Simultaneous generation of multivirus-specific and regulatory T cells for adoptive immunotherapy

Previous studies have established that adoptive immunotherapy with donor-derived virus-specific T cells can prevent/treat viral complications post-stem cell transplant and regulatory T cells show promise as inhibitors of graft-versus-host disease. On the basis of flow cytometric analysis of upregulation of activation markers after stimulation with viral peptide pools, we have developed a rapid and clinically applicable protocol for the simultaneous selection of virus-specific T cells (after stimulation with peptide pools for the immunodominant antigens of cytomegalovirus, Epstein-Barr virus, and adenovirus) and regulatory T cells using CD25 immunomagnetic selection. Using tetramer staining, we detected enrichment of CD8 T cells recognizing peptide epitopes from cytomegalovirus and Epstein-Barr virus antigens after CD25 selection in 6 of 7 donors. Enzyme-linked immunospot assays demonstrated the simultaneous presence of bivirus-specific or trivirus-specific cells in all evaluated donors, with a median 29-fold (6 to 168), 40-fold (1 to 247), and 16-fold (1 to 219) enrichment of cells secreting interferon-γ in response to cytomegalovirus pp65, adenovirus hexon, and Epstein-Barr virus lymphoblastoid cells compared with unmanipulated peripheral blood mononuclear cells from the same donors. Furthermore, the CD25-enriched cells lost alloreactivity in H-thymidine proliferation assays and showed highly effective (median, 98%) suppression of alloreactivity in all evaluated donors. In summary, we have developed a rapid, simple Good Manufacturing Practice compliant methodology for the simultaneous selection of T cells with multiple viral specificities and regulatory T cells. Adoptive transfer of T cells generated using this strategy may enable restoration of cellular immunity to viruses after allogeneic stem cell transplant with a low risk of graft-versus-host disease. Owing to the speed and simplicity of this methodology, this approach may significantly broaden the applicability of adoptive immunotherapy.     

Rapid generation of CMV pp65-specific T cells for immunotherapy

Adoptive immunotherapy with cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) has been shown to be an effective means of restoring cellular immunity to this virus and preventing CMV infection after allogeneic stem cell transplantation. Problems with current strategies include requirements for generating dendritic cells or other antigen presenting cells for stimulating CTL and the time needed for cell culture. The adherent cell fraction of peripheral blood mononuclear cells from 6 CMV seropositive donors were pulsed with pooled CMV pp65 peptides and incubated with nonadherent peripheral blood lymphocytes. CTL lacking specific cytotoxicity to pp65 were restimulated at day 10 of culture using peptide pulsed adherent cells. Of the 6 CMV seropositive donors tested, 5 had specific cytotoxicity to CMV pp65 (range 31% to 75%), with no alloreactivity. The resulting pp65-specific CTL consisted of a mixture of CD4 and CD8 cells, with 1% to 29% of CD8 cells and 0.5% to 10% CD4 cells making interferon-gamma (IFN-gamma) in response to pp65. The donor from whom we could not detect CMV-specific cytotoxicity had detectable CD4 and CD8 CMV pp65 CTL by intracellular cytokine analysis for IFN-gamma. Using this simplified strategy for expanding CMV pp65 CTL, adoptive immunotherapy with pp65-specific CTL could be made available in a more timely manner for patients who have persistent or therapy refractory CMV infections.     

Generation of highly proliferative,rejuvenated cytotoxic T cell clones through pluripotency reprogramming for adoptive immunotherapy

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Selection bias: maintaining less-differentiated T cells for adoptive immunotherapy

The clinical application of T cell immunotherapy depends on ex vivo modification and expansion of T cells for adoptive transfer. In preclinical models, the use of a purified, naive T cell subset enhances persistence and antitumor immunity; however, the majority of clinical studies rely on modification of mixed populations of T cells that contain only a small subset of highly functional T cells with less-differentiated phenotype. In this month’s issue of the JCI, Klebanoff and colleagues uncover a Fas-mediated interaction between naive T cells and antigen-experienced T cells that drives differentiation and impairs adoptive immunotherapy. Further, they show that blockade of Fas signaling enhances antitumor immunity and increases survival in a mouse model of melanoma. Their work supports a growing body of evidence that the use of naive T cells enhances the efficacy of adoptive T cell therapy and suggests a new therapeutic strategy for preserving less-differentiated T cell populations.     

Generation of trispecific cytotoxic T cells recognizing cytomegalovirus, adenovirus, and Epstein-Barr virus: an approach for adoptive immunotherapy of multiple pathogens

Cytomegalovirus (CMV), adenovirus (Ad), and Epstein-Barr virus (EBV) are a major cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Adoptive immunotherapy with donor-derived cytotoxic T cells (CTLs) directed against EBV or CMV prevents the clinical manifestations of these viruses. We have designed a protocol for the simultaneous generation of polyclonal CTL specific for CMV, Ad, and EBV, which could be used to restore immunity to multiple viruses after SCT. EBV-transformed lymphoblastoid cell lines (LCLs), transduced with an adenoviral vector carrying a transgene for the immunodominant CMV antigen pp65 (Ad5f35-pp65GFP), were used to stimulate peripheral blood mononuclear cells in 6 normal donors. We detected the simultaneous presence of CD8 CTL recognizing peptide epitopes from all 3 viruses by pentamer staining. Enzyme-linked immunospot assays demonstrated a median 29-fold (8 to 248), 47-fold (2 to 137), or 18-fold (5 to 29) increase in cells secreting interferon-gamma in response to CMV, adenoviral, or EBV antigens, respectively, compared with unmanipulated peripheral blood mononuclear cell, with concomitant loss of alloreactivity. The CTL lines showed cytotoxicity against autologous LCL alone and increased cytotoxicity to autologous LCLs pulsed with CMV pp65 peptides or infected with Ad. In summary, we have developed a protocol for the generation of CTL with trivirus specificity, enabling adoptive transfer of CTL recognizing multiple viruses to restore cellular immunity after SCT.     

Exploiting cytokine secretion to rapidly produce multivirus-specific T cells for adoptive immunotherapy

Viral infections remain a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT), and conventional small-molecule therapeutics often have modest benefit, high cost, and adverse effects. Adoptive transfer of donor-derived virus-specific T cells has proved feasible and safe after HSCT and to reconstitute immunity against cytomegalovirus, Epstein-Barr virus, and adenovirus. Current protocols to generate these cytotoxic T cell lines are lengthy, taking up to 12 weeks. As viral infections often occur <30 days after HSCT, speedy production of virus-specific cytotoxic T cells lacking alloreactivity is highly desirable. We now describe a modified rapid selection method for production and characterization of CD4 and CD8 T cells specific for cytomegalovirus, Epstein-Barr virus, and adenovirus in a single infusate. We use Ad5f35-pp65/latent membrane protein 2 vectors in a single procedure over a 48-hour time period and manufacture a product suited for clinical use. By simultaneously expanding a portion of the selected product, we can characterize phenotype and function of the infused product and link them with subsequent in vivo outcome.     

Genetic modification of T lymphocytes for adoptive immunotherapy.

Adoptive transfer of T lymphocytes is a promising therapy for malignancies-particularly of the hemopoietic system-and for otherwise intractable viral diseases. Efforts to broaden the approach have been limited by the physiology of the T cells themselves and by a range of immune evasion mechanisms developed by tumor cells. In this review we show how genetic modification of T cells is being used preclinically and in patients to overcome these limitations, by incorporation of novel receptors, resistance mechanisms, and control genes. We also discuss how the increasing safety and effectiveness of gene transfer technologies will lead to an increase in the use of gene-modified T cells for the treatment of a wider range of disorders.     

肽刺激培养法体外扩增巨细胞病毒特异细胞毒性T淋巴细胞

目的研制一种扩增特异细胞毒性T淋巴细胞频数的方法。方法用不同浓度的巨细胞病毒（CMV）特异的表位肽pp65刺激人外周血单个核细胞（PBMCs）使CMV特异CTLs在PBMCs中的百分比增高。用四聚体一PE和CD。一FITC双标记PBMCs中的CMV特异CTLs，然后用流式细胞仪分析。结果这个方法能有效扩增CMV特异CTLs，使在外周血中占淋巴细胞百分比不到l％的CMV特异CTLs迅速增加到接近20％，占CDsT淋巴细胞的40％以上，并能从单份血样中获得大量特异CTLs用于流式分析。结论肽刺激培养法简便、易操作，能有效扩增CMV特异CTLs，使不同人的数据更容易对比和连续的评价。该方法也为过继性免疫疗法治疗巨细胞病毒疾病打下了基础。     

CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.

Multiple clinical trials have shown the efficacy of adoptively transferred allogeneic antigen-specific T cells for the treatment of viral infections and relapsed hematologic malignancies. In contrast, the therapeutic potential of autologous antigen-specific T cells has yet to be established since it has been technically difficult to generate sufficient numbers of these T cells, ex vivo. A major obstacle to the success of this objective derives from our inability to simply and rapidly isolate and/or expand large numbers of highly efficient antigen presenting cells (APCs) for repetitive stimulations of antigen-specific T cells in vitro. We show that autologous CD40-activated B cells represent a readily available source of highly efficient APC that appear to have several important advantages over other APCs for ex vivo T cell expansion including: (a) methodological simplicity necessary to generate continuously large numbers of APCs from just 50 cm3 of peripheral blood without loss of APC function; (b) capacity to induce high peak T cell proliferation and interferon-gamma production without IL-10 production; (c) ease in cryopreservation; and (d) markedly reduced cost. We, therefore, contend that CD40-activated B cells are an alternative source of highly efficient APCs with which to generate antigen-specific T cells ex vivo for autologous adoptive immunotherapy.     

Interleukin 7 generates antitumor cytotoxic T lymphocytes against murine sarcomas with efficacy in cellular adoptive immunotherapy

Interleukin 7 (IL-7) is a 25-kD cytokine that was initially described as a pre-B cell growth factor. This cytokine has also been shown to have T cell proliferative and differentiation effects. In this report, we demonstrate that antitumor cytotoxic T lymphocytes (CTL) generated by secondary in vitro sensitization of draining lymph node cells in IL-7 are effective in treating 3-day syngeneic methylcholanthrene (MCA) sarcoma pulmonary metastases in mice. In vivo titrations comparing IL-7 to IL-2 antitumor CTL show that they have equivalent potency in adoptive immunotherapy. IL-7 antitumor CTL generated against MCA sarcomas of weak immunogeneity are also tumor specific in their in vivo efficacy. This study represents the first successful use of a cytokine other than IL-2 for the generation of cells with in vivo efficacy in cellular adoptive transfer.     

Generation of melanoma-specific cytotoxic T lymphocytes for allogeneic immunotherapy

To exploit alloreactive T-cell responses known as the graft-versus-tumor effect, allogeneic hematopoietic stem cell transplantation is being explored as experimental therapy in selected solid tumors, including metastatic melanoma. However, donor T-cell responses are often delayed and associated with severe graft-versus-host disease. Posttransplant adoptive immunotherapy using tumor-specific cytotoxic T lymphocytes (CTL) of donor origin might provide immediate graft-versus-tumor effects but not graft-versus-host disease. Therefore, the aim of the current study was to define in vitro conditions for the expansion of allogeneic major histocompatibility complex-matched CTLs targeting melanoma-associated antigens (MAA). The CTLs were generated from peripheral blood mononuclear cells (PBMCs) of HLA-A*0201+ healthy donors by repetitive stimulations with HLA-A*0201-restricted MAA-derived peptides. Melanoma reactivity, as determined by lysis of peptide-pulsed T2 cells and HLA-A2+/Ag+ melanoma cells, was detected using in vitro expanded CTL targeting MAA peptides AAGIGILTV(MT(27-35)), IMDQVPFSV(G(209-2M)), and YMDGTMSQV(T(368-376)). In contrast, FLWGPRALV(MAGE3(271)-(279)) and VLPDVFIRCV(GnT-V(nt38-67)) induced peptide-specific recognition of T2 target cells only, whereas ITDQVPFSV(G(209-217)), KTWGQYWQV(G9(154)), MLLAVLYCL(T(1-9)), and tumor lysate could not induce specific CTLs. Specific cytolytic activity was accompanied by interferon-gamma secretion. Peptide-pulsed dendritic cells were required only for the initial stimulation of CTLs and could be substituted by PBMCs during restimulations. The median expansion rate of CTL was five to six times, regardless of whether dendritic cells or PBMCs were used after the initial stimulation. The results delineate the conditions for effective ex vivo expansion of melanoma-specific CTLs from PBMCs of healthy donors to be used as an adjunct in allogeneic cell therapy of metastatic melanoma.     

New adoptive immunotherapy strategies for solid tumours with CIK cells

INTRODUCTION: Despite development and introduction of new and innovative drugs, a large number of malignant diseases are associated with unfavourable prognosis. In recent years, considerable progress in cancer treatment has been obtained by the application of cytokine-induced killer (CIK) cells. AREAS COVERED: This review provides an overview and summary of recent advances in adoptive immunotherapy strategies in cancer treatment using CIK cells. A selective literature search has been performed. EXPERT OPINION: The application of CIK cells as adoptive immunotherapy plays an important role in cancer treatment. Combining CIK cells with other conventional and established therapy options represents an innovative approach and will hopefully provide new insight for the future.     

A requirement for antigen-specific helper T cells in the generation of cytotoxic T cells from thymocyte precursors

Thymocytes cultured with irradiated, allogeneic stimulator cells yield no cytotoxic effector cells after a period in culture. If, however, a population of irradiated spleen cells syngeneic to the responder cells are added to these cultures, cytotoxicity is generated. The helper activity present in the irradiated syngeneic spleen cells was found to be mediated by a cell bearing theta antigens. Furthermore, it was found to be antigen specific; helper cells which were tolerant of the stimulator cell antigens were unable to help the thymocyte responder cells, although these tolerant cells did contain helpers specific for a third party antigen. These experiments are consistent with a requirement for associative recognition of linked determinants in the induction of killer precursors which is thus strictly analogous to the induction of B-cell precursors via collaboration with helper T cells. In more extensive studies, it was found that histoincompatible helper cells (H-2b, H-2p, H-2q) were able to help a cytotoxic T cell (H-2k) response to a third party stimulator cell antigen (H-2d); that is, the helper T cells which interact with cytotoxic T-cell precursors are not strain specific. It seems likely that the histocompatible helper cells induce killer precursors in an antigen-specific cooperation event similar or identical to normal syngeneic cooperation.     

Generation of EBV-specific T cells for adoptive immunotherapy: a novel protocol using formalin-fixed stimulator cells to increase biosafety

Adoptive immunotherapy with in vitro generated Epstein-Barr virus (EBV)-specific T cells is a safe and effective treatment in patients with EBV-related complications after transplantation. More frequent use of EBV-specific T cells is held back by their cost and time-intensive generation under good manufacturing practice (GMP) conditions. Currently, EBV-specific T cells are produced by repetitive stimulation of peripheral blood mononuclear cells with EBV-infected lymphoblastoid cell lines (LCLs), a protocol that requires several open GMP-handling steps. The aim of the present study was to improve T-cell generation under GMP conditions. We introduce a novel generation protocol that replaces repetitive with short-term LCL stimulation of PMBCs. Vital and formalin-fixed LCLs were used to further increase biosafety. Stimulated T cells were selected by the clinically approved cytokine secretion assay followed by nonspecific expansion. Sufficient numbers of EBV-specific T-cell lines were generated with all protocols. Specific recognition and killing of EBV-infected targets was found and was independent of the generation protocol applied. The novel protocol based on formalin-fixed cells, selection, and expansion reduced open GMP-handling steps and increased biosafety. Furthermore, fixation will allow the use of transgenic LCLs (eg, with cytomegalovirus or tumor antigens) and thereby facilitate the generation of antigen-specific T cells directed against pathogens other than EBV.     

A novel approach to generate host antitumor T cells: adoptive immunotherapy by T cells maturing in xenogeneic thymus

Mouse or human T cells developing in xenogeneic porcine thymus are functional. With efficient peripheral repopulation of mouse T cells by grafting fetal pig thymus (FP THY), B6 nude mice were immunized with inactivated syngeneic melanoma, B16 cells. Splenocytes from B16-immunized FP THY-grafted B6 nude mice efficiently killed B16, but not EL4 target cells in cytotoxicity assays in vitro. Adoptive transfer of splenocytes from B16-immunizd FP THY-grafted B6 nude mice to B16-bearing B6 mice significantly prolonged recipient survival and inhibited B16 solid tumor growth when B16 cells were injected IV or SC, respectively, compared with the identical controls. Splenocytes from nonimmunized FP THY-grafted B6 nude mice failed to protect B6 mice from B16-induced mortality. The present data have demonstrated that mouse T cells maturing in xenogeneic thymus have the ability to kill syngeneic tumor cells. This study may offer a novel resource to produce host antitumor T cells for adoptive immunotherapy of tumor patients.     

Collection of large-scale expanded lymphocyte cultures for adoptive immunotherapy using a COBE Spectra apheresis machine

Adoptive cell transfer represents an important mode of cancer immunotherapy and posttransplant associated viral infections. This technique involves massive volume reduction, as the procedure starts with large-scale ex vivo expansion of lymphocyte cultures (volume up to 60 L), which are harvested at the end of the in vitro process and terminates in a small volume to be infused into the patient. Toward this end, we develop a novel efficient process based on the COBE Spectra apheresis machine usually used for apheresis process. As the COBE Spectra cell separator is easy to use and often available at medical centers, this novel technique is applicable to many transplant and cell processing centers. Our results show a high recovery yield (98%+/-15%) and viability (ranged 79% to 99%) of the large-scale expanded lymphocytes. It preserves sterility of the product and is therefore suitable for immunotherapy treatments.     

Cyclophosphamide-facilitated adoptive immunotherapy of an established tumor depends on elimination of tumor-induced suppressor T cells

On the basis of preceding studies showing that tumor-induced, T cell- mediated immunosuppression serves as an obstacle to adoptive immunotherapy of the Meth A fibrosarcoma, it was predicted that cyclophosphamide treatment of tumor bearers would remove this obstacle and allow passively transferred immune T cells to cause tumor regression. It was found that infusion of immune spleen cells alone had no effect on tumor growth, and cyclophosphamide alone caused a temporary halt in tumor progression. In contrast, combination therapy consisting of intravenous injection of 100 mg/kg of cyclophosphamide followed 1 h later by intravenous infusion of tumor-immune spleen cells caused small, as well as large tumors, to completely and permanently regress. Tumor regression caused by combination therapy was completely inhibited by intravenous infusion of splenic T cells from donors with established tumors, but not by spleen cells from normal donors. These suppressor T cells were eliminated from the spleen by treating the tumor-bearing donors with 100 mg/kg of cyclophosphamide. Immune T cells, in contrast, were resistant to this dose of cyclophosphamide. These results show that failure of intravenously-infused, tumor- sensitized T cells to cause regression of the Meth A fibrosarcoma growing in its syngeneic or semi-syngeneic host is caused by the presence of a tumor-induced population of cyclophosphamide-sensitive suppressor T cells.     

DC-CIK细胞过继免疫细胞治疗的护理

DC-CIK细胞过继免疫效应细胞是将人体外周血单个核细胞在体外用多种细胞因子共同培养一段时间后获得的一群异质细胞.具有T淋巴细胞强大的抗肿瘤活性和NK细胞的非MHC限制性杀瘤优点,增殖速度快,杀瘤活性高,杀瘤谱广,对多重耐药肿瘤细胞同样敏感,杀瘤活性不受常用免疫抑制剂的影响,对人体正常细胞无毒性,能抵抗肿瘤细胞引发的效应细胞凋亡.随着肿瘤免疫学研究的深入,过继免疫细胞治疗因具有高效低毒的杀瘤作用而受到重视.     

DC-CIK细胞过继免疫治疗病人的护理

恶性肿瘤是危害人类健康的疾病之一,死亡率居第2位.1985年美国国家癌症中心把生物治疗列为肿瘤综合治疗的第4种模式,作为手术、放疗、化疗3大常规模式的有益补充.