DLGH1 is a negative regulator of T-lymphocyte proliferation

Abstract

Discs large homolog 1 (DLGH1), a founding member of the membrane-associated guanylate kinase family of proteins containing PostSynaptic Density-95/Discs large/Zona Occludens-1 domains, is an ortholog of the Drosophila tumor suppressor gene Discs large. In the mammalian embryo, DLGH1 is essential for normal urogenital morphogenesis and the development of skeletal and epithelial structures. Recent reports also indicate that DLGH1 may be a critical mediator of signals triggered by the antigen receptor complex in T lymphocytes by functioning as a scaffold coordinating the activities of T-cell receptor (TCR) signaling proteins at the immune synapse. However, it remains unclear if DLGH1 functions to enhance or attenuate signals emanating from the TCR. Here, we used Dlgh1 gene-targeted mice to determine the requirement for DLGH1 in T-cell development and activation. Strikingly, while all major subsets of T cells appear to undergo normal thymic development in the absence of DLGH1, peripheral lymph node Dlgh1(-/-) T cells show a hyper-proliferative response to TCR-induced stimulation. These data indicate that, consistent with the known function of Discs large proteins as tumor suppressors and attenuators of cell division, in T lymphocytes, DLGH1 functions as a negative regulator of TCR-induced proliferative responses.

FIG. 1.

Proliferation of Dlgh1^−/− T lymphocytes. (A and B) Purifed CD8⁺ and CD4⁺ T cells from Rag-2-null mice reconstituted with WT or KO fetal liver cells were stimulated with the indicated concentrations of stimulatory anti-CD3 (μg/ml) with or without the addition of anti-CD28 (1 μg/ml) and analyzed for [³H]thymidine incorporation. Shown are raw cpm values from one experiment, representative of three, all with similar results. The error bars indicate standard deviations. (C and D) Lymph node cells from mice reconstituted with WT or KO fetal liver cells were loaded with CFSE and stimulated with the indicated concentrations of anti-CD3 or anti-CD3 plus anti-CD28 (1 μg/ml). The cells were analyzed 72 h poststimulation. Shown is one representative sample from the indicated CD4⁺ or CD8⁺ gate (n > 5), all with similar results. Shown in bar graphs below the histograms is one representative experiment (in which the lymphocytes were stimulated with 0.1 μg/ml anti-CD3) showing the percentage of cells in each generation peak as calculated from the cell counts per peak.

FIG. 2.

Lymphocyte development in Rag-2-null mice reconstituted with KO fetal liver cells. Single-cell suspensions of thymocytes, splenocytes, or lymph node cells were counted, stained with the indicated fluorescent-antibody conjugates, and analyzed by flow cytometry as described in Materials and Methods. For analyses of CD44, CD62L, and Foxp3, the populations shown are from the indicated CD4 and CD8 gates. The data shown are from a representative experiment (n > 5).

FIG. 3.

Actin accumulation and recruitment of polarity and signaling proteins to the immune synapse in KO lymphocytes. (A to C) Purifed T cells from chimeric mice were incubated with anti-CD3-coated latex beads for 30 min. The cells were stained with phalloidin and antibodies against Scribble, Vav1, and Zap-70, followed by analysis by confocal microscopy. Shown is one experiment, representative of three. In each experiment, >60 conjugates were analyzed. DIC, differential interference contrast.

FIG. 4.

Proximal signaling in KO T lymphocytes. (A) Lymph node cells from RAG chimeric mice were loaded with Fluo-4-AM and then stained with antibodies against CD4 (left) or CD8 (right). Cell suspensions were analyzed by flow cytometry for 20 seconds to determine the background and then for an additional 5 min after anti-CD3 (1 μg/ml) stimulation. Ionomycin was added at 5 min to achieve maximal Ca²⁺ signaling. In each plot, the first arrow indicates the addition of anti-CD3 and the second arrow indicates the addition of ionomycin. (B) Purified T cells from mice reconstituted with WT or KO donor cells were stimulated with 1 μg/ml anti-CD3 for the indicated times. Proteins were detected by Western blotting. Shown is one experiment, representative of three, all with similar results.

FIG. 5.

IL-2 and IFN-γ production from Dlgh1-deficient T lymphocytes. The supernatants from purified T cells stimulated with the indicated concentrations of anti-CD3 antibodies for 24 h were assayed for IL-2 and IFN-γ production. Shown are the mean concentrations plus standard deviations from five experiments.

FIG. 6.

KO CD8⁺ T-cell entry into S phase following anti-CD3 stimulation. Lymph node T cells from mice reconstituted with WT or KO fetal liver cells were stimulated with the indicated concentrations of stimulatory anti-CD3 and anti-CD28 antibodies (1 μg/ml). The cells were stimulated for 18 h, pulsed with BrdU, and stained with antibodies against BrdU. Shown are the mean values plus standard deviations from five experiments.

FIG. 7.

c-myc transcripts in KO CD8⁺ T cells. (A) Semiquantitative reverse transcription-PCR analysis of c-myc induction in purified CD8⁺ T cells from WT- or KO-reconstituted mice stimulated for 16 h with 1 μg/ml anti-CD3 and anti-CD28. Fourfold serial dilutions of cDNA from one representative experiment of three is shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were used for normalization of cDNAs. (B) Quantitative RT-PCR analysis of purified CD8⁺ T cells from WT- or KO-reconstituted mice stimulated for 16 h with 1 μg/ml anti-CD3 and anti-CD28. The data shown are mean values (n = 3) normalized to GAPDH.