T cell receptor-alpha beta lacking the beta-chain V domain can be expressed at the cell surface but prohibits T cell maturation.

A TCR-beta gene lacking V domain sequences (delta V-TCR-beta) was inserted into the germline of mice. Expression of the transgene inhibited endogenous TCR-beta, but not TCR-alpha gene rearrangement and expression. The mutated TCR-beta gene affected alpha beta T cell development: the common thymocyte pool was normal in cell number, with cells expressing CD4 and CD8, but the mature, "CD3bright" population expressing either CD4 or CD8 molecules was reduced by 90%. To help understand these effects on TCR-beta gene rearrangement and T cell development, biosynthesis of the delta V-TCR-beta protein was analyzed in a tumor cell line derived from a transgenic mouse. Despite absence of the V domain, the delta V-TCR-beta chain paired with endogenous TCR-alpha chains and assembled with CD3 gamma, -delta, -epsilon, and -zeta components in the endoplasmatic reticulum, followed by transport through the Golgi complex to the plasma membrane. Therefore, assembly of the complex, and even cell surface expression, may be relevant for allelic exclusion of the TCR-beta gene. In the common thymocyte population, the CD3 components, endogenous TCR-alpha, and the delta V-TCR-beta gene product were expressed at the RNA level, but endogenous TCR-beta was not. The TCR-alpha delta beta/CD3 complex was present at the cell surface at low levels and was functional in terms of anti-CD3-induced Ca2+ mobilization. The observed arrest of alpha beta T cell development at the CD4+8+ thymocyte stage indicates that ligand recognition by the TCR, with contribution of the beta-chain V domain, is not required for transition of CD4-8- thymocytes to the CD4+8+ phenotype, but necessary for entry into the "single positive," CD3bright differentiation stage.     

Identification of a new type of invariant V alpha 14+ T cells and responsiveness to a superantigen, Yersinia pseudotuberculosis-derived mitogen.

We examined the expression of the H4 T cell activation marker in thymic T cell subpopulations and found that TCR-alpha beta+ CD4+ thymic T cells are segregated into three subpopulations based upon H4 levels. Thymic T cells with either no or low H4 expression differentiate via the mainstream differentiation pathway in the thymus. H4int thymic T cells, which express a skewed V beta repertoire of V beta 2, -7, and -8 in their TCRs, show the phenotype of NKT cells: CD44high, Ly6Chigh, NK1.1+, and TCR-alpha beta low. H4high thymic T cells also show a skewed V beta repertoire, V beta 2, -7, and -8, and predominantly express an invariant V alpha 14-J alpha 281+ alpha-chain in their TCRs but constitute a distinct population in that they are CD44int, Ly6C-, NK1.1-, and TCR-alpha beta high. Thus, invariant V alpha 14+ thymic T cells consist of ordinary NKT cells and a new type of T cell population. V beta 7+ and V beta 8.1+ invariant V alpha 14+ thymic T cells are present in DBA/2 mice, which carry mammary tumor virus-7-encoded superantigens, in comparable levels to those in BALB/c mice. Furthermore, V beta 7+ invariant V alpha 14+ thymic T cells in DBA/2 mice are in the immunologically responsive state, and Yersinia pseudotuberculosis-derived mitogen-induced V beta 7+ invariant V alpha 14+ thymic T cell blasts from DBA/2 and BALB/c mice exhibited equally enhanced responses upon restimulation with Y. pseudotuberculosis-derived mitogen. Thus, invariant V alpha 14+ thymic T cells that escape negative selection in DBA/2 mice contain T cells as functionally mature as those in BALB/c mice.     

Phenotypic and functional characterization of mouse hepatic CD8 alpha+ lymphoid-related dendritic cells

Recently, attention has focussed on phenotypic and functional differences between classic myeloid dendritic cells (DC), and DC that reportedly develop from an early, committed lymphoid precursor. In mice, DC from these separate hemopoietic lineages differ by their surface expression of CD8 alpha. We undertook a comparative study of CD8 alpha+ (CD11blow; lymphoid-related) and CD8 alpha- (CD11bhigh; myeloid) DC isolated from mouse liver. CD8 alpha+ and CD8 alpha- DC each constituted 相似文献   

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.     

Human T cell gamma-chain gene rearrangements in acute lymphoid and nonlymphoid leukemia: comparison with the T cell receptor beta-chain gene

Rearrangement of germ-line genes coding for T and B cell antigen receptor molecules is an early event in lymphoid development which eventually leads to the generation of clonal diversity in receptor-positive lymphocytes. Three T cell-associated rearranging genes have been described. Two, T alpha and T beta, code for the two polypeptide chains that form the T cell receptor heterodimer. The function of the third gene, the gamma-gene (T gamma), is not known. To learn more about the behavior of T gamma during lymphoid ontogeny, we compared rearrangement of T gamma and T beta genes in leukemic cells arrested at varied stages of lymphoid and myeloid development. We analyzed 38 fresh cell lines and 15 established cell lines from a total of 53 leukemic patients. Cells were immunophenotyped with a panel of monoclonal antibodies recognizing T-, B-, or myeloid-associated surface markers. Sixteen T-lineage cases were studied; 15 displayed both T beta and T gamma rearrangements. The exception (germ-line for T beta and T gamma) was an immature CD2(T11)+, CD3(T3)-, CD7(3A1)+, CD1(T6)+, CD5(T101)+ phenotype. Fourteen non-T non-B leukemias were analyzed; eight were germ-line for both T beta and T gamma, four had rearrangements involving both T beta and T gamma, and two were germ-line for T beta and rearranged to T gamma. Four cases with acute biphenotypic leukemia were studied; two had rearrangements of T beta and T gamma, and two were germ-line for both genes. Cells from nonlymphocytic leukemias were studied in 19 cases. All were found to be germ-line for both T beta and T gamma. Fifty-one of 53 genomic DNA samples were concordant for T gamma and T beta rearrangement. These results indicate that rearrangement of T gamma can occur in leukemic cells of B cell as well as T cell precursor origin, as has been reported previously for T beta.     

Differentiation of monocytic cell clones into CD8 alpha+ dendritic cells (DC) suggests that monocytes can be direct precursors for both CD8 alpha+ and CD8 alpha- DC in the mouse

Dendritic cells (DC) are the professional APCs that initiate T cell immune responses. DC can develop from both myeloid and lymphoid progenitors. In the mouse, the CD8alpha(+) DC had been designated as "lymphoid" DC, and CD8alpha(-) DC as "myeloid" DC until recently when it was demonstrated that common myeloid progenitors can also give rise to CD8alpha(+) DC in bone marrow chimera mice. However, it is still not clear which committed myeloid lineages differentiate into CD8alpha(+) DC. Because monocytes can differentiate into DC in vivo, the simplest hypothesis is that the CD8alpha(+) DC can be derived from the monocyte/macrophage. In this study we show that cell clones, isolated from CD8alpha(+) DC lymphoma but with a monocytic phenotype (CD11c(low/-)D11b(high)CD8alpha(-)I-A(low)), can redifferentiate into CD8alpha(+) DC either when stimulated by LPS and CD40L or when they migrate into the lymphoid organs. Maturation of DC in vivo correlated with strong priming of allogeneic T cells. Moreover, the monocytes from cultured splenocytes or peritoneal exudates macrophages of wild-type mice are also capable of differentiating into CD11c(+)CD8alpha(+) DC after their migration into the draining lymph nodes. Our results suggest that monocytes can be direct precursors for CD11c(+)CD8alpha(+) DC in vivo. In addition, the monocyte clones described in this study may be valuable for studying the differentiation and function of CD8alpha(+) DC that mediate cross-presentation of Ag to CD8 T cells specific for cell-associate Ags.     

Introduction of T cell receptor (TCR)-alpha cDNA has differential effects on TCR-gamma delta/CD3 expression by PEER and Lyon-1 cells

A TCR heterodimer composed of a TCR gamma-chain and a TCR delta-chain was found to be expressed in association with CD3 by a small population of human peripheral blood T cells, thymocytes, and certain leukemic T cell lines. The leukemic T cell lines PEER and Lyon-1 express such a TCR-gamma delta/CD3 complex at the cell surface. In addition, PEER and Lyon-1 cells transcribe a productively rearranged TCR-beta gene. Introduction of TCR alpha-chain cDNA of human or murine origin resulted in cell surface expression of a TCR-alpha beta/CD3 complex on PEER and Lyon-1 cells. The expression of the TCR-gamma delta/CD3 complex on PEER cells was not affected by introduction of TCR-alpha cDNA. In contrast, introduction of a TCR-alpha cDNA and expression of the TCR-alpha beta/CD3 complex in Lyon-1 cells resulted in the disappearance of the TCR-gamma delta/CD3 complex. These data were confirmed by indirect immunofluorescence, at the protein level and by gene expression analysis. Triggering of the TCR-alpha beta/CD3 complexes by anti-CD3 mAb or anti-TCR mAb resulted in increased internal Ca2+ levels, indicating that these receptors were functional in signal transduction. These results indicate that, besides TCR gene rearrangements, membrane expression of TCR-alpha beta heterodimers may be important in regulating TCR-gamma delta cell surface expression.     

Quantitative and qualitative in vitro analysis of the stem cell potential of hematopoietic cells purified from murine skeletal muscle

The murine skeletal muscle contains hematopoietic stem cells, but this potential has so far not been studied quantitatively or qualitatively in vitro. To quantify the hematopoietic stem cell potential, we have used highly purified SP/CD45^＋ cells in long-term culture initiating cell （LTC-IC） assays. The SP/CD45^＋ cell population purified from murine muscle was found to have significant stem cell activity with an LTC-IC frequency of 1/640. Single-cell-sorted SP/CD45^＋ cells from muscle exhibited robust proliferative activity in vitro at day 16 （380-fold amplification）, especially after culture with OP-9 layers that also support embryonic stem cells. Amplified cell populations originating from single cells exhibited multilineage differentiation ability with evidence of myeloid, lymphoid and NK cell markers. Thus, our results demonstrate that hematopoietic stem cells that can be quantified by LTC-IC assays exist in the murine skeletal muscle and show also for the first time, at the single-cell level, that these cells exhibit multilineage differentiation ability and major proliferative potential.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

T regulatory cells control numbers of NK cells and CD8alpha+ immature dendritic cells in the lymph node paracortex

The spleen contains numerous NK cells whose differentiation profile is characterized by a preponderance of mature elements located mainly in the red pulp. In contrast, lymph nodes (LNs) contain few NK cells and they are sited mostly in T cell zones and skewed toward immature developmental stages. We show that, in mice, naturally occurring CD4+ Foxp3+ regulatory T (Treg) cells are both necessary and sufficient to repress accumulation of NK cells in resting LNs. Moreover, we present evidence that Treg cells hamper generation of mature NK cells through short-range interactions with NK precursors. In turn, mature NK cells specifically regulate the amount of CD8alpha+ phenotypically immature dendritic cells present in LN T cell zones. We propose that the dominant influence of Treg cells on NK cell precursors and CD8alpha+ immature dendritic cells explains why "quiescent" LNs in the absence of infection function as privileged sites for induction and maintenance of tolerance to peripheral Ags.     

Flt3-ligand, IL-4, GM-CSF, and adherence-mediated isolation of murine lung dendritic cells: assessment of isolation technique on phenotype and function

Lung dendritic cells (DCs) are difficult to study due to their limited quantities and the complexities required for isolation. Although many procedures have been used to overcome this challenge, the effects of isolation techniques on lung DCs have not been reported. The current study shows that freshly isolated DCs (CD11c+) have limited ability to induce proliferation in allogeneic T cells, and are immature as indicated by low cell surface expression of costimulatory molecules compared with liver or splenic DCs. DCs isolated after overnight culture or from mice treated with Flt3L are phenotypically mature and potent stimulators of allogeneic T cells. DCs could not be propagated from lung mononuclear cells in response to IL-4 and GM-CSF. Contrary to data reported for nonpulmonary DCs, expression of CCR6 was decreased on mature lung DCs, and only a subset of mature DCs expressed higher levels of CCR7. Absence of CD8alpha expression indicates that freshly isolated DCs are myeloid-type, whereas mature DCs induced by overnight culture are both "lymphoid" (CD8alpha+) and "myeloid" (CD8alpha-). DCs from mice genetically deficient in CD8alpha expression were strong simulators of allogeneic T cells which was consistent with data showing that CD8alpha- DCs from CD8alpha-sufficient mice are better APCs compared with CD8alpha+ DCs from the same mice. These data show that freshly isolated lung DCs are phenotypically and functionally distinct, and that the isolation technique alters the biology of these cells. Therefore, lung DC phenotype and function must be interpreted relative to the technique used for isolation.     

Phenotype, ontogeny, and repertoire of CD4-CD8- T cell receptor alpha beta + thymocytes. Variable influence of self-antigens on T cell receptor V beta usage

We have characterized CD4-CD8- double negative (DN) thymocytes that express TCR-alpha beta and represent a minor thymocyte subpopulation expressing a markedly skewed TCR repertoire. We found that DN TCR-alpha beta + thymocytes resemble mature T cells in that they (a) are phenotypically CD2hiCD5hiQa2+HSA-, (b) appear late in ontogeny, and (c) are susceptible to cyclosporin A-induced maturation arrest. In addition, we found that DNA sequences 5' to the CD8 alpha gene were demethylated relative to their germline state, suggesting that DN TCR-alpha beta + thymocytes are derived from cells that had at one time expressed their CD8 alpha gene locus. Because DN TCR-alpha beta + thymocytes are known to express an unusual TCR repertoire with significant overexpression of V beta 8, we were interested in examining the possible role played by self-Ag in shaping their TCR repertoire. It has been suggested that DN TCR-alpha beta + thymocytes are derived from potentially self-reactive thymocytes that have escaped clonal deletion by down-regulating their surface expression of CD4 and/or CD8 determinants. However, apparently inconsistent with such an hypothesis, we found that the frequency of DN thymocytes expressing various anti-self TCR (V beta 6, V beta 8.1, V beta 11, V beta 17a) were not increased in strains expressing their putative self-Ag, but instead were either unaffected or significantly reduced in those strains. With regard to V beta 8 expression among DN TCR-alpha beta + thymocytes, V beta 8 overexpression in DN TCR-alpha beta + thymocytes appeared to be independent of, and superimposed on, the developmental appearance of the basic DN thymocyte repertoire. Even though V beta 8 overexpression appeared to be generated by a mechanism distinct from that generating the rest of the DN TCR-alpha beta + thymocyte repertoire, we found that super-Ag against which V beta 8 TCR react introduced into the neonatal differentiation environment also significantly reduced, rather than increased, the frequency of DN TCR-alpha beta + V beta 8+ thymocytes. Thus, the present study is consistent with DN TCR-alpha beta + thymocytes being mature cells derived from CD8+ precursors, and documents that their TCR repertoire can be influenced, at least negatively, by either self-Ag or Ag introduced into the neonatal differentiation environment. However, we found no evidence to support the hypothesis that DN TCR-alpha beta + thymocytes are enriched in cells expressing TCR reactive against self-Ag.     

Synergistic and antagonistic effects of IL-1 alpha and IL-4, respectively, on the IL-2-dependent growth of a T cell receptor-gamma delta+ human T leukemia cell line

The TALL-103/2 cell line was derived from an immature acute T lymphocytic leukemia with T-myeloid differentiating capacity. The leukemic cells were first expanded in recombinant human IL-3 in which they acquired a myeloid phenotype, and subsequently were adapted to grow in human rIL-2 in which they became lymphoid committed. The TALL-103/2 cell line expresses only T cell-specific differentiation Ag (CD2, CD3, CD7, and CD8) but has retained the CD33 myeloid Ag originally present on the IL-3 expanded population. By using mAb directed at the TCR-alpha beta or specific for framework determinants on human TCR-gamma and -delta chains, the TALL-103/2 cells were shown to be WT31-, TCR delta 1+, TCS-1+, and Ti gamma A-, thus representing a T cell subset expressing the nondisulfide-linked form of the TCR-gamma delta. The TALL-103/2 cells have been maintained for more than 1 y in the presence of human rIL-2 on which they are strictly dependent. Chemical cross-linking and immunofluorescence studies indicate the presence of both high and intermediate affinity IL-2R on the TALL-103/2 cells. Whereas mAb antiTac and H-31 with reactivity to the IL-2R alpha-chain (p55) compete only partially for the IL-2-induced proliferation of these cells, mAb TU27, specific to the IL-2R beta-subunit (p75), inhibits such growth completely even at high concentrations of IL-2. The interactions of the two T cell-stimulating factors IL-1 and IL-4 on the IL-2-dependent growth of TALL-103/2 cells were investigated. IL-1 alpha synergizes with IL-2 in supporting the short and long term growth of this cell line, whereas IL-4 abrogates its growth. These effects are, at least in part, due to the modulation of IL-2R expression induced by the two lymphokines. Functionally, the TALL-103/2 cells display MHC-nonrestricted cytotoxic activity that is significantly enhanced by addition of either IL-4, IL-6, or IFN-gamma. Because of its properties and its stable requirement for IL-2 for continuous growth, this T lymphocytic leukemia-derived cell line represents an interesting model to analyze ontogeny and function of leukemic T cells.     

IL-3 increases production of B lymphoid progenitors from human CD34+CD38- cells

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38- cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38- cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34-CD19+ B cells. B cell production was increased whether CD34+CD38- cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Ralpha expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Ralpha protein expression was largely restricted to myeloid progenitors. CD34+CD38- cells had low to undetectable levels of IL-3Ralpha by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Ralpha protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38- cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.     

Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step, we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells, and then, expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype, expressed myeloperoxidase, and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules, CD1a, CD11c, CD80, CD86, DC-SIGN, and CD40; and were capable of Ag processing, triggering naive T cells in MLR, and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14, and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition, because ES cells can be expanded without limit, they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance.