Thymocytes can tolerize thymocytes by clonal deletion in vitro

Clonal deletion of thymocytes bearing TCR for self antigens is one major mechanism of T cell tolerance induction. Peptide antigen-induced deletion of thymocytes from αβ TCR transgenic mice has been studied using single cell suspension cultures. The results show that antigen-presenting immature CD4+CD8+ thymocytes can tolerize antigen-reactive immature thymocytes in vitro by programmed cell death (apoptosis) 6-8 h after antigen exposure. Antigen-induced apoptosis of immature thymocytes was inhibited by antibodies specific for the αβ TCR, CD3, CD8, and LFA-1 molecules. This implies that clonal elimination of self-reactive CD4+CD8+ thymocytes does not depend on specialized deleting cell types in the thymus and occurs whenever the TCR of immature thymocytes bind antigen fragments presented by MHC molecule

Dynamic Changes During the Immune Response in T Cell–Antigen-presenting Cell Clusters Isolated from Lymph Nodes

Activation of antigen-specific T cells by mature dendritic cells in secondary lymphoid organs is a key control point of the adaptive immune response. Here we describe the ex vivo isolation of preformed multicellular clusters between T cells and antigen-presenting cells. Adoptively transferred, antigen-specific T cells segregated into individual clusters where their activation and proliferation was initiated in vivo. Transit of the T cell cohort through the cluster compartment required 32–36 h. The precise timing of the response to agonistic epitopes was remarkably invariant regardless of the T cell lineage, the major histocompatibility complex haplotype, and the antigen dose. Interestingly, initiation of cell division of T cells specific for a subdominant epitope and a weak agonist was delayed by 6 h. The results provide a basis for the analysis of short range, mutual cell–cell interactions within such confined microenvironments

Myasthenia gravis thymus: complement vulnerability of epithelial and myoid cells, complement attack on them, and correlations with autoantibody status.

Am J Pathol. 2007 Sep;171(3):893-905. Epub 2007 Aug 3. Myasthenia gravis thymus: complement vulnerability of epithelial and myoid cells, complement attack on them, and correlations with autoantibody status. Leite MI, Jones M, Ströbel P, Marx A, Gold R, Niks E, Verschuuren JJ, Berrih-Aknin S, Scaravilli F, Canelhas A, Morgan BP, Vincent A, Willcox N. Department of Clinical Neurology, University of Oxford, Oxford, United Kingdom. Abstract In early-onset myasthenia gravis, the thymus contains lymph node-type infiltrates with frequent acetylcholine receptor (AChR)-specific germinal centers. Our recent evidence/two-step hypothesis implicates hyperplastic medullary thymic epithelial cells (expressing isolated AChR subunits) in provoking infiltration and thymic myoid cells (with intact AChR) in germinal center formation. To test this, we screened for complement attack in a wide range of typical generalized myasthenia patients. Regardless of the exact serology, thymi with sizeable infiltrates unexpectedly showed patchy up-regulation of both C5a receptor and terminal complement regulator CD59 on hyperplastic epithelial cells. These latter also showed deposits of activated C3b complement component, which appeared even heavier on infiltrating B cells, macrophages, and especially follicular dendritic cells. Myoid cells appeared particularly vulnerable to complement; few expressed the early complement regulators CD55, CD46, or CR1, and none were detectably CD59(+). Indeed, when exposed to infiltrates, and especially to germinal centers, myoid cells frequently labeled for C1q, C3b (25 to 48%), or even the terminal C9, with some showing obvious damage. This early/persistent complement attack on both epithelial and myoid cells strongly supports our hypothesis, especially implicating exposed myoid cells in germinal center formation/autoantibody diversification. Remarkably, the similar changes place many apparent AChR-seronegative patients in the same spectrum as the AChR-seropositive patients. PMID: 17675582 [PubMed - indexed for MEDLINE]PMCID: PMC1959483Free PMC Article Images from this publication.See all images (6) Free text Figure 1 Distribution of complement receptors C3aR, C5aR, and CR1 (receptor for C3b and C4b) (all in red) in epithelial areas and/or infiltrates in thymi from non-MG controls (A and B), AChRAb+ (C–E), or SNMG (F) MG patients. A and B: In control thymi, occasional mTECs are weakly C5aR+, as in some areas in MG thymi, bu... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905.Figure 2 Distribution of complement regulators CD46, CD55, and CD59 (all in red) in epithelial areas and infiltrates in control (A and D) and MG thymi (B, C, and E–I). Cytokeratin (CK, green). A: In controls, CD46 (A) and CD55 (not shown) expression is minimal; in MG, both are much stronger in the MEBs than in the nMe... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905.Figure 3 Labeling for C1q and C3b complement fragments (both in red) in epithelial areas and infiltrates in MG and control thymi. Cytokeratin (CK, green). A and B: In MG, there is extensive patchy labeling for C1q in mTECs and other cells in MEBs and in infiltrates and GC in AChRAb+ (A) or SNMG (B) samples. C: In co... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905.Figure 4 Rarity of complement regulators on myoid cells. In both control (not shown) and MG thymi (A), myoid cells (MC) are uniformly CD59− (red), even when exposed to infiltrates, but ∼5% of the latter express detectable CD55 (red) (B, inset). (Donors both female: A, 20 years of age; B, 16 years of age). Desmin (De, ... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905.Figure 5 Labeling for C1q, C3b, or C9 (all in red) on exposed myoid cells (MC) in MG thymi. Desmin (De, green). A and B: Some exposed myoid cells label for C1q in AChRAb+ (A) or SNMG (B) MG samples, in which many of them label for C3b (C and D; enlarged in insets) and some for C9 in AChRAb+ (E) or SNMG (F) samples. Note aggr... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905.Figure 6 Percentages of myoid cells exposed to the infiltrates in non-MG controls and MG patient subgroups. Their rarity in the control and MuSKAb+ samples reflects the paucity of infiltrates. There were significantly fewer myoid cells/mm2 in the AChRAb+ group than in the controls (see mini-table below; *P < 0.0... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905

A threshold level of NFATc1 activity facilitates thymocyte differentiation and opposes notch-driven leukaemia development.

International audienceNFATc1 plays a critical role in double-negative thymocyte survival and differentiation. However, the signals that regulate Nfatc1 expression are incompletely characterized. Here we show a developmental stage-specific differential expression pattern of Nfatc1 driven by the distal (P1) or proximal (P2) promoters in thymocytes. Whereas, preTCR-negative thymocytes exhibit only P2 promoter-derived Nfatc1beta expression, preTCR-positive thymocytes express both Nfatc1beta and P1 promoter-derived Nfatc1alpha transcripts. Inducing NFATc1alpha activity from P1 promoter in preTCR-negative thymocytes, in addition to the NFATc1beta from P2 promoter impairs thymocyte development resulting in severe T-cell lymphopenia. In addition, we show that NFATc1 activity suppresses the B-lineage potential of immature thymocytes, and consolidates their differentiation to T cells. Further, in the pTCR-positive DN3 cells, a threshold level of NFATc1 activity is vital in facilitating T-cell differentiation and to prevent Notch3-induced T-acute lymphoblastic leukaemia. Altogether, our results show NFATc1 activity is crucial in determining the T-cell fate of thymocytes

The impact of negative selection on thymocyte migration in the medulla

Developing thymocytes are screened for self-reactivity before they exit the thymus, but how thymocytes scan the medulla for self antigens is unclear. Using two-photon microscopy, we observed that medullary thymocytes migrated rapidly and made frequent, transient contacts with dendritic cells. In the presence of a negative selecting ligand, thymocytes slowed, became confined to areas of approximately 30 mum in diameter and had increased contact with dendritic cells surrounding confinement zones. One third of polyclonal medullary thymocytes also showed confined, slower migration and may correspond to autoreactive thymocytes. Our data suggest that many autoreactive thymocytes do not undergo immediate arrest and death after encountering a negative selecting ligand but instead adopt an altered migration program while remaining in the medullary microenvironment

Essential role of CCL21 in establishment of central self-tolerance in T cells.

The chemokine receptor CCR7 directs T cell relocation into and within lymphoid organs, including the migration of developing thymocytes into the thymic medulla. However, how three functional CCR7 ligands in mouse, CCL19, CCL21Ser, and CCL21Leu, divide their roles in immune organs is unclear. By producing mice specifically deficient in CCL21Ser, we show that CCL21Ser is essential for the accumulation of positively selected thymocytes in the thymic medulla. CCL21Ser-deficient mice were impaired in the medullary deletion of self-reactive thymocytes and developed autoimmune dacryoadenitis. T cell accumulation in the lymph nodes was also defective. These results indicate a nonredundant role of CCL21Ser in the establishment of self-tolerance in T cells in the thymic medulla, and reveal a functional inequality among CCR7 ligands in vivo

Identifying Individual T Cell Receptors of Optimal Avidity for Tumor Antigens.

Cytotoxic T cells recognize, via their T cell receptors (TCRs), small antigenic peptides presented by the major histocompatibility complex (pMHC) on the surface of professional antigen-presenting cells and infected or malignant cells. The efficiency of T cell triggering critically depends on TCR binding to cognate pMHC, i.e., the TCR-pMHC structural avidity. The binding and kinetic attributes of this interaction are key parameters for protective T cell-mediated immunity, with stronger TCR-pMHC interactions conferring superior T cell activation and responsiveness than weaker ones. However, high-avidity TCRs are not always available, particularly among self/tumor antigen-specific T cells, most of which are eliminated by central and peripheral deletion mechanisms. Consequently, systematic assessment of T cell avidity can greatly help distinguishing protective from non-protective T cells. Here, we review novel strategies to assess TCR-pMHC interaction kinetics, enabling the identification of the functionally most-relevant T cells. We also discuss the significance of these technologies in determining which cells within a naturally occurring polyclonal tumor-specific T cell response would offer the best clinical benefit for use in adoptive therapies, with or without T cell engineering

Promiscuous gene expression in thymic epithelial cells is regulated at multiple levels

The role of central tolerance induction has recently been revised after the discovery of promiscuous expression of tissue-restricted self-antigens in the thymus. The extent of tissue representation afforded by this mechanism and its cellular and molecular regulation are barely defined. Here we show that medullary thymic epithelial cells (mTECs) are specialized to express a highly diverse set of genes representing essentially all tissues of the body. Most, but not all, of these genes are induced in functionally mature CD80hi mTECs. Although the autoimmune regulator (Aire) is responsible for inducing a large portion of this gene pool, numerous tissue-restricted genes are also up-regulated in mature mTECs in the absence of Aire. Promiscuously expressed genes tend to colocalize in clusters in the genome. Analysis of a particular gene locus revealed expression of clustered genes to be contiguous within such a cluster and to encompass both Aire-dependent and –independent genes. A role for epigenetic regulation is furthermore implied by the selective loss of imprinting of the insulin-like growth factor 2 gene in mTECs. Our data document a remarkable cellular and molecular specialization of the thymic stroma in order to mimic the transcriptome of multiple peripheral tissues and, thus, maximize the scope of central self-tolerance