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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 Rag2−/− and  CD4 transgenic Rag2−/− mice. Two parameter fluorescence histograms  of lymphocyte gated events are shown after cell surface staining of thymocytes from age matched mice with anti-CD4-PE and anti-CD8-FITC  (top), or anti-CD8-PE and anti-CD25-FITC (middle). Forward light scatter profiles (FSC) are also shown (bottom) as an indication of relative cell  size. The percentage of cells in each population is indicated. Under identical staining conditions, wild-type CD4+ thymocytes lie at ∼100 units  on the logarithmic scale.
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Figure 1: Flow cytometric analysis of thymocytes from Rag2−/− and CD4 transgenic Rag2−/− mice. Two parameter fluorescence histograms of lymphocyte gated events are shown after cell surface staining of thymocytes from age matched mice with anti-CD4-PE and anti-CD8-FITC (top), or anti-CD8-PE and anti-CD25-FITC (middle). Forward light scatter profiles (FSC) are also shown (bottom) as an indication of relative cell size. The percentage of cells in each population is indicated. Under identical staining conditions, wild-type CD4+ thymocytes lie at ∼100 units on the logarithmic scale.

Mentions: To determine whether we could effect CD4-mediated p56lck activation in pre-T cells in vivo, a CD4 minigene under the control of the lck proximal promoter (24) was placed on a Rag2−/− background. By flow cytometry, CD4Tg+ Rag2−/− thymocytes express ∼10-fold higher levels of CD4 on their surfaces as compared with those found on more mature, wild-type CD4+ thymocytes (Fig. 1). An effect of the CD4 transgene was readily apparent: thymuses from the CD4 transgenic animals were larger than those of Rag2−/− littermate controls, and showed a fivefold increase in cell number (∼1 × 107 vs. ∼2 × 106 thymocytes in Rag2−/− mice). Remarkably, the majority of thymocytes in CD4Tg-bearing Rag2−/− mice acquired surface expression of endogenously encoded CD8 (Fig. 1, top). These cells also lost expression of CD25 (Fig. 1, middle), and were smaller in size (Fig. 1, bottom), both phenotypes associated with the DN to DP transition in normal thymocytes (8). Together these results indicate that the simple expression of CD4 (albeit at high levels) in the DN compartment faithfully reproduces signals ordinarily associated with satisfactory assembly of the pre-TCR. This occurred without any measurable change in the surface representation of CD3 components (judged by flow cytometric analysis of CD3ε expression), and hence suggests that the presence of transgene-derived CD4 was directly responsible for this phenomenon.


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 Rag2−/− and  CD4 transgenic Rag2−/− mice. Two parameter fluorescence histograms  of lymphocyte gated events are shown after cell surface staining of thymocytes from age matched mice with anti-CD4-PE and anti-CD8-FITC  (top), or anti-CD8-PE and anti-CD25-FITC (middle). Forward light scatter profiles (FSC) are also shown (bottom) as an indication of relative cell  size. The percentage of cells in each population is indicated. Under identical staining conditions, wild-type CD4+ thymocytes lie at ∼100 units  on the logarithmic scale.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196103&req=5

Figure 1: Flow cytometric analysis of thymocytes from Rag2−/− and CD4 transgenic Rag2−/− mice. Two parameter fluorescence histograms of lymphocyte gated events are shown after cell surface staining of thymocytes from age matched mice with anti-CD4-PE and anti-CD8-FITC (top), or anti-CD8-PE and anti-CD25-FITC (middle). Forward light scatter profiles (FSC) are also shown (bottom) as an indication of relative cell size. The percentage of cells in each population is indicated. Under identical staining conditions, wild-type CD4+ thymocytes lie at ∼100 units on the logarithmic scale.
Mentions: To determine whether we could effect CD4-mediated p56lck activation in pre-T cells in vivo, a CD4 minigene under the control of the lck proximal promoter (24) was placed on a Rag2−/− background. By flow cytometry, CD4Tg+ Rag2−/− thymocytes express ∼10-fold higher levels of CD4 on their surfaces as compared with those found on more mature, wild-type CD4+ thymocytes (Fig. 1). An effect of the CD4 transgene was readily apparent: thymuses from the CD4 transgenic animals were larger than those of Rag2−/− littermate controls, and showed a fivefold increase in cell number (∼1 × 107 vs. ∼2 × 106 thymocytes in Rag2−/− mice). Remarkably, the majority of thymocytes in CD4Tg-bearing Rag2−/− mice acquired surface expression of endogenously encoded CD8 (Fig. 1, top). These cells also lost expression of CD25 (Fig. 1, middle), and were smaller in size (Fig. 1, bottom), both phenotypes associated with the DN to DP transition in normal thymocytes (8). Together these results indicate that the simple expression of CD4 (albeit at high levels) in the DN compartment faithfully reproduces signals ordinarily associated with satisfactory assembly of the pre-TCR. This occurred without any measurable change in the surface representation of CD3 components (judged by flow cytometric analysis of CD3ε expression), and hence suggests that the presence of transgene-derived CD4 was directly responsible for this phenomenon.

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