possibility that intrathymic negative selection may be defective. Here, we directly compared central T cell tolerance in response to a conventional peptide Ag in lupus-prone MRL/MpJ mice with a nonautoimmune strain using an MHC class II-restricted TCR transgene. Our results did not demonstrate any defects after Ag exposure in the induction of intrathymic deletion of immature CD4+ CD8+ thymocytes, in TCR down-regulation, or in the number of apoptotic thymocytes in MRL/MpJ compared with nonautoimmune mice. Furthermore, we found that the lpr mutation had no influence upon the Ag-induced thymic deletion of immature thymocytes. These data support the notion that T cell autoreactivity in MRL/MpJ mice is caused by defects in peripheral control mechanisms.     

Frequency analysis of the T cell precursors in the thymus

A frequency determination of the T cell precursors in murine adult and fetal thymuses as well as in the bone marrow and fetal liver was made. Cells were serially diluted and injected into deoxyguanosine-treated fetal thymus lobes with a microinjector, and the lobes were cultured for 12 to 21 days. The lobes in which T cell development did not occur were discriminated from those in which T cells developed, and the precursor frequency was determined by Poisson probability distribution analysis. The precursor cell frequencies in adult bone marrow cells (2.4 x 10(-5)) and fetal liver cells (1.7 x 10(-4)) were comparable to those determined previously in in vivo intrathymus transfer experiments. The present study further shows that only a small fraction of fetal thymus cells (0.9-5.0 x 10(-2)), CD4-8- adult thymocytes (1.6 x 10(-2)), and Thy-1 low positive adult thymocytes (3.3 x 10-4)) retain the precursor activity.     

Expansion of mature thymocyte subsets before emigration to the periphery

A small population of DNA-synthesizing mature thymocytes could be defined by analyzing cell surface markers and 5-bromo-2'-deoxyuridine (BrdUrd) labeling by four-color cytofluorometry. These cells have a completely mature phenotype (CD4- CD8+ or CD4+ CD8- TCR(high), HSA-, Qa-2(high)) and expand only weakly after BrdUrd incorporation. They recovered immediately in total number and in DNA synthesis rate after treatment with the antimitotic drug demecolcin, thus much faster than immature CD4+ CD8+ thymocytes. These data demonstrate the existence of a late intrathymic expansion phase, independent of that of developing CD4+ CD8+ immature cells, and involving phenotypically mature cells renewed each day. In mixed chimeras prepared by transfer of bone marrow and lymph node cells into RAG-2(-/-) mice, all cycling mature thymocytes were bone marrow derived. They are thus produced in situ and do not correspond to peripheral T cells reentering the thymus. Double FITC/BrdUrd detection showed that a high proportion (10-20%) of recent thymic emigrants were BrdUrd+ just postcycling cells and that around 50% of cycling mature thymocytes are just ready to emigrate to the periphery in the few hours after DNA synthesis. The late intrathymic expansion phase demonstrated here increases the daily thymic cell export by at least 30%. It could play a role in the adjustment of the T cell repertoire before emigration and in the regulation of the thymic cell output into the peripheral T cell pool.     

Characterization of CD34+ thymic stromal cells located in the subcapsular cortex of the human thymus

In this paper we report that suspensions of human fetal thymocytes contain cells that express high levels of CD34 and Thy-1. These cells were characterized with regard to location within the thymus, phenotype, and function. Confocal laser scan analysis of frozen sections of fetal thymus with anti-CD34 and Thy-1 antibodies revealed that the double-labeled cells were located in the pericortical area. In addition, it was found that the CD34+Thy-1+ cells lacked CD45 and CD50, indicating that these cells are not of hematopoietic origin; this was confirmed by the finding that these cells could be cultured as adherent cells in a medium with cholera toxin and dexamethasone, but failed to grow in mixtures of hematopoietic growth factors. Further analysis indicated that most cultured CD34+Thy-1+ cells expressed cytokeratin (CK) 14 but lacked CK 13, suggesting that these cells are immature epithelial cells. Cultured CD34+Thy-1+ cells were able to induce differentiation of CD1-CD34+CD3-CD4-CD8- thymic precursors into CD4+CD8+ cells in a reaggregate culture in the absence of exogenous cytokines. The CD4+CD8+ cells that developed in these cultures did not express CD3, indicating that CD34+Thy-1+ thymic stromal cells are not capable of completing full T cell differentiation of thymic hematopoietic progenitor cells.     

CXCR4 and CCR5 on human thymocytes: biological function and role in HIV-1 infection

Thymocyte infection with HIV-1 is associated with thymic involution and impaired thymopoiesis, particularly in pediatric patients. To define mechanisms of thymocyte infection, we examined human thymocytes for expression and function of CXCR4 and CCR5, the major cell entry coreceptors for T cell line-tropic (T-tropic) and macrophage-tropic (M-tropic) strains of HIV-1, respectively. CXCR4 was detected on the surface of all thymocytes. CXCR4 expression on mature, high level TCR thymocytes was similar to that on peripheral blood T cells, but was much lower than that on immature thymocytes, including CD34+ thymic progenitors. Consistent with this, stroma-derived factor-1 (SDF-1) induced calcium flux primarily in immature thymocytes, with CD34+ progenitors giving the strongest response. In addition, SDF-1 mRNA was detected in thymic-derived stromal cells, and SDF-1 induced chemotaxis of thymocytes, suggesting that CXCR4 may play a role in thymocyte migration. Infection of immature thymocytes by the T-tropic HIV-1 strain LAI was 10-fold more efficient than that in mature thymocytes, consistent with their relative CXCR4 surface expression. Anti-CXCR4 antiserum or SDF-1 blocked fusion of thymocytes with cells expressing the LAI envelope. In contrast to CXCR4, CCR5 was detected at low levels on thymocytes, and CCR5 agonists did not induce calcium flux or chemotaxis in thymocytes. However, CD4+ mature thymocytes were productively infected with the CCR5-tropic strain Ba-L, and this infection was specifically inhibited with the CCR5 agonist, macrophage inflammatory protein-1beta. Our data provide strong evidence that CXCR4 and CCR5 function as coreceptors for HIV-1 infection of human thymocytes.