TLR agonists are effective at treating superficial cancerous lesions, but their use internally for other types of tumors remains challenging because of toxicity. In this article, we report that murine and human naive CD4+ T cells that sequester Pam3Cys4 (CD4+ TPam3) become primed for Th1 differentiation. CD4+ TPam3 cells encoding the OVA-specific TCR OT2, when transferred into mice bearing established TGF-β–OVA–expressing thymomas, produce high amounts of IFN-γ and sensitize tumors to PD-1/programmed cell death ligand 1 blockade–induced rejection. In contrast, naive OT2 cells without Pam3Cys4 cargo are prone to TGF-β–dependent inducible regulatory Foxp3+ CD4+ T cell conversion and accelerate tumor growth that is largely unaffected by PD-1/programmed cell death ligand 1 blockade. Ex vivo analysis reveals that CD4+ TPam3 cells are resistant to TGF-β–mediated gene expression through Akt activation controlled by inputs from the TCR and a TLR2-MyD88–dependent PI3K signaling pathway. These data show that CD4+ TPam3 cells are capable of Th1 differentiation in the presence of TGF-β, suggesting a novel approach to adoptive cell therapy.
This article is featured in In This Issue, p.373
Toll-like receptors promote host defense through recognizing pathogen-associated molecular patterns released by microorganisms (1). TLR activation initiates potent inflammatory cytokine production and dendritic cell activation that drive the expansion and differentiation of Ag-specific T cells. These observations have led to the clinical use of TLR agonists to promote antitumor responses. These include the use of the TLR7 agonist imiquimod and live preparations of Mycobacterium bovis bacillus of the Calmette-Guérin strain to treat superficial skin and bladder carcinomas, respectively (2, 3). However, TLR agonist therapy has been largely restricted to mucosal lesions because of potential systemic toxicity (4).
Although most studies have focused on TLR2 in APCs, it has been recognized for over a decade that human and mouse T lymphocytes express TLR2 and directly respond to its agonists following TCR stimulation (5). TLR2 on T lymphocytes is primarily thought to function as a costimulatory molecule that controls effector function (6). This activity has best been described in CD8+ T cells, in which TLR2 was shown to stimulate the clonal expansion of long-lived memory cells (5). The expression of Tbx21 (T-bet), a transcription factor that directs Th1 lineage commitment (7), is upregulated by TLR2 agonist stimulation of CD8+ T cells (8). However, T-bet is not required for IFN-γ expression in CD8+ T cells (9), and it remains unclear how TLR2 promotes T-bet expression or Th1 lineage development in CD4+ T cells.
Th1 development is strongly opposed by TGF-β, an immunosuppressive cytokine that is often found in the tumor microenvironment (10). TGF-β inhibits T-bet expression, as well as limits effector cell expression of IFN-γ (11), a critical mediator of antitumor immunity (12). TGF-β also facilitates the conversion of peripheral naive CD4+ T cells into inducible regulatory Foxp3+ CD4+ T cells (iTregs) (13), which, in turn, blunt CD8+ T cell effector cytotoxic activity (14). In T lymphocytes, the transcription factors SMAD2 and SMAD3 play redundant roles in TGF-β−mediated inhibition of IFN-γ expression and iTreg development (15). In addition to being inhibited by TGF-β, Th1 cells may become functionally impaired through the development of exhaustion due to chronic Ag exposure. In particular, high expression of programmed cell death ligand 1 (PD-L1), an immune checkpoint inhibitor, has been strongly linked to poor outcomes in solid tumors (16). PD-L1/PD-1 signaling can inhibit IFN-γ expression, along with the expression of other Th1 effector molecules important in controlling tumor progression (17). These observations have led to the use of strategies to block PD-L1/PD-1 engagement, although such approaches have not always proved successful due to the coexpression of other immune checkpoint inhibitors that promote T cell dysfunction, such as T cell Ig mucin-3 (TIM-3) (18).
Adoptive cell therapy (ACT) using tumor-infiltrating T cells expanded ex vivo or with lymphocytes expressing engineered AgRs have been used successfully to treat metastasis (19). The majority of ACT studies have described the activity of ex vivo–differentiated CD8+ T cells. However, CD8+ T cells require CD4+ T cell help to maintain functionality in vivo (20). This has been exemplified by ACT protocols rendered more effective with the addition of CD4+ T cells (21). Optimal priming and differentiation of CD4+ T cells are likely to occur within tumor-draining lymph nodes (TDLN), as evidenced by the potent antitumor activity of TDLN-derived Th1 cells (22). Previous observations have shown that adoptively transferred naive CD4+ T cells preferentially home to draining lymph nodes (23), suggesting that in vivo priming of tumor-specific naive CD4+ T cells may improve ACT protocols. In this article, using a model of TGF-β–mediated tumor immune evasion, we report that naive tumor Ag–specific CD4+ T cells that carry the synthetic TLR2 agonist Pam3Cys4 differentiate into Th1 cells and control tumor growth. We also show that resistance to TGF-β–directed effects on Th1 development is mediated through a TLR2-MyD88–dependent Akt activation pathway that antagonizes Foxp3 expression.
Materials and Methods
Mice and humans
C57BL/6 (B6), CD45.1, TLR2−/−, CD14−/−, MyD88−/− T-bet−/−, Nur77EGFP, and SBE-luc mice were purchased from The Jackson Laboratory (Bar Harbor, ME). OT2 and OT1 mice were purchased from Charles River Labs (Wilmington, MA). CD45.1+ OT2 mice were generated by intercrossing with CD45.1 mice. Experiments were conducted with mice between 6 and 9 wk age in accordance with an approved Institutional Animal Care and Use Committee protocol. Human CD4+ T cells were isolated under an approved Institutional Review Board protocol (ID number 201012829)
Preparation of naive CD4+ T cells that sequester Pam3Cys4
Spleen and lymph nodes were gently disrupted through a 70-μm filter, lysed for RBCs with ACK Lysing Buffer (Lonza), and stained with the following Abs (all obtained from eBioscience): anti–NK-1.1–FITC (clone PK136), anti-CD45R–FITC (clone RA3-6B2), anti-CD11c–FITC (clone N418), anti-CD11b–FITC (clone M1/70), anti-CD69–FITC (clone H1.2F3), anti-CD4–PerCP–Cy 5.5 (clone RM4-5), anti-CD90.2–allophycocyanin (clone 53-2.1), anti-CD62L–allophycocyanin–Alexa Fluor 780 (clone MEL-14), anti-CD44–FITC (clone IM7), anti-Gr1–FITC (clone RB6-8C5), and anti-CD25–FITC (clone PCS1). Cells were FACS sorted through a CD90.2+ CD4+ CD62L+ CD25− CD69− CD44− CD11c− CD11b− NK1.1− CD45R− Gr1− gate using a Synergy 3200 BSC machine (Sony Biotechnology) into complete culture medium (CM; RPMI 1640, 1% Penicillin/Streptomycin (Life Technologies), 10% FBS, 28.6 μM 2-ME [Sigma]). A Naive CD4+ T Cell Isolation Kit II, human (Miltenyi Biotec) was used to isolate CD4+ T cells from the peripheral blood of healthy human volunteers. CD4+ T cells were coincubated with 10 μg/ml unconjugated, biotinylated, or rhodamine-conjugated Pam3CSK4 (InvivoGen) at 37°C for 3 h in CM, centrifuged at 250 × g, and washed three times with 15 ml of CM prior to use in experiments. Plasma membrane staining was conducted with CellMask Deep Red (Thermo Fisher Scientific), in accordance with the manufacturer’s recommendations.
APCs, CD4+ T cell activation, and polarization
APCs were prepared from TLR2−/− disrupted spleens that were depleted of T lymphocytes using anti-CD90.2 MicroBeads and LS Columns (Miltenyi Biotec) and then gamma irradiated (2000 rad). For polyclonal activation, 0.3 μg/ml CD3ε Ab (clone 2C11; BioLegend) or 0.3 μM OVA peptide (OVAp; ISQAVHAAHAEINEAGR; Sigma) was used for OT2 cells. Plate-bound stimulation was conducted in 96-well flat-bottom plates (Corning) with 0.3 μg/ml CD3ε Ab and 1 μg/ml soluble CD28 Ab (clone CD28.2; BioLegend). Th1 polarization for mouse CD4+ T cells was conducted with 10 μg/ml murine (m)IL-4 (clone 11B11; BioLegend), 10 ng/ml mIFN-γ (PeproTech), and 10 ng/ml mIL-12 (PeproTech) Abs. Th1 polarization for human CD4+ T cells was accomplished using a CellXVivo Human Th1 Cell Differentiation Kit (R&D Systems), in accordance with the manufacturer’s recommendations. For iTreg development, 0.5 ng/ml mTGF-β1 (R&D Systems) was used alone or in combination with Th1-polarizing medium.
Tumors, CD4+ T cell transfer, and TGF-β and PD-L1 neutralization
EG.7-OVA (American Type Culture Collection) was cultured in CM prior to s.c. injection into the left flank of B6 or TLR2−/− mice. Tumors were allowed to progress to ∼250 mm3 in volume prior to i.v. administration of 106 naive CD4+ T cells. Tumors were evaluated for changes in tumor volume every 3 d using the equation 3.14 × (largest diameter × [perpendicular diameter]2)/6. In some experiments, mice were injected i.p. with 1 mg/kg of a pan-specific neutralizing Ab against TGF-β1, TGF-β2, and TGF-β3 (R&D Systems) or control rabbit polyclonal IgG for 1 d before and every other day until the conclusion of the experiment. PD-L1 neutralization was conducted with i.p. administration of 100 μg of the clone 10F.9G2 (Bio X Cell) or control mouse IgG every 3 d after tumor establishment until completion of the study. Tumors were minced with a razor and digested with DNase I (Sigma) and Collagenase/Dispase (Roche) for 1 h at 37°C, and cell suspensions were passed through a 70-μm filter. Tumor cells and infiltrating lymphocytes were quantitated using fluorescent flow cytometric counting beads (BD Biosciences).
FACS, ELISA, and CFSE assays
Cell suspensions were surface stained with fluorochrome-conjugated Abs specifically for mouse CD45.1 (clone A20; BioLegend), CD45.2 (clone 104; BioLegend), CD4 (clone RM4-5; eBioscience), CD90.2 (clone 53-2.1; eBioscience), PD-1 (clone RMP1-30; eBioscience), PD-L1 (clone MIH-5; BD Biosciences), TIM-3 (clone 88.2C12; eBioscience), and CD8α (clone 53-6.7; BioLegend). For intracellular IFN-γ staining, cell suspensions were stimulated for 4 h with 20 ng/ml PMA and 1 μg/ml ionomycin (both from Sigma) in the presence of 1 μg/ml Golgi Plug (BD Biosciences) for 4 h. Cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) and stained with IFN-γ Ab (clone XMG1.2; eBioscience), IL-17A Ab (clone TC11-18H10.1; BioLegend), or SMAD7-specific rabbit polyclonal Ab and Alexa Fluor 647–conjugated Goat anti-Rabbit IgG (Thermo Fisher Scientific). For detection of Foxp3 and T-bet, clone MF-14 (BioLegend) and clone 4B10 (BioLegend) were used, respectively, with a Foxp3/Transcription Factor Staining Buffer Set (eBioscience). IFN-γ accumulation in supernatant was measured with a Ready-SET-Go! ELISA kit (eBioscience). For Akt activation, cells were treated with DMSO vehicle or with the indicated concentration of Ly2924002 (Sigma) 30 min prior to initiation of an assay. At the conclusion of an assay, cells were fixed with 70% ethanol for 10 min and permeabilized with 0.4% Triton X-100, blocked with 2.5% BSA, incubated with rabbit phospho-specific Ab to serine 473 of Akt (clone 700392; Thermo Fisher Scientific) for 5 h at room temperature, and labeled with Alexa Fluor 647–conjugated Goat anti-Rabbit IgG.
Regulator CD45.1+ OT2 cells were isolated from primary cultures by positive selection with CD45.1 biotinylated Abs (clone 820; BioLegend) and anti-biotin MicroBeads and LS Columns (Miltenyi Biotec), in accordance with the manufacturer’s instructions. A total of 1.5 × 105 OT2 regulator cells was added to 96-well round-bottom plates with 1.5 × 105 APCs, pulsed with SIINFEKL (0.1 μg/ml) and SQAVHAAHAEINEAGR (0.1 μg/ml) peptides, and cocultured in triplicate with CFSE-labeled responder CD45.2+ OT1 cells at regulator/responder ratios of 0:1, 1:9, 1:3, and 1:1. Suppression was measured as the percentage retention of CFSE intensity after 5 d of culture.
Semiquantitative real-time RT-PCR
Cells were lysed and fractionated for total RNA with TRIzol Reagent (Invitrogen), in accordance with the manufacturer’s recommendations. RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Four hundred nanograms of cDNA were distributed equally in quadruplicate wells and amplified with TaqMan Assays (Thermo Fisher Scientific) and a 7900HT Fast Real-Time PCR System (Applied Biosystems) in accordance with the manufacturers’ recommended cycling conditions and using the following gene-specific primers: bactin (Mn00607939_s1), tbx21 (Mn00450960_m1), ifng (Mn01168134_m1), gata3 (Mm00484683_m1), rorc (Mm01261022_m1), foxp3 (Mm00475162_m1), Eomes (Mm01351984_m1), prdm1 (Mm00476128_m1), il12rb2 (Mm00434200_m1), icos (Mm00497600_m1), cxcr3 (Mm99999054_s1), cxcr4 (Mm01996749_s1), Fasl (Mm00438864_m1), prfn (Mm00812512_m1), gzmb (Mm00442837_m1), and smad3 (Mm01170760_m1).
Software and statistical analysis
FACS analysis was conducted with FlowJo software version 9 (TreeStar). Statistics were performed with GraphPad Prim Software using one-way ANOVA and the Tukey–Kramer multiple-comparison posttest. Results were considered significant at p < 0.05.
Naive CD4+ T cells sequester TLR2 ligands, leading to enhanced IFN-γ expression
Myeloid cells can sequester TLR agonists (24), but it remains unclear whether this occurs in lymphocytes. We incubated naive B6 CD4+ T cells with biotin-labeled Pam3Cys4 for 3 h at 37°C. Cells were then washed extensively to remove unbound agonist (Fig. 1A). Streptavidin staining revealed that CD4+ T cells take up Pam3Cys4 intracellularly, with about half remaining on the outer plasma membrane. Because naive CD4+ T cells are reported to express low levels of TLR2 (25), we also determined whether Pam3Cys4 is sequestered by naive TLR2−/− and CD14−/− CD4+ T cells, because both receptors have been shown to promote ligand binding (26). Surprisingly, Pam3Cys4 uptake in TLR2−/− and CD14−/− CD4+ T cells was comparable to B6 wild-type CD4+ T cells. Using a rhodamine conjugate of Pam3Cys4, we also visualized ligand binding in naive mouse and human CD4+ T cells (Fig. 1B, 1C).
Based on previous reports that T lymphocytes incubated with Pam3Cys4 during activation enhance IFN-γ production (6, 8), we asked whether we could detect similar responses in naive CD4+ T cells that sequester Pam3Cys4 (CD4+ TPam3). To this end, TLR2−/− and B6 CD4+ TPam3 cells were polyclonally activated with CD3ε Ab and TLR2−/− APCs under Th1-polarizing conditions (Fig. 2A). B6 CD4+ TPam3 cell IFN-γ expression was markedly greater compared with B6 CD4+ T cells without Pam3Cys4 cargo. Pam3Cys4-mediated IFN-γ expression was dependent on TLR2, irrespective of whether ligand was coincubated or sequestered prior to activation. Pam3Cys4 cargo also promoted naive CD4+ T cells encoding the OVA-specific TCR (OT2Pam3 cells) and naive human CD4+ T cells isolated from healthy human volunteers to produce high amounts of IFN-γ when activated under Th1-polarizing conditions (Fig. 2B, 2C). Moreover, two alternative TLR2-specific ligands, Pam2Cys4 and FSL-1, also enhanced IFN-γ expression, demonstrating that augmented Th1 differentiation is not specific to Pam3Cys4. (Supplemental Fig. 1).
Naive tumor-specific CD4+ TPam3 cells differentiate into Th1 cells in vivo
We next determined the fate of naive CD4+ TPam3 cells in vivo. One million naive CD45.1+ OT2 cells, with or without Pam3Cys4 cargo, were administered i.v. into CD45.2+ B6 or CD45.2+ TLR2−/− hosts bearing established EG.7-OVA tumors (Fig. 3A). OT2 cells preferentially homed to TDLN, independent of Pam3Cys4 sequestration (Supplemental Fig. 2). However, only OT2Pam3 cells substantially controlled tumor growth compared with recipients that received untreated OT2 or B6 CD4+ TPam3 cells. Quantitation of tumor-infiltrating OT2 cells showed comparable accumulation, irrespective of treatment with Pam3Cys4 (Fig. 3B), but only hosts that received OT2Pam3 cells had high numbers of OT2 IFN-γ+ cells in their tumors (Fig. 3C). OT2Pam3 cell–treated hosts also had few tumor-infiltrating IL-17A+ OT2 cells (Supplemental Fig. 3A). In line with these observations, tumor-infiltrating OT2Pam3 cells had significantly elevated gene transcripts linked to Th1 lineage determination, including T-bet and its target genes that direct early steps in differentiation (il12rβ2) (27) and trafficking into inflammatory sites (cxcr3) (28). Additionally, we detected known T-bet–targeted genes that stimulate cytotoxicity (Fasl, Prfn, and Gzmb) (29), suggesting that infiltrating OT2Pam3 cells acquire direct antitumor activity (Fig. 3D). In TDLN, we observed elevated IFN-γ+ OT2 and IFN-γ+ CD8+ T cell accumulation in hosts that received OT2Pam3 cells (Fig. 3E, Supplemental Fig. 3B). TDLN CD4+ T cells from hosts that received OT2Pam3 cells also produced high amounts of IFN-γ when challenged with OVAp (Fig. 3F). In contrast, we observed little OVAp-mediated IFN-γ expression by TDLN CD4+ T cells from hosts that received B6 or untreated OT2 cells. Taken collectively, these data demonstrate that tumor-specific CD4+ TPam3 cells differentiate into Th1 cells in vivo.
OT2Pam3 cells induce PD-L1 expression in the tumor microenvironment
The absence of tumor rejection, despite the high frequency of IFN-γ+ OT2Pam3 tumor-infiltrating cells, suggested that alterations within the tumor microenvironment could be inhibiting optimal Th1 effector function. To examine this possibility, tumor-bearing hosts were analyzed for expression of PD-L1, a well-established stimulator of T cell exhaustion that can be upregulated by IFN-γ (30) (Fig. 4A–C). PD-L1 expression on tumor cells at 12 and 24 d after OT2Pam3 cell treatment was significantly higher than in hosts that received OT2 cells and was linked to an increase in late-stage tumorigenesis. PD-L1 engagement of its cognate receptor, PD-1, has been shown to impede Th1 effector responses (31). We examined expression levels of PD-1 on tumor-infiltrating OT2 and OT2Pam3 cells, along with another coinhibitory receptor that negatively regulates Th1 cell survival, TIM-3 (32) (Fig. 4D, 4E). In tumor-infiltrating OT2Pam3 cells, PD-1 and TIM-3 expression levels increased between 12 and 24 d after treatment. Interestingly, coinhibitory receptor expression changes were coupled to the loss of T-bet, suggesting the late-stage development of T cell exhaustion. To explore this relationship further, we analyzed IFN-γ expression in PD-1− TIM-3−, PD-1+ TIM-3−, and PD-1+ TIM-3+ tumor-infiltrating OT2Pam3 cells at 12 and 24 d after treatment (Fig. 4F). Irrespective of PD-1 or TIM-3 expression patterns, most OT2Pam3 tumor-infiltrating cells expressed IFN-γ 12 d after treatment. However, at the 24-d time point, the bulk of tumor-infiltrating OT2Pam3 cells were IFN-γ− PD-1+ Tim-3+ or IFN-γ− PD-1+ Tim-3−, whereas some IFN-γ expression could be still found in the PD-1− Tim-3− cell compartment. Because the loss of IFN-γ expression could be explained by repolarization, we assessed tumor-infiltrating PD-1+ and PD-1− OT2Pam3 cell mRNA for evidence of transcription factor expression changes indicative of alternative CD4+ Th cell lineage determination (Fig. 4G). Rorc, Gata3, and Foxp3 transcript levels were nearly equivalent in PD-1+ or PD-1− cells, indicating the decrease in IFN-γ expression was not due to repolarization into Th17, Th2, or iTreg fates. In contrast, Ifng, Prf1, Gzmb1, and Fasl mRNA levels were lower in the PD-1+ fraction at 24 d after treatment, suggesting the progressive loss of a Th1 effector phenotype. Additionally, we observed the upregulation of transcripts encoding blimp-1 (Prdm1) and Eomes, transcription factors recently linked to PD-1–mediated CD4+ T cell exhaustion (33). Given that these observations suggested that the development of Th1 exhaustion was predominantly associated with high PD-1 expression we evaluated the effect of PD-L1–neutralizing Abs in tumor-bearing recipients that were left untreated or received OT2 or OT2Pam3 cells (Fig. 4H). Although PD-1/PD-L1 blockade had a mild effect on tumor growth in untreated and OT2-treated recipients, it led to the complete rejection of tumors in OT2Pam3 cell–treated hosts. Analysis of isolated tumor-infiltrating OT2Pam3 cells (Supplemental Fig. 4) demonstrated that IFN-γ expression was significantly higher in PD-L1 Ab–treated recipients compared with hosts that received control Abs, indicating that PD-1 engagement in the tumor microenvironment limits ACT-mediated Th1 effector responses.
CD4+ TPam3 cells are resistant to TGF-β–mediated iTreg development
In light of several reports showing that EG.7 tumors produce TGF-β (34, 35), we compared the effects of administering TGF-β–neutralizing Abs to tumor-bearing hosts that received untreated OT2 or OT2Pam3 cells (Fig. 5A). In hosts that received untreated OT2 cells, TGF-β Ab treatment significantly slowed tumor growth and inhibited infiltration of Foxp3+ CD4+ T cells compared with control Ig–treated recipients that received the same cells (Fig. 5B). Surprisingly, hosts that received OT2Pam3 cells and TGF-β Abs did not exhibit significant increases in tumor-infiltrating IFN-γ+ CD4+ T cells relative to OT2Pam3 and control Ig–treated hosts, suggesting that CD4+ T cells carrying Pam3Cys4 cargo are resistant to the regulatory effects of TGF-β on Th1 development (Fig. 5C). A similar pattern was observed in the TDLN (Fig. 5D). Analysis of CD45.1 expression within the IFN-γ+ and Foxp3+ CD4+ T cell compartments demonstrated that conversion of OT2Pam3 cells into Th1 cells was not markedly affected by treatment with TGF-β Abs, whereas differentiation of OT2 cells into Foxp3+ CD4+ T cells was sharply attenuated by TGF-β neutralization.
We next asked whether resistance of CD4+ TPam3 cells to TGF-β–mediated attenuation of IFN-γ expression is mediated by TLR2 signaling pathways. B6, TLR2−/−, and MyD88−/− CD4+ T cells carrying Pam3Cys4 cargo were activated under Th1-polarizing conditions in the absence or presence of graded amounts of TGF-β1 (Fig. 6A). Consistent with previous reports, TGF-β1 inhibited IFN-γ production in all cultures (11). However, at concentrations of up to 0.5 ng/ml TGF-β1, IFN-γ production was significantly higher in B6 CD4+ TPam3 cells compared with analogous cultures of untreated CD4+ T cells. A similar pattern of TGF-β1 resistance was also detected in OT2 cell cultures. In contrast, TLR2−/− and MyD88−/− CD4+ T cells showed no resistance to TGF-β1–mediated suppression of IFN-γ expression. We next analyzed the effects of TGF-β1 on T-bet and Foxp3 expression levels (Fig. 6B). In agreement with previous observations, TGF-β1 upregulated Foxp3 expression and suppressed T-bet expression in cultures with untreated OT2 cells, independent of the application of Th1 polarization (34). However, the addition of TGF-β1 to the Th1-polarization milieu had only a mild effect on T-bet suppression in OT2Pam3 cells. To test the functional significance of these observations, we also performed suppression assays (Fig. 6C). Untreated OT2 cells differentiated with TGF-β1 alone or in combination with Th1 polarization were potent suppressors of responder OVA-specific CD8+ T cell–proliferative responses, whereas OT2Pam3 cells differentiated under the same conditions exhibited significantly less regulatory activity.
Pam3Cys4 antagonizes TCR-mediated suppression of TGF-β signaling
Low levels of Ag stimulation or suboptimal costimulation favors naive CD4+ T cell differentiation into iTregs. TGF-β is thought to mimic the effects of low Ag engagement by antagonizing TCR signaling (36). Given our observations of CD4+ TPam3 cell resistance to conversion into iTregs, we asked whether Pam3Cys4 cargo alters TCR signaling strength. To examine this possibility, we used Nur77EGFP CD4+ T cells, which encode an orphan nuclear hormone receptor Nur77-EGFP transgene that is expressed in proportion to TCR signaling strength (37) (Fig. 7A). In the absence of TGF-β1, TCR signaling strength was moderately higher in CD4+ TPam3 cells relative to CD4+ T cells without Pam3Cys4 cargo, and it became more elevated with TGF-β1 concentrations ranging up to 0.5 ng/ml.
We next examined the effects of Pam3Cys4 cargo on TGF-β–mediated signaling. In CD4+ T cells, the transcription factors SMAD2 and SMAD3 promote TGF-β–mediated iTreg development (15). We loaded Pam3Cys4 cargo into reporter CD4+ T cells that encode a SMAD2/3 promoter–driven luciferase transgene (SBE-luc) (38) (Fig. 7B). TGF-β1–stimulated luciferase expression was significantly lower in SBE-luc CD4+ TPam3 cells compared with untreated SBE-luc CD4+ T cells in response to low to moderate TCR engagement strength. Additionally, SMAD7, which antagonizes TGF-β signaling, in part by promoting the degradation of internalized TGF-β receptor complexes (39), accumulated in higher amounts in CD4+ TPam3 cells (Fig. 7C). Taken together, these data show that TGF-β receptor signaling is blunted in CD4+ TPam3 cells.
TLR2-MyD88–dependent Akt activation pathway antagonizes TGF-β1–mediated suppression of Th1 development
TCR signaling triggers PI3K-mediated activation of protein kinase B (Akt). Several reports have shown that inhibiting Akt activation by blocking PI3K or premature cessation of TCR signaling promotes iTreg development (40–42). We measured the effects of Pam3Cys4 cargo on Akt activation in B6 wild-type and CD4+ T cells deficient in TLR2 and MyD88 following TCR engagement (Fig. 8A, 8B). Relative to untreated CD4+ T cells, CD4+ TPam3 cells had significantly higher Akt activation that could be largely abated by TLR2 or MyD88 ablation or treatment with Ly2924002, a pharmacological inhibitor of PI3K that prevents Akt activation. Blunting Akt activation also inhibited CD4+ TPam3 cell bias toward Th1 differentiation in favor of gene-expression patterns consistent with iTreg development, as reflected by the suppression of T-bet, IL-12β2 receptor, and CXCR3 transcripts and the upregulation of Foxp3, CXCR4, and SMAD2 mRNA (Fig. 8C). Additionally, compared with TLR2−/− or MyD88−/− CD4+ T cells with Pam3Cys4 cargo, B6 CD4+ TPam3 cells were more responsive to the inhibition of Akt activation. Relative to TLR2−/− and MyD88−/− CD4+ TPam3 cells, Ly2924002 treatment induced larger changes in the upregulation of Foxp3 protein and the suppression of T-bet protein in B6 CD4+ TPam3 cells (Figs. 8D, 9).
To the best of our knowledge, this is the first report of a TLR2 agonist taken up by T lymphocytes. TLRs can directly bind pathogen-associated molecular patterns, but they may require other pattern recognition receptors, such as CD14, to induce optimal responses (43). Our data show that TLR2 and CD14 are not required for Pam3Cys4 uptake by CD4+ T cells. This is consistent with a similar observation made in dendritic cells (24). However, in contrast to this single report, pharmacological inhibition of endocytosis or pinocytosis had only a negligible effect on Pam3Cys4 accumulation in naive CD4+ T cells (data not shown). Given that Pam3Cys4, Pam2Cys4, and FSL-1 contain lipid soluble moieties, it is conceivable that agonist sequestration is mediated by nonspecific hydrophobic interactions with the outer plasma membrane, which is followed by translocation to the inner membrane leaflet as part of the normal homeostatic turnover of outer membrane lipids.
Our results show a direct role for TLR2 expression on CD4+ T cells in Th1 development. A potential complication in dissecting TLR function on T cells is the indirect effects mediated by TLR expression on APCs. To minimize this possibility in ex vivo studies, we FACS sorted through a negative selection gate that removes macrophages, dendritic cells, granulocytes, monocytes, NK cells, and B cells. In vivo, we assessed the contribution of TLR2 expression in host cells to antitumor responses. In TLR2−/− hosts that received OT2Pam3 cells, there was a moderate, but significant, late-stage increase in tumor growth compared with wild-type hosts treated with the same cells. We also observed a reduction in tumor-infiltrating IFN-γ+ OT2 cells and less OT2-mediated IFN-γ production in TLR2−/− hosts. These data suggest that some Pam3Cys4 cargo is delivered to TLR2 on host cells, possibly from an agonist located on the outer plasma cell membrane. Because TLR2 agonists promote APC IL-12 expression (44), priming by Pam3Cys4-carrying CD4+ T cells may boost Th1 differentiation and subsequent effector responses against tumors. This effect has been attributed to CD8+ T cell effector responses when Pam3Cys4 has been injected i.v. or around a tumor site (45, 46).
We chose to study naive CD4+ T cells based on previous observations that in vivo activation of naive lymphocytes induces differential responses that lead to better immunity against tumors. These include the maintenance of longer telomeres, less metabolic exhaustion, and augmented resistance to TGF-β–mediated apoptosis (47). Although such factors are likely to have contributed to early OT2Pam3 cell–mediated tumor growth control, there was a marked increase in tumorigenesis 18 d after treatment that correlated with high PD-L1 expression. This observation was linked to PD-1 expression on infiltrating OT2Pam3 cells, which also lacked IFN-γ production and mRNA transcripts that promote Th1 effector function. Nevertheless, PD-1 expression in itself may not be a reliable marker for exhaustion, because previous observations suggest that the PD-1 expression level or its coexpression with other immune checkpoint regulators may be more predictive of dysfunction (18). Indeed, we observed that PD-1+ OT2Pam3 cells maintained most of their IFN-γ expression relative to their PD-1− counterparts early after ACT. However, when OT2Pam3 cells expressed high levels of PD-1 and TIM-3, there was a sharp loss in IFN-γ expression linked to a sudden increase in late-stage tumor growth. The functional relevance of these observations was revealed when we administered PD-L1–neutralizing Abs and assessed tumor growth. Blockade of PD-L1 led to the complete rejection of tumors in OT2Pam3 cell–treated hosts, whereas it had only a small effect on tumor growth in saline- and OT2 cell–treated recipients. Additionally, analysis of IFN-γ production by tumor-infiltrating OT2Pam3 cells showed sharply higher production from recipients treated with PD-L1 Abs when compared with hosts that received control Abs. Therefore, these data suggest that tumor-infiltrating CD4+ TPam3 cells progressively lose effector function through PD-1/PD-L1 engagement.
In line with previous findings, TGF-β blockade suppressed Foxp3+ OT2 cell accumulation within tumors and the TDLN (35). Nevertheless, we initially found it surprising that OT2Pam3 cells are substantially resistant to acquiring Foxp3 expression, irrespective of TGF-β blockade. To better understand these results, we conducted a series of ex vivo studies in which we measured the impact of TGF-β on Th1 and iTreg differentiation from naive CD4+ TPam3 cells. Consistent with previous reports, TGF-β1 also blocked Th1 polarization–mediated expression of T-bet and IFN-γ in untreated OT2 cells (11, 13). However, at TGF-β1 concentrations that normally induced untreated OT2 cell conversion into iTregs, OT2Pam3 cells were largely able to differentiate into Th1 cells. Because TCR stimulation is known to blunt TGF-β–mediated gene activity (39, 41), we also investigated whether Pam3Cys4 cargo increases TCR signaling strength. Using CD4+ T cells that report TCR signaling strength, we observed that CD4+ TPam3 cells perceive more TCR signal for a given amount of engagement compared with untreated CD4+ T cells. Importantly, these differences were most pronounced for low-strength TCR engagement, a point at which iTreg generation would be most favored (41). Additionally, TGF-β1–stimulated CD4+ TPam3 cells had markedly higher levels of SMAD7. Thus, our results suggest that Pam3Cys4 cargo blunts TGF-β signaling through controlling TCR-mediated regulation of SMAD activity.
Our observations of Akt activation in CD4+ TPam3 cells are consistent with several reports of MyD88-dependent PI3K activation in T lymphocytes (8, 48). In this context, MyD88 may play a role synonymous with CD28 where Akt activation is initiated by the recruitment of the PI3K p85 regulatory subunit to an SH2 domain that has been reported on the intracellular domains of both molecules (48, 49). Akt activation promotes Th1 differentiation (50) through activating mTOR complex 1 (51, 52), which, in turn, augments STAT4-mediated T-bet–dependent transcriptional activity. In contrast, Akt activation antagonizes iTreg development (41, 42) through stimulating the nuclear export of Foxo3a and Foxo1, transcription factors critical to drive TGF-β–mediated Foxp3 expression (53). In agreement with our observations of decreased SMAD2/3-mediated reporter activity in CD4+ TPam3 cells, Akt has been reported to directly interact with SMAD3 to prevent its activation and subsequent promotion of iTreg generation (54). These observations led us to that hypothesize that decisions between iTreg and Th1 lineage commitment are regulated by Akt activation levels driven by signaling inputs from TCR and TLR2 signaling pathways. We observed that when both fates were possible, CD4+ TPam3 cells were significantly more sensitive to Akt activation inhibition compared with TLR2−/− and MyD88−/− CD4+ TPam3 cells, as reflected by comparatively larger changes in lineage-specific gene expression. Taken collectively, our findings support a model in which TLR2 activation plays a pivotal role in negatively regulating TGF-β suppression on Th1 development through bolstering Akt activation (Fig. 9). In summary, we report in an ACT model of TGF-β–driven tumor immune evasion that naive tumor-specific CD4+ T cells carrying a TLR2 agonist are able to differentiate into Th1 cells in vivo and control tumor growth. The sequestration of TLR ligands by engineered lymphocytes may be useful to enhance adoptive cell immunotherapies.
This work was supported by the Barnes Jewish Foundation, the Jacqueline G. and William E. Maritz Chair in Immunology and Oncology, and National Institutes of Health Grants P01AI116501-01, R01HL113436-01A1, and R01HL121218-01.
The online version of this article contains supplemental material.
Abbreviations used in this article:
The authors have no financial conflicts of interest.