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.     

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.     

The majority of CD4+8- thymocytes are functionally immature.

The thymus is the major site of T cell development and repertoire selection. During these processes, T cells segregate into two subsets that express either CD4 or CD8 accessory molecules, the phenotype of peripheral T cells. Analysis of CD4+8- thymocytes revealed that the majority of these cells express the heat-stable Ag (HSA) but not the nonclassical class I Ag, Qa-2. This HSA+, Qa-2- phenotype is similar to that of the less mature, CD4+8+ thymocytes. The remaining CD4+8- thymocytes possess the HSA-, Qa-2+ phenotype of peripheral T cells. To determine whether the Qa-2-, CD4+8- thymic subset is fully mature, we have analyzed the functional status of these CD4+8- subpopulations. The results indicate that only those thymocytes which express Qa-2 are fully responsive to anti-TCR stimulation in a manner analogous to peripheral T cells. The Qa-2- subset is nonresponsive to stimulation by anti-TCR antibodies that have been immobilized to plastic, even in the presence of lymphokines or syngeneic APC. This subset is, however, capable of proliferating to allogeneic cells or to anti-TCR on the surface of syngeneic APC, although not to the levels achieved by Qa-2+ thymocytes. Thus, the Qa-2- subset appears to require additional interactions which are not necessary for peripheral T cells or Qa-2+ thymocytes. Relevant to this issue, the Qa-2+ thymocyte subset does not appear until relatively late in development, and does not reach adult frequencies until several weeks after birth. These results would suggest that there is a progression from HSA+, Qa-2- to HSA-, Qa-2+ which parallels the maturation of functional responsiveness. These findings are important to understanding T cell selection since thymocytes with such a decreased responsiveness may have a differential capacity for tolerance induction. The results presented suggest that the bulk of CD4+8- thymocytes are not fully mature and that Qa-2 may serve as a marker for T cells with a more complete functional competence.     

Staphylococcal superantigens induce lymphotactin production by human CD4+ and CD8+ T cells.

Lymphotactin is a potent chemotactic cytokine (chemokine) that is produced by and also attracts T and natural killer (NK) cells. We are studying whether chemokines that affect mainly T cells might also regulate immune responses by preferentially recruiting individual subsets or by affecting cytokine or other chemokine responses. In order to pursue these questions, we need to learn more about the mechanisms regulating lymphotactin production and the cell types capable of releasing this factor. We used new monoclonal antibodies against human lymphotactin to develop a sensitive antigen-capture enzyme linked immunoabsorbent assay (ELISA) that measures chemokine levels in culture fluids. Using this capture ELISA, we showed that lymphotactin could be produced by CD4+ and CD8+ T cells, but only after T cell-receptor-dependent stimulation using bacterial superantigens and not after treatment by inflammatory cytokines or lipopolysaccharide (LPS). Our data show that lymphotactin production responds mainly to T cell-receptor signals in CD4+ and CD8+ T cells, and suggests a mechanism whereby this chemokine could help to regulate T cell immune responses.     

T cell-induced Ig allotypic suppression in mice. II. Both CD4+ CD8- and CD4- CD8+ T cell subsets from sensitized Igha mice are required to induce suppression of Igh-1b allotype expression

We established the phenotype of T splenocytes (Ts) from Igha/a BALB/c mice sensitized against B splenocytes from the Ighb/b CB20 congenic mice that induce Igh-1b (IgG2a of the Ighb haplotype) suppression. This was achieved by studying the action of anti-T cell subset mAb on the capacity of Ts to induce this chronic allotypic suppression in Igha/b (BALB/c x CB20)F1 mice. The Ts were treated with cytotoxic anti-mouse CD4 or anti-mouse CD8 rat mAb in vitro before their injection into the Igha/b newborns or in vivo after their injection into the Igha/b newborns. Exposure to either anti-CD8 or anti-CD4 mAb in vitro or in vivo leads to loss of the capacity of Ts to induce Igh-1b allotypic suppression. Mixing CD4+-cell-depleted Ts and CD8+-cell-depleted Ts preparations restored the capacity of the cells to induce Igh-1b suppression. Thus, both CD4+ CD8- Ts and CD4- CD8+ Ts are required for the induction of this allotypic suppression. Bone marrow cells and B splenocytes from Igh-1b-suppressed adult Igha/b mice were shown to be able to durably express Igh-1b when transferred into irradiated Igha/a BALB/c hosts whereas whole spleen cells from such donors failed to do it. Abrogation of Igh-1b suppression by in vivo anti-CD8 mAb treatment was achieved in adult Igha/b heterozygotes but with a lower efficiency than in adult Ighb/b homozygotes, all being chronically Igh-1b suppressed. The CD4- CD8+ cell population essential for maintaining this suppression is resistant to in vivo 600 rad irradiation and seems to be slightly inhibited by in vivo administration of free Igh-1b.     

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.     

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.     

Thymic nurse cells exclusively bind and internalize CD4+CD8+ thymocytes.

Thymic nurse cells (TNC) contain 20-200 thymocytes within specialized vacuoles in their cytoplasm. The purpose of the uptake of thymocytes by TNCs is unknown. TNCs also have the capacity to present self-antigens, which implies that they may serve a function in the process of thymic education. We have recently reported the development of thymic nurse cell lines that have the ability to bind and internalize T cells. Here, we use one of these TNC lines to identify the thymocyte subpopulation(s) involved in this internalization process. TNCs exposed to freshly isolated thymocytes bind and internalize CD4 and CD8 expressing thymocytes (CD4+CD8+ or double positives) exclusively. More specifically, a subset of the double-positive thymocyte population displayed binding capacity. These double-positive cells express cell surface alpha beta type T cell antigen receptor (TCR), as well as CD3 epsilon. Binding was not inhibited in the presence of antibodies against CD3, CD4, CD8, Class I antigens, or Class II antigens. These results describe two significant events in T cell development. First, TNCs exclusively bind and internalize a subset of alpha beta TCR expressing double-positive T cells. Also, binding is facilitated through a mechanism other than TCR recognition of major histocompatibility complex antigens. This suggests that thymocyte internalization may be independent of the process used by TNCs to present self-antigen.     

Activation of CD 4-, CD 8- thymocytes with IL 4 vs IL 1 + IL 2

Thymocytes from C57BL/6 mice were highly purified to obtain the CD 4-, CD 8- subpopulation which constitutes only 5% of all thymocytes. Substantial proliferation was induced in vitro with either IL-1 + IL-2 or with IL-4 in the presence of PMA. IL-1 and IL-2 synergized in inducing proliferation of these purified CD 4-, CD 8- thymocytes whereas neither synergized with IL-4. In order to determine whether stimulation with IL-1 + IL-2 acted via IL-4 or vice versa, cultures were treated reciprocally with affinity-purified anti-IL-2 or anti-IL-4 antibodies. Cultures with IL-4 were inhibited by anti-IL-4 but were unaffected by anti-IL-2. The CD 4-, CD 8- thymocytes cultured with IL-1 + IL-2 + anti-IL-2 were inhibited to baseline IL-1 stimulation. At low concentrations of IL-1 (1 U/ml) and IL-2 (100 U/ml), anti-IL-4 had no effect, whereas at higher levels of IL-1 (2 U/ml IL-1), and 100 or 200 U/ml IL-2, anti-IL-4 significantly reduced DNA synthesis. This result suggests that at higher concentrations the combination of IL-1 + IL-2 can induce cells to produce IL-4 which then contributes to overall proliferation. When CD 4-, CD 8- thymocytes were cultured with the low doses of IL-1 + IL-2 for 72 h, 62% expressed cell surface T3 complex (vs 11% at initiation) and 27% were F23.1+ (vs 5% at initiation). In contrast, culture with IL-4 led to no increase in numbers of T3+ cells and none were F23.1+; however, there was coexpression of Thy1 and 6B2 on 20% of cells at the end of culture (vs 4% at initiation). Thus, IL-1 + IL-2 causes expansion of a CD 4-, CD 8- thymocyte population expressing the alpha, beta-T cell receptor, whereas IL-4 induces cells to express a phenotype present in small numbers in the periphery of normal mice and in larger numbers in mice bearing the lpr gene.     

B7 costimulates proliferation of CD4-8+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes.

In addition to TCR-derived signals, costimulatory signals derived from stimulation of the CD28 molecule by its natural ligand, B7, have been shown to be required for CD4+8- T cell activation. We investigate the ability of B7 to provide costimulatory signals necessary to drive proliferation and differentiation of virgin CD4-8+ T-cells that express a transgenic TCR specific for the male (H-Y) Ag presented by H-2Db class I MHC molecules. Virgin male-specific CD4-8+ T cells can be activated either with B7 transfected chinese hamster ovary (CHO) cells and T3.70, a mAb specific for the transgenic TCR-alpha chain that is associated with male-reactivity, or by male dendritic cells (DC). Activated CD4-8+ T cells proliferated in the absence of exogenously added IL-2. IL-2 activity was detected in supernatants of CD4-8+T3.70+ cells that were stimulated with T3.70 and B7+CHO cells. The response of CD4-8+T3.70+ cells to T3.70/B7+CHO or to male DC stimulation were inhibited by CTLA4Ig, a fusion protein comprising the extracellular portion of CTLA4 and human IgG C gamma 1. It has been previously shown that CTLA4Ig binds B7 with high affinity. Staining with CTLA4Ig revealed that DC express about 50 times more B7 than CD4-8+ T cells. CTLA4Ig also specifically blocked the proliferation of male-reactive cells in vivo. We have also used an in vitro deletion assay whereby immature CD4+8+ thymocytes expressing the transgenic male-specific TCR are deleted by overnight incubation with either immobilized T3.70 or male DC to investigate the participation of the CD28/B7 pathway in the negative selection of immature thymocytes. Staining with B7Ig established that both immature murine CD4+8+ and mature CD4-8+ thymocytes express a high level of CD28. However, despite the high expression of CD28 on CD4+8+ thymocytes, it was found that deletion of CD4+8+ thymocytes expressing the male-specific TCR by the T3.70 mAb was not inhibited by B7+CHO cells. Furthermore, the deletion of these thymocytes by DC also was not inhibited by CTLA4Ig. These findings provide evidence that although signaling through CD28 can costimulate a primary anti-male response in mature CD4-8+ T cells, the CD28/B7 pathway does not appear to participate in the negative selection of immature CD4+8+ thymocytes.     

MEK activity regulates negative selection of immature CD4+CD8+ thymocytes

CD4+CD8+ thymocytes are either positively selected and subsequently mature to CD4 single positive (SP) or CD8 SP T cells, or they die by apoptosis due to neglect or negative selection. This clonal selection is essential for establishing a functional self-restricted T cell repertoire. Intracellular signals through the three known mitogen-activated protein (MAP) kinase pathways have been shown to selectively guide positive or negative selection. Whereas the c-Jun N-terminal kinase and p38 MAP kinase regulate negative selection of thymocytes, the extracellular signal-regulated kinase (ERK) pathway is required for positive selection and T cell lineage commitment. In this paper, we show that the MAP/ERK kinase (MEK)-ERK pathway is also involved in negative selection. Thymocytes from newborn TCR transgenic mice were cultured with TCR/CD3epsilon-specific Abs or TCR-specific agonist peptides to induce negative selection. In the presence of the MEK-specific pharmacological inhibitors PD98059 or UO126, cell recovery was enhanced and deletion of DP thymocytes was drastically reduced. Furthermore, development of CD4 SP T cells was blocked, but differentiation of mature CD8 SP T cells proceeded in the presence of agonist peptides when MEK activity was blocked. Thus, our data indicate that the outcome between positively and negatively selecting signals is critically dependent on MEK activity.     

Effects of CD4+ and CD8+ T cells in tumor-bearing mice on antibody production

The function of T cell subsets in tumor-bearing mice was examined using an in vitro culture system of anti-(sheep red blood cell) antibody production, which is known to be dependent on T cells. The helper function of T cells of fibrosarcoma-MethA-bearing mice in antibody production decreased with the tumor stage of the mice. T cells were separated into CD4⁺ and CD8⁺ cells for further analysis of T cell subsets by the panning method using monoclonal antibodies. The helper function of CD4⁺ T cells in antibody production began to decrease significantly in tumor-bearing mice 1 week after the tumor transplantation. On the other hand, the suppressive function of CD8⁺ T cells was retained and had not decreased in the mice even 3 weeks after the transplantation. The same changes in function of CD4⁺ and CD8⁺ T cells were also observed in Methl-bearing mice. These results suggested that this tumor-associated immunosuppression in antibody production is attributable to the decrease in helper activity of CD4⁺ T cells and the maintenance of the suppressive activity of CD8⁺ T cells.     

Soluble factor-mediated differentiation of CD4+CD8+ thymocytes to single positives in vitro

Although the thymic microenvironment provides the necessary elements for T-cell differentiation, the precise role of individual components remains to be determined. In this paper, attempts were made to address the possibility that CD4 or CD8 single-positive (SP) thymocytes could be developed from immature CD4+CD8+ (double-positive; DP) thymocytes in a suspension culture in the presence of soluble factors. We observed that IL-4 and IFN-gamma weakly induced DP cells to differentiate to CD4 cells, but not to CD8. In contrast, IL-2 weakly induced differentiation to CD8. Interestingly, Con A sup strongly induced differentiation to CD8 SP from the purified DP thymocytes prepared from C57BL/6 or LCMV TCRtg mice. In particular, it was found that thymocyte culture with Con A sup generated CD69+DP cells, and the CD69+DP differentiated to CD8 SP under the suspension culture with soluble factors. Thus, Con A sup or combinations of IL-2, IL-4 and IL-7 strongly induced differentiation of CD69+DP to CD8 SP, whereas individual cytokines did not. These results suggest that soluble factors like cytokines play an important role in the generation of SP thymocytes in the absence of thymic stromal cells, at least from a distinctive subpopulation like CD69+DP thymocytes, and perhaps from those of broader range when in conjunction with TCR/MHC interaction.     

CD8+ T lymphocytes protect SCID mice against Encephalitozoon cuniculi infection

Microsporidia are obligate intracellular parasites that cause opportunistic infections in immunocompromised patients. The role of two main T cell subsets in anti-microsporidial immunity has been studied using an Encephalitozoon cuniculi-severe combined immunodeficient (SCID) mouse model. Whereas SCID mice reconstituted with CD4+ T lymphocyte-depleted naive BALB/c splenocytes resolved the infection, adoptive transfer of CD8+ T cell-depleted splenocytes failed to protect the animals against a lethal E. cuniculi infection. Splenocytes from E. cuniculi-immune mice specifically killed syngeneic infected macrophages in a short-term 51Cr-release assay. These results suggest the crucial role of cytotoxic T lymphocytes in the protection against E. cuniculi infection.     

Functional characterization of MHC class II-restricted CD8+CD4- and CD8-CD4- T cell responses to infection in CD4-/- mice

Classical CD4(+) and CD8(+) T cells recognize Ag presented by MHC class II (MHCII) and MHC class I (MHCI), respectively. However, our results show that CD4(-/-) mice mount a strong, readily detectable CD8(+) T cell response to MHCII-restricted epitopes after a primary bacterial or viral infection. These MHCII-restricted CD8(+)CD4(-) T cells are more similar to classical CD8(+) T cells than to CD4(+) T cells in their expression of effector functions during a primary infection, yet they also differ from MHCI-restricted CD8(+) T cells by their inability to produce high levels of the cytolytic molecule granzyme B. After resolution of a primary infection, epitope-specific MHCII-restricted T cells in CD4(-/-) mice persist for a long period of time as memory T cells. Surprisingly, upon reinfection the secondary MHCII-restricted response in CD4(-/-) mice consists mainly of CD8(-)CD4(-) T cells. In contrast to CD8(+) T cells, MHCII-restricted CD8(-)CD4(-) T cells are capable of producing IL-2 in addition to IFN-gamma and thus appear to have attributes characteristic of CD4(+) T cells rather than CD8(+) T cells. Therefore, MHCII-restricted T cells in CD4(-/-) mice do not share all phenotypic and functional characteristics with MHCI-restricted CD8(+) T cells or with MHCII-restricted CD4(+) T cells, but, rather, adopt attributes from each of these subsets. These results have implications for understanding thymic T cell selection and for elucidating the mechanisms regulating the peripheral immune response and memory differentiation.     

Extracellular ATP induces cell death in CD4+/CD8+ double-positive thymocytes in mice infected with Trypanosoma cruzi

In the acute phase of Trypanosoma cruzi infection, there is dramatic atrophy of the thymus. However, the pathways involved in this change have not yet been identified. This event is mainly characterized by a massive loss of cortical CD4+/CD8+ double-positive cells, but also by other structural and functional alterations in the organ. A number of molecules, including extracellular ATP, have been suggested to play a role in the selective processes that take place in the thymus. ATP and analogues trigger many different cellular responses in thymocytes and other cell types, such as the opening of plasma membrane cation channels and a pore that may induce cell death. Herein, we investigated the possible involvement of extracellular ATP in thymus atrophy induced by infection with T. cruzi. We observed that ATP induces an increase in plasma membrane permeabilization and cellular death in CD4+/CD8+ double-positive thymocytes collected from infected mice during the atrophy phase. No differences were observed prior to the atrophy phase or during the chronic phase. Our results indicate that P2Z/P2X7 receptors may play a central role in thymus atrophy during T. cruzi infection.