Effect of cyclosporin A on lymphopoiesis. I. Absence of mature T cells in thymus and periphery of bone marrow transplanted mice treated with cyclosporin A

In this report, we investigate the effect of cyclosporin A (CsA) on lymphopoiesis, and demonstrate that CsA selectively abrogates the development of CD4+CD8- and CD4-CD8+ T cells (single positive cells) in the thymus. This developmental arrest results in the complete absence of mature T cells (assessed both by phenotypic and functional analyses) in the spleen of syngeneic bone marrow transplanted mice subsequently treated with CsA. In contrast to its remarkable effect on T cells, CsA had no detectable effect on B cells differentiation. In the thymus, the generation of CD4+CD8+ thymocytes was not affected by CsA treatment, and CD4-CD8- thymocytes of CsA-treated mice expressed surface markers characteristic of normal CD4-CD8- thymocytes, and exhibited normal functional activity when stimulated with anti-CD3 antibody. Thus, CsA appears to prevent the generation of mature, single positive T cells without affecting the development of immature T cells in the thymus. In addition to its immunosuppressive effect on immunocompetent cells, these results indicate a novel feature of CsA, which involves arrest of T cell differentiation, a finding that may be important for applications in clinical bone marrow transplantation.     

IL-7 maintains the T cell precursor potential of CD3-CD4-CD8- thymocytes.

We and other investigators have reported that IL-4 (in the presence of PMA) or IL-7 (used alone) induce proliferation of both adult and fetal (gestation day 15) CD4-CD8- thymocytes. These results suggested that these cytokines may be growth factors for pre-T cells. However, we recently observed that among adult CD4-CD8- thymocytes, only the CD3+ subset proliferates in response to IL-7, whereas IL-4 + PMA induces proliferative responses in both CD3- and CD3+ subsets. Thus, we concluded that IL-7 used alone is not a potent growth stimulus for adult thymic CD3-CD4-CD8- triple negative (TN) T cell precursors. Interestingly, the viability of adult TN thymocytes in culture was improved by IL-7 for up to 1 wk, in spite of the inability of IL-7 to induce significant [3H]TdR incorporation in these cells. After culture in IL-7 for 4 days, the viable cells remained CD4-CD8-, but 25 to 35% expressed CD3 whereas the rest remained CD3-. In contrast, most of the cells cultured with IL-4 + PMA for 4 days remained TN. To investigate whether adult TN thymocytes that survive in vitro in the presence of IL-4 + PMA or IL-7 retain T cell progenitor potential, we tested whether they could reconstitute lymphoid cell-depleted (2-deoxyguanosine-treated) fetal thymus organ cultures. Our results demonstrate that TN cells cultured in IL-7 retain T cell progenitor potential.     

Thymic shared antigen-2: a novel cell surface marker associated with T cell differentiation and activation

Thymic shared Ag-2 (TSA-2) is a 28-kDa, glycophosphatidylinitosol-linked cell surface molecule expressed on various T cell and thymic stromal cell subsets. It is expressed on most CD3-CD4-CD8-, CD4+CD8+, and CD3highCD4-CD8+ thymocytes but is down-regulated on approximately 40% of CD3highCD4+CD8- thymocytes. Expression on peripheral TCR-alphabeta+ T cells is similar to that of CD3+ thymocytes, although a transient down-regulation occurs with cell activation. Consistent with the recent hypothesis that emigration from the thymus is an active process, recent thymic emigrants are primarily TSA-2-/low. TSA-2 expression reveals heterogeneity among subpopulations of CD3highCD4+CD8- thymocytes and TCR-gamma delta+ T cell previously regarded as homogenous. The functional importance of TSA-2 was illustrated by the severe block in T cell differentiation caused by adding purified anti-TSA-2 mAb to reconstituted fetal thymic organ culture. While each CD25/CD44-defined triple-negative subset was present, differentiation beyond the TN stage was essentially absent, and cell numbers of all subsets were significantly below those of control cultures. Cross-linking TSA-2 on thymocytes caused a significant Ca2+ influx but no increase in apoptosis, unless anti-TSA-2 was used in conjunction with suboptimal anti-CD3 mAb. Similar treatment of mature TSA-2+ T cells had no effect on cell survival or proliferation. This study reveals TSA-2 to be a functionally important molecule in T cell development and a novel indicator of heterogeneity among a variety of developing and mature T cell populations.     

In vitro induction of CD8 expression on thymic pre-T cells. I. Transforming growth factor-beta and tumor necrosis factor-alpha induce CD8 expression on CD8- thymic subsets including the CD25+CD3-CD4-CD8- pre-T cell subset.

We previously reported that IL-7 maintains the viability and differentiation potential of CD25 (IL-2R p55) positive CD3-CD4-CD8- thymic pre-T cells in vitro. This culture system is suitable for studying signals that regulate differentiation of T cell precursors in the thymus. In this study, we screened cytokines for their capacity to induce CD4 or CD8 in murine thymic pre-T cells cultured with IL-7. Of 15 cytokines tested, only transforming growth factor (TGF-beta) and TNF-alpha induced CD8 (Lyt-2), while no cytokine was able to induce CD4 on CD25+CD3-CD4-CD8- thymocytes. The combination of TGF-beta and TNF-alpha was synergistic, and the majority of cells recovered after 2 to 3 days in culture expressed CD8 (but not CD3 or CD4). A similar effect of TGF-beta and TNF-alpha was observed using day-15 fetal thymocytes, CD3+CD4-CD8- or CD3+CD4+CD8- adult thymocytes, although the combination of these cytokines resulted in an additive rather than a synergistic effect in these subsets. In contrast, neither TGF-beta nor TNF-alpha induced CD8 expression on splenic CD4+CD8- T cells. These observations suggest a role for these cytokines in the induction of CD8 expression in CD8- thymocyte subsets including CD3-CD4-CD8- thymic pre-T cells.     

Clonal analysis of human CD4-CD8-CD3- thymocytes highly purified from postnatal thymus.

Human triple-negative (CD4-CD8-CD3-) thymocytes purified from postnatal thymus by the use of magnetic bead columns and cell sorting were cultured in bulk or cloned with a feeder cell mixture of irradiated PBL, irradiated JY cells, and PHA. Triple-negative thymocytes proliferated well under these culture conditions, and after 12 days in bulk culture they remained triple negative. Limiting dilution experiments revealed that the frequency of clonogenic cells in fresh triple-negative thymocytes was less than 1%. Of 40 clones obtained in a representative experiment, 37 were triple negative and 3 were CD4+ TCR-alpha beta+. No TCR-gamma delta+ clones were isolated. Some of the triple-negative clones expressed CD16 and were apparently NK cells. Seven representative CD16-triple-negative clones were expanded and characterized in detail. These clones shared the common cell surface phenotype of CD1-CD2+CD3-CD4--CD8-CD5-CD7+CD16-CD56+. One of them expressed cytoplasmic CD3 delta and CD3 epsilon Ag, but these Ag were not detected in any peripheral blood-derived CD16- NK clones examined for comparison. The seven CD16- thymus-derived clones exhibited significant cytolytic activity against K562. The clone that expressed cytoplasmic CD3 Ag was shown to have the germ-line configuration of the TCR-beta and TCR-gamma genes. Thus, it is suggested that in vitro culture of triple-negative thymocytes can give rise to NK-like cells, including those that express cytoplasmic CD3 Ag. In contrast to previous reports, our results gave no evidence of differentiation of triple-negative thymocytes into TCR-alpha beta+ or TCR-gamma delta+ T cells.     

Signals from the IL-9 receptor are critical for the early stages of human intrathymic T cell development

Highly purified human CD34+ hemopoietic precursor cells differentiate into mature T cells when seeded in vitro in isolated fetal thymic lobes of SCID mice followed by fetal thymus organ culture (FTOC). Here, this chimeric human-mouse FTOC was used to address the role of IL-9 and of the alpha-chain of the IL-9 receptor (IL-9Ralpha) in early human T cell development. We report that addition of the mAb AH9R7, which recognizes and blocks selectively the human high affinity alpha-chain of the IL-9R, results in a profound reduction of the number of human thymocytes. Analysis of lymphoid subpopulations indicates that a highly reduced number of cells undergo maturation from CD34+ precursor cells toward CD4+CD3-CD8-CD1+ progenitor cells and subsequently toward CD4+CD8+ double positive (DP) thymocytes. Addition of IL-9 to the FTOC resulted in an increase in cell number, without disturbing the frequencies of the different subsets. These data suggest that IL-9Ralpha signaling is critical in early T lymphoid development.     

Age-associated rapid and Stat6-independent IL-4 production by NK1-CD4+8- thymus T lymphocytes.

The source of IL-4 required for priming naive T cells into IL-4-secreting effectors has not been clearly identified. Here we show that upon TCR stimulation, thymus NK1-CD4+8- T cells produced IL-4, the magnitude of which was inversely correlated with age. This IL-4 production response by Th2-prone BALB/c mice was approximately 9-fold that of Th1-prone C57BL/10 mice. More than 90% of activated NK1-CD4+8- thymocytes did not use the invariant V alpha 14-J alpha 281 chain characteristic of typical CD1-restricted NK1+CD4+ T cells. Stat6-null NK1-CD4+8- thymocytes produced bioactive IL-4, with induction of IL-4 mRNA expression within 1 h of stimulation. Our results support the possibility that TCR repertoire-diverse conventional NK1-CD4+ T cells are a potential IL-4 source for directing naive T cells toward Th2/type 2 CD8+ T cell (Tc2) effector development.     

Inhibitory effect of murine recombinant IL-4 on thymocyte development in fetal thymus organ cultures

The effect of rIL-4 on early T cell development in fetal thymus organ culture was studied by two-color immunofluorescence and by proliferation assays. Addition of IL-4 to an organ culture of 14-day-old fetal BALB/c thymus resulted in a decreased cell yield (less than 15% as compared to the control culture) and in a complete inhibition of CD4+CD8+ thymocytes after 12 days of culture. This IL-4 effect was time dependent as shorter times (less than 6 days) resulted in a thymic development comparable to the untreated control. When thymuses at later stages of gestation were treated with IL-4, the inhibition was less pronounced. The inhibitory activity of IL-4 was abrogated by the anti-IL-4 mAb 11B11, ruling out the possibility of a contaminant. Organ cultured 14-day-old fetal thymocytes treated with IL-4 during 12 days responded vigorously to IL-2 alone and to IL-4 + PMA + ionomycin. Taken together, these results indicate that IL-4 affects the intrathymic T cell development at an early stage.     

Treatment of fetal thymic organ culture with IL-1 leads to accelerated differentiation of subsets of CD4-CD8- cells.

Using fetal thymic organ culture (FTOC), we describe the effects of IL-1 on T cell differentiation, particularly within the CD4-CD8- subset. While treatment of FTOC with IL-1 led to a modest reduction in total thymocyte yield, it induced an increase in the percentage of CD4-CD8- cells that express IL-2R early in culture and a decrease in the number of their precursors (CD44+IL-2R- cells). The increase in the percentage of cells expressing IL-2R was not accompanied by an increase in the number of these cells. At later time points these IL-2R+ cells (and their precursors) were reduced relative to controls. The total number of CD4-CD8-CD3- precursor cells in IL-1-treated cultures was reduced to approximately half that in controls at Day 12 of culture. However, only minor inhibition of total cell number was observed, which, taken together with the greater frequency of IL-2R+ precursors, suggests that this depletion of the pool of precursors may have been due to the induction of premature differentiation rather than to its inhibition.     

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.     

Tcl-30, a new T cell-specific gene expressed in immature glucocorticoid-sensitive thymocytes.

Glucocorticoid treatment induces programmed cell death (apoptosis) in susceptible T lymphocytes. We have previously isolated and characterized 13 genes induced by agents that cause apoptosis in the murine thymoma cell line, WEHI-7TG. One of these genes, now designated Tcl-30, encodes a 2.4-kb mRNA that is specifically expressed in thymus. Sequence analysis of a full-length cDNA for Tcl-30 reveals a potential open reading frame of 454 amino acids that shares sequence identity to a human placental-specific protein of unknown function, PP11. The putative protein encoded by Tcl-30 also contains a cysteine-rich somatomedin B-like domain found in vitronectin, PP11, and the plasma cell membrane glycoprotein, PC-1. Subpopulations of murine thymocytes sorted on the basis of their expression of the CD4, CD8, and surface heat-stable Ag (HSA) were analyzed by an RNase protection assay to determine the expression of Tcl-30 as a function of T cell development. Tcl-30 was expressed exclusively in the HSA+ T cell populations (CD4-CD8-HSA+; CD4+CD8-HSA+; CD4-CD8+HSA+; and CD4+CD8+HSA+) and not in the HSA- single positive T cell populations (CD4+CD8- or CD4-CD8+) of the thymus or spleen. Therefore, we conclude that Tcl-30 expression is lost during T cell maturation and is absent at the most mature stages of T cell development. The function of Tcl-30 is unknown; however, the CD4+CD8+ double-positive subpopulation expressing Tcl-30 represents thymocytes destined to undergo massive intrathymic cell death. The possibility that Tcl-30 expression may define a population of T lymphocytes that is sensitive to glucocorticoid-mediated apoptosis is discussed.     

Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

Effects of cyclosporine A on thymocyte differentiation in fetal thymus organ culture

During the course of differentiation in the thymus, precursor T cells are negatively selected by a self-tolerance mechanism or positively selected to acquire restriction specificity to self major histocompatibility complexes. We investigated the process of T cell differentiation and those selections using a fetal thymus organ culture with or without cyclosporine A. The agent blocked the maturation step from CD4+8+ double positive cells to mature CD4-8+ or CD4+8- single positive cells. On the other hand, the agent did not inhibit the development of CD3+4-8- T cell receptor (TCR)alpha beta- cells, which were supposed to be T cells bearing gamma delta-TCR chains. These results suggest that the development of thymocytes bearing alpha beta- or gamma delta-TCR chains differ in requirement for thymocyte-stromal cell interaction.     

Expression of an unusual T cell receptor (TCR)-V beta repertoire by Ly-6C+ subpopulations of CD4+ and/or CD8+ thymocytes. Evidence for a developmental relationship between Ly-6C+ thymocytes and CD4-CD8-TCR-alpha beta+ thymocytes.

A novel thymocyte subpopulation expressing an unusual TCR repertoire was identified by high surface expression of the Ly-6C Ag. Ly-6C+ thymocytes were distributed among all four CD4/CD8 thymocyte subsets, and represented a readily identifiable subpopulation within each one. Ly-6C+ thymocytes express TCR-alpha beta, arise late in ontogeny, and appear in the CD4/CD8 developmental pathway after birth in a sequence that resembles that followed by conventional Ly-6C- cells during fetal ontogeny. Most interestingly, adult Ly-6C+ thymocytes express an unusual TCR-V beta repertoire that is identical to that expressed by CD4-CD8-TCR-alpha beta+ thymocytes in its overexpression of TCR-V beta 8 and in its expression of some potentially autoreactive TCR-V beta specificities. This unusual TCR-V beta repertoire was even expressed by Ly-6C+ thymocytes contained within the CD4+ CD8- 'single positive' thymocyte subset. Thus, expression of this unusual TCR-V beta repertoire is not limited to CD4-CD8-thymocytes, and is unlikely to be a consequence of their double negative phenotype. Rather, we think that Ly-6C+TCR-alpha beta+ thymocytes and CD4-CD8-TCR-alpha beta+ are developmentally interrelated, a conclusion supported by several lines of evidence including the selective failure of both Ly-6C+ and CD4-CD8-TCR-alpha beta+ thymocyte subsets to appear in TCR-beta transgenic mice. In contrast, peripheral Ly-6C+ T cells are developmentally distinct from Ly-6C+ thymocytes in that peripheral Ly-6C+ T cells expressed a conventional TCR-V beta repertoire and developed normally in TCR-beta transgenic mice in which Ly-6C+ thymocytes failed to arise. We conclude that: 1) expression of a skewed TCR-V beta repertoire is a characteristic of Ly-6C+TCR-alpha beta+ thymocytes as well as CD4-CD8-TCR-alpha beta+ thymocytes, and is not unique to thymocytes expressing neither CD4 nor CD8 accessory molecules; and 2) Ly-6C+ thymocytes are developmentally linked to CD4-CD8-TCR-alpha beta+ thymocytes, but not to Ly-6C+ peripheral T cells. We suggest that Ly-6C+TCR-alpha beta+ thymocytes are not the developmental precursors of Ly-6C+ peripheral T cells, but rather may be the developmental precursors of CD4-CD8-TCR-alpha beta+ thymocytes.     

Phenotypic and functional characterization of c-kit expression during intrathymic T cell development.

We have studied the expression and function of c-kit on subsets of mouse thymocytes. c-kit was primarily expressed on subpopulations of CD4-CD8-CD3- triple negative (TN) cells. The strongest c-kit expression was associated with subsets that represent the least mature TN cells, including CD44+CD25- TN, and a subpopulation of CD25+ TN. These cells were also Thy-1lo, H-2Khi TSA-1hi, HSAlo, B220-, Mac-1-, and Gr-1-. Additionally, the recently described pre-TN thymocyte population (CD4loCD3-CD8-) was also c-kit+. CD25+ TN thymocytes proliferated in the presence of IL-7 and stem cell factor (the ligand for c-kit), and this proliferation was completely inhibited in the presence of anti-c-kit. Furthermore, the addition of anti-c-kit to 2-deoxyguanosine-treated fetal thymic lobes undergoing reconstitution with fetal liver-derived precursor cells inhibited their T cell differentiation potential. These observations indicate an important role for c-kit/stem cell factor interactions during early thymocyte development.     

Lunatic fringe controls T cell differentiation through modulating notch signaling

T cells differentiate from bone marrow-derived stem cells by expressing developmental stage-specific genes. We here searched arrays of genes that are highly expressed in mature CD4-CD8+ (CD8 single-positive (SP)) T cells but little in CD4+CD8+ (double-positive (DP)) cells by cDNA subtraction. Lunatic fringe (Lfng), a modulator of Notch signaling, was identified to be little expressed in DP cells and highly expressed in CD8SP T cell as well as in CD4-CD8- (double-negative (DN)) and mature CD4+CD8- (CD4SP) T cells. Thus, we examined whether such change of expression of Lfng plays a role in T cell development. We found that overexpression of Lfng in Jurkat T cells strengthened Notch signaling by reporter gene assay, indicating that Lfng is a positive regulator for Notch signaling in T cells. The enforced expression of Lfng in thymocytes enhanced the development of immature CD8SP cells but decreased mature CD4SP and CD8SP cells. In contrast, the down-regulation of Lfng in thymocytes suppressed DP cells development due to the defective transition from CD44+CD25- stage to subsequent stage in DN cells. The overexpression of Lfng in fetal liver-derived hemopoietic stem cells enhanced T cell development, whereas its down-regulation suppressed it. These results suggested that the physiological high expression of Lfng in DN cells contributes to enhance T cell differentiation through strengthening Notch signaling. Shutting down the expression of Lfng in DP cells may have a physiological role in promoting DP cells differentiation toward mature SP cells.     

A population of early fetal thymocytes expressing Fc gamma RII/III contains precursors of T lymphocytes and natural killer cells.

We have identified a dominant fetal thymocyte population at day 14.5 of gestation in the mouse that lacks CD4 and CD8 but expresses Fc gamma RII/III several days prior to acquisition of the T cell receptor (TCR) in vivo. If maintained in a thymic microenvironment, this population of CD4-CD8-TCR-Fc gamma RII/III+ thymocytes differentiates first into CD4+CD8+TCRlowFc gamma RII/III- thymocytes and subsequently CD4+CD8-TCRhighFc gamma RII/III- and CD4-CD8+TCRhighFc gamma RII/III- mature Ti alpha-beta lineage T cells. However, if removed from the thymus, the CD4-CD8-TCR-Fc gamma RII/III+ thymocyte population selectively generates functional natural killer (NK) cells in vivo as well as in vitro. These findings show that a cellular pool of Fc gamma RII/III+ precursors gives rise to T and NK lineages in a microenvironment-dependent manner. Moreover, they suggest a hitherto unrecognized role for Fc receptors on primitive T cells.     

Selection of CD4+CD8+ thymocytes by complex formation with medulla-derived epithelial cells

The role of lymphostromal complexes in T-cell differentiation is far from elucidated, mainly because a clear association of a particular stromal cell type with a distinct thymocyte subset has never been identified. Using an in vitro system, detecting the adherence of thymocytes to a thymic medullary epithelial cell line (E-5), we showed that the phenotype of these thymocytes was that of cortical type: Thy-1hi, LFA-1+, PNAhi, CD4+CD8+, MEL-14-/lo, IL-2R-, CD3-/lo, and TcR V beta 8-/lo. They were enriched in cells in G2/M at the time of complex formation, showed a higher basal proliferation in culture, and did not respond to PHA, IL-2 and only marginally to Con A. These data show that complex formation with mouse thymic medullary epithelium selects for CD4+CD8+ thymocytes, as shown by the marked decrease in CD4+CD8-/CD4-CD8+ thymocytes, and the incapacity of CD4-CD8- thymocytes to adhere.     

Effect of cyclosporin A on the ontogeny of different T cell sublineages in chickens

We have used a panel of murine mAb against chicken TCR and associated molecules to study the effect of cyclosporin A (CsA) on the ontogeny of the different sublineages of T cells. After injection of CsA (20 mg/kg/day from day 0 to 20) we observed a significant suppression of the normal maturation of the TCR2 (alpha beta TCR) cells in their transition from cortical CD4+CD8+ thymocytes to the mature single positive cells in the thymus medulla. The TCR3 subpopulation, a distinct form of alpha beta-like TCR in chickens, was inhibited from initially developing within the cortex by CsA, indicating that the TCR3 subpopulation is functionally distinct from the TCR2+ cells. In contrast, the maturation and peripheral emigration of TCR1 (gamma delta TCR) cells was unaffected by CsA treatment. Mature splenic T cells sorted for either TCR1+ or TCR2+ subsets were equally sensitive to CsA blockade of Con A-stimulated mitogenesis, indicating that there is no inherent difference in CsA sensitivity between these sublineages. Furthermore, no difference was detected in the expression of class II MHC Ag in thymi of birds treated with olive oil vs CsA. Inasmuch as the mechanism of CsA action appears to involve inhibition of TCR initiated signal transduction for lymphokine synthesis, these data indicate that a similar signaling is involved in thymic repertoire selection for TCR2. The lack of an effect on TCR1 cell maturation suggests that the TCR1 repertoire may not undergo selection in the thymus as do TCR2+ cells.