Immunotherapy in liver transplantation for hepatocellular carcinoma:Pros and cons

Liver transplantation(LT)has emerged as a curative strategy for hepatocellular carcinoma(HCC),but contributes to a higher predisposition to HCC recurrence in the immunosuppression context,especially for tumors beyond the Milan criteria.Although immunotherapy has dramatically improved survival for immunocompetent patients and has become the standard of care for a variety of tumors,including HCC,it is mainly used outside the scope of organ transplantation owing to potentially fatal allograft rejection.Nevertheless,accumulative evidence has expanded the therapeutic paradigms of immunotherapy for HCC,from downstaging or bridging management in the pretransplant setting to the salvage or adjuvant strategy in the posttransplant setting.Generally,immunotherapy mainly includes immune checkpoint inhibitors(ICIs),adoptive cell transfer(ACT)and vaccine therapy.ICIs,followed by ACT,have been most investigated in LT,with some promising results.Because of the complex tumor microenvironment and immunoreactivity when immunosuppressants are combined with immunotherapy,it is difficult to reach formulations for immunosuppressant adjustment and the optimal selection of immunotherapy as well as patients.In addition,the absence of effective biomarkers for identifying rejection and tumor response is still an unresolved barrier to successful clinical immunotherapy applications for LT.In this review,we comprehensively summarize the available evidence of immunotherapy used in LT that is specific to HCC.Moreover,we discuss clinically concerning issues regarding the concurrent goals of graft protection and antitumor response.

Prevention of recurrent hepatitis B infection after liver transplantation

BACKGROUND:Recurrence of hepatitis B virus(HBV) infection after liver transplantation can lead to graft loss and a reduction in long-term survival.The purpose of this review is to summarize the current therapeutic options for preventing HBV recurrence in liver transplant recipients.DATA SOURCES:Up to January 2013,studies that were published in MEDLINE and EMBASE on prevention of HBV recurrence after liver transplantation were reviewed.RESULTS:There have been remarkable advancements in the past two decades on the prevention of HBV recurrence after liver transplantation,from the discovery of hepatitis B immune globulin(HBIG) and lamivudine monotherapy to the combination therapy using HBIG and lamivudine.With the development of newer and stronger antiviral agents,the need for life-long HBIG is doubtful.With their low resistance profile,oral antiviral prophylaxis using these new agents alone is sufficient and is associated with excellent outcome.CONCLUSIONS:Restoration of host HBV immunity with adoptive immunity transfer and vaccination may represent the ultimate strategy to withdraw prophylactic treatment and to achieve a drug free regimen against HBV recurrence after liver transplantation.

Technical advances in NK cell-based cellular immunotherapy

Natural killer(NK)cells represent a promising future for tumor immunotherapy because of their unique biological functions and characteristics.This review focuses on technical advances in NK cell-based cellular immunotherapy and summarizes the developments of recent years in cell sources,genetic modification,manufacturing systems,clinical programs,and outcomes.Future prospects and challenges in NK cell immunotherapy are also discussed,including off-the-shelf NK cell exploitation,automatic and closed manufacturing systems,cryopreservation,and therapies involving regulatory checkpoints.

Effective adoptive transfer of haploidentical tumor-specific T cells in B16-melanoma bearing mice

Background Adoptive transfer of allogeneic tumor-specific T cells often results in severe graft-versus-host disease （GVHD）. Here, we sought to maximize graft-versus-tumor and minimize GVHD by using haploidentical T cells in pre-irradiated B16-melanoma bearing mice.

Adoptive transfer of FTY720-treated immature bone marrowderived dendritic cells(BMDCs) significantly reduced the spontaneous resorption rate in the CBA/J ×DBA/2 mouse model

Objective To investigate the effect of FTY720-treated immature bone marrow-derived dendritic cells（BMDCs） on the embryo resorption rate in the CBA/J× DBA/2 abortion mouse model.Methods The dendritic cells（DCs） were derived from bone marrow of DBA/2 mice, and then co-cultured with FTY720. The abortion mouse models were established by mating female CBA/J mice with DBA/2 mice. Via the CBA/J×DBA/2 abortion mouse model, six groups were established, group A： normal pregnancy model; group B： abortion mouse model with no treatment; group C： abortion mouse model injected with DC culture medium（DCCM）; group D： abortion mouse model injected with DC; group E： abortion mouse model injected with FTY720; group F： abortion model mouse injected with FTY720-DC. The differences were compared in the embryo resorption rates of the CBA/J ×DBA/2 abortion mouse model treated with FTY720-DC or different controls observed on gestation day 12 to 14, and then the microenvironment in murine pregnancy was investigated.Results The embryo resorption rate was statistically significantly decreased in group D and group E when they compared with group B and group C（P〈0.05, respectively）.Furthermore, the embryo resorption rate in group F showed a statistically significant decrease when compared with the other groups except group A（P〈0.01）. These resultssuggest that FTY720-DCs possess a notable advantage over DCs or FTY720 in reducing the embryo resorption rate of the abortion mouse model. The percentage of Th17（IL-17＋CD4＋T cells） in peripheral blood mononuclear cell（PBMC） in the abortion mouse model was 4.35%±0.34% before treated with FTY720-DC, and was1.34%±0.28% after treated with FTY720-DC（P〈0.01）. The percentage of Tregs（CD4~＋CD25~＋Foxp3~＋T cells） in PBMC was significantly increased in group F（8.35%±1.80%） as compared with group B（2.68%±0.65%）（P〈0.01）.Conclusion Adoptive transfer of FTY720-DC can statistically significantly reduce the embryo resorption rate in the CBA/J×DBA/2 abortion mouse model. The lower embryo resorption rate in the FTY720-DC treated abortion mouse model may be caused by the imbalance of Treg/Th17.

Adoptive transfer of metabolically reprogrammed macrophages for atherosclerosis treatment in diabetic ApoE^(-/-) mice

Atherosclerosis is characterized by inflammation in the arterial wall,which is known to be exacerbated by diabetes.Therapeutic repression of inflammation is a promising strategy for treating atherosclerosis.In this study,we showed that diabetes aggravated atherosclerosis in apolipoproteinE knockout(ApoE^(-/-))mice,in which increased expression of long-chain acyl-CoA synthetase 1(Acsl1)in macrophages played an important role.Knockdown of Acsl1 in macrophages(Mφ^(shAcsl1))reprogrammed macrophages to an anti-inflammatory phenotype,especially under hyperglycemic conditions.Injection of Mφ^(shAcsl1) reprogrammed macrophages into streptozotocin(STZ)-induced diabetic ApoE^(-/-) mice(ApoE^(-/-)+STZ)alleviated inflammation locally in the plaque,liver and spleen.Consistent with the reduction in inflammation,plaques became smaller and more stable after the adoptive transfer of reprogrammed macrophages.Taken together,our findings indicate that increased Acsl1 expression in macrophages play a key role in aggravated atherosclerosis of diabetic mice,possibly by promoting inflammation.Adoptive transfer of Acsl1 silenced macrophages may serve as a potential therapeutic strategy for atherosclerosis.

Regulatory T cells and cardiovascular diseases

Inflammation is a major underlying mechanism in the progression of numerous cardiovascular diseases(CVDs).Regulatory T cells(Tregs)are typical immune regulatory cells with recognized immunosuppressive properties.Despite the immunosuppressive properties,researchers have acknowledged the significance of Tregs in maintaining tissue homeostasis and facilitating repair/regeneration.Previous studies unveiled the heterogeneity of Tregs in the heart and aorta,which expanded in CVDs with unique transcriptional phenotypes and reparative/regenerative function.This review briefly summarizes the functional principles of Tregs,also including the synergistic effect of Tregs and other immune cells in CVDs.We discriminate the roles and therapeutic potential of Tregs in CVDs such as atherosclerosis,hypertension,abdominal arterial aneurysm,pulmonary arterial hypertension,Kawasaki disease,myocarditis,myocardial infarction,and heart failure.Tregs not only exert anti-inflammatory effects but also actively promote myocardial regeneration and vascular repair,maintaining the stability of the local microenvironment.Given that the specific mechanism of Tregs functioning in CVDs remains unclear,we reviewed previous clinical and basic studies and the latest findings on the function and mechanism of Tregs in CVDs.

Biological drug and drug delivery-mediated immunotherapy

The initiation and development of major infammatory diseases,i.e.,cancer,vascular infammation,and some autoimmune diseases are closely linked to the immune system.Biologics-based immunotherapy is exerting a critical role against these diseases,whereas the usage of the immunomodulators is always limited by various factors such as susceptibility to digestion by enzymes in vivo,poor penetration across biological barriers,and rapid clearance by the reticuloendothelial system.Drug delivery strategies are potent to promote their delivery.Herein,we reviewed the potential targets for immunotherapy against the major infammatory diseases,discussed the biologics and drug delivery systems involved in the immunotherapy,particularly highlighted the approved therapy tactics,and finally offer perspectives in this feld.

Challenges of NK cell-based immunotherapy in the new era

Natural killer cells （NKs） have a great potential for cancer immunotherapy because they can rapidly and directly kill transformed cells in the absence of antigen presensitization. Various cellular sources, including peripheral blood mononuclear cells （PBMCs）, stem cells, and NK cell lines, have been used for producing NK cells. In particular, NK cells that expanded from allogeneic PBMCs exhibit better efficacy than those that did not. However, considering the safety, activities, and reliability of the cell products, researchers must develop an optimal protocol for producing NK cells from PBMCs in the manufacture setting and clinical therapeutic regimen. In this review, the challenges on NK cell-based therapeutic approaches and clinical outcomes are discussed.

Emerging role of natural products in cancer immunotherapy

Cancer immunotherapy has become a new generation of anti-tumor treatment,but its indications still focus on several types of tumors that are sensitive to the immune system.Therefore,effective strategies that can expand its indications and enhance its efficiency become the key element for the further development of cancer immunotherapy.Natural products are reported to have this effect on cancer immunotherapy,including cancer vaccines,immune-check points inhibitors,and adoptive immune-cells therapy.And the mechanism of that is mainly attributed to the remodeling of the tumorimmunosuppressive microenvironment,which is the key factor that assists tumor to avoid the recognition and attack from immune system and cancer immunotherapy.Therefore,this review summarizes and concludes the natural products that reportedly improve cancer immunotherapy and investigates the mechanism.And we found that saponins,polysaccharides,and flavonoids are mainly three categories of natural products,which reflected significant effects combined with cancer immunotherapy through reversing the tumor-immunosuppressive microenvironment.Besides,this review also collected the studies about nano-technology used to improve the disadvantages of natural products.All of these studies showed the great potential of natural products in cancer immunotherapy.

The protective role of myeloid-derived suppressor cells in concanavalin A-induced hepatic injury

The mechanism underlying T cell-mediated fulminant hepatitis is not fully understood. In this study, we investigated whether myeloid derived suppressor cells （MDSCs） could prevent the concanavalin A （ConA）- induced hepatitis through suppressing T cell proliferation. We observed an increase in the frequencies of MDSCs in mouse spleen and liver at early stage of ConA treatment, implicating that the MDSCs might be involved in the initial resistance of mice against ConA- mediated inflammation. Subpopulation analysis showed that the MDSCs in liver of ConA-induced mice were mainly granulocytic MDSCs. Adoptive transfer of the bone marrow-derived MDSCs into ConA-treated mice showed that the MDSCs migrated into the liver and spleen where they suppressed T cell proliferation through ROS pathway. In addition, the frequencies of MDSCs in mice were also significantly increased by the treatment with immune suppressor glucocorticoids. Transfer of MDSCs into the regulatory T cell （Treg）- depleted mice showed that the protective effect of MDSCs on ConA-induced hepatitis is Treg-independent. In conclusion, our results demonstrate that MDSCs possess a direct protective role in T cell-mediated hepatitis, and increasing the frequency of MDSCs by either adoptive transfer or glucocorticoid treatment represents a potential cell-based therapeutic strategy for the acute inflammatory disease.

Tracking in vivo migration and distribution of antigen-specific cytotoxic T lymphocytes by 5,6-carboxyfluorescein diacetate succinimidyl ester staining during cancer immunotherapy

Background Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte （CTL） numbers,immunophenotype,tumor-specificity,and in vivo residence time,migration,and distribution.Therefore,tracing in vivo persistence,migration,and distribution of CTLs is important for cancer immunotherapy.Methods Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 （CCK-8） assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester （CFSE）-labeled melanoma antigen-specific cytotoxic T lymphocytes （CFSE-CTLs） for malignant melanoma cells in vitro were compared.Additionally,CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma,and their residence time,migration,and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue （％FI/g） in various tissues and analyzing tumor/non-tumor （T/NT） values.Anti-tumor effects of transferred CTLs and correlation between ％FI/g and D-value of tumor size were analyzed.Results Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity.No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing （P=0.849） or interleukin-2 （P=0.318） and interferon-y （P=0.201）levels.Distribution of CTLs in vivo varied with time.A negative correlation between ％FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed.CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion.CTLs were observed up to three weeks later in the tumor,liver,kidneys,and spleen; this was related to the abundant blood supply or the nature of immune organs.Conclusions CCK-8 assay is a novel method to select optimal CFSE staining concentrations.Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors.CFSE fluorescent markers can trace in vivo CTL persistence,migration,and distribution because of its stability,long half-life,and low toxicity.

Vitamin C promotes the proliferation and effector functions of human γδ T cells

γδT cells are of interest as effector cells for cellular immunotherapy due to their HLA-non-restricted lysis of many different tumor cell types.Potential applications include the adoptive transfer of in vitro-expandedγδT cells.Therefore,it is important to optimize the culture conditions to enable maximal proliferative and functional activity.Vitamin C(L-ascorbic acid)is an essential vitamin with multiple effects on immune cells.It is a cofactor for several enzymes,has antioxidant activity,and is an epigenetic modifier.Here,we investigated the effects of vitamin C(VC)and its more stable derivative,L-ascorbic acid 2-phosphate(pVC),on the proliferation and effector function of humanγδT cells stimulated with zoledronate(ZOL)or synthetic phosphoantigens(pAgs).VC and pVC did not increaseγδT-cell expansion within ZOL-or pAg-stimulated PBMCs,but increased the proliferation of purifiedγδT cells and 14-day-expandedγδT-cell lines in response toγδT-cell-specific pAgs.VC reduced the apoptosis ofγδT cells during primary stimulation.While pVC did not prevent activation-induced death of pAg-restimulatedγδT cells,it enhanced the cell cycle progression and cellular expansion.Furthermore,VC and pVC enhanced cytokine production during primary activation,as well as upon pAg restimulation of 14-day-expandedγδT cells.VC and pVC also increased the oxidative respiration and glycolysis ofγδT cells,but stimulus-dependent differences were observed.The modulatory activity of VC and pVC might help to increase the efficacy ofγδT-cell expansion for adoptive immunotherapy.

Breast Cancer Immunotherapy

Breast cancer is a leading cause of cancer-related deaths in women worldwide.Although tumorectomy, radiotherapy,chemotherapy and hormone replacement therapy have been used for the treatment of breast cancer,there is no effective therapy for patients with invasive and metastatic breast cancer.Immunotherapy may be proved effective in treating patients with advanced breast cancer.Breast cancer immunotherapy includes antibody based immunotherapy,cancer vaccine immunotherapy,adoptive T cell transfer immunotherapy and T cell receptor gene transfer immunotherapy.Antibody based immunotherapy such as the monoclonal antibody against HER-2/neu (trastuzumab) is successfully used in the treatment of breast cancer patients with over-expressed HER-2/neu,however,HER-2/neu is over-expressed only in 25-30% of breast cancer patients.Cancer vaccine immunotherapy is a promising method to treat cancer patients.Cancer vaccines can be used to induce specific anti-tumor immunity in breast cancer patients,but cannot induce objective tumor regression.Adoptive T cell transfer immunotherapy is an effective method in the treatment of melanoma patients.Recent advances in anti-tumor T cell generation ex vivo and limited clinical trial data have made the feasibility of adoptive T cell transfer immunotherapy in the treatment of breast cancer patients.T cell receptor gene transfer can redirect the specificity of T cells.Chimeric receptor,scFv(anti-HER-2/neu)/zeta receptor,was successfully used to redirect cytotoxic T lymphocyte hybridoma cells to obtain anti-HER-2/neu positive tumor cells,suggesting the feasibility of treatment of breast cancer patients with T cell receptor gene transfer immunotherapy.Clinical trials will approve that immunotherapy is an effective method to cure breast cancer disease in the near future.Cellular & Molecular Immunology.2004;1(4):247-255.

Effector cells derived from naive T cells used in tumor immunotherapy of mice bearing B16 melanoma

Background Adoptive cell transfer （ACT） immunotherapy has been used clinically for years to treat malignancies.Improving the killing efficiency of effector cells,such as tumor-specific cytotoxic T lymphocytes （CTLs）,is an important component for enhancing the clinical response of cancer immunotherapy.Hence,we explored a novel method for preparing cancer-specific CTLs using naive T lymphocytes.Methods C57BL/6 mice bearing B16 melanoma tumors were pretreated with cyclophosphamide （CTX） by peritoneal injection.The immunosuppressive influence of CTX on tumor regression and the tumor microenvironment was assessed.Naive T cells and T cell pools were isolated via negative selection using immunomagnetic beads.The proliferative potential and cytokine production of different T cell subpopulations were evaluated in vitro.Tumor-specific CTLs derived from naive T cells （naive CD4＋ T cells：naive CD8＋ T cells=2：1） and pooled T cells were generated in vitro,respectively.B16 melanoma-bearing C57BL/6 mice were pretreated with CTX,followed by ACT immunotherapy using dendritic cell-induced CTLs.The homing abilities of the effector cells and interleukin-2 （IL-2）,interferon-y,granzyme B,and perforin mRNA levels in tumor tissues were evaluated,and the change in tumor volume was measured.Results Mice receiving CTX peritoneal pretreatment injections did not display tumor regression compared with control mice.However,a significant downregulation of splenic Tregs and tumor growth factor-β1 （TGF-β1） and interleukin-10 （IL-10） serum levels was observed （P ＜0.05）.Naive T cells showed a stronger proliferative capacity and elevated cytokine production than did pooled T cells （P ＜0.05）.In addition,effector cells generated from naive T cells displayed more potent antitumor activity in vivo than those derived from pooled T cells （P ＜0.05）.Conclusion Effector cells derived from the naive T cells possess a stronger proliferative potential,homing capacity,and enhanced cytokine production,which leads to a superior antitumor response.

The dark side of immunotherapy:pancreatic cancer

Since the journal Science deemed cancer immunotherapy as the“breakthrough of the year”in 2014,there has been an explosion of clinical trials involving immunotherapeutic approaches that,in the last decade-thanks also to the renaissance of the immunosurveillance theory(renamed the three Es theory)-have been continuously and successfully developed.In the latest update of the development of the immuno-oncology drug pipeline,published last November by Nature Review Drug Discovery,it was clearly reported that the immunoactive drugs under study almost doubled in just two years.Of the different classes of passive and active immunotherapies,“cell therapy”is the fastest growing.The aim of this review is to discuss the preclinical and clinical studies that have focused on different immuno-oncology approaches applied to pancreatic cancer,which we assign to the“dark side”of immunotherapy,in the sense that it represents one of the solid tumors showing less response to this type of therapeutic strategy.