Enhancing the Efficacy of Anti-tumor Immunotherapy by Targeting Tcell Metabolism

Tumor immunotherapy has become the fourth major type of cancer therapy, being used alone or in combination with surgery, radiotherapy or chemotherapy. In recent years, immunotherapies, especially immune checkpoint blockade (ICB) therapy and chimeric antigen receptor T-Cell (CAR-T), have achieved surprising curative effects in both preclinical studies and clinical practice. However, with the expansion of clinical cases and tumor types treated, the limitations of immunotherapy have gradually emerged. For example, the clinical positive response rate of ICB therapy is only 20%-30%, and is ineffective against or may even promote the progression and metastasis of certain types of tumors. CAR-T cells therapy is very effective against hematological tumors, but its application for treating solid tumors has encountered a bottleneck. Therefore, combination therapeutic strategies have emerged to overcome the drawbacks associated with the different treatments. At present, research on immunotherapy combined with radiotherapy, chemotherapy and targeted therapy is booming. Investigations about the metabolism of tumors and immune cells have become one of the hotspots in recent years. Regulating the metabolism of effector T cells in the tumor microenvironment represents an effective way to improve immunotherapy, resulting in the restoration or enhancement of the ability of effector T cells to produce an anti-tumor immune response. In this review, we discuss recent progress in this field, with an emphasis on the metabolic characteristics of tumor and immune cells, especially T cells in the tumor microenvironment. We also provide a snapshot of how T cell metabolic reprogramming can be regulated to restore or enhance the efficacy of tumor immunotherapy, as well as the challenges and solutions associated with this metabolic reprogramming.

Highlights of the advances in basic immunology in 2011

In this review, we summarize the major fundamental advances in immunological research reported in 2011. The highlights focus on the improved understanding of key questions in basic immunology, including the initiation and activation of innate responses as well as mechanisms for the development and function of various T-cell subsets. The research includes the identification of novel cytosolic RNA and DNA sensors as well as the identification of the novel regulators of the Toll-like receptor （TLR） and retinoic acid-inducible gene I （RIG-I）-Iike receptor （RLR） signaling pathway. Moreover, remarkable advances have been made in the developmental and functional properties of innate lymphoid cells （I LCs）. Helper T cells and regulatory T （Treg） cells play indispensable roles in orchestrating adaptive immunity. There have been exciting discoveries regarding the regulatory mechanisms of the development of distinct T-cell subsets, particularly Th17 cells and Treg cells. The emerging roles of microRNAs （miRNAs） in T cell immunity are discussed, as is the recent identification of a novel T-cell subset referred to as follicular regulatory T （TFR） cells.

Cellular and molecular regulation of innate inflammatory responses

Innate sensing of pathogens by pattern-recognition receptors （PRRs） plays essential roles in the innate discrimination between self and non-self components, leading to the generation of innate immune defense and inflammatory responses. The initiation, activation and resolution of innate inflammatory response are mediated by a complex network of interactions among the numerous cellular and molecular components of immune and non- immune system. While a controlled and beneficial innate inflammatory response is critical for the elimination of pathogens and maintenance of tissue homeostasis, dysregulated or sustained inflammation leads to pathological conditions such as chronic infection, inflammatory autoimmune diseases. In this review, we discuss some of the recent advances in our understanding of the cellular and molecular mechanisms for the establishment and reJzulation of innate immunity and inflammatory responses.

TGF-β1 gene-modified,immature dendritic cells delay the development of inflammatory bowel disease by inducing CD4^(+)Foxp3^(+)regulatory T cells

Inflammatory bowel disease(IBD)is caused by an uncontrolled immune response in the intestinal lumen,leading to inflammation in genetically predisposed individuals.Immunotherapy may be a promising approach to the treatment of IBD.Here,we show that transforming growth factor-β1(TGF-β1)gene-modified immature dendritic cells(imDCs)could enhance the inhibitory function of imDCs and delay the progress of IBD induced by dextran sodium sulfate in mice.The results of fluorescence-activated cell sorter(FACS)demonstrated that this protective effect is mediated partially by inducing CD4^(+)Foxp3^(+)regulatory T cells(Tregs)in mesentery lymph nodes to control inflammation.In vitro experiments also supported this hypothesis.In conclusion,we provide evidence that TGF-b1-modified bone marrow-derived imDCs may have a therapeutic effect to IBD.

Activated cytotoxic lymphocytes promote tumor progression by increasing the ability of 3LL tumor cells to mediate MDSC chemoattraction via Fas signaling

The Fas/FasL system transmits intracellular apoptotic signaling, inducing cell apoptosis. However, Fas signaling also exerts non-apoptotic functions in addition to inducing tumor cell apoptosis. For example, Fas signaling induces lung cancer tumor cells to produce prostaglandin E2 （PGE2） and recruit myeloid-derived suppressor cells （MDSCs）. Activated cytotoxic T lymphocytes （CTLs） induce and express high levels of FasL, but the effects of Fas activation initiated by FasL in CTLs on apoptosis-resistant tumor cells remain largely unclear. We purified activated CD8^＋ T cells from OT-1 mice, evaluated the regulatory effects of Fas activation on tumor cell escape and investigated the relevant mechanisms. We found that CTLs induced tumor cells to secrete PGE2 and increase tumor cell-mediated chemoattraction of MDSCs via Fas signaling, which was favorable to tumor growth. Our results indicate that CTLs may participate in the tumor immune evasion process. To the best of our knowledge, this is a novel mechanism by which CTLs play a role in tumor escape. Our findings implicate a strategy to enhance the antitumor immune response via reduction of negative immune responses to tumors promoted by CTLs through Fas signaling.

The function and regulation of TET2 in innate immunity and inflammation

TET2,a member of ten-eleven translocation(TET)family as a-ketoglutarate-and Fe2+-dependent dioxygenase catalyzing the iterative oxidation of 5-methylcytosine(5mC),has been widely recognized to be an important regulator for normal hematopoiesis especially myelopoiesis.Mutation and dysregulation of TET2 contribute to the development of multiple hematological malignancies.Recent studies reveal that TET2 also plays an important role in innate immune homeostasis by promoting DNA demethylation or independent of its enzymatic activity.Here,we focus on the functions of TET2 in the initiation and resolution of inflammation through epigenetic regulation and signaling network.In addition,we highlight regulation of TET2 at various molecular levels as well as the correlated inflammatory diseases,which will provide the insight to intervene in the pathological process caused by TET2 dysregulation.

Conditional Knockout of Src Homology 2 Domain-containing Protein Tyrosine Phosphatase-2 in Myeloid Cells Attenuates Renal Fibrosis after Unilateral Ureter Obstruction

Background：Src homology 2 domain-containing protein tyrosine phosphatase-2 （SHP-2） is a kind of intracellular protein tyrosine phosphatase.Studies have revealed its roles in various disease,however,whether SHP-2 involves in renal fibrosis remains unclear.The aim of this study was to explore the roles of myeloid cells SHP-2 in renal interstitial fibrosis.Methods：Myeloid cells SHP-2 gene was conditionally knocked-out （CKO） in mice using loxP-Cre system,and renal interstitial fibrosis was induced by unilateral ureter obstruction （UUO）.The total collagen deposition in the renal interstitium was assessed using picrosirius red stain.F4/80 immunostaing was used to evaluate macrophage infiltration in renal tubular interstitium.Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to analyze the production of cytokines in the kidney.Transferase-mediated dUTP nick-end labeling stain was used to assess the apoptotic renal tubular epithelial cells.Results：Src homology 2 domain-containing protein tyrosine phosphatase-2 gene CKO in myeloid cells significantly reduced collagen deposition in the renal interstitium after UUO.Macrophage infiltration was evidently decreased in renal tubular interstitium of SHP-2 CKO mice.Meanwhile,the production of pro-inflammatory cytokines was significantly suppressed in SHP-2 CKO mice.However,no significant difference was observed in the number of apoptotic renal tubular epithelial cells between wild-type and SHP-2 CKO mice.Conclusions：Our observations suggested that SHP-2 in myeloid cells plays a pivotal role in the pathogenesis of renal fibrosis,and that silencing of SHP-2 gene in myeloid cells may protect renal from inflammatory damage and prevent renal fibrosis after renal injury.

Platelets promote allergic asthma through the expression of CD154

Platelet activation is associated with multiple immune responses and the pathogenesis of various immune-related diseases. However, the exact role and the underlying mechanism of platelets in the progression of allergic asthma remain largely unclear. In this study, we demonstrate that during antigen sensitization, platelets can be activated by ovalbumin （OVA） aerosol viathe upregulation of CD154 （CD4OL） expression. Platelet transfer promoted allergic asthma progression by inducing more severe leukocyte infiltration and lung inflammation, elevated IgE production and strengthened T helper 2 （Th2） responses in asthma-induced mice. Accordingly, platelet depletion compromised allergic asthma progression. CdI54-deficient platelets failed to promote asthma development, indicating the requirement of CD154 for platelets to promote asthma progression. The mechanistic study showed that platelets inhibited the induction of Foxp3 ＋ regulatory T cells both in vivoand in vitroat least partially through CD154, providing an explanation for the increase of Th2 responses by platelet transfer. Our study reveals the previously unknown role of platelet CD154 in the promotion of asthma progression by polarizing Th2 responses and inhibiting regulatory T-cell generation and thus provides a potential clue for allergic disease interventions.

Apoptotic cell administration enhances pancreatic islet engraftment by induction of regulatory T cells and tolerogenic dendritic cells

cell transfer has been found to be able to facilitate engraftment of allograft. However, the underlying mechanisms remain to be fully understood. Here we demonstrate that intravenous administration of donor apoptotic splenocytes can promote pancreatic islet engraftment by inducing generation of tolerogenic dendritic cells （ToI-DCs） and expansion of CD4＋Foxp3＋ regulatory T cells （Tregs）. In vivo clearance of either dendritic cells （DCs） or Tregs prevented the induction of immune tolerance by apoptotic cell administration. Transient elimination of Tregs using anti-CD25, monoclonal antibody （mAb） abrogated the generation of ToI-DCs after administration of apoptotic splenocytes. Reciprocally, depletion of DCs within CD1 lc-DTR mice using diphtheria toxin （DT） prevented the generation of Tregs in the recipients with administration of apoptotic splenocytes. Induction of Tregs by ToI-DCs required direct cell contact between the two cell types, and programmed death 1 ligand （PD-L1） played important role in the Tregs expansion. Apoptotic cell administration failed to induce ToI-DCs in IL-lO-deficient and Smad3-deficient mice, suggesting that IL-10 and transforming growth factor-β （TGF-β） are needed to maintain DCs in the tolerogenic state. Therefore, we demonstrate that ToI-DCs promote the expansion of Tregs via PD-L1 on their surface and reciprocally Tregs facilitate ToI-DCs to maintain transplantation tolerance induced by apoptotic cells via secreting IL-IO and TGF-β.

Efficient induction of a Her2-specific anti-tumor response by dendritic cells pulsed with a Hsp70L1–Her2_(341–456) fusion protein

Heat shock proteins(HSPs)have been shown to interact with antigen-presenting cells(APCs),especially dendritic cells(DCs).HSPs act as potent adjuvants,inducing a Th1 response,as well as antigen-specific CD8^(+) cytotoxic T lymphocytes(CTL)via cross-presentation.Our previous work has demonstrated that Hsp70-like protein 1(Hsp70L1),a new member of the Hsp70 subfamily,can act as a powerful Th1 adjuvant in a DC-based vaccine.Here we report the efficient induction of tumor antigen-specific T cell immune response by DCs pulsed with recombinant fusion protein of Hsp70L1 and Her2_(341–456),the latter of which is a fragment of Her2/neu(Her2)containing E75(a HLA-A2 restricted CTL epitope).The fusion protein Hsp70L1–Her2_(341–456) promotes the maturation of DCs and activates them to produce cytokines,such as IL-12 and TNF-a,and chemokines,such as MIP-1a,MIP-1b and RANTES.Taken together,these results indicate that the adjuvant activity of Hsp70L1 is maintained in the fusion protein.Her2-specific HLA-A2.1-restricted CD8^(+) CTLs can be generated efficiently either from the Peripheral blood lymphocytes(PBL)of healthy donors or from the splenocytes of immunized HLA-A2.1/K^(b) transgenic mice by in vitro stimulation or immunization with DCs pulsed with the Hsp70L1–Her2_(341–456) fusion protein.This results in more potent target cell killing in an antigen-specific and HLA-A2.1-restricted manner.Adoptive transfer of splenocytes from transgenic mice immunized with Hsp70L1–Her2_(341–456)-pulsed DCs can markedly inhibit tumor growth and prolong the survival of nude mice with Her2^(+)/HLA-A2.1^(+) human carcinomas.These results suggest that Hsp70L1–Her2_(341–456)-pulsed DCs could be a new therapeutic vaccine for patients with Her2^(+) cancer.

Extracellular calcium elicits feedforward regulation of the roll-like receptor-triggered innate immune response

Despite the expanding knowledge on feedback regulation of Toll-like receptor （TLR） signaling, the feedforward regulation of TLR signaling for the proper innate response to invading microbes is not fully understood. Here, we report that extracellular calcium can coordinate the activation of the small GTPases Ras and Ras-proximate-1 （Rap1） upon TLR stimulation which favors activation of macrophages through a feedforward mechanism. We show that different doses of TLR agonists can trigger different levels of cytokine production, which can be potentiated by extracellular calcium but are impaired by the chelating reagent ethylene glycol tetraacetic acid （EGTA） or by knockdown of stromal interaction molecule 1 （STIM1）. Upon TLR engagement, GTP-bound Ras levels are increased and GTP-bound Rap1 is decreased, which can be reversed by EGTA-mediated removal of extracellular calcium. Furthermore, we demonstrate that Rap1 knockdown rescues the inhibitory effects of EGTA on the TLR-triggered innate response. Examination of the TLR signaling pathway reveals that extracellular calcium may regulate the TLR response via feedforward activation of the extracellular signal-regulated kinase signaling pathway. Our data suggest that an influx of extracellular calcium, mediated by STIM 1-operated calcium channels, may transmit the information about the intensity of extracellular TLR stimuli to initiate innate responses at an appropriate level. Our study may provide mechanistic insight into the feedforward regulation of the TLR-triggered innate immune response.

HSP70L1-mediated intracellular priming of dendritic cell vaccination induces more potent CTL response against cancer

Heat-shock protein(HSP)-based immunotherapy is established on its adjuvant effects when applied via an extracellular approach to pulse and activate dendritic cells(DCs).Our previous studies indicate that DCs pulsed with recombinant fusion proteins of antigenic fragment and HSP70-like protein 1(HSP70L1)are potent in stimulating antigen-specific Th1 responses.We herein evaluated the cytotoxic T cell(CTL)response by an intracellular approach of priming DCs with transfection of recombinant adenovirus-expressing the fusion gene of the 576–699 fragment of carcinoembryonic antigen(CEA)and HSP70L1.As compared with DCs pulsed with extracellular fusion protein,the DCs transfected with recombinant adenovirus expressing the fusion gene displayed equivalent mature phenotypes but less inflammatory appearance.However,the transfected DCs were superior to the pulsed DCs in inducing CEA-specific CTLs.Consistently,immunization of HLA-A2.1/H-2Kb transgene mice with the transfected DCs could induce more quantities of HLA-A2.1-restricted CEA-specific CTLs,protecting nude mice more significantly from human CEA-expressing colon tumor challenge when adoptively transferred.Mechanistic investigation indicated that intracellular expression of the fusion protein empowered the transfected DCs by activation of STAT1 possibly via inducing IFN-βand ERK pathways.Therefore,the more potent ability to induce anti-CEA CTL responses enables the DCs,which transfected with recombinant adenovirus expressing the fusion gene of antigenic CEA fragment and Th1 adjuvant,as an alternative promising approach for the immunotherapy of CEA-positive tumors.

Reversing epigenetic repression of transposable elements for improving tumor immunogenicity

In a recent study published in Nature,Zhang et al.[1]revealed a new epigenetic mechanism for restraining tumor intrinsic immunogenicity:Lysine demethylase 5B(KDM5B)recruits SET domain bifurcated histone lysine methyltransferase 1(SETDB1)to repress the transcription of transposable elements(TEs)by H3K9me3 modifica-tion,while TE de-repression by targeting KDM5B conse-quently activated nucleic acid sensing pathways and anti-gen presentation to elicit tumor intrinsic immunity which enhanced host anti-tumor immune response.

eutrophil sensing of cytoplasmic, pathogenic DNA in a GAS-STING-independent manner

The innate immune system is an integral part of the host res- ponse to invading pathogens and endogenous danger signals. Pattern recognition receptors （PRRs）, which are utilized by the innate immune system, can detect pathogen-associated molecular patterns （PAMPs） of invading pathogens and damage-associated molecular patterns （DAMPs） and signal to initiate the innate immune response.

Reversing the mitochondrial stress-induced exhaustion of CD8^(+)T cells for improving cancer immunotherapy

Antigen-specific CD8+T cells that infiltrate tumor tissue have been considered the primary cytotoxic lymphocytes that exert antitumor effects,building a foundation for current immunotherapies against cancer.However,in the face of continuous antigenic stimulation during chronic infections and cancer,T-cell differentiation leads to a state of incapacitation called T-cell exhaustion,which is a critical barrier to successful T-cell immunotherapy of cancer.The explosive growth of malignant tumor cells accompanied by a dysfunctional vascular system and metabolite competition leads to a stressful tumor microenvironment characterized by low-oxygen tension,acidic interstitial pH,and insufficient nutrients.The evidence for the contribution of the tumor microenvironment to the metabolic reprogramming of exhausted CD8+T cells has been increasing,but the response of intratumoral T cells to metabolic stress signals is still not fully understood.Findings of a study by Scharping et al.,1 together with other recent evidence,have highlighted that the tumor microenvironment can influence the mitochondrial stress of T cells and drive the trajectory of T-cell differentiation toward exhaustion.These findings add new perspectives on and insights into the molecular initiators of T-cell exhaustion and the combination of strategies that should be utilized to improve the efficacy of immunotherapy,and these perspectives are discussed in this review.

Arsenic trioxide elicits prophylactic and therapeutic immune responses against solid tumors by inducing necroptosis and ferroptosis

Boosting tumor immunosurveillance with vaccines has been proven to be a feasible and cost-effective strategy to fight cancer. Although major breakthroughs have been achieved in preventative tumor vaccines targeting oncogenic viruses, limited advances have been made in curative vaccines for virus-irrelevant malignancies. Accumulating evidence suggests that preconditioning tumor cells with certain cytotoxic drugs can generate whole-cell tumor vaccines with strong prophylactic activities. However, the immunogenicity of these vaccines is not sufficient to restrain the outgrowth of existing tumors. In this study, we identified arsenic trioxide (ATO) as a wide-spectrum cytotoxic and highly immunogenic drug through multiparameter screening. ATO preconditioning could generate whole-cell tumor vaccines with potent antineoplastic effects in both prophylactic and therapeutic settings. The tumor-preventive or tumor-suppressive benefits of these vaccines relied on CD8^(+) T cells and type I and II interferon signaling and could be linked to the release of immunostimulatory danger molecules. Unexpectedly, following ATO-induced oxidative stress, multiple cell death pathways were activated, including autophagy, apoptosis, necroptosis, and ferroptosis. CRISPR‒Cas9-mediated knockout of cell death executors revealed that the absence of Rip3, Mlkl, or Acsl4 largely abolished the efficacy of ATO-based prophylactic and therapeutic cancer vaccines. This therapeutic failure could be rescued by coadministration of danger molecule analogs. In addition, PD-1 blockade synergistically improved the therapeutic efficacy of ATO-based cancer vaccines by augmenting local IFN-γ production.

Aged hematopoietic stem cells entrap regulatory T cells to create a prosurvival microenvironment

Although DNA mutation drives stem cell aging,how mutation-accumulated stem cells obtain clonal advantage during aging remains poorly understood.Here,using a mouse model of irradiation-induced premature aging and middle-aged mice,we show that DNA mutation accumulation in hematopoietic stem cells(HSCs)during aging upregulates their surface expression of major histocompatibility complex class II(MHCII).MHCII upregulation increases the chance for recognition by bone marrow(BM)-resident regulatory T cells(Tregs),resulting in their clonal expansion and accumulation in the HSC niche.On the basis of the establishment of connexin 43(Cx43)-mediated gap junctions,BM Tregs transfer cyclic adenosine monophosphate(cAMP)to aged HSCs to diminish apoptotic priming and promote their survival via activation of protein kinase A(PKA)signaling.Importantly,targeting the HSC–Treg interaction or depleting Tregs effectively prevents the premature/physiological aging of HSCs.These findings show that aged HSCs use an active self-protective mechanism by entrapping local Tregs to construct a prosurvival niche and obtain a clonal advantage.

TLR4 is essential for dendritic cell activation and anti-tumor T-cell response enhancement by DAMPs released from chemically stressed cancer cells

The combination of immunotherapy and chemotherapy is regarded as a promising approach for the treatment of certain types of cancer. However, the underlying mechanisms need to be fully investigated to guide the design of more efficient protocols for cancer chemoimmunotherapy. It is well known that danger-associated molecular patterns （DAMPs） can activate immune cells, including dendritic cells （DCs）, via Toll-like receptors （TLRs）; however, the role of DAMPs released from chemical drug-treated tumor cells in the activation of the immune response needs to be further elucidated. Here, we found that colorectal cancer （CRC） cells treated with oxaliplatin （OXA） and/or 5-fluorouracil （5-Fu） released high levels of high-mobility group box 1 （HMGB1） and heat shock protein 70 （HSP70）. After OXA/5-Fu therapy, the sera of CRC patients also exhibited increased levels of HMGB1 and HSP70, both of which are well-known DAMPs. The supernatants of dying CRC cells treated with OXA/5-Fu promoted mouse and human DC maturation, with upregulation of HLA-DR, CD80 and CD86 expression and enhancement of IL-lp, TNF-a, MIP-la, MIP-lp, RANTES and IP-IO production. Vaccines composed of DCs pulsed with the supernatants of chemically stressed CRC cells induced a more significant IFN-y-producing Thl response both in vitroand in vivo. However, the supernatants of chemically stressed CRC cells failed to induce phenotypic maturation and cytokine production in TLR4-deficient DCs, indicating an essential role of TLR4 in DAMP-induced DC maturation and activation. Furthermore, pulsing with the supernatants of chemically stressed CRC cells did not efficiently induce an IFN-y-producing Thl response in TLR4-deficient DCs. Collectively, these results demonstrate that DAMPs released from chemically stressed cancer cells can activate DCs viaTLR4 and enhance the induction of an anti-tumor T-cell immune response, delineating a clinically relevant immuno-adjuvant pathway triggered by DAMPs.

CD24 aggravates acute liver injury in autoimmune hepatitis by promoting IFN-γ production by CD4^(+) T cells

The T-cell-mediated immune response is implicated in many clinical hepatic injuries, such as autoimmune hepatitis and acute virus hepatitis. CD24 is widely expressed by different immune cells and plays an important role in the pathogenesis of many autoimmune diseases. However, the role of CD24 in T-cell-mediated liver injury has not been elucidated until now. Here we showed that CD24 deficiency protects mice from concanavalin A (ConA)-induced fulminant liver injury by reducing serum interferon-γ (IFN-γ) levels. CD24 expression by hepatic T cells was markedly increased following ConA challenge. Moreover, decreased IFN-γ production by hepatic CD4^(+) T cells in CD24-deficient mice was detected, which was correlated with downregulated phosphorylation of STAT1 in hepatic tissue. In vitro experiments also supported the conclusion that CD24 deficiency impaired IFN-γ production by CD4^(+) T cells following ConA, CD3/CD28 and phorbol myristate acetate/ionomycin stimulation. Our study suggests that CD24 deficiency confers hepatoprotection by decreasing CD4^(+) T-cell-dependent IFN-γ production in vivo, which suggests that CD24 might be a potential target molecule for reducing clinical hepatitis.

Inflammation-induced CD69^+ Kupffer cell feedback inhibits T cell proliferation via membrane-bound TGF-β1

Kupffer cells,tissue-resident macrophage lineage cell,are enriched in vertebrate liver.The mouse F4/80^+ Kupffer cells have been subclassified into two subpopulations according to their phenotype and function:CD68^+ subpopulation with potent reactive oxygen species(ROS) production and phagocytic capacities,and CD11b^+ subpopulation with a potent capacity to produce T helper 1 cytokines.In addition,CD11b^+ Kupffer cells/macrophages may be migrated from the bone marrow or spleen,especially in inflammatory conditions of the liver.For analyzing diverse Kupffer cell subsets,we infected mice with Listeria monocytogenes and analyzed the phenotype variations of hepatic Kupffer cells.During L.monocytogenes infection,hepatic CD69^+ Kupffer cells were significantly induced and expanded,and CD69^+ Kupffer cells expressed higher level of CD11 b,and particularly high level of membrane-bound TGF-β1(mTGF-β1) but lower level of F4/80.We also found that clodronate liposome administration did not eliminate hepatic CD69^+ Kupffer cell subset.We consider the hepatic CD69^+ Kupffer cell population corresponds to CD11b^+Kupffer cells,the bone marrow-derived population.Hepatic CD69^+ Kupffer cells suppressed Ag-nonspecific and OVA-specific CD4 T cell proliferation through mTGF-β1 both in vitro and in vivo,meanwhile,they did not interfere with activation of CD4 T cells.Thus,we have identified a new subset of inflammation-induced CD69^+ Kupffer cells which can feedback inhibit CD4 T cell response via cell surface TGF-β1 at the late stage of immune response against infection.CD69^+ Kupffer cells may contribute to protect host from pathological injure by preventing overactivation of immune response.