Cell Development In Thymus Research Articles

IL-9 Expression by Invariant NKT Cells Is Not Imprinted during Thymic Development.

Invariant NKT (iNKT) cell thymic development can lead to distinct committed effector lineages, namely NKT1, NKT2, and NKT17. However, following identification of IL-9-producing iNKT cells involved in mucosal inflammation, their development remains unaddressed. In this study, we report that although thymic iNKT cells from naive mice do not express IL-9, iNKT cell activation in the presence of TGF-β and IL-4 induces IL-9 secretion in murine and human iNKT cells. Acquisition of IL-9 production was observed in different iNKT subsets defined by CD4, NK1.1, and neuropilin-1, indicating that distinct functional subpopulations are receptive to IL-9 polarization. Transcription factor expression kinetics suggest that regulatory mechanisms of IL-9 expression are shared by iNKT and CD4 T cells, with Irf4 and Batf deficiency deeply affecting IL-9 production. Importantly, adoptive transfer of an enriched IL-9(+) iNKT cell population leads to exacerbated allergic inflammation in the airways upon intranasal immunization with house dust mite, confirming the ability of IL-9-producing iNKT cells to mediate proinflammatory effects in vivo, as previously reported. Taken together, our data show that peripheral iNKT cells retain the capacity of shaping their function in response to environmental cues, namely TGF-β and IL-4, adopting an IL-9-producing NKT cell phenotype able to mediate proinflammatory effects in vivo, namely granulocyte and mast cell recruitment to the lungs.

MicroRNA-183/96/182 cluster regulates iNKT cell development and function (IRM7P.706)

Abstract MicroRNAs (miRNAs), a class of non-coding small RNAs. Invariant Natural Killer T (iNKT) cells are potent regulators of diverse immune responses. Our previous study indicated that lack of miRNAs following the deletion of Dicer, a miRNAs-processing enzyme, affect iNKT cell development and function (PNAS, 2009). However, the roles of specific miRNA in iNKT cell development and function remain unclear. The miR-183/96/182 cluster, composed of 3 miRNA genes, is significantly upregulated upon T cell activation. In this study, we investigate the role of miR-183/96/182 cluster on development and function of iNKT cells, using miR-183/96/182 knockout (KO) mice. We found that the frequency of immature iNKT cells significantly increased, while mature iNKT cells significantly decreased in thymus of KO mice compared to littermate control (P<0.05), indicating the blockage of iNKT cell development in thymus. Furthermore, lack of miR-183/96/182 resulted in a substantial reduction of iNKT cell number (p<0.05) in the thymus and in spleen. The mutant iNKT cells displayed defective cytokine production, including IL-4 and IFNγ, compared to iNKT cells from littermate control (P<0.05). Finally, bone marrow chimera transferring experiments further suggested the miR-183/96/182 cluster controlled iNKT cell development and function by cell-intrinsic characteristics. Thus, our data define a specific role of miR-183/96/182 cluster in the development and function of iNKT cells.

Generation of Potent Tumor-Specific CTLs from High-Dose IL-2 Activated naïve CD8 T Cells with Overcoming the Tumor-Derived Immune Suppression

(Background) Adoptive T cell therapy using tumor-infiltrating lymphocytes (TILs) is a promising treatment for cancer patients unresponsive to conventional therapies. However, clonal expansion of T cells easily faced with T cell exhaustion or terminal differentiation which related to suppression of the tumor-killing function in tumor microenvironment. Naïve CD8+T cells have an astounding capacity to react to antigens by massive expansion and differentiation into cytotoxic effector cells. However, it is the main key factor that how can increase the limited number of tumor-specific naïve CD8 T cells due to the negative selection during T cell thymic development.(Methods) In human, naïve, memory T cells and TILs were activated with CD3/CD28 dynabeads and culture on CD2-coated plate to generate various effector T cell populations (Teff N, Teff M and activated TILs). Those different effectors were analyzed the expression of surface exhaustion phenotypes, intracellular transcription factors (T-bet/Eomes) and granzyme/perforin secretions using FACS and western blot assay. Telomere length of three kinds of effector cells was analyzed. High-dose IL-2 (1ug/mL) activated naïve CD8 T cells were stimulated with tumor antigen-loaded dendritic cells (DCs) and then, ELISPOT/cytokine ELISA assay was performed to evaluate tumor-specific (TA) CTL function. In murine model, we also checked the functional difference of each effector cells as like the same methods. In addition, tumor challenging test using EG7-EL4 cell line (OVAp expression) was performed in C57BL/6 mice which adoptively transferred with OT-I thy1.1 Teff N, Teff M and activated TILs.(Results) In vitro expansion of all human naïve, memory and TIL CD8+ T cells was induced successfully. After 3-5 days of expansion, effectors from different progenitors were assessed for the several activation markers CD44, OX40 and CD27 and were considered as the CD62LlowCD44highOX40highCD27high populations. Population frequency of Teff N was significantly higher than Teff M (p < 0.05) or activated TILs (p < 0.005). Telomere length, which correlates with replicative capacity, was greatest in TeffN, shorter in TeffM and shortest in aTILs. When compared the T cell exhaustion phenotypes in three different effector cells, TeffN showed the very low expression of inhibitory markers including PD-1, CTLA-4, and KLRG-1.aTILs expressed most high exhaustion phenotypes with shorter telomere length. Moreover, the secretion of cytotoxic granules such as granzyme B and perforin gradually increased in all effector cells but, among the fully effector cells status, TeffN possess the highest expression level compared to TeffM and aTILs wheras similar level of IFN-r. To further confirm expression profile of T-box transcription factors, the expression of both T-bet and Eomes in TeffN increased at relatively early time point (D+3) and sustained high expression levels during effector status (D+5 and D+8). High expression of T-bet in TeffN promoted the full effector generation and Eomes expression linked to formation of long-term tumor-specific memory population. In CTL function, high-dose IL2-activated naïve T cells were successfully increased and generated the tumor-specific CTLs co-cultured with TA DCs, which inducing the outstanding CTL function compared to those from memory CD8 T cells or aTILs.(Discussion) High-dose IL-2 could increase the tumor-specific naïve CD8 T cells without resulting in clonal exhaustion and could generate the potent tumor-specific CTLs with overcoming the tumor-derived immune suppression compared with CD8+ TILs or secondary effectors from memory T cells. DisclosuresNo relevant conflicts of interest to declare.

Cutting edge: Discrete functions of mTOR signaling in invariant NKT cell development and NKT17 fate decision.

Invariant NKT (iNKT) cells recently were classified into NKT1, NKT2, and NKT17 lineages with distinct transcription factor and cytokine profiles, but the mechanisms underlying such fate decisions remain elusive. In this article, we report crucial roles for mechanistic target of rapamycin (mTOR) signaling, especially mTORC2, in iNKT cell development and fate determination of NKT17 cells. Loss of Rictor, an obligatory component of mTORC2, decreased thymic and peripheral iNKT cells, which was associated with defective survival. Strikingly, Rictor deficiency selectively abolished the NKT17 lineage, as indicated by a marked reduction in RORγt and IL-17 expression. Moreover, deletion of phosphatase and tensin homolog (Pten) upregulated mTORC2 activity and enhanced NKT17 generation, but concomitant loss of Rictor reversed the NKT17 dysregulation. In contrast, mTORC1 regulators Raptor and Rheb are dispensable for NKT17 differentiation, despite their importance in iNKT cell thymic development. Our findings establish pivotal and unique roles for mTORC2 signaling, which is reciprocally regulated by Rictor and Pten, in NKT17 lineage determination.

Critical role for invariant chain in CD1d-mediated selection and maturation of Vα14-invariant NKT cells

The development and maturation of Vα14 invariant (i)NKT cells in mice requires CD1d-mediated lipid antigen presentation in the thymus and the periphery. Cortical thymocytes mediate positive selection, while professional APCs are involved in thymic negative selection and in terminal maturation of iNKT cells in the periphery. CD1d requires entry in the endosomal pathway to allow antigen acquisition for assembly as lipid/CD1d complexes for display to iNKT cells. This process involves tyrosine-based sorting motifs in the CD1d cytoplasmic tail and invariant chain (Ii) that CD1d associates with in the endoplasmic reticulum. The function of Ii in iNKT cell thymic development and peripheral maturation had not been fully understood. Using mice deficient in Ii and the Ii-processing enzyme cathepsin S (catS), we addressed this question. Ii−/− mice but not catS−/− mice developed significantly fewer iNKT cells in thymus, that were less mature as measured by CD44 and NK1.1 expression. Ii−/− mice but not catS−/− mice developed fewer Vβ7+ cells in their iNKT TCR repertoire than WT counterparts, indicative of a change in endogenous glycolipid antigen/CD1d-mediated iNKT cell selection. Finally, using a Mycobacterium tuberculosis infection model in macrophages, we show that iNKT developed in Ii−/− but not catS−/− mice have defective effector function. Our data support a role for professional APCs expressing Ii, but no role for catS in the thymic development and peripheral terminal maturation of iNKT cells.

The role of deltex1 in regulating T cell tolerance (63.20)

Abstract Deltex1 (DTX1) is an E3 ubiquitin ligase and is suggested to be a mediator of Notch signaling. Our previous study found that DTX1 supressses T cell activation and promotes MEKK1 degradation. In this study, we found that T cell activation was partially suppressed by DTX1 with either inactive mutation of RING finger domain or deletion of WWE domain. In addition, we identified another suppressive mechanism of DTX1 by upregulation of Gadd45β, an inhibitor of JNK activation. DTX1 may promote the expression of Gadd45β in the absence of RF domain or WWE domain. Therefore, DTX1 inhibit T cell activation by either E3-dependent or E3-independent mechanisms. To further investigate the physiological function of DTX1 in lymphocyte development and activation, Dtx1-deficient mice were generated. T cell development in thymus and spleen was normal. B cell development in bone marrow was not affected but margine zone B cell population in spleen was reduced in Dtx1-deficient mice. T cell proliferation, cytokine production and MAPK activation were enhanced in Dtx1-deficient T cells. In addition, enhanced autoantibody production, elevated immune complex deposition in kidney and increased leukocyte infiltration in lung and liver were observed in aged Dtx1-deficient mice. These results suggest that DTX1 plays a role in T cell tolerance induction.

Novel function of perforin in regulating CD4+ T cell activation by affecting calcium signaling (35.7)

Abstract Perforin is a pore forming protein engaged mainly in mediating target-cell death and is employed by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. However, whether it also plays a role in conventional CD4+ T cell function remains unclear. Here we report that in perforin-deficient(PKO) mice, CD4+ T cells are hyperproliferative in response to TCR stimulation. This feature of activated CD4+ T cells is accompanied by an enhancement both in cell division and in IL-2 secretion. The perforin deficiency does not appear to influence T cell development in thymus and lymph node. In vivo, perforin deficiency results in increased antigen-specific T cell proliferation and antibody production. Furthermore, PKO mice appear more susceptible to experimental autoimmune uveitis. Examination of the mechanisms involved demonstrate that in PKO CD4+ T cells TCR stimulation increases intracellular calcium flux and subsequently enhances activation of transcription factor NF-AT1. Our results indicate that perforin plays a negative role in regulating CD4+ T cell activation and immune response by affecting TCR-dependent Ca2+ signaling.

CD1d presentation of glycolipids.

The CD1 family of antigen-presenting molecules consists of five members, CD1a to e. Of these molecules CD1d has been the subject of much interest over the past 10 years following the discovery that this molecule presents antigens to a group of T cells known as invariant natural killer T cells (iNKT). iNKT cells carry an invariant T cell receptor which contains homologous gene segments in mouse and man. iNKT cells are positively selected in the thymus in the same manner as major histocompatibility complex restricted T cells, except iNKT cells require CD1d to be presented by thymocytes rather than epithelial cells. Once in peripheral organs, iNKT cells appear to play multiple roles in host defence against pathogens and cancer. If the numbers of iNKT cells are not correctly regulated it can result in autoimmune disorders, such as diabetes. The ligands for iNKT cells have been the subject of much research but identifying physiologically relevant candidate ligands for positive selection or activation has proved technically very challenging. This is largely due to the fact that the ligands for iNKT cells are lipids. The lipid ligands for thymic selection and some of those involved in peripheral activation are self-derived. Glycosphingolipids are suggested to be the class of lipid for iNKT cell thymic development. For peripheral activation it appears multiple classes of self-derived lipids may play a role, in addition to pathogen-derived lipids. This review will cover essential background to iNKT cell and CD1d biology with emphasis on the candidate iNKT cell ligands proposed to date.

T 세포 발생 연구를 위한 유전자 적중 마우스

T 세포의 발생은 흉선에서 이루어지는데, 골수나 태아의 간으로부터 유래한 전구세포가 여러 단계의 복잡한 발달단계를 거쳐서 단일 CD4나 CD8 분자를 세포 표면에 발현하는 성숙한 T세포로 분화하여 2차 림프기관으로 이동한다. T세포 수용체로부터 유래하는 신호와 하위 신호전달 경로는 성숙한 T 세포 및 흉선 T 세포의 발생과 분화에 중요하다. 유전자 적중 마우스를 이용한 연구들을 통해서 복잡한 T 세포 발생을 조절하는 중요한 신호전달 경로를 파악하여 메카니즘에 대한 이해가 깊게 되었다. 유전자의 발현 시기와 조직이나 세포를 고려한 신호전달을 이해하기 위해 보다 정교한 유전자 조작로 기법의 개발이 중요하다고 사료된다. T cells develop in thymus, where immature progenitors undergo multi-stage developmental processes until mature CD4+ and CD8+ single positive T cells are generated and egress out to the periphery. Signals from T cell receptor are pivotal for T cell development in thymus as well as in periphery. Gene targeted mutant mice have contributed the understanding underlying mechanisms by dissecting the signaling pathways which governing these complex processes. Development of refined mutational system will be imperative to analyze the signaling pathways by taking consideration of context of 'timing and/or space' of gene function.

The Expression of Retinoic Acid Receptors in Thymus of Young Children and the Effect of All-Transretinoic Acid on the Development of T Cells in Thymus

To explore the effect of retinoic acid on the development of T cells in thymus, we examined the expression and distribution of retinoic acid receptors mRNA in thymus of young children (< or =5 years of age) and investigated the affection of all-transretinoic acid on the differentiation and maturation of the T cells in thymus and cultured in vitro. Twenty thymus samples were collected. By in situ hybridization, we found that four retinoic acid receptor genes expressed in thymus, and the levels of mRNA for retinoic acid receptor genes (detected by reverse transcriptase, fluorescent quantitative PCR), were changed with age. In in vitro culture, all-transretinoic acid promoted the maturation from CD4+CD8+ cells to CD4+ cells but inhibited the differentiation from CD4+CD8+ cells to CD8+ cells. Thus, retinoic acid likely plays important roles in T cell development in thymus and perhaps by affecting the relative expression of retinoic acid receptors.

T cell development in the thymus: From periodic seeding to constant output

T cell development occurs in the thymus throughout life. Recent experimental findings show that the seeding of the thymus by multi-potent stem cells from the bone marrow is periodic rather than continuous, as previously assumed. However it is well known that the output rate of cells from the thymus is relatively constant. A quantitative model is used to verify the current hypotheses regarding T cell development in the steady state mouse thymus. The results show that the thymus could be at a periodic steady state with out-of-phase thymocyte populations. Experiments to examine possible periodic fluctuations in the thymus are proposed and methods for further analysis are outlined.

IL-7 Effects on Thymocyte Progenitors.

IL-7 is a non-redundant cytokine in T cell development. We studied the role of IL-7 in early T-cell development using a model of transgenic (Tg) mice with the murine IL-7 gene under control of the lck proximal promoter. At high IL-7 over-expression (x39 fold increase at day 1 in total thymic tissue), we observed a disruption of TCRαβ development along with increased B cell development in the thymus (7- to 13-fold increase) (El Kassar, Blood, 2004). In order to further explore abnormal T and B cell thymic development in these mice, we first confirmed that they both arise in parallel and were non-cell autonomous, by in vivo injection of neutralizing anti-IL-7 MAb and mixed bone marrow chimera experiments. Using a six color flow cytometry analysis, we found a dramatic decrease of the early thymocyte progenitors (ETPs, lin−CD44+CD25−c-kithiIL-7R−/lo) in the adult Tg mice (x4.7 fold decrease). Lin−CD44+CD25−c-kit+ thymocytes were sorted and cultured on OP9 and OP9 delta-like1 (OP9-DL1) stromal cells (kindly provided by Pr Zuniga Pflucker). At day 14, we observed an important decrease of T cell development (54% vs. 1% of DP cells) and an increase of NK cells (x5 fold increase) in the Tg-derived DN1 cell culture. DN2 (Lin−CD44+CD25−c-kit+) Tg thymocytes showed the same, but less dramatic abnormalities. While DN1 progenitors developed effectively into B220+CD19+ cells on OP9 stromal cells, no B cell development was observed on OP-DL stromal cells from DN1-Tg derived progenitors or by addition of increasingly high doses of IL-7 (x10, x40, x160) to normal B6-derived DN1 progenitors. Instead, a block of T-cell development was observed with increased IL-7. We hypothesized a down regulation of Notch signaling by IL-7 over-expression and analyzed by FACS Notch expression in the DN thymocytes. By staining the intra-cellular part of Notch cleaved after Notch 1/Notch ligand activation, Tg-derived DN2 cells showed decreased Notch signaling. More importantly, HES expression was decreased in the DN2, DN3 and DN4 fractions by semi-quantitative PCR. Sorted Pro/Pre B cells from Tg thymi showed TCR Dβ1-Jβ1 rearrangement indicating their T specific origin, in opposition to Pro/Pre B cells sorted from the bone marrow of the same mice. We suggest that more than one immature progenitor seeds the thymus from the bone marrow. While ETPs had T and NK proliferative capacity, another thymic progenitor with B potential may be responsible for thymic B cell development in normal and IL-7 Tg mice. Finally, IL-7 over-expression may induce a decreased Notch signaling in thymic progenitors, inducing a switch of T vs. B lineage development.

Coronin-1 expression in T lymphocytes: insights into protein function during T cell development and activation

Coronin has been described as an actin-binding protein of Dictyostelium discoideum, and it has been demonstrated to play a role in cell migration, cytokinesis and phagocytosis. Coronin-related proteins are found in many eukaryotic species, including Coronin-1 in mammals whose expression is enriched in the hematopoietic tissues. Here, we characterize Coronin-1 gene and protein expression in mouse embryonic and adult T lymphocytes. Coronin-1 is expressed throughout T cell ontogeny and in peripheral alphabeta T cells. Expression varies along thymic cell development, with maximum levels observed in embryonic early thymocytes and, in the adults, the selected TCRalphabeta(+) single-positive thymocytes. Subcellular localization analysis indicates that Coronin-1 is in equilibrium between the cytosol and the cell cortex, where it accumulates in F-actin-rich membrane protrusions induced by polarized activation of TCR-CD3-stimulated T cells. These data are consistent with a role of Coronin-1 in T cell differentiation/activation events involving membrane dynamisms and the cortical actin cytoskeleton.

The reproductive system at the neuroendocrine-immune interface: focus on LHRH, estrogens and growth factors in LHRH neuron–glial interactions

Bidirectional communication between the neuroendocrine and immune systems plays a pivotal role in health and disease. Signals generated by the hypothalamic–pituitary–gonadal (HPG) axis (i.e. luteinizing hormone-releasing hormone, LHRH, and sex steroids) are major players coordinating the development immune system function. Conversely, products generated by immune system activation exert powerful and longlasting effects on HPG axis activity. In the central nervous system (CNS), one chief neuroendocrine-immune (NIE) compartment is represented by the astroglial cell population and its mediators. Of special interest, the major supporting cells of the brain and the thymus, astrocytes and thymic epithelial cells, share a similar origin and a similar set of peptides, transmitters, hormones and cytokines functioning as paracrine/autocrine regulators. This may explain some fundamental analogies in LHRH regulation of both cell types during ontogeny and in adult life. Hence, the neuropeptide LHRH significantly modulates astrocyte and thymic cell development and function. Here we focus this work on LHRH neuron–glial signaling cascades which dictate major changes during LHRH neuronal differentiation and growth as well as in response to hormonal manipulations and pro-inflammatory challenges. The interplay between LHRH, growth factors, estrogens and pro-inflammatory mediators will be discussed, and the potential physiopathological implications of these findings summarized. The overall study highlights the plasticity of this intersystem cross-talk and emphasize neuron–glial interactions as a key regulatory level of neuroendocrine axes activity.

Enterocyte expression of interleukin 7 induces development of gammadelta T cells and Peyer's patches.

The intestinal mucosa is suggested to support extrathymic T cell development, particularly for T cell receptor (TCR)-gammadelta intraepithelial lymphocytes (IELs). TCR-gammadelta cell development requires interleukin (IL)-7; IL-7(-/)- or IL-7 receptor(-/)- mice lack TCR-gammadelta cells. Using the intestinal fatty acid binding protein (iFABP) promoter, we reinstated expression of IL-7 to mature enterocytes of IL-7(-/)- mice (iFABP-IL7). In iFABP-IL7 mice, TCR-gammadelta IELs were restored, as were cryptopatches and Peyer's patches. TCR-gammadelta cells remained absent from all other tissues. Likewise, T cell development in thymus and B cell maturation in the bone marrow and spleen retained the IL-7(-/)- phenotype. Thus, IL-7 expression by enterocytes was sufficient for extrathymic development of TCR-gammadelta cells in situ within the intestinal epithelium and was crucial for organization of mucosal lymphoid tissue.

A Mouse Carrying Genetic Defect in the Choice Between T and B Lymphocytes

Transgenic mice with human CD3epsilon gene have been shown to exhibit early arrest of T cell development in the thymus. The present study shows that, instead of T cells, B cells are generated in the thymus of a line, tg epsilon26, of the human CD3epsilon transgenic mice. The accumulation of mature B cells in the thymus was found only in tg epsilon26 mice, not in other human CD3epsilon transgenic mouse lines or other T cell-deficient mice, including CD3-epsilon knockout mice and TCR-beta/TCR-delta double knockout mice. Hanging drop-mediated transfer into 2-deoxyguanosine-treated thymus lobes showed that lymphoid progenitor cells rather than thymus stromal cells were responsible for abnormal B cell development in tg epsilon26 thymus, and that tg epsilon26 fetal liver cells were destined to become B cells in normal thymus even in the presence of normal progenitor cells undergoing T cell development. These results indicate that lymphoid progenitor cells in tg epsilon26 mice are genetically defective in thymic choice between T cells and B cells, generating B cells even in normal thymus environment. Interestingly, tg epsilon26 thymocytes expressed GATA-3 and TCF-1, but not LEF-1 and PEBP-2alpha, among T cell-specific transcription factors that are involved in early T cell development, indicating that GATA-3 and TCF-1 expressed during thymocyte development do not necessarily determine the cell fate into T cell lineage. Thus, tg epsilon26 mice provide a novel mouse model in that lineage choice between T and B lymphocytes is genetically defective.

Molecular Cloning of a Novel T Cell-directed CC Chemokine Expressed in Thymus by Signal Sequence Trap Using Epstein-Barr Virus Vector

Precursors of most secreted and cell surface molecules carry signal sequences at their amino termini. Here we describe an efficient signal sequence trap method and isolation of a novel CC chemokine. An expression library was constructed by inserting 5' portion-enriched cDNAs from phytohemagglutinin-stimulated peripheral blood mononuclear cells into upstream of signal sequence-deleted CD4 cDNA in an Epstein-Barr virus shuttle vector. After electroporation into Raji cells, CD4 antigen-positive cells were enriched by repeated cell sorting and plasmids were recovered in Escherichia coli. Out of 100 plasmid clones examined, 42 clones directed expression of CD4 antigen on the cell surface. Among them were signal sequences of CD6, beta2-microglobulin, MGC-24, and T cell receptor epsilon-chain, and at least four novel potential signal sequences. A cDNA clone encoding a novel CC chemokine was isolated by using one of the trapped fragments. The gene designated as TARC from Thymus and Activation-Regulated Chemokine was expressed transiently in phytohemagglutinin-stimulated peripheral blood mononuclear cells and constitutively in thymus. Radiolabeled recombinant TARC specifically bound to T cell lines and peripheral T cells but not to monocytes or granulocytes. The binding of radiolabeled TARC to the high-affinity receptor (Kd, 2.1 nM) on Jurkat was displaced by TARC but not by interleukin-8, MIP-1alpha, RANTES, or MCP-1. TARC also bound to the promiscuous chemokine receptor on erythrocytes (Kd, 17 nM). TARC induced chemotaxis in T cell lines Hut78 and Hut102. Pretreatment of Hut78 with pertussis toxin abolished the TARC-induced cell migration. Collectively, T cells express a highly selective receptor for TARC that is coupled to pertussis toxin-sensitive G-protein. TARC may a factor playing important roles in T cell development in thymus as well as in trafficking and activation of mature T cells.

Characterization of human thymic lymphocytes forming rosettes with stromal cells.

The interaction of thymic lymphocytes and stromal cells is believed to be important for T cell development in thymus. In this study, thymic rosettes (TR), which are cell-cell complexes of thymic lymphocytes and stromal cells, were isolated from human thymic tissue, and were characterized. Treating human thymus with collagenase in mild condition, human TR were successfully isolated. Subsequently, TR were purified by the 1G sedimentation method. Human TR consisted of a stromal cell in center surrounded by lymphocytes. The stromal cells were positive for CD14, CD11b, and HLA-DR but negative for thymic epithelial cell specific mAb, UH-1, suggesting that they are macrophage/dendritic cells. The lymphocytes which formed TR (TRL) were mainly double positive (CD4+CD8+) and CD1+ cells, and few of them expressed bright CD3, indicating that TRL are in the intermediate maturation stage. TRL expressed activation markers (Ta1 and HLA-DR) in a significantly higher percentage of cells than did unselected thymocytes. Blocking test revealed that CD11a and CD2 are involved in the binding of TRL and the stromal cells as adhesion molecules.

Clonal deletion of self-reactive T cells at the early stage of T cell development in thymus of radiation bone marrow chimeras.

Sequential appearance of T cell subpopulations occurs in the thymocytes of irradiated C3H/He mice (H-2k, Mls-1b2a, Thy-1.2) after transplantation with bone marrow cells of AKR/J mice (H-2k, Mls-1a2b, Thy-1.1) (AKR----C3H chimeras). The donor-derived thymocytes of AKR----C3H chimeras on day 14 after bone marrow transplantation (BMT) contained a large number of blastlike CD4+CD8+ cells which represent relatively immature thymocytes, whereas those on day 21 after BMT consisted of small sized CD4+,CD8+ cells which represent a great part in normal thymocytes. To define the developmental stage at which clonal deletion of self-reactive T cells occurs in adult thymus, we followed the fate of V beta 6- or V beta 11-bearing T cells in the donor-derived thymocytes at the early stage of AKR----C3H chimeras. Mature thymocytes expressing high intensity of V beta 6 or V beta 11, which are involved in recognition of Mls-1a or MHC I-E gene products, respectively, were deleted from the donor-derived thymocytes on day 21. Immature thymocytes expressing low intensity of V beta 6 in CD3low thymocyte fraction decreased in proportion, whereas those expressing low intensity of V beta 11 rather increased in proportion in the donor-derived thymocytes of AKR----C3H chimeras from day 14 to day 21 after BMT. These results suggest that the clonal deletion of V beta 6-positive cells occurs just at the stage of immature CD3lowCD4+CD8+ cells, whereas the clonal deletion of V beta 11-positive cells may begin at the transitional stage from CD3lowCD4+CD8+ cells to CD3high single positive cells. Timing of negative selection of thymocytes may vary in distinct T cells capable of recognizing different self-Ag.