Murine dendritic cell rapamycin-resistant and rictor-independent mTOR controls IL-10, B7-H1, and regulatory T-cell induction

Dendritic cells (DCs) are innate professional antigen-presenting cells (APCs) that initiate and regulate adaptive immunity.^1,2  DCs control T-cell reactivity by coordinating display of Ag to T cells in the context of major histocompatibility class molecules with the delivery of costimulation and cytokines that dictate T-cell differentiation and function. Although costimulatory molecules support T-cell responses, coinhibitory molecules restrain T-cell reactivity. Our understanding of the precise molecular mechanisms regulating expression of proinflammatory vs regulatory signals by DCs remains unclear.

B7-homolog 1 (B7-H1, programmed death 1 [PD-1] ligand 1; CD274) is a B7 family coinhibitory molecule expressed on DCs in a regulated manner that binds to PD-1 (CD279) on activated T cells, thereby reducing their proliferation and proinflammatory cytokine production.^3,4  The B7-H1/PD-1 pathway plays a crucial role in the maintenance of peripheral tolerance.⁵  B7-H1 stimulates T-cell secretion of anti-inflammatory interleukin (IL) 10⁶  and promotes the induction, maintenance, and function of regulatory T cells (Tregs) from naive T cells.⁷  Importantly, the precise upstream mechanisms regulating B7-H1 expression remain elusive, and the differential regulation of costimulatory vs coinhibitory molecule expression is poorly understood, despite their central role in the activation and constraint of adaptive T-cell responses by DCs.

Mammalian target of rapamycin (mTOR) is a highly conserved, serine/threonine kinase that controls APC and T-cell function.^8,9  The mTOR kinase performs the catalytic function of 2 independent complexes: mTOR complex (mTORC) 1 and mTORC2.^10,11  mTORC1 consists of mTOR, regulatory associated protein of mTOR (raptor), mammalian lethal with Sec13 protein 8 (mLST8), and proline-rich substrate of Akt of 40 kD (PRAS40), whereas mTORC2 contains mTOR, rapamycin (RAPA)-insensitive companion of mTOR (rictor), mLST8, mSIN1, and protein associated with rictor (PROTOR).¹²  Although RAPA is a potent allosteric inhibitor of mTORC1, it exerts little activity against RAPA-insensitive mTORC2.^10,11  However, novel, highly selective adenosine triphosphate (ATP)–competitive active site mTOR inhibitors that block both mTOR-containing complexes have revealed RAPA-resistant mTORC1 and mTORC2 signaling in nonimmune cells.^13,14  mTORC1 inhibition suppresses conventional DC maturation and promotes their tolerogenicity.^2,8,15  Conversely, RAPA has paradoxical, proinflammatory effects on DCs, including increased secretion of IL-12p70 and IL-1β, with concomitant reduced secretion of IL-10 and expression of B7-H1.^16-21  These effects on DCs are mediated by augmentation of nuclear factor κB (NF-κB) activity and reduction in signal transducer and activator of transcription (STAT) 3 activity.^17,20,21 

RAPA-insensitive mTORC2 regulates the actin cytoskeleton in nonimmune cells,^10,11  and insight is emerging into its function in T lymphocytes. Selective deletion of mTORC2 in T cells impairs their differentiation into T helper (Th) 1 and Th2²²  or only Th2 subsets.²³  In contrast to the well-defined role of mTORC1, little is known about the function of mTORC2 in APCs or innate immunity. In this study, we sought to define the role of RAPA-resistant mTOR in molecular regulation of the ability of DCs to promote T-cell immunity.

We find that RAPA-resistant mTOR negatively regulates conventional DC STAT3-mediated IL-10 and B7-H1 expression. Deletion of the mTORC2 subunit rictor had the opposite effect, suggesting that residual RAPA-resistant mTORC1 activity or dual mTORC1 and 2 inhibition mediates this central anti-inflammatory pathway in DCs. Enhanced STAT3 activation in DCs exposed to ATP-competitive mTOR inhibitors correlated with a reduction in suppressor of cytokine signaling (SOCS) 3. Functionally, mTORC1-inhibited DCs were unable to stimulate proliferation of forkhead box P3 (Foxp3^–) effector T cells (Teffs), whereas ATP-competitive mTOR inhibition additionally promoted the induction of Foxp3⁺ Tregs in a B7-H1–dependent manner that also required IL-1β. These data reveal a novel, RAPA-resistant anti-inflammatory pathway in DCs that regulates IL-10 and B7-H1 and identifies divergent regulation of T_eff and T_reg responses by DCs due to RAPA-sensitive and RAPA-resistant mTOR.

Male C57BL/6 (B6; H2K^b), BALB/c (H2K^d), B6.129S2-Irf1^tm1Mak/J (interferon regulatory factor 1; IRF-1 null), B6.129P2-Il10^tm1Cgn/J (IL-10^−/−), B6.129X1-Ebi3^tm1Rsb/J (Epstein-Barr virus–induced gene 3; Ebi3^−/−), and B6.129S1-Il12b^tm1Jm/J (IL-12/23p40^−/−) mice were purchased from The Jackson Laboratory. Male B6.129S2-Il6^tm1Kopf/J (IL-6^−/−) and IL-10–green fluorescent protein (IL-10-gfp) mice were kindly provided by Dr A. Jake Demetris and Dr David Rothstein, respectively (University of Pittsburgh). Femurs and tibiae from mice containing loxP-flanked O class forkhead box (FoxO) 1, FoxO3, and FoxO4 crossed to mice containing tamoxifen-inducible Cre recombinase under the ROSA26 promoter (FoxO1/3/4^fl/fl × ROSA26-CreERT2) were used to generate bone marrow (BM)–derived DCs. B7-H1^−/− mouse pairs on a B6 background were kindly provided by Dr Lieping Chen (Johns Hopkins University) and bred at the University of Pittsburgh. Mice containing loxP-flanked rictor (rictor^fl/fl) and ROSA26-CreERT2 were maintained at the University of Pittsburgh. WYE-125132 (50 mg/kg²⁴  intraperitoneally; Selleck Chemicals) was dissolved in dimethylsulfoxide (DMSO) and administered in a vehicle composed of 5% Tween 80 and 54% polyethylene glycol (Sigma-Aldrich; average M_n 300) at the time of lipopolysaccharide (LPS) injection (Escherichia coli 0111:B4; Sigma-Aldrich; 100 μg/kg intraperitoneally). The work performed in this study was covered under University of Pittsburgh Institutional Animal Care and Use Committee (IACUC) protocol 1102281.

DCs were generated from BM cells, as described^19,25  using recombinant (r) mouse granulocyte macrophage–colony-stimulating factor and r mouse IL-4 (both 1000 U/mL; R&D Systems). On day 8 of culture, myeloid DCs were selected from nonadherent cells by anti-CD11c immunomagnetic bead purification (Miltenyi Biotec). Torin1¹³  (kindly provided by Dr Nathanael S. Gray [Dana-Farber Cancer Institute] or purchased from Tocris Bioscience), AZD8055²⁶  (Selleck Chemicals), WYE-125132, or RAPA (LC Laboratories) was added to cultures on day 2 at the concentration indicated and refreshed on day 4 and day 6. Where indicated, Toll-like receptor (TLR) 4–specific LPS (Salmonellaminnesota R595; 100 ng/mL; Alexis Biochemicals) was used to stimulate DC cultures on day 7 for 16 to 18 hours. In some experiments, 250 nM STAT3 inhibitor VII (EMD Chemicals) was added on day 7, 2 hours before LPS stimulation.

Cell surface and intracellular staining was performed as described.^19,25  Fluorochrome-conjugated monoclonal antibodies were purchased from eBioscience, BD Bioscience, or Biolegend. P-STAT3 quantification by flow cytometry was performed as described.²⁰  Data were acquired using an LSR II or LSR Fortessa flow cytometer (BD Bioscience) and analyzed using FlowJo 8.8.6 (Tree Star). Percent P-STAT3 positive cells was determined using Overton Subtraction (FlowJo) by comparing experimental samples with corresponding control samples stained with secondary antibody (Ab) only.

ϒ-irradiated (20 Gy) DCs (B6; 1 × 10⁴) were used as stimulators in 5-day allogeneic MLR as described.²⁷  Normal CD4⁺CD25^– BALB/c T-cell responders (1 × 10⁵) were isolated by negative selection and labeled with carboxyfluorescein succinimidyl ester (CFSE) according to the manufacturer’s protocol (Invitrogen). In some experiments, neutralizing Ab was added to cultures at 10 μg/mL (αIL-1β [B122], αIL-10 [JES5-16E3; BD Bioscience], αB7-H1 [MIH5], or αPD-1 [RMP1-14; eBioscience]).

Enzyme-linked immunosorbent assays were performed on cell-free DC supernatants according to the manufacturer’s instructions to quantify IL-12p40 and IL-6 (Biolegend). IL-1β was quantified by cytometric bead array (BD Bioscience) according to the manufacturer’s protocol.

One hundred nanomolar (Z)-4-hydroxytamoxifen (4OHT; Sigma-Aldrich) was added to BM cultures on day 0 to delete FoxO transcription factors from FoxO1/3/4^fl/fl × ROSA26-CreERT2 BM-derived DCs. Tamoxifen (15 mg/mL; Sigma-Aldrich) was administered intraperitoneally in sunflower seed oil (Sigma-Aldrich) every 2 days for 3 doses to rictor^fl/fl × ROSA26-CreERT2 mice. BM cells were harvested from these mice 7 days later and cultured to generate rictor^−/− BM-derived DCs.

Immunoblotting was performed on DC lysates as described^19,25  using primary Abs from Cell Signaling, with the exception of glyceraldehyde-3-phosphate dehydrogenase (Novus Biologicals) and SOCS5 (Thermo Scientific). Densitometry was performed using ImageJ (National Institutes of Health).

Results are expressed as means +1 standard deviation. The unpaired, 2-tailed Student t test was used to determine the significance of differences between means (GraphPad Prism).

We first examined the surface phenotype of B6 BM-derived conventional DCs propagated under conditions of either mTORC1 inhibition with RAPA or mTORC1 and 2 inhibition with the ATP-competitive mTOR inhibitor Torin1. As reported previously for RAPA,¹⁶  DCs differentiated in Torin1 were small and homogenous compared with control DCs (Figure 1A). Although mTOR inhibition did not affect CD11c⁺ DC differentiation (Figure 1C), both RAPA and Torin1 reduced the DC yield from BM cell cultures (Figure 1D). In agreement with previous studies,^17,20  RAPA reduced the expression of CD80, CD86, B7-H1, and B7-DC on both unstimulated and LPS-stimulated DCs (Figure 1B,E; supplemental Figure 1, see the Blood Web site). Although Torin1 also reduced CD80, CD86, and B7-DC expression, by contrast with RAPA, Torin1 selectively and dose-dependently enhanced B7-H1 expression on DCs (Figure 1B,E). Expression of major histocompatibility class molecules, CD40, CD54, CD80, and B7RP-1 was similarly regulated by RAPA and Torin1 (supplemental Figure 1). B7-H3 and B7-H4 were not detected (data not shown). Torin1 reduced CD86 expression similarly to RAPA on human monocyte-derived DCs and CD34⁺ cell-derived DCs; however, B7-H1 expression was spared by Torin1, resulting in significantly higher expression than on RAPA-exposed DCs (supplemental Figure 2).

AZD8055, another ATP-competitive mTOR inhibitor, similarly increased B6 DC B7-H1 expression (Figure 1F; supplemental Figure 3A), thus confirming that B7-H1 upregulation was due to on-target mTOR inhibition. Surprisingly, rictor^−/− DCs lacking mTORC2 activity displayed diminished B7-H1 expression following LPS stimulation (Figure 1G-H). DC differentiation was not affected, whereas the DC yield was reduced slightly in rictor^−/− BM cultures (supplemental Figure 3B-C). Together, these data suggest that RAPA-resistant mTORC1 is a negative regulator of DC B7-H1 expression downstream of TLR4, whereas mTORC2 is a positive regulator of B7-H1 expression.

Further analysis revealed that enhanced B7-H1 expression on Torin1-conditioned DCs occurred predominantly on immature CD86^lo cells (Figure 2A-B). Similarly, AZD8055-conditioned DCs were mainly CD86^loB7-H1^hi (supplemental Figure 3A,D-E). The ratio of coinhibitory (B7-H1) to costimulatory (CD86) B7 family molecule expression was enhanced on Torin1- and AZD8055-conditioned DCs to a greater extent than on RAPA-conditioned DCs, with or without LPS stimulation (Figure 2C; supplemental Figure 3F). Human monocyte-derived DCs, but not CD34⁺ cell-derived DCs, differentiated in Torin1 exhibited a trend toward an elevated ratio of B7-H1 to CD86 expression compared with those differentiated in RAPA (supplemental Figure 2C,F).

The B7-H1 promoter contains 2 IRF-1 binding sites that are required for constitutive expression and interferon ϒ–induced upregulation of B7-H1 in cancer cell lines.²⁸  We analyzed B7-H1 expression on wild-type and IRF-1–null Torin1-conditioned DCs, but IRF-1 was not required for augmented DC B7-H1 expression (Figure 3A-B; supplemental Figure 4A-C).

STAT3 binds the B7-H1 promoter and is required for B7-H1 expression by lymphoma cell lines.²⁹  Importantly, STAT3 has also been identified as a critical regulator of B7-H1 on human APC.³⁰  STAT3 inhibition reversed enhanced B7-H1 expression (Figure 3C-D), eliminated concomitant CD86^loB7-H1^hi expression (supplemental Figure 4D-E), and reduced the B7-H1/CD86 ratio on Torin1-conditioned DCs (supplemental Figure 4F). These data demonstrate that enhanced DC B7-H1 expression induced by Torin1 occurs through an IRF-1–independent and STAT3-dependent pathway.

FoxO transcription factors mediate immune homeostasis³¹  and control the ability of DCs to limit T-cell expansion following viral infection.³²  FoxO promotes STAT3 activity, and FoxO3 transcription correlates with elevated B7-H1 expression by tumor-associated DCs.³³  Torin1-conditioned DCs were generated from FoxO1/3/4^−/− BM cells by exposing FoxO1/3/4^fl/fl × ROSA26-CreERT2 BM cells to 4OHT.³⁴  Deletion of FoxO3a was confirmed by immunoblot (data not shown). FoxO was not required for B7-H1 upregulation by Torin1 (Figure 3E-F), thus suggesting a STAT3-dependent but FoxO-independent mechanism of RAPA-resistant mTOR negative regulation of B7-H1.

Studies with RAPA have implicated mTORC1 as a positive regulator of STAT3 in DCs^17,20 ; however, reduced IL-10 secretion by RAPA-exposed DCs leads to reduced autocrine STAT3 activation.²¹  DCs were preincubated with RAPA or Torin1 for 2 hours before LPS stimulation (hereby referred to as “short-term” mTOR inhibition). As reported,¹²  RAPA abolished mTORC1 signaling (P-S6K T389), but only Torin1 blocked mTORC2 signaling (P-Akt S473; Figure 4A). Both short-term RAPA and Torin1 inhibited STAT3 phosphorylation following LPS stimulation (Figure 4B-C). In contrast, DC differentiated in the presence of Torin1, but not RAPA, from day 2 to day 8 (hereby referred to as “RAPA- or Torin1-conditioned DC”) demonstrated augmented STAT3 phosphorylation 3 hours after LPS stimulation (Figure 4D-F). SOCS proteins regulate cytokine signaling by inhibiting Janus kinase (JAK)–STAT pathways.³⁵  RAPA conditioning led to augmented SOCS1 and SOCS5, which are negative regulators of STAT1³⁶  and STAT6,³⁷  respectively (Figure 4G). Torin1 suppressed expression of the key STAT3 negative regulator SOCS3 (Figure 4E,G).³⁸  As such, we make the novel observation that extended mTORC1 and 2 inhibition by Torin1, but not mTORC1 inhibition by RAPA, downregulates SOCS3 and enhances STAT3 signaling.

Rictor^−/− DCs were unable to phosphorylate Akt S473; however, mTORC1 signaling was intact (Figure 4H). These cells exhibited diminished P-STAT3 following LPS stimulation, and there was no change in SOCS3 expression (Figure 4I-J). Importantly, these data establish that RAPA-resistant mTORC1 negatively regulates STAT3, whereas mTORC2 is a positive regulator of STAT3.

STAT3 phosphorylation occurs late (1-3 hours) after LPS stimulation of DCs (Figure 4), suggesting autocrine signaling that induces phosphorylation. Autocrine IL-10 signaling is required for STAT3 phosphorylation following LPS stimulation.²¹  IL-10 production was increased markedly in B7-H1^hi Torin1-conditioned IL-10-gfp reporter DCs (Figure 5A,C). The IL-10 mean fluorescence intensity (MFI) was increased in Torin1-conditioned DCs compared with RAPA-conditioned and control DCs (Figure 5B,D). Both RAPA- and Torin1-conditioned DCs secreted increased IL-12/23p40 (Figure 5E). Rictor^−/− DCs also secreted elevated IL-12p40 (data not shown). IL-6 and IL-1β secretion by DCs after LPS stimulation was reduced and increased, respectively, by RAPA, but unaffected by Torin1 (Figure 5F-G).

IL-6, IL-10, IL-23, and IL-27 all stimulate STAT3 phosphorylation.^38-40  Neither autocrine IL-6 nor IL-10 was required for enhanced B7-H1 expression by Torin1 on control or LPS-stimulated DCs (Figure 5H-K). Autocrine IL-23 and IL-27 were also not required, as determined using IL-12/23p40^−/− and Ebi3^−/− BM cell cultures, respectively (supplemental Figure 5A-D). Collectively, these results identify a RAPA-resistant mTOR pathway that coregulates B7-H1 and IL-10 expression that does not depend on autocrine cytokine stimulation.

We next sought to determine how ATP-competitive mTOR inhibition modulates the T-cell stimulatory function of DCs. RAPA reduces the T-cell stimulatory capacity of DCs.^{15,16,19,27,41}  Torin1 shared this ability, although its effect was less than that of RAPA (Figure 6A-B). Although B7-H1 has been shown to restrain T-cell proliferation,³  B7-H1^−/− RAPA-DCs that express low CD80 and CD86 were less stimulatory than wild-type DCs (Figure 6A-B). These data are in agreement with early reports describing a T-cell proliferation–promoting effect of B7-H1.⁶ 

B7-H1 is a key regulator of T_reg induction and function.⁷  Because Torin1-conditioned DCs express elevated levels of B7-H1, we determined their ability to induce Tregs (Figure 6C-F). Torin1-conditioned DCs induced a significantly greater frequency of Tregs compared with control and RAPA-conditioned DCs (Figure 6C-D). Enhanced induction of Tregs by Torin1-conditioned DCs was dependent on B7-H1 (Figure 6C-D) but independent of PD-1 (supplemental Figure 6A-B). Although Torin1-conditioned DCs made increased IL-10 (Figure 5A-D), it was not required for their ability to induce Tregs (supplemental Figure 6A-B). Neither genetic deletion nor neutralization of B7-H1 reduced T_reg induction by control DCs (Figure 6C-D; supplemental Figure 6A-B). The absolute number of Tregs induced by RAPA-conditioned DCs was reduced dramatically compared with control (supplemental Figure 6C). Torin1-conditioned DCs induced a greater number of Tregs compared with RAPA-conditioned DCs, which was reduced in the absence of B7-H1 (supplemental Figure 6C).

CFSE dilution profiles of Foxp3⁺ and Foxp3^– T cells were used to determine the relative contribution of inhibition of T_eff (Foxp3^–) proliferation to induction of Tregs (Figure 6E-F). Consistent with our previous report,²⁷  RAPA-conditioned DCs stimulated Teffs poorly but stimulated CFSE^loFoxp3⁺ T cells similarly to control DCs (Figure 6E-F). Torin1-conditioned DCs also stimulated Teffs less than control DCs; however, they approximately doubled the frequency of CFSE^loFoxp3⁺ T cells (Figure 6E-F). These data demonstrate that ATP-competitive mTOR inhibition in DCs enhances their ability to induce Tregs in a B7-H1–dependent manner and augment CFSE^loFoxp3⁺ T cells, whereas RAPA-conditioned DCs specifically reduce proliferation of Teffs without affecting CFSE^loFoxp3⁺ T cells.

There is evidence that IL-1β promotes T_reg induction⁴²  and mTORC1 regulates IL-1β production by DCs.^16,18  Although Torin1 did not alter IL-1β secretion by DCs (Figure 5G), neutralization of IL-1β suppressed T_reg induction by Torin1-conditioned DCs (Figure 6G-H). T_reg induction and augmentation of CFSE^loFoxp3⁺ T cells were further reduced when IL-1β–inhibited Torin1-conditioned DCs were B7-H1 deficient (Figure 6G-H; supplemental Figure 6D-F). These data suggest that B7-H1 and IL-1β act cooperatively to promote T_reg induction by Torin1-conditioned DCs.

To determine if RAPA-resistant mTOR modulates DC B7-H1 expression in a more physiological setting, we investigated in vivo modulation of DCs by ATP-competitive mTOR inhibition. WYE-125132 is an ATP-competitive mTOR inhibitor with in vivo bioavailability.²⁴  We first verified that WYE-125132 augmented DC B7-H1 expression while reducing CD86 expression in vitro (Figure 7A-D). When given as a single dose at the time of LPS administration, WYE-125132 enhanced splenic DC B7-H1 expression while reducing CD86 expression (Figure 7E-G). Furthermore, the ratio of B7-H1 to CD86 expression was increased (Figure 7H). These DCs augmented T_reg induction ex vivo compared with control DCs (Figure 7I-J). Together, these findings confirm that RAPA-resistant mTOR controls B7-H1 expression and modulates the ability of DCs to induce Tregs in vivo.

Herein we describe a novel RAPA-resistant and mTORC2-independent signaling pathway in conventional DCs that controls B7-H1 and IL-10 expression downstream of TLR4. This novel signaling network coordinates the ability of DCs to stimulate T_eff and T_reg responses. DCs conditioned in ATP-competitive mTOR inhibitors dose-dependently and selectively upregulated B7-H1 expression. Elevated IL-10 production by Torin1-conditioned DCs was found in a B7-H1^hi population. B7-H1 upregulation by Torin1 was STAT3 dependent but did not require FoxO1/3/4 or autocrine IL-6, IL-10, IL-12/23, or IL-27 signaling. Augmented STAT3 phosphorylation correlated with a reduction in SOCS3 expression. Rictor^−/− DCs did not exhibit augmented STAT3 phosphorylation or B7-H1 expression. ATP-competitive mTOR inhibition resulted in DCs that markedly enhanced B7-H1–dependent but PD-1–independent T_reg induction in the absence of exogenous TGFβ. IL-1β, but not IL-10, was required for enhanced T_reg induction by DCs conditioned in Torin1 and acted additively with B7-H1. Together, these findings establish how distinct mTORC signaling coordinates to regulate DC stimulatory function at a molecular level.

mTOR is a central regulator of T-cell function⁹  that is targeted for clinical immunosuppression, yet the impact of mTOR, particularly RAPA-resistant mTOR, on innate immune cells is poorly understood.^8,43  RAPA is a potent and selective mTORC1 inhibitor; however, prolonged exposure can inhibit mTORC2 by preventing its assembly in certain cell types.⁴⁴  After verifying the mTORC specificity of RAPA and ATP-competitive mTOR inhibitors, we used this strategy to dissect the function of RAPA-sensitive and RAPA-resistant pathways in conventional DCs. RAPA-sensitive mTOR (mTORC1) promoted costimulatory CD80 and CD86 expression and reduced IL-12 secretion. RAPA inhibits phosphorylation of inhibitory residues on glycogen synthase kinase 3 leading to NF-κB p65 activation and increased IL-12 secretion after TLR4 ligation.^17,19-21,45  Our data support these findings where RAPA-sensitive mTORC1 suppresses IL-12 production. A recent study demonstrated a similar role for mTORC2 in DCs following LPS activation where rictor knockdown enhanced proinflammatory cytokine and reduced IL-10 secretion. However, elevated IL-12 secretion in these cells did not require NF-κB but was due to hyperactive FoxO1.⁴⁶  The present study identifies, for the first time, a RAPA-resistant mTORC1 pathway that is critical for controlling the immune regulatory properties of DCs and further elaborates on signaling events downstream of TLR4.

ATP-competitive mTOR inhibition promoted DC IL-10 and B7-H1 expression and enhanced STAT3 activation. Enhanced STAT3 activation and reduced SOCS3 expression were only seen following prolonged ATP-competitive mTOR inhibition. These data demonstrate that the kinetics of mTOR inhibition (short-term exposure vs long-term conditioning), in addition to the complexes being targeted, are critical to understanding the function of mTOR in DCs. Initial publications describing ATP-competitive mTOR inhibitors revealed, unexpectedly, that RAPA did not inhibit mTORC1 completely; however, ATP-competitive mTOR inhibitors fully inhibited residual, RAPA-resistant mTORC1.^13,14  To our knowledge, the present report describes the first RAPA-resistant mTOR pathway that is not dependent on RAPA-sensitive mTORC1. Contrastingly, RAPA produces the opposite effects, whereby B7-H1 and IL-10 are diminished. Raptor-deficient DCs lacking RAPA-sensitive and RAPA-resistant mTORC1 activity display reduced IL-10 production.⁴⁷  Extended exposure of rictor^−/− DCs to RAPA was not sufficient to upregulate B7-H1 (data not shown). Our findings can therefore be ascribed to a novel, RAPA-resistant mTORC1 pathway that is subordinate to RAPA-sensitive mTORC1 or to concomitant inhibition of RAPA-resistant mTORC1 and mTORC2.

Our data highlight the distinct function of mTOR-containing complexes in DCs when compared with T cells. mTOR-deficient T cells show reduced STAT3 phosphorylation,⁴⁸  similarly to DCs exposed to short-term mTOR inhibition. However, extended exposure of DCs to ATP-competitive mTOR inhibition augmented STAT3 activation. Although rictor^−/− DCs demonstrate reduced STAT3 activation, rictor^−/− T cells exhibit normal STAT3 activation.^22,23  Furthermore, resting rictor^−/− T cells express reduced SOCS3.²³  Our data show that extended ATP-competitive mTOR inhibitor conditioning reduces DC SOCS3 expression, but rictor deletion has no effect on SOCS3 in DCs. These apparent inconsistencies support how mTOR signaling occurs differentially in DCs and T cells.

Evidence is accumulating for a role of mTOR in DCs in shaping T-cell responses. Our data demonstrate that, although RAPA-sensitive mTORC1 promotes costimulatory molecule expression and Foxp3^– T_eff proliferation, RAPA-resistant and rictor-independent mTOR reduces IL-10 secretion and restrains T_reg induction by downregulating coinhibitory B7-H1. Interestingly, although DC B7-H1 expression was required for augmented T_reg induction by Torin1-conditioned DCs, the only known receptor for B7-H1, PD-1, was not required. PD-1–independent B7-H1 activity has been reported,⁴⁹  and our data also suggest that unidentified B7-H1 receptors may exist. IL-1β was required for T_reg induction and functioned additively with B7-H1 on Torin1-conditioned DCs. Neutralization of IL-1β (data not shown) and genetic deletion of B7-H1 also reduced T_reg induction by RAPA-conditioned DCs but not control DCs, which suggests that other effects of mTORC1 inhibition are required that function collectively with IL-1β and B7-H1 to promote T_reg induction.

ATP-competitive inhibition to target RAPA-resistant mTOR may be useful for the treatment of inflammatory disorders. mTORC2 signaling is required for Th1 and Th2²²  or only Th2²³  differentiation, and deletion of mTOR in T cells causes diversion to Foxp3⁺ Tregs following stimulation.⁴⁸  Our data support the development of ATP-competitive mTOR inhibitors for clinical use, especially because similar findings regarding B7-H1 expression were obtained in human DC cultures and following administration of an ATP-competitive mTOR inhibitor to mice. Further development of ATP-competitive mTOR inhibitors will be required because some have brief half-lives,²⁶  and their in vivo pharmacology is poorly understood. Given the paradoxical enhancement of IL-12p70^17,19,45  and IL-1β^16,18  production by RAPA-conditioned DCs and the reported pulmonary inflammation that can be associated with RAPA,⁵⁰  augmentation of DC-derived IL-10 secretion and lack of increased IL-1β production by ATP-competitive mTOR inhibitors may mitigate untoward side effects of mTORC1 inhibition. Together, these studies demonstrate a novel, immune regulatory pathway mediated by RAPA-resistant mTOR downstream of TLR4 that is relevant for clinical applications of mTOR inhibition.

The authors thank Ms Gabriela Michel for technical assistance with the human CD34⁺ cell-derived DC experiments.

This work was supported by an American Heart Association Predoctoral Fellowship (11PRE7070020) (B.R.R.) and National Institutes of Health grants R01AI67541 (A.W.T.), R00HL97155 (H.R.T.), and T32AI74490 (A.W.T.). D.R.-R. was supported by a European Society for Organ Transplantation/American Society of Transplantation grant, and B.G. was supported by a Leukemia & Lymphoma Society fellowship.

Contribution: B.R.R. designed the research, performed experiments, analyzed results, made the figures, and wrote the paper; D.R.-R., B.M.M., and H.H. designed the research, performed experiments, and analyzed data; K.L., B.G., R.A.D., and M.B. analyzed data and wrote the paper; and H.R.T. and A.W.T. designed the research, analyzed data, and wrote the paper.

Conflict-of-interest disclosure: A.W.T. is an inventor of US patents for the generation of DCs to promote organ allograft tolerance. The remaining authors declare no competing financial interests.

Correspondence: Angus W. Thomson, Thomas E. Starzl Transplantation Institute, Departments of Surgery and Immunology, University of Pittsburgh School of Medicine, 200 Lothrop St, Biomedical Science Tower W1540, Pittsburgh, PA 15213; e-mail: thomsonaw@upmc.edu; and Hēth R. Turnquist, Thomas E. Starzl Transplantation Institute, Departments of Surgery and Immunology, University of Pittsburgh School of Medicine, 200 Lothrop St, Biomedical Science Tower E1554, Pittsburgh, PA 15213; e-mail: turnquisthr@upmc.edu.