Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells

Foxp3(+)CD4(+)CD25(+) regulatory T cells can differentiate from Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) naive T cells. However, the impact of these two processes on size and composition of the peripheral repertoire of regulatory T cells is unclear. Here we followed the fate of individual Foxp3(+)CD4(+)CD25(+) thymocytes and T cells in vivo in T cell receptor (TCR) transgenic mice that express a restricted but polyclonal repertoire of TCRs. By utilizing high-throughput single-cell analysis, we showed that Foxp3(+)CD4(+) peripheral T cells were derived from thymic precursors that expressed a different TCRs than Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) T cells. Furthermore, the diversity of TCRs on Foxp3(+)CD4(+) regulatory T cells exceeded the diversity of TCRs on Foxp3(-)CD4(+) naive T cells, even in mice that lack expression of tissue-specific antigens. Our results imply that higher TCR diversity on Foxp3(+) regulatory T cells helps these cells to match the specificities of autoreactive and naive T cells.     

The Src-like adaptor protein downregulates the T cell receptor on CD4+CD8+ thymocytes and regulates positive selection

In this report, we show that the Src-like adaptor protein (SLAP) plays an important role in thymocyte development. SLAP expression is developmentally regulated; it is low in CD4-CD8- thymocytes, it peaks in the CD4+CD8+ subset, and it decreases to low levels in more mature cells. Disruption of the SLAP gene leads to a marked upregulation of TCR and CD5 expression at the CD4+CD8+ stage. The absence of SLAP was also developmentally significant because it enhanced positive selection in mice expressing the DO11.10 transgenic T cell receptor. Moreover, SLAP deletion at least partially rescued the development of ZAP-70-deficient thymocytes. These results demonstrate that SLAP participates in a novel mechanism of TCR downregulation at the CD4+CD8+ stage and regulates positive selection.     

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.     

Evidence for down-regulation of highly expressed TCR by CD4 and CD45 on non-selected CD4+CD8+thymocytes

Immature CD4+CD8+ double-positive (DP) thymocytes are positivelyselected for further development if they express TCR reactingwith thymic ligands of low affinity. However, the majority ofDP thymocytes express low TCR levels. This low level of TCRmay be insufficient to recognize thymic ligands. To understandthe basis for the low expression of TCR on DP thymocytes, wedetermined the density of TCR expression at various stages oftheir development using TCR transgenic (TCR-Tg) mice. We foundthat TCR expression was high in the thymocytes that had recentlytransited into the DP stage but then gradually decreased onDP cells if they were not selected by TCR interaction with MHCmolecules. However, such TCR suppression was not observed inpositively selected DP cells and in the non-selected DP cellsobtained from CD45 deficient mice or from mice receiving anti-CD4mAb. These findings suggest that the once highly expressed TCRat the DP stage is suppressed by CD45 and/or CD4 on non-selectedthymocytes. Furthermore, TCR suppression is prevented by TCR-mediatedsignals. The maintenance of high TCR levels on positively selectedDP thymocytes may facilitate their selection.     

Memory/effector T cells in TCR transgenic mice develop via recognition of enteric antigens by a second, endogenous TCR.

The majority of clonotypic CD4(+) T cells in the intestinal lamina propria of DO11.10 TCR transgenic mice have an activated/memory phenotype and produce effector cytokines despite the absence of prior exposure to ovalbumin (OVA), the transgene-specific antigen. A small number of splenic T cells have a similar phenotype. Clonotypic T cells from Peyer's patch are intermediate in both phenotype and effector cytokine production. Flow cytometric analysis of cells isolated from thymectomized, OVA-naive DO11.10 mice treated with continuous administration of BrdU indicated that a significant fraction of clonotype-positive T cells in the lamina propria and Peyer's patch were in the cell cycle, with significantly fewer cycling cells in the spleen. Most of the cycling cells from each anatomic site expressed low levels of CD45RB. Effector cytokine expression was enriched in the CD45RB(low) populations. These memory/effector cell populations were eliminated in DO11.10/SCID and DO11.10/RAG-2(-/-) mice, suggesting that recognition of non-OVA antigens through a second, non-clonotypic TCR was driving differentiation of memory/effector cells in naive BALB/c DO11.10 mice. Clonotypic CD4(+) T cells isolated from DO11.10, but not from DO11.10/SCID or DO11.10/RAG-2(-/-) mice, were stimulated to enter the cell cycle by antigen-presenting cells pulsed with an intestinal bacterial antigen extract. These data provide direct evidence that enteric bacterial antigens can activate transgenic T cells through a second, non-clonotypic TCR, and support the notion that the development and turnover of memory/effector cells in vivo is driven by the intestinal flora.     

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

How T cell receptor (TCR) avidity influences CD8⁺ T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP^?/? mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP^?/? Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8⁺ T cell development and repertoire selection. In comparing SLAP^?/? OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP^?/? Vβ5 mice. We have found that SLAP^?/? OT-1 mice have fewer CD8⁺ thymocytes but have increased CD5 expression. SLAP^?/? OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8⁺ splenocytes upon tetramer staining. Our data demonstrate that SLAP^?/? Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8⁺ T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8⁺ T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8⁺ T cell development influences repertoire selection.     

Evidence for a repertoire of functional untolerized CD4+ T cells specific for melanoma-associated antigens

Active vaccination against melanoma requires tolerance break as melanoma-associated antigens (MAA) used in vaccine formula are mostly self-antigens. While tolerance to MAA in the CD8(+) T cell compartment is well characterized, it is still not the case for the CD4(+) T cell compartment. Here, we analysed CD4(+) T cell tolerance to such antigens in mice genetically engineered to express ovalbumin (OVA) in melanocytes (Tyr-OVA mice). When we crossed Tyr-OVA mice with DO11.10 and OT-II mice transgenic for an OVA-specific TCR restricted by MHC class II, we observed different tolerization levels. Central tolerance was complete for high avidity DO11.10 CD4(+) T cells, but absent for low avidity OT-II CD4(+) T cells. OT-II CD4(+) T cells also ignored OVA in the periphery of Tyr-OVA mice, albeit being potently reactive to vaccination. OVA challenge in single transgenic Tyr-OVA mice confirmed the existence of OVA-reactive CD4(+) T cells with the induction of efficient T helper cells for antibody production and anti-tumour T cell response. In total, our study demonstrates the existence of low avidity MAA-specific CD4(+) T cells escaping by ignorance central and peripheral tolerance, but valuable in the context of vaccination against melanoma.     

Positive and negative thymic selection in T cell receptor-transgenic mice correlate with Nur77 mRNA expression

The orphan nuclear receptor Nur77 has been implicated in thymic negative selection. We studied the effect of two T cell receptor (TCR) transgenes on positive selction and Nur77 mRNA expression in thymus. DO11.10 mice, expressing a transgenic TCR specific for an ovalbumin (OVA) 323–339 peptide presented by I-A^d, were found to have an enlarged thymus with a reduced apoptotic activity, measured by flow cytometry, reduced mitochondrial membrane potential and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) techniques. In contrast, in F5 mice expressing a transgenic TCR recognizing the influenza virus nucleoprotein (NP) 366–374 peptide restricted by D^b, this positive selection effect was much less pronounced. Positive thymic selection in DO11.10 TCR⁺ mice correlated with a reduced level of Nur77 mRNA expression shown by Northern blot. F5 mice expressed levels close to those expressed by the wild type. Both transgenic mouse strains responded with extensive cortical apoptosis, and with up-regulation of Nur77 mRNA, to injection of cognate peptides. As 9-cis-Retinoic acid (9-cis-RA) inhibits Nur77-dependent apoptosis in T cell hybridomas in vitro, mice were pretreated with the drug to investigate a similar effect in vivo. However, the drug itself, at saturating concentrations, caused extensive apoptosis in immature CD4⁺/CD8⁺ thymocytes. The result demonstrates a correlation between Nur77 expression and thymic apoptotic activity, both during positive and negative selection events.     

MHC-restricted T cell receptor signaling is required for alpha beta TCR replacement of the pre T cell receptor

A developmental block is imposed on CD25(+)CD44(-) thymocytes at the beta-selection checkpoint in the absence of the pre T cell receptor (preTCR) alpha-chain, pTalpha. Early surface expression of a transgenic alphabeta TCR has been shown to partially circumvent this block, such that thymocytes progress to the CD4(+)CD8(+) double-positive stage. We wanted to analyze whether a restricting MHC element is required for alphabeta TCR-expressing double-negative (DN) thymocytes to overcome the developmental block in pTalpha-deficient animals. We used the HY-I knock-in model that endows thymocytes with alphabeta TCR expression in the DN compartment but has the advantage of physiological expression levels, in contrast to conventional TCR transgenes. On a pTalpha-deficient background, this HY-I TCR transgene 'rescued' CD25(+)CD44(-) thymocytes from apoptosis and enabled progression to later differentiation stages. On a non-selecting MHC background, however, pTalpha-deficient HY-I mice presented a pronounced reduction in numbers of splenocytes and thymocytes when compared to animals of selecting MHC genotype, showing that MHC restriction is necessary to drive HY-TCR-mediated rescue of pTalpha-deficient thymocytes.     

CD53, a thymocyte selection marker whose induction requires a lower affinity TCR-MHC interaction than CD69, but is up-regulated with slower kinetics

The molecular mechanisms that govern the survival, maturation and export of thymocytes are the subject of intense study, and candidates for involvement in these processes might be identified by their differential expression during thymocyte selection. One such molecule is the tetraspanin CD53, which is not expressed on most CD4(+)CD8(+) double-positive (DP) cells in the normal mouse. We have examined CD53 expression on DP from several class I- and class II-restricted TCR transgenic (Tg) mice, and have found a strong correlation between CD53 expression and positive selection. CD53 expression in DP was formally demonstrated to be dependent upon MHC recognition as evidenced by studying DP from MHC-deficient mice which totally lack expression of this molecule. This link between selection and CD53 expression was reminiscent of CD69, and indeed the majority of selected DP from normal mice that express CD53 also express CD69. We compared CD53 and CD69 induction in vitro using pre-selected thymocytes from TCR-Tg mice that were stimulated either with mAb against TCR or with antigen-presenting cells (APC) pulsed with peptides. The data shows that with either stimulus, CD69 is induced rapidly on the thymocyte surface with expression detected in as little as 2 h. CD53 induction is slower with maximal expression taking up to 20 h. We also stimulated pre-selected thymocytes from the OT-1 TCR-Tg strain with APC pulsed with peptides of varying affinities for the TCR. Here low-affinity peptides which induce CD69 expression poorly were able to induce significant levels of CD53 expression. These data demonstrate that the induction of CD53 and CD69 upon selection is not identical. Thus a combination of the CD69 and CD53 selection markers may be a powerful tool to isolate thymocytes that have either been very recently selected or have arisen from differing MHC--TCR affinity interactions during selection.     

Daily subcutaneous injections of peptide induce CD4+ CD25+ T regulatory cells

Peptide immunotherapy is being explored to modulate varied disease states; however, the mechanism of action remains poorly understood. In this study, we investigated the ability of a subcutaneous peptide immunization schedule to induce of CD4(+) CD25(+) T regulatory cells. DO11.10 T cell receptor (TCR) transgenic mice on a Rag 2(-/-) background were injected subcutaneously with varied doses of purified ovalbumin (OVA(323-339)) peptide daily for 16 days. While these mice have no CD4(+) CD25(+) T regulatory cells, following this injection schedule up to 30% of the CD4(+) cells were found to express CD25. Real-time quantitative polymerase chain reaction (QPCR) analysis of the induced CD4(+) CD25(+) T cells revealed increased expression of forkhead box P3 (FoxP3), suggesting that these cells may have a regulatory function. Proliferation and suppression assays in vitro utilizing the induced CD4(+) CD25(+) T cells revealed a profound anergic phenotype in addition to potent suppressive capability. Importantly, co-injection of the induced CD4(+) CD25(+) T cells with 5,6-carboxy-succinimidyl-fluorescence-ester (CFSE)-labelled naive CD4(+) T cells (responder cells) into BALB/c recipient mice reduced proliferation and differentiation of the responder cells in response to challenge with OVA(323-339) peptide plus adjuvant. We conclude that repeated subcutaneous exposure to low-dose peptide leads to de novo induction of CD4(+) CD25(+) FoxP3(+) T regulatory cells with potent in vitro and in vivo suppressive capability, thereby suggesting that one mechanism of peptide immunotherapy appears to be induction of CD4(+) CD25(+) Foxp3(+) T regulatory cells.     

Restriction of the CD4+ T-cell receptor repertoire prevents immune pathology in TGF-beta1 knockout mice

Mice with a targeted deletion in TGF-beta1 spontaneously develop CD4+ T-cell-dependent multifocal inflammatory disease and autoimmune pathology. T cells from TGF-beta1-/- mice are strongly activated, but the mechanisms that lead to T-cell activation and organ pathology are not well understood. Recent work shows that TGF-beta1 raises the threshold for signaling through the TCR, suppressing the response of T cells to mitogenic stimuli. This suggests the possibility that CD4+ T cells in TGF-beta1-/- mice become aberrantly activated and cause damage in response to physiologic inputs that ordinarily are not sufficient for cell activation, such as homeostatic MHC-TCR interactions, cytokines, or adhesion molecules. This model predicts that pathology is largely antigen-independent, and that CD4+ T cells, regardless of antigen specificity, will become activated in TGF-beta1-/- mice, with subsequent organ pathology. To test this model, we crossed BALB/c-TGF-beta1-/- mice with the DO11.10 TCR transgenic mouse. To obviate the possible development of nonclonotypic TCRs, we also bred in a deficiency in RAG-1. Cohorts of highly inbred BALB/c background TGF-beta1-/- mice with an increasingly restricted CD4+ T-cell repertoire (TGF-beta1-/- mice; DO11.10-TGF-beta1-/- mice; DO11.10-RAG-1-/-TGF-beta1-/- mice) were then analyzed for inflammatory organ pathology and T-cell activation. The data show that progressively restricting the CD4+ T-cell repertoire improved survival, ameliorated target organ pathology, and reduced T-cell activation to control levels. Therefore, these results find no support for the involvement of atypical T-cell activation pathways in disease in TGF-beta1-/- mice. Rather, T-cell activation and pathology in TGF-beta1-/- mice appear to be functions of typical TCR activation pathways. This supports the hypothesis that immune pathology in TGF-beta1-/- mice is self-antigen triggered.     

Antigen-specific CD4 T cell clonal expansion and differentiation in the aged lymphoid microenvironment. I. The primary T cell response is unaffected

Aging is associated with changes in the immune system that lead to decreased immunity in the elderly. Prior studies from humans and mice have shown that aged T cells exhibit numerous defects, including decreased proliferation following in vitro stimulation, suggesting that intrinsic defects exist within aged T cells, leading to defective T cell activation and clonal expansion. In vivo, however, cellular and soluble factors in the lymphoid microenvironment influence T cell function. To investigate the effects of the aged lymphoid microenvironment on T cell function, we monitored the immune response of CD4 T cells from DO11.10 TCR transgenic mice following adoptive transfer into young and aged hosts. After immunization with specific antigen similar rates of donor DO11.10 T cell division were observed in the two host types. However, at the peak of the response, greater numbers of DO11.10 T cells were found in the aged hosts. Regardless of the age of the host, the donor DO11.10 T cell population differentiated into functional effector cells. Despite the increased CD4 T cell growth in aged hosts, similar numbers of memory DO11.10 T cells were found in young and in aged hosts. As CD4 T cell clonal expansion and differentiation is not impaired in the aged microenvironment, our data suggest that diminished T cell immunity during aging is largely due to intrinsic T cell defects, rather than to extrinsic influences associated with the aged lymphoid microenvironment.     

Development of T cells expressing an altered TCR complex

Normal mouse T cells may express alternative TCR complexes containing the FcepsilonR gamma chain (FcRgamma) rather than the zeta homodimer that is present in conventional TCR complexes. While these T cells could play critical roles in regulating immunity, the role of alternative TCR complexes and their requirement for signaling molecules in T cell development remains unknown. We show thatexpression of an FcRgamma transgene in zeta chain-deficient mice (FcRgammaTG, zetaKO mice) reduced the percentage and number of CD4(+) T cells present in these animals, when compared to C57BL/6 mice. Further studies of FcRgammaTG, zetaKO mice expressing the DO11.10 TCR (DOTCR) transgene showed that, when compared to mice expressing conventional TCR complexes, the development of CD4(+), DOTCR(+) thymocytes was altered in mice of different MHC backgrounds and required the presence of zeta-associated protein (ZAP)-70 and lck kinases. The CD4(+), DOTCR(+) T cells bearing alternative TCR complexes have impaired Ca(2+) flux and proliferative response to stimulation. Altogether, these results suggest that the altered development of CD4(+) T cells is not due to qualitative differences in TCR-mediated signals, but more consistent with the hypothesis that it is due to reduced signaling strength mediated through the FcRgamma chain containing only one immunoreceptor tyrosine-based activation motif.