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Replacement of pre-T cell receptor signaling functions by the CD4 coreceptor.

Norment AM, Forbush KA, Nguyen N, Malissen M, Perlmutter RM - J. Exp. Med. (1997)

Bottom Line: However, the biochemical mechanisms governing p56lck activation remain poorly understood.In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation.We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle 98195, USA.

ABSTRACT
An important checkpoint in early thymocyte development ensures that only thymocytes with an in-frame T cell receptor for antigen beta (TCR-beta) gene rearrangement will continue to mature. Proper assembly of the TCR-beta chain into the pre-TCR complex delivers signals through the src-family protein tyrosine kinase p56lck that stimulate thymocyte proliferation and differentiation to the CD4+CD8+ stage. However, the biochemical mechanisms governing p56lck activation remain poorly understood. In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation. To study the effect of synchronously inducing p56lck activation in immature CD4-CD8- thymocytes, we generated mice expressing a CD4 transgene in Rag2-/- thymocytes. Remarkably, without further experimental manipulation, the CD4 transgene drives maturation of Rag2-/- thymocytes in vivo. We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules. Together these results indicate that binding of MHC class II molecules to CD4 can deliver a biologically relevant, Lck-dependent activation signal to thymocytes in the absence of the TCR-alpha or -beta chain.

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Flow cytometric analysis of thymocytes from wild-type CD4Tg+ Rag2−/− and tailless CD4Δ Rag2−/− transgenic mice. (A) Representative  fluorescence staining profiles of CD4 and CD8 are shown for thymocytes from age matched CD4Tg+ Rag2−/− mice (CD4, left panel) or tailless CD4  (CD4Δ, middle panel) Rag2−/− mice (n = 6). A single parameter histogram better shows the relative levels of CD4 expression (right panel) for these mice  as compared to a transgene negative littermate control of CD4Δ (Neg. Cont.). (B) A similar immunofluorescense analysis is shown for thymocytes from a  CD4Tg+ Rag2−/− mouse (left panel), and two of seven CD4Δ Rag2−/− positive progeny from a CD4Δ × CD4Δ mating (middle two panels). Two parameter fluorescence profiles are shown for wild-type CD4Tg+ and the mice expressing the lowest (CD4Δlo) and highest (CD4Δhi) level of CD4Δ in the litter. The relative levels of CD4 expression are again shown by a single parameter histogram (right panel). The percentage of cells in each population is indicated. While some CD4Δ Rag2−/− mice (7/21 analyzed) contained a very small fraction of CD8+CD25− thymocytes (1–2%), this did not correlate  with the level of CD4Δ expression. The relative percentage of CD8+CD25− thymocytes in wild-type CD4 Rag 2−/−/CD4Δ Rag 2−/− mice was >50fold.
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Figure 2: Flow cytometric analysis of thymocytes from wild-type CD4Tg+ Rag2−/− and tailless CD4Δ Rag2−/− transgenic mice. (A) Representative fluorescence staining profiles of CD4 and CD8 are shown for thymocytes from age matched CD4Tg+ Rag2−/− mice (CD4, left panel) or tailless CD4 (CD4Δ, middle panel) Rag2−/− mice (n = 6). A single parameter histogram better shows the relative levels of CD4 expression (right panel) for these mice as compared to a transgene negative littermate control of CD4Δ (Neg. Cont.). (B) A similar immunofluorescense analysis is shown for thymocytes from a CD4Tg+ Rag2−/− mouse (left panel), and two of seven CD4Δ Rag2−/− positive progeny from a CD4Δ × CD4Δ mating (middle two panels). Two parameter fluorescence profiles are shown for wild-type CD4Tg+ and the mice expressing the lowest (CD4Δlo) and highest (CD4Δhi) level of CD4Δ in the litter. The relative levels of CD4 expression are again shown by a single parameter histogram (right panel). The percentage of cells in each population is indicated. While some CD4Δ Rag2−/− mice (7/21 analyzed) contained a very small fraction of CD8+CD25− thymocytes (1–2%), this did not correlate with the level of CD4Δ expression. The relative percentage of CD8+CD25− thymocytes in wild-type CD4 Rag 2−/−/CD4Δ Rag 2−/− mice was >50fold.

Mentions: Because activated Lck (containing a phenylalanine-for-tyrosine substitution at position 505) can stimulate the DN to DP transition in Rag−/− thymocytes (17), and because Lck binds directly to CD4 (18), we reasoned that expression of transgene-derived CD4 in Rag2−/− thymocytes had permitted assembly of a signaling complex leading to Lck activation in vivo. This hypothesis predicts that the ability of CD4 to stimulate thymocyte development would depend upon its interaction with Lck. To test this conjecture, we generated an additional line of transgenic animals expressing a mutant CD4 transgene (CD4Δ) in which sequences encoding all but the eight most membrane-proximal cytoplasmic residues were truncated. Prior studies have demonstrated that this truncation yields a molecule that cannot interact with p56lck (25). Fig. 2 A shows that although the CD4Δ transgene was expressed under the lck proximal promoter at levels comparable to those achieved using the wild-type CD4 transgene, Rag2−/− thymocytes were not induced to mature by the presence of this mutant protein. Indeed, even when the CD4Δ transgenic mice were intercrossed in the effort to obtain progeny with increased expression of truncated CD4, no improvement in thymocyte maturation was noted (Fig. 2 B). We conclude that the CD4 cytoplasmic tail, almost certainly reflecting its ability to bind p56lck, is required to permit a CD4-derived signal to drive the DN to DP transition in thymocytes.


Replacement of pre-T cell receptor signaling functions by the CD4 coreceptor.

Norment AM, Forbush KA, Nguyen N, Malissen M, Perlmutter RM - J. Exp. Med. (1997)

Flow cytometric analysis of thymocytes from wild-type CD4Tg+ Rag2−/− and tailless CD4Δ Rag2−/− transgenic mice. (A) Representative  fluorescence staining profiles of CD4 and CD8 are shown for thymocytes from age matched CD4Tg+ Rag2−/− mice (CD4, left panel) or tailless CD4  (CD4Δ, middle panel) Rag2−/− mice (n = 6). A single parameter histogram better shows the relative levels of CD4 expression (right panel) for these mice  as compared to a transgene negative littermate control of CD4Δ (Neg. Cont.). (B) A similar immunofluorescense analysis is shown for thymocytes from a  CD4Tg+ Rag2−/− mouse (left panel), and two of seven CD4Δ Rag2−/− positive progeny from a CD4Δ × CD4Δ mating (middle two panels). Two parameter fluorescence profiles are shown for wild-type CD4Tg+ and the mice expressing the lowest (CD4Δlo) and highest (CD4Δhi) level of CD4Δ in the litter. The relative levels of CD4 expression are again shown by a single parameter histogram (right panel). The percentage of cells in each population is indicated. While some CD4Δ Rag2−/− mice (7/21 analyzed) contained a very small fraction of CD8+CD25− thymocytes (1–2%), this did not correlate  with the level of CD4Δ expression. The relative percentage of CD8+CD25− thymocytes in wild-type CD4 Rag 2−/−/CD4Δ Rag 2−/− mice was >50fold.
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Figure 2: Flow cytometric analysis of thymocytes from wild-type CD4Tg+ Rag2−/− and tailless CD4Δ Rag2−/− transgenic mice. (A) Representative fluorescence staining profiles of CD4 and CD8 are shown for thymocytes from age matched CD4Tg+ Rag2−/− mice (CD4, left panel) or tailless CD4 (CD4Δ, middle panel) Rag2−/− mice (n = 6). A single parameter histogram better shows the relative levels of CD4 expression (right panel) for these mice as compared to a transgene negative littermate control of CD4Δ (Neg. Cont.). (B) A similar immunofluorescense analysis is shown for thymocytes from a CD4Tg+ Rag2−/− mouse (left panel), and two of seven CD4Δ Rag2−/− positive progeny from a CD4Δ × CD4Δ mating (middle two panels). Two parameter fluorescence profiles are shown for wild-type CD4Tg+ and the mice expressing the lowest (CD4Δlo) and highest (CD4Δhi) level of CD4Δ in the litter. The relative levels of CD4 expression are again shown by a single parameter histogram (right panel). The percentage of cells in each population is indicated. While some CD4Δ Rag2−/− mice (7/21 analyzed) contained a very small fraction of CD8+CD25− thymocytes (1–2%), this did not correlate with the level of CD4Δ expression. The relative percentage of CD8+CD25− thymocytes in wild-type CD4 Rag 2−/−/CD4Δ Rag 2−/− mice was >50fold.
Mentions: Because activated Lck (containing a phenylalanine-for-tyrosine substitution at position 505) can stimulate the DN to DP transition in Rag−/− thymocytes (17), and because Lck binds directly to CD4 (18), we reasoned that expression of transgene-derived CD4 in Rag2−/− thymocytes had permitted assembly of a signaling complex leading to Lck activation in vivo. This hypothesis predicts that the ability of CD4 to stimulate thymocyte development would depend upon its interaction with Lck. To test this conjecture, we generated an additional line of transgenic animals expressing a mutant CD4 transgene (CD4Δ) in which sequences encoding all but the eight most membrane-proximal cytoplasmic residues were truncated. Prior studies have demonstrated that this truncation yields a molecule that cannot interact with p56lck (25). Fig. 2 A shows that although the CD4Δ transgene was expressed under the lck proximal promoter at levels comparable to those achieved using the wild-type CD4 transgene, Rag2−/− thymocytes were not induced to mature by the presence of this mutant protein. Indeed, even when the CD4Δ transgenic mice were intercrossed in the effort to obtain progeny with increased expression of truncated CD4, no improvement in thymocyte maturation was noted (Fig. 2 B). We conclude that the CD4 cytoplasmic tail, almost certainly reflecting its ability to bind p56lck, is required to permit a CD4-derived signal to drive the DN to DP transition in thymocytes.

Bottom Line: However, the biochemical mechanisms governing p56lck activation remain poorly understood.In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation.We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle 98195, USA.

ABSTRACT
An important checkpoint in early thymocyte development ensures that only thymocytes with an in-frame T cell receptor for antigen beta (TCR-beta) gene rearrangement will continue to mature. Proper assembly of the TCR-beta chain into the pre-TCR complex delivers signals through the src-family protein tyrosine kinase p56lck that stimulate thymocyte proliferation and differentiation to the CD4+CD8+ stage. However, the biochemical mechanisms governing p56lck activation remain poorly understood. In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation. To study the effect of synchronously inducing p56lck activation in immature CD4-CD8- thymocytes, we generated mice expressing a CD4 transgene in Rag2-/- thymocytes. Remarkably, without further experimental manipulation, the CD4 transgene drives maturation of Rag2-/- thymocytes in vivo. We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules. Together these results indicate that binding of MHC class II molecules to CD4 can deliver a biologically relevant, Lck-dependent activation signal to thymocytes in the absence of the TCR-alpha or -beta chain.

Show MeSH
Related in: MedlinePlus