Expression of high molecular weight isoforms of CD45 by mouse thymic progenitor cells

We have studied the expression of isoforms of CD45 (leukocyte common antigen, LCA) among T cell precursors using the organ culture system of Jenkinson et al. (Eur. J. Immunol. 1982. 12: 583). These experiments show that cells capable of recolonizing alymphoid embryonic thymic lobes in vitro can be detected in the thymus of fetal and adult mice and are enriched when thymocytes are depleted of cells bearing CD4 or CD8. These data are consistent with results from in vivo experiments of Fowlkes et al. (J. Exp. Med. 1985. 162: 802) indicating that T cell precursors lie within the double-negative (CD4-CD8-) compartment. No precursors were detected among the reciprocal populations of cells bearing CD4 and/or CD8 (single and double positives). Double-negative cell fractions were then divided on the basis of reactivity with monoclonal antibodies RA3-2C2 and RA3-3A1. These antibodies recognize the high molecular weight species of the LCA or, more accurately, a product defined by exon A of the CD45 gene. Recolonizing cells were found predominantly in the CD45RA+ (RA3-2C2 and RA3-3A1 reactive) fraction of double-negative thymocytes; CD45RA- enriched populations had increased efficiency of recolonization and CD45RA- depleted populations had decreased ability to recolonize as compared with the whole CD4-CD8- fraction. To clarify whether progenitors enriched in the CD45RA+ fraction were capable of giving rise to mature CD4+, CD8+ and CD4+ CD8+ cells, we analyzed the progeny of lobes seeded with CD4-CD8-CD45RA+ fractions. After 7-9 days in organ culture the proportion of CD4+, CD8+ or CD4+ CD8+ cells had increased to 35.2%, 18.6% and 23.7%, respectively (mean of five experiments), indicating that progenitors among the CD45RA+ population were indeed multipotent. These results suggest that the majority of T stem cells in the thymus are among thymocytes that express the CD45RA molecule, an hypothesis supported by our finding that removal of CD45RA-expressing cells (using complement and antibody) eliminated recolonizing capacity of thymic cell fractions.     

CD45RA antibodies split the CD3bright T cell subset.

Thymocyte subsets have been well characterized on the basis of CD4 and CD8 antigen expression. Recently, the use of anti-CD3 antibodies has allowed more precise phenotyping of these subsets. The most immature T cell precursors are largely CD3-CD4-CD8-, while the most mature are CD3brightCD4+CD8- or CD3brightCD4-CD8+. Moreover, the expression of CD45RA on thymocytes appears to define a progenitor population and may define a continuous lineage of cells. Using a panel of CD45RA antibodies, we have further characterized the CD45RA+ thymocyte population in the murine system. The size of this subset is greatly enhanced in cortisone-treated mice and in sublethally irradiated mice. Moreover, the CD45RA+ population is present early in foetal life and is maintained thereafter. Using three-colour immunofluorescence, we show that (i) while most CD45RA+ cells are present amongst the CD4-CD8- thymocyte subset in the normal thymus, after cortisone treatment or irradiation, all four thymocyte subsets co-express significant amounts of CD45RA. This suggests that not only progenitor cells but also the mature population which can survive such manipulation are CD45RA+; and (ii) a large proportion of CD45RA+ cells are CD3bright and this subset is represented in the thymus at all stages of maturation tested. These data suggest that a proportion of TCR-gamma delta + CD3+ cells in the fetus as well as of TCR-alpha beta+ CD3+ cells in the adult co-express CD45RA.     

Functional and phenotypic delineation of two subsets of CD4 single positive cells in the thymus

CD4 single positive thymocytes are the fraction of mature thymocytes that contains precursors of MHC class II restricted T cells. In the experiments presented here, we demonstrate phenotypic and functional heterogeneity amongst CD4 single positive thymocytes from adult mice. Approximately 70% of these cells adhere to anti-CD8 antibody-coated dishes and therefore express low levels of CD8 molecules. They are referred to here as CD8loCD4hi. The remaining 30% are CD8-CD4hi. The CD8loCD4hi subset also expresses 3-fold higher surface levels of heat-stable antigen (HSA) than CD8-CD4hi thymocytes. Both CD4hi subsets express high levels of the alpha beta TCR/CD3 complex on the cell surface, and can proliferate in response to allogeneic cells expressing MHC class II differences but not to cells expressing only class I disparate molecules. However, CD8-CD4hi thymocytes are self-sufficient in such a proliferative response, whereas CD8loCD4hi thymocytes require exogenous IL-2 for optimal proliferation. The results suggest that CD8loCD4hi thymocytes are not completely mature. Their phenotype suggests that they might be descendants from CD8hiCD4hi double positive thymocytes, and that they have begun to down-regulate gradually CD8 and HSA molecules. The relationship between the two CD4hi single positive subsets is discussed.     

CD45 isoform expression during T cell development in the thymus.

Various isoforms of leukocyte common antigen, or CD45, are expressed differentially on T cells at different stages of development and activation. We report studies on CD45 isoform expression on various subsets of human T cells using two- and three-color flow cytometry and cell depletion. Bone marrow cells that were depleted of CD3+ and HLA-DR+ cells were CD45RA-RO-. The earliest CD3-CD4-CD8-CD19- thymocytes were CD45RO- with 20%-30% CD45RA+ cells. The most prominent population of CD4+CD8+ double-positive thymocytes were CD45RA-RO+. Even the CD4+CD8+ blasts were greater than 90% CD45RO+. About 80% of single-positive thymocytes (CD4+CD8- or CD4-CD8+) were also CD45RO+. Only 4.3% of CD4+ and 18% of CD8+ single-positive thymocytes were CD45RA+. In contrast, cord blood T cells which represent the stage that immediately follows single-positive thymocytes, contained 90% CD45RA+ cells. Thus, in terms of CD45 isoform expression, single-positive thymocytes are more like double-positive cells than cord blood T cells. These results suggest the following sequence of CD45 isoform switching during T cell development: CD45RA-RO- or RA+RO- (double-negative thymocytes)----RA-RO+ (double-positive and most single-positive thymocytes)----RA+RO- (cord blood T cells), the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus.     

Interleukin-4 induces expression of the CD45RA antigen on human thymocyte subpopulations

Interleukin-4 (IL-4) is a multifunctional lymphokine which promotes the growth and/or maturation of multiple cell types. We have examined the ability of IL-4 to promote the phenotypic maturation of subsets of human thymocytes. When cultured in serum-free medium supplemented with recombinant IL-4, a subset of immature CD3-CD45RA- human thymocytes ceased to express the CD1 common thymocyte antigen and acquired phenotypic features characteristic of relatively mature thymocytes, such as high-density expression of the CD3 antigen and de novo expression of the CD45RA isoform of the common leukocyte antigen family. These changes, which were not seen in cells cultured in medium alone, occurred over an 8-9 day period and were accompanied by a significant increase in cell size. The CD45RA+ cells that derived from these immature CD3-CD45RA- precursors were mainly CD4-CD8- or CD8+ cells, and a significant proportion of these cells expressed the T cell receptor delta chain. IL-4 also increased expression of the CD45RA antigen on the more mature CD3+ thymocyte population. However, the CD45RA+ cells derived from IL-4 stimulated CD3+ thymocyte precursors expressed either the CD4 or the CD8 antigen, and virtually all expressed alpha/beta TCR chains. Studies of cell viability and cell growth indicated that these findings were due to direct changes in the phenotype of responsive cells rather than the growth or selective survival of a small number of mature thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Timing of deletion of autoreactive V{beta}6+ cells and down-modulation of either CD4 or CD8 on phenotypically distinct CD4+8+ subsets of thymocytes expressing intermediate or high levels of T cell receptor

In this paper we describe a differentiation sequence amongst adult murine thymocytes which goes from CD4+8+3lo(low) to CD4+8+3int(intermediate) to CD4+8+3hi(high) and then to mature single positive CD3hi thymocytes. Phenotypic characterization of CD4+8+3int/hi cells for a number of other surface markers is consistent with them being in transition from CD4+8+3lo phenotype to mature phenotype. The same observation was made for sensitivity towards ionomycin-mediated apoptosis. In the thymus of Mls-1a mice, where autoreactive TCR-V beta 6+ cells are negatively selected, deletion of TCR-V beta 6+ cells was first detected in the CD4+8+3int subset, and was complete by the CD4+8+3hi stage, suggesting that up-regulation of the TCR/CD3 complex is required for deletion of Mls-1a autoreactive thymocytes. No sign of apoptosis was detected among any fresh thymocyte subsets suggesting that apoptotic cells are rapidly cleared from the thymus. The CD4+8+3int/CD4+8+3hi cells are therefore populations in transit from the typical cortical thymocytes to the mature T-cells.     

CD4-CD8- thymocytes that express the T cell receptor may have previously expressed CD8

Amongst CD4-CD8- (double negative) thymocytes there is a sizeable population (variable from strain to strain) of cells expressing surface T cell receptor (TCR). These TCR+ double negatives are predominantly non-cycling, have very little precursor activity, and, unlike the TCR-CD4-CD8- thymocytes, appear not to be part of the mainstream of thymocyte development. A unique feature of this population is the biased V beta-gene region usage. In CBA mice, 60-70% of TCR+ CD4-CD8- cells express receptors that utilize V beta 8 gene products, compared with peripheral T cells from the same strain which are only 20-30% V beta 8+. This suggests that the high V beta 8 usage may be the result of some selective process. A growing body of experimental data suggests that TCR specificity selection occurs at the CD4+CD8+ stage of thymocyte development. In order to gain some insight into the previous history of the TCR+ double negatives, in particular whether or not they have previously expressed CD8 and therefore been eligible for selection, we have determined the methylation state of the CD8 gene and compared it to other thymocyte populations. We show that the TCR+ CD4-CD8- thymocytes are demethylated at some sites in the CD8 gene, consistent with previous CD8 expression. However, the demethylation pattern is distinct from that seen on typical peripheral T cells or on mature thymocytes, suggesting that the TCR+ CD4-CD8- thymocytes are not derived from mature thymocytes or peripheral T cells which have returned to the thymus and downregulated CD8 expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Delineation of chicken thymocytes by CD3-TCR complex, CD4 and CD8 antigen expression reveals phylogenically conserved and novel thymocyte subsets.

To further define the relationship between thymocyte subsets and their developmental sequence, multi-parameter flow cytometry was used to determine the distribution of the CD3-TCR complex and the accessory molecules CD4 and CD8 on chicken thymocytes. As in mammals, adult thymocytes could be subdivided into CD3-, CD3lo, and CD3hi staining populations. CD4 and CD8 distribution on such populations revealed the presence of CD3-CD4+CD8- and CD3-CD4-CD8+ thymocytes, putative precursors to CD4+CD8+ cells, detectable in the adult and at high frequency during ontogeny. Of particular interest was the existence of CD3lo expression on CD4+CD8- and CD4-CD8+, and in some instances, on CD4-CD8- thymocytes. Such phenotypes are not easily detectable in the mammalian thymus but were readily observed in both adult and embryonic chicken thymus from 16 days of embryogenesis. Further analysis of the TCR lineage of these CD3lo cells revealed that they were essentially all of the alpha beta TCR type. Mature CD3hi thymocytes were found within the CD4+CD8+ and CD4+CD8- and CD4-CD8+ subsets. Both alpha beta and gamma delta TCR lineage thymocytes were detected within all CD4- and CD8-defined subsets, thus identifying novel thymocyte subsets in the chicken thymus, namely alpha beta TCR+CD4-CD8- and gamma delta TCR+ CD4+CD8- cells. Hence, this analysis of chicken thymocytes, while confirming the phylogenically conserved nature of the thymus, has revealed novel T cell subsets, providing further insight into the complexity of mainstream thymocyte maturation pathways.     

The variable occurrence of CD45RA on CD8+ thymocytes correlates with the presence of Mtv sequences; its expression on other thymocytes is rare

While all thymocytes express CD45, only a small fraction (<3% in mice) bear the high molecular weight isoform, CD45RA. It has been suggested that these cells alone constitute the generative thymocyte lineage and should, therefore, occur within every ontogenic subset. To test this, we determined CD45RA expression among thymocyte subsets defined by CD4 and CD8. In some strains, exemplified by C57BL/Icrf, very few (< 0.2%) Tthymocytes expressed CD45RA and were mostly CD4^?CD8^? or CD4⁺CD8⁺, inconsistent with them constituting the generativelineage. In others, exemplified by BALB/c, CD45RA was expressed on up to 3% of T thymocytes, which were mostly CD4^?CD8⁺. The limited occurrence of CD4^?CD8⁺CD45RA⁺ thymocytes suggests that they are a nonconstitutive subset playing a role in the thymus of only some strains. Their occurrence correlates with that of Mtv proviruses within the mouse genome; however, their T cell receptor V_β repertoire is diverse, suggesting they are not uniquely selected by Mtv superantigens. We propose that they may be mature CD8 T cells, possibly responsible for introducing viral superantigens into the thymus.     

Ontogeny of T cell maturation in LEC mutant rats which bear a congenital arrest of maturation from CD4+CD8+ to CD4+CD8- thymocytes.

LEC rats bear a congenital deficiency in CD4+CD8- thymocytes and peripheral CD4+ T cells, and consequently a deficiency in Th cell functions. Ontogeny of T cell maturation in normal and LEC mutant rats was, therefore, investigated. Prenatal development of thymocytes in normal rat strains, with respect to the expression of CD4/CD8 and TcR antigens, was similar to that of mice except that its kinetics was delayed by approximately 24 h. The kinetics of T cell maturation in LEC rats was comparable with that of normal rats up to day 19 of gestation, at which stage double-negative thymocytes (CD4-CD8-) developed into double positives (CD4+CD8+) through immature CD4-CD8+ subset. At day 19 of gestation in LEC as well as normal rats, double positives occupied approximately 80% of the total thymocytes, half of which were TcR-dull positive, indicating that TcR was normally rearranged and then expressed in LEC rat thymocytes. These data indicate that double negatives normally mature into at least double positives in LEC rats. Both single positives appeared after day 19 of gestation in normal rats, while in LEC rats CD4+CD8- cells did not appear, suggesting that the deficiency in CD4+CD8- cells is due to a congenital arrest of maturation from CD4+CD8+ to CD4+CD8- cells, but not due to a postnatal deletion.     

T cell receptor-negative thymocytes from SCID mice can be induced to enter the CD4/CD8 differentiation pathway

In order to investigate the role of T cell receptor (TcR) expression in thymocyte maturation, we have analyzed thymocytes from C.B-17/SCID mice, which are unable to productively rearrange their antigen receptor genes and fail to express TcR. Despite this defect, SCID thymocytes are functional as they produce lymphokines and proliferate in response to a variety of stimuli. Phenotypic analysis revealed that thymocyte populations from young adult SCID mice resemble thymocyte populations from normal embryonic mice in that they are large, Thy-1.2+, CD4-, CD8-, TcR- and enriched in CD5lo, IL2R+ and Pgp1+ cells. However, other TcR- populations normally present in adult mice (i.e., CD4-CD8+ cells and CD4+CD8+ cells) are absent from the thymus of TcR- adult SCID mice. To understand the basis of the developmental arrest of TcR- SCID thymocytes at the CD4-CD8- stage of differentiation, we analyzed thymi from the occasional "leaky" SCID mouse which possesses small numbers of TcR+ thymocytes. We found that the presence of TcR+ cells within a SCID thymus was invariably associated with the presence of CD4+ and/or CD8+ SCID thymocytes. Interestingly, however, the CD4+/CD8+ SCID thymocytes were not themselves necessarily TcR+. That is, emergence of SCID thymocytes expressing CD4/CD8 was tightly linked to the presence of TcR+ cells within that SCID thymus, but the SCID thymocytes that expressed CD4/CD8 were not necessarily the same cells that expressed TcR. Finally, we found that the introduction into TcR- SCID mice of normal bone marrow cells that give rise to TcR+ cells within the SCID thymus promoted the differentiation of SCID thymocytes into CD4-CD8+ and CD4+CD8+ TcR- cells. These data indicate that TcR+ cells within the thymic milieu provide critical signals which promote entry of CD4-CD8-TcR- precursor T cells into the CD4/CD8 differentiation pathway. When applied to differentiation of normal thymocytes, these findings may imply a critical role for early appearing CD4-CD8- TcR (gamma/delta)+ cells in initiating normal thymic ontogeny.     

Intrathymic selection of murine TCR alpha beta+CD4-CD8- thymocytes

The CD4-CD8- thymocyte population contains the precursors of all other thymocytes. However, it also contains a significant proportion of cells which express surface TCR alpha beta, and have little or no precursor activity. Like peripheral T cells, but unlike most other thymocytes, these TCR alpha beta+CD4-CD8- thymocytes do not express heat stable antigen. Both the origin and developmental status of these cells are unclear, and are the subject of this report. We have measured the proportion of V beta 8.1+ cells amongst TCR+HSA-CD4-CD8- thymocytes in MIs-1a versus MIs-1b mice, in order to determine whether they have undergone negative selection. The proportions were similar in both strains, in contrast to mature T cells, indicating that neither they nor their precursors had undergone clonal deletion. We also measured the accumulation of these cells over the early life of the animal and found that it was extremely slow. Our data also show that although TCR-V beta 8.1+ cells are reactive to MIs-1a in association with MHC class II, most mature TCR-V beta 8.1+ cells in MIs-1b mice are CD8+, suggesting an additional reactivity with MHC class I. We raise the possibility that TCR-V beta 8.1+CD4-CD8- thymocytes are derived from TCR-V beta 8.1+CD4+CD8+ thymocytes, and that the reactivity of TCR-V beta 8.1 with both MHC classes I and II has resulted in the down-regulation of both CD4 and CD8.     

The generation and fate of thymocytes

In the adult thymus the majority of thymocytes are at a non-proliferating end stage, during which 3% of all CD4+CD8+ cortical thymocytes are selected on the basis of appropriate T-cell antigen-receptor (TcR) specificity to become CD4-CD8+ or CD4+CD8- mature thymocytes. The selected mature cells gradually emigrate to the periphery. The 97% unselected, or positively rejected, CD4+CD8+ thymocytes die in the thymus. This 3-day end stage is, however, the product of around 2 weeks of proliferation by less mature thymocytes, which gives about a 10(5)-fold expansion from the hundred prothymocytes estimated to seed the thymus each day. Rearrangement and expression of TcR genes and a sequence of changes in surface antigens occurs during this prolonged period. Two sequential waves of expansion and differentiation appear to be involved. The first, about 1 week long, involves the less than 0.1% of thymocytes which still resemble the prothymocyte; this leads via a non-dividing interval to the second 1-week stage involving the 3% of CD4-CD8- thymocytes and then the CD4+CD8+ blasts.     

T cell receptor expression on immature thymocytes with in vivo and in vitro precursor potential

Immature CD8-CD4- double-negative (DN) thymocytes differentiate intrathymically into CD8+CD4- and CD8-CD4+ thymocytes and migrate to the periphery. This differentiation proceeds through several intermediate phenotypic changes in the expression of CD8 and CD4. We have recently established the existence of a CD8loCD4lo cell population in murine thymus that can repopulate the irradiated thymus in vivo and differentiate rapidly in vitro to CD8+CD4+ double-positive (DP) cells. The CD8loCD4lo cells score as DN upon direct cytofluorometric analysis, yet are distinct from true DN cells by various criteria. Experimental evidence strongly suggests that they are descendants of true DN in the maturation pathway. In the experiments presented here, we further characterize this CD8loCD4lo thymocyte population. Northern blot and RNA protection analysis reveal that these cells transcribe full length mRNA for the T cell receptor (TcR)alpha chain, unlike the less mature interleukin 2 receptor-positive DN thymocytes. Surface expression of the TcR-associated CD3 molecule occurs on approximately 15% of these cells at low levels characteristic of immature cells. In the course of in vitro differentiation a vast majority (approximately 80%) of these cells convert to CD8+CD4+ and significant numbers of the brightly staining DP convertants (11%-34% on day 1 and 48%-68% on day 2) express immature levels of CD3. Our results indicate that CD8lo, CD4lo cells might be the first thymic subset to rearrange TcR alpha chain genes and express TcR alpha/beta heterodimer on the surface at levels characteristic of immature cells. Furthermore, the surface expression of TcR persists on the in vitro progeny of these thymocytes.     

Origin and selection of peripheral CD4-CD8- T cells bearing alpha/beta T cell antigen receptors in autoimmune gld mice

We have analyzed the origin and development of unusual CD4-CD8- alpha/beta T cell receptor-positive peripheral T cells produced in large numbers by mice homozygous for the gld mutation (C3H-gld/gld). These mice may be an important model for investigating processes controlling T cell development. Bone marrow transfers demonstrated that the gld defect was intrinsic to bone marrow-derived cells. Clonal deletion of potentially autoreactive cells was observed in peripheral gld CD4-CD8-, CD4+CD8-, and CD4-CD8+ T cells, as well as mature thymocytes. This suggests that gld CD4-CD8- T cells have passed through the thymus in ontogeny and that gld autoimmunity does not result from a general defect in elimination of self-reactive thymocytes. These observations, combined with demethylation of the CD8 gene in the CD4-CD8- population, support prior expression of CD4 and/or CD8 in gld CD4-CD8- T cell ontogeny, perhaps at a CD4+CD8+ stage. Steroid sensitivity of gld thymocytes and CD4-CD8- T cells was normal. Therefore, we found no gross abnormalities in two major mechanisms of inducible cell death in the gld thymus, the clonal deletion process associated with tolerance and the steroid-inducible endogenous endonuclease thought to be involved in apoptosis of unselected thymocytes. The data suggest that if gld CD4-CD8- T cells arise via escape from normal elimination in the thymus, they must do so by a novel defect in thymic selection (perhaps related to aberrant positive signals) and/or are expanded by an extrathymic process which allows clonal deletion to occur.     

CD45RA is detected in all thymocyte subsets defined by CD4 and CD8 by using three-colour flow cytometry.

In the mouse, using three-colour flow cytometry, the presence of CD45RA+ cells is demonstrated amongst all of the thymocyte subsets defined by expression of CD4 and CD8, i.e. amongst the double negatives, immature CD8 single positives, double-positive blasts and CD4 and CD8 single positives. This evidence is compatible with the existence of a continuous lineage of T cells expressing CD45RA which would develop from double-negative to mature single-positive T cells.     

Different subpopulations of developing thymocytes are associated with adherent (macrophage) or nonadherent (dendritic) thymic rosettes.

Thymic rosettes (ROS), structures consisting of thymic lymphoid cells attached to a central stromal cell, were isolated from mouse thymus by collagenase digestion and unit-gravity elutriation. The ROS were then separated into those where the stromal cells were either macrophage-like (M-ROS) or dendritic cell-like (D-ROS), on the basis of the differences in adherence properties or in the level of MAC-1 surface antigen. The ROS were then dissociated and the thymocyte content analyzed by immunofluorescent staining and flow cytometry. M-ROS and D-ROS differed in thymocyte composition, although the major component of both was the CD4+CD8+ cortical thymocyte. D-ROS were enriched in thymocytes expressing high levels of surface T-cell antigen receptor (TcR) and the associated CD3 complex, and these included both CD4+CD8-CD3++ and CD4-CD8+CD3++ mature thymocytes. M-ROS were enriched in CD4-CD8- thymocytes and had a reduced content of thymocytes expressing high TcR-CD3 levels; they nevertheless contained some mature thymocytes, but only of the CD4+CD8-CD3++ category. Several lines of evidence indicated that the mature thymocytes in ROS were cells recently formed in the cortex, and were not from the medullary pool. ROS-associated mature thymocytes expressed lower levels of H-2K than free, mature thymocytes. The CD4+CD8+CD3++ subpopulation, believed to be a developmental intermediate between cortical thymocytes and mature T cells, was present in both ROS populations. Further, late intermediates leading to both mature T-cell categories were evident in D-ROS, but only those leading to CD4+CD8-CD3++ T cells were evident in M-ROS. The results are compatible with a role for ROS in TcR-specificity selection and in the final maturation steps in the thymic cortex.     

Analysis of recent thymic emigrants with subset- and maturity-related markers

The thymus plays an integral role in the development and production of T lymphocytes. However, thymocytes differ markedly in their phenotypic characteristics from the T cells normally found in the peripheral lymphoid organs. We have examined the phenotypic characteristics of recent thymic emigrants and compared them with both mature phenotype thymocytes (CD4+ CD8-CD3+ and CD4-CD8+ CD3+) and lymph node T cells. Recent thymic emigrants were defined as those fluorescein-positive cells found in the lymph node up to 16 h after intrathymic injection of fluorescein. Most cells emigrating from the thymus expressed CD3 and either CD4 or CD8, indicating maturity. Recent thymic emigrants, like mature phenotype thymocytes, were slightly larger on average than peripheral T cells, but this differential was lost within 24 h of emigration. Also like mature thymocytes but unlike peripheral T cells, some recent emigrants expressed heat-stable antigen. This did not change within 24 h of emigration. The antigen CD44 (Pgp-1, Ly-24) was expressed on a proportion of mature thymocytes, recent thymic emigrants, and peripheral T cells, and its expression did not show any clear relationship to maturity. The antigen CD45R also did not show marked changes associated with maturity, but our data do not parallel the published data of the expression of CD45R in the human. We conclude that recent thymic emigrants are phenotypically mature with respect to some antigens but not others. None of the antigens we investigated could have been used to uniquely distinguish recent thymic emigrants from peripheral T cells or from mature thymocytes.     

Emigration of mature T cells from the thymus is inhibited by the imidazole-based compound 2-acetyl-4-tetrahydroxybutylimidazole.

The primary role of the thymus is to provide mature T cells for the peripheral immune system. The mechanisms involved in the cellular export processes are as yet unknown. In this study, we examined the ability of 2-acetyl-4-tetrahydroxybutylimidazole (THI), an agent widely used as a component of ammonia caramel food colouring, to inhibit T-cell export from the thymus. BALB/c mice were maintained on drinking water containing THI for 5 days. The mice showed a twofold increase in the total number of mature medullary thymocytes (CD4+CD8- and CD4-CD8+) as well as a slight decrease in the total number of immature double-positive cells (CD4+CD8+). The mature single-positive thymocytes were found to express high levels of the homing molecule L-selectin, suggesting that these potential emigrants were prevented from leaving the thymus. To confirm this, THI-treated mice were injected intrathymically with fluorescein isothiocyanate and the number of labelled T cells appearing in the lymph nodes and spleen was determined 16 hr later. A 10-fold decrease in the number of CD4+ and CD8+ recent thymic emigrants in the lymph nodes and spleen of THI-treated mice was observed. Previous studies have shown that THI does not affect other aspects of thymocyte development, such as proliferation and differentiation. Taken together, these results suggest that the immunosuppressive effects of THI may be due, in part, to preventing of the final step of T-cell export out of the thymus.