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Prussian blue nanoparticle (PBNP), a new type of theranostic nanomaterial, had been used for cancer magnetic resonance imaging and photothermal therapy. However, their long-term toxicity after short exposure in vivo was still unclear. In this study, we investigated the dynamic changes of the biochemical and immunity indicators of mice after PBNPs injection through tail vein. Histological results showed that the PBNPs were mainly accumulated in liver and spleen. In the spleen, we found the frequency of T cells was starting to decrease after 1 day of PBNPs injection, but then slowly recovered to normal level after 60 days of injection. Meanwhile, the frequency of T cells in the blood was firstly decreased after the PBNPs injection, and then the T cell frequency kept increasing and recovered back to normal levels after 7 days of injection. The serum indexes of liver functions (alanine transaminase, aspartate transaminase, total bilirubin, and alkaline phosphatase) increased rapidly to a relatively high level only after 1 h of injection, which meant certain acute liver damage, but these indexes were gradually decreased to normal levels after 60 days of injection. These results indicate that PBNPs have acute toxicity in vivo, however, their long-term toxicity after short-time exposure is low, which might provide guidance for further applications of PBNPs in future., (© The Author(s) 2015.)

The severity of superantigen (SAg)-mediated shock is associated with T cell infiltration in major organs. We postulated that endothelial cell inflammation and damage in sepsis might be mediated by chemotaxis and adherence of SAg-activated T cells of vascular endothelium. We therefore investigate whether staphylococcal enterotoxin B (SEB) could modulate chemokine receptors expression on T cells as well as cytokine release, and then we examined the up-modulation of chemokine-associated affect on T cell-mediated damage of endothelial cells. We consistently observed that SEB could upregulate expression of CCR5 on T cells and induce a panel of cytokines release from T cell, the latter could further induce increased release of chemokine such as MCP-1, MIP-1α and RANTES from human pulmonary artery endothelial cell (HPAEC). Both upregulation of CCR5 on SEB-activated T cell and increased release of chemokine from HPAEC contribute to higher chemoattraction and adherence of T cell to HPAEC and ultimately led to more extensive endothelial damage. (These data suggest that T cell migration and acute inflammatory response in bacterial SAg-mediated shock maybe explained partly by upregulation of chemotaxis of T cell of endothelium.) Revised as following: Our results demonstrate that upregulation of chemotaxis of T cell of endothelium is SEB specific, and this mechanism could account in part for T cell migration and acute inflammatory response in bacterial SAg-mediated shock., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

To determine the effect of dexamethasone on the antimyeloma effects of lenalidomide, we tested in vitro proliferation, tumor suppressor gene expression, caspase activity, cell cycling, and apoptosis levels in a series of multiple myeloma (MM) and plasma cell leukemia cell lines treated with lenalidomide and dexamethasone, alone or in combination. The effect of dexamethasone on the immunomodulatory activities of lenalidomide such as T cell and natural killer (NK) cell activation was measured via interleukin [IL]-2 production, and interferon-gamma and granzyme B production respectively. Lenalidomide inhibited proliferation in most cell lines tested, and this effect was enhanced by dexamethasone. This effect was observed in MM cells containing the high-risk cytogenetic abnormalities t(4;14), t(14;16), del17p, del13, and hypodiploidy. Mechanistically, lenalidomide plus dexamethasone synergistically induced expression of the tumor suppressor genes Egr1, Egr2, Egr3, p15, p21, and p27 in MM cell lines and MM patient cells. The combination activated caspases 3, 8, and 9; and induced cell cycle arrest and apoptosis. Lenalidomide alone increased T cell production of IL-2, and NK cell production of interferon-gamma and granzyme B. Notably, dexamethasone antagonized these immunostimulatory effects of lenalidomide in a dose-dependent manner. These data further elucidate the mechanism of action of lenalidomide and dexamethasone in MM, and suggest that use of low-dose dexamethasone with lenalidomide may retain the antiproliferative effect of lenalidomide while permitting greater immunomodulatory effects of this combination regimen.

Current concepts of chemokine receptor (CKR) association with Th1 and Th2 cell polarization and effector function have largely ignored the diverse nature of effector and memory T cells in vivo. Here, we systematically investigated the association of 11 CKRs, singly or in combination, with CD4 T cell polarization. We show that Th1, Th2, Th0, and nonpolarized T cells in blood and tissue can express any of the CKRs studied but that each CKR defines a characteristic pool of polarized and nonpolarized CD4 T cells. Certain combinations of CKRs define populations that are markedly enriched in major subsets of Th1 versus Th2 cells. For example, although Th0, Th1, and Th2 cells are each found among blood CD4 T cells coordinately expressing CXCR3 and CCR4, Th1 but not Th2 cells can be CXCR3(+)CCR4(-), and Th2 but only rare Th1 cells are CCR4(+)CXCR3(-). Contrary to recent reports, although CCR7(-) cells contain a higher frequency of polarized CD4 T cells, most Th1 and Th2 effector cells are CCR7(+) and thus may be capable of lymphoid organ homing. Interestingly, Th1-associated CKRs show little or no preference for Th1 cells except when they are coexpressed with CXCR3. We conclude that the combinatorial expression of CKRs, which allow tissue- and subset-dependent targeting of effector cells during chemotactic navigation, defines physiologically significant subsets of polarized and nonpolarized T cells.

Chemokine receptors mediate the migration of lymphocytes through the binding of soluble ligands, and their expression is differentially regulated in lymphocyte subsets. The pattern of chemokine receptor expression in T-cell non-Hodgkin lymphoma has not been previously studied. Using a panel of mouse monoclonal antibodies, we studied the immunohistochemical expression of the Th1-associated chemokine receptor CXCR3 in 141 patients with T-cell lymphoma, and we studied the receptors CCR4 and CCR5 and some of their ligands in a subset of these tumors. Expression of CXCR3 was typical of the smaller T cells in angioimmunoblastic lymphoma (15 of 18 patients), angiocentric lymphoma (3 of 3 patients), histiocyte-rich tumors (4 of 5 patients), and unspecified T-cell lymphomas (17 of 39 patients). CXCR3 expression was seen in only 1 of 15 patients with anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma. In contrast, all ALK-positive tumors showed diffuse reactivity for the Th2-associated receptor CCR4 (5 of 5 patients). CCR4 expression was also a consistent feature of the large-cell transformation of mycosis fungoides. CCR5 expression showed no consistent association with any T-cell tumor type. The chemokines Mig (CXCR3 ligand), TARC (CCR4 ligand), and MCP-2 (CCR5 ligand) were detected in intratumoral blood vessels and histiocytes. Mig was also coexpressed by a subset of CXCR3-positive tumor cells in 6 of 20 lymphomas. MCP-2 was highly expressed in stromal cells in 3 patients with nodal involvement by cutaneous T-cell lymphoma. As with normal T-cell subsets, we demonstrated that there is frequent differential expression of chemokine receptors in T-cell tumors, which may explain, in part, the distinctive patterns of spread in different tumor subtypes. (Blood. 2000;96:685-690)

T cell differentiation relies on pre-T cell receptor (TCR) and TCR signaling events that take place at successive steps of the pathway. Here, we show that two of these T cell differentiation checkpoints are regulated by Ikaros. In the absence of Ikaros, double negative thymocytes can differentiate to the double positive stage without expression of a pre-TCR complex. Subsequent events in T cell development mediated by TCR involving transition from the double positive to the single positive stage are also regulated by Ikaros. Nonetheless, in Ikaros-deficient thymocytes, the requirement of pre-TCR expression for expansion of immature thymocytes as they progress to the double positive stage is still maintained, and the T cell malignancies that invariably arise in the thymus of Ikaros-deficient mice are dependent on either pre-TCR or TCR signaling. We conclude that Ikaros regulates T cell differentiation, selection, and homeostasis by providing signaling thresholds for pre-TCR and TCR.

In the present review, a series of studies on the origins of dendritic cells of mice and humans are summarized. Several subsets of mature dendritic cells found in vivo are described and these may correspond to distinct lineages. There is evidence that some dendritic cells are myeloid-derived and that others are lymphoid-derived. The different ways of generating dendritic cells are examined and an attempt to reconcile the differences seen using mouse and human culture models is made. The particular case of Langerhans cells is discussed and an historical overview of the biology of the plasmacytoid T cells, which may represent a distinct 'lymphoid-related' dendritic cell lineage, is given. It is concluded that three or four different pathways lead to the development of different subtypes of dendritic cells.

Lymphocytes that are responsible for regional (tissue-specific) immunity home from the blood to the intestines, inflamed skin or other sites through a multistep process involving recognition of vascular endothelial cells and extravasation. Chemoattractant cytokine molecules known as chemokines regulate this lymphocyte traffic, in part by triggering arrest (stopping) of lymphocytes rolling on endothelium. Here we show that many systemic memory T cells in blood carry the chemokine receptor CCR4 and therefore respond to its ligands, the chemokines TARC and MDC. These cells include essentially all skin-homing cells expressing the cutaneous lymphocyte antigen and a subset of other systemic memory lymphocytes; however, intestinal (alpha4beta7+) memory and naive T cells respond poorly. Immunohistochemistry reveals anti-TARC reactivity of venules and infiltration of many CCR4+ lymphocytes in chronically inflamed skin, but not in the gastrointestinal lamina propria. Moreover, TARC induces integrin-dependent adhesion of skin (but not intestinal) memory T cells to the cell-adhesion molecule ICAM-1, and causes their rapid arrest under physiological flow. Our results suggest that CCR4 and TARC are important in the recognition of skin vasculature by circulating T cells and in directing lymphocytes that are involved in systemic as opposed to intestinal immunity to their target tissues.

To investigate the influence of endogenous peptides on the developmental processes that occur during thymocyte selection, we have used monoclonal antibodies that preferentially recognize the major histocompatibility complex (MHC) molecule I-Ek when it is bound to the moth cytochrome c peptide (88-103). One of these antibodies (G35) specifically blocks the positive selection of transgenic thymocytes expressing a T cell receptor that is reactive to this peptide- MHC complex. Furthermore, G35 does not block the differentiation of transgenic T cells bearing receptors for a different I-Ek-peptide complex. This antibody recognizes a subset of endogenous I-Ek-peptide complexes found on a significant fraction of thymic antigen-presenting cells, including cortical and medullary epithelial cells. The sensitivity of G35 to minor alterations in peptide sequence suggests that the thymic peptide-MHC complexes that mediate the positive selection of a particular class II MHC-restricted thymocyte are structurally related to the complexes that can activate it in the periphery.

We have previously shown that immunologically different mouse mammary cancer cell lines induce antitumor responses after IL2 or IL4 gene transfection. We now report the ability of cytokine-transfected tumors to induce eradication of established wild-type tumor. Animals with 6-day-old tumor treated with IL2-transfected cells also had significantly smaller tumors 2.8 and 1.7 cm2 (EMT6 and 410.4). Findings were similar for IL4-transfected cells. Tumor infiltrating lymphocytes or cells from draining lymph nodes demonstrated tumor-specific in vitro cytotoxicity. Immunohistochemical studies revealed T cell infiltrates in transfected tumors.

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic HIV-1 strains to enter the host cells. In this study, we aim to better understand the ligand-binding profiles of CCR5 and the chemokine-receptor usage on leukocyte cells. We found that MCP-2 could bind to CCR5 transfectants with high affinity and cross-compete effectively with RANTES, MIP-1alpha, and MIP-1beta. MCP-2 is a true agonist for CCR5, eliciting a robust chemotactic response in CCR5 transfectants similar to that of the three known CCR5 ligands and exhibiting cross-desensitization with RANTES in the Ca2+ flux response. MCP-4 also bound to CCR5 with high affinity and was efficiently displaced by other CCR5 ligands. However, MCP-4 only partially displaced the binding of radiolabeled MIP-1alpha and caused a chemotactic response only at high concentrations. Furthermore, MCP-2 inhibited the binding of the M-tropic HIV-1 gp120 envelope glycoprotein to CCR5 and HIV-1 infection of peripheral blood mononuclear cells. More importantly, we found that MCP-2 could bind and elicit chemotaxis in CD3-activated and IL-2-maintained T cells, and most of these functions could be specifically inhibited by the anti-CCR5 mAb 2D7, whereas the responses mediated by MIP-1alpha or MCP-4 were only partially inhibited by 2D7. Thus, although MCP-2 can bind to and signal through CCR1, CCR2b, and CCR5, among which both CCR2 and CCR5 are expressed at high levels on activated T cells, it appears to preferably utilize CCR5 on these cells. In contrast, MIP-1alpha and MCP-4 seem to activate multiple receptors on the same cells., (Copyright 1998 Academic Press.)

Thymic dendritic cells (DC) mediate negative selection at a relatively late stage of the T-cell developmental pathway. We present evidence that the development of thymic DC and of T-lineage cells is linked via a common precursor at an early stage of thymocyte development. T-lineage precursor populations from the adult mouse thymus, prior to T-cell receptor gene rearrangement, display a capacity to produce DC as well as T cells in the thymus, and are very efficient precursors of DC in culture. These lymphoid/DC precursors have little capacity to form myeloid cells, indicating that thymic DC are a lymphoid-related rather than myeloid-related lineage. In contrast to myeloid-related DC, granulocyte-macrophage colony-stimulating factor is not required for the development of these lymphoid-related DC in vivo or in vitro. DC can develop in mutant mice lacking mature T cells, provided the common precursors are present. However, in mutant mice lacking functional Ikaros transcription factors, there are deficiencies in lymphoid precursor cells, in mature lymphoid cells and in DC.

The earliest T precursor population in the adult mouse thymus (CD4lo8-3-44+25-c-kit+) was previously shown to be lymphoid-restricted (T, B, NK) but to have a capacity to form dendritic cells (DC). This led to the concept of a lineage of lymphoid-derived DC. DC could be generated with high efficiency in culture from this low CD4 precursor, using a complex mix of cytokines, a mix that notably did not include GM-CSF, the cytokine normally used for development in culture of myeloid-derived DC. Using this new culture system we now show that the capacity to form DC extends to the pro-T precursor population (CD4-8-3-44+25+c-kit+) but is lost by the pre-T precursor stage (CD4-8-3-44-25+c-kit+), the point of T-cell antigen-receptor beta-gene rearrangement. The DC generated in the cultures resemble mature thymic DC by most markers, but differ in their lack of expression of BP-1 and CD8 alpha.

Chemokine receptors serve as coreceptors for HIV entry into CD4+ cells. Their expression is thought to determine the tropism of viral strains for different cell types, and also to influence susceptibility to infection and rates of disease progression. Of the chemokine receptors, CCR5 is the most important for viral transmission, since CCR5 is the principal receptor for primary, macrophage-tropic viruses, and individuals homozygous for a defective CCR5 allele (delta32/delta32) are highly resistant to infection with HIV-1. In this study, CCR5-specific mAbs were generated using transfectants expressing high levels of CCR5. The specificity of these mAbs was confirmed using a broad panel of chemokine receptor transfectants, and by their non-reactivity with T cells from delta32/delta32 individuals. CCR5 showed a distinct pattern of expression, being abundant on long-term activated, IL-2-stimulated T cells, on a subset of effector/memory T cells in blood, and on tissue macrophages. A comparison of normal and CCR5 delta32 heterozygotes revealed markedly reduced expression of CCR5 on T cells from the heterozygotes. There was considerable individual to individual variability in the expression of CCR5 on blood T cells, that related to factors other than CCR5 genotype. Low expression of CCR5 correlated with the reduced infectability of T cells with macrophage-tropic HIV-1, in vitro. Anti-CCR5 mAbs inhibited the infection of PBMC by macrophage-tropic HIV-1 in vitro, but did not inhibit infection by T cell-tropic virus. Anti-CCR5 mAbs were poor inhibitors of chemokine binding, indicating that HIV-1 and ligands bind to separate, but overlapping regions of CCR5. These results illustrate many of the important biological features of CCR5, and demonstrate the feasibility of blocking macrophage-tropic HIV-1 entry into cells with an anti-CCR5 reagent.

Dendritic cells (DCs) are specialized for presentation of antigen to T cells and are essential for primary T cell activation. Although DCs are generally considered to be myeloid derived, we now have evidence that a subgroup are of lymphoid origin. In particular, the DCs of the adult mouse thymus appear to be derived from the same early, lymphoid-restricted precursor cells that generate T lymphocytes. Purified early thymic T precursors have the capacity to produce T cells, B cells, NK cells and DCs, but not myeloid cells, on transfer to irradiated recipients. They also produce thymic DCs on culture with a mix of cytokines; this mix does not include GM-CSF, needed to generate myeloid-derived DCs. A subgroup of DCs in other lymphoid organs, which like thymic DCs express CD8 as an alpha alpha homodimer, may likewise be of lymphoid origin. These CD8+ DCs in mouse spleen differ functionally from the conventional CD8+ DCs. CD8+ DCs efficiently activate CD4+ T cells but then kill them via Fas ligand on the DC surface. CD8+ DCs efficiently recruit CD8+ T cells into the cell cycle, but their proliferation is then restricted by an inadequate production of interleukin 2. This subgroup of CD8+ DCs therefore appears to have a regulatory role.

The earliest lymphoid precursor population in the adult mouse thymus had previously been shown to produce not only T cells, but also dendritic cell (DC) progeny on transfer to irradiated recipients. In this study, culture of these isolated thymic precursors with a mixture of cytokines induced them to proliferate and to differentiate to DC, but not to T lineage cells. At least 70% of the individual precursors had the capacity to form DC. The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures. The cultured DC also expressed high levels of class I and class II major histocompatibility complex, together with CD11c, DEC-205, CD80, and CD86, markers characteristic of mature DC in general. However, they did not express CD8 alpha or BP-1, markers characteristic of normal thymic DC. The optimized mixture of five to seven cytokines required for DC development from these thymic precursors did not include granulocyte/macrophage colony stimulating factor (GM-CSF), usually required for DC development in culture. The addition of anti-GM-CSF antibody or the use of precursors from GM-CSF-deficient mice did not prevent DC development. Addition of GM-CSF was without effect on DC yield when interleukin (IL) 3 and IL-7 were present, although some stimulation by GM-CSF was noted in their absence. In contrast, DC development was enhanced by addition of the Flt3/Flk2 ligand, in line with the effects of the administration of this cytokine in vivo. The results indicate that the development of a particular lineage of DC, probably those of lymphoid precursor origin, may be independent of the myeloid hormone GM-CSF.

Successive T-precursors isolated from adult mouse thymus were examined for their developmental potential, by transfer to irradiated Ly 5-disparate recipients. The earliest, "low CD4" precursors formed T, B, and dendritic cells (DC), but not myeloid cells, in accordance with earlier studies. Surprisingly, the next downstream CD4-8-3 44+25+ precursor population still formed DC as well as T cells although it no longer formed B or myeloid cells. Further down-stream, the CD4-8 3-44-25+ population formed only T cells. The thymic and splenic DC progeny of the early thymic precursors all expressed high levels of CD8 alpha, in contrast with normal splenic DC and the splenic DC progeny of bone marrow stem cells, which consisted of both CD8 and CD8+ DC. A common precursor of T cells and of a subclass of DC is proposed, with CD8 alpha as a marker of the lymphoid-related DC lineage.

The earliest T precursor population in the adult mouse thymus, considered to have the surface phenotype CD4lo8-3-44+25-Thy-1lo c-kit+ (termed the low CD4 precursor), has been shown to have the capacity to produce B cells and dendritic cells, as well as T cells, and to have the T cell antigen receptor (TCR) C beta gene region in germ-line configuration. Because of evidence that this precursor population may have low levels of CD8 as well as CD4 on the cell surface, it was isolated, stained for surface CD4 and CD8 and assayed for the expression of messenger RNA (mRNA) for CD4 and CD8 by the reverse transcriptase polymerase chain reaction (RT-PCR). The low CD4 precursors gave definite, moderate levels of staining for both CD8 and CD4, in contrast to downstream precursors which showed only marginal staining and so could be considered as genuine CD4-8-3- triple negatives. The low CD4 precursor expressed a significant level of mRNA for CD4, indicating that the surface CD4 was produced by these cells. However, the low CD4 precursor did not express a detectable level of mRNA for CD8, suggesting that the surface CD8 was acquired from other cells. Since the low CD4 precursor population was found already to express mRNA for enzymes involved in TCR gene rearrangement, including in this study terminal deoxynucleotidyl transferase (TdT), a PCR procedure was used to assay early precursors for D-J rearrangements at the TCR beta gene locus. However, the low CD4 precursor had the TCR beta D-J genes in germ-line configuration, D-J gene rearrangements being first detected several stages downstream in the CD3-4-8-44-25-+ precursor population. We conclude that a transient synthesis of CD4, but not of CD8, characterize these early thymus precursors. Although they have initiated synthesis of some recombination-associated enzymes, full commitment to the T lineage and TCR gene rearrangement is a later event.

The earliest steps along the pathway leading to T cells in mice and humans are reviewed. These are the steps between the multipotent hemopoietic stem cell (HSC) and the fully committed precursors undergoing T cell receptor (TCR) gene rearrangement. At this level significant differences between adult and fetal lymphopoiesis have been demonstrated. The extent of lymphoid commitment of precursors within bone marrow is still unresolved, although HSCs clearly undergo developmental changes before migration to the thymus. Both multipotent and T-restricted precursors have now been isolated from fetal blood, suggesting both may seed the thymus. Within the thymus, several minute but discrete populations of T precursors precede the stage of TCR gene rearrangement. They include precursors that are not exclusively T-lineage committed, although they are distinct from HSCs. These precursors have a potential to form NK cells, B cells, dendritic cells, and sometimes other myeloid cells. Some factors that control early lymphoid development are discussed, including IL-7 and the Ikaros transcription factors. These will eventually help to clarify the process of T-lineage commitment.

Analysis of kinetics of thymic repopulation by Rh123low, Lin-, Ly6A/E+, c-kit+ (Rh123low) cells, highly enriched for long-term in vivo hematopoietic repopulating cells, reveals that this population is deficient in thymic repopulation at week 3 after intravenous transplantation when compared to normal bone marrow cells. This suggests that the marrow prothymocytes have been depleted from this population, and analysis of thymic repopulation at week 3 can therefore be used to differentiate prothymocytes and their precursors. Using this short-term assay, the Rh123high, Lin-, Ly6A/E+, c-kit+ (Rh123high) population has been found to be relatively more efficient at early thymic repopulation, suggesting that this population contains the prothymocytes. In addition, the differentiation potential and reconstitution behavior of the Rh123high population observed after intravenous and intrathymic transfer strongly indicates that this population is at the transitional stage between the marrow primitive pluripotential and thymic more mature lymphoid restricted stem cells. We propose that the thymic repopulating ability of the Rh123low population is through generation of the more mature Rh123high progeny, presumably in the marrow.

Dendritic cells, a minor cell population in lymphoid tissues, are specialized for presentation of antigenic peptides to T lymphocytes. Thymic dendritic cells are involved in the deletion of self-reactive T lymphocytes. Although all dendritic cells are ultimately of bone-marrow origin, it has not been clear whether thymic dendritic cells are produced in the adult thymus from a precursor cell or whether they migrate there preformed from the periphery. Recently we isolated from adult mouse thymus a population of early T precursors that could still form B lymphocytes, but not erythroid or myeloid cells, when transferred intravenously. Here we show that these thymic lymphoid precursor cells, as well as bone-marrow haematopoietic stem cells, are able to form both dendritic cells and T-cell progeny when transferred into an irradiated thymus. Such linked development may ensure that developing T cells are negatively selected predominantly by self antigens presented on newly formed thymic dendritic cells.

A new, numerically minute population of cells representing the earliest T precursor cells in the adult mouse thymus has recently been isolated. This population has been shown to be similar to bone marrow hemopoietic stem cells in surface antigenic phenotype and to express moderate levels of CD4. We now show, by fluorescence-activated cell sorting and intrathymic transfer to irradiated mice, that this apparently homogeneous population differs from multipotent stem cells in expressing the surface stem cell antigen 2 (Sca-2), that it differs from most early B lineage cells in lacking B220 and class II major histocompatibility complex expression, and that it binds rhodamine 123 like an activated rather than a quiescent cell. Irradiated recipient mice differing at the Ly 5 locus were used to compare the developmental potential of these early intrathymic precursors with bone marrow stem cells. Only T lineage product cells were detected when the intrathymic precursor population was transferred back into an irradiated thymus. However, when the intrathymic precursor population was transferred intravenously, it displayed the capacity to develop into both B and T lymphoid cells in recipient bone marrow, spleen, and lymph nodes, but no donor-derived myeloid cells were detected. The absence of myeloid and erythroid precursor activity was confirmed by showing that the intrathymic precursor population was unable to develop into myeloid or erythroid spleen colonies on intravenous transfer or to form colonies in an agar culture. These findings indicate that this earliest intrathymic precursor population has become restricted (or strongly biased) to lymphoid lineage development, but not exclusively to T lymphocytes.

Precursors of all T-lineage cells are found in a population of thymocytes that lack the CD4 and CD8 surface glycoproteins. These "double-negative" thymocytes are markedly heterogeneous in their expression of other surface markers and include cells at various stages of development. In this study, CD4- CD8- adult murine thymocytes were separated into subsets based on the expression of the "heat stable antigen" (HSA) and of Ly 1 (CD5). The sorted subsets were analyzed directly (without prior expansion in culture) for T-cell antigen receptor (TcR) gene rearrangement and mRNA expression and for TcR and CD3 cell-surface protein expression. Very little surface CD3 or TcR expression was detected on the major HSA+ Ly 1low subset. However, the HSA+ Ly 1high, HSA- Ly 1high, and HSA- Ly 1low subsets all contained cells with surface expression of CD3 and TcR. In contrast to previous studies, we found no subset that exclusively expressed either the alpha beta or gamma delta heterodimer, although the ratio of alpha beta+ to gamma delta+ varied widely. Two of these three subsets (HSA- Ly 1low and HSA- Ly 1high) showed very high usage of V beta 8 gene products in the alpha beta heterodimer, but nevertheless included approximately equal to 15% non-V beta 8 alpha beta forms. All CD4- CD8- subsets were found to have extensively rearranged their TcR gamma genes and to express gamma mRNA. Expression of a high ratio of mature [1.3 kilobases (kb)] to truncated (1.0 kb) beta message and presence of alpha message was largely restricted to subsets with TcR alpha beta surface expression.

Precursors of all T-lineage cells are found in the population of thymocytes that lacks the CD4 and CD8 surface markers. These "double-negative" thymocytes are heterogeneous and can be divided into discrete subpopulations based on their expression of other surface markers. We have determined the relative maturity of these subpopulations based on the extent of rearrangement and expression of their T-cell receptor genes, their cell cycle status, and their thymus reconstitution capacity. Within the subpopulation of double negatives expressing high levels of the heat-stable antigen, the additional markers phagocytic glycoprotein 1 (Pgp-1) and interleukin 2 receptor (IL-2R) can be used to define the sequence IL-2R- Pgp-1+----IL-2R+ Pgp-1-----IL-2R- Pgp-1-, which occurs before the expression of CD4 and CD8. Transient expression of the IL-2R marks an important developmental point in the sequence just prior to a burst of cell proliferation and a loss of thymus reconstitution ability. The earliest cells in this sequence are already partially rearranged for genes in the C beta 1 region. IL-2R expression marks a second wave of T-cell antigen receptor of beta-chain gene rearrangement and the initiation of T-cell antigen receptor alpha- and beta-chain gene expression.

In this chapter we have summarized our view of the subsets of murine CD4- CD8- thymocytes which can be identified with a range of monoclonal antibodies. We have shown the division rate and turnover time of the main subsets and have listed what we know of the TcR gene rearrangement, and expression at the RNA and protein levels. We have been unable to completely segregate gamma delta-TcR-expressing cells from alpha beta-TcR-expressing cells by any of the markers we have used, although the proportions of the two receptor forms vary widely in the different subsets. Experiments involving intrathymic transfer of the CD4- CD8- subsets are described, which indicate that all the TcR- subsets of the CD4- CD8- thymocytes display some precursor activity and which suggest a progression of at least five stages through the TcR- subpopulations of CD4- CD8- cells. The earliest precursor is a Thy 1 low, HSA low, Pgp-1 high cell which has unrearranged C beta and is non-dividing and which closely resembles the bone marrow prothymocyte. The later precursors are Thy 1 high, HSA high, Pgp-1 low, have rearranged C beta and are rapidly dividing. We tentatively conclude that none of the TcR+ CD4- CD8- cells are precursors of the major thymocyte subsets or of typical peripheral T cells, and we have found no evidence so far of separate precursors for the different mature subsets of thymocytes or peripheral T cells.