In vitro differentiation and commitment of CD4+ thymocytes to the CD4+ lineage without TCR engagement

Thymocyte positive selection is based on protection of immatureCD4/CD8 double-positive (DP) thymocytes from apoptosis and theirdifferentiation into CD4 or CD8 single-positive (SP) cells.Intracellular signals essential for positive selection appearto be induced through the TCR and some of the accessory moleculesincluding LFA-1, CD4 and CD8 upon Interaction with thymic stromalcells. The signals, however, still remain to be identified.Since physiological levels of glucocorticoids potentially induceor enhance thymocyte apoptosis even in vivo, the signals arelikely to inhibit the apoptotic effect of glucocorticoids. Wehave previously shown that proper cross-linking of TCR-CD3 withLFA-1, CD4 or CD8 inhibited glucocortlcold-lnduced thymocyteapoptosis in vitro, and that a proper combination of the calciumionophore, ionomycin and the protein kinase C (PKC) activator,phorbol 12-myrlstate 13-acetate (PMA), mimicked the inhibitoryeffect. Here we determined whether this combination of ionomycinand PMA induces differentiation of isolated DP thymocytes fromnormal and TCR transgenic mice. We found that pretreatment ofDP thymocytes with ionomycin and PMA followed by 1 day cultureof the cells without the reagents resulted in the differentiationof the cells into CD4 SP and CD4⁺ CD8^lo T cells that have mostlycommitted to the CD4 lineage. The changes in expression of otherdifferentiation markers were also in good accordance with thoseassociated with positive selection, except the final maturation.The results indicate that moderate and transient increases inintracellular Ca²⁺ level and PKC activity induce differentiationand commitment of DP thymocytes to the CD4 lineage, and suggestedthat the biochemical pathway leading to positive selection isbased on a similar mechanism.     

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 human single-positive fetal thymocytes in vitro into IL-4- and/or IFN-{gamma}-producing CD4+ and CD8+ T cells

In this study we have investigated the capacity of human fetalthymocytes to differentiate in vitro into subsets of T cellswith polarized T_h1 or T_h2 cytokine profiles. Stimulation offreshly isolated human fetal thymocytes with anti-CD3 mAb, cross-linkedonto CD32,CD58,CD80-expressing mouse fibroblasts and subsequentculture in the presence of exogenous rIL-2 for 6 days, inducedthe production of both IL-4 and IFN-, which was mainly producedby CD4⁺ single-positive (SP) and CD8⁺ SP cells respectively.Addition of rIL-4 during priming augmented IL-4 production incultures of human fetal thymocytes, which was mainly due toan increased production of IL-4 by CD8SP cells. In contrast,addition of IL-4 to the cultures only slightly enhanced IL-4production and had little effect on frequencies of IL-4-producingCD4SP cells. Both CD4SP and CD8SP cells produced IL-5, IL-10and IL-13 at comparable levels, following priming in the presenceof rIL-4. Priming in the presence of rIL-12 strongly enhancedthe production of IFN- in both CD4SP and CD8SP cells. No correlationbetween expression of CD27, CD30 and CD60, and a particularcytokine profile of differentiated thymocytes could be demonstrated.Together, these results demonstrate the full capacity of fetalhuman thymocytes to differentiate into cytokine-producing Tcells in a priming milieu with appropriate stimulatory moleculesand exogenous cytokines. In addition, CD4SP thymocytes rapidlydifferentiate into polarized T_h2 cells following stimulationin vitro in the absence of exogenous rIL-4.     

Generation of mature T cell populations in the thymus: CD4 or CD8 down-regulation occurs at different stages of thymocyte differentiation

We have studied the differentiation and repertoire selection during the maturation of CD4⁺CD8⁺ (DP) thymocytes into CD4⁺CD8^- (CD4SP) and CD8⁺CD4^- (CD8SP) T cells, in normal mice, mice transgenic for T cell receptor (TcR)-αβ restricted by either class I or class II major histocompatibility (MHC), and in mice deficient in class I or class II MHC expression. Our data suggest that mature CD4 and CD8 T cells derive from different pathways of T cell differentiation in the thymus. Thus, interaction of DP thymocytes with MHC class II leads to the immediate down-regulation of CD8, which occurs simultaneously with an increase in TcR expression; DPTcR^loHSA^hi thymocytes mature into a CD4⁺CD8^lo TcR^hiHSA^hi intermediate population. This cell population generates CD4SP thymocytes, the majority of which are still HSA^hi. In contrast, interaction with MHC class I induces the up-regulation of TcR, which precedes the down-regulation of CD4; DPTcR^lo generate DPTcR^hi thymocytes, the majority of which are the committed precursors of CD8SP cells. Further differentiation results in CD4 down-regulation and the transition from DPTcR^hi into CD8⁺CD4^lo TcR^hiHSA^lo and CD8SPTcR^hiHSA^- T cells. Since down-regulation of CD4 and CD8 occurs at different stages of thymocyte differentiation, our results do not support a stochastic/selective model of lineage commitment in the thymus.     

CD69 cell surface expression identifies developing thymocytes which audition for T cell antigen receptor-mediated positive selection

CD69, an ‘activation marker’ that is rapidly inducedon mature T cells after stimulation through the T cell antigenreceptor (TCR) was found to be expressed on 10% of normal thymocytes.All of these CD69⁺ thymocytes express ß TCR, and theyinclude both TCR^lowCD4⁺CD8⁺ and TCR^highCD4⁺CD8^– or CD4^–CD8⁺thymocytes. The CD69⁺ cells can be further segregated into heat-stableantigen (HSA)⁺TCR^*HSA^–TCR^high and HSA⁺TCR^high thymocytepopulations. None of CD69⁺ cells express the mature T cell markerQa-2. Thus CD69⁺ cells present in vivo appear phenotyplcallyto represent transitional cell populations between immatureTCR^lowHSA⁺Qa-2^– double-positive cells and mature TCR^highHSA^–QA-2⁺single-positive cells. In addition, TCR engagement by MHC moleculesis required for CD69 expression in the thymus. Taken together,the CD69 ⁺ thymocytes appear to represent the cells auditioningin positive selection process or they are the cells that havebeen positively selected recently. Analysis of a TCR transgenicmouse model revealed an increased number of CD69⁺ thymocytesin a positively selecting thymus, whereas no CD69⁺ transgenicTCR⁺ thymocytes were observed in the non-selecting thymus. Basedon the results of this study, we suggest that the surface expressionof CD69 serves as a useful marker to identify and trace thosethymocytes that are engaged in the TCR-mediated positive selectionprocess in the thymus.     

Cross-Linking the TCR Complex Induces Apoptosis in CD4+8+ Thymocytes in the Presence of Cyclosporin A

Although it is generally agreed that TCR ligation is a minimal requirement for negative selection in the CD⁺8⁺ double-positive (DP) thymocyte subset, the costimulatory requirements and specific signaling events necessary to induce apoptosis are not well defined. We have explored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports, we demonstrate that culturing DP thymocytes with plate-bound anti-TCR antibody induces downregulation of CD4 and CD8 and upregulation of CD69 expression. Nevertheless, the activated cells did not undergo apoptosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion of the DP subset occurred within 24 hr when thymocytes were incubated in the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP thymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the CsA-induced death of TCR-stimulated DP thymocytes. These data verify that TCR cross-linking alone is insufficient to induce apoptosis of DP thymocytes and further suggest that TCR stimulation activates a CsA-sensitive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes.     

Lck dependence of signaling pathways activated by {gamma}irradiation and CD3{varepsilon} engagement in RAG-1-/-immature thymocytes

The src family tyrosine kinase, Lck, transduces signals fromthe pre-TCR complex which regulate the development and expansionof CD4/CD8 double-positive (DP) thymocytes from CD25⁺ CD4/CD8double-negative progenitors. We and others have recently shownthat sublethal irradiation bypasses the need for TCRßexpression to promote the development and expansion of DP thymocytesin acid or recombinase-activating gene (RAG)-deficient mice.Here we demonstrate that -irradiation activates an Lck-dependentsignaling process in Immature thymocytes similar to that initiatedphysiologically by the pre-TCR complex.     

Entry of CD4 CD8 immature thymocytes into the CD4/CD8 developmental pathway is controlled by tyrosine kinase signals that can be provided through TCR components

Entry of thymus-migrated precursor cells into the CD4/CD8 developmentalpathway was analyzed by using the short-term organ culturesof day 14 fetal mouse thymus lobes. Organ cultures of CD4^–CD8^–day 14 fetal thymocytes for 1-2 days resulted in the generationof CD4^–CD8⁺ cells, which were mostly immediate precursorcells for CD4⁺CD8⁺ thymocytes. This differentiation of CD4^–CD8^–thymocytes into CD4^–CD8⁺ cells was strongly enhanced byanti-CD3 antibodies. The anti-CD3-induced generation of CD4^–CD8⁺cells was even found in the immunodeficient scid fetal thymuscultures, and the cell surface CD3 expression on the scid fetalthymocytes could be directly visualized, indicating that functionalCD3 could be expressed on CD4^–CD8^– immature thymocyteswithout being associated with rearranged TCR components. Theanti-CD3-lnduced generation of CD4^–CD8⁺ cells from scidand normal fetal thymus cultures was inhibited by tyrosine kinaseinhibitors Herblmycin A and Tyrphostin. The generation of CD4^–CD8⁺cells in unstimulated normal fetal thymus cultures was alsomarkedly inhibited by the tyrosine kinase inhibitors but notby Cyclosporin A, suggesting that tyrosine klnase-dependentbut calclneurin-lndependent signals were essential for the differentiationof CD4^–CD8^– thymocytes. Interestingly, the generationof CD4^–CD8⁺ cells from the normal fetal thymus cultureswas modestly but consistently enhanced by anti-TCRßantibody, suggesting that functional TCRß in additionto CD3 was expressed on normal CD4^–CDS⁺ immature thymocytes.On the other hand, anti-TCR antibody did not affect this differentiationin the normal fetal thymus cultures and the generation of CD4^–CD8⁺cells from the normal fetal thymus cultures of TCR-deficientmice was still enhanced by anti-TCRß or anti-CD3 antibodies,indicating that either TCR chains or TCR⁺ cells were not involvedin the control of the differentiation into CD4^–CD8⁺ cells.These results indicate that the entry of CD4^–CD8^–immature thymocytes into the CD4/CD8 developmental pathway iscontrolled by tyrosine kinase signals and that these signalscan be provided through the engagement of TCR-CD3 complexeswith or without TCRß chains expressed on the CD4^–CD8^–immature thymocytes.     

Implication of the common {gamma} chain of the IL-7 receptor in intrathymic development of pro-T cells

The effects of IL-7 on the growth and differentiation of thymocyteswere analyzed using murine fetal thymua organ cultures (FTOC)in the presence of mAbs specific for the conventional IL-7 receptor(1L-7R) and for the common (c) chain. In FTOC, the developmentof CD4^–CD8^– double-negative thymocytes to CD4+CD8+double-positive (DP) and CD4+ or CD8+ single-positive (SP) cellswas not completely blocked by adding these mAbs, although cellgrowth was reduced by the treatment. To define a developingstage sensitive to the mAbs, most immature thymocytes, Pgp-1⁺c-kit cells, were cultured in the 2-deoxyguartosine treatedfetal thymus. In the presence of both mAbs in the culture, neitherDP nor SP thymocytes developed whereas either of the mAbs partiallyblocked their development. These results indicate that the Cchain is involved in early T cell development as an indispensablesubunlt of the functional IL-7 receptor complex.     

Irreversible Suppression of CD8 Expression in CD4-CD8+ Thymocytes Upon in vitro Stimulation

CD8 (Ly-2) expression was suppressed in purified murine CD4-CD8⁺ thymocytes at the mRNA level upon continuous stimulation with PMA and ionomycin in the presence of rIL-2. The level of CD8 expression on CD4^?CD8⁺ thymocytes was reduced gradually during the culture and a majority of them turned into CD4^? CD8^? cells after 48 hr. This suppression was not transient, since CD8 expression was not recovered on these cells in additional 48 hr of culture without PMA and ionomycin. The suppression was dependent on the concentrations of PMA and ionomycin, and inhibited by adding an immunosuppressant, CsA to the culture. Treatment with either PMA or ionomycin alone did not induce suppression of CD8. Crosslinking of CD3-? chains also induced suppression of CD8 for a part of CD4^?CD8⁺ thymocytes. Interestingly, CD8 expression was hardly suppressed in CD4^?CD8⁺ peripheral T lymphocytes, suggesting that the mechanisms of suppression of CD8 is developmentally regulated. We propose that the suppression of CD8 expression at CD4^?CD8⁺ stage involves an additional mechanism of negative selection of thymic T cells.     

Role of CD4+CD45RA+ T cells in the development of autoimmune diabetes in the non-obese diabetic (NOD) mouse

The non-obese diabetic (NOD) mouse spontaneously develops aT cell-mediated autoimmune disease, sharing many features withhuman insulin-dependent diabetes mellltus (IDDM), leading toinsulin-secreting ß cell destruction. The role ofCD4⁺ T cells has been evidenced at two levels. First, CD4⁺ Tcells from diabetic animals are required to transfer diabetesto non-diabetic recipients in conjunction with CD8⁺ effectorT cells. Second, suppressive CD4⁺ T cells have been characterizedin non-diabetic NOD mice. T cells with different functions canthus share the CD4⁺ phenotype. Since CD4⁺ T cells can be dividedinto at least two subgroups on the basis of CD45 isoform expression,we evaluated the distribution of CD4⁺ T cells expressing theCD45RA isoform on NOD mouse thymocytes and peripheral T cells.The percentage of CD45RA⁺ cells was dramatically increased amongthe most mature CD3^bright thymocytes and among CD4⁺ T cellsin lymph nodes of the NOD mouse as compared with control strains.This increase was related to the development of insulitls. Interestingly,the CD45RA isoform was expressed on most CD4⁺ T cells invadingthe islets. In vivo treatment with an antl-CD45RA mAb preventedthe development of insulitls and spontaneous diabetes in femaleanimals but not the transfer of diabetes by T cells collectedfrom diabetic NOD donors. These results indicate that anti-CD45RAmAb is only effective if given before the full commitment ofeffector T cells to the destruction of islet ß cells.ThusCD4⁺CD45RA⁺ T cells play a key role in early activation stepsof anti-islet immunity.     

Thymic education curtailed: defective immune responses in nude rats reconstituted with immature thymocyte subsets

We have studied the ability of thymocyte subsets from allotypemarked donors to populate athymlc nude rats with T cells andto restore immune responsiveness. Following adoptive transfer,CD4⁺CD8^– double-negative (DN) thymocytes (lymphold precursorcells) or the CD4⁺CD8⁺ double-positive (DP) subset (Intermediatethymocytes) or CD4⁺CD8^– single-positive (CD4 SP) cells(mature thymocytes) each generated a permanent population ofCD4⁺ progeny in syngeneic nude recipients. DN and DP thymocytesalso produced small numbers of CD8⁺ cells; there was no evidenceof a CD4^–CD8^– or CD4⁺CD8⁺ donor cell population.CD4 SP thymocytes conferred T cell functions [graft-versus-host(GVH) responses, allograft rejection and thymus-dependent antibodyresponses] on nude rats that were almost indistinguishable fromthose conferred by mature peripheral recirculating CD4 T cells.Transfer of DP thymocytes extended the life-span of the immunolncompetentnudes and produced CD4⁺ progeny with near normal GVH responsiveness.However, DP-derived CD4⁺ cells were deficient at inducing allograftrejection and provided little or no help for antibody synthesis.The CD4⁺ progeny of DN thymocytes did not prolong the survivalof nude recipients, gave reduced GVH reactivity, showed almostno capacity to initiate skin allograft rejection and failedto help B cells produce antibody. The results suggest that intrathymicdevelopment proceeds stepwlse; each stage is accompanied byacquisition of additional properties that are reflected by Tcell responses in the periphery. Thymic education does not becomecomplete until the SP stage is reached when thymocytes becomefully independent of the thymic mlcroenvironment. Received 22 September 1993, accepted 22 December 1993.     

Preferential requirement of CD3{zeta}-mediated signals for development of immature rather than mature thymocytes

Antigen recognition signals by the TCR are transduced throughactivation motifs present in the cytoplasmic region of CD3 chains.In vitro analysis has suggested that the CD3 chain mediatesdifferent signals from other CD3 chains. To analyze the in vivofunction of CD3-mediated signals for T cell development, miceexpressing a mutant CD3 chain lacking all the activation motifswere generated by introducing the transgene into -knockout mice.Mature CD4⁺ single-positive (SP) thymocytes in these mice weregreater in number than in -deficient mice, and the promoteddifferentiation was indicated by the changes of CD69 and HSAphenotypes. We found that even in the absence of activationmotifs in CD3, these mature cells became functional, being ableto induce Ca²⁺ mobilization and proliferation upon stimulation.On the other hand, CD4^-CD8^- double-negative (DN) thymocytes,most of which were arrested at the CD44^-CD25⁺ stage similarlyto those in -deficient mice, could not be promoted for differentiationinto CD4⁺CD8⁺ double-positive thymocytes in these mice in spiteof the fact that the expression of the transgene in DN thymocyteswas higher than that of in wild-type mice. These results demonstratethe preferential dependence of the promotion of developmentand/or expansion of DN thymocytes rather than mature thymocytesupon the activation signals through the chain and suggest differentialrequirements of TCR signaling for mature SP and immature DNthymocyte developments in vivo.     

Developmentally regulated expression of the PD-1 protein on the surface of double-negative(CD4 CD8 ) thymocytes

PD-1, a member of the Ig superfamily, was previously isolatedfrom an apoptosis-induced T cell hybridoma 2B4.11 by subtractivehybridization. Expression of the PD-1 mRNA is restricted tothymus in adult mice. Using an anti-PD-1 mAb (J43), we examinedexpression of the PD-1 protein during differentiation of thymocytesin normal adult, fetal and RAG-2^-/- mice with or without anti-CD3mAb stimulation. While PD-1 was expressed only on 3–5%of total normal thymocytes, –34% of the CD4^-CD8^- double-negative(DN) fraction are PD-1⁺ cells with two distinct expression levels(low and high). PD-1^high thymocytes belonged to TCR lineagecells. In the DN compartment of the TCR ß lineage,PD-1 expression started at the low level from the CD44⁺CD25⁺stage and the majority of thymocytes expressed PD-1 at the CD44^-CD25^-stage in which thymocytes express TCR ß chains. Theanti-CD3 antibody administration augmented the PD-1 expressionas well as the differentiation of the CD44^-CD25⁺ DN cells intothe CD44^-CD25^- DN stage, not only in normal mice but also inRAG-2-deficient mice. The fraction of the PD-1^low cells in theCD4⁺CD8⁺ double-positive (DP) compartment was very small (>5%)but increased by stimulation with the anti-CD3 antibody, althoughthe total number of DP cells was drastically reduced. The resultsshow that PD-1 expression is specifically induced at the stagespreceding clonal selection.