Early growth response gene-2 (Egr-2) regulates the development of B and T cells

Abstract

Background: Understanding of how transcription factors are involved in lymphocyte development still remains a challenge. It has been shown that Egr-2 deficiency results in impaired NKT cell development and defective positive selection of T cells. Here we investigated the development of T, B and NKT cells in Egr-2 transgenic mice and the roles in the regulation of distinct stages of B and T cell development.

Methods and findings: The expression of Egr1, 2 and 3 were analysed at different stages of T and B cell development by RT-PCT and results showed that the expression was strictly regulated at different stages. Forced expression of Egr-2 in CD2(+) lymphocytes resulted in a severe reduction of CD4(+)CD8(+) (DP) cells in thymus and pro-B cells in bone marrow, which was associated with reduced expression of Notch1 in ISP thymocytes and Pax5 in pro-B cells, suggesting that retraction of Egr-2 at the ISP and pro-B cell stages is important for the activation of lineage differentiation programs. In contrast to reduction of DP and pro-B cells, Egr-2 enhanced the maturation of DP cells into single positive (SP) T and NKT cells in thymus, and immature B cells into mature B cells in bone marrow.

Conclusions: Our results demonstrate that Egr-2 expressed in restricted stages of lymphocyte development plays a dynamic, but similar role for the development of T, NKT and B cells.

Figure 1. Defective thymus development in Egr-2 transgenic mice.

A). confirmation of presence of transgene by PCR. B). expression of Egr-2 in thymocytes from wild type, Egr-2 cKO and two Egr-2 cTg mouse lines (14 and 18) determined by quantitative RT-PCR. The relative expression was measured after normalization against b-actin. _* Egr-2 was not detected in Egr-2 cKO T cells. C). Thymus size in wild type, Egr-2 cTg and Egr-2 cKO mice. D). Thymocytes from wild type and Egr-2 transgenic mice were stained with anti-CD4 and anti-CD8. E). Expression of CD3, TCRβ and CD5 by gated CD4⁺ or CD8⁺SP T cells in thymus. The data are representative of three experiments in which 3 to 5 mice from each group were used.

Figure 2. Development of thymocytes.

A). Absolute number of thymocyte subpopulations in thymus. The ISP cells were defined as CD4⁻CD8⁺CD3⁻ (see Fig. 1E). B). The subpopulations of DN cells in thymus were examined by analysis of CD25 and CD44 expression on gated CD4⁻CD8⁻ DN cells. C). Absolute number of DN subsets in thymus. D). Percentage of CD3⁺DP cells that develop into CD3⁺CD4⁺ or CD3⁺CD8⁺ cells in thymus. E). Subpopulations of T cells in lymph nodes and spleen. F). Expression of CD3, TCRβ and CD5 on CD4⁺ or CD8⁺ cells in spleens. * p<0.001. The data are representative of more than three experiments in which 3 to 5 mice from each group were used.

Figure 3. Thymocyte apoptosis.

A). Tunel assay on thymus of wild type, Egr-2 cTg and Egr-2 cKO mice. B). Percentage of Annexin V⁺ and PI⁻ thymocytes in wild type, Egr-2 cTg and Egr-2cKO mice. The data are representative of three to five mice.

Figure 4. Development of NKT cells in thymus.

A). Thymocytes from wild type, Egr-2 cKO and Egr-2 cTg mice were stained with NK1.1 and TCRβ antibodies (top) or with TCRvβ and CD1 tetramer (bottom). B). Absolute numbers of NKT cells in thymus of Egr-2 cKO, Egr-2 cTg and wild type mice. C). Percentages of DP cells that develop into NKT cells in thymus of Egr-2 cKO, Egr-2 cTg and wild type mice. * p<0.001. The data are representative of more than two experiments in which 3 to 5 mice from each group were used.

Figure 5. B cell development.

A). Cells from bone marrow, lymph nodes and spleen of wild type, Egr-2 cKO and Egr-2 cTg mice were co-stained with anti-CD19 and anti-B220 (left) or anti-IgM and anti-B220 (right). B). Absolute number of B cell precursor subpopulations in bone marrow of wild type, Egr-2 cKO and Egr-2 cTg mice. Pre-pro-B cells defined as NK1.1⁻B220⁺CD19⁻CD43⁺, pro-B cells defined as NK1.1⁻B220⁺CD19⁺CD43⁺, pre-B cells as NK1.1⁻B220⁺CD43⁻, immature B cells as NK1.1⁻B220⁺IgM⁺ and mature B cells defined as NK1.1-B220highIgM+. C). Percentages of immature B cells that develop into mature B cells in bone marrow. * p<0.001. The data are representative of more than two experiments in which 3 to 5 mice from each group were used.

Figure 6. Kinetic expression of Egr in wild type and expression of regulatory genes in thymocytes and bone marrow B cell precursors from Egr-2 cTg mice.

Egr expression as determined by quantitative RT-PCR in subpopulations of thymocytes (A) or bone marrow B cell precursors (B) of wild type mice. CD8⁺CD4⁻CD3⁻ ISP cells and CD4⁺CD8⁺ DP cells were sorted by FACS. DN cell subpopulations were isolated based on their expression of CD25 and CD44 after first gating on CD4⁻CD8⁻ cells. B cell subsets were sorted by FACS based on triple staining with CD19⁻B220⁻CD127⁺ for CLP cells, NK1.1⁻B220⁺CD19⁻CD43⁺ for pre-pro-B cells, NK1.1⁻B220⁺CD19⁺CD43⁺ for pro-B cells and NK1.1⁻B220⁺CD43⁻ for pre-B cells. C). Expression of Notch1 and NUR77 genes in DN (CD4⁻CD8⁻) and ISP (CD8⁺CD3⁻) thymocytes from wild type and Egr-2 cTg mice determined by quantitative RT-PCR. D). Expression of Pax5 in sub-populations of B cells from bone marrow of wild type and Egr-2 cTg mice. The relative expression is shown as mean ± s.e.m. for duplicate determinations, relative to actin. * p<0.001. The data are representative of more than two experiments in which 3 to 5 mice from each group were used.