Malignant pleural effusions and ascites were collected from cancer patients pathologically diagnosed with multiple cancer types. The clinicopathological characteristics of the enrolled patients are presented in Additional file 1: Table S1. The study was approved by the Ethics Committee for Human Studies of Southeast University (protocol 2016ZDKYSB112).

C57BL/6 female mice were purchased from the Comparative Medicine Center of Yangzhou University. Tlr4 ^−/−, Tlr2 ^−/−, Myd88 ^−/− and OT-I mice were purchased from the Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). Il6 ^−/− mice were gifts from Dr. Jinping Zhang (Institutes of Biology and Medical Sciences, Soochow University, Suzhou, China). Mice were maintained in the barrier facility at Southeast University. All animal experiments were approved by the Institutional Animal Care and Use Committee of Southeast University.

The murine hepatic carcinoma line Hepa1–6, melanoma line B16F10, Lewis lung carcinoma line LLC, lymphoma line EL4, and the human melanoma line A375, hepatic carcinoma line HepG2 and breast carcinoma line MDA-MB-231 were cultured in complete RPMI-1640 medium with 10% FBS (Gibco), 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 °C in a 5% CO₂ incubator. Becn1 knockdown (Becn1 KD) and negative control B16F10 cells (Becn1 NC) were established by using lentivirus expressing Becn1-targeting (5′- GCGGGAGUAUA GUGAGUUUTT-3′) and scrambled (5′-TTCTCCGAACGTGTCACGTAA-3′) shRNA (Hanbio Biotechnology, Shanghai, China), respectively.

The inhibitors PD98059, SP600125, SB203580, LY294002, BAY11–7082, and Stattic were purchased from MCE (Shanghai, China). Recombinant murine IL-2 and IL-12 were purchased from PeproTech (Rocky Hill, USA). CFSE were purchased from Invitrogen/Thermo Fisher Scientific. IL-6, IL-10 and IL-21 neutralizing antibodies were purchased from R&D Systems. Lymphocyte separation media were purchased from MultiSciences (Hangzhou, China). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Tumor cells were seeded in a T175 flask in complete RPMI-1640 culture medium supplemented with 10% heat-inactivated FBS (Gibco), 100 U/ml penicillin, and 0.1 mg/ml streptomycin and incubated for 3–4 days at 37 °C, 5% CO₂ until 100% confluency was reached. Tumor cell culture supernatants were collected for TRAPs isolation as described previously [18, 20]. Briefly, supernatants were centrifuged at 2000 rpm for 10 min to remove whole cells and debris. The supernatants were further centrifuged at 12,000 g for 30 min to harvest the TRAPs-containing pellet. The TRAPs-containing pellet was washed three times with PBS and isolated with magnetic beads (Miltenyi Biotec) combined with LC3b antibody (Cell Signaling Technology) for TRAPs. The purity of TRAPs was analyzed by flow cytometry and western blot. The size of TRAPs was determined by dynamic light scattering using a Malvern Instrument.

Mouse splenic B cells (Invitrogen, 11422D), CD4⁺ T cells (Invitrogen, 11415D), CD8⁺ T cells (Invitrogen, 11417D) and human peripheral blood CD4⁺ T cells (Miltenyi Biotec, 130–045-101) were purified by magnetic-activated cell sorting (MACS) following the manufacturer’s instructions. After the MACS, the purity of T and B cells were > 95% as assessed by flow cytometry.

Purified CD4⁺ T or CD8⁺ T cells were cultured in a 24-well plate pre-coated with 2 μg/ml anti-CD3 (BD Biosciences, 550,275) and 2 μg/ml anti-CD28 mAb (BD Biosciences, 553,294) in the presence of 50 U/ml IL-2 (PeproTech), purified TRAPs and 30% culture supernatants from CD4⁺ T cells or B cells. In some cases, culture supernatants from CD4⁺ T cells or B cells were pretreated with neutralizing mAbs against IL-6, IL-10, or IL-21 for 1 h at 4 °C and subsequently exposed to T cells or B cells. Three days later, IFN-γ⁺ CD4⁺ T, IFN-γ⁺ CD8⁺ T or IL-10⁺ B cells were evaluated by flow cytometry. For intracellular staining, the cells were stimulated with the ovalbumin (OVA) protein or anti-CD3 and anti-CD28 mAbs at 37 °C for 24 or 72 h. Leukocyte activation cocktail and GolgiPlug (BD Biosciences) were added to the culture 5 h prior to flow cytometric analysis. Subsequently, the cells were stained with antibodies specific to the various surface molecules, fixed and permeabilized with a Fixation/Permeabilization Kit (BD Biosciences), and finally stained with antibodies against the various intracellular molecules. To detect Bcl-6 and Foxp3, the cells were fixed and permeabilized using a Transcription Factor Buffer Set (BD Biosciences). Data were acquired using a FACS Calibur analyzer (BD Biosciences) and analyzed by FlowJo. The gates were set according to the staining by isotype-matched control antibodies of the respective cells. The fluorochrome-conjugated Abs used are listed in Additional file 1: Table S2.

Total RNA from CD4⁺ T cells was isolated with TRIzol reagent (Invitrogen) and reverse-transcribed using 5 × PrimeScriptRT Master Mix (Takara), following the manufacturer’s instructions. The specific primers used to amplify the genes are listed in Additional file 1: Table S3. The PCR was performed in triplicate using Fast Start Universal SYBR Green Master (ROX) (Roche Life Science) in a StepOne Real-Time PCR System (Thermo Fisher Scientific). GAPDH was used as an internal standard.

Cytokines in the sera or cell culture supernatants were quantified using ELISA kits according to the manufacturer’s protocol. ELISA sets were purchased from eBioscience (IL-6 and IL-10) and R&D Systems (IL-21).

The proteins samples were extracted from CD4⁺ T cells with RIPA lysis buffer. They were separated and transferred as previously described [21]. The membranes were blocked with 5% BSA in TBST for 1 h and separately incubated with the primary antibodies overnight at 4 °C. After washing with TBST buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. The results were visualized by enhanced chemiluminescence according to the manufacturer’s protocol. The primary antibodies used are listed in Additional file 1: Table S4.

Wild type C57BL/6 mice or Il6 ^−/− C57BL/6 mice were subcutaneously inoculated with B16F10, B16F10 Becn1 NC or B16F10 Becn1 KD cells (2 × 10⁵ cells/mouse). Tumor growth was measured using a caliper. On day 21, draining lymph nodes (dLN), spleens or tumor tissues were harvested from tumor-free or tumor-bearing mice. The frequencies of IL-10⁺ CD4⁺ T cells, IL-21⁺ CD4⁺ T cells, or IL-10⁺ B cells were evaluated by flow cytometry after ex vivo stimulation with the leukocyte activation cocktail and GolgiPlug (BD Biosciences) for 5 h. In the subcutaneous tumor model, B16F10 tumor cells (2 × 10⁵ cells/mouse) and CD4⁺ T cells treated with TRAPs, or B cells treated with the indicated culture conditions (2 × 10⁶ cells/mouse) were subcutaneously injected into the right flank of C57BL/6 mice. Subcutaneous tumor growth was monitored and measured using vernier calipers. In the tumor metastasis model, B16F10 tumor cells (5 × 10⁵ cells/mouse) were intravenously injected into C57BL/6 mice and TRAPs-treated or untreated CD4⁺ T cells or B cells (5 × 10⁶ cells/mouse) treated with the indicated culture conditions were injected every other day for 3 times. Three weeks later, mice were sacrificed, and the tumor nodules in the lungs were examined. To evaluate the role of CD4⁺ T cells and B cells treated with the indicated culture conditions in OVA-loaded DC₋mediated specific immune response, C57BL/6 mice were adoptively transferred with OT-I splenocytes (1 × 10⁷ cells/mouse) on day 0 and vaccinated with OVA-loaded DCs (1 × 10⁶ cells/mouse) on days 1, 4, and 7. After intravenous administration of CD4⁺ T cells and B cells on days 2, 5, and 8, mice from each group were sacrificed on day 14 and the frequency and number of CD8⁺Vβ5.1⁺ T cells were evaluated by flow cytometry. The frequency of IFN-γ⁺ CD4⁺ and CD8⁺ T cells in the spleens was determined by intracellular cytokine staining after ex vivo stimulation with the OVA protein for 24 h.

C57Bl/6 mice (n = 5/group) were inoculated subcutaneously in the flank with 1 × 10⁶ Becn1-NC or Becn1-KD B16F10 cells. On day 9, the tumor-bearing mice were subsequently depleted of either CD4⁺ T cells, CD8⁺ T cells or CD20⁺ B cells by intravenous administration of 250 μg/mouse of anti-mouse CD4 (clone GK1.5, BioXCell) or anti-mouse CD8 (clone 2.43, BioXCell) twice weekly throughout the course of tumor growth, or 250 μg of anti-mouse CD20 (clone SA271G2, BioLegend), respectively. Control mice were treated similarly but with isotype-matched control antibodies. Depletion was confirmed by staining of peripheral blood cells with anti-mouse CD4 (RM4–5, BD Pharmingen), anti-mouse CD8 (clone 53–6.7, BioLegend), or anti-mouse CD19 (clone 6D5, BioLegend).

Data were derived from at least 3 independent experiments and analyzed using GraphPad Prism 5.0 software. Multiple group comparisons were performed by one-way ANOVA and the Tukey-Kramer multiple test. Comparisons between 2 groups were performed using unpaired Student’s t-test or Mann-Whitney U test. P < 0.05 was considered significant.

To determine whether TRAPs impact CD4⁺ T cell function, we first isolated TRAPs from the culture supernatants of mouse B16F10 melanoma cells [18, 19]. The TRAPs preparation specifically contained the mature autophagosome marker LC-3II (Fig. 1a, b) and exhibited an average size of 436.3 nm, which was distinct from isolated exosomes that had an average size of 85.6 nm (Fig. 1c) and expressed the exosome markers CD63 and TSG101 (Fig. 1d). Treatment of mouse splenic CD4⁺ T cells with TRAPs during activation by anti-CD3 and anti-CD28 resulted in the induction of the transcripts encoding Il6, Il21, Il10, and Il17, but not Il1b, Il2, Il4, Il9, Tnf, Ifng, Foxp3 or Tgfb1 (Additional file 2: Figure S1a). Consistently, the frequency of IL-6⁺, IL-10⁺ or IL-21⁺ CD4⁺ T cells and the secretion of IL-6, IL-10 or IL-21 by CD4⁺ T cells were increased by TRAPs treatment (Fig. 1e, f). TRAPs-induced IL-21⁺ CD4⁺ T cells expressed elevated levels of the follicular helper T cell (Tfh)-associated molecules CXCR5 and Bcl-6 (Additional file 2: Figure S1b, c). In contrast, TRAPs reduced the frequency of IFN-γ⁺ CD4⁺ T cells (Fig. 1e) and suppressed IL-12-mediated induction of IFN-γ⁺ Th1 cells (Additional file 2: Figure S1d). Depletion of TRAPs from the culture media via ultracentrifugation (Additional file 2: Figure S1e) resulted in a significant reduction of IL-6, IL-10 and IL-21 production by CD4⁺ T cells (Fig. 1g). Intriguingly, we also found that LC3B⁺ EVs (TRAPs) purified from B16F10 culture supernatant were more potent than LC3B⁻ EVs and exosomes in upregulating IL-6 expression, suggesting that LC3B⁺ EVs (TRAPs) are the dominant large EVs that instruct CD4⁺ T cells (Additional file 2: Figure S2a-d). In order to ascertain the role of TRAPs in inducing IL-6, IL-10 and IL-21 production by CD4⁺ T cells in vivo, normal saline (NS) or TRAPs were administered intravenously (i.v.) into C57BL/6 mice every other day for 3 times. The frequencies of IL-6⁺, IL-10⁺ and IL-21⁺ CD4⁺ T cells in the inguinal lymph node and spleen increased markedly after TRAPs administration (Fig. 1h). Consistently, in B16F10 tumor-bearing mice, the frequency of IL-6⁺, IL-21⁺ and IL-10⁺ CD4⁺ T cells in the draining lymph node and spleen were also increased (Fig. 1i). Taken together, these results show that TRAPs could modulate CD4⁺ T cell differentiation by inducing IL-6, IL-10, and IL-21 expression and suppressing their IFN-γ production.Fig1Fig. 1

TRAPs induce IL-6, IL-10, and IL-21 expression by CD4⁺ T cells. a–d Characterization of TRAPs from B16F10 cells, including Western blot (a, d), flow cytometric (b), and dynamic light scattering (c) analyses of the expression of the autophagosome-specific marker LC-3II, the percentage, and the size distribution of the isolated TRAPs. e Flow cytometric determination of the percentage of IL-6-, IL-21-, IL-10-, IL-4-, IL-17-, IFN-γ-, and Foxp3-expressing CD4⁺ T cells treated with control media (CM) or 3 μg/ml TRAPs in the presence of anti-CD3 and anti-CD28 for 72 h. f ELISA of IL-6, IL-10, and IL-21 secretion by CD4⁺ T cells treated with CM or 1, 3, or 10 μg/ml TRAPs in the presence of anti-CD3 and anti-CD28 for 72 h. g ELISA of IL-6, IL-10 and IL-21 secretion by CD4⁺ T cells cultured in B16F10 tumor cell-conditioned culture media, TRAP-depleted tumor cell culture media or TRAPs purified from the equal cell culture media in the presence of anti-CD3 and anti-CD28 for 72 h. h Flow cytometric and statistical analyses of the percentage of IL-6⁺, IL-10⁺ or IL-21⁺ CD4⁺ T cells in the inguinal lymph nodes (iLN) and spleens of C57BL/6 mice (n = 6) 7 days after i.v. administration of normal saline (NS) or TRAPs (30 μg/mouse) every other day for 3 times. i Flow cytometric and statistical analyses of the percentage of IL-6⁺, IL-10⁺ or IL-21⁺ CD4⁺ T cells in the draining lymph nodes (dLN) and spleens of C57BL/6 tumor-bearing (TB) mice (n = 6) 21 days after s.c. inoculation of B16F10 cells, in comparison to the tumor-free (TF) mice. Data (mean ± SEM) represent 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant, by one-way ANOVA with the Tukey-Kramer multiple test, 2-tailed unpaired t-test or Mann-Whitney U test

We then investigated the mechanism by which TRAPs induce IL-6, IL-10, and IL-21 in CD4⁺ T cells. Within the time frame of the induction of these cytokines, TRAPs adhered to the surface of CD4⁺ T cells in a dose-dependent manner without being internalized (Fig. 2a, b), suggesting the involvement of surface molecules on TRAPs that interact with receptors on CD4⁺ T cells. TRAPs are enriched with various danger-associated molecular patterns (DAMPs) capable of stimulating pattern recognition receptors (PRRs) [17, 18]. CD4⁺ T cells expressed appreciable levels of TLR2 and TLR4 (Additional file 2: Figure S3a). We therefore examined whether TLR2 or TLR4 was involved in sensing TRAPs by CD4⁺ T cells. While TRAPs-induced IL-6, IL-10 and IL-21 secretion by CD4⁺ T cells was independent of TLR4, Tlr2 ^−/− and Myd88 ^−/− CD4⁺ T cells were completely defective in producing these cytokines in response to TRAPs (Fig. 2c). Consistently, TLR2 on the surface of CD4⁺ T cells was in direct contact with TRAPs (Fig. 2d). In agreement with the above finding, Tlr2 ^−/− mice bearing B16F10 tumors had a significant reduction of IL-21⁺ and IL-10⁺ CD4⁺ T cells in the tumor tissue compared to WT tumor-bearing mice (Fig. 2e, f). Collectively, these results show that TRAPs induce CD4⁺ T cells to produce IL-6, IL-10, and IL-21 in a TLR2- and MyD88-dependent manner.Fig2Fig. 2

TRAPs induce IL-6/IL-10/IL-21 production of CD4⁺ T cells via TLR2-MyD88 pathway. a Confocal microscopy analysis of CFSE-labeled TRAPs (3 μg/ml) and mouse splenic CD4⁺ T cells (stained with anti-CD4-PE) after 24 h of co-culture. Scale bar: 5 μm. b Flow cytometric determination of the proportion of CFSE⁺ CD4⁺ T cells after incubated with CFSE-labeled TRAPs (0, 1, 3, or 10 μg/ml) in the presence of anti-CD3 and anti-CD28 for 24 h. c ELISA of IL-6, IL-10, and IL-21 secretion by WT, Tlr2 ^−/−, Tlr4 ^−/− or Myd88 ^−/− CD4⁺ T cells treated with TRAPs (3 μg/ml) or control media (CM) in the presence of anti-CD3 and anti-CD28 for 72 h. d Purified CD4⁺ T cells were co-cultured with CFSE-labeled TRAPs (3 μg/ml) for 24 h, and then stained for TLR2 and analyzed by confocal microscopy. e, f Flow cytometric and statistical analyses of the percentage of IL-10⁺ CD4⁺ T cells (e) or IL-21⁺ CD4⁺ T cells (f) in the tumor tissues of WT or Tlr2 ^−/− C57BL/6 mice (n = 6) 21 days after s.c. inoculation of B16F10 cells. Data (mean ± SEM) represent 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant, by 2-tailed unpaired t-test or Mann-Whitney U test

We further sought to determine the signals downstream of TLR2 in the induction of IL-6, IL-10, and IL-21 by TRAPs. TRAPs treatment of WT CD4⁺ T cells resulted in the phosphorylation of NF-κB, Akt, p38 and STAT3, but not ERK1/2 or JNK1/2 (Fig. 3a), whereas TRAPs failed to induce NF-κB, Akt, p38 and STAT3 phosphorylation in Tlr2 ^−/− or Myd88 ^−/− CD4⁺ T cells when compared to CD4⁺ T cells from WT or Tlr4 ^−/− mice (Additional file 2: Figure S3b). Pretreatment of CD4⁺ T cells with an inhibitor of NF-κB, Akt or p38 attenuated TRAPs-induced secretion of IL-6, IL-10 and IL-21, whereas the inhibition of JNK1/2 or ERK1/2 had no effect (Fig. 3b). Of note, pretreatment of CD4⁺ T cells with a STAT3 inhibitor diminished the production of IL-10 and IL-21, but not IL-6, in a dose-dependent manner (Fig. 3b, c), indicating that NF-κB, Akt, and p38 activation was needed for TRAPs-induced IL-6, IL-10 and IL-21 production but STAT3 activation was only required for IL-10 and IL-21 production.Fig3Fig. 3

TRAPs promote CD4⁺ T cell expression of IL-6 in an NF-κB/p38/Akt-dependent manner and induce IL-10 and IL-21 via IL-6–STAT3 signaling. a Western blot analyses of the phosphorylation of JNK, ERK, p38, Akt, IKKα/β, IκBα, p65 and STAT3 in CD4⁺ T cells treated with TRAPs (3 μg/ml) for the indicated time. b CD4⁺ T cells pre-treated with the indicated inhibitors for 1 h and then co-cultured with TRAPs (3 μg/ml) for 72 H. IL-6, IL-10 and IL-21 levels in the supernatants were determined by ELISA. c Western blot analyses of the phosphorylation of STAT3 in CD4⁺ T cells pre-treated with the STAT3 inhibitor Stattic at the indicated concentrations (0.5, 1 or 2 μM) for 1 h, and then co-cultured with TRAPs (3 μg/ml) for 2 h. ELISA of IL-10 and IL-21 secretion by CD4⁺ T cells treated as above for 72 h. d Western blot analyses of the phosphorylation of STAT3 in CD4⁺ T cells treated with anti-IL-6 neutralizing antibody (1 μg/ml) and TRAPs (3 μg/ml) for 2 h. ELISA of IL-10 and IL-21 secretion by CD4⁺ T cells treated as above for 72 h. e Western blot analyses of the phosphorylation of STAT3 in WT or Il6 ^−/− CD4⁺ T cells treated with TRAPs (3 μg/ml) for 2 h and ELISA of IL-10 and IL-21 secretion by WT or Il6 ^−/− CD4⁺ T cells for 72 h. f, g Flow cytometric and statistical analyses of the percentage of IL-21⁺ CD4⁺ T cells (f) or IL-10⁺ CD4⁺ T cells (g) in the iLN and spleens of WT or Il6 ^−/− C57BL/6 mice (n = 6) 7 days after i.v. administration of normal saline (NS) or TRAPs (30 μg/mouse) every other day for 3 times. Data (mean ± SEM) represent 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant by one-way ANOVA with the Tukey-Kramer multiple test, 2-tailed unpaired t-test or Mann-Whitney U test

The IL-6–STAT3 pathway plays a crucial role in Th cell differentiation [22]. Upon IL-6 neutralization with a blocking antibody, the induction of IL-21 and IL-10 mRNA and proteins by TRAPs was completely abolished, with a concomitant decline of STAT3 phosphorylation (Fig. 3d, Additional file 2: Figure S4a). Consistently, TRAPs failed to induce IL-10 and IL-21 expression or STAT3 phosphorylation in Il6 ^−/− CD4⁺ T cells (Fig. 3e, Additional file 2: Figure S4b). Moreover, following i.v. administration of TRAPs, the frequencies of IL-10⁺ and IL-21⁺ CD4⁺ T cells in the inguinal lymph node and spleen were much lower in Il6 ^−/− mice than in WT mice (Fig. 3f, g). Collectively, these results support a TRAPs-initiated regulatory cascade of CD4⁺ T cell differentiation involving TLR2–NF-κB/p38/Akt-dependent induction of autocrine IL-6 which then promotes IL-10 and IL-21 expression via STAT3.

To identify the molecular components in TRAPs that are responsible for stimulating CD4⁺ T cell production of IL-6, we first subjected TRAPs to proteinase K digestion or sonication. These treatments impaired the ability of TRAPs to induce IL-6 from CD4⁺ T cells (Fig. 4a, b), indicating that proteins on the surface, but not the soluble contents, of TRAPs are largely responsible for IL-6 induction in CD4⁺ T cells. In addition, TRAPs from the hepatic carcinoma Hepa1–6, lung cancer LLC or lymphoma EL4 cells also potently enhanced IL-6 secretion in CD4⁺ T cells (Additional file 2: Figure S5a). Several ligands of TLR2, including HMGB1, Hsp60, Hsp70, and Hsp90α [18, 23], were enriched in and present on the surface of TRAPs (Fig. 4c, Additional file 2: Figure S5b). Blocking of Hsp90α, but not HMGB1, Hsp60 or Hsp70, on the surface of TRAPs partially diminished TRAPs-induced IL-6 secretion by CD4⁺ T cells, indicating that other molecules on TRAPs may also play a role (Fig. 4d). Accordingly, an anti-Hsp90α antibody dose-dependently inhibited TRAPs binding to CD4⁺ T cells (Fig. 4e, f), reduced TRAPs-induced IL-6 secretion (Fig. 4g), and suppressed the activation of NF-κB, Akt and p38 (Additional file 2: Figure S5c). Remarkably, compared to intact TRAPs, tumor cell lysates containing an equal amount of total protein but much more Hsp90α, or sonicated TRAPs containing an equal amount of Hsp90α, or proteinase K-treated TRAPs were much less effective in inducing IL-6 secretion from CD4⁺ T cells (Fig. 4h). Taken together, these results show that membrane-bound Hsp90α on intact TRAPs effectively induces IL-6 expression from CD4⁺ T cells.Fig4Fig. 4

Hsp90α on intact TRAPs is essential for IL-6 induction in CD4⁺ T cells. a, b ELISA of IL-6 secretion by CD4⁺ T cells treated with TRAPs (3 μg/ml), proteinase K-digested TRAPs (a) or sonicated TRAPs (b) for 72 h in the presence of anti-CD3/CD28. c Flow cytometric determination of the Hsp60, Hsp70, Hsp90α, or HMGB1 expression levels on the surface of TRAPs from B16F10 tumor cells. d ELISA of IL-6 secretion by CD4⁺ T cells treated with TRAPs (3 μg/ml) or blocking antibody-pretreated TRAPs (anti-HMGB1, anti-Hsp60, anti-Hsp70, anti-Hsp90α antibodies) for 72 h in the presence of anti-CD3/CD28. e-g CFSE-labeled TRAPs were pretreated with the indicated dose of functional an anti-Hsp90α antibody or an isotype-matched control antibody overnight at 4 °C and then co-cultured with purified CD4⁺ T cells in the presence of anti-CD3/CD28. Twenty-four hour later, the percentage of CFSE⁺ CD4⁺ T cells was assessed by flow cytometry (e, f). Seventy-two hour later, IL-6 levels in supernatants were determined by ELISA (g). h The Hsp90α level in tumor cell lysates, an equal amount of TRAPs, sonicated TRAPs, and proteinase K-pretreated TRAPs was determined by western blot. Purified CD4⁺ T cells were co-cultured with the above stimulators for 72 H. IL-6 levels in the supernatants were determined by ELISA. i ELISA of IL-6 secretion by human CD4⁺ T cells treated with 3 μg/ml human TRAPs (hTRAPs) from 3 human tumor cell lines (A375, MDA-MB-231 and HepG2 cells) or 8 tumor patient effusions and ascites, respectively for 72 h in the presence of anti-CD3/CD28. j ELISA of IL-6 secretion by human CD4⁺ T cells treated with hTRAPs (3 μg/ml) or anti-Hsp90α-pretreated hTRAPs for 72 h in the presence of anti-CD3/CD28. Data (mean ± SEM) represent 3 independent experiments. *, P < 0.05; **, P < 0.01; ns, not significant by one-way ANOVA with the Tukey-Kramer multiple test

To characterize the function of TRAPs-elicited CD4⁺ T cells (T_TRAP), we activated CD4⁺ and CD8⁺ T cells with anti-CD3 and anti-CD28 in culture supernatants harvested from T_TRAP or control CD4⁺ T cells. T_TRAP supernatants (SN/T_TRAP) strongly suppressed the secretion of IFN-γ by activated CD4⁺ and CD8⁺ T cells (Fig. 5a). Pretreatment of SN/T_TRAP with a neutralizing antibody against IL-6 or IL-10, but not IL-21, abolished its suppressive effect on IFN-γ production by activated CD4⁺ and CD8⁺ T cells (Fig. 5b). We then transferred control CD4⁺ T cells or T_TRAP into C57BL/6 mice that had received OVA-specific Vβ5.1⁺CD8⁺ OT-I T cells and vaccinated with OVA-loaded dendritic cells (DC_OVA). DC_OVA vaccination induced the expansion of Vβ5.1⁺CD8⁺ OT-I T cells in the host, which was suppressed by the adoptive transfer of T_TRAP but not control CD4⁺ T cells (Fig. 5c). Moreover, the transfer of T_TRAP but not control CD4⁺ T cells led to a decrease of IFN-γ⁺ CD8⁺ and CD4⁺ T cells induced by DC_OVA vaccination (Fig. 5d). Therefore, T_TRAP could suppress T cell IFN-γ response in vivo.Fig5Fig. 5

IL-6/IL-10 from T_TRAP is responsible for tumor growth and metastasis. a Flow cytometric and statistical analyses of the percentage of IFN-γ⁺ CD4⁺ and CD8⁺ T cells treated with the supernatants from T_TRAP (SN/T_TRAP) or control CD4⁺ T cells for 3 d in the presence of anti-CD3/CD28. b SN/T_TRAP was pretreated with anti-IL-6, IL-10, IL-21 neutralizing antibodies respectively, and then co-cultured with the purified CD4⁺ T cells and CD8⁺ T cells in the presence of anti-CD3/CD28 for 3 d. The percentages of IFN-γ⁺ CD4⁺ T cells and CD8⁺ T cells were evaluated by flow cytometry. c C57BL/6 mice were adoptively transferred i.v. with OT-I spleen cells and then vaccinated s.c. with OVA-loaded DC on day 1, 4 and 7, following injection i.v. with T_TRAP or control CD4⁺ T cells on day 2, 5 and 8. On the 15th day, the frequencies and the number of Vβ5.1⁺CD8⁺ T cells in spleen were analyzed by flow cytometry. d C57BL/6 mice were vaccinated with OVA-loaded DC and following adoptively transferred with T_TRAP or CD4⁺ T cells. On the 15th day, the splenocytes were re-stimulated with OVA-protein for 24 h, and the frequencies of IFN-γ⁺ T cells were determined by flow cytometry. e B16F10 tumor cells were mixed with T_TRAP or control CD4⁺ T cells and injected s.c. into C57BL/6 mice (n = 6 per group). The growth of the tumor was monitored. f B16F10 tumor cells were intravenously injected into C57BL/6 mice (n = 4 to 6 per group) to establish a lung metastasis model. Subsequently, T_TRAP or control CD4⁺ T cells were adoptively transferred i.v. 3 times with 1 d of interval. Three weeks later, the tumor nodules in the lungs were examined. Data (mean ± SEM) represent 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant, by 1-way ANOVA with the Tukey-Kramer multiple test, 2-tailed unpaired t-test or Mann-Whitney U test

To better define the immunosuppressive capacity of T_TRAP, we examined the impact of T_TRAP on regulatory B cell (Breg) differentiation. In accordance with our earlier findings [18], TRAPs induced B cell differentiation into IL-10-producing Bregs (Fig. 6a). Moreover, co-culture of B cells and CD4⁺ T cells in the presence of TRAPs led to a marked increase in Bregs differentiation (Fig. 6a). Consistently, SN/T_TRAP could directly promote IL-10⁺ Bregs differentiation and IL-10 secretion (Additional file 2: Figure S6a, b). Next, adoptive transfer of T_TRAP, but not control CD4⁺ T cells, also significantly increased the frequency and number of IL-10⁺ Bregs in vivo (Fig. 6b). Therefore, TRAPs can promote IL-10⁺ Breg differentiation directly by activating on B cells and indirectly by conditioning CD4⁺ T cells.Fig6Fig. 6

T_TRAP enhance Breg differentiation and function via IL-6, IL-10, and IL-21. a Flow cytometric assessment of IL-10 expression by splenic B cells after 3 d of co-culture with 3 μg/ml TRAPs or 3 μg/ml TRAPs and an equal number of CD4⁺ T cells. b T_TRAP were adoptively (i.v.) transferred into C57BL/6 mice (n = 3 per group) every other day for 3 times. The frequency and number of splenic IL-10⁺ Bregs 7 days after the last transfer of T_TRAP was determined by flow cytometry. c SN/T_TRAP was pretreated with an anti-IL-6, −IL-10 or -IL-21 neutralizing antibody and co-cultured with splenic B cells and TRAPs for 72 h. The numbers of IL-10⁺ Bregs and IL-10 secretion were determined by flow cytometry and ELISA, respectively. d The supernatants from B cells stimulated with 3 μg/ml TRAPs and SN/T_TRAP (SN/B_{TRAP + SN/TTRAP}) were untreated or pre-treated with an anti-IL-10 neutralizing antibody and then used to culture anti-CD3/28-activated CD4⁺ or CD8⁺ T cells for 3 days. The percentage of IFN-γ⁺ T cells was determined by flow cytometry. e C57BL/6 mice were i.v. injected with OT-I splenocytes and vaccinated s.c. with DC_OVA on day 1, 4 and 7, following adoptive transfer of B cells induced by TRAPs (B_TRAP) or by TRAPs and SN/T_TRAP (B_{TRAP + SN/TTRAP}) on day 2, 5 and 8. On day 15, the frequency and the number of Vβ5.1⁺CD8⁺ T cells in spleens were analyzed by flow cytometry. f C57BL/6 mice were vaccinated with DC_OVA and transferred with B_TRAP or B_{TRAP + SN/TTRAP}. At day 15, the frequencies of splenic IFN-γ⁺ CD4⁺ and CD8⁺ T cells were determined after ex vivo re-stimulation. g, h B16F10 tumor cells were mixed with B_TRAP, B_{TRAP + SN/CD4+ T}, or B_{TRAP + SN/TTRAP} and injected s.c. into C57BL/6 mice (n = 4 per group). The growth of tumor was monitored (g). B16F10 tumor cells were injected i.v. into C57BL/6 mice (n = 4 per group) to establish a lung metastasis model. Subsequently, the above-prepared B cells were i.v. transferred every other day for 3 times. Three weeks later, tumor nodules in the lungs were examined (h). Data (mean ± SEM) represent 3 independent experiments. *, P < 0.05; **, P < 0.01, ***, P < 0.001; ns, not significant

Having shown a critical role of TRAPs in the inhibition of anti-tumor immunity, we explored whether inhibition of TRAPs formation by targeting Becn1, a gene essential for autophagosome formation, could abolish the generation of tumor-promoting T_TRAP (Additional file 2: Figure S7a, b). Becn1 knock-down in B16F10 cells diminished intracellular LC3-II accumulation and markedly reduced TRAPs secretion (Additional file 2: Figure S7b, c). The culture media collected from Becn1 knock-down B16F10 cells had reduced ability to induce IL-6, IL-10, and IL-21 in CD4⁺ T cells (Fig. 7a). In the mice bearing knock-down B16F10 tumors, the frequency of IL-21⁺ and IL-10⁺ CD4⁺ T cells in the tumor draining lymph node and tumor tissue and the serum IL-6 level were significantly reduced as compared to those in the mice bearing control tumors (Fig. 7b–d). Moreover, the frequency of IL-10⁺ B cells and IFN-γ⁺CD4⁺ T cells in mice bearing Becn1 knock-down tumors was markedly decreased and increased, respectively (Fig. 7e, f). Additionally, Becn1 knock-down B16F10 cells exhibited significantly slower growth in vivo (Additional file 2: Figure S7d). These results indicate that inhibition of tumor autophagosome formation and release could enhance anti-tumor immunity and inhibit tumor growth in vivo.Fig7Fig. 7

Knockdown of Beclin1 or knockout of IL-6 inhibits tumor growth and alters CD4⁺T and B cells polarization. a ELISA of IL-6, IL-10, and IL-21 secretion by CD4⁺ T cells treated with CM, or media from negative control (NC) or Becn1-knockdown (KD) B16F10 cells in the presence of anti-CD3/CD28 for 72 h. b-f Control (NC) or Becn1-KD B16F10 cells were inoculated s.c. into C57BL/6 mice (n = 6 per group). Twenty-one days later, serum IL-6 level was measured by ELISA (b). The frequency of IL-10⁺ CD4⁺ T cells (c), IL-21⁺ CD4⁺ T cells (d), IFN-γ⁺ CD4⁺ T cells (e), and IL-10⁺ B cells (f) in tumor-draining lymph nodes (dLN) or tumor tissues (Tumor) were analyzed by flow cytometry. g-j The tumor-bearing mice (n = 5 per group) were depleted of either CD4⁺ or CD8⁺ T cells or CD20⁺ B cells by intravenous administration of 250 μg/mouse of anti-mouse CD4 or anti-mouse CD8 antibodies twice weekly throughout the course of tumor growth, or 250 μg of anti-mouse CD20 antibodies, respectively. Control mice were treated with isotype-matched control antibodies. g Tumor growth was monitored by calipers. h, i The frequency of CD4⁺ IFN-γ⁺ and CD8⁺ IFN-γ⁺ T cells in tumor infiltrating lymphocytes were determined by intracellular staining. j The frequency of B cells in tumor infiltrating lymphocytes were determined by flow cytometry. k-n WT or Il6 ^−/− C57BL/6 mice were inoculated s.c. with B16F10 cells. On day 21, the frequencies of IL-10⁺ CD4⁺ T cells (k), IL-21⁺ CD4⁺ T cells (l), and IL-10⁺ B cells (m) in dLN and tumor tissues were evaluated by flow cytometry. n Tumor size was measured by caliper. o, p B16F10 tumor cells were mixed with WT T_TRAP or Il6 ^−/− T_TRAP and injected s.c. into C57BL/6 mice (n = 6 per group). The growth of the tumor was monitored. o, q B16F10 tumor cells were i.v. injected into C57BL/6 mice (n = 6 per group). Subsequently, WT T_TRAP or Il6 ^−/− T_TRAP were transferred i.v. every other day for 3 times. Three weeks later, tumor nodules in the lungs were examined. r A proposed model for the mechanisms and immunosuppressive functions of TRAPs-induced CD4⁺ T cells. Data (mean ± SEM) represent 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant, by 1-way ANOVA with the Tukey-Kramer multiple test, 2-tailed unpaired t-test or Mann-Whitney U test