A novel Zap70 mutation with reduced protein stability demonstrates the rate‐limiting threshold for Zap70 in T‐cell receptor signalling

Loss of ζ‐associated protein 70 (Zap70) results in severe immunodeficiency in humans and mice because of the critical role of Zap70 in T‐cell receptor (TCR) signalling. Here we describe a novel mouse strain generated by N‐ethyl‐N‐nitrosourea mutagenesis, with the reduced protein stability (rps) mutation in Zap70. The A243V rps mutation resulted in decreased Zap70 protein and a reduced duration of TCR‐induced calcium responses, equivalent to that induced by a 50% decrease in catalytically active Zap70. The reduction of signalling through Zap70 was insufficient to substantially perturb thymic differentiation of conventional CD4 and CD8 T cells, although Foxp3⁺ regulatory T cells demonstrated altered thymic production and peripheral homeostasis. Despite the mild phenotype, the Zap70^A243V variant lies just above the functional threshold for TCR signalling competence, as T cells relying on only a single copy of the Zap70^rps allele for TCR signalling demonstrated no intracellular calcium response to TCR stimulation. This addition to the Zap70 allelic series indicates that a rate‐limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null.     

Positive selection of CD4+ T cells by TCR-specific antibodies requires low valency TCR cross-linking: implications for repertoire selection in the thymus

The developmental fate of immature CD4⁺ 8⁺ thymocytes is determined by intrathymic signals transduced by surface TCR complexes. In particular, TCR signals are required for immature CD4⁺ 8⁺ thymocytes to further differentiate into CD4⁺ 8⁻ or CD4⁻ 8⁺ T cells, a process referred to as positive selection. It is generally thought that positive selection results from low affinity TCR interactions with self antigens which engage the relatively few surface TCR complexes that are on immature CD4⁺ 8⁺ thymocytes. However, we now demonstrate with TCR-specific antibodies that positive selection of CD4⁺ T cells requires low valency cross-linking of surface TCR complexes on immature thymocytes. That is, positive selection signals are only generated within a narrow range of TCR cross-linking: cross-linking either too few or too many surface TCR complexes fails to signal positive selection. We interpret these results as indicating that positive selection of CD4⁺ T cells is not signaled by low affinity TCR interactions per se, but rather can be signaled by any combination of TCR affinity and ligand density that induces low valency TCR cross-linking on immature thymocytes.     

A ZAP‐70 kinase domain variant prevents thymocyte‐positive selection despite signalling CD69 induction

Quantitative reductions in T‐cell receptor (TCR) signalling are associated with severe immunodeficiency, yet in certain cases can lead to autoimmunity. Mutation of the tyrosine kinase ZAP‐70 can cause either of these outcomes, yet the limits of its signal transducing capacity are not well defined. To investigate these limits we have made use of mrtless: a chemically induced mutation of Zap70 associated with T‐cell deficiency. Unlike cells devoid of ZAP‐70, mrtless thymocytes showed partial induction of CD5 and CD69, and were sensitive to TCR stimulation with a dose–response shifted approximately 10‐fold. However, essentially no T cells were able to compensate for the mrtless mutation and mature beyond the CD4⁺ CD8⁺ stage. This outcome contrasts with a ZAP‐70 Src Homology 2 domain mutant strain, where high‐affinity self‐reactive TCR are positively selected rather than deleted. We discuss these data with respect to current models of TCR signalling in thymocyte selection.     

Targeted loss of SHP1 in murine thymocytes dampens TCR signaling late in selection

SHP1 is a tyrosine phosphatase critical to proximal regulation of TCR signaling. Here, analysis of CD4‐Cre SHP1^fl/fl conditional knockout thymocytes using CD53, TCRβ, CD69, CD4, and CD8α expression demonstrates the importance of SHP1 in the survival of post selection (CD53⁺), single‐positive thymocytes. Using Ca²⁺ flux to assess the intensity of TCR signaling demonstrated that SHP1 dampens the signal strength of these same mature, postselection thymocytes. Consistent with its dampening effect, TCR signal strength was also probed functionally using peptides that can mediate selection of the OT‐I TCR, to reveal increased negative selection mediated by lower‐affinity ligand in the absence of SHP1. Our data show that SHP1 is required for the survival of mature thymocytes and the generation of the functional T‐cell repertoire, as its absence leads to a reduction in the numbers of CD4⁺ and CD8⁺ naïve T cells in the peripheral lymphoid compartments.     

Effects of PGE2 upon differentiation and programmed cell death of suspension cultured CD4− CD8− thymocytes

Recently, several works have focused on the modulation of the immune response by arachidonic acid metabolites. Some of these metabolites, such as prostaglandins, have been shown to influence thymocyte “education” in vitro. However, the effect of one of them, prostaglandin E₂ (PGE₂), in the education of CD4⁻ CD8⁻ double negative immature thymocytes (DN cells) remained unclear. Using a flow cytometry analysis of DN cells cultured for 24 h in the presence of PGE₂, we observed, compared with DN thymocytes cultured without PGE₂, an increase in the CD4⁺ CD8⁻ CD3⁻ immature thymocytes and in the CD4⁺ CD8⁻ and CD8⁺ CD4⁻ mature single positive thymocytes and a decrease in the DN and CD4^high CD8^high double positive thymocytes. Other differentiation thymocyte surface markers, such as CD3, CD5, TCRαß, TCRδγ and HSAg, revealed an increasing number of thymocytes bearing these first four markers and a lower expression of the HSAg. Furthermore, we observed an accumulation of CD4^low CD8^low thymocytes and an increasing proportion of hypodiploid nuclei. These two findings have been shown to be markers of the programmed cell death process. These findings suggest that PGE₂ probably acts on thymocyte differentiation through at least two distinct pathways. On the one hand, PGE₂ seems to promote differentiation of DN cells into CD4⁺ CD8⁻ CD3⁻ immature cells and drive CD4⁺ CD8⁺ CD3⁺ thymocyte to a CD4⁺ CD8⁻ and CD8⁺ CD4⁻ mature phenotype. On the other hand, PGE₂ is probably implicated directly or indirectly in the increase or the acceleration of the programmed cell death process of immature CD4⁺ CD8⁺ CD3⁺ thymocytes, which is linked to the positive and/or negative selection.     

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.     

EBI2 contributes to the induction of thymic central tolerance in mice by promoting rapid motility of medullary thymocytes

Maturing thymocytes enter the thymic medulla, where they encounter numerous self‐antigens presented by antigen presenting cells (APCs). Those thymocytes that are strongly self‐reactive undergo either negative selection or diversion into the regulatory T‐cell lineage. Although the majority of the proteome is expressed in the medulla, many self‐antigens are expressed by only a minor fraction of medullary APCs; thus, thymocytes must efficiently enter the medulla and scan APCs to ensure central tolerance. Chemokine receptors promote lymphocyte migration, organization within tissues, and interactions with APCs in lymphoid organs. The chemokine receptor EBI2 governs localization of T cells, B cells, and dendritic cells (DCs) during immune responses in secondary lymphoid organs. However, the role of EBI2 in thymocyte development has not been elucidated. Here, we demonstrate that EBI2 is expressed by murine CD4⁺ single positive (CD4SP) thymocytes and thymic DCs. EBI2 deficiency alters the TCR repertoire, but does not grossly impact thymocyte cellularity or subset distribution. EBI2 deficiency also impairs negative selection of OT‐II TCR transgenic thymocytes responding to an endogenous self‐antigen. Two‐photon imaging revealed that EBI2 deficiency results in reduced migration and impaired medullary accumulation of CD4SP thymocytes. These data identify a role for EBI2 in promoting efficient thymic central tolerance.     

Strength of TCR signal from self‐peptide modulates autoreactive thymocyte deletion and Foxp3+ Treg‐cell formation

Autoreactive CD4⁺CD8^? (CD4SP) thymocytes can be subjected to deletion when they encounter self‐peptide during their development, but they can also undergo selection to become CD4SPFoxp3⁺ Treg cells. We have analyzed the relationship between these distinct developmental fates using mice in which signals transmitted by the TCR have been attenuated by mutation of a critical tyrosine residue of the adapter protein SLP‐76. In mice containing polyclonal TCR repertoires, the mutation caused increased frequencies of CD4SPFoxp3⁺ thymocytes. CD4SP thymocytes expressing TCR Vβ‐chains that are subjected to deletion by endogenous retroviral superantigens were also present at increased frequencies, particularly among Foxp3⁺ thymocytes. In transgenic mice in which CD4SP thymocytes expressing an autoreactive TCR undergo both deletion and Treg‐cell formation in response to a defined self‐peptide, SLP‐76 mutation abrogated deletion of autoreactive CD4SP thymocytes. Notably, Foxp3⁺ Treg‐cell formation still occurred, albeit with a reduced efficiency, and the mutation was also associated with decreased Nur77 expression by the autoreactive CD4SP thymocytes. These studies provide evidence that the strength of the TCR signal can play a direct role in directing the extent of both thymocyte deletion and Treg‐cell differentiation, and suggest that distinct TCR signaling thresholds and/or pathways can promote CD4SP thymocyte deletion versus Treg‐cell formation.     

Thymus‐specific serine protease regulates positive selection of a subset of CD4+ thymocytes

Thymus‐specific serine protease (TSSP) was initially reported as a putative protease specifically expressed in the endosomal compartment of cortical thymic epithelial cells (cTEC). As such, TSSP is potentially involved in the presentation of the self‐peptides that are bound to MHC class II molecules expressed at the cTEC surface and are involved in the positive selection of CD4⁺ thymocytes. We tested this hypothesis by generating mutant mice deprived of Prss16, the gene encoding TSSP. TSSP‐deficient mice produced normal numbers of T cells, despite a decrease in the percentage of cTEC expressing high surface levels of MHC class II. By using sensitive transgenic models expressing MHC class II‐restricted TCR transgenes (Marilyn and OT‐II), we showed that the absence of TSSP markedly impaired the selection of Marilyn and OT‐II CD4⁺ T cells. In contrast, selection of CD8⁺ T cells expressing an MHC class I‐restricted TCR transgene (OT‐I) was unaffected. Therefore, TSSP is involved in the positive selection of some CD4⁺ T lymphocytes and likely constitutes the first serine protease to play a function in the intrathymic presentation of self‐peptides bound to MHC class II complexes.     

CD4+CD8− thymocytes that express L-selectin protect rats from diabetes upon adoptive transfer

Previous studies have shown that insulin-dependent diabetes can be induced in normal PVG.RT1^u rats by a protocol of adult thymectomy and irradiation. The injection of CD4⁺ T cells from non-irradiated syngeneic donors prevents the onset of disease in approximately 50 % of pre-diabetic recipients but all rats are protected if a particular subset of CD4⁺ cells is transferred. These protective cells express TCRαβ and have a memory phenotype, being CD45RC^low RT6⁺. Further studies have demonstrated that the transfer of CD4⁺CD8⁻ thymocytes, like that of unfractionated CD4⁺ peripheral T cells, also protects approximately half of recipients from diabetes suggesting that, as with the peripheral T cells, a functional heterogeneity may exist amongst CD4⁺CD8⁻ thymocytes. In this study, we show that L-selectin is expressed by 50–60 % of all CD4⁺CD8⁻ thymocytes from 6-week-old rats. Adoptive transfer of these populations into thymectomized and irradiated rats revealed that the protection from diabetes observed by CD4⁺CD8⁻ thymocytes was mediated almost entirely by the L-selectin⁺ subset. Cells with this phenotype were also able to mediate both humoral and cell mediated responses, providing primed B cells with help for secondary antibody responses and mediating local graft-versus-host reactions. L-selectin⁻ CD4⁺CD8⁻ thymocytes failed to mediate these responses. These data indicate that CD4⁺CD8⁻ thymocytes must mature to the stage of L-selectin expression, before they can mediate normal T cell function. The implications of these results are discussed with respect to the possible role of murine NK1.1⁺ thymocytes in the control of autoimmunity.     

Hypomorphic mutation of ZAP70 in human results in a late onset immunodeficiency and no autoimmunity

Complete lack of function of the tyrosine kinase ZAP70 in humans results in a severe immunodeficiency, characterized by a lack of mature CD8⁺ T cells and non‐functional CD4⁺ T cells. We report herein an immunodeficiency with an inherited hypomorphic mutation of ZAP70 due to a single G‐to‐A substitution in a non‐coding intron. This mutation introduces a new acceptor splice site and allows low levels of normal alternative splicing and of WT ZAP70 expression. This partial deficiency results in a compromised TCR signaling that was totally restored by increased expression of ZAP70, demonstrating that defective activation of the patient T cells was indeed caused by the low level of ZAP70 expression. This partial ZAP70 deficiency was associated with an attenuated clinical and immunological phenotype as compared with complete ZAP70 deficiency. CD4⁺ helper T‐cell populations including, follicular helper T cells, Th1, Th17 and Treg were detected in the blood. Finally, the patient had no manifestation of autoimmunity suggesting that the T‐cell tolerogenic functions were not compromised, in contrast to what has been observed in mice carrying hypomorphic mutations of Zap70. This report extends the phenotype spectrum of ZAP70 deficiency with a residual function of ZAP70.     

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.     

Two separable T cell receptor signals reconstitute positive selection of CD4 lineage T cells in vivo

Positive selection is an obligatory step during intrathymic T cell differentiation. It is associated with rescue of short-lived, self major histocompatibility complex (MHC)-restricted thymocytes from programmed cell death, CD4/CD8 T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TCR) signaling during positive selection can be closely mimicked by targeting TCR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. We show that selection of CD4 T cell lineage cells in mice deficient for MHC class I and MHC class II expression can be reconstituted in vivo by two separable T cell receptor signaling steps, whereas a single TCR signal leads only to induction of short-lived CD4⁺CD8^la intermediates. These intermediates remain susceptible to a second TCR signal for 12-48 h providing an estimate for the duration of positive selection in situ. While both TCR signals induce differentiation steps, only the second one confers long-term survival on immature thymocytes. In further support of the two-step model of positive selection we provide evidence that CD4 T cell lineage cells rescued by a single hybrid antibody pulse in MHC class II-deficient mice are pre-selected by MHC class 1.     

Thymocyte‐derived BDNF influences T‐cell maturation at the DN3/DN4 transition stage

Brain‐derived neurotrophic factor (BDNF) promotes neuronal survival, regeneration, and plasticity. Emerging evidence also indicates an essential role for BDNF outside the nervous system, for instance in immune cells. We therefore investigated the impact of BDNF on T cells using BDNF knockout (KO) mice and conditional KO mice lacking BDNF specifically in this lymphoid subset. In both settings, we observed diminished T‐cell cellularity in peripheral lymphoid organs and an increase in CD4⁺CD44⁺ memory T cells. Analysis of thymocyte development revealed diminished total thymocyte numbers, accompanied by a significant increase in CD4/CD8 double‐negative (DN) thymocytes due to a partial block in the transition from the DN3 to the DN4 stage. This was neither due to increased thymocyte apoptosis nor defects in the expression of the TCR‐β chain or the pre‐TCR. In contrast, pERK but not pAKT levels were diminished in DN3 BDNF‐deficient thymocytes. BDNF deficiency in T cells did not result in gross deficits in peripheral acute immune responses nor in changes of the homeostatic proliferation of peripheral T cells. Taken together, our data reveal a critical autocrine and/or paracrine role of T‐cell‐derived BDNF in thymocyte maturation involving ERK‐mediated TCR signaling pathways.     

Role of TCR specificity in CD4+CD25+ regulatory T-cell selection

Summary: CD4⁺CD25⁺ regulatory T cells play a crucial role in preventing autoimmune disease and can also modulate immune responses in settings such as transplantation and infection. We have developed a transgenic mouse system in which the role that T‐cell receptor (TCR) specificity for self‐peptides plays in the formation of CD4⁺CD25⁺ regulatory T cells can be examined. We have shown that interactions with a single self‐peptide can induce thymocytes bearing an autoreactive TCR to undergo selection to become CD4⁺CD25⁺ regulatory T cells and that thymocytes bearing TCRs with low affinity for the selecting peptide do not appear to undergo selection into this pathway. In addition, thymocytes with identical specificity for the selecting self‐peptide can undergo overt deletion versus abundant selection to become CD4⁺CD25⁺ regulatory T cells in response to variations in expression of the selecting peptide in different lineages of transgenic mice. Finally, we have shown that CD4⁺CD25⁺ T cells proliferate in response to their selecting self‐peptide in the periphery, but these cells do not proliferate in response to lymphopenia in the absence of the selecting self‐peptide. These studies are determining how the specificity of the TCR for self‐peptides directs the thymic selection and peripheral expansion of CD4⁺CD25⁺ regulatory T cells.     

Identification and cellular distribution of the rat interleukin-2 receptor β chain: induction of the IL-2Rα−β+ phenotype by major histocompatibility complex class I recognition during T cell development in vivo and by T cell receptor stimulation of CD4+8+ immature thymocytes in vitro

A mouse monoclonal antibody (mAb) to the rat interleukin-2 receptor β (IL-2Rβ) chain was generated using IL-2Rβ cDNA-transfected mouse L929 cells for immunization and differential screening. This antibody, called L316, detects a cell surface protein with an apparent molecular mass of about 80 kDa. In peripheral lymphoid organs of young adult rats, IL-2Rβ expression is restricted to T and natural killer (NK) cells, and less than 10% of IL-2Rβ⁺ cells co-express the IL-2Rα chain. IL-2Rβ was detected on all NKRP-1^hi (NK⁻) and NKRP-1^lo cells (T-lineage cells of unknown function), most peripheral γδ T cells and on 30–40% of CD8 and 10% of CD4 αβ T cells. In the adult rat thymus, mAb L316 detects a small subset (about 1%) of predominantly IL-2Rα⁻ cells which express cell surface markers characteristic of mature T lymphocytes and contain a high proportion of CD4⁻8⁻ and CD4⁻8⁺ αβ T cell receptor (TCR)⁺ thymocytes. TCR-V usage suggests that major histocompatibility complex (MHC) class I plays a more important role than MHC class II in the selection of these cells. On immature CD4⁺8⁺ rat thymocytes, IL-2Rβ cell surface expression is readily induced by TCR stimulation in vitro, supporting the idea that in vivo, the IL-2Rβ⁺ phenotype is the result of TCR engagement during thymic selection.     

A thymic epithelial cell line induces both positive and negative selection in the thymus

TCR engagement in the thymus results in both survival and eliminationsignals for developing thymocytes. To examine whether both signalscan be provided by the same cell type, we investigated the abilityof a thymic epithelial cell (TEC) line 427.1, previously shownto allow positive selection in the thymus, to induce clonaldeletion of Immature thymocytes. [H-2^b/s–H-2^s] bone marrowchimeras are non-responsive to antigens in the context of H-2b.However, chimeras that underwent intrathymic injection of H-2^b/s427.1 cells expressing vesicular stomatitis virus (VSV) nucleocapsidantigen acquired the ability to raise influenza, but not VSVspecific H-2^b restricted cytotoxic T lymphocyte (CTL) responses.The ability of 427.1 cells to delete CD4⁺CD8⁺ thymocytes wasdetermined using mice transgenlc for the TCR specific for ovalbumln(OVA) in the context of H-2K^b. OVA transfected, but not mocktransfected 427.1 TECs, Induced in vitro deletion of CD4⁺CD8⁺TCR transgenlc thymocytes manifested as a down-modulation ofCD4 and CD8 molecules, a shift in the side versus forward scattercharacteristics of thymocytes, and appearance of thymocyteswith subdlplold content of DNA indicated the ongoing processof DNA fragmentation. The finding that the same TEC line Iscapable of inducing both positive and negative selection inthe thymus suggests that thymocytes bearing TCRs specific forself peptldes expressed by positively selecting thymic epitheliumcan be deleted. Therefore the expression of a unique set ofMHC associated peptldes by TECs does not appear to be the basisfor the positive outcome of the TCR llgatlon on immature thymocytes.     

Aire controls the recirculation of murine Foxp3+ regulatory T‐cells back to the thymus

In the thymus, medullary thymic epithelial cells (mTEC) determine the fate of newly selected CD4⁺ and CD8⁺ single positive (SP) thymocytes. For example, mTEC expression of Aire controls intrathymic self‐antigen availability for negative selection. Interestingly, alterations in both Foxp3⁺ Regulatory T‐cells (T‐Reg) and conventional SP thymocytes in Aire^?/? mice suggest additional, yet poorly understood, roles for Aire during intrathymic T‐cell development. To examine this, we analysed thymocytes from Aire^?/? mice using Rag2GFP and Foxp3 expression, and a recently described CD69/MHCI subset definition of post‐selection CD4⁺ conventional thymocytes. We show that while Aire is dispensable for de novo generation of conventional αβT‐cells, it plays a key role in controlling the intrathymic T‐Reg pool. Surprisingly, a decline in intrathymic T‐Reg in Aire^?/? mice maps to a reduction in mature recirculating Rag2GFP^? T‐Reg that express CCR6 and re‐enter the thymus from the periphery. Furthermore, we show mTEC expression of the CCR6 ligand CCL20 is reduced in Aire^?/? mice, and that CCR6 is required for T‐Reg recirculation back to the thymus. Collectively, our study re‐defines requirements for late stage intrathymic αβT‐cell development, and demonstrates that Aire controls a CCR6‐CCL20 axis that determines the developmental makeup of the intrathymic T‐Reg pool.