IL-7 maintains the T cell precursor potential of CD3-CD4-CD8- thymocytes.

We and other investigators have reported that IL-4 (in the presence of PMA) or IL-7 (used alone) induce proliferation of both adult and fetal (gestation day 15) CD4-CD8- thymocytes. These results suggested that these cytokines may be growth factors for pre-T cells. However, we recently observed that among adult CD4-CD8- thymocytes, only the CD3+ subset proliferates in response to IL-7, whereas IL-4 + PMA induces proliferative responses in both CD3- and CD3+ subsets. Thus, we concluded that IL-7 used alone is not a potent growth stimulus for adult thymic CD3-CD4-CD8- triple negative (TN) T cell precursors. Interestingly, the viability of adult TN thymocytes in culture was improved by IL-7 for up to 1 wk, in spite of the inability of IL-7 to induce significant [3H]TdR incorporation in these cells. After culture in IL-7 for 4 days, the viable cells remained CD4-CD8-, but 25 to 35% expressed CD3 whereas the rest remained CD3-. In contrast, most of the cells cultured with IL-4 + PMA for 4 days remained TN. To investigate whether adult TN thymocytes that survive in vitro in the presence of IL-4 + PMA or IL-7 retain T cell progenitor potential, we tested whether they could reconstitute lymphoid cell-depleted (2-deoxyguanosine-treated) fetal thymus organ cultures. Our results demonstrate that TN cells cultured in IL-7 retain T cell progenitor potential.     

A novel T cell-activating molecule (THAM) highly expressed on CD4-CD8- murine thymocytes

Recent studies have focused on the potential role of accessory molecules such as CD2, CD28, Thy-1, or TAP in the delivery of activating signals to thymocytes through antigen-independent pathways. To better understand the molecular interactions involved in the expansion of early thymic immigrants, rat mAb were raised against murine thymocyte-surface molecules and screened for their capacity to trigger thymocyte proliferation. One of these mAb (H194-112, IgG2a) was found to recognize a novel heterodimeric thymocyte-activating molecule (THAM) of Mr = 110,000 to 128,000. Flow cytometric analyses and staining patterns on frozen thymus sections subdivided adult thymocytes in three subsets expressing THAM at either low (10%), moderate (80%), or high (5 to 8%) cell-surface density; these cell groups were found to correspond, respectively, to the medullary, the cortical, and the immature CD4-CD8-, J11d+ thymocytes, in which the T cell precursor pool is included. Moreover, most (90%) day 16 fetal thymocytes were also found to upregulate THAM cell-surface expression. The THAMhigh cells were localized in the subcapsular area of the neonatal thymus and scattered throughout the adult organ. Cross-linked mAb H194-112 induced the proliferation of both immature and mature thymocytes in the presence of either PMA or IL-1 and IL-2. The observation that early thymocytes up-regulate THAM along with the IL-2R suggests that this molecule might be involved in an important activation pathway during thymocyte differentiation.     

Subpopulations of mature murine thymocytes: properties of CD4-CD8+ and CD4+CD8- thymocytes lacking the heat-stable antigen

The heat-stable antigen (HSA), recognized by the monoclonal antibodies M1/69, B2A2, and J11d, is low or absent on the surface of most murine peripheral T cells but present on all but 3% of thymocytes. The CD4-CD8+ and CD4+CD8- or "single positive" thymic populations may be divided into further subgroups based on surface HSA expression. One group, CD4-CD8+ and expressing very high levels of HSA (HSA++), is an immature, T cell antigen receptor (TcR) negative, outer cortical blast cell. However, a further subdivision of CD4-CD8+ and CD4+CD8- single positives may be made, into those negative to low for HSA (HSA-) and those expressing moderate amounts of HSA (HSA+). The proportion of HSA- single positives is low in the thymus of young mice, whereas the proportion of HSA+ single positives is similar to that of the adult. Both the HSA- and the HSA+ subsets of single positive thymocytes from adult mice are CD3+ and express the normal peripheral T cell incidence of V beta 8 determinants on the TcR. On stimulation with concanavalin A in limit-dilution culture both HSA- and HSA+ subsets of single positive thymocytes give a high frequency of proliferating clones, and the clones from both HSA- and HSA+ subsets of CD4-CD8+ thymocytes are cytotoxic. Thus both HSA- and HSA+ single positive thymocytes are functionally mature. The HSA- subsets of single positive thymocytes differ from the HSA+ subsets in being slightly larger in size, in expressing higher levels of MEL-14, in binding more peanut agglutinin, and in including a proportion of cells expressing high levels of the Pgp-1 glycoprotein. It is suggested that HSA- CD4-CD8+ and HSA- CD4+CD8- thymocytes are more mature than their HSA+ counterparts, and might represent a previously activated or "memory" thymic subpopulation.     

Sister-chromatid exchange in highly purified human CD4+ and CD8+ lymphocytes.

Sister-chromatid exchange (SCE) frequencies were determined in human peripheral blood CD₄⁺ and CD₈⁺ T lymphocyte subpopulations which were rapidly and highly purified from pooled T lymphocytes by immunological methods. The purified lymphocytes were stimulated with phytohemagglutinin (PHA) for 4 days. CD₄⁺ lymphocytes showed significantly higher SCE frequencies than autologous CD₈⁺ lymphocytes when measured simultaneously after identical bromodeoxyuridine (BrdU) incubation times. Differences in SCE frequencies between CD₄⁺ and CD₈⁺ lymphocytes were also detected when mitomycin C (MMC) was added to the cultures. Higher SCE frequencies in CD₄⁺ lymphocytes were associated with lower proliferating rate indices (PRI) as compared to autologous CD₈⁺ lymphocytes. Abnormalities in CD₄⁺ T lymphocyte function and number in peripheral blood have been observed in several diseases characterized by immunological disorders. Thus, our data may suggest a link between some immunological disturbances and abnormal SCE frequencies in T lymphocyte subsets.     

Subpopulations of early thymocytes. A cross-correlation flow cytometric analysis of adult mouse Ly-2-L3T4-(CD8-CD4-) thymocytes using eight different surface markers

Putative early thymocytes, the Ly-2-L3T4-(CD8-CD4-) cells representing 3 to 4% of adult CBA mouse thymic lymphocytes, were isolated in high purity (99.5%). They were then stained by using mAb and analyzed by flow cytometry for the expression of six additional surface antigenic markers. Cross-correlation of the data obtained from a complete series of successive two-parameter analyses revealed the existence of about 11 discrete subsets, falling into four-main groups, within the Ly-2-L3T4- population. All subsets consisted of relatively large lymphoid cells. The most numerous group of Ly-2-L3T4- cells was Ly-1 low B2A2-M1/69 high Thy-1 high Pgp-1 low and by these markers resembled Ly-2+L3T4+ cortical blasts. Many of the cells in this group were positive for the IL-2R and/or for MEL-14. A second major group of Ly-2-L3T4- cells was Ly-1 high B2A2-M1/69 low Pgp-1 high, and resembled in some respects activated mature T cells. This group had previously been shown to be absent from the embryonic thymus. The group could be divided into Thy-1 high and Thy-1 low subsets. None of the cells in this group were positive for the IL-2R and very few expressed MEL-14. A third group, 13% of the Ly-2-L3T4- population, was Ly-1 low B2A2-M1/69 low Pgp-1 high, and could also be divided into Thy-1 high and Thy-1 low subsets. A final minor group, 9% of the Ly-2-L3T4- population, was Ly-1 high B2A2-M1/69 high Pgp-1 low Thy-1 high. The particular pattern of markers on these subsets, combined with subsequent information on their properties, makes it unlikely that they all represent sequential steps in one continuous developmental stream, and indicates that complex developmental steps have occurred, even at this supposedly early stage of T cell differentiation.     

T cell antigen receptor expression by subsets of Ly-2-L3T4- (CD8-CD4-) thymocytes

The V beta 8-specific mAb F23.1 and KJ16 were used as fluorescent stains to test for TCR expression on the surface of subpopulations of early, CD4-CD8- (L3T4-Ly-2-) thymocytes from adult CBA mice. A surprisingly high proportion (27%) of Ly-2-L3T4- thymocytes were strongly F23.1 and KJ16 positive. No positive cells were detected among Ly-2-L3T4- thymocytes from V beta 8-negative SJL mice. In contrast to the adult thymus, Ly-2-L3T4- cells from embryonic CBA thymus lacked F23.1-positive cells. Subsets of adult CBA Ly-2-L3T4- thymocytes were separated to determine which expressed V beta 8. The major subset, Ly-1 low B2A2-M1/69+Thy-1+Pgp-1-, representing a phenotype similar to embryonic Ly-2-L3T4- thymocytes and the phenotype commonly isolated from adult thymocytes as Ly-1 "dull," lacked cells strongly positive for F23.1. In contrast, a series of subsets of adult CBA Ly-2-L3T4- thymocytes which were B2A2-M1/69- and Pgp-1+ all included strongly F23.1-positive cells. A minor subset, negative for most markers except Pgp-1 and presumed on the basis of this phenotype and some reconstitution studies to include the earliest intrathymic precursors, contained 28% F23.1-positive cells. However, no F.23.1-positive cells were detected in equivalent "prethymic" populations from bone marrow or from athymic mouse spleen. The subsets of Ly-2-L3T4- thymocytes which were Ly-1 high, B2A2-M1/69-, and Pgp-1+ all contained about 70% F23.1-positive cells, indicating a V beta 8 usage much higher than the mature T cell average. These results indicate that a series of distinct developmental events have occurred within these CD4-CD8- thymocytes previously considered as a single group of early precursor cells, and that some aspects of repertoire selection may be occurring amongst thymocytes which lack CD4 or CD8.     

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.     

Gads-/- mice reveal functionally distinct subsets of TCRbeta+ CD4-CD8- double-negative thymocytes

TCRbeta expression in CD4(-)CD8(-) double-negative (DN) thymocytes induces signaling pathways that promote survival and proliferation, as well as differentiation into CD4(+)CD8(+) double-positive thymocytes. The signaling pathways that regulate survival, proliferation, and differentiation remain unclear. We used Gads-deficient mice to investigate the signaling pathways that regulate these cell fates. During this investigation, we focused on TCRbeta(+) DN thymocytes and found that there are at least three functionally distinct subsets of TCRbeta(+) DN thymocytes: TCRbeta(+) DN3E, TCRbeta(+) DN3L, and TCRbeta(+) DN4. Survival and proliferation of TCRbeta(+) DN3E were independent of Gads, but survival and proliferation of TCRbeta(+) DN3L cells were Gads dependent. Likewise, expression of Bcl-2 in TCRbeta(+) DN3E cells was Gads independent, but Gads was necessary for Bcl-2 expression in TCRbeta(+) DN3L cells. Bcl-2 expression was not dependent on Gads in TCRbeta(+) DN4 cells, but proliferation of TCRbeta(+) DN4 cells was Gads dependent. Gads was not required for the differentiation of DN thymocytes into DP thymocytes. In fact, Gads(-/-) DN3E cells differentiated into DP thymocytes more readily than wild-type cells. We conclude that signaling pathways required to initiate TCRbeta-induced survival and proliferation are distinct from the pathways that maintain survival and proliferation. Furthermore, signaling pathways that promote survival and proliferation may slow differentiation.     

Immature CD4- CD8+ murine thymocytes

Mature thymocytes are usually defined and separated from other less mature thymocytes on the basis of their mutually exclusive expression of either CD4 or CD8. However, such murine "single positives" include a subpopulation of immature cells with properties resembling CD4- CD8- thymocytes or CD4+ CD8+ cortical blasts. Most of these immature single positives are CD4- CD8+, some expressing relatively low levels of CD8. They are large, dividing cortisone-sensitive cells found in the outer cortex. They express high levels of the heat-stable antigen (recognized by the monoclonals M1/69, B2A2, and J11d) but they are MEL-14-. The absence of detectable surface CD3, the absence of alpha-chain messenger RNA, and the predominance of the truncated form of the beta-chain messenger RNA all indicate that they do not express the T-cell antigen-receptor complex. Strategies for eliminating such immature cells from preparations of mature thymocytes are given, and their developmental significance is discussed.     

Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

CD4+CD8- thymocytes from neonatal mice induce IgM production in SCID mice.

Defective recombination of both the TCR and Ig genes results in the absence of mature lymphocytes in mice with the scid mutation. We have shown previously that the transfer of neonatal, but not adult, thymocytes results in high levels of Ig production in 100% of C.B-17-scid (SCID) mice, in contrast to the 10 to 25% of SCID mice spontaneously producing low levels of oligoclonal Ig. In this report we demonstrate that neonatal CD4+8- thymocytes were able to induce this response; the CD4+8+ and CD4-8+ subpopulations were totally inactive and CD4-8- T cells had only limited activity several weeks after transfer. The stimulation of IgM production in SCID mice was detectable by 1 wk posttransfer of CD4+8- thymocytes or splenic T cells, and could be achieved with as few as 300 cells. The ability of neonatal CD4+8- thymocytes to induce Ig diminished gradually to insignificant levels at 3 wk postbirth; this loss of function was not associated with differential survival of neonatal T cells. Neonatal CD4+8- thymocytes from C.B-17 and other H-2d strains rescued Ig production, whereas cells from H-2b, H-2a, and H-2k strains were much less effective. These results suggest that a CD4+8- subpopulation found in both neonatal thymus and peripheral lymphoid tissues is able to induce the expansion or differentiation of the small numbers of functional B lymphocytes in SCID mice, and that the inducing T cell disappears shortly after birth, perhaps during the acquisition of self-tolerance.     

B7-CD28 interaction promotes proliferation and survival but suppresses differentiation of CD4-CD8- T cells in the thymus

Costimulatory molecules play critical roles in the induction and effector function of T cells. More recent studies reveal that costimulatory molecules enhance clonal deletion of autoreactive T cells as well as generation and homeostasis of the CD25(+)CD4(+) regulatory T cells. However, it is unclear whether the costimulatory molecules play any role in the proliferation and differentiation of T cells before they acquire MHC-restricted TCR. In this study, we report that targeted mutations of B7-1 and B7-2 substantially reduce the proliferation and survival of CD4(-)CD8(-) (double-negative (DN)) T cells in the thymus. Perhaps as a result of reduced proliferation, the accumulation of RAG-2 protein in the DN thymocytes is increased in B7-deficient mice, which may explain the increased expression of TCR gene and accelerated transition of CD25(+)CD44(-) (DN3) to CD25(-)CD44(-) (DN4) stage. Qualitatively similar, but quantitatively less striking effects were observed in mice with a targeted mutation of CD28, but not CTLA4. Taken together, our results demonstrate that the development of DN in the thymus is subject to modulation by the B7-CD28 costimulatory pathway.     

TCRαβ^＋CD^＋4CD^—8胸腺髓质细胞的表型分析

Phenotypic analysis of the medullary-type CD4+CD8- (CD4SP) thymocytes have revealed phenotypic heterogeneity within these cells. The phenotype of mature peripheral T cells is Qa-2+ HSA- CD69-, whereas in the medullary-type CD4SP thymocytes, the expression pattern of many markers were quite different, suggesting that the medullary-type CD4SP thymocytes may undergo phenotypic maturation. According to the results of two-color cytometry, seven discrete phenotypes were defined by the relative expression of Qa-2, HSA, CD69, 3G11 and 6C10: 3G11-6C10+CD69+HSAhi-->3G11+6C10+CD69+ HSAhi-->3G11+6C10-CD69+HSAint-->3G11+6C10- CD69-HSAint Qa-2(-)-->3G11+HSAlo/-Qa-2lo, at the same time, 3G11+6C10-CD69-HSAint Qa-2(-)-->3G11-HSAlo Qa-2(-)-->3G11-HSAlo/- Qa-2hi, the last two Qa-2 positive subsets could exit the thymus and home into periphery.     

NK1.1+ thymocytes. Adult murine CD4-, CD8- thymocytes contain an NK1.1+, CD3+, CD5hi, CD44hi, TCR-V beta 8+ subset.

CD4-, CD8- thymocytes were purified from thymi obtained from normal C57BL/6 mice. By flow cytometry analysis, 5 to 10% of these double negative (DN) thymocytes were found to express NK1.1 on their surface. The NK1.1+ DN thymocytes were demonstrated, by two-color fluorescence, to be CD3lo, CD5hi, CD44hi, J11d-, B220-, MEL 14-, IL2R- with 60% expressing TCR-V beta 8 as determined by the mAb F23.1. In contrast, splenic and peripheral blood NK cells were NK1.1+, CD3-, CD5-, TCR-V beta 8- with 40 to 60% being MEL 14+. Unlike peripheral NK cells, fresh DN thymocytes enriched for NK1.1+ cells were unable to kill YAC-1, the classical murine NK cell target. However, these cells were able to mediate anti-CD3 redirected lysis even when they were assayed immediately after purification, i.e., with no culture or stimulation. These data demonstrate that adult murine thymocytes contain NK1.1+ cells which are distinct, both by function and phenotype, from peripheral NK cells. These data also raise the issue of a possible NK/T bipotential progenitor cell.     

Generation of CD3+CD8low thymocytes in the HIV type 1-infected thymus

Infection with the HIV type 1 (HIV-1) can result both in depletion of CD4(+) T cells and in the generation of dysfunctional CD8(+) T cells. In HIV-1-infected children, repopulation of the peripheral T cell pool is mediated by the thymus, which is itself susceptible to HIV-1 infection. Previous work has shown that MHC class I (MHC I) molecules are strongly up-regulated as result of IFN-alpha secretion in the HIV-1-infected thymus. We demonstrate in this study that increased MHC I up-regulation on thymic epithelial cells and double-positive CD3(-/int)CD4(+)CD8(+) thymocytes correlates with the generation of mature single-positive CD4(-)CD8(+) thymocytes that have low expression of CD8. Treatment of HIV-1-infected thymus with highly active antiretroviral therapy normalizes MHC I expression and surface CD8 expression on such CD4(-)CD8(+) thymocytes. In pediatric patients with possible HIV-1 infection of the thymus, a low CD3 percentage in the peripheral circulation is also associated with a CD8(low) phenotype on circulating CD3(+)CD8(+) T cells. Furthermore, CD8(low) peripheral T cells from these HIV-1(+) pediatric patients are less responsive to stimulation by Ags from CMV. These data indicate that IFN-alpha-mediated MHC I up-regulation on thymic epithelial cells may lead to high avidity interactions with developing double-positive thymocytes and drive the selection of dysfunctional CD3(+)CD8(low) T cells. We suggest that this HIV-1-initiated selection process may contribute to the generation of dysfunctional CD8(+) T cells in HIV-1-infected patients.