Functional maturation of mouse CD4~+CD8~- thymocytes induced by medullary-type thymus epithelial cells

Murine CD4+CD8- (CD4SP) thymocyte subset is a heterogeneous population, in which the Qa-2- cells are less functional, whereas the Qa-2+ cells are fully functional. Evidence is provided here that the transition from Qa-2- to Qa-2+ CD4SP thymocytes is an intrathymic process of differentiation induced by thymic medullary-type epithelial cells. The separated Qa-2-CD4SP could be induced to express Qa-2 molecules up to 84%- 89% of the total viable celb after cocultured for 3d with MTEC1 cells, a murine thymic medullary type epithelial cell line established in our laboratory. Kinetic study showed that both the percentage of Qa-2+ cells and the density of the expressed Qa-2 molecules on CD4SP thymocytes induced by MTEC1 were progressively increasing in 72-h cultures. The MTECl-induced Qa-2+CD4SP thymocytes were fully functional, which exhibited capabilities of proliferation and cytokine secretion in response to Con A stimulation as high as those of freshly isolated Qa-2+CD4SP thymocytes. The profile of cytokine     

Specialisation of thymic epithelial cells for positive selection of CD4+8+ thymocytes.

Following their migration into the thymus, hemopoeitic stem cell precursors enter a complex developmental pathway involving proliferation, differentiation and alphabetaT-cell receptor (alphabetaTCR)-mediated selection procedures, in order to generate mature T-cell populations ready for export to the periphery. Thus, a critical stage during intrathymic T-cell development involves the generation of functionally mature CD4+8- and CD4-8+ cells from immature CD4+8- precursor thymocytes, a poorly understood process referred to as positive selection. While interactions between the alphabetaTCR and MHC-peptide complexes are known to be essential for the initiation of positive selection, additional unknown signals are also required. Using an in vitro reaggregate thymic organ culture system which allows comparison of the abilities of various cell types to induce maturation of CD4+8+ precursors, we provide evidence that both MHC-peptide complexes and specialised accessory molecules must be provided by thymic epithelium for efficient mediation of positive selection. Moreover, analysis of positive selection in the presence of thymic and non-thymic stromal cells expressing MHC class II molecules with the same limited peptide array suggests that this unique ability of thymic epithelium to mediate positive selection of CD4+8- cells is not solely due to presentation of a specialised peptide repertoire, but is dependent upon provision of specialised accessory interactions.     

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.     

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.     

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.     

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.     

Functional maturation of mouse CD4+CD8- thymocytes induced by medullary-type thymus epithelial cells

Murine CD4⁺CD8^- (CD4SP) thymocyte subset is a heterogeneous population, in which the Qa-2^- cells are less functional, whereas the Qa-2⁺ cells are fully functional. Evidence is provided here that the transition from Qa-2^- to Qa-2⁺ CD4SP thymocytes is an intrathymic process of differentiation induced by thymic medullary-type epithelial cells. The separated Qa-2^-CD4SP could be induced to express Qa-2 molecules up to 84%- 89% of the total viable celb after cocultured for 3d with MTEC1 cells, a murine thymic medullary type epithelial cell line established in our laboratory. Kinetic study showed that both the percentage of Qa-2⁺ cells and the density of the expressed Qa-2 molecules on CD4SP thymocytes induced by MTEC1 were progressively increasing in 72-h cultures. The MTECl-induced Qa-2⁺CD4SP thymocytes were fully functional, which exhibited capabilities of proliferation and cytokine secretion in response to Con A stimulation as high as those of freshly isolated Qa-2⁺CD4SP thymocytes. The profile of cytokines secreted by MTECl-induced Qa-2⁺CD4SP thymocytes was Thy 0 type specified by the production of IL-2, IL-4 and IL-6. The results suggest that Qa-2^-CD4SP thymocytes may give rise to the Qa-2⁺CD4SP thymocytes, and acquire fully functional competence in thymic medulla under the foster of local epithelial cells.     

Reduced generation but efficient TCR beta-chain selection of CD4+8+ double-positive thymocytes in mice with compromised CD3 complex signaling

Maturation to the CD4+8+ double-positive (DP) stage of thymocyte development is restricted to cells that have passed TCRbeta selection, an important checkpoint at which immature CD4-8- double-negative (DN) cells that express TCRbeta polypeptide chains are selected for further maturation. The generation of DP thymocytes following TCRbeta selection is dependent on cellular survival, differentiation, and proliferation, and the entire process appears to be mediated by the pre-TCR/CD3 complex. In this study, we investigate the signaling requirements for TCRbeta selection using mice single deficient and double deficient for CD3zeta/eta and/or p56lck. While the numbers of DP cells are strongly reduced in the single-deficient mice, a further drastic reduction in the generation of DP thymocytes is seen in the double-deficient mice. The poor generation of DP cells in the mutant mice is primarily due to an impaired ability of CD25+ DN thymocytes to proliferate following expression of a TCRbeta-chain. Nevertheless, the residual DP cells in all mutant mice are strictly selected for expression of TCRbeta polypeptide chains. DN thymocytes of mutant mice expressed TCRbeta and CD3epsilon at the cell surface and contained mRNA for pre-Talpha, but not for clonotypic TCRalpha-chains, together suggesting that TCRbeta selection is mediated by pre-TCR signaling in all cases. The data suggest differential requirements of pre-TCR signaling for cell survival on the one hand, and for the proliferative burst associated with TCRbeta selection on the other.     

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.     

Heterodimeric complex formation with CD8 and TCR by bispecific antibody sustains paracrine IL-2-dependent growth of CD3+ CD8+ T cells.

During physiologic activation of mature CD8+ T cells, TCR and CD8 bind to the same Ag-complexed MHC class I molecule. Thereby, close proximity is induced between CD8 and the TCR/CD3 complex. During this engagement, CD8 may deliver TCR-independent signals via its associated protein tyrosine kinase, p56lck. We studied the potential biologic effects of close association between CD8 and TCR/CD3 complexes by using a bispecific antibody (bsAb) directed against both TCR and CD8 molecules. This hybrid hybridoma (quadroma)-produced bsAb binds as a monomeric molecule to CD3+ CD8+ but not CD3+ CD4+ T cells. The bsAb proved capable of inducing the cytotoxic effector function of cloned CD3+ CD8+ T cells but not of CD3+ CD4+ T cells. When the bsAb was presented to resting T cells by monocytes, proliferation of the CD3+ CD4+ but not the CD3+ CD8+ subset of T lymphocytes was induced. Parental anti-TCR antibody induced vigorous growth of cells of both subsets. Essentially identical results were obtained when bsAb was presented in an immobilized fashion. The unresponsiveness of the CD3+ CD8+ T cells with respect to mitogenesis could be restored by exogenous rIL-2. The data suggest that bsAb-induced activation differs from activation by monospecific anti-TCR antibody. The former appears to more closely mimic physiologic Ag-induced signaling, because it leads to a similar paracrine IL-2-dependent growth pattern. The bsAb may, therefore, be instrumental in studying T cell signaling pathways, in particular the role of CD8-associated p56lck therein.     

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.     

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.     

Inefficient cell spreading and cytoskeletal polarization by CD4+CD8+ thymocytes: regulation by the thymic environment

CD4(+)CD8(+) double-positive (DP) thymocytes express a lower level of surface TCR than do mature T cells or single-positive (SP) thymocytes. Regulation of the TCR on DP thymocytes appears to result from intrathymic signaling, as in vitro culture of these cells results in spontaneous TCR up-regulation. In this study, we examined cell spreading and cytoskeletal polarization responses that have been shown to occur in response to TCR engagement in mature T cells. Using DP thymocytes stimulated on lipid bilayers or nontransgenic thymocytes added to anti-CD3-coated surfaces, we found that cell spreading and polarization of the microtubule organizing center and the actin cytoskeleton were inefficient in freshly isolated DP thymocytes, but were dramatically enhanced after overnight culture. SP (CD4(+)) thymocytes showed efficient responses to TCR engagement, suggesting that releasing DP thymocytes from the thymic environment mimics some aspects of positive selection. The poor translation of a TCR signal to cytoskeletal responses could limit the ability of DP thymocytes to form stable contacts with APCs and may thereby regulate thymocyte selection during T cell development.     

Frequency analysis of CD4+CD8+ T cells cloned with IL-4

The coexpression of both CD4 and CD8 molecules on T cells occurs in the peripheral blood at a low frequency and can be generated transiently on CD4+ peripheral blood T cells by treatment with lectin which induces CD8 biosynthesis and cell surface expression. We have cloned T cells in a nonselective fashion from normal subjects in the presence of either IL-2, rIL-4 and IL-2, or rIL-4 and have examined the phenotypic expression of CD4 and CD8. The addition of excess rIL-4 increased the expression of CD8 on the surface of CD4+ T cell clones but did not increase CD4 expression on CD8+ T cell clones. There were three patterns of CD4 and CD8 expression observed: high density CD8 with no CD4 expression; high density CD4 with low CD8 expression; or high density CD4 with higher cell surface CD8 expression which was regulated by the presence of rIL-4. CD4+ T cell clones originally cultured in IL-2 and rIL-4 and subsequently grown in IL-2 alone exhibited decreased expression of the CD8 molecule. The increased expression of CD8 did not correlate with NK activity or lectin-dependent cytotoxicity in an antigen independent system. In addition, rIL-4 alone or in combination with IL-2 appeared to accelerate the growth curve of T cell clones as compared to IL-2 alone. These results show that IL-4 can upregulate CD8 expression on CD4+ T cell clones while not effecting CD4 expression on CD8+ T cell clones. As class I MHC is the ligand for the CD8 molecule, expression of CD8 induced by IL-4 on CD4+ T cells may allow for increased nonspecific cell to cell contact during the course of an inflammatory response.     

p53 and thymic 'death by neglect': thymic epithelial cell-induced apoptosis of CD4+8+ thymocytes is p53-independent

The involvement of the tumor suppressor protein, p53, in thymic epithelial cell-induced apoptosis of CD4+8+ (double positive) thymocytes, was studied in an in vitro model consisting of a thymic epithelial cell line (TEC) and thymocytes. p53 expression was not augmented in double positive (DP) thymocytes upon co-culturing with TEC, although extensive apoptosis was observed. In the same cells, p53 expression was upregulated in response to low ionizing irradiation, which was accompanied with massive apoptosis. Moreover, TEC induced apoptosis in two DP thymomas, derived from p53(-/-) mice, and in a double positive thymoma clone expressing mutant p53. The extent and kinetics of TEC-induced apoptosis was not affected by the status of p53 in the thymocytes tested. We conclude that thymic epithelial cell-induced apoptosis of immature DP thymocytes is p53-independent and apparently, involves a different apoptotic pathway than that triggered by DNA damage.     

Selective support of a mouse thymus epithelial cell line （MTEC1） to the viability and proliferation of CD4＋CD8—,CD4^—CD8^—,and CD4^＋CD8^＋thymocytes in vitro

The MTEC1 cell line,established in our laboratory,is a normal epithelial cell line derived from thymus medulla of Balb/c mice and these cells constituteively produce multiple cytokines.The selection of thymic microenvironment on developing T cells was investigated in an in vitro system.Unseparated fresh thymocytes from Balb/c mice were cocultured with MTEC1 cells or/and MTEC1-SN,then,the viability,proliferation and phenotypes of cultured thymocytes were assessed.Without any exogenous stimulus,both MTEC1 cells and MTEC1-SN were able to maintain the viability of thymocytes,while only the MTEC1 cells,not the MTEC1-SN,could directly activate thymocytes to exhibit moderate proliferation,indicating that the proliferative signal is delivered through cell surface interatcions of MTEC1 cells and thymocytes.Phenotype analysis on FACS of viable thymocytes after coculture revealed that MTEC1 cells preferentially activate the subsets of CD4^ CD8^-,CD4^ CD^8 and CD^4- CD^8- thymocytes;whereas MTEC1-SN preferentially maintained the viability of CD4^ CD^8- and CD4^-CD8^ thymocyte subsets.For the Con A-activated thymocytes.both MTEC1 cells and MTEC1-SN provided accessory signal(s) to significantly increase the number of viable cells and to markedly enhance the proliferation of thymocytes with virtually equal potency,phenotyped as CD4^ CD8^-,CD4^-CD8^ ,and CD^4-CD8^-subests,In summary,MTEC1 cells displayed Selection of thymic epithelial cells on thymocyte subsets. selective support to the different thymocyte subsets,and the selectivity is dependent on the status of thymocytes.     

Immature CD4+CD8+ thymocytes and mature T cells regulate Nur77 distinctly in response to TCR stimulation

The orphan steroid receptor, Nur77, is thought to be a central participant in events leading to TCR-mediated clonal deletion of immature thymocytes. Interestingly, although both immature and mature murine T cell populations rapidly up-regulate Nur77 after TCR stimulation, immature CD4+CD8+ thymocytes respond by undergoing apoptosis, whereas their mature descendants respond by dividing. To understand these developmental differences in susceptibility to the proapoptotic potential of Nur77, we compared its regulation and compartmentalization and show that mature, but not immature, T cells hyperphosphorylate Nur77 in response to TCR signals. Nur77 resides in the nucleus of immature CD4+CD8+ thymocytes throughout the course of its expression and is not found in either the organellar or cytoplasmic fractions. However, hyperphosphorylation of Nur77 in mature T cells, which is mediated by both the MAPK and PI3K/Akt pathways, shifts its localization from the nucleus to the cytoplasm. The failure of immature CD4+CD8+ thymocytes to hyperphosphorylate Nur77 in response to TCR stimulation may be due in part to decreased Akt activity at this developmental stage.     

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.     

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.