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- CD4–CD8– double-negative (DN)
- CD4+CD8+ double-positive (DP)
- CD4+ or CD8+ single-positive (SP) subsets.
A number of studies have revealed that the MAPK pathway (consisting of p21Ras, Raf, MEK and ERK) represents one of the important signaling cascades coupled to TCR-mediated responses, including the determination of cell fate at various stages of thymic selection [3, 4].
Expression of dominant-negative or active forms of Ras, Raf or MEK1 revealed that the MAPK pathway is involved in positive selection at the CD4+CD8+ DP stage .
Consistent with this, ERK activation is not only involved in the critical events during TCR signaling and plays a crucial role in positive selection, but is also known to be vital for intracellular signals promoting the first β-selection checkpoint and lineage commitment of mature CD4+ or CD8+ SP cells [3, 4].
Therefore, understanding how TCR signaling in thymocytes results in ERK activation is important for uncovering the mechanisms of thymocyte development.
Raf Kinase Family
Different phenotypes of each Raf kinase-deficient mouse suggest that each has a specific function due to their distinct expression patterns and thresholds of activation
Previous research suggests that B-Raf may be an important signaling molecule in TCR-mediated responses [6-9]
However, T cells cannot be obtained from B-Raf-null mice, since disruption of B-Raf gene results in embryonic lethality. So, the in vivo role of B-Raf on development and activation of T cells remains unclear.
Expression and activation of B-Raf in thymocytes
DN-arrested thymocytes from RAG2-/- mice, purified CD4+CD8+ DP thymocytes and peripheral T cells from wild-type (WT) C57BL/6 mice immunoblotted with anti-B-Raf Ab (Fig. 1A):
- Expression experiments reveal high expression of B-Raf protein in all subsets of immature thymocytes and peripheral T cells.
- B-Raf+/- mice express less B-Raf protein than T cells from WT mice
- B-Raf-/- T cells did not exhibit B-Raf expression but did express Raf-1 and ERK
- B-Raf high expressed in PC12 cells and B-Raf transfected Jurkat cells (positive controls)
Is B-Raf activity regulated by TCR stimulation?
- TCR stimulation induced up-regulation of kinase activity of B-Raf
- Activation prolonged for up to 60 mins and was coincident with ERK activation (Fig. 1B,C)
- In TCR-stimulated peripheral T cells, activation of Raf-1 and B-Raf was induced (Fig. 1D)
These findings of TCR stimulation-inducible B-Raf activation prompted hypothesis that B-Raf could play a role in TCR-mediated T cell responses in thymocytes
β-selection proceeds normally in vitro and in vivo in B-Raf-deficient T cell progenitors
T cell progenitors enter the thymus as CD44+CD25- cells and acquire expression of CD25. The next transition, from CD44-CD25+ (DN3 stage) to CD44-CD25- (DN4 stage) is called β-selection, which requires pre-TCR-mediated signals and is coincident with proliferation
Using in vitro co-culture system Tsukamoto et al. assessed requirement of B-Raf during developmental progression of the DN subpopulation and in β-selection:
- Regardless of B-Raf status, T cell progenitors, CD4+CD8+ DP cells were efficiently induced (Fig. 2A)
- B-Raf deficiency still permits TCR-mediated proliferation during transition from CD25+ DN3 to CD25- DN4 stage (Fig. 2B)
- In vivo analysis reveals that B-Raf deficient thmyocytes undergo normal phenotypic progression to DN4 stage
These results suggest that B-Raf is not necessary for T cell development at the DN stages and pre-TCR-mediated β-selection in vitro and in vivo.
Requirement of B-Raf for the development of single-positive thymocytes
Examining the CD4 and CD8 expression profiles of B-Raf+/+ and B-Raf-/- thymocytes in fetal liver (FL) chimeras, reveals that B-Raf-/- FL chimeras showed accumulation of CD4+CD8+ DP cells, and decreased CD4+ SP and CD8+ SP thymocytes compared with B-Raf+/+ controls (Fig. 3A)
Results here suggest that the abnormal phenotype of B-Raf-/- thymocytes is T cell autonomous, and B-Raf-/- DP thymocytes do not transmit sufficient TCR signals to lead to thymocyte maturation, resulting in the DP arrest
B-Raf is a positive regulator of ERK activation in thymocytes
Thymocyte differentiation at the DP stage is dependent on TCR-mediated intracellular signaling.
Since ERK activity is regulated by TCR-mediated signaling and acts as an important regulator of thymic selection, researchers measured the ERK activation status following TCR stimulation to evaluate whether the inability of B-Raf-/- to diferentiate into the SP stage is due to impaired signaling through the TCR (Fig. 4)
- TCR stimulation induced ERK activation in WT DP thymocytes
- ERK activation was drastically weakened in B-Raf-/- DP cells at 5 and 20 min after stimulation
- TCR-mediated ERK activation in WT and B-Raf-/- thymocytes was completely blocked by the treatment of cells with MEK inhibitor U0216
- In contrast, TCR stimulation with phorbol myristate acetate (PMA) evoked comparable ERK activation in both thymocytes
- B-Raf deficiency specifically reduced TCR-mediated ERK activation in the DP thymocytes, while PMA stimulation bypassed B-Raf function and induced full ERK activation
Results suggest that development arrest of SP transition in B-Raf deficient thymocytes reflects a defective cellular response to the TCR-mediated ERK activation pathway
- B-Raf is expressed in thymocytes and peripheral T cells
- B-Raf is a positive regulator of T cell development
- B-Raf contributes to the promotion of TCR-mediated ERK activation at the DP stage
- B-Raf plays a specific and crucial role in T cell development in vivo
- Clearly sets the stage for the conducted research with thorough background and significance
- Logical approach to experimentally determining if B-Raf plays a role in T cell development in mice
- Strong experimental support for conclusions made in the paper
- This paper does effectively show that B-Raf deficiency results in depletion of CD4+ and CD8+ SP thymocytes, but does not yet solve the direct action of B-Raf in T cell development
- Future research should be geared towards identifying the specific action of B-Raf in T cell development in mice
- Mason, C. S., Springer, C. J., Cooper, R. G., Superti-Furga, G., Marshall, C. J. and Marais, R., Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation. EMBO J. 1999. 18: 2137–2148. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1171298/
- Germain, R. N., T-cell development and the CD4-CD8 lineage decision. Nat. Rev. Immunol. 2002. 2: 309–322. http://www.ncbi.nlm.nih.gov/pubmed/12033737
- Fischer, A. M., Katayama, C. D., Pages, G., Pouyssegur, J. and Hedrick, S. M., The role of erk1 and erk2 in multiple stages of T cell development. Immunity 2005. 23: 431–443. http://www.ncbi.nlm.nih.gov/pubmed/16226508
- Pages, G., Guerin, S., Grall, D., Bonino, F., Smith, A., Anjuere, F., Auberger, P. and Pouyssegur, J., Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice. Science 1999. 286: 1374–1377. http://www.ncbi.nlm.nih.gov/pubmed/10558995
- Alberola-Ila, J., Hogquist, K. A., Swan, K. A., Bevan, M. J. and Perlmutter, R. M., Positive and negative selection invoke distinct signaling pathways. J. Exp. Med. 1996. 184: 9–18. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2192689/
- Pritchard, C. A., Bolin, L., Slattery, R., Murray, R. and McMahon, M., Post-natal lethality and neurological and gastrointestinal defects in mice with targeted disruption of the A-Raf protein kinase gene. Curr. Biol. 1996. 6: 614–617. http://www.ncbi.nlm.nih.gov/pubmed/8805280
- Brummer, T., Shaw, P. E., Reth, M. and Misawa, Y., Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling. EMBO J. 2002. 21: 5611–5622. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC131085/
- Hagemann, C. and Rapp, U. R., Isotype-specific functions of Raf kinases. Exp. Cell Res. 1999. 253: 34–46. http://www.ncbi.nlm.nih.gov/pubmed/10579909
- Marais, R., Light, Y., Paterson, H. F., Mason, C. S. and Marshall, C. J., Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases. J. Biol. Chem. 1997. 272: 4378–4383. http://www.ncbi.nlm.nih.gov/pubmed/9020159
- Tsukamoto, H., Irie, A., Senju, S., Hatzopoulos, A.K., Wojnowski, L., and Nishimura, Y., B-Raf-mediated signaling pathway regulates T cell development. Eur J Immunol. 38(2):518-27 (2008). http://www.ncbi.nlm.nih.gov/pubmed/18228248