CD 69 expression during selection and maturation of CD4+8+ thymocytes

We have analyzed the inducibility of protein kinase C (PKC)-dependent expression of CD 69 molecules in T cell receptor (TCR) transgenic thymocytes developing in the presence or absence of selecting, class I major histocompatibility complex (MHC) molecules. Small CD4⁺8⁺ thymocytes developing in the absence of selecting MHC molecules could not be induced to express CD 69 by TCR cross-linking even after spontaneous in vitro up-regulation of their TCR level which resulted in enhanced Ca⁺⁺ flux. In contrast, a small proportion of CD4⁺8⁺TCR^low and most TCR^high (CD4⁺8⁺ and CD4⁺8⁺) thymocytes developing in the presence of selecting MHC ligands could be induced to express CD 69 upon TCR cross-linking. Unlike the anti-TCR antibody, phorbol 12-myristate 13-acetate - a direct activator of PKC - induced the expression of CD 69 on all thymocytes. These results suggest that positive selection of CD4⁺8⁺ thymocytes results in coupling of TCR-mediated signals to the CD 69 expression pathway. In vitro analysis of thymocytes before and after positive selection suggests that (1) positive selection does not immediately result in resistance to deletion and (2) that sustained TCR ligation is needed to promote maturation of positively selected CD4⁺8⁺ thymocytes resulting in gradual loss of the sensitivity to deletion and acquisition of the ability to proliferate in response to TCR-mediated signals.     

Differential induction of helper and killer T cells from isolated CD4+CD8+ thymocytes in suspension culture

Thymocytes of T cell receptor transgenic mice with nonselecting and RAG-2^−/− backgrounds were developmentally arrested at the CD4⁺CD8⁺ stage before positive selection. These thymocytes underwent lineage commitment upon transient stimulation with a combination of ionomycin, a calcium ionophore, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, in suspension culture. The effective drug doses were limited within narrow ranges and much lower than those which induce proliferation of mature T cells. The doses corresponded to those which inhibit glucocorticoid-induced apoptosis in these thymocytes. CD4 lineage commitment required longer duration, higher intensity of the stimulation, or both, than CD8 lineage commitment. Functional helper T cells (Th1 and Th2) were induced from the CD4 lineage-committed cells upon secondary stimulation with a combination of ionomycin and PMA followed by lymphokine treatment. Cytotoxic T cells were induced from the CD8 lineage-committed cells upon incubation with concanavalin A and irradiated splenic dendritic cells, but not with the combination of ionomycin and PMA. These results indicate that positive selection is mimicked by the pharmacological stimulation in the absence of other cell types, but that final maturation of CD8 T cells may require a different signal.     

Thymic Nurse Cells Support CD4^-CD8^＋ Thymocytes to Differentiate into CD4^＋CD8^＋ Cells

Thymic nurse cells （TNCs） represent a unique microenvironment in the thymus for T cell maturation. In order to investigate the role of thymic nurse cells during T cell differentiation, a TNC clone, RWTE-1, which formed a typical complex with fetal thymocytes in vitro was established from normal Wistar rat. Hanging drop culture method was applied to reveal the interaction between TNCs and thymocytes. Our result revealed that eighty percent of immature CD4^-CD8^＋ cells differentiated into CD4^＋CD8^＋ cells after a 12-hour hanging drop culture with RWTE-1. However, in a 12-hour culture of immature CD4^-CD8^＋ cells with or without RWTE-1 supernatant, only 30% of the cells differentiated into CD4^＋CD8^＋ cells spontaneously. This observation led to the conclusion that RWTE-1 cell has the capacity to facilitate immature CD4^-CD8^＋ thymocytes to differentiate into CD4^＋CD8^＋ T cells by direct interaction.     

In vivo and in vitro repertoire of CD3+CD4 CD8 thymocytes

CD4-CD8- thymocytes contain a cell subset which expresses thecomplete CD3-TCR complex (/ or /) of which the ontogeny andfiliation are unknown. One of the questions is whether thispopulation can be intrathymically selected, an obligatory stepfor the mature CD4⁺ and CD8⁺ cell differentiation pathway, orif the absence of CD4 and CD8 allows them to escape thymic selection.The repertoire of CD3 ⁺ CD4 - CD8 - (CD3 ⁺ DN) thymocytes wasanalyzed in different strains of mice with different combinationsof H-2 and Mls expression. The expression of V_ß8.1in freshly isolated CD3⁺ DN cells is the highest in Mls-l^b miceand the lowest in MIS-l^a and F₁ mice, suggesting that selectionagainst this specificity can be achieved in vivo. By contrast,a low percentage of V_ß6⁺ cells is found in all thestrains, with no correlation according to MIS expression. Invitro culture of DN thymocytes with IL-1 and IL-2 leads to theproliferation of CD3⁺ DN cells. In vitro culture amplifies thein vivo pattern of V_ß8.1 expression, while V_ß6⁺cells only expand in DN cells of 66 and 61002 Mls-l^b mice withthe same genetic background. These results show: (i) CD3⁺ DNthymic cells can be intrathymically selected but the repertoireis different from that of mature T cells; (ii) V_ß6and V_ß8.1 selection do not follow the same pattern;(iii) this repertoire can be modified by In vitro culture, towarda more mature type; and (iv) comparison of DN cell repertoireof BALBlc, BALB.D2 (congenic for MLs), and other strains ofmice suggests a multigenic control for this selection, and aninvolvement of background genes.     

Differentiation of CD4highCD8low coreceptor-skewed thymocytes into mature CD8 single-positive cells independent of MHC class I recognition

Thymocytes with a CD4^hiCD8^lo coreceptor-skewed (CRS) phenotype have been shown to contain precursors for CD8 single-positive (SP) thymocytes, in addition to precursors for CD4 SP cells. The selection mechanisms that stimulate CD4^hiCD8^lo cells to revert to the CD8 lineage are not known. Mice transgenic (tg) for the major histocompatibility complex (MHC) class I-restricted P14 T cell receptor (TCR), on the H-2^bm13 background, generate a large number of CD4^hiCD8^lo CRS thymocytes. We analyzed the developmental potential and the differentiation requirements of the CD4^hiCD8^lo population of these mice. Using reaggregate thymic organ cultures (RTOC), we observed that these cells efficiently and almost exclusively differentiate into CD8 SP cells. Differentiation occurred independent of whether or not the MHC haplotype of the thymic stroma corresponds to the MHC restriction of the tg TCR. Loss of CD4 was independent of thymic stroma, up-regulation of CD8 to full levels was dependent on thymic stroma but independent of MHC haplotype. After trypsin treatment and overnight incubation, these CRS cells re-expressed CD8 but failed to re-express CD4, indicating that they are in the process of terminating CD4 synthesis. CD8 SP cells derived from the CRS cells proliferate in response to peptide-pulsed antigen-presenting cells. Our data suggest that CD4^hiCD8^lo CRS thymocytes bearing the P14 tg TCR have completed positive selection and differentiate autonomously into functionally competent CD8 SP cells.     

The acquisition of CD4 and CD8 during the differentiation of early thymocytes in short-term culture

Subpopulations of thymocytes known to represent early stages of T cell development were isolated from the adult mouse thymus, and their ability to differentiate during short periods of culture was assessed by their acquisition of surface CD4 and CD8. Virtually all cells of the most mature of the CD4-CD8- thymocyte subpopulations (other surface markers CD3- HSA++ IL-2R-Pgp-1-) and of the immature CD4-CD8+ thymocyte subpopulation (other surface markers also CD3- HSA++ IL-2R- Pgp-1-) became CD4+CD8+ in less than 1 day of culture without added stimuli or growth factors. This suggested they had already received signals initiating CD4 and CD8 acquisition. However, stimulation of these precursor cells with phorbyl ester and ionomycin prevented this acquisition of CD4 and CD8. No distinct CD4-CD8+ intermediate was detected as the CD4-CD8- cells became CD4+CD8+ in the non-stimulated cultures, thus questioning the assumption that these three groups of cells are sequential steps in one lineage. In contrast to this pre-programmed acquisition of CD4 and CD8, the less mature CD4-CD8- IL-2R+ subpopulation did not progress to the CD4+CD8+ stage in culture, although it is able to develop further on intrathymic transfer. It is likely that this subpopulation represents a control point requiring specific differentiation signals for further development.     

Delineation of human thymocytes with or without functional potential by CD1-specific antibodies

Hematopoietic precursors lacking T cell antigen receptors (TCR-CD3-) and CD4 and CD8 surface markers (i.e. double-negative thymocytes) give rise to functionally mature T lymphocytes. Yet their major progeny are immunologically unresponsive thymocytes in spite of having acquired TCR-CD3 and CD4-CD8. Because only mature thymocytes migrate to peripheral lymphoid organs and most thymocytes die in situ, the knowledge of the events associated with functional maturation in the double-negative thymocyte progeny is a fundamental question in T cell development. We reasoned that a clue to trace the fate of early human thymocytes may perhaps come from the study of the developmental acquisition of CD1 antigen, currently used to define better the functionally inert CD4+8+ (double-positive) stage and absent in mature, medullary thymocytes and peripheral T cells. By using antibodies specific for CD1 (HTA 1/T6) we show here that a large fraction of double-negative thymocytes also express CD1. CD1+3-, CD1+3+, CD1-3+, and CD1-3- subsets all exist. The CD1+3- subset generates CD1+3-4-8+ precursors of CD1+ double-positive cells. A large portion of the CD1+3+ subset bears TCR gamma delta-CD3 complexes. The CD1- subsets are responsive in assays of function, in which they can be stimulated to use the interleukin 2 pathway of proliferation and to mediate cytotoxicity. In contrast, all CD1+ thymocytes behave as functionally inert cells. Thus, the CD1 surface marker delineates human thymocyte precursors and their products which lack, or possess, functional potential in vitro, on both alpha beta and gamma delta lineages.     

Parainfluenza type 1 virus-infected cells are killed by both CD8+ and CD4+ cytotoxic T cell precursors.

The memory cytotoxic T lymphocyte (CTL) response to human parainfluenza type 1 virus (hPIV-1), a prominent cause of respiratory infection in young children, has been analysed for a panel of healthy adults. The CTL response to the parainfluenza viruses has not been investigated previously. Precursor CTL (CTLp) with activity against hPIV-1-infected Epstein-Barr virus (EBV)-transformed B lymphoblastoid target cells were found at a relatively high precursor frequency (approximately 1/2500-1/4700 CD8+ and CD4+ subsets respectively) in peripheral blood. Both CD4+ and CD8+ CTLp were detected by the analysis of individual microcultures set up under limiting dilution conditions from freshly isolated blood, the phenotype of the responder cell from individual wells being determined by flow cytometry. Further characterization of the CTL response demonstrated MHC restriction by the HLA-A2 glycoprotein in 3/4 HLA-A2+ donors. The presence of effective, hPIV-1-directed T cell memory may explain, in part, the protection observed in the adult population.     

Interleukin‐4 downregulates CD127 expression and activity on human thymocytes and mature CD8+ T cells

Signaling via the IL‐7 receptor complex (IL‐7Rα/CD127 and IL‐2Rγ/CD132) is required for T‐cell development and survival. Decreased CD127 expression has been associated with persistent viral infections (e.g. HIV, HCV) and cancer. Many IL‐2Rγ‐sharing (γ_C) cytokines decrease CD127 expression on CD4⁺ and CD8⁺ T cells in mice (IL‐2, IL‐4, IL‐7, IL‐15) and in humans (IL‐2, IL‐7), suggesting a common function. IL‐4 is of particular interest as it is upregulated in HIV infection and in thyroid and colon cancers. The role of IL‐4 in regulating CD127 expression and IL‐7 activity in human thymocytes and mature CD8⁺ T cells is unknown and was therefore investigated. IL‐4 decreased CD127 expression on all thymocyte subsets tested and only on naïve (CD45RA⁺) CD8⁺ T cells, without altering membrane‐bound CD127 mRNA expression. Pre‐treatment of thymocytes or CD8⁺ T cells with IL‐4 inhibited IL‐7‐mediated phosphorylation of STAT5 and decreased proliferation of CD8⁺ T cells. By downregulating CD127 expression and signaling on developing thymocytes and CD8⁺ T cells, IL‐4 is a potential contributor to impaired CD8⁺ T‐cell function in some anti‐viral and anti‐tumor responses. These findings are of particular consequence to diseases such as HIV, HCV, RSV, measles and cancer, in which CD127 expression is decreased, IL‐7 activity is impaired and IL‐4 concentrations are elevated.     

CD3-induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4⁺CD8⁺ double positive thymocyte stage. Immature CD4⁺CD8⁺ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4⁺CD8⁺ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4⁺CD8⁺ thymocytes can occur even in TCRα^? mutant mice which do not express the TCRαβ/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4⁺CD8⁺ thymocytes in TCRα^? mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4⁺CD8⁺ thymocytes from TCRα^? mice are not associated with clonotypic TCR chains, including TCRβ. Thus, apoptosis of CD4⁺CD8⁺ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4⁺CD8⁺ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.     

Two separable T cell receptor signals reconstitute positive selection of CD4 lineage T cells in vivo

Positive selection is an obligatory step during intrathymic T cell differentiation. It is associated with rescue of short-lived, self major histocompatibility complex (MHC)-restricted thymocytes from programmed cell death, CD4/CD8 T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TCR) signaling during positive selection can be closely mimicked by targeting TCR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. We show that selection of CD4 T cell lineage cells in mice deficient for MHC class I and MHC class II expression can be reconstituted in vivo by two separable T cell receptor signaling steps, whereas a single TCR signal leads only to induction of short-lived CD4⁺CD8^la intermediates. These intermediates remain susceptible to a second TCR signal for 12-48 h providing an estimate for the duration of positive selection in situ. While both TCR signals induce differentiation steps, only the second one confers long-term survival on immature thymocytes. In further support of the two-step model of positive selection we provide evidence that CD4 T cell lineage cells rescued by a single hybrid antibody pulse in MHC class II-deficient mice are pre-selected by MHC class 1.     

Pertussis toxin can replace T cell receptor signals that induce positive selection of CD8 T cells

CD4⁺ helper T lymphocytes and CD8⁺ killer T lymphocytes are both generated in the thymus from common precursor cells expressing CD4 and CD8. The development of immature CD4⁺ CD8⁺ thymocytes into mature ‘single-positive’ T cells requires T cell antigen-receptor (TCR)-mediated positive selection signals. Although it is known that the recognition specificity of TCR expressed by CD4⁺ CD8⁺ thymocytes determines their fate to become either CD4⁺ or CD8⁺ T cells, the molecular signals that direct precursor thymocytes to become CD4⁺ and CD8⁺ T cells are unclear. By using ZAP-70^? mutant thymus organ cultures in which T cell development is arrested at the CD4⁺ CD8⁺ thymocyte stage, the present study shows that distinct biochemical treatments can selectively restore the generation of mature CD4⁺ and CD8⁺ T cells, bypassing TCR-induced positive selection signals. The combination of phorbol ester and ionomycin selectively restores the generation of CD4⁺CD8^? TCR^high cells consistent with previous results. On the other hand, we find that the generation of CD4^? CD8⁺ TCR^high cells is selectively induced by pertussis toxin. Interestingly, the signals generated by pertussis toxin, which increase Notch expression, can dominate the signals by phorbol ester and ionomycin, steering thymocyte development to CD8 lineage. These results indicate that distinct biochemical signals replace TCR signals that selectively induce positive selection of CD4⁺ and CD8⁺ T cells, and that biochemical treatment can manipulate the development and choice of CD4⁺ and CD8⁺ T cells.     

MHC class II-independent CD25+ CD4+ CD8alpha beta+ alpha beta T cells attenuate CD4+ T cell-induced transfer colitis

CD4+ alpha beta T cell populations that develop in mice deficient in MHC class II (through 'knockout' of either the Aalpha, or the Abeta chain of the I-A(b) molecule) comprise a major 'single-positive' (SP) CD4+ CD8- subset (60-90%) and a minor 'double-positive' (DP) CD4+ CD8alpha beta+ subset (10-40%). Many DP T cells found in spleen, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) express CD25, CD103 and Foxp3. Adoptive transfer of SP but not DP T cells from Aalpha(-/-) or Abeta(-/-) B6 mice into congenic RAG(-/-) hosts induces colitis. Transfer of SP T cells repopulates the host with only SP T cells; transfer of DP T cells repopulates the host with DP and SP T cells. Anti-CD25 antibody treatment of mice transplanted with DP T cells induces severe, lethal colitis; anti-CD25 antibody treatment of mice transplanted with SP T cells further aggravates the course of severe colitis. Hence, regulatory CD25+ T cells within (or developing from) the DP T cell population of MHC class II-deficient mice control the colitogenic potential of CD25- CD4+ T cells.     

{gamma}{delta} T cells promote CD4 and CD8 expression by SCID thymocytes

Molecular studies of the TCR, which is expressed by a minorsubpopulatlon of T lymphocytes in all vertebrate species, havedefined a subset which expresses a receptor with extreme junctionaldiversity and a second subset, most commonly found in eplthella,which expresses a receptor of very limited diversity. In thedeveloping murine thymus, T cells appear in an ordered sequenceof specific v rearrangements, V3V, 1 on day 14, V2V1 on day17, and subsequently V4V5, V6, or V7. We demonstrate that thetransfer of expanded populations of cells from newborn thymusand cell lines expressing the invariant V3V1 receptor into SCIDmice, which lack T and B cells, results in the appearance ofCD3^–CD4⁺CD8⁺ thymocytes. Thus, one role of the early appearingV3V1 T cells in thymlc development in vivo is to promote CD4and CO8 surface expression on precursor cells.     

免疫磁珠负性分离纯化小鼠脾脏CD8+ T细胞

目的 探讨小鼠脾脏CD8 T细胞的免疫磁珠负性分选方法,并对分选后所得细胞进行纯度、活力及功能检测.方法 以免疫磁珠负性分选法从小鼠脾脏细胞中分离CD8 T细胞,流式细胞术检测所得细胞的纯度,台盼蓝检测细胞活力并用ConA刺激检测增殖能力. 结果 经过流式细胞仪测定免疫磁珠负性分选后的小鼠脾脏CD8 T细胞纯度达到(91.6±3.6)%,台盼兰染色细胞活力为(94.9±3.2)%,ConA刺激72 h后有(56.3±1.7)%的细胞增殖.结论 免疫磁珠负性分选法能够分选出高纯度的CD8 T细胞,并且不影响分选靶细胞的细胞活力和功能.     

Developmental status of CD4-CD8+ and CD4+CD8- thymocytes with medium expression of CD3

In normal mice, more than 10% of thymocytes in the CD4+CD8- and CD4-CD8+ single-positive (SP) subsets express a medium level of CD3 on the cell surface. However, the fate of CD3medium cells is unclear. The CD3medium SP subpopulations might contain (i) cells in an immature stage of the pathways leading to CD3high cells, (ii) cells in developmental pathways that do not lead to CD3high cells, or (iii) cells that have been negatively selected. We found that sorted CD3medium CD4+CD8- thymocytes from adult mice up-regulated CD3 to high levels in reaggregation thymus organ culture. Unlike their CD3high counterparts, CD3medium CD4+CD8- thymocytes were unable to undergo chemotaxis towards the chemokines CCL19 and CCL21. CD3medium thymocytes of both CD4+CD8- and CD4-CD8+ subsets were also considerably more responsive than CD3high SP cells to apoptotic signals induced in vitro by ligation of CD95 (Fas/APO-1) or by dexamethasone. In both SP subsets, a higher frequency of thymocytes expressing forbidden Vbeta+ T cell receptors reactive with endogenous mammary tumor virus superantigens was found in CD3medium subpopulations than in CD3high subpopulations. These findings argue that the CD3medium SP thymocyte subpopulations contain apoptosis-susceptible precursor cells of CD3high SP cells and are subject to negatively selecting pressures.     

'Radioresistant' CD4-CD8- intrathymic T cell precursors differentiate into mature CD4+CD8- and CD4-CD8+ T cells. Development of 'radioresistant' CD4-CD8- intrathymic T cell precursors

Using lectin (PNA) and monoclonal antibodies for Pgp-1, IL-2R, H-2k, CD3, and F23.1 (T cell receptor V beta 8), we characterized the 'radioresistant' CD4-CD8- double negative thymocytes at an early stage after 800 rad irradiation. Most of the CD4-CD8- cells on day 8 after irradiation expressed a high level of Thy-1, H-2k, and PNA, while a small proportion of these cells were CD3+ and/or F23.1+. The appearance of Pgp-1 and IL-2R on the 'radioresistant' double negative precursors was also sequentially examined from day 5 to day 9 after irradiation. The double negative thymocytes at day 5 expressed the highest level of Pgp-1 antigens and these cells gradually decreased in number from day 7 to day 9. By contrast, IL-2R was transiently expressed on the double negative cells on the day 7 and 8 after irradiation. These results indicate that progression of thymocyte development occurred within the CD4-CD8- thymocytes after irradiation. We further examined the homing ability of the double negative 'radioresistant' intrathymic T cell precursors to the periphery by intrathymic cell transplantation method. The double negative thymocytes proliferate and differentiate into CD4+CD8+ cells and CD4+CD8- cells but few CD4-CD8+ cells in the thymus, while only CD4-CD8+ cells were detected in the peripheral lymphoid organs 14 days after intrathymic transplantation of the double negative cells in the H-2 compatible Thy-1 congenic mice. These results suggest that the 'radioresistant' intrathymic precursors differentiate and mature in the thymus and migrate to the periphery.