Massive clonal expansion of polycytotoxic skin and blood CD8+ T cells in patients with toxic epidermal necrolysis.

Toxic epidermal necrolysis (TEN) is a life-threatening cutaneous adverse drug reaction. To better understand why skin symptoms are so severe, we conducted a prospective immunophenotyping study on skin and blood. Mass cytometry results confirmed that effector memory polycytotoxic CD8+ T cells (CTLs) are the main leucocytes in TEN blisters at the acute phase. Deep T cell receptor (TCR) repertoire sequencing identified massive expansion of unique CDR3 clonotypes in blister cells. The same clones were highly expanded in patient's blood, and the degree of their expansion showed significant correlation with disease severity. By transducing α and ß chains of the expanded clonotypes into a TCR-defective cell line, we confirmed that those cells were drug specific. Collectively, these results suggest that the relative clonal expansion and phenotype of skin-recruited CTLs condition the clinical presentation of cutaneous adverse drug reactions.

T cell receptor gene rearrangement lineage analysis reveals clues for the origin of highly restricted antigen-specific repertoires.

Due to ordered, stage-specific T cell receptor (TCR)-beta and -alpha locus gene rearrangements and cell division during T cell development, a given, ancestral TCR-beta locus VDJ rearrangement might be selected into the mature T cell repertoire as a small cohort of "half-sibling" progeny expressing identical TCR-beta chains paired with different TCR-alpha chains. The low frequency of such a cohort relative to the total alphabeta TCR repertoire precludes their direct identification and characterization in normal mice. We considered it possible that positive selection constraints might limit the diversity of TCR-alpha chains selected to pair with beta chains encoded by an ancestral VDJ-beta rearrangement. If so, half-sibling T cells expressing structurally similar, but different TCR-alpha chains might recognize the same foreign antigen. By single cell polymerase chain reaction analysis of antigen-specific TCRs selected during a model anti-tumor response, we were able to identify clusters of T cells sharing identical VDJ-beta rearrangements but expressing different TCR-alpha chains. The amplification of residual DJ-beta rearrangements as clonal markers allowed us to track T cells expressing different TCR-alpha chains back to a common ancestral VDJ-beta rearrangement. Thus, the diversity of TCR-alpha's selected as partners for a given VDJ-beta rearrangement into the mature TCR repertoire may indeed be very limited.

CD1c-Related DCs that Express CD207/Langerin, but Are Distinguishable from Langerhans Cells, Are Consistently Present in Human Tonsils.

Several subsets of dendritic cells (DCs) are present in the oropharyngeal tonsillar tissues and are thought to behave as major actors in development and regulation of immunity by acting as a first line of recognition for airborne and alimentary antigens. We previously discovered in human adult tonsils infected with Epstein-Barr virus (EBV), a subset of DCs that expressed langerin/CD207, a lectin usually recognized as a hallmark of epidermal Langerhans cells (LCs). In the present study, we analyzed the content of several child and adult tonsils in order to characterize in more detail the phenotype of these tonsillar CD207-expressing DCs (tCD207 DCs) and to compare it with that of other human DC subsets. We showed that all the human tonsils studied (n = 12) contained significant proportions of tCD207 DCs among tonsillar cells expressing HLA-DR. Moreover, the presence of tCD207 DCs in tonsils from young children free of EBV infection indicated that these cells could be established early in the tonsil independently of EBV infection. We also showed that tCD207 DCs, that were found mainly located within the tonsillar lymphoid stroma, were distinguishable from LCs by the level of expression of CD1a and EpCAM, and also from human inflammatory DCs by the lack of CD1a, CD206, and CD14 expression. Detailed analysis of cell surface DC markers showed that tCD207 DCs were unrelated to CD141(+) DCs or macrophages, but defined a subtype of tonsillar DCs closely related to myeloid resident CD1c DCs. Since it was established that blood CD1c myeloid DCs exhibit plasticity and are capable of expressing CD207 notably in the presence of inflammatory cytokines, it is tempting to speculate that CD207(+) CD1c(+) DCs may play a specific immune role.

Multiple T-cell clones specific for the same foreign pMHC ligand can be generated from a single, ancestral TCR-VDJbeta precursor.

Owing to ordered, stage-specific T-cell receptor (TCR) gene rearrangements and cell division during T-cell development, small cohorts of "half-sibling" T cells sharing an ancestral TCR VDJbeta rearrangement but expressing different TCR alpha-locus rearrangements may be selected into the mature T-cell repertoire. We wondered whether different alphabetaTCRs expressed by T cells from the same ancestral VDJbeta cohort might be capable of recognizing the same foreign peptide-major histocompatibility complex complex (pMHC). By a combined flow cytometric and single-cell polymerase chain reaction (PCR) approach to analyze TCRs selected by the previously defined foreign antigen, pCW3170-179/H-2Kd, we were able to identify cohorts of half-sibling antigen-specific CD8 T cells after their expansion in immunized mice. We amplified residual DJbeta rearrangements as clonal markers to confirm that the shared VDJbeta sequences represent ancestral rearrangements rather than identical but independent ones. An intriguing explanation of our findings would be that only a very limited repertoire of TCR alpha-chains is selected to pair with a given TCR beta-chain during T-cell development.

EBV infection is common in gingival epithelial cells of the periodontium and worsens during chronic periodontitis.

An amplifying role for oral epithelial cells (ECs) in Epstein-Barr Virus (EBV) infection has been postulated to explain oral viral shedding. However, while lytic or latent EBV infections of oro/nasopharyngeal ECs are commonly detected under pathological conditions, detection of EBV-infected ECs in healthy conditions is very rare. In this study, a simple non-surgical tissue sampling procedure was used to investigate EBV infection in the periodontal epithelium that surrounds and attaches teeth to the gingiva. Surprisingly, we observed that the gingival ECs of the periodontium (pECs) are commonly infected with EBV and may serve as an important oral reservoir of latently EBV-infected cells. We also found that the basal level of epithelial EBV-infection is significantly increased in chronic periodontitis, a common inflammatory disease that undermines the integrity of tooth-supporting tissues. Moreover, the level of EBV infection was found to correlate with disease severity. In inflamed tissues, EBV-infected pECs appear to be prone to apoptosis and to produce larger amounts of CCL20, a pivotal inflammatory chemokine that controls tissue infiltration by immune cells. Our discovery that the periodontal epithelium is a major site of latent EBV infection sheds a new light on EBV persistence in healthy carriers and on the role of this ubiquitous virus in periodontitis. Moreover, the identification of this easily accessible site of latent infection may encourage new approaches to investigate and monitor other EBV-associated disorders.

A natural structural variant of the mouse TCR beta-chain displays intrinsic receptor function and antigen specificity.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (<35%) of TCRbeta-Cbeta0 expression among CD8 T cells selected by Ag in vivo. To study the potential activity of the TCRbeta-Cbeta0 splice variant, we cloned full-length TCR cDNAs by single-cell RT-PCR into retroviral expression vectors. We found that the TCRbeta-Cbeta0 splice isoform can function during an early stage of T cell development normally dependent on TCR beta-chain expression. We also demonstrate that T hybridoma-derived cells expressing a TCRbeta-Cbeta0 isoform together with the clonally associated TCR alpha-chain recognize the same cognate peptide-MHC ligand as the corresponding normal alphabetaTCR. This maintenance of receptor function and specificity upon insertion of the Cbeta0 peptide cassette signifies a remarkable adaptability for the TCR beta-chain, and our findings open the possibility that this splice isoform may function in vivo.

Obligatory role for cooperative signaling by pre-TCR and Notch during thymocyte differentiation.

The first checkpoint during T cell development, known as beta selection, requires the successful rearrangement of the TCR-beta gene locus. Notch signaling has been implicated in various stages during T lymphopoiesis. However, it is unclear whether Notch receptor-ligand interactions are necessary during beta selection. Here, we show that pre-TCR signaling concurrent with Notch receptor and Delta-like-1 ligand interactions are required for the survival, proliferation, and differentiation of mouse CD4(-)CD8(-) thymocytes to the CD4(+)CD8(+) stage. Furthermore, we address the minimal signaling requirements underlying beta selection and show a hierarchical positioning of key proximal signaling molecules. Collectively, our results demonstrate an essential role for Notch receptor-ligand interactions in enabling the autonomous signaling capacity of the pre-TCR complex.