Tryptophan starvation inhibits T-cell proliferation through activation of GCN2 and induction of ER-stress.

(A) Proliferation of αCD3/CD28-activated CFSE labelled PBMC co-cultured with either bmMSC or dpMSC in the presence of L-1-MT, D-1-MT, indomethacin, or TGF-β neutralizing antibody (n = 6). (B) Proliferation of αCD3/CD28-activated CFSE labelled CD4+ T-cells cultured in 100% non-activated dpMSC conditioned medium (CM) or IFN-γ activated dpMSC conditioned medium (γCM), supplemented with Kynurenine (Kyn) or Tryptophan (Trp) (n = C) αCD3/CD28-activated CD4+ T-cells cultured in 100% γCM supplemented with increasing concentrations of Tryptophan (Trp). Proliferation was determined by CFSE dilution (n = 4). (D) Representative immunoblot of phopsho-mTOR (pmTOR) in αCD3/CD28-activated CD4+ T-cells (72 hours) cultured with Rapamycin (Rapa), Tryptophan (Trp) free conditions or in the presence of dpMSC. (E) Representative histograms showing CFSE proliferation of αCD3/CD28-activated CD4+ T-cells (72 hours) cultured with Rapamycin, conditioned medium γCM or with dpMSC. Data are pooled from at least two independent experiments. (F) Flow cytometry analysis of phospho-ATF2 (MFI) in αCD3/CD28-activated CD4+ T cells cultured for 48 and 72 hours in the presence or not of dpMSC. (White) = non-activated T cells; (Blue) = αCD3/CD28; (Orange) = dpMSC.</p

Tryptophan starvation inhibits T-cell proliferation through activation of GCN2 and induction of the cellular stress response.

(A) Representative histograms showing the proliferation of αCD3/CD28-activated CFSE-labelled CD4+ T-cells co-cultured with dpMSC, conditioning medium from IFNγ licenced IDO+ dpMSC cultures (γCM) with or without Tryptophan (Trp), Rapamycin (Rapa), or in the presence of the ER-stress inducer Tunicamycin (Tun). Data are representative of at least two independent experiments. (B) Representative immunoblot showing the expression of pGCN2 and ATF4 in αCD3/CD28-activated CD4+ T-cells upon direct co-culture with dpMSC, Rapamycin (Rapa), CM, γCM in the presence or absence of Tryptophan (Trp) or in the presence of the ER-stress inducer Tunicamycin (Tun). (C) Proliferation of αCD3/CD28-activated CD4+ T-cells, as measured by CFSE dilution, in the presence of varying concentrations of three ER-stress inducers (Bref-A, Thap, DNJ). Data are pooled from at least two independent experiments. (D) Representative immunoblot showing the expression of ATF4 in αCD3/CD28-activated CD4+ T-cells in response to ER-stress inducing compounds at concentrations that straddle their inhibition of T-cell proliferation.</p

Mouse MSCs inhibit T-cell proliferation via NO production and induction of cellular stress response.

(A) Proliferation of αCD3/CD28-activated CFSE-labelled CD4+ T-cells cultured in direct contact with mMSC ± iNOS inhibitor LNMMA (n = 4). (B) Representative immunoblot and (C) quantification of GCN2 phosphorylation (pGCN2) in αCD3/CD28-activated CD4+ T-cells co-cultured with mMSC or mMSC in the presence of L-Arg. (D) Proliferation of αCD3/CD28-activated CFSE-labelled CD4+ T-cells cultured in 100% non-activated or IFN-γ/TNF-α activated mMSC-conditioned medium.</p

Mesenchymal stem cells inhibit T-cell function through conserved induction of cellular stress

The physiological role of mesenchymal stem cells (MSCs) is to provide a source of cells to replace mesenchymal-derivatives in stromal tissues with high cell turnover or following stromal tissue damage to elicit repair. Human MSCs have been shown to suppress in vitro T-cell responses via a number of mechanisms including indoleamine 2,3-dioxygenase (IDO). This immunomodulatory capacity is likely to be related to their in vivo function in tissue repair where local, transient suppression of immune responses would benefit differentiation. Further understanding of the impact of locally modulated immune responses by MSCs is hampered by evidence that IDO is not produced or utilized by mouse MSCs. In this study, we demonstrate that IDO-mediated tryptophan starvation triggered by human MSCs inhibits T-cell activation and proliferation through induction of cellular stress. Significantly, we show that despite utilizing different means, immunomodulation of murine T-cells also involves cellular stress and thus is a common strategy of immunoregulation conserved between mouse and humans.</div

Cellular stress inhibits T-cell activation.

(A) Flow cytometry analysis of T-cell activation marker expression (MFI) in αCD3/CD28-activated CD4+ T-cells cultured alone or in the presence of dpMSC (n = 4). (B) Representative histograms showing T-cell activation marker expression at 72 hours in αCD3/CD28 activated CD4+ T-cells cultured alone, in the presence of dpMSC, Tunicamycin (Tun) 100% IFNγ-activated dpMSC CM or in IFNγ activated dpMSC supplemented with tryptophan and rapamycin (Rapa). (C) Principal component analysis of activated cell subset phenotypes in CD4+ T cells cultured under the conditions indicated.</p

Components in hemidesmosome and Sox18 protein in <i>Ra</i> mice.

<p>TEM image of wild-type hemidesmosomes in the oral cavity (arrows in A). Schematic representation of molecular organization of basement membrane zone including hemidesmosomes (B). (C) Schematic representation of the Sox18 proteins in <i>Ra</i> mice. The numbering indicates the amino acid coordiates of the represented boxes.</p

<i>Sox7</i> and <i>Sox17</i> expression in developing heads.

<p>Expression of <i>Sox7</i> (A, C) and <i>Sox17</i> (B, D) in heads at E11.5 (A, B) and E12.5 (C, D). Radioactive <i>in situ</i> hybridisation on wild type frontal sections. Tooth germ epithelium is outlined in red. E; RT-PCR analysis showed the presence of <i>Sox17</i> and <i>Sox7</i> expression and absence of <i>Msx1</i> or <i>Lhx7</i> in epithelium. Epi, total RNA extracted from only epithelium of mandible; Epi+Mesen, total RNA extracted from whole mandibles.</p

mRNA level in <i>Ra^op</i> mice.

<p>Quantitative PCR analysis of Plectin (upper) and integrin β4 (lower) expression level. <i>Ra^opHE</i>; heterozygous <i>Ra^op</i> mice. <i>Ra^opHO</i>; homozygous <i>Ra^op</i> mice.</p

<i>Sox18</i> expression in head.

<p>Radioactive <i>in situ</i> hybridisation showed strong expression of <i>Sox18</i> in mesenchyme (m) at E9.5 (B), E10.5 (D), E11.5 (F) and E13.5 (G) of wild-type. Insets of A, C and E represent histological images of B, D and F, respectively. Arrows indicate mandible (B–F). A, B; Sagittal section, C–K; Frontal sections. B, D, F; Epithelia of jaws outlined by red dots. (G) <i>Sox18</i> expression is observed outside of condensed mesenchyme of tooth germs (arrowheads) and the buccal regions of maxillary jaws (arrows). (H, I) Blood cells were observed at the buccal side of maxillary mesenchyme (arrows in H) and outside the condensed mesenchyme of tooth germs (arrowheads in I). von-Willbrand factor proteins were detected in the same regions (arrowheads and arrows in J). The endothelial marker gene, <i>Claudin 5</i> showed a similar expression pattern to <i>Sox18</i> (arrowheads and arrows in K). (G, J, K) Tooth germ epithelium is outlined by red dots. L; RT-PCR analysis showed the presence of <i>Sox18</i> expression and absence of <i>Msx1</i> or <i>Lhx7</i> in epithelium. Epi, total RNA extracted from only epithelium of mandible; Epi+Mesen, total RNA extracted from whole mandibles. Scale bars: 100 µm (C–F); 125 µm (G, I–K).</p

The epithelial phenotype of <i>Ra^op</i> mice.

<p>Normal attached epithelium was observed in wild-type head (A, C, E, G). By contrast detached epithelium was observed in the skin of the snout (B), the mucosa of the incisor region (D), molar region (F), diastema (H) and the skin of trunk (L) of <i>Ra^op</i> mice. (D, F) Arrowheads indicate tooth germs. E12.5 (A–D, I, J) and E14.5 (E–H, K, L). Detachment of epithelium was found before histological processing (green arrowheads in J). Scale bars: 125 µm (C, D); 300 µm (E–H).</p