[show abstract][hide abstract] ABSTRACT: Immune homeostasis is dependent on tight control over the size of a population of regulatory T (T(reg)) cells capable of suppressing over-exuberant immune responses. The T(reg) cell subset is comprised of cells that commit to the T(reg) lineage by upregulating the transcription factor Foxp3 either in the thymus (tT(reg)) or in the periphery (iT(reg)). Considering a central role for Foxp3 in T(reg) cell differentiation and function, we proposed that conserved non-coding DNA sequence (CNS) elements at the Foxp3 locus encode information defining the size, composition and stability of the T(reg) cell population. Here we describe the function of three Foxp3 CNS elements (CNS1-3) in T(reg) cell fate determination in mice. The pioneer element CNS3, which acts to potently increase the frequency of T(reg) cells generated in the thymus and the periphery, binds c-Rel in in vitro assays. In contrast, CNS1, which contains a TGF-beta-NFAT response element, is superfluous for tT(reg) cell differentiation, but has a prominent role in iT(reg) cell generation in gut-associated lymphoid tissues. CNS2, although dispensable for Foxp3 induction, is required for Foxp3 expression in the progeny of dividing T(reg) cells. Foxp3 binds to CNS2 in a Cbf-beta-Runx1 and CpG DNA demethylation-dependent manner, suggesting that Foxp3 recruitment to this 'cellular memory module' facilitates the heritable maintenance of the active state of the Foxp3 locus and, therefore, T(reg) lineage stability. Together, our studies demonstrate that the composition, size and maintenance of the T(reg) cell population are controlled by Foxp3 CNS elements engaged in response to distinct cell-extrinsic or -intrinsic cues.
[show abstract][hide abstract] ABSTRACT: Because the deletion of self-reactive T cells is incomplete, thymic development of natural Foxp3+CD4+ regulatory T cells (Treg cells) is required for preventing autoimmunity. However, the function of T cell antigen receptor (TCR) specificity in thymic Treg cell development remains controversial. To address this issue, we generated a transgenic line expressing a naturally occurring Treg cell-derived TCR. Unexpectedly, we found that efficient thymic Treg cell development occurred only when the antigen-specific Treg cell precursors were present at low clonal frequency (o1%) in a normal thymus. Using retroviral vectors and bone marrow chimeras, we observed similar activity with two other Treg cell-derived TCRs. Our data demonstrate that thymic Treg cell development is a 'TCR-instructive' process involving a niche that can be saturable at much lower clonal frequencies than is the niche for positive selection.
[show abstract][hide abstract] ABSTRACT: The CD4 and CD8 coreceptor molecules on immature thymocytes participate in T cell repertoire selection. To examine more definitively the role of CD4 and CD8 in the negative and positive selection of immature thymocytes, we generated transgenic mice with elevated surface CD4 expression and mated them with mice expressing a transgenic T cell receptor. Augmented CD4 expression was found to markedly alter CD8-dependent negative and positive selection of T cells specific for the male (H-Y) antigen presented by H-2Db major histocompatibility complex class I molecules. Moreover, the cytoplasmic tail of CD4 was essential for effecting these alterations, since the overexpression of tailless CD4 molecules failed to influence the outcome of CD8-dependent selection. The inhibition of positive and negative selection in double-transgenic mice expressing the full-length CD4 molecule was associated with a decreased interaction between the protein tyrosine kinase p56lck and CD8. These results strongly implicate p56lck in T cell repertoire selection.
European Journal of Immunology 11/2005; 22(3):735 - 743. · 4.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: The class II-associated invariant chain peptide (CLIP) region of the invariant chain (Ii) directly influences MHC class II presentation by occupying the MHC class II peptide-binding groove, thereby preventing premature loading of peptides. Different MHC class II alleles exhibit distinct affinities for CLIP, and a low affinity interaction has been associated with decreased dependence upon H-2M and increased susceptibility to rheumatoid arthritis, suggesting that decreased CLIP affinity alters the MHC class II-bound peptide repertoire, thereby promoting autoimmunity. To examine the role of CLIP affinity in determining the MHC class II peptide repertoire, we generated transgenic mice expressing either wild-type human Ii or human Ii containing a CLIP region of low affinity for MHC class II. Our data indicate that although degradation intermediates of Ii containing a CLIP region with decreased affinity for MHC class II do not remain associated with I-A(b), this does not substantially alter the peptide repertoire bound by MHC class II or increase autoimmune susceptibility in the mice. This implies that the affinity of the CLIP:MHC class II interaction is not a strong contributory factor in determining the probability of developing autoimmunity. In contrast, in the absence of H-2M, MHC class II peptide repertoire diversity is enhanced by decreasing the affinity of CLIP for MHC class II, although MHC class II cell surface expression is reduced. Thus, we show clearly, in vivo, the critical chaperone function of H-2M, which preserves MHC class II molecules for high affinity peptide binding upon dissociation of Ii degradation intermediates.
The Journal of Immunology 05/2004; 172(7):4142-50. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. However, ECM components can also serve as an integral part of the innate immunity. Mice lacking expression of mindin (spondin 2), a highly conserved ECM protein, have an impaired ability to clear bacterial infection, and mindin-deficient macrophages show defective responses to a broad spectrum of microbial stimuli. Moreover, mindin binds directly to bacteria and their components and functions as an opsonin for macrophage phagocytosis of bacteria. Thus, mindin is essential in the initiation of the innate immune response and represents a unique pattern-recognition molecule in the ECM for microbial pathogens.
[show abstract][hide abstract] ABSTRACT: T helper (Th) cell activation, differentiation, and immune function are regulated by costimulatory molecules. Inducible costimulator (ICOS) is a recently identified costimulatory receptor expressed on activated T cells. A ligand for ICOS, B7h, is expressed on B cells and other types of antigen-presenting cells (APC). Although ICOS has been shown to be essential in T cell activation and differentiation, the regulatory roles of B7h at different stages of T cell immune responses have not been examined genetically. In this study, we generated and analyzed B7h-deficient mice. We present evidence that B7h is the only ligand for ICOS, and ICOS, its only corresponding receptor. Th cells, when activated with B7h-deficient APC, exhibited reduced proliferation and IL-2 production. In addition, Th cells produced significantly reduced amounts of IL-4 and -13 after differentiation at the presence of B7h-/- APC. This cytokine defect was associated with a deficiency in c-Maf expression and could be rescued completely by c-Maf overexpression in T cells. Furthermore, we showed that effector T cells, when restimulated in the presence of B7h-deficient APC, exhibited reduced Th2 cytokine production. Therefore, B7h is required for proper Th cell activation, differentiation, and effector cytokine expression.
Proceedings of the National Academy of Sciences 12/2003; 100(24):14163-8. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cathepsin S (catS) and cathepsin L (catL) mediate late stages of invariant chain (Ii) degradation in discrete antigen-presenting cell types. Macrophages (Mphis) are unique in that they express both proteases and here we sought to determine the relative contribution of each enzyme. We observe that catL plays no significant role in Ii cleavage in interferon (IFN)-gamma-stimulated Mphis. In addition, our studies show that the level of catL activity is significantly decreased in Mphis cultured in the presence of IFN-gamma whereas catS activity increases. The decrease in catL activity upon cytokine treatment occurs despite the persistence of high levels of mature catL protein, suggesting that a specific inhibitor of the enzyme is up-regulated in IFN-gamma-stimulated peritoneal Mphis. Similar inhibition of activity is observed in dendritic cells engineered to overexpress catL. Such enzymatic inhibition in Mphis exhibits only partial dependence upon Ii and therefore, other mechanisms of catL inhibition are regulated by IFN-gamma. Thus, during a T helper cell type 1 immune response catL inhibition in Mphis results in preferential usage of catS, such that major histocompatibility complex class II presentation by all bone marrow-derived antigen-presenting cell is regulated by catS.
Journal of Experimental Medicine 02/2003; 197(2):169-79. · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: CD1d antigen presentation to natural killer T (NKT) cells expressing the semi-invariant T cell receptor V(alpha)14J(alpha)18 requires CD1d trafficking through endosomal compartments; however, the endosomal events remain undefined. We show that mice lacking the endosomal protease cathepsin L (catL) have greatly reduced numbers of V(alpha)14(+)NK1.1(+) T cells. In addition, catL expression in thymocytes is critical not only for selection of these cells in vivo but also for stimulation of V(alpha)14(+)NK1.1(+) T cells in vitro. CD1d cell-surface expression and intracellular localization appear normal in catL-deficient thymocytes, as does the lysosomal morphology; this implies a specific role for catL in regulating presentation of natural CD1d ligands mediating V(alpha)14(+)NK1.1(+) T cell selection. These data implicate lysosomal proteases as key regulators of not only classical major histocompatibility complex class II antigen presentation but also nonclassical CD1d presentation.
[show abstract][hide abstract] ABSTRACT: If T cells require specific interactions with MHC-bound peptides during positive selection, then the specificities of T cells selected by one peptide should be distinct from those selected by another. We have examined positive selection of CD4 T cells in four strains of mice, each overexpressing a different peptide-1-A(b)(A(b)) complex. We show that a subset of CD4 T cells is selected by the overexpressed peptide and that the specificities of the CD4 T cells, as measured by reactivity to wild-type antigen-presenting cells, vary greatly depending on which peptide is overexpressed. These differences in specificity are mediated through positive selection not negative selection. Each of the four peptide-A(b) complexes appears to adopt a different conformation, and these differences correlate with the differences in reactivity. Our results suggest that individual peptide-MHC complexes positively select different subsets of self-MHC-reactive T cells and that the conformation of the peptide-MHC complex may contribute to this process.
Proceedings of the National Academy of Sciences 06/2002; 99(10):6937-42. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have identified a novel LIM gene encoding the thymus LIM protein (TLP), expressed specifically in the thymus in a subset of cortical epithelial cells. TLP was identified as a gene product which is upregulated in a thymus in which selection of T cells is occurring (Rag(-/-) OT-1) compared to its expression in a thymus in which selection is blocked at the CD4+ CD8+ stage of T-cell development (Rag(-/-) Tap(-/-) OT-1). TLP has an apparent molecular mass of 23 kDa and exists as two isomers (TLP-A and TLP-B), which are generated by alternative splicing of the message. The sequences of TLP-A and TLP-B are identical except for the C-terminal 19 or 20 amino acids. Based on protein sequence alignment, TLP is most closely related to the cysteine-rich proteins, a subclass of the family of LIM-only proteins. In both medullary and cortical thymic epithelial cell lines transduced with TLP, the protein localizes to the cytoplasm but does not appear to be strongly associated with actin. In immunohistochemical studies, TLP seems to be localized in a subset of epithelial cells in the cortex and is most abundant near the corticomedullary junction. We generated mice with a targeted disruption of the Tlp locus. In the absence of TLP, thymocyte development and thymus architecture appear to be normal but thymocyte cellularity is reduced by approximately 30%, with a proportional reduction in each subpopulation.
Molecular and Cellular Biology 01/2002; 21(24):8592-604. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Intrathymic self-peptide-major histocompatibility complex class II (MHC) molecules shape the T cell repertoire through positive and negative selection of immature CD4(+)CD8(+) thymocytes. By analyzing the development of MHC class II-restricted T cell receptor (TCR) transgenic T cells under conditions in which the endogenous peptide repertoire is altered, we show that self-peptide-MHC complexes are also involved in setting T cell activation thresholds. This occurs through changes in the expression level of molecules on thymocytes that influence the sensitivity of TCR signaling. Our results suggest that the endogenous peptide repertoire modulates T cell responsiveness in the thymus in order to enforce tolerance to self-antigens.
Journal of Experimental Medicine 06/2001; 193(10):1179-87. · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: p56(lck) is a protein tyrosine kinase expressed throughout T cell development. It associates noncovalently with the cytoplasmic domains of the CD4 and CD8 coreceptor molecules and has been implicated in TCR signaling in mature T cells. Its role in early thymocyte differentiation has been demonstrated in vivo, both by targeted gene disruption and by transgene expression. Previously, we showed that expression of a dominant-negative form of p56(lck) in double-positive thymocytes inhibits positive selection. We now demonstrate that expression of constitutively activated p56(lck) (p56(lck)F505) accelerates the transition from the double-positive to the single-positive stage. Importantly, p56(lck)F505 drives survival and lineage commitment of thymocytes in the absence of TCR engagement by appropriate MHC molecules. These results indicate that activation of p56(lck) constitutes an early step in conveying maturational signals after TCR ligation by a positively selecting ligand. Our study provides direct in vivo evidence for the role of p56(lck) in regulating TCR signaling.
The Journal of Immunology 03/2001; 166(4):2209-17. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Considerable evidence supports a role for the Src family protein tyrosine kinase Lck in regulating multiple aspects of thymocyte development. In this report, we establish that early events in T lymphopoiesis are restored to Lck-deficient mice by provision of a transgene encoding a version of Lck that cannot interact with the coreceptors CD4 and CD8. In addition, we demonstrate that later events in thymocyte development, specifically, the processes of positive and negative selection, are compromised in mice where the only Lck available cannot associate with either CD4 or CD8. We conclude that not only is Lck activity required for positive and negative selection, but that that activity must be coupled to the CD4 and CD8 coreceptors.
The Journal of Immunology 02/2001; 166(2):809-18. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Notch proteins regulate many developmental processes. Notch1 is highly expressed on thymocytes, but its role in regulating their development is not known. We show that activation of Notch1 signaling in CD4+CD8+ double positive thymocytes promotes the maturation of both CD4+ and CD8+ single positive thymocytes and that this occurs in the absence of interactions between the T cell receptor and MHC molecules expressed on thymic epithelial cells. We have also identified several genes that are transcriptionally regulated by Notch1 in T cells and show that they are upregulated during maturation into both single positive lineages. These observations suggest that Notch1 signaling plays a role in promoting maturation into both the CD4 and CD8 T cell lineages.
[show abstract][hide abstract] ABSTRACT: Thymocyte development is a tightly regulated process. CD4+CD8+ double-positive (DP) immature thymocytes exhibit distinct phenotypic features from mature T cells; they express only 10% of surface TCR that are found on mature T cells and do not proliferate and produce IL-2 in response to stimulation. In this report we show that transgenic expression of the orphan nuclear receptor ROR gamma t in mature T cells down-regulates their surface TCR expression. The ROR gamma t transgene inhibits IL-2 production by mature T cells, and this inhibition may be partially due to the inhibitory effect of ROR gamma t on c-Rel transcription. Furthermore, ectopic expression of ROR gamma t inhibits the proliferation of mature and immature T cells. These results, together with its predominant expression in DP thymocytes, suggest that ROR gamma t controls these distinct phenotypic features of DP thymocytes. Our data suggest that down-regulation of ROR gamma t expression in thymocytes is essential for their maturation.
The Journal of Immunology 07/2000; 164(11):5668-74. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Thymocyte development is a tightly regulated process. CD4+CD8+ double-positive (DP) immature thymocytes exhibit distinct phenotypic features from mature T cells; they express only 10% of surface TCR that are found on mature T cells and do not proliferate and produce IL-2 in response to stimulation. In this report we show that transgenic expression of the orphan nuclear receptor RORγt in mature T cells down-regulates their surface TCR expression. The RORγt transgene inhibits IL-2 production by mature T cells, and this inhibition may be partially due to the inhibitory effect of RORγt on c-Rel transcription. Furthermore, ectopic expression of RORγt inhibits the proliferation of mature and immature T cells. These results, together with its predominant expression in DP thymocytes, suggest that RORγt controls these distinct phenotypic features of DP thymocytes. Our data suggest that down-regulation of RORγt expression in thymocytes is essential for their maturation.
The Journal of Immunology 06/2000; 164(11):5668-5674. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Caspases, a family of cysteine proteases, are critical mediators of apoptosis. To address the importance of caspases in thymocyte development, we have generated transgenic mice that express the baculovirus protein p35, a viral caspase inhibitor, specifically in the thymus. p35 expression inhibited Fas (CD95)-, CD3-, or peptide-induced caspase activity in vitro and conferred resistance to Fas-induced apoptosis. However, p35 did not block specific peptide-induced negative selection in OT1 and HY TCR transgenic mouse models. Even the potent pharmacological caspase inhibitor zVAD-FMK (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone) could not prevent peptide-induced deletion of OT1 thymocytes, although it improved basal thymocyte survival in vitro. Moreover, the developmental block observed in rag1-/- thymocytes, which lack pre-TCR signaling, was also not rescued by p35 expression. These results indicate that caspase-independent signal transduction pathways can mediate thymocyte death during normal T cell development.
The Journal of Immunology 05/2000; 164(8):4071-9. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Successful in-frame rearrangement of immunoglobulin heavy chain genes or T cell antigen receptor (TCR) beta chain genes in lymphocyte progenitors results in formation of pre-BCR and pre-TCR complexes. These complexes signal progenitor cells to mature, expand in cell number, and suppress further rearrangements at the immunoglobulin heavy chain or TCRbeta chain loci, thereby ensuring allelic exclusion. We used transgenic expression of a constitutively active form of c-Raf-1 (Raf-CAAX) to demonstrate that activation of the Map kinase pathway can stimulate both maturation and expansion of B and T lymphocytes, even in the absence of pre-TCR or pre-BCR formation. However, the same Raf signal did not mediate allelic exclusion. We conclude that maturation of lymphocyte progenitors and allelic exclusion require distinct signals.
[show abstract][hide abstract] ABSTRACT: The stress-activated protein kinases (SAPK) are a group of dual-specificity kinases with potential roles in the control of apoptosis and proliferation. In most cells they are regulated through phosphorylation by MKK-4. We have investigated the role of MKK-4 in T cell development and function by generating transgenic animals expressing catalytically inactive MKK-4 (dMKK-4) in the thymus. Our results show that overexpression of dMKK-4 does not interfere with normal T cell development. Furthermore, expression of dMKK-4 inhibits Fas- but not phorbol ester plus ionomycin-induced activation of SAPK, suggesting that a SAPK kinase different from MKK-4 is responsible for the regulation of SAPK activation after stimulation of T cells with phorbol ester plus ionomycin. We then analyzed the effect of dMKK-4 on Fas-induced apoptosis of thymocytes. Our results show that activation of SAPK is not a necessary event in Fas-induced apoptosis of thymocytes.
International Immunology 09/1998; 10(8):1077-82. · 3.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: The tyrosine kinase Jak3 plays a key role in transducing signals from the IL-2, -4, -7, -9, and -15 receptors. Mice lacking Jak3 exhibit a profound, early block in both B and T cell development. To examine the mechanisms whereby Jak3 influences T cell function, we have reconstituted thymic development in Jak3-/- animals by introducing a Jak3 transgene in which expression was driven by the lck proximal promoter. Thymic reconstitution required Jak3 kinase activity, as catalytically inactive Jak3 did not restore early thymic development. Furthermore, the thymus-restricted expression pattern of the transgene allowed us to assess the requirement for Jak3 in peripheral T cells. In these mice, loss of Jak3 expression was associated with a failure to proliferate in response to antigen receptor crosslinking, the accumulation of T cells manifesting an activated cell surface phenotype, and an increased CD4/CD8 ratio among peripheral T cells, all of which are characteristics that were observed in Jak3-/- animals. Finally, we present data which suggest that peripheral T cells proliferate more rapidly in vivo and also undergo apoptosis more rapidly, upon loss of Jak3. Hence Jak3 exerts effects on mature peripheral T lymphocytes, as well as on thymocytes, resulting in the proper maintenance of circulating, quiescent cells.
The Journal of Immunology 03/1998; 160(5):2130-8. · 5.52 Impact Factor