Limits...
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.

Show MeSH

Related in: MedlinePlus

Flow cytometric  analysis of thymocytes from control and CD4Tg+ Rag2−/− MHC  class II−/− littermates treated  with anti-CD3ε or anti-CD4  mAb. (A) 4-wk-old mice were  treated intraperitoneally with 100  μg of the anti-CD3ε mAb 2C11  (top panels), or 20 μg of the antiCD4 mAb GK1.5 (bottom panels).  After 4 d, thymocytes were  stained and analyzed by flow cytometry using anti-CD4-PE and  anti-CD8-FITC. Identical results  were obtained for CD4Tg+  Rag2−/− MHC II−/− mice  treated with 100 μg or 500 μg  GK1.5. The percentage of cells  in each population is indicated.  Total thymocyte numbers were  as follows: anti-CD3ε–treated  Rag2−/− MHC II−/− (1.2 × 107)  or CD4 Rag2−/− MHC II−/−  mice (3.2 × 107). (B) Single parameter fluorescence histograms  show CD69 expression 20 h after treatment with 150 μg of  mAb 2C11 (gray line) or GK1.5  (thick black line) as compared to  untreated controls (thin black  line).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196103&req=5

Figure 4: Flow cytometric analysis of thymocytes from control and CD4Tg+ Rag2−/− MHC class II−/− littermates treated with anti-CD3ε or anti-CD4 mAb. (A) 4-wk-old mice were treated intraperitoneally with 100 μg of the anti-CD3ε mAb 2C11 (top panels), or 20 μg of the antiCD4 mAb GK1.5 (bottom panels). After 4 d, thymocytes were stained and analyzed by flow cytometry using anti-CD4-PE and anti-CD8-FITC. Identical results were obtained for CD4Tg+ Rag2−/− MHC II−/− mice treated with 100 μg or 500 μg GK1.5. The percentage of cells in each population is indicated. Total thymocyte numbers were as follows: anti-CD3ε–treated Rag2−/− MHC II−/− (1.2 × 107) or CD4 Rag2−/− MHC II−/− mice (3.2 × 107). (B) Single parameter fluorescence histograms show CD69 expression 20 h after treatment with 150 μg of mAb 2C11 (gray line) or GK1.5 (thick black line) as compared to untreated controls (thin black line).

Mentions: Previous studies demonstrate that administration of antiCD3 antibodies to Rag2−/− mice stimulates the DN to DP transition, an effect which depends upon the presence of Lck (35). Because administration of anti-CD4 antibodies to T cell clones (22) as well as murine thymocytes (21) stimulates Lck activity, and because expression of MHC class II molecules stimulates CD4-driven thymocyte maturation in Rag2−/− mice, we wished to determine whether anti-CD4 mAb treatment would similarly drive maturation of CD4Tg+ Rag2−/−MHC class II−/− thymocytes. Fig. 4 A demonstrates that anti-CD3 treatment effectively stimulates thymocyte maturation in Rag2−/− MHC class II−/− mice, irrespective of the presence of the CD4 transgene. In contrast, administration of the anti-CD4 mAb GK1.5 did not stimulate maturation of CD4Tg-bearing thymocytes. However, all CD4Tg+ Rag2−/− MHC class II−/− thymocytes clearly are capable of receiving a GK1.5 stimulated signal: GK1.5 or anti-CD3 induce comparable surface expression of the activation marker CD69 (36) within 24 h of mAb treatment (Fig. 4 B). CD69 expression is believed to increase after receipt of normal pre-TCR signals (35). Moreover, antiCD3-stimulated CD69 induction is impaired in Lck−/− mice (35). Hence treatment with anti-CD4 mAb delivers a signal to CD4Tg+ Rag2−/− MHC class II−/− thymocytes that recapitulates one feature of the Lck-dependent preTCR signaling process. This signal may differ qualitatively or quantitatively from that which devolves after interaction of CD4 with class II molecules (see below).


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 control and CD4Tg+ Rag2−/− MHC  class II−/− littermates treated  with anti-CD3ε or anti-CD4  mAb. (A) 4-wk-old mice were  treated intraperitoneally with 100  μg of the anti-CD3ε mAb 2C11  (top panels), or 20 μg of the antiCD4 mAb GK1.5 (bottom panels).  After 4 d, thymocytes were  stained and analyzed by flow cytometry using anti-CD4-PE and  anti-CD8-FITC. Identical results  were obtained for CD4Tg+  Rag2−/− MHC II−/− mice  treated with 100 μg or 500 μg  GK1.5. The percentage of cells  in each population is indicated.  Total thymocyte numbers were  as follows: anti-CD3ε–treated  Rag2−/− MHC II−/− (1.2 × 107)  or CD4 Rag2−/− MHC II−/−  mice (3.2 × 107). (B) Single parameter fluorescence histograms  show CD69 expression 20 h after treatment with 150 μg of  mAb 2C11 (gray line) or GK1.5  (thick black line) as compared to  untreated controls (thin black  line).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Flow cytometric analysis of thymocytes from control and CD4Tg+ Rag2−/− MHC class II−/− littermates treated with anti-CD3ε or anti-CD4 mAb. (A) 4-wk-old mice were treated intraperitoneally with 100 μg of the anti-CD3ε mAb 2C11 (top panels), or 20 μg of the antiCD4 mAb GK1.5 (bottom panels). After 4 d, thymocytes were stained and analyzed by flow cytometry using anti-CD4-PE and anti-CD8-FITC. Identical results were obtained for CD4Tg+ Rag2−/− MHC II−/− mice treated with 100 μg or 500 μg GK1.5. The percentage of cells in each population is indicated. Total thymocyte numbers were as follows: anti-CD3ε–treated Rag2−/− MHC II−/− (1.2 × 107) or CD4 Rag2−/− MHC II−/− mice (3.2 × 107). (B) Single parameter fluorescence histograms show CD69 expression 20 h after treatment with 150 μg of mAb 2C11 (gray line) or GK1.5 (thick black line) as compared to untreated controls (thin black line).
Mentions: Previous studies demonstrate that administration of antiCD3 antibodies to Rag2−/− mice stimulates the DN to DP transition, an effect which depends upon the presence of Lck (35). Because administration of anti-CD4 antibodies to T cell clones (22) as well as murine thymocytes (21) stimulates Lck activity, and because expression of MHC class II molecules stimulates CD4-driven thymocyte maturation in Rag2−/− mice, we wished to determine whether anti-CD4 mAb treatment would similarly drive maturation of CD4Tg+ Rag2−/−MHC class II−/− thymocytes. Fig. 4 A demonstrates that anti-CD3 treatment effectively stimulates thymocyte maturation in Rag2−/− MHC class II−/− mice, irrespective of the presence of the CD4 transgene. In contrast, administration of the anti-CD4 mAb GK1.5 did not stimulate maturation of CD4Tg-bearing thymocytes. However, all CD4Tg+ Rag2−/− MHC class II−/− thymocytes clearly are capable of receiving a GK1.5 stimulated signal: GK1.5 or anti-CD3 induce comparable surface expression of the activation marker CD69 (36) within 24 h of mAb treatment (Fig. 4 B). CD69 expression is believed to increase after receipt of normal pre-TCR signals (35). Moreover, antiCD3-stimulated CD69 induction is impaired in Lck−/− mice (35). Hence treatment with anti-CD4 mAb delivers a signal to CD4Tg+ Rag2−/− MHC class II−/− thymocytes that recapitulates one feature of the Lck-dependent preTCR signaling process. This signal may differ qualitatively or quantitatively from that which devolves after interaction of CD4 with class II molecules (see below).

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