T cell development in mice regulated by B-Raf-mediated signaling pathway

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Introduction

Thymocyte development, in the thymus, is responsible for generation of mature T cells. Developing thymocytes can be classified into three stages of increasing maturity [1, 2]:

  • CD4–CD8– double-negative (DN)
  • CD4+CD8+ double-positive (DP)
  • CD4+ or  CD8+ single-positive (SP) subsets.

http://www.nature.com/nri/journal/v2/n5/fig_tab/nri798_F1.html

T Cell Development Video

T Cell Selection Animation

T-cell receptor (TCR) signaling mediates these processes, especially positive and negative selection at the DP stage.

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].

Interactive Animation of MAP Kinase Signal Transduction

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 [5].

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

The Raf kinase family (A-Raf, B-Raf, C-Raf (or Raf-1)) contributes to activation of MEK/ERK pathway

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

Figure 1. Expression and TCR-mediated activation of B-Raf in T cells (A) Western blotting analysis of B-Raf expression in thymocytes fromRAG2−/− mice, DP and SP thymocytes sorted from C57BL/6 mice, peripheral (splenic) T cells fromRAG2−/− chimeras reconstituted with B-Raf+/+, B-Raf +/− or B-Raf−/− FL cells, as well as mock- and B-Raf-transfected Jurkat cells. Raf-1 and ERK blottings are shown as loading controls. PC12 and NIH3T3 were utilized as positive and negative controls of B-Raf expression, respectively. (B, C) In vitro kinase assays for Raf-1 and B-Raf isolated from thymocytes stimulated with 10 µg/mL anti-CD3 Ab or 25 ng/mL PMA for 10 min (B) or anti-CD3Ab for the indicated times (C). Recombinant GST-MEK was used as a substrate, and incorporated 32P radioactivity was visualized by autoradiography (upper left panel). ERK phosphorylation was determined by blotting with an antibody specific for phosphorylated ERK. Equal loading of each Raf isoform and ERK was confirmed (lower left panels). (D) Splenic T cells were stimulated with 5 µg/mL anti-CD3 Ab for the indicated times in an in vitro kinase assay for B-Raf and Raf-1 performed as described in (B) (ref. 10).

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.

Figure 2 Pre-TCR-mediated β-selection is not affected by B-Raf deficiency (A) Phenotypic changes of DN cells differentiated from wild-type and B-Raf−/− FL-derived progenitors in co-culture with OP9-DL1 for various times. Developmental progression of the T cell lineage was assessed on days 4, 8 and 12 by CD25 and CD44 expression profiles on CD4−CD8− DN-gated cells. In the lowest panels, percentages of CD4+CD8+ DP cells co-cultured with OP9-DL1 for 15 days are indicated. Lymphoid cells were gated based on their SSC and FSC profiles. (B) DN T cell progenitors derived from B-Raf+/+ and B-Raf−/− FL cells co-cultured with OP9-DL1 for 8 days were loaded with CFSE and re-seeded on OP9 GFP (upper panels) or OP9 DL1 (lower panels). After 48 h, proliferation indicated by CFSE dilution and CD25 expression on DN cells during β-selection were determined. Dot plots show cells gated to eliminate CD4+CD8+ DP and OP9-DL1 cells. (C) Thymocytes from B-Raf+/+ and B-Raf−/− chimeric mice 7 wk after transfer of FL cells into RAG2−/− mice were isolated by positively sorting with anti-Thy-1 magnetic beads. The plots for CD44 and CD25 expression were gated on the CD4−CD8− DN population. The data are representative of three animals per group in two independent experiments with similar results, and the percentages of gated cells that fall into each quadrant are shown (ref. 10).

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

Figure 3. Inefficient production of CD4+ and CD8+ SP cells in B-Raf-deficient thymocytes. (A) Single-cell suspensions of thymocytes (upper panels) and spleen cells (lower panels) were isolated from age-matched B-Raf+/+ and B-Raf−/− FL chimeric mice 8 wk after transfer, stained with Ab against CD4, CD8, B220 and TCR-β chain, and analyzed by flow cytometry. Lymphoid cells were gated based on SSC and FSC. The percentages of total thymocytes and spleen cells that fall into each region are indicated. (B) Cell surface expression of CD44 on peripheral T cells isolated from spleen and lymph nodes of B-Raf +/+ (filled gray) and B-Raf−/− (bold line) FL chimeric mice. (C) Thymi were harvested 8–12 wk after transfer and the cell suspension enumerated. After calculating the percentage of lymphoid cells (~85% of total) on the basis of FSC/SSC, the numbers of the indicated population were calculated (mean ± SD). Data are based on four to five samples of thymus (*p<0.01). All data are representative of at least three B-Raf +/+ and B-Raf−/− chimeric mice in two independent experiments (ref. 10).

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

Figure 4. Total thymocytes were stimulated by crosslinking of CD3 with 10 µg/mL anti-CD3 Ab and anti-hamster Ig Ab for 5 or 20 min (upper panels) or by incubation with 25 ng/mL PMA for 20 min (lower right panel). Cells were treated for 30 min with 10 µM U0126 prior to TCR stimulation (lower left panel). Histograms show profiles of phospho-ERK in B-Raf+/+ (filled gray) and B-Raf−/− (bold line) cells gated on CD4+CD8+ DP cells from DN cell-chimeric mice. Dotted lines indicate the status of phospho-ERK in unstimulated B-Raf+/+ DP cells. The data are from one representative of three independent experiments (ref. 10).

Conclusions

  • 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

Paper Strengths

  • 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

Paper Weaknesses

  • 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

References:

  1. 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/
  2. 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
  3. 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
  4. 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
  5. 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/
  6. 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
  7. 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/
  8. 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
  9. 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
  10. 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

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