T cell receptor-mediated signaling events in CD4+CD8+ thymocytes undergoing thymic selection: requirement of calcineurin activation for thymic positive selection but not negative selection.

The goal of this study was to identify the differences of intracellular signals between the processes of thymic positive and negative selection. The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocked the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression were inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation did not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) was not inhibited by in vivo treatment with FK506 and that there was no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. DNA fragmentation induced by TCR activation of DP thymocytes in vitro was not affected by FK506. In addition, different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes.

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A Kinetic Threshold between Negative and Positive Selection Based on the Longevity of the T Cell Receptor–Ligand Complex

Journal of Experimental Medicine ◽

10.1084/jem.189.10.1531 ◽

1999 ◽

Vol 189 (10) ◽

pp. 1531-1544 ◽

Cited By ~ 92

Author(s):

Calvin B. Williams ◽

Deborah L. Engle ◽

Gilbert J. Kersh ◽

J. Michael White ◽

Paul M. Allen

Keyword(s):

T Cells ◽

T Cell ◽

Positive Selection ◽

T Cell Receptor ◽

Cell Receptor ◽

Ligand Complex ◽

Altered Peptide Ligands ◽

Self Peptides ◽

Selection Of

We have developed a unique in vivo system to determine the relationship between endogenous altered peptide ligands and the development of major histocompatibility complex class II– restricted T cells. Our studies use the 3.L2 T cell receptor (TCR) transgenic mouse, in which T cells are specific for Hb(64–76)/I-Ek and positively selected on I-Ek plus self-peptides. To this endogenous peptide repertoire, we have individually added one of six well-characterized 3.L2 ligands. This transgenic approach expands rather than constrains the repertoire of self-peptides. We find that a broad range of ligands produce negative selection of thymocytes in vivo. When compared with the in vitro TCR–ligand binding kinetics, we find that these negatively selecting ligands all have a half-life of 2 s or greater. Additionally, one of two ligands examined with no detectable binding to the 3.L2 TCR and no activity on mature 3.L2 T cells (Q72) enhances the positive selection of transgenic thymocytes in vivo. Together, these data establish a kinetic threshold between negative and positive selection based on the longevity of TCR–ligand complexes.

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FK506 inhibits the differentiation of developing thymocytes but not negative selection of T cell receptor Vβ5+ and Vβ;11+ T lymphocytes in vivo

Transplant Immunology ◽

10.1016/0966-3274(94)90072-8 ◽

1994 ◽

Vol 2 (1) ◽

pp. 11-21 ◽

Cited By ~ 6

Author(s):

Jacky Woo ◽

Suzanne T. Ildstad ◽

Angus W. Thomson

Keyword(s):

T Cell ◽

T Lymphocytes ◽

T Cell Receptor ◽

Negative Selection ◽

Cell Receptor ◽

Selection Of

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Quantitation of the Cell Surface Level of Ld Resulting in Positive Versus Negative Selection of the 2C Transgenic T Cell Receptor In Vivo

Immunity ◽

10.1016/s1074-7613(00)80526-9 ◽

1997 ◽

Vol 7 (2) ◽

pp. 233-241 ◽

Cited By ~ 35

Author(s):

James R Cook ◽

Eva-Marie Wormstall ◽

Tara Hornell ◽

John Russell ◽

Janet M Connolly ◽

...

Keyword(s):

T Cell ◽

Cell Surface ◽

T Cell Receptor ◽

Negative Selection ◽

Cell Receptor ◽

Surface Level ◽

Cell Surface Level ◽

Selection Of

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The T cell receptor repertoire of CD4-8+ thymocytes is altered by overexpression of the BCL-2 protooncogene in the thymus.

Journal of Experimental Medicine ◽

10.1084/jem.179.1.145 ◽

1994 ◽

Vol 179 (1) ◽

pp. 145-153 ◽

Cited By ~ 59

Author(s):

W Tao ◽

S J Teh ◽

I Melhado ◽

F Jirik ◽

S J Korsmeyer ◽

...

Keyword(s):

Cell Death ◽

T Cell ◽

Transgenic Mice ◽

Positive Selection ◽

T Cell Receptor ◽

Negative Selection ◽

Cell Receptor ◽

Target Cells ◽

Cell Death Pathway ◽

Selection Of

The bcl-2 gene encodes an intracellular, membrane-associated protein that protects immature cortical thymocytes from a wide variety of apoptotic stimuli, including glucocorticoids, radiation, and anti-CD3 treatment. Since cortical thymocytes are the primary target cells for thymic positive and negative selection processes, and since these processes are associated with cell death, we evaluated the role of bcl-2 in T cell development in two ways. In the first approach, transgenic mice expressing high levels of Bcl-2 in cortical thymocytes were mated with H-Y T cell receptor (TCR) transgenic mice, the latter being a well-defined system for the study of positive and negative selection of T cells. We found that the bcl-2 transgene had a dramatic effect on positive selection. This was manifested by a greatly increased production of mature thymocytes that were highly skewed towards the CD4-8+ lineage. The change involving CD4-8+ thymocytes occurred not only in bcl-2 transgenic mice, but was also observed in H-Y TCR/bcl-2 doubly transgenic mice, regardless of whether the H-Y TCR was expressed in the selecting (H-2b) or nonselecting (H-2d) environments. Furthermore, a large proportion of CD4-8+ thymocytes produced in H-2b H-Y TCR/bcl-2 doubly transgenic female mice expressed endogenous TCR alpha chains rather than the transgenic TCR alpha chain. These observations are consistent with the model that high expression of Bcl-2 in cortical thymocytes overrides the normal apoptotic pathway. This then allows the selection of CD4-8+ thymocytes expressing TCRs that are otherwise nonselectable. However, the bcl-2 transgene did not protect CD4+8+ thymocytes expressing the male-specific TCR from deletion in male doubly transgenic mice. In the second approach, we determined the level of bcl-2 mRNA expression in populations of thymocytes defined by their CD4/CD8 phenotypes using quantitative reversed transcriptase PCR techniques. Our results indicate that bcl-2 mRNA was expressed at a high level in immature CD4-8- thymocytes and in mature CD4+8- thymocytes. There is a dramatic downregulation of bcl-2 mRNA in CD4+8+ thymocytes, particularly those expressing a low level of TCR. CD4+8+ thymocytes that upregulated their TCR, likely as a result of receiving positive selection signals, also upregulated bcl-2 mRNA. This observation suggests that rescue of immature thymocytes from the programmed cell death pathway by positive selection signals is accompanied by the upregulation of bcl-2 mRNA.

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Mechanism for cotolerance in nonlethally conditioned mixed chimeras: negative selection of the Vbeta T-cell receptor repertoire by both host and donor bone marrow-derived cells

Blood ◽

10.1182/blood.v88.12.4601.bloodjournal88124601 ◽

1996 ◽

Vol 88 (12) ◽

pp. 4601-4610 ◽

Cited By ~ 47

Author(s):

YL Colson ◽

J Lange ◽

K Fowler ◽

ST Ildstad

Keyword(s):

Bone Marrow ◽

T Cell ◽

T Cell Receptor ◽

Negative Selection ◽

Cell Receptor ◽

Mixed Chimerism ◽

Clonal Deletion ◽

Receptor Repertoire ◽

Total Body ◽

Selection Of

Bone marrow (BM) chimeras prepared by complete recipient ablation (A-->B) exhibit donor-specific tolerance, yet survival is often limited by graft-versus-host disease (GVHD). Negative selection of potentially donor-reactive T cells, as assessed by relative T-cell receptor (TCR)- Vbeta expression, is dependent on donor BM-derived deleting ligands. Mixed chimerism and tolerance for both donor and host antigens can be achieved using partial recipient myeloablation with 500 cGy total-body irradiation (TBI) before transplantation followed by cyclophosphamide (CyP) on day +2. To examine the influence of residual host elements on negative selection, the peripheral TCR-Vbeta repertoire was analyzed in partially ablated C57BL/10SnJ (B10) recipients reconstituted with BM from major histocompatibility complex (MHC)-disparate B10.BR/SgSnJ or MHC, Hh-1 and Mls-disparate BALB/cByJ donors, which delete Vbeta5+ and 11+ or Vbeta3+, 5+, and 11+ TCR subsets, respectively. As in myeloblated recipients, donor-reactive subfamilies were deleted in B10.BR-->B10 and BALB/c-->B10 chimeras, suggesting that donor I-E and minor lymphocyte-stimulating (Mls) antigens contribute to the deleting ligands in the nonmyeloablated host. In striking contrast to completely ablated B10-->B10.BR chimeras, partially ablated recipients showed intramedullary I-E expression in the thymus and deleted host-reactive Vbeta5+ and Vbeta11+ subfamilies. These data demonstrate that efficient clonal deletion occurs after partial myeloablation and that both donor and host ligands contribute to TCR repertoire selection.

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