Tolerance induction by clonal deletion of CD4+8+ thymocytes in vitro does not require dedicated antigen-presenting cells

The cellular requirements of T cell tolerance induction in the thymus by clonal deletion was investigated by using an in vitro assay: thymocytes from mice expressing a transgenic TcR specific for lymphocytic choriomeningitis virus (LCMV) and H-2D^b were co-cultured with various H-2^b cell types as antigen-presenting cells in the presence of the antigenic LCMV peptide. The results revealed that all cell lines examined including embryonic and transformed fibroblasts, melanoma cells, cortical thymic epithelial cells, lymphomas and neuronal cells induced an antigen dose-dependent deletion of CD4⁺8⁺ thymocytes. Similarly, highly enriched accessory cell populations from thymus and spleen (macrophages, dendritic and cortical epithelial cells, i.e. thymic nurse cells) could induce antigen-specific depletion of immature CD4⁺8⁺ thymocytes. Depending on the cell type examined micromolar to picomolar concentration of LCMV peptide were required to induce deletion. The effectiveness of deletion by the different cell types did not correlate with their major histocompatibility class I expression level; it was, however, influenced by the presence of ICAM-1 adhesion molecules.     

Tolerance in TCR/Cognate Antigen Double-Transgenic Mice Mediated by Incomplete Thymic Deletion and Peripheral Receptor Downregulation

Influenza nucleoprotein (NP)-specific T-cell receptor transgenic mice (F5) were crossed with transgenic mice expressing the cognate antigenic protein under the control of the H- 2K^b promoter. Double-transgenic mice show negative selection of thymocytes at the CD4⁺8⁺TCR¹⁰ to CD4⁺8⁺TCR^hi transition stage. A few CD8 T cells, however, escape clonal deletion, and in the peripheral lymphoid organs of these mice, they exhibit low levels of the transgenic receptor and upregulated levels of the CD44 memory marker. Such cells do not proliferate upon exposure to antigen stimulation in vivo or ex vivo, however, they can develop low but detectable levels of antigen-specific cytotoxic function after stimulation in vitro in the presence of IL-2.     

Gads‐deficient thymocytes are blocked at the transitional single positive CD4+ stage

Positive selection of T‐cell precursors is the process by which a diverse T‐cell repertoire is established. Positive selection begins at the CD4⁺CD8⁺ double positive (DP) stage of development and involves at least two steps. First, DP thymocytes down‐regulate CD8 to become transitional single positive (TSP) CD4⁺ thymocytes. Then, cells are selected to become either mature single positive CD4⁺ or mature single positive CD8⁺ thymocytes. We sought to define the function of Gads during the two steps of positive selection by analyzing a Gads‐deficient mouse line. In Gads^+/+ mice, most TSP CD4⁺ thymocytes are TCR^hiBcl‐2^hiCD69⁺, suggesting that essential steps in positive selection occurred in the DP stage. Despite that Gads^?/? mice could readily generate TSP CD4⁺ thymocytes, many Gads^?/? TSP CD4⁺ cells were TCR^loBcl‐2^loCD69^?, suggesting that Gads^?/? cells proceeded to the TSP CD4⁺ stage prior to being positively selected. These data suggest that positive selection is not a prerequisite for the differentiation of DP thymocytes into TSP CD4⁺ thymocytes. We propose a model in which positive selection and differentiation into the TSP CD4⁺ stage are separable events and Gads is only required for positive selection.     

The balance between deletion and activation of CD4+8+ thymocytes is controlled by T cell receptor-antigen interactions and is affected by cyclosporin A

The sensitivity of immature thymocytes to antigen-induced deletion has been shown to correlate with their differentiation status. By using an in vitro approach we have investigated whether parameters of antigenic stimulation may also affect the response of thymocytes. Two T cell receptor (TcR)-transgenic (Tg) mouse models have been compared, both of which recognize the allo-antigen H-2K^b but are functionally CD8“-dependent” (KB5.C20-Tg) and “-independent” (BM3.3-Tg). Presentation of the antigen H-2K^b on the surface of fibroblasts, to thymocytes in vitro, resulted in the apoptosis of CD4⁺8⁺ thymocytes. In contrast to in vivo deletion, in vitro deletion was much greater for KB5.C20-Tg than for BM3.3-Tg. In the absence of engagement of CD8 (using an altered H-2K^b-α3 domain or CD8-specific antibodies), the H-2K^b-induced deletion of CD4⁺8⁺ thymocytes was decreased for KB5.C20-Tg, but no change in the pattern of deletion for BM3.3-Tg occurred. CD4⁺8⁺ thymocytes which remained viable after in vitro exposure to antigen, were shown to have been activated. Cyclosporin A (CsA), which has been reported to inhibit activation-induced cell death, did not affect antigen-induced deletion of CD4⁺8⁺ thymocytes from KB5.C20-Tg. More strikingly, deletion of CD4⁺8⁺ thymocytes from BM3.3-Tg increased, whilst activation was partially inhibited by CsA. These results provide direct evidence that presentation of antigen to thymocytes can result in deletion or activation, depending on not only the differentiation status of the cell, but also parameters of TcR-antigen interaction. Additionally, the effects of CsA suggest that activation can prevent the induction of deletion.     

CD 69 expression during selection and maturation of CD4+8+ thymocytes

We have analyzed the inducibility of protein kinase C (PKC)-dependent expression of CD 69 molecules in T cell receptor (TCR) transgenic thymocytes developing in the presence or absence of selecting, class I major histocompatibility complex (MHC) molecules. Small CD4⁺8⁺ thymocytes developing in the absence of selecting MHC molecules could not be induced to express CD 69 by TCR cross-linking even after spontaneous in vitro up-regulation of their TCR level which resulted in enhanced Ca⁺⁺ flux. In contrast, a small proportion of CD4⁺8⁺TCR^low and most TCR^high (CD4⁺8⁺ and CD4⁺8⁺) thymocytes developing in the presence of selecting MHC ligands could be induced to express CD 69 upon TCR cross-linking. Unlike the anti-TCR antibody, phorbol 12-myristate 13-acetate - a direct activator of PKC - induced the expression of CD 69 on all thymocytes. These results suggest that positive selection of CD4⁺8⁺ thymocytes results in coupling of TCR-mediated signals to the CD 69 expression pathway. In vitro analysis of thymocytes before and after positive selection suggests that (1) positive selection does not immediately result in resistance to deletion and (2) that sustained TCR ligation is needed to promote maturation of positively selected CD4⁺8⁺ thymocytes resulting in gradual loss of the sensitivity to deletion and acquisition of the ability to proliferate in response to TCR-mediated signals.     

Secondary but not primary T cell responses are enhanced in CTLA-4-deficient CD8+ T cells

Negative as well as positive co-stimulation appears to play an important role in controlling T cell activation. CTLA-4 has been proposed to negatively regulate T cell responses. CTLA-4-deficient mice develop a lymphoproliferative disorder, initiated by the activation and expansion of CD4⁺ T cells. To assess the function of CTLA-4 on CD8⁺ T cells, CTLA-4^−/- animals were crossed to an MHC class I-restricted 2C TCR transgenic mouse line. We demonstrate that although the primary T cell responses were similar, the CTLA-4-deficient 2C TCR⁺ CD8⁺ T cells displayed a greater proliferative response upon secondary stimulation than the 2C TCR⁺ CD8⁺ T cells from CTLA-4 wild-type mice. These results suggest that CTLA-4 regulates antigen-specific memory CD8⁺ T cell responses.     

Tolerance induction by elimination of subsets of self-reactive thymocytes

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

Separately expressed T cell receptor α and β chain transgenes exert opposite effects on T cell differentiation and neoplastic transformation

Two aspects of T cell differentiation in T cell receptor (TCR)-transgenic mice, the generation of an unusual population of CD4^?CD8^?TCR⁺ thymocytes and the absence of γδ cells, have been the focus of extensive investigation. To examine the basis for these phenomena, we investigated the effects of separate expression of a transgenic TCR α chain and a transgenic TCR β chain on thymocyte differentiation. Our data indicate that expression of a transgenic TCR α chain causes thymocytes to differentiate into a CD4^?CD8^?TCR⁺ lineage at an early developmental stage, depleting the number of thymocytes that differentiate into the αβ lineage. Surprisingly, expression of the TCR α chain transgene is also associated with the development of T cell lymphosarcoma. In contrast, expression of the transgenic TCR β chain causes immature T cells to accelerate differentiation into the αβ lineage and thus inhibits the generation of γδ cells. Our observations provide a model for understanding T cell differentiation in TCR-transgenic mice.     

Identification of an HLA-DQ6-derived peptide recognized by mouse MHC class I H-2Db-restricted CD8+ T cells in HLA-DQ6 transgenic mice

Summary CD8⁺ T cells from C57BL/6(B6) mice show cytotoxicity to B cell blasts prepared from syngeneic transgenic mice expressing HLA-DQ6 molecules in a mouse MHC class I H-2D^b restricted manner. Although these results suggest that CD8⁺ T cells recognize peptides derived from DQ6 molecule bound to H-2D^b on target cells, no direct evidence so far has been obtained. To clarify this, we synthesized 23 peptides corresponding to DQ6α orβ chain and carrying the motifs of D^b-binding peptides, and examined their capacity to induce cytotoxicity in the CD8⁺ T cell line. We show here that DQA1-2, one of these peptides, induced cytotoxicity of the CD8⁺ T cells when this peptide was pulsed to H-2D^b expressing target cells, as efficiently as HLA-DQ6 expressing target cells did. Thus, our results suggest that DQA1-2 can be naturally processed from DQ6 molecules and recognized by the CD8⁺ T cells in the context of H-2D^b molecules. These results suggest that allogeneic HLA class II molecules are involved in the rejection not only as the ligand for T cell receptor of alloreactive CD4⁺ T cells but also as self-peptides bound to HLA class I molecules recognized by CD8⁺ T cells.     

Assessment of alloreactive T cell subpopulations of aged mice in vivo. CD4+ but not CD8+ T cell-mediated rejection response declines with advanced age

The present investigation explored age-related alterations in T cell populations mediating allospecific responses in vivo. Healthy aged and young H-2^b and H-2^bxH-2^k mice were engrafted with major histocompatibility complex (MHC) class II-disparate bm12 skin, rejection of which requires CD4⁺ T cells, and MHC class I-disparate bm1 skin, rejection of which requires CD8⁺ T cells. Aged mice of both genders exhibited prolonged survival of bm12 skin grafts relative to their young counterparts but rejected bm1 skin grafts at a rate equivalent to that of young mice. Consistent with prolonged survival of bm12 skin grafts, markedly diminished levels of Ia^bm12 CTL activity were elicited from T cells of aged mice in vitro. However, no such decline was observed in the level of K^bm1 CTL from T cells of aged mice. The alterations in Ia^bm12 allospecific responses were not attributable to quantitative changes in CD4⁺ T cells of aged mice, and addition of soluble T cell helper factors to response cultures of aged mice did not augment Ia^bm12 cytotoxic T lymphocytes activity. These data demonstrate that aging fundamentally affects CD4⁺ T cell-mediated allospecific responses particularly in vivo, and that deficient generation of soluble T cell helper factors alone cannot explain this deficit.     

Characterization of mouse CD53: Epitope mapping,cellular distribution and induction by T cell receptor engagement during repertoire selection

The pan-leukocyte antigen CD53 is a member of the poorly understood transmembrane 4 superfamily (TM4SF) of cell membrane glycoproteins. CD53 is proposed to play a role in thymopoiesis, since rat CD53 is expressed on immature CD4^?8^?thymocytes and the functionally mature single-positive subset, but is largely absent from the intermediate CD4⁺8⁺ cells. We have characterized CD53 in the mouse through the production of two new monoclonal antibodies, MRC OX-79 and OX-80, which were raised against the RAW 264 cell line and screened on recombinant CD53 fusion proteins. The epitopes recognized by both antibodies are dependent on disulfide bonding and map to the major extracellular region of CD53, requiring the presence of a single threonine residue at position 154. Mouse CD53 has a molecular mass of 35-45 kDa and is expressed on virtually all peripheral leukocytes, but not on cells outside the lymphoid or myeloid lineages. CD53 expression distinguishes subpopulations of thymocytes in the mouse and resembles the expression pattern of rat CD53. Amongst the immature CD4^?8^? thymocytes, mouse CD53 is clearly detectable on the earliest CD44^high25^? subset, but down-regulated on the later CD44^high25⁺, CD44^low25⁺ and CD44^low25^? stages. Also, the subsequent transient TcR^?/low CD4^?8⁺ cells and most CD4⁺8⁺ thymocytes express little or no CD53. This is consistent with the idea that cells which are committed to enter the selectable CD4⁺8⁺ compartment switch off CD53. The effect of T cell receptor (TcR) engagement on the reexpression of CD53 on CD4⁺8⁺ thymocytes was studied both ex vivo and in vitro using F5 mice, transgenic for the H-2^b/influenza nucleoprotein-peptide-specific TcR, back-crossed onto an H-2^q or H-2^b background of RAG-2-deficient mice. CD4⁺8⁺ thymocytes from non-selecting H-2^q F5 mice are CD53 negative, but in vitro stimulation through the TcR dramatically induces CD53 expression. In contrast, a fraction of CD4⁺8⁺ thymocytes from positively selecting H-2^b F5 transgenic mice express CD53. Therefore TcR engagement by selecting major histo-compatibility complex peptide complexes, or surrogate ligands, induces CD53 expression on otherwise CD53-negative, non-selected CD4⁺8⁺ thymocytes. Whether CD53 itself participates as a signaling molecule in further stages of thymic selection is still a matter of speculation.     

Co-expression of CD8α in CD4+ T cell receptor αβ+ T cells migrating into the murine small intestine epithelial layer

We investigated the surface phenotype of CD3⁺CD4⁺ T cell receptor (TCR) αβ⁺ T cells repopulating the intestinal lymphoid tissues of C.B-17 scidlscid (severe-combined immunodeficient; scid) (H-2^d, L^d+) mice. CD4⁺ CD8^? T cells were cell sorter-purified from various secondary and tertiary lymphoid organs of congenic C.B-17 +/+ (H-2^d, L^d+) or semi-syngeneic dm2 (H-2^d, L^d?) immunocompetent donor mice. After transfer of 10⁵ cells into young scid mice, a mucosa-homing, memory CD44^hi CD45RB^lo CD4⁺ T cell population was selectively engrafted. Large numbers of single-positive (SP) CD3⁺ CD2⁺ CD28⁺ CD4⁺ CD^8? T cells that expressed the α₄ integrin chain CD49d were found in the spleen, the mesenteric lymph nodes, the peritoneal cavity and the gut lamina propria of transplanted scid mice. Unexpectedly, large populations of donor-type doublepositive (DP) CD4⁺ CD8α⁺ CD8β^? T cells with high expression of the CD3/TCR complex appeared in the epithelial layer of the small intestine of transplanted scid mice. In contrast to SP CD4⁺ T cells, the intraepithelial DP T cells showed low expression of the CD2 and the CD28 co-stimulator molecules, and of the α₄ integrin chain CD49d, but expressed high levels of the α_IEL integrin chain CD103. The TCR-Vβ repertoire of DP but not SP intraepithelial CD4⁺ T cells was biased towards usage of the Vβ6 and Vβ8 viable domains. Highly purified populations of SP and DP CD4⁺ T cell populations from the small intestine epithelial layer of transplanted scid mice had different abilities to repopulate secondary scid recipient mice: SP CD4⁺ T cells repopulated various lymphoid tissues of the immunodeficient host, while intraepithelial DP CD4⁺ T cells did not. Hence, a subset of CD3⁺ CD4⁺ TCRαβ⁺ T cells apparently undergoes striking phenotypic changes when it enters the microenvironment of the small intestine epithelial layer.     

Clearance of Sendai virus by CD8+ T cells requires direct targeting to virus-infected epithelium

Minimal numbers of CD8⁺ T cells are found in bronchoalveolar lavage (BAL) populations recovered from Sendai virus-infected mice that are homozygous (?/?) for β2-microglobulin (β2-m) gene disruption. The prevalence of the CD8⁺ set was substantially increased in the pneumonic lungs of 8?12-week radiation chimeras made using substantially class I major histocompatibility complex (MHC) glycoprotein-negative β2-m (?/?) recipients and normal β2-m (+/+) bone marrow. Even so, the CD8⁺ (but not the CD4⁺) lymphocyte counts were still much lower than in the (+/+)→(+/+) controls. The (+/+)→(+/+) and (+/+)→(?/?) chimeras cleared Sendai virus and potent virus-immune CD8⁺ cytotoxic T lymphocytes (CTL) specific for H-2K^b + viral nucleoprotein peptide were found in the BAL from both groups. However, following in vivo depletion of the CD4⁺ population, only the (+/+)→(+/+) mice were able to deal with the infection. Similarly, adoptively transferred, H-2K^b-restricted CD8⁺ T cells from previously-primed (+/+) mice also failed to clear virus from the lungs of (+/+)→(?/?) chimeras infected within 2 weeks of reconstitution with bone marrow, though they were effective in the (+/+)→(+/+) controls. Sendai virus-immune CD8⁺ T cells are thus unable to eliminate virus-infected β2-m (?/?) lung epithelial cells that might be thought to be expressing very small amounts of either isolated class I heavy chain, or class I MHC glycoprotein that has bound β2-m derived from β2-m (+/+) T cells or macrophages present in the pneumonic lung. Furthermore, the CD8⁺ CTL that are being exposed to β2-m (+/+) stimulators in the BAL population cannot operate in some bystander mode to clear virus from respiratory epithelium.     

T lymphocyte development in the absence of Fcϵ receptor Iγ subunit: analysis of thymic-dependent and independent αβ and γδ pathways

During fetal development, early thymocyte progenitors transiently express low affinity Fc receptors for IgG (FcγR) of both FcγRII and III isoforms. Only the FcγRIII isoform requires association of an FcγRIII (CD16) α subunit with an FcϵRIγ homodimer for surface expression. To address the role of FcγR in ontogeny, we studied thymic development in FcϵRIγ^−/− mice. We find that day 14.5 CD4⁻CD8⁻ double-negative (DN) fetal thymocytes of FcϵRIγ^−/− mice express mRNA of both FcγRIIb₁ and FcγRIII. Surface expression of FcγRII/III is readily detected on these cells. It appears that FcγRIIb₁, whose surface expression is FcϵRIγ independent, replaces FcγRIII during thymic development in these animals. Moreover, subsequent development into CD4⁺CD8⁺ double-positive and CD4⁺CD8⁻ and CD4⁻CD8⁺ single-positive subsets appears normal even in the absence of FcϵRIγ. However, alterations were noted in adult animals among the DN αβ TCR⁺ thymocytes and peripheral splenic DN T cells as well as CD8αα⁺ intestinal intraepithelial lymphocytes (iIEL). In contrast to conventional T lymphocytes, which do not express either FcγRIII or FcϵRIγ, DN αβ TCR⁺ thymocytes and extrathymically derived αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL express TCR which incorporate FcϵRIγ as one of their subunits. Consistent with this, the TCR levels of these cells are lower than the TCR levels on cells from wild-type C57BL/6 mice. Despite the reduction in the level of surface TCR, the development of these cells was unaltered by the absence of FcϵRIγ. Thus, we observed alterations in adult DN αβ TCR⁺ thymocytes, splenic DN αβ TCR⁺ and DN γδ TCR⁺ large granular lymphocytes (LGL), and αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL, but no detectable changes in their major fetal thymic developmental pathways. Cultivation of peripheral DN αβ TCR⁺ and DN γδ TCR⁺ cells from FcϵRIγ^−/− mice with interleukin-2 generates LGL which mediate natural killer activity. Unlike LGL from wild-type C57BL/6 mice, LGL from FcϵRIγ^−/− mice lack FcγRIII expression and could not mediate antibody-dependent cellular cytotoxicity through FcγRIII.     

Leishmania major infection in mice primes for specific major histocompatibility complex class I-restricted CD8+ cytotoxic T cell responses

This report shows that lymphoid tissues of mice which have resolved a primary infection with Leishmania major contain parasite-specific major histocompatibility complex (MHC) class I-restricted cytolytic CD8⁺ T cell precusors that can be expanded after specific restimulation in vitro with syngeneic antigen-presenting cells pulsed with a cyanogen bromide digest of L. major. In H-2^b mice, two distinct populations of CD8⁺ T cells were identified which both lysed target cells pulsed with L. major-derived peptides but were restricted by a different H-2^b class I gene product. Interestingly, these two populations appear to recognize different parasite-derived peptides. It is noteworthy that one K°-restricted CD8⁺ T cell line was able to specifically lyse syngeneic macrophages infected with viable L. major, indicating that some L. major-derived peptides may reach the MHC class I pathway of presentation from the phagolysosomal compartment where the parasites are confined in infected macrophages. The importance of these parasite-specific MHC class I restricted cytolytic CD8⁺ T cells for the elimination of L. major by the infected host remains to be determined.     

CD8/CD4 lineage commitment occurs by an instructional/default process followed by positive selection

In the present study, we investigated the developmental potential of purified populations of transitional CD4ⁱⁿ CD8^hi and CD4^hi CD8ⁱⁿ thymocytes that were further defined according to their differentiation stage by their levels of T cell receptor (TCR) expression into TCR^lo, TCRⁱⁿ and TCR^hi subpopulations. The differentiation potential of each of these subsets was tested in vitro in a single-cell suspension culture assay that showed that CD4ⁱⁿ CD8^hi TCR^hi are precursors of CD8 single-positive cells, whereas CD4^hi CD8ⁱⁿ TCR^in/hi are precursors of both CD4 and CD8 single-positive thymocytes. The analysis of transitional subsets in mutant mice for either β2-microglobulin or major histocompatibility complex (MHC) class II further revealed that lineage commitment to the CD8 lineage requires a TCR-MHC class I engagement, presumably at the immature double-positive stage of thymic development, while CD4 commitment does not require an MHC class II-mediated signal, but rather occurs by default. Using the addition of MHC class I- or class II-expressing cells or the addition of total thymocytes to purified sorted transitional precursors for the duration of the cultures in vitro, we identified an additional stage of differentiation for both CD4 and CD8 lineages that requires a positive selection signal. Examination of protein tyrosine phosphorylation of transitional precursors revealed that CD4ⁱⁿ CD8^hi transitional cells contain a high level of a 70-kDa phosphorylated protein consistent with a role for ZAP70 in the signal transduction during the positive selection of CD8⁺ cells.     

CD3-induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4⁺CD8⁺ double positive thymocyte stage. Immature CD4⁺CD8⁺ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4⁺CD8⁺ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4⁺CD8⁺ thymocytes can occur even in TCRα^? mutant mice which do not express the TCRαβ/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4⁺CD8⁺ thymocytes in TCRα^? mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4⁺CD8⁺ thymocytes from TCRα^? mice are not associated with clonotypic TCR chains, including TCRβ. Thus, apoptosis of CD4⁺CD8⁺ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4⁺CD8⁺ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.     

Discrimination between maintenance- and differentiation-inducing signals during initial and intermediate stages of positive selection

As well as signaling through the αβ T cell receptor complex, positive selection of immature CD4⁺ 8⁺ thymocytes involves additional ill-defined accessory interactions provided by thymic epithelial cells. Here, we have isolated CD4⁺ 8⁺ thymocytes at a pre-positive selection stage of development (TCR^? CD69^? 4⁺ 8⁺ cells), or after initiation of positive selection (CD69⁺ 4⁺ 8⁺ cells), from mice where the normal lifespan of thymocytes is extended by the presence of a bcl-2 transgene, to allow us to discriminate between requirements for maintenance and differentiation signals during positive selection. We find that MHC class II⁺ thymic epithelial cells drive positive selection of TCR^? CD69^? 4⁺ 8⁺ bcl-2 tg thymocytes to the CD4⁺ and CD8⁺ stage, while no such mature subsets are observed when thymocytes are cultured alone or with major histocompatibility complex (MHC) class II⁺ salivary epithelial cells. However, CD4⁺ 8⁺ cells remain in such cultures in considerable numbers, and retain the potential for positive selection if re-cultured with thymic epithelium, suggesting that thymic epithelial cells provide specific differentiation-inducing signals for positive selection. In contrast, intermediate CD69⁺ 4⁺ 8⁺ thymocytes show some capacity for phenotypic conversion in the absence of thymic stromal cells although strikingly the single-positive CD4⁺ and CD8⁺ cells generated are not functionally competent. Finally, we show that prior culture of thymic epithelial cells under monolayer conditions abrogates their ability to support the initiation of positive selection, suggesting that the epithelial cell molecules necessary for the provision of differentiation signals during positive selection are down-regulated under such conditions.     

Differential reactivity of residual CD8+ T lymphocytes in TAP1 and β2-microglobulin mutant mice

TAP1 -/- and β2-microglobulin (β2m) -/- mice (H-2^b background) express very low levels of major histocompatibility complex (MHC) class I molecules on the cell surface. Consequently these mice have low numbers of mature CD8⁺ T lymphocytes. However, TAP1 -/- mice have significantly higher numbers of CD8⁺ T cells than β2m -/- mice. Alloreactive CD8⁺ cytotoxic T lymphocyte (CTL) responses were also stronger in TAP1 -/- mice than in β2m -/- mice. Alloreactive CTL generated in TAP1 -/- and β2m -/- mice cross-react with H-2^b-expressing cells. Surprisingly, such cross-reactivity was stronger with alloreactive CTL from β2m -/- mice than with similar cells from TAP1 -/- mice. The β2m -/- mice also responded more strongly when primed with and tested against cells expressing normal levels of H-2^b MHC class I molecules. Such H-2^b-reactive CD8⁺ CTL from β2m -/- mice but not from TAP1 -/- mice also reacted with TAP1 -/- and TAP2-deficient RMA-S cells. In contrast, H-2^b-reactive CD8⁺ CTL from neither β2m -/- mice nor TAP1 -/- mice killed β2m -/- cells. In line with these results, β2m -/- mice also responded when primed and tested against TAP1 -/- cells. We conclude that the reactivity of residual CD8⁺ T cells differs between TAP1 -/- and β2m -/- mice. The MHC class I-deficient phenotype of TAP1 -/- and β2m -/- mice is not equivalent: class I expression differs between the two mouse lines with regard to quality as well as quantity. We propose that the differences observed in numbers of CD8⁺ T cells, their ability to react with alloantigens and their cross-reactivity with normal H-2^b class I are caused by differences in the expression of MHC class I ligands on selecting cells in the thymus.