Thymic anlage is colonized by progenitors restricted to T, NK, and dendritic cell lineages

It remains controversial whether the thymus-colonizing progenitors are committed to the T cell lineage. A major problem that has impeded the characterization of thymic immigrants has been that the earliest intrathymic progenitors thus far identified do not necessarily represent the genuine thymic immigrants, because their developmental potential should have been influenced by contact with the thymic microenvironment. In the present study, we examined the developmental potential of the ontogenically earliest thymic progenitors of day 11 murine fetus. These cells reside in the surrounding mesenchymal region and have not encountered thymic epithelial components. Flow cytometric and immunohistochemical analyses demonstrated that these cells are exclusively Lin(-)c-kit(+)IL-7R(+). Limiting dilution analyses disclosed that the progenitors with T cell potential were abundant, while those with B cell potential were virtually absent in the region of day 11 thymic anlage. Clonal analyses reveled that they are restricted to T, NK, and dendritic cell lineages. Each progenitor was capable of forming a large number of precursors that may clonally accommodate highly diverse TCRbeta chains. These results provide direct evidence that the progenitors restricted to the T/NK/dendritic cell lineage selectively immigrate into the thymus.     

Prethymic T-cell development defined by the expression of paired immunoglobulin-like receptors

T cells are produced in the thymus from progenitors of extrathymic origin. As no specific markers are available, the developmental pathway of progenitors preceding thymic colonization remains unclear. Here we show that progenitors in murine fetal liver and blood, which are capable of giving rise to T cells, NK cells and dendritic cells, but not B cells, can be isolated by their surface expression of paired immunoglobulin-like receptors (PIR). PIR expression is maintained until the earliest intrathymic stage, then downregulated before the onset of CD25 expression. Unlike intrathymic progenitors, generation of prethymic PIR(+) progenitors does not require Hes1-mediated Notch signaling. These findings disclose a prethymic stage of T-cell development programmed for immigration of the thymus, which is genetically separable from intrathymic stages.     

Characterization of developmental pathway of natural killer cells from embryonic stem cells in vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(-) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(-) EB cells showed that CD45(+)Mac-1(-)Ter119(-) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(-)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(-) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(-) and they rapidly acquire CD122 as they differentiate along the NK lineage.     

Generation of macrophages from early T progenitors in vitro

Early T progenitors in the thymus have been reported to have the capacity to develop into B cells, thymic dendritic cells, and NK cells. Here we describe conditions that induce early T progenitors to develop into macrophages. Initially, we observed that early T progenitors could be induced to develop into macrophages by cytokines produced from a thymic stromal cell line, TFGD, and later we found that the cytokine mixture of M-CSF plus IL-6 plus IL-7 also induced macrophage differentiation from pro-T cells. M-CSF by itself was unable to induce macrophage differentiation from early T progenitors. To correlate this observation with the developmental potential of early T progenitors, mouse embryonic thymocytes were sorted into four populations, pro-T1 to pro-T4, based on the expression of CD44 and CD25, and then cultured with TFGD culture supernatant. We found that pro-T1 and pro-T2 cells, but not pro-T3 and pro-T4 cells, generate macrophages. Limiting dilution analysis of the differentiation capability of sorted pro-T2 cells also confirmed that pro-T2 cells could generate macrophages. These results suggest that T cells and thymic macrophages could originate from a common intrathymic precursor.     

The earliest thymic progenitors in adults are restricted to T, NK, and dendritic cell lineage and have a potential to form more diverse TCRbeta chains than fetal progenitors

T cell progenitors in the adult thymus (AT) are not well characterized. In the present study, we show that the earliest progenitors in the murine AT are, like those in fetal thymus (FT), unable to generate B or myeloid cells, but still retain the ability to generate NK cells and dendritic cells. However, AT progenitors are distinct from those in FT or fetal liver, in that they are able to produce approximately 100 times larger numbers of T cells than progenitors in fetuses. Such a capability to generate a large number of T cells was mainly attributed to their potential to extensively proliferate before the TCRbeta chain gene rearrangement. We propose that the AT is colonized by T/NK/dendritic cell tripotential progenitors with much higher potential to form diversity in TCRbeta chains than FT progenitors.     

Clonal analysis reveals uniformity in the molecular profile and lineage potential of CCR9(+) and CCR9(-) thymus-settling progenitors

The entry of T cell progenitors to the thymus marks the beginning of a multistage developmental process that culminates in the generation of self-MHC-restricted CD4(+) and CD8(+) T cells. Although multiple factors including the chemokine receptors CCR7 and CCR9 are now defined as important mediators of progenitor recruitment and colonization in both the fetal and adult thymi, the heterogeneity of thymus-colonizing cells that contribute to development of the T cell pool is complex and poorly understood. In this study, in conjunction with lineage potential assays, we perform phenotypic and genetic analyses on thymus-settling progenitors (TSP) isolated from the embryonic mouse thymus anlagen and surrounding perithymic mesenchyme, including simultaneous gene expression analysis of 14 hemopoietic regulators using single-cell multiplex RT-PCR. We show that, despite the known importance of CCL25-CCR9 mediated thymic recruitment of T cell progenitors, embryonic PIR(+)c-Kit(+) TSP can be subdivided into CCR9(+) and CCR9(-) subsets that differ in their requirements for a functional thymic microenvironment for thymus homing. Despite these differences, lineage potential studies of purified CCR9(+) and CCR9(-) TSP reveal a common bias toward T cell-committed progenitors, and clonal gene expression analysis reveals a genetic consensus that is evident between and within single CCR9(+) and CCR9(-) TSP. Collectively, our data suggest that although the earliest T cell progenitors may display heterogeneity with regard to their requirements for thymus colonization, they represent a developmentally homogeneous progenitor pool that ensures the efficient generation of the first cohorts of T cells during thymus development.     

Human thymic epithelial cells inhibit IL-15- and IL-2-driven differentiation of NK cells from the early human thymic progenitors

T/NK progenitors are present in the thymus; however, the thymus predominantly promotes T cell development. In this study, we demonstrated that human thymic epithelial cells (TEC) inhibit NK cell development. Most ex vivo human thymocytes express CD1a, indicating that thymic progenitors are predominantly committed to the T cell lineage. In contrast, the CD1a(-)CD3(-)CD56(+) NK population comprises only 0.2% (n = 7) of thymocytes. However, we observed increases in the percentage (20- to 25-fold) and absolute number (13- to 71-fold) of NK cells when thymocytes were cultured with mixtures of either IL-2, IL-7, and stem cell factor or IL-15, IL-7, and stem cell factor. TEC, when present in the cultures, inhibited the increases in the percentage (3- to 10-fold) and absolute number (3- to 25-fold) of NK cells. Furthermore, we show that TEC-derived soluble factors inhibit generation of NK-CFU and inhibit IL15- or IL2-driven NK cell differentiation from thymic CD34(+) triple-negative thymocytes. The inhibitory activity was found to be associated with a 8,000- to 30,000 Da fraction. Thus, our data demonstrate that TEC inhibit NK cell development from T/NK CD34(+) triple negative progenitors via soluble factor(s), suggesting that the human thymic microenvironment not only actively promotes T cell maturation but also controls the development of non-T lineage cells such as the NK lineage.     

CCR7 ligands, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are chemoattractants for CD56(+)CD16(-) NK cells and late stage lymphoid progenitors

Two human CC chemokines, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are previously reported as efficacious chemoattractants for T- and B-cells and dendritic cells. SLC and CKbeta-11 share only 32% amino acid identity, but are ligands for the same chemokine receptor, CCR7. In this study, we examined chemotactic activity of SLC and CKbeta-11 for NK cells and lymphoid progenitors in bone marrow and thymus. It was found that these two CCR7 ligands are chemoattractants for neonatal cord blood and adult peripheral blood NK cells and cell lines. SLC and CKbeta-11 preferentially attract the CD56(+)CD16(-) NK cell subset over CD56(+)CD16(+) NK cells. SLC and CKbeta-11 also demonstrate selective chemotactic activity on late stage CD34(-)CD19(+)IgM- B-cell progenitors and CD4(+) and CD8(+) single-positive thymocytes, but not early stage progenitors. It was noted that SLC is an efficient desensitizer of CKbeta-11-dependent NK cell chemotaxis, while CKbeta-11 is a weak desensitizer of SLC-dependent chemotaxis. Taken together, these results suggest that SLC and CKbeta-11 have the potential to control trafficking of NK cell subsets and late stage lymphoid progenitors in bone marrow and thymus.     

A common pathway for dendritic cell and early B cell development

B cells and dendritic cells (DCs) each develop from poorly described progenitor cells in the bone marrow (BM). Although a subset of DCs has been proposed to arise from lymphoid progenitors, a common developmental pathway for B cells and BM-derived DCs has not been clearly identified. To address this possibility, we performed a comprehensive analysis of DC differentiative potential among lymphoid and B lymphoid progenitor populations in adult mouse BM. We found that both the common lymphoid progenitors (CLPs), shown here and elsewhere to give rise exclusively to lymphocytes, and a down-stream early B-lineage precursor population devoid of T and NK cell precursor potential each give rise to DCs when exposed to the appropriate cytokines. This result contrasts with more mature B-lineage precursors, all of which failed to give rise to detectable numbers of DCs. Significantly, both CLP and early B-lineage-derived DCs acquired several surface markers associated with functional DCs, and CLP-derived DCs readily induced proliferation of allogeneic CD4(+) T cells. Surprisingly, however, DC differentiation from both lymphoid-restricted progenitors was accompanied by up-regulation of CD11b expression, a cell surface molecule normally restricted to myeloid lineage cells including putative myeloid DCs. Together, these data demonstrate that loss of DC developmental potential is the final step in B-lineage commitment and thus reveals a previously unrecognized link between early B cell and DC ontogeny.     

Emergence of T cell progenitors without B cell or myeloid differentiation potential at the earliest stage of hematopoiesis in the murine fetal liver

It has been unclear whether the progenitors colonizing the thymus are multipotent or T cell lineage restricted. We investigated the developmental potential of hematopoietic progenitors in various populations of liver and blood cells from day 12 fetuses using the recently established in vitro experimental system effective in determining the capability of individual progenitors to generate T, B, and myeloid cells. Multipotent progenitors (p-Multi) were exclusively found in the Sca-1 high-positive (Sca-1high) subpopulation of lineage marker (Lin)-c-kit+CD45+ fetal liver cells. Restriction of developmental capacity begins at the Sca-1high stage, and a large majority of progenitors in the Sca-1low or Sca-1- population are restricted to generate T, B, or myeloid cells. Such a lineage commitment or restriction taking place in the fetal liver is independent of the thymus, because no difference in the proportion of different types of progenitors were seen between nu/nu and nu/+ fetuses. T cell lineage-restricted progenitors (p-T) were abundant in the blood of day 12 fetuses, whereas p-Multi were undetectable. It was further shown that the p-Multi generated a large number of B and myeloid cells in the thymic lobe. These results strongly suggest that it is p-T but not p-Multi that migrate into the thymus.     

Cutting edge: an essential role for Notch-1 in the development of both thymus-independent and -dependent T cells in the gut

Whereas most T cells arise in the thymus, a distinct lineage of extrathymically derived T cells is present in the gut mucosa. The developmental origin of extrathymic T cells is poorly understood. We show here that Notch-1, a transmembrane receptor involved in T cell fate specification of bipotential T/B precursors in the thymus, is absolutely required for the development of extrathymic (as well as thymus-derived) mature T cells in the intestinal epithelium. In the absence of Notch-1, CD117(+) T cell precursors are relatively more abundant in the gut than the thymus, whereas immature B cells accumulate in the thymus but not the gut. Collectively, these data demonstrate that Notch-1 is essential for both thymic and extrathymic T cell fate specification and further suggest that bipotential T/B precursors that do not receive a Notch-1 signal adopt a B cell fate in the thymus but become developmentally arrested in the gut.     

TCR gamma delta+ and CD161+ thymocytes express HIV-1 in the SCID-hu mouse,potentially contributing to immune dysfunction in HIV infection

The vast diversity of the T cell repertoire renders the adaptive immune response capable of recognizing a broad spectrum of potential antigenic peptides. However, certain T cell rearrangements are conserved for recognition of specific pathogens, as is the case for TCRgammadelta cells. In addition, an immunoregulatory class of T cells expressing the NK receptor protein 1A (CD161) responds to nonpeptide Ags presented on the MHC-like CD1d molecule. The effect of HIV-1 infection on these specialized T cells in the thymus was studied using the SCID-hu mouse model. We were able to identify CD161-expressing CD3(+) cells but not the CD1d-restricted invariant Valpha24/Vbeta11/CD161(+) NK T cells in the thymus. A subset of TCRgammadelta cells and CD161-expressing thymocytes express CD4, CXCR4, and CCR5 during development in the thymus and are susceptible to HIV-1 infection. TCRgammadelta thymocytes were productively infectable by both X4 and R5 virus, and thymic HIV-1 infection induced depletion of CD4(+) TCRgammadelta cells. Similarly, CD4(+)CD161(+) thymocytes were depleted by thymic HIV-1 infection, leading to enrichment of CD4(-)CD161(+) thymocytes. Furthermore, compared with the general CD4-negative thymocyte population, CD4(-)CD161(+) NK T thymocytes exhibited as much as a 27-fold lower frequency of virus-expressing cells. We conclude that HIV-1 infection and/or disruption of cells important in both innate and acquired immunity may contribute to the overall immune dysfunction seen in HIV-1 disease.     

IL-15Rα of radiation-resistant cells is necessary and sufficient for thymic invariant NKT cell survival and functional maturation

The development of invariant NKT (iNKT) cells depends on the thymus. After positive selection by CD4(+)CD8(+)CD1d(+) cortical thymocytes, iNKT cells proceed from CD44(low)NK1.1(-) (stage 1) to CD44(high)NK1.1(-) (stage 2), and then to CD44(high)NK1.1(+) (stage 3) cells. The programming of cytokine production occurs along the three differentiation stages, whereas the acquisition of NK receptors occurs at stage 3. Stage 3 thymic iNKT cells are specifically reduced in Il15ra(-/-) mice. The mechanism underlying this homeostatic deficiency and whether the IL-15 system affects other thymic iNKT cell developmental events remain elusive. In this study, we demonstrate that increased cell death contributed to the reduction of stage 3 cells in Il15ra(-/-) mice, as knockout of Bim restored this population. IL-15-dependent upregulation of Bcl-2 in stage 3 cells affected cell survival, as overexpression of hBcl-2 partially restored stage 3 cells in Il15ra(-/-) mice. Moreover, thymic iNKT cells in Il15ra(-/-) mice were impaired in functional maturation, including the acquisition of Ly49 and NKG2 receptors and the programming of cytokine production. Finally, IL-15Rα expressed by radiation-resistant cells is necessary and sufficient to support the survival as well as the examined maturation events of thymic iNKT cells.     

Functional and phenotypic analysis of thymic CD34+CD1a- progenitor-derived dendritic cells: predominance of CD1a+ differentiation pathway

Whether thymic dendritic cells (DC) are phenotypically and functionally distinct from the monocyte lineage DC is an important question. Human thymic progenitors differentiate into T, NK, and DC. The latter induce clonal deletion of autoreactive thymocytes and therefore might be different from their monocyte-derived counterparts. The cytokines needed for the differentiation of DC from thymic progenitors were also questioned, particularly the need for GM-CSF. We show that various cytokine combinations with or without GM-CSF generated DC from CD34+CD1a- but not from CD34+CD1a+ thymocytes. CD34+ thymic cells generated far fewer DC than their counterparts from the cord blood. The requirement for IL-7 was strict whereas GM-CSF was dispensable but nonetheless improved the yield of DC. CD14+ monocytic intermediates were not detected in these cultures unless macrophage-CSF (M-CSF) was added. Cultures in M-CSF generated CD14-CD1a+ DC precursors but also CD14+CD1a- cells. When sorted and recultured in GM-CSF, CD14+ cells down-regulated CD14 and up-regulated CD1a. TNF-alpha accelerated the differentiation of progenitors into DC and augmented MHC class II transport to the membrane, resulting in improved capacity to induce MLR. The trafficking of MHC class II molecules was studied by metabolic labeling and immunoprecipitation. MHC class II molecules were transported to the membrane in association with invariant chain isoforms in CD14+ (monocyte)-derived and in CD1a+ thymic-derived DC but not in monocytes. Thus, thymic progenitors can differentiate into DC along a preferential CD1a+ pathway but have conserved a CD14+ maturation capacity under M-CSF. Finally, CD1a+-derived thymic DC and monocyte-derived DC share very close Ag-processing machinery.     

Potential role of NKG2D/MHC class I-related chain A interaction in intrathymic maturation of single-positive CD8 T cells

The nonclassical MHC class I molecule MHC class I-related chain A (MICA) interacts with the NKG2D receptor expressed at the surface of most peripheral CD8 T cells, gammadelta T cells, and NK cells. We investigated the role of MICA-NKG2D interactions in the selection or maturation of the T cell repertoire within the thymus using MICA tetramers and anti-MICA mAbs. MICA tetramers identified a small population of late stage CD8 single-positive, CD45RA(+) CD62L(+) CCR7(+) CD69(-) thymocytes, a phenotype compatible with that of fully mature CD8(+) cells ready to emigrate to the periphery as naive cells. MICA molecules were expressed in the outer layer of Hassal's corpuscles within the medulla of normal thymus. In thymomas, an overexpression of MICA in cortical and medullar epithelial cells was observed. This was associated with a decreased percentage of NKG2D-positive thymocytes, which expressed a less mature phenotype than in normal thymus. These results indicate that CD8(+) thymocytes up-regulate NKG2D as they complete their developmental program before leaving the thymic medulla to seed the periphery, and identify NKG2D as a potential regulator of the developmental processes in T cells that are essential for immune homeostasis.     

Enhanced tumor responses to dendritic cells in the absence of CD8-positive cells

Wild-type mice immunized with MART-1 melanoma Ag-engineered dendritic cells (DC) generate strong Ag-specific immunity that has an absolute requirement for both CD8(+) and CD4(+) T cells. DC administration to CD8 alpha knockout mice displayed unexpectedly enhanced levels of protection to tumor challenge despite this deficiency in CD8(+) T cells and the inability to mount MHC class I-restricted immune responses. This model has the following features: 1) antitumor protection is Ag independent; 2) had an absolute requirement for CD4(+) and NK1.1(+) cells; 3) CD4(+) splenocytes are responsible for cytokine production; 4) lytic cells in microcytotoxicity assays express NK, but lack T cell markers (NK1.1(+) alpha beta TCR(-) CD3(-)); and 5) the lytic phenotype can be transferred to naive CD8 alpha knockout mice by NK1.1(+) splenocytes. Elucidation of the signaling events that activate these effective cytotoxic cells and the putative suppressive mechanisms in a wild-type environment may provide means to enhance the clinical activity of DC-based approaches.     

Dendritic cell precursors are permissive to dengue virus and human immunodeficiency virus infection

CD14(+) interstitial cells reside beneath the epidermis of skin and mucosal tissue and may therefore play an important role in viral infections and the shaping of an antiviral immune response. However, in contrast to dendritic cells (DC) or blood monocytes, these antigen-presenting cells (APC) have not been well studied. We have previously described long-lived CD14(+) cells generated from CD34(+) hematopoietic progenitors, which may represent model cells for interstitial CD14(+) APC. Here, we show that these cells carry DC-SIGN and differentiate into immature DC in the presence of granulocyte-macrophage colony-stimulating factor. We have compared the CD14(+) cells and the DC derived from these cells with respect to dengue virus and human immunodeficiency virus type 1 (HIV-1) infection. Both cell types are permissive to dengue virus infection, but the CD14(+) cells secrete the anti-inflammatory cytokine interleukin 10 and no tumor necrosis factor alpha. Regarding HIV, the CD14(+) cells are permissive to HIV-1, release higher p24 levels than the derived DC, and more efficiently activate HIV Pol-specific CD8(+) memory T cells. The CD14(+) DC precursors infected with either virus retain their DC differentiation potential. The results suggest that interstitial CD14(+) APC may contribute to HIV-1 and dengue virus infection and the shaping of an antiviral immune response.