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.     

Negative selection by endogenous antigen and superantigen occurs at multiple thymic sites

The site of negative selection in the thymus has been inferredfrom a range of different experiments. Analysis of thymic deletionof Vß5⁺, V_ß11⁺ or Vß17a⁺ cellsH-2E transgenic mice led to the theory that negative selectionoccurs predominantly in the medulla (specifically, through presentationby medullary dendritic cells). Other experiments investigatedwhether transgenic TCR are deleted at the double-positive (DP)or single-positive stage following encounter with peptide ligand:by flow cytometric analysis deletion is generally found to occurat the DP thymocyte stage and as these cells are found predominantlyin the cortex, it has been inferred that this is the key siteof negative selection. The visualization of apoptotic thymocytesin situ has recently been reported for specific examples ofnegative selection. Using a panel of TCR transgenic lines inwhich negative selection occurs at different stages of thymocytedevelopment, we have used TUNEL staining to analyse the anatomicalsites of thymocyte apoptosis. For the first time we have beenable to compare directly the sites of deletion induced by theendogenous cognate peptides or by endogenous superantigen. Weshow that generalization from the medullary deletion of V_ß5⁺,V_ß11⁺ or V_ß17a⁺ cells by the endogenoussuperantigens Mtv 8 and 9 and from limited examples of corticaldeletion by exogenous peptide administered to TCR transgenicmice is over-simplified. Apoptotic thymocytes in mice lackingMtv superantigens are indeed localized in the cortex. However,when deletion is induced by cognate self peptide, apoptosiscan occur in the cortex, the medulla or at the junction betweenthe two.     

Cyclosporin A inhibits the decrease of CD4/CD8 expression induced by protein kinase C activation

Cyclosporin A (CsA) is a powerful immunosuppressive drug widelyused in transplantation medicine. A major effect of CsA is inhibitionof the differentiation of immature double-positive (DP) CD4⁺CD8⁺thymocytes into mature single-positive (SP) CD4⁺CD8^– orCD4^–CD8⁺thymocytes. The mechanisms underlying the changesin CD4/CD8 expression during normal differentiation of thymocytesand the way CsA interferes with this differentiation processare still unknown. Here we show that protein kinase C (PKC)activation by phorbol 12-myristate 13-acetate (PMA) causes adecrease of both CD4 and CD8 expression at the cell surfacelevel and at the mRNA level in a CD4⁺CD8⁺ T cell line and infreshly isolated thymocytes. A PKC inhibitor, staurosporin,interferes with the differentiation from DP to SP in fetal thymusorgan culture system. These data suggest that the alternationof CD4/CD8 expression from DP to SP is dependent on PKC activation.CsA blocks this decrease of CD4/CD8 expression by PMA in vitro.Moreover, this PMA effect is also blocked by treatment withcycloheximlde. These results suggest that the reduction of CD4/CD8expression requires de novo synthesis of a protein(s) inducedin response to a signal conveyed by activated PKC. CsA may blockthe transition from DP to SP by inhibition of CD4/CD8 down-regulationinduced by PKC activation.     

Antigen-specific and nonspecific deletion of immature cortical thymocytes caused by antigen injection

Analysis of antigen-induced negative selection of thymocytes in T cell receptor (TCR)-transgenic mice is complicated by the presence of an antigen-responsive peripheral T cell compartment. Our experiments address the question of whether and how peripheral T cell activation can affect immature thymocytes. Following three daily injections of peptide antigen into mice expressing a peptide-specific transgenic TCR and deficient for TAP1, we and others have found profound deletion of the CD4⁺CD8⁺ (DP) thymocyte subset. However, our work shows that even though mature CD8⁺ T cells are inefficiently selected in TAP1-deficient mice, there was a striking degree of peripheral expansion and activation of CD8⁺ peripheral T cells. Furthermore, when cells from TCR-transgenic mice were adoptively transferred, we found that deletion of non-transgenic DP thymocytes occurred in Thy-1-congenic and even more efficiently in TAP1-deficient recipients after repeated peptide injection resulting in peripheral T cell activation. In the adoptive transfer experiments the degree of deletion of immature bystander thymocytes was decreased upon blocking of TNF. These data show that deletion of DP thymocytes can result from excessive peripheral T cell activation and identify TNF as an important effector molecule for this process. When steps are taken to avoid peripheral T cell activation, peptide antigen can induce TCR-mediated thymocyte deletion, presumably in the thymus cortex, since injection of TAP1-deficient TCR-transgenic mice resulted in deletion of immature DP thymocytes prior to detectable peripheral T cell expansion and activation. This effect was not blocked by inhibiting tumor necrosis factor activity. In addition, DP depletion was seen in the absence of peripheral T cell activation when antibody-mediated depletion of CD8⁺ T cells was performed. Our work clearly shows that two mechanisms for deletion of DP thymocytes exist: deletion induced by antigen presentation in the thymus and deletion as a consequence of repeated stimulation of mature peripheral T cells.     

CD3{varepsilon}-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2 / mice in the absence of TCR {beta} chain expression

Recent studies have shown that TCR ß chain expressioncan effect the differentiation of CD4^–CD8^– double-negative(DN) thymocytes to CD4⁺CD8⁺ double-positive (DP) thymocytes.The TCR ß chain is expressed on the surface of DPthymocytes in association with CD3, and chains, suggestinga potential role for CD3 components in this signaling process.We now report detection of a very tow level of surface expressionof CD3 on adult DN RAG-2^–/–; thymocytes. This surfaceCD3 was associated with CD3 and chains, as detected by anti-CD3immunoprecipitation analyses. Significantly, injection of anti-CD3mAb into RAG-2^–/– mice led to the accumulation ofan IL-2R^– CD2⁺ DP cell population and a nearly 100-foldincrease in thymic cellularity to essentially normal levels.Together, these data strongly indicate that TCR ßchain-mediated developmental signals are transduced by CD3 componentsand provide potential insights into mechanisms by which TCRß chain expression may effect this process.     

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

How T cell receptor (TCR) avidity influences CD8⁺ T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP^?/? mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP^?/? Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8⁺ T cell development and repertoire selection. In comparing SLAP^?/? OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP^?/? Vβ5 mice. We have found that SLAP^?/? OT-1 mice have fewer CD8⁺ thymocytes but have increased CD5 expression. SLAP^?/? OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8⁺ splenocytes upon tetramer staining. Our data demonstrate that SLAP^?/? Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8⁺ T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8⁺ T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8⁺ T cell development influences repertoire selection.     

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.     

Tolerance induction by elimination of subsets of self-reactive thymocytes

Immature thymocytes expressing TCRs which confer reactivityto self-MHC molecules are subject to efficient elimination asa result of negative selection. Previously, we have identifieda lineage of H-2K^b Tg mice, CD2K^b-3, which fails to reject skingrafts from mice expressing H-2K^b even though H-2K^b-specrficcytotoxlc T cells can be generated in vitro. We now show thatbone marrow derived cells are responsible for tolerance inductionand that tolerance is acquired, at toast in part, by negativeselection in CD2K^b-3 mice. Thymocytes expressing two differenttransgenic TCR (TCR-T_g) clonotypes conferring reactivity toH-2K^b are eliminated prior to the CD8⁺CD4⁺ stage of differentiationin double Tg (CD2K^b-3xTCR-T_g)F₁ mice. As in other cases wherethymocytes from TCR-T_g mice develop in the presence of deletingligands, large numbers of TCR⁺ CD8^–CD4^– T cellsaccumulate in double Tg mice. However, these T cells fail torespond to H-2K^b in vitro but can be activated with immobilizedanti-clonotyplc antibody. Consequently, thymocytes expressingthese types of TCR molecules represent a fraction of H-2K^b-reactivethymocytes which are unable to mature into T cells capable ofmounting H-2K^b-specific cytotoxic responses. Presumably, precursorsof H-2K^b-spicific cytotoxlc T cells found in the periphery ofCD2K^b-3 mice express a distinct repertoire of TCR moleculesconferring reactivity to H-2K^b. We consider potential explanationsto account for this discrepancy and their wider implications,including the possibility that the repertoire of thymocyteaable to recognize setf-H-2Kb molecules In CD2K ^b-3 mice is dividedinto distinct subsets; those which are, and those which arenot, subject to negative selection.     

Expression of an ovalbumin-specific V{beta}8.2 TCR transgene inhibits collagen arthritis in B10.Q mice

Previous studies have illustrated the importance of T cellsbearing ß TCRs in the induction and development ofcollagen induced arthritis (CIA) in mice. However, the scopeof TCR usage in CIA has yet to be clearly defined. Given theinherent diversity of the TCR repertoire, the relative flexibilityof the arthritogenic TCR repertoire specific for type II collagen(CII) is not clear. Therefore, we chose to examine the influenceof a highly skewed TCR repertoire on CIA. Arthritis susceptibleB10.Q (H-2^q) mice were mated with C57L (H-2^b) animals expressingan ovalbuminspecific V_ß8.2 TCR transgene (Tg) andTg⁺ offspring were further backcrossed to B10.Q. HomozygousH-2^a/q, V_ß8.2 Tg⁺ mice displayed a high level of V_ß8.2⁺T cells in peripheral blood. However, expression of some endogenousV_ß TCR, such as V_ß14, was still detected.Upon immunization with bovine CII in adjuvant, V_ß8.2Tg⁺ mice were highly resistant to CIA when compared with Tg^–littermates. Analysis of sera demonstrated a marked reductionin antibody specific for homologous mouse CII as well as heterologousbovine CII in Tg⁺ animals. Interestingly, V_ß8.2 Tg⁺mice still mounted good antibody responses following immunizationwith human thyroglobulin, indicating that the skewed TCR repertoireaffected anti-CII but not antithyroglobulin responses. Thus,our findings show that constraints placed on the TCR repertoireInhibit pathogenic responses against CII and suggest that inH-2^q mice the arthritogenlc TCR repertoire bears only limitedflexibility.     

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.     

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.     

NK1.1+ T cells from IL-7-deficient mice have a normal distribution and selection but exhibit impaired cytokine production

Particular subsets of T cells expressing the NK1.1 antigen havebeen proposed to play an immune regulatory role by their fastand strong production of cytokines, in particular IL-4. We soughtto determine factors driving the functional differentiationof NK1.1⁺ T cells. Since NK1.1⁺ T cells are exquisitely sensitiveto IL-7 stimulation, we analyzed the development, selectionand IL-4 production of NK1.1⁺ T cells in IL-7 deficient mice(IL-7–/–mice). Besides a sharp reduction of allT cell subsets, NK1.1⁺ T cells develop at normal relative frequenciesin IL-7–/–;mice. They also undergo a normal selectionprocess, as revealed by the biased V_ß TCR repertoireidentical to the one in IL-7+/+ mice. However, NK1.1₊ T cellsfrom IL-7+/+ mice were found to be impaired in IL-4 and IFN-production in in vitro and in vivo models. In addition, IL-7was able to restore IL-4 production by NK1.1⁺ thymocytes fromIL-7–/– mice. Finally, IL-7 but not IL-4 was ableto maintain and increase IL-4 production by NK1.1⁺ thymocytesfrom normal mice. These data suggest that the functional maturationof NK1.1⁺ T cells requires a cytokine-driven differentiationprocess, in which IL-7 plays a major role.     

A thymic epithelial cell line induces both positive and negative selection in the thymus

TCR engagement in the thymus results in both survival and eliminationsignals for developing thymocytes. To examine whether both signalscan be provided by the same cell type, we investigated the abilityof a thymic epithelial cell (TEC) line 427.1, previously shownto allow positive selection in the thymus, to induce clonaldeletion of Immature thymocytes. [H-2^b/s–H-2^s] bone marrowchimeras are non-responsive to antigens in the context of H-2b.However, chimeras that underwent intrathymic injection of H-2^b/s427.1 cells expressing vesicular stomatitis virus (VSV) nucleocapsidantigen acquired the ability to raise influenza, but not VSVspecific H-2^b restricted cytotoxic T lymphocyte (CTL) responses.The ability of 427.1 cells to delete CD4⁺CD8⁺ thymocytes wasdetermined using mice transgenlc for the TCR specific for ovalbumln(OVA) in the context of H-2K^b. OVA transfected, but not mocktransfected 427.1 TECs, Induced in vitro deletion of CD4⁺CD8⁺TCR transgenlc thymocytes manifested as a down-modulation ofCD4 and CD8 molecules, a shift in the side versus forward scattercharacteristics of thymocytes, and appearance of thymocyteswith subdlplold content of DNA indicated the ongoing processof DNA fragmentation. The finding that the same TEC line Iscapable of inducing both positive and negative selection inthe thymus suggests that thymocytes bearing TCRs specific forself peptldes expressed by positively selecting thymic epitheliumcan be deleted. Therefore the expression of a unique set ofMHC associated peptldes by TECs does not appear to be the basisfor the positive outcome of the TCR llgatlon on immature thymocytes.     

Evidence for selective in vivo expansion of synovial tissue-infiltrating CD4+ CD45RO+ T lymphocytes on the basis of CDR3 diversity

In this study we have analyzed the TCR V and V_ß regionsat the DNA level in the CD4⁺CD45RO⁺ memory T cell populationof synovial tissue infiltrating T lymphocytes of three rheumatoidarthritis (RA) patients and one patient with chronic arthritis.Cell lines of CD4⁺CD45RO⁺, CD4⁺CD45RO^–, CD8⁺CD45RO⁺ andCD8⁺CD45RO^– T lymphocyte populations were generated followingFACS cell sorting of freshly isolated synovial tissue mononuclearcell infiltrates (STMC) and of freshly isolated peripheral bloodmononuclear cells (PBMC) of these patients. The phenotyplc andmolecular analyses have revealed the following. (I) The TCRrepertoires of tissue infiltrating T lymphocytes in the varioussubsets were extensive on the basis of TCR V gene family usage.(II) Furthermore, each patient displayed individual specificTCR V gene expression patterns in the various STMC and PBMCderived T cell subsets. However, the majority of these arthritispatients manifested increased expression of multiple TCR V genefamilies in the synovial tissue derived CD4⁺CD45RO^– Tcell population when compared with the peripheral blood derivedCD4⁺CD45RO⁺ subset. Of these gene families, we found enhancedexpression of the TCR V7 and V_ß11 gene segments insynovial tissue to be shared by all four patients analyzed.OH) Nucleotlde sequence analysis of the CDR3 regions of a numberof TCR V regions in the CD4⁺CD45RO⁺ T cell subsets has revealedthat the CDR3 regions comprised within synovial tissue derivedTCR V regions differed from those found in peripheral bloodderived TCR V regions. These differences in CDR3 diversity mightbe the consequence of a specific interaction with particularMHC-peptlde complexes expressed at the site of inflammation.(Iv) The CDR3 region analysis also showed individual specificamlno acid motifs within the N-D-N regions of all analyzed TCRV_ß genes derived from PBMC as well as STMC.     

Common gamma chain cytokines promote regulatory T cell development and survival at the CD4+ CD8+ stage in the human thymus

Thymic commitment of human FOXP 3⁺ regulatory T cells begins at the double‐positive (DP ) CD 4⁺ CD 8⁺ stage. In the current study, we show that interleukin‐2 promotes the development of FOXP 3⁺ thymocytes and enhances their survival at the DP phase. IL ‐2 increases the frequency of FOXP 3⁺ cells and promotes the Treg phenotype after TCR ‐mediated positive selection at the most mature DP stage. However, it has no effect on FOXP 3⁺ cells at the earlier maturation steps before positive selection. DP FOXP 3⁺ thymocytes are highly susceptible to cell death but IL ‐2 promotes their survival. The anti‐apoptotic protein BCL ‐2 (B Cell Lymphoma 2) is also upregulated by IL ‐2 at the most mature DP stage. In addition to IL ‐2, we identify IL ‐15 to have a significant role in the upregulating FOXP 3 and survival of Tregs at the DP phase. IL ‐7 also increases the expression of BCL ‐2 in the DP FOXP 3⁺ thymocytes. Our results indicate that common gamma chain cytokines IL ‐2, IL ‐7 and IL ‐15 promote the development of regulatory T cells at the most mature DP stage after TCR ‐mediated positive selection through suppressing cell death.     

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.     

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.