The in vitro generation of multi-tumor antigen-specific cytotoxic T cell clones: Candidates for leukemia adoptive immunotherapy following allogeneic stem cell transplantation

Adoptive T-cell immunotherapy is a promising approach to manage and maintain relapse-free survival of leukemia patients, especially following allogeneic stem cell transplantation. Post-transplant adoptive immunotherapy using cytotoxic T lymphocytes (CTLs) of the donor origin provide graft-versus-tumor effects, with or without graft–versus-host disease. Myeloid leukemias express immunogenic leukemia associated antigens (LAAs); such as WT-1, PRAME, MAGE, h-TERT and others, most of them are able to induce specific T cell responses whenever associated with the proper co-stimulation. We investigated the ability of a LAA-expressing hybridoma cell line to induce CTL clones in PBMCs of HLA-matched healthy donors in vitro. The CTL clones were induced by repetitive co-culture with LAAs-expressing, HLA-A*0201⁺ hybrid cell line, generated by fusion of leukemia blasts to human immortalized APC (EBV-sensitized B-lymphoblastoid cell line; HMy2). The induced cytotoxic T cell clones were phenotypically and functionally characterized by pentamer analysis, IFN-γ release ELISPOT and cellular cytotoxicity assays. All T cell lines showed robust peptide recognition and functional activity when sensitized with HLA-A*0201-restricted WT-1_235–243, hTERT_615–624 or PRAME_100–108 peptides-pulsed T2 cells, in addition to partially HLA-matched leukemia blasts. This study demonstrates the feasibility of developing multi-tumor antigen-specific T cell lines in allogeneic PBMCs in vitro, using LAA-expressing tumor/HMy2 hybrid cell line model, for potential use in leukemia adoptive immunotherapy in partially matched donor-recipient setting.     

T细胞受体基因转染过继性免疫治疗的研究进展

利用T细胞进行过继性免疫治疗是治疗病毒感染性疾病和肿瘤的理想方法,但是用于治疗的T细胞的特异性、亲和性和数量等限制了其应用,如何获得特异、高效、一定数量的T细胞是目前亟待解决的问题。采用TCR基因转染的方法,将特异性高亲和力TCR转移到受体的T细胞中,可以特异性杀伤受体体内的肿瘤细胞。     

Role of memory T cell subsets for adoptive immunotherapy

Adoptive transfer of primary (unmodified) or genetically engineered antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. Besides the definition of optimal targets and antigen receptors, the differentiation status of transferred T cells is emerging as a crucial parameter for generating cell products with optimal efficacy and safety profiles.Long-living memory T cells subdivide into phenotypically as well as functionally different subsets (e.g. central memory, effector memory, tissue-resident memory T cells). This diversification process is crucial for effective immune protection, with probably distinct dependencies on the presence of individual subsets dependent on the disease to which the immune response is directed as well as its organ location.Adoptive T cell therapy intends to therapeutically transfer defined T cell immunity into patients. Efficacy of this approach often requires long-term maintenance of transferred cells, which depends on the presence and persistence of memory T cells. However, engraftment and survival of highly differentiated memory T cell subsets upon adoptive transfer is still difficult to achieve. Therefore, the recent observation that a distinct subset of weakly differentiated memory T cells shows all characteristics of adult tissue stem cells and can reconstitute all types of effector and memory T cell subsets, became highly relevant. We here review our current understanding of memory subset formation and T cell subset purification, and its implications for adoptive immunotherapy.     

肿瘤过继性细胞免疫治疗进展

过继性细胞免疫治疗(ACI)是肿瘤治疗的重要手段,通过回输体外扩增及激活的肿瘤特异性免疫细胞,达到控制肿瘤的目的.虽然ACI在恶性黑色素瘤的治疗中取得了可喜的疗效,但患者完全缓解率仍不足10％,而且ACI对其他肿瘤的临床疗效欠佳.近年来研究表明,改善细胞培养方法、优化成分组成、增强细胞本身功能以及联合其他治疗等是进一步提高肿瘤细胞免疫治疗的关键.     

Therapeutic efficacy of adoptive immunotherapy is predicated on in vivo antigen-specific proliferation of donor T cells

Activated T cells with down-regulated L-selectin expression (L-sel(-)) from tumor-draining lymph nodes represent a potent source of specific immune effectors in adoptive immunotherapy. Using congenic pairs of mice and carboxyfluorescein diacetate succinimidyl ester-labeled L-sel(-) T cells, the current study analyzed in vivo proliferation of transferred cells. In the lung of MCA205 tumor-bearing mice, 6% or 0.3 x 10(6) of the 5 x 10(6) donor cells were identified 24 h after transfer. Vigorous proliferation of donor cells was evident on day 2, reaching a maximum on day 6. The proliferation was tumor-specific and CD4 T cells divided with greater magnitude than CD8 cells. Successful adoptive immunotherapy also required sublethal whole-body irradiation (WBI) of the recipient. WBI exerted its effects on facilitating specific T cell proliferation at the tumor site. Taken together, our results demonstrate that adoptively transferred T cells undergo extensive proliferation in response to the tumor and this response is associated with therapeutic efficacy.     

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4(+) and CD8(+) T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-gamma-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4(+) and CD8(+) lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.     

T cell targeted immunotherapy for autoimmune disease

Much emphasis has been placed on the so-called “biologics” in the treatment of immune disorders within the last few years. Here we discuss the expanding horizon of potential strategies for immunotherapies targeting T lymphocytes as key effectors and regulators of autoimmunity. We review emerging reagents in a variety of animal models and human disorders that may offer new therapeutic options in current or modified iterations.     

Synergistic antitumor effect of combining metronomic chemotherapy with adoptive cell immunotherapy in nude mice

Adoptive cell immunotherapy with cytokine‐induced killer cell (CIK cell) represents a promising non‐toxic anticancer therapy. However, the clinical efficacy of CIK cells is limited because of abnormal tumor vasculature. Metronomic chemotherapy shows promising anticancer activity by its potential antiangiogenic effect and reduced toxicity. We hypothesized that metronomic chemotherapy with paclitaxel could improve the antitumor effect of adoptive CIK cell immunotherapy. Mice health status was analyzed by measuring mice weight and observing mice behavior. Immunohistochemistry was used to investigate the recruitment of CIK cells, the expression of endothelial cell molecules, as well as the hypoxic tumor area. Metronomic paclitaxel synergized with adoptive CIK cell immunotherapy to inhibit the growth of non‐small cell lung cancer (NSCLC). Metronomic paclitaxel reduced hypoxic tumor area and increased CIK cell infiltration. Hypoxia impeded the adhesion of CIK cells and reduced the expression of endothelial cell adhesion molecules. In vivo studies demonstrated that more CIK cells were found in endothelial cell adhesion molecules high expressed area. Our study provides a new rationale for combining metronomic chemotherapy with adoptive cell immunotherapy in the treatment of xenograft NSCLC tumors in immunodeficient mice. Further clinical trials integrating translational research are necessary to better evaluate the clinical benefit of this promising approach.     

Dual T cell receptor T cells with two defined specificities mediate tumor suppression via both receptors

Grafting T cells with new antigen specificity by T cell receptor (TCR) gene transfer could greatly facilitate adoptive T cell immunotherapy. Little is known about how two TCR on one T cell influence each other. Among other reasons, this is often due to the fact that only one TCR specificity is known. We have genetically generated murine dual TCR T cells (OT-I/P14), specific for ovalbumin(ova257) and lymphocyte choriomeningitis virus glycoprotein (gp33). These cells retain both specificities and can be stimulated by either antigenic peptide to proliferate and produce IFN-gamma. Even though one TCR (P14) is expressed at reduced levels on dual TCR T cells, the peptide sensitivity of these cells is similar to that of single TCR T cells of the same specificity. TCR down-modulation on dual TCR T cells depends primarily on binding of the specific ligand. Adoptively transferred dual TCR T cells suppress the growth of both B16-ova and B16-gp33 melanoma cells, regardless of the peptide used for in vitro activation. Taken together, despite a certain dominance of expression between two TCR on the same T cell, this need not necessarily have functional consequences.     

T cell vaccination as an immunotherapy for autoimmune diseases

Immunization with inactivated autoreactive T cells(T cell vaccination) selected from individual's own T cellrepertoire provides a unique in vivo setting for testing immune regulation that is known to involve interactionsof a variety of related surface molecules(1).It induces regulatory immune responses that closely resemble thein vivo situation where the immune system is challenged by clonal activation and expansion of given T cellpopulations in various autoimmune diseases.T cell vaccination provides a powerful means of eliciting naturalreactions of the immune system in response to clonal expansion of T cells,which can be used as a therapeuticapproach to suppress or eliminate specific pathogenic autoreactive T cells in autoimmune conditions.Clinicaltrials using T cell vaccination to deplete autoreactive T cells in human autoimmune conditions have begun toreveal the pathologic relevance of various autoimmune T cell populations in the disease processes,providing aunique opportunity to test the autoimmune theories in a clinical setting.Cellular & Molecular Immunology.2004;1(5):321-327.     

Minimally manipulated murine regulatory T cells purified by reversible Fab Multimers are potent suppressors for adoptive T‐cell therapy

The transfer of regulatory T cells, either freshly isolated, or modified, represents a promising therapeutic approach to dampen misdirected immune responses, like autoimmune diseases, chronic inflammatory syndromes and graft versus host disease. Clinical isolation of highly pure regulatory T cell (Treg) populations is still challenging and labeling reagents can influence their viability and functionality, potentially altering the potency of isolated Treg cell products. Here we show that reversible Fab multimer‐based Treg purification can prevent conventional antibody label‐induced interferences in vitro and in vivo. Remaining isolation reagents negatively interfere with Treg engraftment efficacy in C57BL/6 wild‐type mice due to Fcγ‐receptor‐ as well as IL‐2 receptor‐mediated mechanisms. Using a preclinical model for acute GvHD, we further show that purified ‘label‐freed’ Tregs are protective at substantially lower cell numbers as compared to conventional nonreversible antibody staining, translating into significantly improved survival of mice treated with minimally manipulated Tregs. These findings have important clinical relevance for future Treg‐based cell therapies.     

抗原特异性T细胞受体基因转导技术优化策略的研究进展

肿瘤抗原特异性T细胞受体（TCR）基因转导技术作为肿瘤过继性免疫治疗的新手段之一,细胞和动物实验都证明它有很好的抗肿瘤作用,但临床试验结果却不尽人意。影响临床疗效主要原因是肿瘤抗原特异性T细胞受体不能有效地表达于T淋巴细胞表面,因而探索提高抗原特异性TCR高效表达的方法可能是解决这种问题的必要途径。     

Robust T cell responses to aspergillosis in chronic granulomatous disease: implications for immunotherapy

Chronic granulomatous disease (CGD) patients are highly susceptible to invasive aspergillosis and might benefit from aspergillus‐specific T cell immunotherapy, which has shown promise in treating those with known T cell defects such as haematopoietic stem cell transplant (HSCT) recipients. But whether such T cell defects contribute to increased risks for aspergillus infection in CGD is unclear. Hence, we set out to characterize the aspergillus‐specific T cell response in CGD. In murine CGD models and in patients with CGD we showed that the CD4⁺ T cell responses to aspergillus were unimpaired: aspergillus‐specific T cell frequencies were even elevated in CGD mice (P < 0·01) and humans (P = 0·02), compared to their healthy counterparts. CD4‐depleted murine models suggested that the role of T cells might be redundant because resistance to aspergillus infection was conserved in CD4⁺ T cell‐depleted mice, similar to wild‐type animals. In contrast, mice depleted of neutrophils alone or neutrophils and CD4⁺ T cells developed clinical and pathological evidence of pulmonary aspergillosis and increased mortality (P < 0·05 compared to non‐depleted animals). Our findings that T cells in CGD have a robust aspergillus CD4⁺ T cell response suggest that CD4⁺ T cell‐based immunotherapy for this disease is unlikely to be beneficial.     

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 breastcancer,there is no effective therapy for patients with invasive and metastatic breast cancer.Immunotherapymay be proved effective in treating patients with advanced breast cancer.Breast cancer immunotherapyincludes antibody based immunotherapy,cancer vaccine immunotherapy,adoptive T cell transferimmunotherapy and T cell receptor gene transfer immunotherapy.Antibody based immunotherapy such as themonoclonal antibody against HER-2/neu (trastuzumab) is successfully used in the treatment of breast cancerpatients with over-expressed HER-2/neu,however,HER-2/neu is over-expressed only in 25-30% of breastcancer patients.Cancer vaccine immunotherapy is a promising method to treat cancer patients.Cancervaccines can be used to induce specific anti-tumor immunity in breast cancer patients,but cannot induceobjective tumor regression.Adoptive T cell transfer immunotherapy is an effective method in the treatment ofmelanoma patients.Recent advances in anti-tumor T cell generation ex vivo and limited clinical trial data havemade the feasibility of adoptive T cell transfer immunotherapy in the treatment of breast cancer patients.T cellreceptor gene transfer can redirect the specificity of T cells.Chimeric receptor,scFv(anti-HER-2/neu)/zetareceptor,was successfully used to redirect cytotoxic T lymphocyte hybridoma cells to obtain anti-HER-2/neupositive tumor cells,suggesting the feasibility of treatment of breast cancer patients with T cell receptor genetransfer immunotherapy.Clinical trials will approve that immunotherapy is an effective method to cure breastcancer disease in the near future.Cellular & Molecular Immunology.2004;1(4):247-255.     

Adoptive T cell therapy: Boosting the immune system to fight cancer

Cellular therapies have shown increasing promise as a cancer treatment. Encouraging results against hematologic malignancies are paving the way to move into solid tumors. In this review, we will focus on T-cell therapies starting from tumor infiltrating lymphocytes (TILs) to optimized T-cell receptor-modified (TCR) cells and chimeric antigen receptor-modified T cells (CAR-Ts). We will discuss the positive preclinical and clinical findings of these approaches, along with some of the persisting barriers that need to be overcome to improve outcomes.     

T细胞免疫治疗实体肿瘤的临床试验疗效分析

过继性T细胞治疗(adoptive cell transfer therapy,ACT)是以肿瘤浸润淋巴细胞(tumor infiltrating lymphocyte,TIL)、基因工程编辑的T细胞受体T细胞(T cell receptor engineered T cells,TCR-T)和嵌合抗原受体T细胞(ch...     

Harnessing TH9 cells in cancer immunotherapy

CD4 T cell effector subsets not only profoundly affect cancer progression, but recent evidence also underscores their critical contribution to the anticancer efficacy of immune checkpoint inhibitors. In 2012, the two seminal studies suggested the superior antimelanoma activity of T_H9 cells over other T cell subsets upon adoptive T cell transfer. While these findings provided great impetus to investigate further the unique functions of T_H9 cells and explore their relevance in cancer immunotherapy, the following questions still remain outstanding: are T_H9 cell anticancer functions restricted to melanoma? What are the factors favouring T_H9 cell effector functions? What is the contribution of T_H9 cells to cancer immunotherapy treatments? Can T_H9 cells be identified in humans and, if so, what is their clinical relevance? By reviewing the studies addressing these questions, we will discuss how T_H9 cells could be therapeutically harnessed for cancer immunotherapy strategies.     

Harnessing innate and adaptive immunity for adoptive cell therapy of renal cell carcinoma

The development of immunotherapies for renal cell carcinoma (RCC) has been the subject of research for several decades. In addition to cytokine therapy, the benefit of various adoptive cell therapies has again come into focus in the past several years. Nevertheless, success in fighting this immunogenic tumor is still disappointing. RCC can attract a multitude of different effector cells of both the innate and adaptive immune system, including natural killer (NK) cells, γδ T cells, NK-like T cells, peptide-specific T cells, dendritic cells (DC), and regulatory T cells (Tregs). Based on intensive research on the biology and function of different immune cells, we now understand that individual cell types do not act in isolation but function within a complex network of intercellular interactions. These interactions play a pivotal role in the efficient activation and function of effector cells, which is a prerequisite for successful tumor elimination. This review provides a current overview of the diversity of effector cells having the capacity to recognize RCC. Aspects of the functions and anti-tumor properties that make them attractive candidates for adoptive cell therapies, as well as experience in clinical application are discussed. Improved knowledge of the biology of this immune network may help us to effectively harness various effector cells, placing us in a better position to develop new therapeutic strategies to successfully fight RCC.     

Harnessing proteases for T regulatory cell immunotherapy

The immune system is tightly regulated by a subset of T cells defined as regulatory T cells (Tregs). Tregs maintain immune homeostasis by restraining unwarranted immune cell activation and effector function. Here, we discuss an important but underappreciated role of proteases in controlling Treg function. Proteases regulate a number of vital processes that determine T cell immune responses and some of them such as furin, ADAM (through regulating LAG receptor), MALT, and asparaginyl endopeptidase are implicated in Treg immunobiology. Targeted protease inhibition, using either small molecule inhibitors or gene deficient mice has demonstrated their specificity in modulating Treg function in experimental murine models. These data further highlight the ability of proteases to specifically regulate Tregs but no other T effector lineages. Taken together, it is apparent that incorporating proteases as targets within Treg cell engineering protocols may enable generation of robust Treg cellular therapeutics. These engineered Tregs may possess enhanced regulatory function along with resistance to lineage deviation in inflammatory disease such as colitis and graft versus host disease. Within this review, we summarize research on the role of proteases in regulating Treg function and discuss the translational potential of harnessing Treg function by targeting protease driven regulatory pathways.