Selection of phenotypically distinct NK1.1+ T cells upon antigen expression in the thymus or in the liver.

Unlike the main TCR alphabeta T cell lineage in which deletion occurs at the CD4+ CD8+ double-positive (DP) stage upon TCR engagement by antigen in the thymus, some T cells appear to require such engagement for their selection, either in the thymus or extrathymically. We used a transgenic TCR (tgTCR) model which, as we previously showed, led to selection upon expression of the corresponding antigen H-2Kb (Kb) in the thymus, of tgTCR/CD3(lo) CD4- CD8- double-negative (DN) thymocytes that expressed the NK1.1 marker (NK T cells) (Curnow, S. J., et al., Immunity 1995. 3: 427). We now report that antigen expression on medullary epithelial cells of the thymus failed to select the NK T cells, whereas its expression on thymocytes did, although tgTCR DP thymocyte development was affected under both conditions. Antigen expression on hepatocytes (Alb-Kb mice) did not perturb tgTCR DP thymocyte development. No enrichment in tgTCR NK T cells was detected in the periphery, except for the liver of the Alb-Kb/tgTCR mice. When reconstitution of thymectomized and irradiated H-2k hosts expressing or not Kb was performed with bone marrow from tgTCR H-2k mice, an enrichment in tgTCR+ NK T cells was found in the liver, but not in the spleen, of the hosts which expressed Kb, either selectively on hepatocytes or ubiquitously. Surprisingly, the majority of the hepatic tgTCR+ NK T cells also expressed the CD8 alpha/beta heterodimer. These results indicate that thymus-independent NK T cells with unique phenotypic characteristics can be selected upon antigen encounter in the liver.     

Early deletion and late positive selection of T cells expressing a male-specific receptor in T-cell receptor transgenic mice

The ontogeny of T cells in T-cell receptor (TCR) transgenic mice, which express a transgenic alpha beta heterodimer, specific for the male (H-Y) antigen in association with H-2Db, was determined. The transgenic alpha chain was expressed on about 10% of the fetal thymocytes on day 14 of gestation. About 50% of day-15 fetal thymocytes expressed both alpha and beta transchains and virtually all fetal thymocytes expressed the transgenic alpha beta heterodimer by day 17. The early expression of the transgenic TCR on CD4-8- thymocytes prevented the development of gamma delta cells, and led to accelerated growth of thymocytes and an earlier expression of CD4 and CD8 molecules. Up to day 17, no significant differences in T-cell development could be detected between female and male thymuses. By day 18 of gestation, the male transgenic thymus contained more CD4-8- thymocytes than the female transgenic thymus. The preponderance of CD4-8- thymocytes in the male transgenic thymus increased until birth and was a consequence of the deletion of the CD4+8+ thymocytes and their CD4-8+ precursors. By the time of birth, the male transgenic thymus contained half the number of cells as the female transgenic thymus. The deletion of autospecific precursor cells in the male transgenic mouse began only at day 18 of gestation, despite the fact that the ligand could already be detected by day 16. The preferential accumulation of CD4-8+ T cells, which expressed a high density of the transgenic TCR, occurred only after birth and was obvious in 6-week-old female thymus. These data support the hypothesis that the positive selection of T cells expressing this transgenic heterodimer may involve two steps, i.e., the commitment of CD4+8+ thymocytes to the CD4-8+ lineage following the interaction of the transgenic TCR with restricting major histocompatibility molecules, followed by a slow conversion of CD4+8+ thymocytes into CD4-8+ T cells. In normal mice, the precursors of CD4+8+ and single positive thymocytes have the CD4-8- CD3-J11d+ (or M1/69+) phenotype. Because of the early expression of the transgenic alpha beta heterodimer, this population was not detected in adult transgenic mice. All CD4-8- M1/69+ cells expressed the transgenic receptor associated with CD3 and could be readily grown in media containing T-cell lectins and interleukin 2.     

CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes.

T-cell development is arrested at the CD4+CD8+ (DP; double-positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T-cell receptor (TCR) specific for the male antigen presented by H-2Db (H-Y TCR), the up-regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)-restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H-Y TCR transgenic mice. It was found that in female H-Y TCR transgenic mice, MHC-restricted positive selection of CD4- CD8+ H-Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4- CD8+ thymocytes that express H-Y TCR.     

CD4+CD8+ thymocytes develop into CD4 or CD8 single-positive cells in athymic nude mice

A differentiation pathway from CD4+CD8+ cells to CD4+CD8- or CD4-CD8+ cells was investigated in athymic nude mice. Using fluorescence-activated cell sorter, CD4+CD8+ cells were sorted out from AKR thymocytes (H-2k, Thy-1.1) stained with two monoclonal antibodies against CD4 and CD8 (anti-L3T4 and anti-Ly-2). These CD4+CD8+ AKR thymocytes were injected i.v. into CBA or C3H nude mice (H-2k, Thy-1.2) which had received 650 rads and had been reconstituted with syngeneic nude bone marrow cells. The lymph node cells of the nude recipients at 4 wks post-thymocyte transfer were shown to contain 50% AKR-derived Thy-1.1+ cells. The majority of the Thy-1.1+ cells were found to express either CD4 or CD8 alone but not to express both CD4 and CD8. These findings indicate that CD4+CD8+ thymocytes can develop into CD4+CD8- and CD4-CD8+ single-positive cells in extrathymic tissues.     

Spontaneous clustering of Thy-1 antigens on CD4+ CD8+ thymocytes lacking TCR engagement by MHC/peptide complexes

While much is known concerning CD4+ CD8+ thymocytes positively or negatively selected through interaction of their TCR with self peptides bound to self-MHC molecules, little is known of the majority of CD4+ CD8+ thymocytes lacking this interaction. We developed a monoclonal antibody (mAb) 1D11, the ligand of which (1D11-L) is expressed on 60-70% of CD4+ CD8+ thymocytes but not on other subsets of thymocytes and peripheral T cells. 1D11-L expression on CD4+ CD8+ thymocytes reversely correlates with their TCR engagement, in vitro and in vivo. In addition, 1D11-L+ CD4+ CD8+ thymocytes were more susceptible than 1D11-L- CD4+ CD8- thymocytes to apoptosis. We also found that T lineage cells other than CD4+ CD8+ thymocytes and a Thy-1-expressing fibroblast cell line became positive for 1D11-L by cross-linking their Thy-1 antigens with anti-Thy-1 mAb but not with their Fab fragment, suggesting that 1D11 recognizes multimerized Thy-1 antigens. Confocal laser scanning microscopy revealed that Thy-1 antigens as well as 1D11-L are clustered on some CD4+ CD8+ thymocytes but not on the other subsets of thymocytes. Taken together, we suggest that clustering of Thy-1 antigens spontaneously and specifically occurs on CD4+ CD8+ thymocytes lacking TCR engagement by MHC/peptide complexes.     

IL-2 and IL-7 differentially induce CD4-CD8- alpha beta TCR+NK1.1+ large granular lymphocytes and IL-4-producing cells from CD4-CD8- alpha beta TCR+NK1.1- cells: implications for the regulation of Th1- and Th2- type responses

Effector functions of CD4-CD8- double negative (DN) alpha beta TCR+ cells were examined. Among mouse DN alpha beta TCR+ thymocytes, NK1.1+ cells expressing a canonical V alpha 14/J alpha 281 TCR but not NK1.1- cells produce IL-4 upon TCR cross-linking and IFN-gamma upon cross- linking of NK1.1 as well as TCR. Production of IL-4 but not IFN-gamma from DN alpha beta TCR+NK1.1+ cells was markedly suppressed by IL-2. Whereas V alpha 14/J alpha 281 TCR+ cells express NK1.1+, these cells are not the precursor of DN alpha beta TCR+NK1.1+CD16+B220+ large granular lymphocytes (LGL). IL-2 induces rapid proliferation and generation of NK1.1+ LGL from DN alpha beta TCR+NK1.1- but not from DN alpha beta TCR+NK1.1+ cells. LGL cells exhibit NK activity and produce IFN-gamma but not IL-4 upon cross-linking of surface TCR or NK1.1 molecules. In contrast to IL-2, IL-7 does not induce LGL cells or NK activity from DN alpha beta TCR+NK1.1- cells but induces the ability to produce high levels of IL-4 upon TCR cross-linking. Our results show that DN alpha beta TCR+ T cells have several distinct subpopulations, and that IL-2 and IL-7 differentially regulate the functions of DN alpha beta TCR+ T cells by inducing different types of effector cells.      

A novel subset of adult γδ thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire

We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of γδ thymocytes, initially defined by expressing low levels of Thy-1, that represents around 5 % and 30 % of total γδ thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS-sorted Thy-1^dull γδ thymocytes from DBA/2 mice with anti-γδ monoclonal antibodies in the presence of interleukin-2 (IL-2) results in the secretion of high levels of several cytokines, including interferon-γ (IFN-γ), IL-4, IL-10, and IL-3. In contrast, only IFN-γ was detected in parallel cultures of Thy-1^bright γδ thymocytes. Virtually all Thy-1^dull γδ thymocytes express high levels of CD44 and low levels of the heat-stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy-1^dull γδ thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy-1^dull γδ thymocytes from DBA/2 mice express TCR encoded by the Vγ1 gene and a novel Vδ6 gene named Vδ6.4. Sequence analysis of these functionally rearranged γ and δ genes revealed highly restricted Vδ-Dδ-Jδ junctions, and somewhat more diverse Vγ-Jγ junctions. We conclude that Thy-1^dull γδ thymocytes exhibit properties that are equivalent to those of natural killer TCRαβ T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.     

Peptide-activated double-negative T cells can prevent autoimmune type-1 diabetes development

Autoimmune diseases may develop because of defective maturation, activation, differentiation and function of regulatory T cells. Previous studies have shown that exposure to donor antigen activates peripheral TCRalphabeta+CD3+CD4-CD8-NK1.1-, double-negative (DN) T cells, which specifically suppress anti-donor T cells and enhance survival of skin and heart grafts from allogeneic and xenogeneic donors. However, the role of DN T cells in preventing T cell-mediated autoimmune disease is unknown. Here, we analyzed the ability of DN T cells to recognize peptides expressed on self MHC and to suppress peptide-reactive CD8+ T cells, using the P14 mouse model that expresses a transgenic TCR specific for gp33 peptide presented on self MHC class I-Db. We found that injection of gp33 peptide resulted in increased DN and decreased CD8+ T cell numbers in the lymph nodes when compared to untreated mice. Injection of gp33, but not TCR-non-specific AV peptide, increased expression of T cell activation markers on DN T cells. Moreover, gp33-activated DN T cells suppressed proliferation of syngeneic CD8+ T cells via killing activated CD8+ T cells in an antigen-specific fashion in vitro. Furthermore, transferring gp33-activated DN T cells inhibited the development of autoimmune diabetes, suggesting that DN T cells may provide a novel therapy for T cell-mediated autoimmune diseases.     

Intrathymic proliferation of perinatal mouse alpha beta and gamma delta T cell receptor-expressing mature T cells

Using three colour flow microfluorimetry to analyse simultaneously two cell surface molecules and DNA content, CD4 SP cells in the late foetal and early postnatal thymus were found to contain significant numbers of cells in the S and G2 + M phases of the cell cycle. In contrast to neonatal thymocytes, CD4 SP cells in the adult thymus were found to be non-cycling. Two other phenotypic properties, namely expression of J11d and larger cell size, also distinguish perinatal from adult CD4 SP cells. In the perinatal thymus of (B/6 x D2)F1 mice, CD4 SP thymocytes expressing either V beta 6 (Mls-reactive) or V beta 11 (I-E-reactive) TCR are deleted by negative selection yet cell cycle analysis showed that, coincident with deletion, they were cycling. Thus negative selection in the perinatal thymus was operating on a population of cycling cells. Finally, perinatal CD3+, gamma delta TCR-expressing DN thymocytes were also found to be cycling. Taken together, these results show that, prior to migration to peripheral lymphoid tissues, in the perinatal period there is intrathymic proliferation of developing gamma delta- and alpha beta-expressing T cells.     

Death of T cell precursors in the human thymus: a role for CD38

Thymic T cell maturation depends on interactions between thymocytes and cells of epithelial and hematopoietic lineages that control a selective process whereby developing T cells with inappropriate or self-reactive receptors die. Molecules involved in this process are the TCR expressed on thymocytes together with the CD3 complex and MHC-peptide on accessory cells. However, other molecules may favor or prevent death of thymocytes, thus playing a role in selection. CD38 is expressed by the majority of human thymocytes, mainly at the double-positive (DP) stage. In contrast, CD38 is not found on subcapsular double-negative (DN) thymocytes and on a proportion of medullary single-positive (SP) thymocytes. CD38 enhances death of thymocytes when it is cross-linked by goat anti-mouse (GAM) antiserum or by one of its ligands, CD31, expressed by thymic epithelial cells or transfected into murine fibroblasts (L cells). As most thymocytes are at an intermediate (DP) stage of development, it is likely that these cells are most vulnerable to death mediated via MHC-peptide-TCR interactions that is increased by CD38 cross-linking. DN and SP thymocytes are refractory to CD38-induced apoptosis. Accessory molecules, e.g. CD38, are expressed during thymic cell maturation and their presence is relevant for the survival or death of DP T cells in the course of selection. Based on our data, CD38 enhances thymocyte death by interacting with CD31 expressed by accessory cells. In addition, CD28 expression on developing thymocytes also appears to play a role for their selection and it synergizes with CD38 to induce apoptosis of DP thymocytes.     

Strain-specific TCR repertoire selection of IL-4-producing Thy-1 dull gamma delta thymocytes

Thy-1 dull gamma delta thymocytes constitute an unusual subset of mature TCR gamma delta cells which share with NK T cells the expression of cell surface markers usually associated with activated or memory cells and the simultaneous production of high levels of IL-4 and IFN-gamma upon activation. In DBA / 2 mice, Thy-1 dull gamma delta thymocytes express a restricted repertoire of TCR that are composed of the V1 gene product mainly associated with V6.4 chains exhibiting very limited junctional sequence diversity. In this study we have characterized this gamma delta T cell population in different strains of mice and show that Thy-1 dull gamma delta thymocytes are present in every strain tested, albeit at different frequencies. Moreover IL-4 production by gamma delta thymocytes is mainly confined to the Thy-1 dull population in every strain tested. Finally, the repertoire of TCR expressed by Thy-1 dull gamma delta thymocytes varies in different strain of mice, although a biased expression of Vgamma1 and Vdelta6 chains was observed in all strains studied. However, the extent of junctional diversity of the V1 and V6 chains expressed by Thy-1 dull gamma delta thymocytes varied from oligoclonal in DBA/2 mice to polyclonal in FVB/N mice. Thy-1 dull gamma delta thymocytes from mouse strains such as C3H/HeJ and BALB/c contain cells with diverse Vdelta6(D)Jdelta junctions together with cells with relatively homogeneous Vdelta6(D)Jdelta junctions, similar to those found in DBA/2. Thus, the Thy-1 dull gamma delta population appears to contain two subsets of cells which differ in the diversity of their TCR.     

Establishment of efficient reaggregation culture system for gene transfection into immature T cells by retroviral vectors

To overcome low efficiency of retroviral infection into immature T cells, we modified reaggregation fetal thymus organ culture by closely packed co-culture with virus-producing cells (VPC). The viral vector was constructed in chimeric vector, pMX, with IRES and tailless-rat CD2 as a surface marker of infected cells. A rearranged TCR beta gene (Vbeta8.2) was further inserted into the construct for investigating effect of the introduced gene in T cell development. Using this system, we succeeded to transfer the viral vector into immature thymocytes at a remarkably higher efficiency compared to conventional methods using medium containing retrovirus. Moreover, the introduced TCR beta gene was expressed on thymocytes of RAG2-deficient mice to induce in the transition of CD4-CD8- double-negative (DN) into CD4+CD8+ double-positive (DP) cells by transducing beta-selection signaling. Thus, our modified reaggregation culture system is useful for studying the molecular mechanism of T cell development due to a highly efficient gene transfer into immature T cells.     

Co-ordinate expression of the pre-T-cell receptor complex and a novel immature thymocyte-specific antigen, IMT-1, during thymocyte development

Previously we described a monoclonal antibody (mAb) that reacted with a cell-surface antigen, immature thymocyte antigen-1 (IMT-1), which is expressed on thymocytes of late CD4- CD8- (double negative) to early CD4+ CD8+ (double positive) differentiation stages. In this study, we investigated the expression of IMT-1 on various cell lineages in thymus as well as in peripheral lymphoid organs. We found that IMT-1 is expressed on T-cell receptor (TCR)-betalo and TCR-deltalo thymocytes, but not on TCR-betahi, TCR-deltahi or natural killer (NK)1.1+ thymocytes, or on peripheral alpha beta or gamma delta T cells. We also investigated the kinetics of expression of IMT-1 during fetal thymocyte development and compared it with the expression of the pre-TCR complex, comprising CD3, pre-TCR-alpha (pTalpha) and TCR-beta. We found that expression of both was similar, starting at day 14.5 of gestation, peaking on day 16.5 and gradually decreasing thereafter. Furthermore, the expression of both IMT-1 and pTalpha was drastically reduced when DN thymocytes in recombination activating gene (RAG)-2-/- mice were challenged in vivo with anti-CD3 mAb. These results indicate that IMT-1 is expressed on not only immature thymocytes of alpha beta T-cell lineage but also on those of gamma delta T-cell lineage, and that the expression of IMT-1 and the pre-TCR complex is co-ordinately regulated during the alpha beta lineage thymocyte development.     

Functional maturation of recent thymic emigrants in the periphery: development of alloreactivity correlates with the cyclic expression of CD45RC isoforms.

The transition from fully developed CD4+CD8- single-positive (SP) thymocytes into fully mature recirculating peripheral T cells is both poorly understood with regard to the expression of restricted isoforms (CD45R) of the leukocyte common antigen and in terms of T cell function. The present investigation monitored the extrathymic development of CD4+CD8- SP thymocytes in euthymic recipients using allotype-marked donor cells and monoclonal antibody OX22 which recognizes an epitope on the C exon of rat CD45R. We established that donor-derived cells in the blood 1 day later bore the phenotype of the injected SP thymocytes (CD4+ Thy-1+ CD45RC-). T cells with the identical phenotype were also present in the thoracic duct lymph of uninjected rats, suggesting that the Thy-1+ CD45RC- T cells represent recent thymic emigrants (RTE) which have migrated to the periphery of their own accord. During extrathymic maturation donor-derived peripheral RTE lost Thy-1 within 3 days and expressed the CD45RC+ high molecular weight isoform by day 7; between days 8 and 14 a proportion (25%-30%) of the donor cells once again lost the high molecular weight isoform (CD45RC-). The transition of SP (CD45RC-) thymocytes to fully mature CD45RC+ CD4 T cells via intermediate peripheral RTE was accompanied at each stage by an increased ability of the maturing T cells to induce skin allograft rejection. Unexpectedly, the subsequent loss of the high molecular weight isoform, following presumed antigen encounter, was associated with a significant reduction in the ability of this Thy-1-CD45RC- subpopulation to effect graft rejection. The cyclic expression of CD45RC isoforms on both immature and mature CD4 T cells and the fact that the low molecular weight isoform was found in the periphery on both RTE (unquestionably naive) and antigen-experienced CD4 T cells, makes it unlikely that this isoform uniquely identifies memory T cells, at least in the rat.     

Developmentally Regulated Expression of the Transmembrane Adaptor Protein TRIM in Fetal and Adult T Cells

TRIM is a recently identified transmembrane adaptor protein which is exclusively expressed in T cells and natural killer (NK) cells. In peripheral blood T cells TRIM has been reported to coprecipitate, comodulate, and cocap with the T-cell receptor (TCR), suggesting that it is an integral component of the TCR/CD3/zeta complex. Here we investigate the expression of TRIM mRNAs and proteins in developing thymocytes. Two splicing isoforms with open reading frames are observed, namely a full length (TRIM) and a truncated version (DeltaTM-TRIM). The latter lacks the extracellular and transmembrane domains as well as the first 10 cytoplasmic aminoacids and is significantly expressed only as mRNA in early fetal thymocytes. TRIM mRNA is detected in all mainstream thymocyte subsets in adult mice. TRIM protein, in contrast, first appears in the DN2 (CD44+ CD25+) subset of adult double negative (DN) cells. In fetal thymocyte development, TRIM mRNA is seen from dg 14.5 onwards whereas TRIM protein appears first on dg 16.5. In contrast to the adult, the TRIM protein was seen in a subset of fetal DN1 cells. In fetal and adult thymocytes, TRIM protein expression was highest in DN2, DN3 (CD44-25+) and in DP cells, compatible with a functional role at or around phases of thymic selection.     

Modulation of CD4 co-receptor limits spontaneous autoimmunity when high-affinity transgenic TCR specific for self-antigen is expressed on a genetically resistant background

Myelin proteolipid protein (PLP) 139-151 is an immunodominant peptide that induces experimental autoimmune encephalomyelitis (EAE) in H-2(s) SJL/J mice. While PLP 139-151-specific TCR transgenic (tg) 4E3 mice develop fulminant spontaneous disease on the susceptible SJL/J background, spontaneous EAE is dramatically reduced on the H-2(s) congenic B10.S background. On this resistant background, we observed a high frequency of positively selected tg CD4-CD8- (DN) thymocytes and peripheral DN tg T cells. Splenic DN tg T cells responded to anti-CD3 stimulation similarly to CD4+ cells, but proliferative and cytokine responses to PLP 139-151 were blunted, implying that CD4 co-receptor down-regulation modulated T cell responses to the self-antigen in vitro. Adoptive transfer of tg DN CD3hi cells into RAG-deficient wild-type (WT) recipients induced EAE less efficiently than transfer of CD4+ T tg cells indicating the blunted responses of DN tg T cells to self-antigen in vivo. The frequency of tg DN T cells was irrespective of thymic expression of the autoantigen. These data implicate that down-regulation of CD4 co-receptor in the thymus, which is independent from the expression of thymic autoantigen, results in a blunted response to the autoantigen in the periphery and limits the incidence of spontaneous autoimmunity in genetically resistant mice bearing a large autoreactive tg T cell repertoire.