Effects of platelets on characteristics of lymphocytes cultured in vitro and optimization of adoptive immunotherapy

T lymphocytes,the main participants of cellular immunity,can express a variety of surface molecules and form different lymphocyte subsets under the induction of different factors to play the functions of immune regulation and immune killing.Studies have shown that platelets play a crucial role in maintaining the stable differentiation of lymphocytes and the balance in immunomodulation.Therefore,it is necessary to study the effect of platelets on lymphocytes in vitro to better understand the role of platelets in the immune system and broaden the application of adoptive immunotherapy.Methods:Cell counting and microscopic observation were used to detect the effect of activated platelets on lymphocyte proliferation in vitro;Flow cytometry was used to detect whether changes in platelet activity affect the proportion of lymphocyte subpopulations in vitro,and to detect differences in the expression of granzyme B;lactate dehydrogenase assay(LDH)was used to determine the difference in lymphocyte killing activity caused by platelet activity in vitro.Results:This was the first to promote lymphocyte proliferation through the expression or release of certain molecules in vitro,demonstrating that platelet activation is one of the key factors.Secondly,activated platelets or inactivated platelets promoted lymphocyte subset differentiation by enhancing the proportion of CD3+CD8+T lymphocytes(CTL cells)but had a slight effect on the proportion of CD3+CD4+T(Th cells)and CD4+CD25+T lymphocytes(Treg cells).Then,it was found that either activated platelets or inactivated platelets down-regulated the proportion of natural killer(NK)T lymphocytes,while activated platelets significantly enhance the proportion of NK lymphocytes.Therefore,by further detecting the killing activity of PBMCs treated with platelets,it was found that activated platelets promoted the extensive anti-tumor activity of lymphocytes and significantly increased the expression of granzyme B.Conclusion:Our results suggest that activated platelets promote lymphocyte proliferation,optimize lymphocyte subpopulation ratio,and promote cytotoxic effect of lymphocytes in vitro,which may provide a new strategy for optimizing the adoptive immunotherapy culture system and improving its efficacy.

Adoptive immunotherapy for acute leukemia:New insights in chimeric antigen receptors

Relapses remain a major concern in acute leukemia. It is well known that leukemia stem cells(LSCs) hide in hematopoietic niches and escape to the immune system surveillance through the outgrowth of poorly immunogenic tumor-cell variants and the suppression of the active immune response. Despitethe introduction of new reagents and new therapeutic approaches, no treatment strategies have been able to definitively eradicate LSCs. However, recent adoptive immunotherapy in cancer is expected to revolutionize our way to fight against this disease, by redirecting the immune system in order to eliminate relapse issues. Initially described at the onset of the 90's, chimeric antigen receptors(CARs) are recombinant receptors transferred in various T cell subsets, providing specific antigens binding in a non-major histocompatibility complex restricted manner, and effective on a large variety of human leukocyte antigen-divers cell populations. Once transferred, engineered T cells act like an expanding "living drug" specifically targeting the tumor-associated antigen, and ensure long-term antitumor memory. Over the last decades, substantial improvements have been made in CARs design. CAR T cells have finally reached the clinical practice and first clinical trials have shown promising results. In acute lymphoblastic leukemia, high rate of complete and prolonged clinical responses have been observed after anti-CD19 CAR T cell therapy, with specific but manageable adverse events. In this review, our goal was to describe CAR structures and functions, and to summarize recent data regarding pre-clinical studies and clinical trials in acute leukemia.

The status,limitation and improvement of adoptive cellular immunotherapy in advanced urologic malignancies

In recent years, immunotherapy has been gradually established as the fourth frequently adopted antitumor therapy, following surgery, chemotherapy and radiotherapy, for advanced urologic malignancies with an improved understanding of theoretical basis, such as molecular biology and immunology. Thereinto, adoptive cellular immunotherapy （ACI） has become one of the hotspots, which comprises a variety of treatment approaches, such as TIL, CIK cell, ~＇~ T cell, CAR-engineered T cell and Allogeneie stem cell transplantation （alloSCT）. Although preclinical efficacy has been demonstrated remarkably, clinical trials could not consistently show the benefit due to multi-factors in complex immnnosuppressive microenvironment in vivo compared to that of in vitro. Here we review some timely aspects of ACI for advanced urologic malignancies, and describe the current status and limitation of immunotherapy from the cellular level. It＇s our expectation to provide prompting consideration of novel combinatorial ACI strategies and a resurgence of interest in ACI for advanced urologic malignancies.

Immunotherapy for hepatocellular carcinoma: From basic research to clinical use

Hepatocellular carcinoma(HCC) is a common cancer worldwide with a poor prognosis. Few strategies have been proven efficient in HCC treatment, particularly for those patients not indicated for curative resection or transplantation. Immunotherapy has been developed for decades for cancer control and is attaining more attention as a result of encouraging outcomes of new strategies such as chimeric antigen receptor T cells and immune checkpoint blockade. Right at the front of the new era of immunotherapy, we review the immunotherapy in HCC treatment, from basic research to clinical trials, covering anything from immunomodulators, tumor vaccines and adoptive immunotherapy. The mechanisms, efficacy and safety as well as the approach particulars are unveiled to assist readers to gain a concise but extensive understanding of immunotherapy of HCC.

Advances in biological immunotherapy for gastric cancer:a mini-review

Gastric cancer immunotherapy refers to the use of biological technology to mobilize the immune function so that the body has a natural anti-cancer ability.It can be induced in vitro by collecting immune cells and cancer cells from patients with gastric cancer to form specific immune cell groups.Besides,a large number of these immune cell groups are cultured,separated,and then reinfused into patients,to achieve high efficiency,eliminate tumors and mobilize immune mechanisms in patients.In theory,this method can cure tumors because the principle of immunotherapy is to stimulate the body's autoimmune response.However,for some special populations,there may be more severe side effects.At present,the prediction,prevention,and treatment of this severe side effect are not complete.The immunotherapy of gastric cancer has not yet reached the full promotion,but it is a good treatment direction.It can be used clinically with chemotherapy and radiotherapy,surgery and traditional Chinese medicine cooperate,thereby achieving significant curative effects,and even curing gastric cancer.

Effect of DC-CIK cell on the proliferation,apoptosis and differentiation of leukemia cells

Objective:To observe the effect of co-culture cytokine-induced killer cells(CIK) and homologous dendritic cells(DC) on the proliferative activity and phenotype change of the DCCIK cell and the cell killing activity of leukemia HL-60.Methods:50 mL cord blood sample was obtained from infants delivered by full term healthy woman and the cord blood mononuclear cells were isolated by density gradient centrifugal ion.Non-adherent cells were collectedfor the induction culture of CIK.adherent cells were differentiated into mature DC;cultured mature DC was mixed with and CIK in the proportion of 1:5 for 12 d.killing activity of DC-CIK co-cultured cell on leukemia HL-60 was detected by MTT assay.Results:Compared with CIKs.the cocultured DC-CIKs presented a markedly higher proliferation and killing activity.Conclusions:Co-culture of DC-CIK cells led to a significant increase of the proliferation and cytotoxicity of CIK.

Antitumor activities of human autologous cytokineinduced killer(CIK)cells against hepatocellular carcinoma cells in vitro and in vivo

AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptive immunotherapy for the patients with primary hepatocellular carcinoma (HCC), we evaluated the proliferation rate, phenotype and the antitumor activity of human CIK cells from healthy donors and HCC patients in vitro and in vivo. METHODS: Peripheral blood mononuclear cells (PBMC) from healthy donors and patients with primary HCC were incubated in vitro and induced into CIK cells in the presence of various cytokines such as interferon-gamma (IFN-gamma), interleukin-1 (IL-1), IL-2 and monoclonal antibody (mAb) against CD3. The phenotype and characterization of CIK cells were identified by flow cytometric analysis. The cytotoxicity of CIK cells was determined by (51)Cr release assay. RESULTS: The CIK cells were shown to be a heterogeneous population with different cellular phenotypes. The percentage of CD3+/CD56+ positive cells, the dominant effector cells, in total CIK cells from healthy donors and HCC patients, significantly increased from 0.1-0.13% at day 0 to 19.0-20.5% at day 21 incubation, which suggested that the CD3+ CD56+ positive cells proliferated faster than other cell populations of CIK cells in the protocol used in this study. After 28 day in vitro incubation, the CIK cells from patients with HCC and healthy donors increased by more than 300-fold and 500-fold in proliferation cell number, respectively. CIK cells originated from HCC patients possessed a higher in vitro antitumor cytotoxic activity on autologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivo animal experiment, CIK cells had stronger effects on the inhibition of tumor growth in Balb/c nude mice bearing BEL-7402-producing tumor than LAK cells (mean inhibitory rate, 84.7% vs 52.8%, P【0.05) or PBMC (mean inhibitory rate, 84.7% vs 37.1%, P【0.01). CONCLUSION: Autologous CIK cells are of highly efficient cytotoxic effector cells against primary hepatocellular carcinoma cells and might serve as an alternative adoptive therapeutic strategy for HCC patients.

Transduction of human hepatocellular carcinoma cells with human γ-interferon gene via retroviral vector

AIM To investigate the therapeutic potential of gamma interferon (IFN γ) gene modified human hepatocellular carcinoma (HCC) cells. METHODS The IFN γ gene was introduced retrovirally into four HCC cell lines. Secreted IFN γ activity was assessed using bioassay. The expression of MHC molecules was detected by FACS. Tumorigenicity was analysed by tumor formation in nude mice. RESULTS Four IFN γ gene transduced HCC cell lines secreted different amounts of IFN γ, as in the same case of five clones derived from one HCC cell line. Transduction with IFN γ caused significant increase in the expression of major histocompatibility complex (MHC) antigens on HCC cells. The expression of HLA class Ⅰ was increased by 2-3 times in terms of mean fluorescence intensities, while for class Ⅱ expression, the percentage of positive cells augmented from <10% to >50%. When equal amount of tumor cells were injected into nude mice, the tumorigenicity of some transduced cells decreased dramatically. CONCLUSION IFN γ gene transduction can convert weakly immunogenic HCC cells to activate antitumor immune response, and further pave the way for the future use of such gene modified tumor cells as a modality for the cancer immunotherapy.

The clinical effects of DC-CIK cells combined with chemotherapy in the treatment of advanced NSCLC

Objective: The aim of the study was to evaluate the safety and therapeutic effects of autologous dendritic cells co-cultured with cytokine-induced killer cells (DC-CIK) combined with chemotherapy in advanced non-small cell lung cancer (NSCLC) patients. Methods: Fifty patients with advanced NSCLC (stages III to IV), who had received therapies in our Center (Department of Biotherapy, Affiliated to Cancer Hospital of Shanxi Medical University, Taiyuan, China) from August 2008 to January 2010, were treated by DC-CIK + chemotherapy as the combined treatment group; fifty advanced NSCLC patients treated with chemotherapy at the same time served as controls. The immunologic function, short-term therapeutic effects, the 1-year survival rate, the life quality, the chemotherapy side effects were compared between the two groups, the safety and therapeutic effects of DC-CIK cells therapy were observed too. Results: There was no obvious change of subsets of T cells in peripheral blood before and after therapy in DC-CIK + chemotherapy group, and IFN-γ was improved after therapy in this group (P < 0.05); in chemotherapy alone group, the ratios of CD3+CD4+, CD3+CD8+, CD3-CD56+ cells and the secretion of IL-2, TNF-α decreased significantly after therapy (P < 0.05); the ratios of CD3+CD8+, CD3+CD56+ were improved after cell culture (P < 0.05). The disease control rate (DCR) of DC-CIK + chemotherapy group was higher than that in the chemotherapy alone group (78.0% vs 56.0%, P < 0.05); the 1-year survival rates of DC-CIK + chemotherapy group and chemotherapy alone group were 50% and 44% respectively, had no significant difference. Compared with chemotherapy alone group, the occurrence of chemotherapy side effects (including bone marrow suppression, nausea and vomiting, peripheral nerve toxicity) was less in the DC-CIK + chemotherapy group (P < 0.05). The physical and appetite were better in DC-CIK + chemotherapy group after therapy. Conclusion: To compare with simple chemotherapy, DC-CIK + chemotherapy for advanced NSCLC is safe and effective, and it can improve patients' life quality and remission rate, and prolong their survival time.

Engineering advanced dynamic biomaterials to optimize adoptive T-cell immunotherapy

Adoptive T-cell therapy(ACT)is a promising therapeutic approach based on the concept of potent T-cell mediated immunity against the tumor.The outcome of antigen-specific T-cells responses relies on the interaction between T-cells and antigen-presenting cells,which provides signals for generating different T-cell phenotypes with different roles in tumor removal.However,such interaction is often not optimal in vivo and results in low therapeutic efficacy.To reach the full potential of the T-cell response,current research put effort into developing dynamic biomaterials as artificial antigen-presenting cells to study and regulate the T-cell activity for controlling T-cell fate.In this perspective,we provide(1)an overview of ACT and general T-cells behaviors,(2)explore the insight on how biomaterials can be used for studying and regulating T-cell behaviors,(3)and discuss conceptual gaps in knowledge for biomaterials-based immunotherapy.

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.

Advancing cell-based therapy in sepsis:An anesthesia outlook

Sepsis poses a health challenge globally owing to markedly high rates of morbidity and mortality.Despite employing bundle therapy over two decades,approaches including transient organ supportive therapy and clinical trials focusing on signaling pathways have failed in effectively reversing multiple organ failure in patients with sepsis.Prompt and appropriate perioperative management for surgical patients with concurrent sepsis is urgent.Consequently,innovative therapies focusing on remedying organ injuries are necessitated.Cell therapy has emerged as a promising therapeutic avenue for repairing local damage to vital organs and restoring homeostasis during perioperative treatment for sepsis.Given the pivotal role of immune cell responses in the pathogenesis of sepsis,stem cell-based interventions that primarily modulate immune responses by interacting with multiple immune cells have progressed into clinical trials.The strides made in single-cell sequencing and gene-editing technologies have advanced the understanding of disease-specific immune responses in sepsis.Chimeric antigen receptor(CAR)-immune cell therapy offers an intriguing option for the treatment of sepsis.This review provides a concise overview of immune cell therapy,its current status,and the strides made in the context of sepsis research,discussing potential strategies for the management of patients with sepsis during perioperative stages.

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.

ncreasing the safety and efficacy of chimeric antigen receptor T cell therapy

Chimeric antigen receptor （CAR） T cell therapy is a promising cancer treatment that has recently been undergoing rapid development. However, there are still some major challenges, including precise tumor targeting to avoid off-target or ＂on-target/off-tumor＂ toxicity, adequate T cell infiltration and migration to solid tumors and T cell proliferation and persistence across the physical and biochemical barriers of solid tumors. In this review, we focus on the primary challenges and strafegies to design safe and effective CAR T cells, including using novel cutting-edge technologies for CAR and vecfor designs to increase both the safety and efficacy, further T cell modification to overcome the tumorassociated immune suppression, and using gene editing technologies to generate universal CAR T cells. All these efforts promote the development and evolution of CAR T cell therapy and move toward our ultimate goal--curing cancer with high safety, high efficacy, and low cost.

Advancing chimeric antigen receptor T cel therapy with CRISPR/Cas9

The clustered regularly interspaced short palindromic repeats （CRISPR）/CRISPR-associated 9 （CRISPR/Casg） system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/ Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for thera- peutic applications, especially chimeric antigen receptor （CAR） T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared. The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored. We also address the safety concerns associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy. As an integration-free gene insertion method, CRISPR/Cas9 holds great promise as an efficient gene knock-in platform. Given the tremendous progress that has been made in the past few years, we believe that the CRISPPJ Cas9 technology holds immense promise for advancing immunotherapy.

Enhancing Immune Responses for Cancer Therapy

Although the immune system possesses the means to respond to cancer, it often fails to control the spread of malignancy. Nonetheless, equipping endogenous immunity to release a strong antitumor response has significant advantages over conventional therapies. This review explores some of the options available to accomplish this, focusing first on vaccinations with tumor antigens to stimulate the immune system and empower stronger antitumor responses. We then compare and contrast the so-far limited clinical success of vaccination with the well-documented curative potential of adoptive therapy using T lymphocytes transfer. Finally, we highlight novel approaches using T call receptor （TCR） gene transfer strategy to exploit allogeneic T cell repertoires in conjunction with receptors selected in vitro for defined MHC/peptide combinations, as a basis for antigen-specific gene therapy of cancers. Cellular ＆ Molecular Immunology.

Serum-free culture of dendritic cells from patients with chronic myeloid leukemia in vitro and estimation of their cytotoxicity

OBJECTIVE: To establish a serum-free culture system of dendritic cells (DCs) from chronic myeloid leukemia (CML) cells so that DCs vaccine may be applied to the adoptive immunotherapy of CML in the near future. METHODS: Fetal calf serum, serum-free medium and autologous serum were used for culture of DCs. The usage of cytokines was classified into two groups: group A (stem cell factor, granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-alpha and interleukin-4) and group B (granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-alpha and interleukin-4). The phenotypes of DCs were analyzed by using indirect immunofluorescence and flow cytometry. Mixed leukocyte responses were performed by methyl thiazolyl tetrazolium (MTT) assay. Chromosome analysis of DCs can be achieved by displaying G banding. T cells from CML patients were stimulated with autologous DCs and T-cell cytotoxicity was measured by (MTT) assay. RESULTS: CD34(+) cells or mononuclear cells were obtained from peripheral blood or bone marrow samples of eight patients of chronic-phase CML. Group A of serum-free medium was better than group B in expansion of total cell numbers and the rate of DCs. These results of serum-free medium were not significantly different from those of fetal calf serum medium, but the results of autologous serum medium were inferior to two groups above. The expression of major histocompatibility complex class II antigen on the surface of DCs was notable (> 50%), but the expression of CD83 and the costimulatory molecules CD86 was not noticeable (10% - 50%). Although CD1a(+)/CD14(-) DCs were potent stimulators of allogeneic lymphocytes, expansion of T cells from normal volunteers were not significant (average 27.2 fold at DCs: T cells ratio of 1:10). At day 12, CD1a(+) cells from three patients were studied by displaying G banding and Ph(+) cells in these populations were 100%, 98% and 60%, respectively. At an effector: target ratio of 40:1, 32% to 45% cytotoxicity was noted with DC-stimulated T cells against autologous leukemia cells. CONCLUSIONS: A stable serum-free culture system of CML-DCs was established. The expression of CD83 and CD86 on the surface of CML-DCs and DCs' potent stimulation of allogeneic lymphocytes were not notable. DCs in CML patients can be derived from the malignant clone and these malignant DCs could induce anti-leukemic reactivity in autologous T lymphocytes without the necessity for additional exogenous antigens.

CAR T-Cell therapies in lymphoma: current landscape, ongoing investigations, and future directions

Chimeric antigen receptor(CAR)T-cell therapy has significantly improved outcomes for patients with relapsed/refractory large B-cell lymphoma,mantle cell lymphoma,and follicular lymphoma,with multiple FDA-approved CAR T products now commercially available.Ongoing studies seek to move CAR T-cells to earlier lines of therapy and to characterize the efficacy and safety of CAR T-cell approaches in additional lymphoma histologies including relapsed/refractory follicular lymphoma and chronic lymphocytic leukemia.Other areas of active research address CAR T in combination with other lymphoma-directed therapies,and mechanisms of CAR T resistance.This review focuses on the FDA-approved anti-CD19 CAR T products for B-cell lymphomas,management of CAR T-cell-associated toxicities,approaches to bridging therapy,and ongoing clinical trials and future research directions across a broad range of lymphoma histologies.

上期临床病案讨论结果

上期临床病案讨论结果术前诊断左上腹巨大包块：①腹膜后包块（嗜铬细胞瘤？）；②左肝肿瘤？手术情况于１９９４年５月７日在静脉复合麻醉下取双侧肋缘下人字形切口行剖腹探查术。术中见肝脏正常，未扣及结节或肿块，但在左肝下缘与胃小弯之间发现一凸出的巨大肿块。切开...