Martie C. M. Verschuren

T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation

T cell differentiation in the thymus is characterized by a hierarchical order of rearrangement steps in the T cell receptor (TCR) genes, resulting in the joining of V, D, and J gene segments. During each of the rearrangement steps, DNA fragments between rearranging V, D, and J gene segments are deleted as circular excision products, the so-called TRECs (T cell receptor excision circles). TRECs are assumed to have a high over-time stability, but they can not multiply and consequently are diluted during T cell proliferation. It was recently suggested that quantitative detection of TRECs would allow for direct measurement of thymic output. The δRec-ψJα TREC appears to be the best marker, because the majority of thymocyte expansion occurs before this TREC is formed. However, apart from thymic output several other factors determine the TREC content of a T cell population, such as cell division and cell death. Likewise, the number of TRECs depends not only on thymic output, but also on the longevity of naive T cells. This warrants caution with regard to the interpretation of TREC data as measured in healthy and diseased individuals. δRec-ψJα TREC detection is a new and elegant tool for identification of recent thymic emigrants in the periphery, but further research is required for making quantitative estimations of thymic output with the use of TREC analysis.

Early stages in the development of human T, natural killer and thymic dendritic cells

Summary: T‐cell development is initiated when CD34+ pluripotent stem cells or their immediate progeny leave the bone marrow Co migrate to the thymus. Upon arrival in the thymus the stem cell progeny is not yet committed to the T‐cell lineage as it has the capability to develop into T, natural killer (NK) and dendritic cells (DC). Primitive hematopoietic progenitor cells in the human thymus express CD34 and lack CD la. When these progenitor cells develop into T cells they traverse a number of checkpoints. One early checkpoint is the induction of T‐cell commitment, which correlates with appearance of CD la and involves the loss of capacity to develop into NK cells and DC and the initiation of T‐cell receptor (TCR) gene rearrangements, Basic helix‐loop‐helix transcription factors play a role in induction of T‐cell commitment. CDla+CD34+ cells develop into CD4+CD8α+β+ cells by upregulating first CD4, followed by CD8α and then CD8β. Selection for productive TCRβ gene rearrangements (β selection) likely occurs in the CD4+CD8α+β‐ and CD4+CD8α+β+ populations. Although the T and NK‐cell lineages arc closely related to each other, NK cells can develop independently of the thymus. The fetal thymus is most likely one site of NK‐cell development.

Disruption of αβ but not of γδ T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors

Enforced expression of Id3, which has the capacity to inhibit many basic helix–loop–helix (bHLH) transcription factors, in human CD34+ hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRαβ and γδ cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre‐T cells), inhibits development into TCRαβ but not into TCRγδ T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre‐Tα mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre‐T cells into TCRαβ and γδ cells. We also observed that cell surface CD4−CD8−CD3− cells with rearranged TCR genes developed from Id3‐transduced but not from control‐transduced pre‐T cells in an FTOC. These cells had properties of both natural killer (NK) and pre‐T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint.

Antigen receptors on T and B lymphocytes: parallels in organization and function.

Membrane-bound antigen receptors are pivotal in the regulation of lymphocyte survival, growth and differentiation. They already come into play in precursor T and B cells, when receptor gene rearrangements are incomplete and have allowed only the synthesis of the T-cell receptor (TCR) p chain or the immunoglobulin (Ig) ft heavy (H) chain. Throughout development, receptor-mediated signals are thought to regulate gene rearrangement and rescue lymphocytes from programmed cell death (positive selection). In addition, self-reactive receptors may give signals resulting in cell death or non-responsiveness (negative selection). Upon recognition of exogenous antigen, T cells differentiate into cytotoxic or helper cells that maintain a membrane-bound receptor, while B lymphocytes differentiate into plasma cells that secrete their antigen receptors. In mature T cells, antigen recognition by the TCR triggers the effector function. In contrast, signal transduction leading to the cellular effector function of Ig is mediated by Fc receptors (FcR) on, e.g., natural killer (NK) cells, to which secreted Ig molecules bind. Given the parallels in the early stages of Tand B-cell development, it is not surprising that membrane-bound antigen receptors of T and B cells are similarly

Psoriatic lesional skin exhibits an aberrant expression pattern of interferon regulatory factor-2 (IRF-2).

Psoriasis is a T‐cell‐mediated inflammatory skin disease. A Th1 cytokine profile with increased levels of interferon‐gamma (IFN‐γ) is predominant in skin and peripheral blood mononuclear cells (PBMCs) from psoriasis patients. Furthermore, psoriatic keratinocytes exhibit an aberrant sensitivity and response to IFN‐γ. The transcriptional activator interferon regulatory factor‐1 (IRF‐1) plays a crucial role in the activation of IFN‐γ‐induced gene expression. Recently it was shown that mice deficient in IRF‐2, a transcriptional repressor of IFN signalling and thereby acting as an IRF‐1 antagonist, display psoriasis‐like skin abnormalities. It was therefore hypothesized that a dysbalance between IRF‐1 and IRF‐2, the activator and repressor of IFN responses, respectively, contributes to the altered IFN‐γ signalling observed in patients with psoriasis. In the epidermis of patients with psoriasis and healthy controls, similar IRF‐1 and IRF‐2 mRNA expression levels were observed. Furthermore, it was not possible to detect any differences in IRF‐1 and IRF‐2 protein levels in nuclear extracts from the epidermis of controls and psoriasis patients by electrophoretic mobility shift assay and western blot analysis. Using double immunofluorescence labelling, it was observed that in normal skin IRF‐1 was expressed in keratinocytes throughout the epidermis, whereas IRF‐2 was restricted to the basal cell layer. In psoriatic skin, IRF‐1 expression was comparable to normal skin, whereas IRF‐2 was expressed in both basal and suprabasal cell layers. This altered IRF‐2 expression in suprabasal cell layers may therefore result in a dysbalance between the activator and repressor of IFN responses in these cell layers, putatively contributing to aberrant responses to IFN‐γ and eventually to the psoriatic skin phenotype. Copyright

Identification of a new cluster of T-cell receptor delta recombining elements.

Within the human T‐cell receptor δ (TCRD) gene we have identified a new cluster of seven δ recombining elements (δRec2.1–2.7), located 2·6–5·2 kilobases downstream of the Vδ2 gene segment. The δRec2 elements are isolated recombining signal sequences (RSS), which were shown to rearrange with the Dδ3 and Jδ1 segments of the TCRD gene as well as with the ψJα of the TCRA gene. Rearrangements involving the δRec2 elements were found in all peripheral blood (PB) samples from 10 healthy individuals, although their frequency was about 100‐fold lower than that of classical δRec rearrangements. The total frequency of δRec2 rearrangements was lower in PB T lymphocytes, as compared with thymocytes, suggesting that they are deleted during T‐cell development. The decrease of the frequency of the δRec2‐Dδ3 rearrangements was most prominent: 11 times lower in PB T lymphocytes than in thymocytes. Since the δRec2‐Jδ1 rearrangements contained the Dδ3 segment in the junctional region, we assume that they are derived from the δRec2‐Dδ3 rearrangements. In contrast, the majority of δRec2‐ψJα rearrangements did not contain the Dδ3 segment, indicating that they are single step rearrangements. The δRec2‐Jδ1 and δRec2‐ψJα rearrangements seem to be T‐lineage specific, but the δRec2‐Dδ3 rearrangements were also found at very low frequencies in B lymphocytes and natural killer cells. Our results suggest that δRec2 rearrangements are transient steps in the recombinatorial process of the TCRAD locus and are probably deleted by subsequent Vα‐Jα rearrangements. We hypothesize, that in a similar manner to the classical δRec rearrangements, the δRec2 rearrangements might also contribute to T‐cell differentiation towards the TCR‐αβ lineage.