Monoallelic Germline <i>TSC1</i> Mutations Are Permissive for T Lymphocyte Development and Homeostasis in Tuberous Sclerosis Complex Individuals

<div><p>Germline and somatic biallelic mutations of the Tuberous sclerosis complex (<i>TSC</i>) <i>1</i> and <i>TSC2</i> gene products cause TSC, an autosomal dominant multifocal hamartomatosis with variable neurological manifestations. The consequences of TSC1 or TSC2 loss in cells of hematopoietic origin have recently started to be unveiled in mice and showed to hinder the development of proper T cell immunity. To date, the consequences of germline <i>TSC1</i> mutations and/or its loss in mature human T cells remain to be determined. To address these issues, we analyzed subset representation, phenotype and responsiveness to mitogens in T cells from patients with inherited monoallelic <i>TSC1</i> mutations, and induced shRNA-mediated TSC1 down-regulation in primary and transformed human T cells. We report that, the distribution of peripheral CD4 and CD8 T cell subsets, their cytokine-secretion profile, and responsiveness to <i>in vitro</i> stimulation were largely preserved in TSC subjects with monoallelic <i>TSC1</i> germline mutations when compared to healthy controls. Sufficient levels of hamartin and tuberin and proper control of mTOR-dependent signaling in primary T cells from TSC subjects best explained this. In contrast, shRNA-induced down-regulation of <i>TSC1</i>, likely mimicking biallelic inactivation of <i>TSC1</i>, compromised hamartin and tuberin expression and mTORC2/AKT/FoxO1/3 signaling causing both primary and transformed T cells to die by apoptosis. Thus, our results indicate that, while one functional <i>TSC1</i> allele preserves human T lymphocytes development and homeostasis, TSC1 acute down-regulation is detrimental to the survival of both primary and transformed T cells.</p></div

Analysis of apoptosis of thymocytes of Pbx1NT TG mice.

<p>Purified DN thymocytes from Pbx1ΝΤ TG mice and their wild-type controls were stained with PE-conjugated anti-CD44, APC-conjugated anti-CD25, FITC-conjugated annexin V, and 7AAD prior to FACS analysis. The percentage of annexinV-positive 7AAD-negative DN3 and DN4 thymocytes <i>ex vivo</i> (0h) and after 24 hours of incubation at 37°C (24 h) is shown. The cells were from wild-type mice (open bars) or from Pbx1ΝΤ TG mice (black bars). Values that were significantly different are indicated as follows: *, P<0.05.</p

T cells from TSC subjects proliferate and survive to similar extents in response to mitogenic signals.

<p>A) Freshly isolated PBMC of four healthy donors (HD, white bars) and four TSC subjects (Pt, black bars) were left unstimulated (0) or activated on plate-coated anti-CD3 and anti-CD28 mAb or PHA and IL-2 for 4 days. Proliferation was assessed by a 18 h-³H-thymidine incorporation assay. Data depict mean ³H-thymidine incorporation ± SEM. B) freshly isolated PBMC (1 HD, 2 TSC1 Pts) were activated on anti-CD3/CD28 coated beads for 6 days. Thereafter, T cells were isolated and maintained in culture with IL-7 and IL-15 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091952#s4" target="_blank">methods</a>). At the indicated times, viable counts were obtained. Data are depicted as fold expansion over input and represent one out of two independent determinations. C) Freshly isolated frozen PBMC were thawed and cultured overnight in complete medium. Viable cells were counted. Data are expressed as percentage of input and represent the mean of 10 HD and 4 TSC1 Pts (± SEM) analyzed in independent experiments. D) Frozen CD3⁺ cell lines obtained from 2 HD and 3 TSC1 Pts were thawed, and cultured (2×10∧6/ml) in the absence (-) or presence of IL-7 and IL-15 (5 ng/ml). After 24 h cells were analyzed by flow cytometry. CD3⁺, Annexin V⁺ cells are depicted.</p

Correction: Monoallelic Germline <i>TSC1</i> Mutations Are Permissive for T Lymphocyte Development and Homeostasis in Tuberous Sclerosis Complex Individuals

Correction: Monoallelic Germline TSC1 Mutations Are Permissive for T Lymphocyte Development and Homeostasis in Tuberous Sclerosis Complex Individual

shRNA-assisted TSC1 knock down hinders mTORC2 dependent regulation of FoxO1/3.

<p>Human PBMC from 10 independent healthy subjects were activated with anti-CD3 mAb in the presence of IL-2, IL-7 and IL-15 for 48 hours, and transduced with lentiviral vectors overnight (shTSC1). A day after cells were selected for additional 4 days in Puromycin. By then, no viable cells could be recovered from untransduced Puromycin-treated cells. Viable cells in Ctrl and shTSC1 cultures were separated on a Ficoll gradient, counted and re-plated in fresh complete medium supplied with IL-2, IL-7 and IL-15. A) Schematic representation of T cell transduction/selection. B–E) Following viable cell selection at day 5, cells were analyzed by WB (B–D) and real time PCR (E). In B, D cells were left untreated, while in C they were left untreated (-) or stimulated (+) in the presence of anti-CD3 (2 ug/ml) and anti-CD28 (5 ug/ml) coated mAb for 30 min, lysed and analyzed by WB. Representative images (B–C) and quantification (D) of 3-6 independent determinations are depicted. Relative levels (± SEM) refer to expression of given proteins normalized to Actin first, and then to control cells. In E, expression of selected FoxO target genes was normalized to the housekeeping gene (<i>GAPDH</i>) and expressed relatively to control cells by the ΔΔCt method. Results depict 4–5 independent determinations. Statistical significance was evaluated by paired, two-tailed Student t-Test.</p

Analysis of proliferation of thymocytes of Pbx1NT TG mice.

<p>Percentage of BrdU-positive DN1, DN2, DN3 and DN4 thymocytes after <i>in-vivo</i> BrdU staining (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002424#s2" target="_blank">Matherial and Methods</a>) is shown. The cells were from wild-type mice (open bars) or from Pbx1NT TG mice (black bars). (B) Cell cycle analysis of electronically gated DN2, DN3 and DN4 was performed with BrdU-flow kit (BD Biosciences, San Jose, CA) according to the manufacturer's recommendations. The cells were from wild-type mice (open bars) or from Pbx1ΝΤ TG mice (black bars).</p

TSC subjects with germline mutation of <i>TSC1</i> reveal proper representation of CD4 and CD8 mature T cell subsets.

<p>PBMC from healthy donors and TSC subjects were isolated by density gradient centrifugation, counted and surface stained with anti-CD3, CD4, CD8, CD45RA and CD27 mAb and analyzed by FACS. A–B) Viable cell counts per ml of blood and the frequency of CD4⁺ and CD8⁺ cells within CD3⁺ lymphocytes is depicted for individual subjects. Open squares refers to Pt1 and 2 carrying the Leu129Pro mutation, black squares refers to Pt3 and 4 carrying a stop codon mutation in the position of Arg692. C–E) Distribution of naïve (Naïve, CD45RA^highCD27^high), central memory (CM, CD45RA^lowCD27^high), effector memory (EM, CD45RA^lowCD27^low) and terminally differentiated effectors (EF, CD45RA^highCD27^low) within CD3⁺CD4⁺ (C, D) or CD3⁺CD8⁺ (E) cells is depicted. F–G) Freshly isolated PBMC were stimulated for 4 h with PMA and Ionomycin. Expression of IL-2 and IFN-γ was determined by intracellular cytokine staining. Results depict the frequency of cytokine secreting cells after gating on CD3⁺CD4⁺ (F) or CD3⁺CD8⁺ (G) cells (2 HD and Pt3, 4). Cytokine production was also analyzed after a 5d CD3/CD28-driven culture for 8 HD and the 4 TSC1 Pts with no differences (not shown). H) The frequency of CD3⁺CD4⁺CD25⁺FoxP3⁺ cells is shown. Data depicted in D, E, H reflect the mean ± SEM of 8 HD and 4 TSC1 Pts. Statistical significance was evaluated by unpaired two-tailed Student t-Test (A–B, F–H) or Mann-Whitney test (D–E).</p

mTOR-dependent signaling is preserved in human T cells with monoallelic germline <i>TSC1</i> mutations.

<p>A–D) Human CD3⁺ lines derived from healthy donors (HD) and TSC1 subjects (Pt1-4) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091952#s4" target="_blank">methods</a>) were left untreated (-) or stimulated with anti-CD3 and CD28 mAb for 30 min (+). Where indicated cells were pretreated for 30 min with Rapamycin and then stimulated with anti-CD3/CD28 mAb (+R). Cell extracts were analyzed with the indicated antibody. Arrows indicate the specific bands. E-F) Cell size was determined in fresh PBMC isolated from 8 HD and 4 TSC1 Pts by FACS and is depicted after gating on CD4⁺ or CD8⁺ cells. In E a representative overlay is depicted, while data in F represent the average cell size ± SEM.</p

Flow cytometric analysis of thymocytes from Pbx1NT TG mice (TG) and their wild-type controls (WT).

<p>(A) Thymocytes were stained with PE-conjugated anti-CD4, CyChrome-conjugated anti-CD8, and FITC-conjugated anti-TCRβ antibodies. Viable cells were gated according to their forward scatter (FCS) and side scatter (SCS) characteristics. One representative of 8 independent experiments is shown. (B) Absolute numbers of DN, DP and SP (CD4⁺ and CD8⁺) cells. The cells were from wild-type mice (open bars) or from Pbx1NT TG mice (black bars). (C) Flow cytometric analysis of DN (CD4⁻CD8⁻) thymocytes from Pbx1NT TG mice and their wild-type controls. DN cells were enriched by rabbit anti-CD4 and anti-CD8 complement-mediated lysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002424#s2" target="_blank">Materials and Methods</a>). DN thymocytes were stained with PE-conjugated anti-CD44, APC-conjugated anti-CD25 antibodies and CyChrome-conjugated anti-CD4 and anti-CD8 antibodies to negatively gate residual CD4 and CD8 -positive cells. Viable cells were gated according to their FCS and SCS characteristics. One representative of 8 independent experiments is shown. (D) Absolute numbers of DN1, DN2, DN3, and DN4 within DN cells. The cells were from wild-type mice (open bars) or from Pbx1ΝΤ TG mice (black bars). Values that were significantly different are indicated as follows: *, P<0.05; ***, P<0.001. (E) The same as in (C) for TN (CD4⁻CD8⁻CD3⁻) thymocytes. (F) The same as in (D) for TN thymocytes.</p

T cell populations in wild-type (wt) and <i>Pbx1NT</i> (tg) mice

<p>Data are means (bold)±s.e.m. (in parenthesis)</p>*<p>million of cells per organ</p><p>N.D. non determined</p