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

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

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

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

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

shRNA-assisted TSC1 knock down hinders survival of primary human T cells.

<p>shTSC1 cells were obtained by lentiviral-mediated infection and Puromycin selection as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091952#pone-0091952-g004" target="_blank">Figure 4A</a>. Control cells and TSC1 knock down cells were harvested at day 8 (three days after Ficoll) and stained with the Mitotracker Orange dye, or with Annexin V and analyzed by FACS (A and D, respectively), or lysed and analyzed by WB (B–C). In A and D, histograms are representative of 4 independent determinations. E) Cell viability of control cells and cells infected with shScrambled, shTSC1 and shTSC1a-recombinant lentiviral vectors was determined over time by Trypan blue counts. Data are depicted as fold changes over the input, and are representative of mean ± SEM of four independently performed experiments. Statistical significance at day 11 was evaluated by unpaired two-tailed Student t-Test. F) TSC1 levels ± SEM of Ctr, shScrambled, shTSC1 and shTSC1a-infected cells were analyzed by WB analysis in four independent determinations. Statistical significance was evaluated by unpaired two-tailed Student t-Test.</p

Supplementary Data from Targeting Tumor Vasculature with TNF Leads Effector T Cells to the Tumor and Enhances Therapeutic Efficacy of Immune Checkpoint Blockers in Combination with Adoptive Cell Therapy

5 supplementary figures Figure S1. ICB does not prolong survival of TRAMP mice. A) Kaplan-Meier survival curve of 16-18 week-old TRAMP mice untreated (Untreated; dotted line; n=14), or treated with isotype specific antibodies (IgG; gray line; n=5), or ICB (green line; n=5). B) Sixteen-18 week old TRAMP mice received ICB and NGR-TNF. The urogenital organs were recovered at day 40. Coded sections were analyzed by H&E and scored according to signs of complete regression (CR; black area), partial regression (PR; gray area), or no regression (NR; white area). The pie graph shows that all mice (n=3) were scored as NR. Figure S2. Phenotype and functional activity of ACT-derived CD8+ T cells infiltrating melanomas. Mice bearing a 7-day-old B16-OVA melanoma were randomly assigned to the treatments described in Fig. 2. Mice were killed when the mean tumor diameter reached approximately 4 mm. Single cell suspensions from each tumor were analyzed by flow cytometry. The panels show representative contour plots of cells gated on ACT-derived CD3+ CD8+ Vβ5.1+ Vα2+ cells, and analysed for expression of CD44 and CD62L (A), CD44 and PD1 (B), Granzyme B (C) and IFN-γ production (D). Data are representative of two independent experiments Figure S3. Phenotype and functional activity of endogenous CD8+ T cells infiltrating melanomas. Mice bearing a 7-day-old B16-OVA melanoma were randomly assigned to the treatments described in Fig. 2. Mice were killed when the mean tumor diameter reached approximately 4 mm. Single cell suspensions from each tumor were analyzed by flow cytometry. The panels show representative contour plots of cells gated on endogenous CD3+ CD8+ Vβ5.1- Vα2- cells, and analysed for expression of CD44 and CD62L (A), CD44 and PD-1 (B), Granzyme B (C) and IFN-γ production (D). Data are representative of two independent experiments. Figure S4. NGR-TNF improves efficacy of ICB by recruiting endogenous T cells into protective anti-tumor immune responses. Tumors from mice treated as described in the legend to Fig. 3 were processed to single cells and analyzed by flow cytometry. Graphs report percentage (mean {plus minus} SEM;) of OT1 cells and endogenous CD8+ T cell within CD8+ T cells (A, F respectively), and percentages (mean {plus minus} SEM) of CD44+ CD62L- cells (B and G), CD44+ PD-1+ cells (C and H), Granzyme B+ cells (D and I), and IFN-γ + cells (E and L) within the OT1 (A-E) or the endogenous CD8+ T cell subpopulations (F-L). Each symbol represents an individual mouse. Data are aggregated from two independent experiments. Student t-test: * P < 0.05; * * P < 0.01; * * * P < 0.001; * * * * P < 0.0001; ns, not significant. Figure S5. Therapeutic efficacy of the combination between ICB and NGR-TNF in MC38- bearing mice. A) Schematic representation of the experimental treatment. C57BL/6 mice were subcutaneously challenged with MC38 colon cancer cells (5 x105 /mouse; kindly provided by M.P.Colombo, Milan, Italy). Before injection, Mycoplasma infection was excluded by PCR. When the tumor reached 3 mm in mean diameter, mice were randomly assigned to either one of following treatments: vehicle (PBS; n=9), IgG+NGR-TNF (n=8), anti-CTLA-4 (100 µg/mouse) and anti-PD-1 (250 µg/mouse) antibodies (ICB n=12), or ICB+NGR-TNF (n=12). Mice were followed therefore for tumor growth and survival. B-D) Tumor volume of individual mice treated with PBS (B), IgG+NGR-TNF (C), ICB (D), or ICB+NGR-TNF (E). Each line represents one mouse. F) KaplanMeir plot reporting the survival of the mice treated as described in A. Log-rank test: * P < 0.05; * * P < 0.01; * * * P < 0.001; * * * * P < 0.0001; ns, not significant.</p

Additional file 5: Figure S3. of Metabolic determinants of the immune modulatory function of neural stem cells

Co-culturing with LNC and cytokine priming has no effects on NSC survival. LNC were labelled with the vital dye CFSE and co-cultured with NSC, Th1 NSCs or Th2 NSCs for 72 h as in Additional file 2: Fig. S2. Absolute fractions of CFSE− TO-PRO3+ CD4− cells, as an indicator of dead NSCs are expressed as mean % (±SD) from n ≥ 3 independent experiments

Additional file 1: Figure S1. of Metabolic determinants of the immune modulatory function of neural stem cells

NSCs induce downregulation of the activation cell surface marker CD44 on purified CD4+ T cells. CD4+ T cells were purified by negative selection from total LNC, labelled with the vital dye CFSE, and cultured on CD3/CD28 coated mAb in the absence or the presence of NSCs, Th1 NSCs or Th2 NSCs for 72 h. Cells were surface stained with anti-CD4 and CD44 antibodies. Absolute fractions of CFSEdimCD44low CD4+ T cells are depicted. Data are expressed as mean % (±SD) from n ≥ 3 independent experiments. *p ≤ 0.05 and **p ≤ 0.01 vs. T cells