Lsh, an SNF2/helicase family member, is required for proliferation of mature T lymphocytes - PubMed (original) (raw)

Lsh, an SNF2/helicase family member, is required for proliferation of mature T lymphocytes

T M Geiman et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Lsh (Hells) is closely related to SNF2/helicase family members that remodel chromatin and thus regulate gene transcription. In the adult mouse Lsh is expressed primarily in lymphoid tissue, showing the highest level in thymocytes. Lsh gene expression can be induced in thymic pro-T cells by pre-T cell receptor crosslinking and in mature T cells by T cell receptor crosslinking together with costimulation via CD28. The time course of Lsh gene and protein expression correlated closely with the onset of S phase of the cell cycle. To explore the function of Lsh during lymphoid development or activation, we deleted the Lsh gene by homologous recombination in ES cells. Fetal liver cells from Lsh-/- were used as a source of hematopoietic precursors to reconstitute lymphoid development in Rag2-/- mice. Lsh-/- (compared to Lsh+/+ or +/-) chimeras showed a modest reduction in thymocyte numbers due to a partial arrest at the transition from the CD4(-)CD8(-) stage to the CD4(+)CD8(+) stage of T cell development. Mature peripheral lymphocytes were reduced in number to approximately 60% for T cells and 40% for B cells; however, V(D)J recombination of the immune receptor genes was normal. Although polyclonal activation of Lsh-/- T cells induced normal levels of cytokines, cell proliferation was severely suppressed and cells underwent apoptosis. Several genes involved in the regulation of apoptosis were expressed normally with the exception of Bcl-2 that was actually elevated. These findings demonstrate that Lsh is not obligatory for normal lymphoid development but is essential for normal proliferation of peripheral T lymphocytes.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Expression of Lsh mRNA in lymphoid tissue. (A) Lsh mRNA expression in fetal vs. adult lymphoid tissue. Embryonal thymi were removed at day 14, 15, or 16 of gestation (left) or thymus, spleen, and lymph node were removed from 4–8 wk old adults (right) and compared to fetal thymus (day 15). EL4 is a T cell line and 38B9 is a pre-B cell line. Total RNA was extracted and subjected to Northern analysis by using a 2.2-kb Lsh cDNA probe comprising the whole ORF. (B) Lsh mRNA expression in T cell precursor populations. Fetal (day 14) or adult thymus were stained with the indicated antibodies and sorted by flow cytometry. Total RNA was extracted and subjected to RT-PCR analysis (1/5 serial dilutions) for specific detection of Lsh or 18s (control) mRNA. (C) Induction of Lsh mRNA by pre-TCR signaling. Rag2−/− and SCID mice were injected with anti-CD3 antibodies and the thymus removed 6 d later for Northern analysis as in A.

Figure 2

Figure 2

Induction of LSH gene expression in mature T lymphocytes. (A) Induction of Lsh mRNA by CD3 and CD28 costimulation. Splenocytes were cultured for 48 h in the presence of the indicated stimuli and Northern analysis performed as in Fig. 1_A_. (B) Inhibition of Lsh mRNA by immunosuppressive drugs. Splenocytes from animals were cultured for 48 h with ConA in the presence or absence of the immunosuppressive drugs cyclosporine A, FK506, or rapamycin, and Northern analysis was performed as in Fig. 1_A_. (C) Time kinetics of Lsh mRNA expression. Splenocytes were stimulated for 2–72 h with ConA, and Northern analysis was performed as in Fig. 1_A_. (D) Time kinetics of Lsh protein expression. Splenocytes were stimulated for 24–96 h with ConA. Nuclear and cytoplasmic protein was extracted and subjected to Western analysis using rabbit antiserum against the C-terminal peptide of Lsh. (E) Time kinetic of cell cycle induction of ConA-activated splenocytes. Splenocytes were stimulated for 2–72 h with ConA and analyzed by flow cytometry for cell cycle.

Figure 3

Figure 3

Effect of Lsh deletion on lymphoid development. (A) Absence of Lsh protein in Lsh−/− splenocytes. Fetal liver cell suspensions of the indicated Lsh genotype were injected into Rag-2−/− recipients and spleens harvested after 4–5 wk. Splenocytes were cultured for 48 h with ConA, nuclear protein extracted, and subjected to Western analysis using rabbit antiserum against the C-terminal peptide of Lsh. (B) Lsh−/− (○, n = 11) chimeric thymus and spleen cell numbers were evaluated and compared to cell numbers generated from + /+ mice (▵, n = 9). No statistically significant difference in thymic or spleen cell numbers was found between + /+ or ± animals. Statistically relevant differences are indicated as P values below. (C) Lsh−/− chimeras (○, n = 11) were analyzed for the thymic subpopulation surface markers CD4 and CD8 by FACs and compared to control +/+ animals (▵, n = 8). Statistically relevant differences are indicated as P values below. (D) Lsh−/− spleen chimeras (○, n = 9) were analyzed by FACs for expression of T cell surface markers CD3, CD4, and CD8 or B cell surface markers B220 and IgM and compared to spleen from + /+ animals (▵, n = 7). Statistically relevant differences are indicated as P values below. (E) Genomic DNA from either thymus or spleen of Lsh−/− or Lsh+/+ chimeric animals was analyzed for V(D)J recombination at the TCRα, β, or γ locus or the IgH chain locus (D-JH4, V7183, J558). The control reactions amplified the Vγ2 gene independent of the recombination event. Only the control reactions for spleen samples are shown.

Figure 4

Figure 4

Analysis of proliferation and cell cycle in Lsh−/− chimeras. (A) Effect of Lsh on thymidine incorporation. Fetal liver cell suspensions of the Lsh−/− (○) or control embryos (± ▵) were injected into Rag-2−/− recipients and spleens harvested after 5 wk. Splenocytes were stimulated with ConA for the indicated times (left) or with ConA or LPS for 48 h until thymidine incorporation was measured (right; data summarizes two experiments with Lsh−/− (n = 4) and control (n = 7). (B) Lsh−/− or + /+ spleen cells were subjected to cell cycle analysis after stimulation with ConA for 48 h. Number of cells undergoing apoptosis or in G1 phase of cell cycle are indicated in the upper right corner of two representative Lsh−/− individual mice (n = 10) and two + /+ controls (n = 10).

Figure 5

Figure 5

Analysis of expression of genes involved in cell cycle or apoptosis. Fetal liver cell suspensions of the indicated Lsh genotype were injected into Rag-2−/− recipients and spleens harvested after 5 wk. Splenocytes were cultured for 48 h with ConA (a) or for 24 and 48 h (b), total RNA extracted, and subjected to RT-PCR analysis for detection of the indicated genes.

Similar articles

Cited by

References

    1. Eisen J A, Sweder K S, Hanawalt P C. Nucleic Acids Res. 1995;23:2715–2723. - PMC - PubMed
    1. Imbalzano A N. Crit Rev Eukaryotic Gene Expression. 1998;8:225–255. - PubMed
    1. Jarvis C D, Geiman T, Vila-Storm M P, Osipovich O, Akella U, Candeias S, Nathan I, Durum S K, Muegge K. Gene. 1996;169:203–207. - PubMed
    1. Geiman T M, Durum S K, Muegge K. Genomics. 1998;54:477–483. - PubMed
    1. Haks M C, Oosterwegel M A, Blom B, Spits H M, Kruisbeek A M. Semin Immunol. 1999;11:23–37. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources