Tumor Cell-Released Autophagosomes(TRAP) Induce CD4+T Cell-Mediated Immunosuppression And Its Underlying Mechanisms

The cross-talk between cancer cells and immune cells can result in immunoediting of tumors that fosters immune escape and cancer progression.Recently,we have found extracellular secretory autophagosomes from the supernatant of tumor cells,which is a new extracellular vesicles,and have termed such tumor-released autophagosomes TRAPs.We confirmed that TRAPs can be taken up by phagocytes such as neutrophils and macrophages,as well as B cells,and endow them with immunosuppressive activities.CD4⁺T cells are critical effectors of anti-tumor immunity,however,the mechanistic aspects of TRAPs in the modulation of CD4⁺T cell effector function is not fully understood.ObjectivesTo investigate whether TRAPs could regulate CD4⁺T cells differentiation and effector function;to elucidate the immune regulation network between TRAPs-instructed CD4⁺T cells and tumor immune microenvironment,and provide a new target or treatment strategy for tumor immunotherapy in future.Methods1.Study of TRAPs-induced CD4⁺T cells differentiation in vitro and in vivoThe purified splenic CD4⁺T cells were co-culture with different concentrations of TRAPs?0,1,3,or 10?g/ml?in the presence of anti-CD3/CD28 mAbs for 24 h or 72 h.The frequencies of cytokine-or Foxp3-expressing CD4⁺T cells were assessed by qRT-PCR or flow cytometric analysis.The IL-6,IL-10,and IL-21 levels in supernatants were measured by ELISA.WT C57BL/6 mice?n=6?were i.v.injected with TRAPs?30?g/mouse?three times with 1 d of intervals,or s.c.inoculated with B16-F10 cells.On day 7 or 21,draining lymph nodes?dLN?and spleens were harvested to detect the frequencies of IL-10⁺CD4⁺T cells and IL-21⁺CD4⁺T cells using flow cytometric analysis.2.Investigation of the mechanisms underlying TRAPs-induced CD4⁺T cells differentiationThe purified splenic CD4⁺T cells were co-cultured with CFSE-labeled TRAPs for 24 h,and then stained with anti-CD4-PE or anti-TLR2-PE antibody for confocal laser scanning microscopy image analysis.The proportion of CFSE-labeled TRAPs bonded to the CD4⁺T cells performed by flow cytometry.CD4⁺T cells purified from WT,Tlr2^-/-,Tlr4^-/-,Myd88^-/-or Il6^-/-mice stimulated with TRAPs?3?g/mL?or pre-treated with inhibitors for 72 h.IL-6,IL-10,and IL-21 levels in supernatants were measured by ELISA.CD4⁺T cells were incubated with TRAPs?3?g/ml?for the indicated length of time.Cell lysates were analyzed for phosphorylation of JNK,ERK,p38,Akt,IKK?/analysis.TRAPs were pretreated with Proteinase K,sonication,or?HMGB1,?HSP60,?HSP70 and?HSP90?,respectively,followed by incubation with CD4⁺T cells for indicated time.IL-6 was tested by RT-PCR or ELISA.Wild type or Il6^-/-C57BL/6 mice?n=6?were i.v.injected with TRAPs?30?g/mouse?three times with 1 d of intervals.At day 7,lymph node?LN?and spleen?SP?were harvested to detect the frequencies of IL-21⁺CD4⁺T cells and IL-10⁺CD4⁺T cells using flow cytometric analysis.3.Investigation of the effects of TRAPs-elicited CD4⁺T cells(T_TRAP)on immune responses and its mechanisms3.1 Investigation of the effects of TRAPs-elicited CD4⁺T cells(T_TRAP)on T-cell responses and tumorigenesisThe purified CD4⁺T cells and CD8⁺T cells were stimulated with anti-CD3/CD28 mAbs in the presence of supernatants from TRAPs-induced CD4⁺T cells(SN/T_TRAP)or control CD4⁺T cells?SN/CD4⁺T?or SN/T_TRAP pretreated with anti-IL-6,IL-10 or IL-21 neutralizing antibodies for 3 days.The percentage of IFN-?⁺T cells was determined by flow cytometric analysis.C57BL/6 mice were adoptively transferred with OT-I splenocytes?1×10⁷cells/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 on days 2,5,and 8,mice from each group were sacrificed on day 14 and the frequency and number of CD8⁺V?5.1⁺T cells were evaluated by flow cytometry.The frequency of IFN-?⁺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.In the B16F10 subcutaneous tumor model and metastasis model,TRAPs-treated or untreated CD4⁺T or IL-6^-/-CD4⁺T cells were injected.Subcutaneous tumor growth and tumor nodules in the lungs were examined.3.2 Investigation of the effects of TRAPs-elicited CD4⁺T cells(T_TRAP)on B-cell differentiation and tumorigenesisTRAP-induced CD4⁺T cells were adoptively i.v.transferred into C57BL/6 mice three times with 1 d of intervals.The frequencies and number of IL-10⁺B cells in the spleen determined at day 7.The purified splenic B cells were stimulated with or without TRAPs?3?g/ml?or CD4⁺T cells?at ratio of 1:1?,or SN/T_TRAP pretreated with anti-IL-6,IL-10 or IL-21neutralizing antibodies for 3 days,the frequencies and secretion of IL-10 were determined by flow cytometric analysis and ELISA.C57BL/6 mice were adoptively transferred with OT-I splenocytes and vaccinated with OVA-loaded DCs.After intravenous administration of B cells treated with the indicated culture conditions,mice from each group were sacrificed on day 14 and the frequency and number of CD8⁺V?5.1⁺T cells or IFN-?⁺CD4⁺and CD8⁺T cells in the spleens were evaluated by flow cytometry.In the B16F10 subcutaneous tumor model and metastasis model,B cells treated with the indicated culture conditions were injected.Subcutaneous tumor growth and tumor nodules in the lungs were examined.3.3 Investigation of inhibition of autophagosomes formation or IL-6 secretion on CD4⁺T-cell,B-cell differentiation and tumor growth in vivoTumor cell culture media from negative control?Becn1 NC?or Becn1 knockdown?Becn1KD?B16-F10 cells were concentrated and detected the expression of LC-3I/II.WT,Becn1NC or Becn1 KD B16-F10 cells were?s.c.?injected into WT or Il6^-/-C57BL/6 mice,to establish an subcutaneous melanoma tumor model,respectively.At day 21 after injection,the serum,tumor-draining lymph nodes,and tumor tissues were harvested,respectively.The frequencies of IL-10⁺CD4⁺T cells,IL-21⁺CD4⁺T cells,IL-10⁺B cells,and IFN-?⁺CD4⁺T cells were analyzed by flow cytometric analysis.The IL-6 level in serum was examined by ELISA.The growth of tumor was monitored by calipers.Results1.Study of TRAPs-induced CD4⁺T cells differentiation in vitro and in vivoTreatment of mouse splenic CD4⁺T cells with TRAPs during activation by?CD3/CD28resulted in the induction of the transcripts encoding Il6,Il21,Il10,and Il17,but not Il1b,Il2,Il4,Il9,Tnf,Ifng,Foxp3 or Tgfb1.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.TRAPs-induced IL-21⁺CD4⁺T cells expressed the follicular helper T cell?Tfh?-associated molecules CXCR5 and Bcl-6.In contrast,TRAPs reduced the frequency of IFN-?⁺CD4⁺T cells and suppressed Th1 cell differentiation.In vivo results showed that,consistent with the B16F10 tumor-bearing mice,the frequencies of IL-6⁺,IL-10⁺and IL-21⁺CD4⁺T cells in the dLNs and spleens increased markedly after TRAPs administration.2.Investigation of the mechanisms underlying TRAPs-induced CD4⁺T cells differentiationWithin the time frame of the induction of CD4⁺T cells differentiation,TRAPs adhered to the surface of CD4⁺T cells without being internalized.TRAPs-induced IL-6,IL-10 and IL-21secretion by CD4⁺T cells were completely Tlr2 and Myd88–dependent,but not Tlr4deficiency.Consistently,TLR2 on the surface of CD4⁺T cells was in direct contact with TRAPs.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.TRAPs treatment of WT CD4⁺T cells resulted in the phosphorylation of NF-?B,Akt,p38 and STAT3,but not ERK1/2 or JNK1/2.Pretreatment of CD4⁺T cells with inhibitors of NF-?B,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.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.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.Consistently,TRAPs failed to induce IL-10 and IL-21 expression or STAT3 phosphorylation in Il6^–/–CD4⁺T cells.Moreover,following i.v.administration of TRAPs,the frequencies of IL-10⁺and IL-21⁺CD4⁺T cells in the dLNs and spleens were much lower in Il6^–/–mice than in WT mice.Collectively,these results support a TRAPs-initiated regulatory cascade of CD4⁺T cell differentiation involving TLR2–NF-?B/p38/Akt-dependent induction of autocrine IL-6 which then promotes IL-10 and IL-21 expression via STAT3.TRAPs from the hepatic carcinoma Hepa1-6,lung cancer LLC or lymphoma EL4 cells also potently enhanced IL-6 secretion in CD4⁺T cells.Treatment of TRAPs with proteinase K or sonication impaired the ability of TRAPs to induce IL-6 from CD4⁺T cells,indicating that proteins on the surface,but not the soluble contents,of TRAPs are largely responsible for IL-6 induction in CD4⁺T cells.Blocking of Hsp90?,but not HMGB1,Hsp60 or Hsp70,on the surface of TRAPs diminished TRAPs binding to CD4⁺T cells,reduced TRAPs-induced IL-6 secretion,and suppressed the activation of NF-?B,Akt and p38.Similar to mouse TRAPs,hTRAPs-induced IL-6 transcription and secretion by human CD4⁺T were almost completely abolished by pretreatment of hTRAPs with an anti-hsp90?blocking antibody.These results indicate that induction of CD4⁺T cells IL-6 expression by HSP90?on the surface of TRAPs is a common characteristic in humans and mice.3.Investigation of the effects of TRAPs-elicited CD4⁺T cells(T_TRAP)on immune responses and its mechanisms3.1 Investigation of the effects of TRAPs-elicited CD4⁺T cells(T_TRAP)on T-cell responses and tumorigenesisT_TRAP supernatants(SN/T_TRAP),but not SN/CD4⁺T,strongly suppressed the secretion of IFN-?by activated CD4⁺and CD8⁺T cells.Consistently,the transfer of T_TRAP but not control CD4⁺T cells led to a decrease of IFN-?⁺CD8⁺and CD4⁺T cells and the expansion of V?5.1⁺CD8⁺OT-I T cells induced by DC_OVA vaccination.In addition,T_TRAP could promote B16F10 melanoma cells growth and metastasis in vivo.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-?production by activated CD4⁺and CD8⁺T cells.Consistent with the above results,WT T_TRAP showed accelerated growth and lung metastasis as compared to those inoculated with B16F10 cells alone.In contrast,co-inoculation of B16F10 cells with Il6^–/–T_TRAP resulted in no enhancement of tumor growth and lung metastasis,and the mice even exhibited slightly,albeit not statistically significant,retarded tumor growth.These results corroborate the conclusion that T_TRAP rely on IL-6 to dampen T cell-mediated antitumor immunity and foster tumor progression.3.2 Investigation of the effects of TRAPs-elicited CD4⁺T cells(T_TRAP)on B-cell differentiation and tumorigenesisAdoptive transfer of T_TRAP,but not control CD4⁺T cells,significantly increased the frequency and number of IL-10⁺Bregs in vivo.Consistently,SN/T_TRAP could also directly promote IL-10⁺Bregs differentiation and IL-10 secretion.Interestingly,co-culture of B cells and CD4⁺T cells in the presence of TRAPs led to a marked increase in Bregs differentiation than without CD4⁺T cells.The results indicated that TRAPs can promote IL-10⁺Breg differentiation directly by activating on B cells and indirectly by conditioning CD4⁺T cells.In agreement with the above results,culturing B cells in SN/T_TRAP together with TRAPs resulted in a synergistic increase the frequencies of IL-10⁺Bregs and IL-10 secretion as compared to TRAPs or SN/T_TRAP alone.Neutralizing IL-6,IL-10 or IL-21 partially abolished the effect of SN/T_TRAP in promoting IL-10 production of TRAPs-induced B cells,indicated that secreted cytokines,including IL-6,IL-10,and IL-21,from T_TRAP were involved in promoting Bregs differentiation.Adoptive transfer of B cells pretreated by TRAPs(B_TRAP)inhibited the expansion of OT-I T cells,and the transfer of B cells pretreated by TRAPs and SN/T_TRAP(B_{TRAP+SN/TTRAP}),which exhibited a higher proportion of IL-10⁺B cells,resulted in a more pronounced and almost complete inhibition of the expansion of OT-I T cells.Moreover,adoptive transfer of B_{TRAP+SN/TTRAP}RAP+SN/TTRAP decreased the numbers of IFN-?⁺CD8⁺and CD4⁺T cells induced by DC_OVAVA vaccination and promoted the growth of B16F10 melanoma cells and their metastasis to the lung.Taken together,these results suggest that IL-6,IL-10,and IL-21 from T_TRAP augment the differentiation and immunosuppressive function of TRAPs-induced B cells to facilitate tumor growth and metastasis.3.3 Investigation of inhibition of autophagosomes formation or IL-6 secretion on CD4⁺T-cell,B-cell differentiation and tumor growth in vivoBecn1 KD in B16F10 cells diminished intracellular LC3-II accumulation and markedly reduced TRAPs secretion.In the mice bearing Becn1 KD B16F10 tumors,the frequency of IL-21⁺,IL-10⁺CD4⁺T cells and IL-10⁺B 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.In contrast,the frequency of IFN-?⁺CD4⁺T cells in mice bearing Becn1 KD tumors was markedly increased.Additionally,Becn1 KD B16F10 cells exhibited significantly slower growth in vivo.In WT or Il6^–/–mice bearing B16F10 tumors,the frequencies of IL-10⁺and IL-21⁺CD4⁺T cells and IL-10⁺B cells in draining lymph nodes and tumor tissues from Il6^–/–tumor-bearing mice were significantly decreased.Accordingly,B16F10 tumors grew more slowly in Il6^–/–mice than in WT mice.Conclusions1.TRAPs could induce CD4⁺T cells to produce IL-6,IL-10 and IL-21,and suppress Th1 cells differentiation;TRAPs-induced IL-21⁺CD4⁺T cells expressed the follicular helper T cell?Tfh?-associated molecules CXCR5 and Bcl-6.2.Membrane-bound HSP90?on intact TRAPs is crucial for inducing IL-6 production in CD4⁺T cells via a TLR2–MyD88–NF-?B signal cascade.In addition,p38 and PI3K/Akt signaling pathways are also involved in this process.3.Autocrine or paracrine IL-6 induced by TRAPs further stimulates CD4⁺T cells to produce IL-10 and IL-21 via IL-6-STAT3 pathways.4.TRAPs-elicited CD4⁺T cells inhibited CD4⁺and CD8⁺effector T cell function in an IL-6- and IL-10-dependent manner and induced IL-10-producing regulatory B cells?Bregs?via IL-6,IL-10 and IL-21,thereby promoting tumor growth and metastasis.5.Inhibition of tumor autophagosome formation or IL-6 secretion by CD4⁺T cells markedly retarded tumor growth,and inhibited IL-21⁺CD4⁺T cells,IL-10⁺CD4⁺T cells and IL-10⁺B cells differentiation.