A cytokine cocktail directly modulates the phenotype of DC-enriched anti-tumor T cells to convey potent anti-tumor activities in a murine model

Adoptive cell transfer (ACT) using ex vivo-expanded anti-tumor T cells such as tumor-infiltrated lymphocytes or genetically engineered T cells potently eradicates established tumors. However, these two approaches possess obvious limitations. Therefore, we established a novel methodology using total tumor RNA (ttRNA) to prime dendritic cells (DC) as a platform for the ex vivo generation of anti-tumor T cells. We evaluated the antigen-specific expansion and recognition of T cells generated by the ttRNA–DC–T platform, and directly modulated the differentiation status of these ex vivo-expanded T cells with a cytokine cocktail. Furthermore, we evaluated the persistence and in vivo anti-tumor efficacy of these T cells through murine xenograft and syngeneic tumor models. During ex vivo culture, IL-2 preferentially expanded CD4 subset, while IL-7 enabled homeostatic proliferation from the original precursors. T cells tended to lose CD62L during ex vivo culture using IL-2; however, IL-12 could maintain high levels of CD62L by increasing expression on effector T cells (Tem). In addition, we validated that OVA RNA–DC only selectively expanded T cells in an antigen-specific manner. A cytokine cocktail excluding the use of IL-2 greatly increased CD62L^high T cells which specifically recognized tumor cells, engrafted better in a xenograft model and exhibited superior anti-tumor activities in a syngeneic intracranial model. ACT using the ex vivo ttRNA–DC–T platform in conjunction with a cytokine cocktail generated potent CD62L^high anti-tumor T cells and imposes a novel T cell-based therapeutic with the potential to treat brain tumors and other cancers.     

Efficient ex vivo induction of T cells with potent anti-tumor activity by protein antigen encapsulated in nanoparticles

Protein antigen (Ag)-based immunotherapies have the advantage to induce T cells with a potentially broad repertoire of specificities. However, soluble protein Ag is generally poorly cross-presented in MHC class I molecules and not efficient in inducing robust cytotoxic CD8⁺ T cell responses. In the present study, we have applied poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) which strongly improve protein Ag presentation by dendritic cells (DC) in the absence of additional Toll-like receptor ligands or targeting devices. Protein Ag-loaded DC were used as antigen presenting cells to stimulate T cells in vitro and subsequently analyzed in vivo for their anti-tumor effect via adoptive transfer, a treatment strategy widely studied in clinical trials as a therapy against various malignancies. In a direct comparison with soluble protein Ag, we show that DC presentation of protein encapsulated in plain PLGA-NP results in efficient activation of CD4⁺ and CD8⁺ T cells as reflected by high numbers of activated CD69⁺ and CD25⁺, interferon (IFN)-γ and interleukin (IL)-2-producing T cells. Adoptive transfer of PLGA-NP-activated CD8⁺ T cells in tumor-bearing mice displayed good in vivo expansion capacity, potent Ag-specific cytotoxicity and IFN-γ cytokine production, resulting in curing mice with established tumors. We conclude that delivery of protein Ag through encapsulation in plain PLGA-NP is a very efficient and simple procedure to stimulate potent anti-tumor T cells.     

Pertussis toxin-induced cytokine differentiation and clonal expansion of T cells is mediated predominantly via costimulation

Pertussis toxin (PTX) has potent immunologic adjuvant activity in vivo and concomitantly enhances both T helper type (Th1) and Th2 cytokine responses. The PTX-induced enhancement of Th1 and Th2 immunity is mediated via the activation of antigen presenting cells (APCs), but the underlying mechanism is not known. Here we asked whether the adjuvant activity of PTX on T cell immunity was mediated by cytokines and/or costimulatory signals. The results show that in vivo blockade of CD28-CD80/86 costimulation essentially abrogated PTX-mediated enhancement of antigen-specific Th1 and Th2 responses. Blockade of CD40L-CD40 interactions was less efficient in inhibiting PTX-mediated enhancement of Th1 and Th2 responses. In contrast, the adjuvant activity of PTX was not mediated via cytokines, because neither Th1 nor Th2 responses were substantially impaired in mice deficient for IL-12, IFN-gamma, IL-4, IL-5, or IL-6. Collectively, the data suggest that PTX mediates its adjuvant effects on T cell cytokine differentiation and clonal expansion via the modulation of costimulatory molecules on APCs. Understanding the costimulatory pathways targeted by PTX could lead to the design of novel adjuvants that selectively induce Th1 or Th2 immunity.     

Activated self-MHC-reactive T cells have the cytokine phenotype of Th3/T regulatory cell 1 T cells

In the present study, we show that human self-MHC-reactive (autoreactive) T cell clones are functionally distinct from Ag-specific T cell clones. Self-MHC-reactive T cells exhibited helper function for B cell Ig production when cultured with non-T cells alone, and they exhibit suppressor function when cultured with PWM- or rCD40 ligand (rCD40L)-activated non-T cells, whereas tetanus toxoid (TT)-specific clones exhibited only helper function in the presence of TT with or without PWM or rCD40L. Addition of neutralizing Abs to the cultures showed that the suppression was mediated by TGF-beta but not by IL-10 or IFN-gamma. The self-MHC-reactive clones also inhibited proliferation of primary CD4+ T cells and TT-specific T cell clones, but in this case the inhibition was mediated by both IL-10 and TGF-beta. In further studies, the interactions between self-MHC-reactive T cell clones and non-T cells that led to suppressor cytokine production have been explored. We found that prestimulation of non-T cells for 8 h with PWM or for 48 h for rCD40L results in non-T cells capable of inducing self-MHC-reactive T cell to produce high levels of TGF-beta and IL-10. In addition, these prestimulation times coincided with peak induction of HLA-DR and costimulatory B7 molecule (especially CD86) expression on B cells. Finally, addition of CTLA-4/Fc or blocking F(ab')2 anti-CTLA-4 mAb, plus optimally stimulated non-T cells, to cultures of self-MHC-reactive clones inhibited the induction of TGF-beta but not IL-10 or IFN-gamma production. In summary, these studies show that activated self-MHC-reactive T cells have the cytokine phenotype of Th3 or T regulatory cell 1 and thus may be important regulatory cells that mediate oral and peripheral tolerance and prevent the development of autoimmunity.     

Serum enhances the ex vivo generation of HIV-specific cytotoxic T cells

The ex vivo expansion of antigen-specific cytotoxic T lymphocyte (CTL) lines is being developed for immunotherapy of viral infections and cancer and is critically dependent on the precise cell expansion and stimulation conditions. In this article, we investigate medium requirements for the development of HIV-specific CTL in cell lines generated from the peripheral blood of seven asymptomatic HIV-infected individuals. We find that HIV-specific CTL do not readily develop in the serum-free medium AIM V but do develop if the medium is supplemented with 1% plasma or serum. T cell lines with antigen-specific cytolytic activity express more cell-surface CD57 than do cell lines grown in the absence of serum or plasma. Three sources of serum (human autologous, human AB, or fetal calf) are comparable. Human plasma is somewhat less effective than serum from an identical source. (c) 1996 John Wiley & Sons, Inc.     

ROCKing cytokine secretion balance in human T cells

Balanced regulation of cytokine secretion in T cells is critical for maintenance of immune homeostasis and prevention of autoimmunity. The Rho-associated kinase (ROCK) 2 signaling pathway was previously shown to be involved in controlling of cellular movement and shape. However, recent work from our group and others has demonstrated a new and important role of ROCK2 in regulating cytokine secretion in T cells. We found that ROCK2 promotes pro-inflammatory cytokines such as IL-17 and IL-21, whereas IL-2 and IL-10 secretion are negatively regulated by ROCK2 under Th17-skewing activation. Also, in disease, but not in steady state conditions, ROCK2 contributes to regulation of IFN-γ secretion in T cells from rheumatoid arthritis patients. Thus, ROCK2 signaling is a key pathway in modulation of T-cell mediated immune responses underscoring the therapeutic potential of targeted inhibition of ROCK2 in autoimmunity.     

T helper cells in immune mice amplify the primary anti-tumor cytotoxic response

In this report we examine the influence of splenic helper cells in the primary cytotoxic T lymphocyte (CTL) response against syngeneic murine leukemia virus-(MuLV) induced tumor cells. We identify an Lyt-1+ 800 R radiation-resistant helper T cell that will amplify the in vitro generation of CTL against syngeneic tumor cells from nonimmune spleen cells.     

Regulation of T cell cytokine production by dendritic cells

Previous work has established that the dendritic cells (DC) of mouse spleen regulate the IL-2 production, and hence the extent of proliferation, of the CD8 T cells they activate. It is now reported here that interaction of primary CD8 T cells with splenic CD8alpha- DC induced much higher production of IL-3, IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as IL-2, than did interaction with CD8alpha+ splenic DC. Furthermore, the CD8alpha- DC also induced higher levels of IL-2, IL-3 and IL-10 production in primary CD4 T cells, compared with that induced by CD8alpha+ DC. These quantitative differences did not involve qualitative shifts in the type of cytokine produced. Interleukin-4 production remained low in all the primary T cell cultures and restimulation experiments in secondary cultures did not reveal any bias in the cytokine production profile. When exogenous IL-2 was added to the primary cultures to ensure equal proliferation in response to CD8alpha- or CD8alpha+ DC, the higher level of production of IL-3, IFN-gamma and GM-CSF induced by CD8alpha- DC was maintained. Thus, this general control of T cell cytokine production by splenic DC involves factors additional to those that govern activation of T cells into cell cycle.     

Responsiveness of naive CD4 T cells to polarizing cytokine determines the ratio of Th1 and Th2 cell differentiation

The intrinsic features of naive CD4 T cells that affect their ability to respond to polarizing signals for Th cell differentiation are not well understood. In this study, we show that naive CD4 T cells from mice transgenic for the Hlx gene expressed lower levels of IL-4Ralpha. The down-regulation of IL-4Ralpha diminished IL-4 signaling and the Th2 response and enhanced the Th1 response under suboptimal polarizing conditions. In nontransgenic CD4 T cells, blocking IL-4Ralpha with Abs had the same effect in an Ab dose-dependent manner. Conversely, Hlx haploinsufficiency caused higher expression of IL-4Ralpha to favor Th2 cell differentiation. Thus, the IL-4Ralpha level on naive CD4 T cells is genetically controlled by Hlx and determines the ratio of Th1 and Th2 cell differentiation.     

T1/ST2 expression is enhanced on CD4+ T cells from schistosome egg-induced granulomas: analysis of Th cell cytokine coexpression ex vivo

Th cells are categorized into subsets based on the cytokine production of in vitro-differentiated Th populations. For in vivo-differentiated Th subsets, little is known about the heterogeneity of cytokine production in single cells. We recently described a molecule, T1/ST2, that is preferentially expressed on the surface of Th2 cells. Here we combined high-gradient magnetic cell separation with four-color single-cell cytometry to analyze simultaneously three intracellular cytokines and T1/ST2 surface expression on CD4+ cells from lungs containing granulomas induced by Schistosoma mansoni eggs. T1/ST2 was highly up-regulated on CD4+ T cells from hepatic granulomas and granulomatous lungs. T1/ST2+ cells from granulomatous lungs preferentially produced type 2 cytokines ex vivo. In the total CD4+ population, coexpression of type 1 and type 2 cytokines occurred frequently. However, such coproduction was drastically reduced in T1/ST2+ cells compared with T1/ST2- cells. Coexpression of type 1 and type 2 cytokines was also rare in cells simultaneously producing two cytokines of one type. These findings indicate that individual CD4+ T cells in vivo have different levels of commitment to a certain Th phenotype. Coexpression of two type 2 cytokines or production of one type 2 cytokine together with surface expression of T1/ST2 indicate advanced commitment to the Th2 phenotype.     

Lowering glycosphingolipid levels in CD4+ T cells attenuates T cell receptor signaling, cytokine production, and differentiation to the Th17 lineage

Lipid rafts reportedly have a role in coalescing key signaling molecules into the immunological synapse during T cell activation, thereby modulating T cell receptor (TCR) signaling activity. Recent findings suggest that a correlation may exist between increased levels of glycosphingolipids (GSLs) in the lipid rafts of T cells and a heightened response of those T cells toward activation. Here, we show that lowering the levels of GSLs in CD4(+) T cells using a potent inhibitor of glucosylceramide synthase (Genz-122346) led to a moderation of the T cell response toward activation. TCR proximal signaling events, such as phosphorylation of Lck, Zap70 and LAT, as well as early Ca(2+) mobilization, were attenuated by treatment with Genz-122346. Concomitant with these events were significant reductions in IL-2 production and T cell proliferation. Similar findings were obtained with CD4(+) T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4(+) T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th(17) lineage but not to other Th subsets in vitro. Taken together with the recently reported effects of Raftlin deficiency on Th(17) differentiation, these results strongly suggest that altering the GSL composition of lipid rafts modulates TCR signaling activity and affects Th(17) differentiation.     

Sustained activation of the raf-MEK-ERK pathway elicits cytokine unresponsiveness in T cells

Activation of T cells via the TCR and other costimulatory receptors triggers a number of signaling cascades. Among them, the Ras-activated Raf-mitogen-activated protein/extracellular signal-related kinase (ERK) kinase (MEK)-ERK signaling cascade has been demonstrated to be crucial for both T cell development and activation. It has previously been demonstrated that high doses of Ag or anti-CD3 mAb are able to induce in T cells a nonresponsive state to subsequent treatment with cytokines such as IL-2. The precise biochemical mechanisms underlying this effect are not fully characterized. In this study, we demonstrate that cytokine nonresponsiveness is accompanied by the induction of the cyclin-dependent kinase inhibitor p21Cip1 that is mediated, at least in part, by the activation of the Raf-MEK-ERK pathway. Furthermore, we demonstrate that selective activation of the Raf-MEK-ERK signaling pathway in T cells is sufficient to induce cytokine nonresponsiveness in both a T cell clone and naive primary T cells. In this case, nonresponsiveness is accompanied by the induction of p21Cip1 and the prevention of p27Kip1 down-regulation, leading to inhibition of cyclin E/cyclin-dependent kinase 2 activity. These data suggest that anti-CD3 mAb-induced cytokine nonresponsiveness may be a consequence of hyperactivation of the Raf-MEK-ERK pathway, leading to alterations in the expression of key cell cycle regulators. These observations may provide a novel insight into the mechanisms of induction of peripheral tolerance.     

Spontaneous differentiation of periodontal ligament stem cells into myofibroblast during ex vivo expansion

Periodontitis is characterized by the chronic inflammation and destruction of tooth-supporting tissues. Periodontal ligament stem cell (PDLSC) is the mesenchymal stem cell (MSC) population isolated from periodontal ligament, which is the key tissue for regeneration of periodontal tissues. Although transplantation of PDLSCs is proposed as novel regenerative therapy, limited information is available, regarding the characteristic change of PDLSCs during ex vivo expansion. In this study, we encountered morphological change of PDLSCs during standard cell culture and aimed to investigate the change of PDLSCs in stem cell characteristics and to search for the culture condition to maintain stem cell properties. Characteristics of PDLSCs were examined using in vitro osteoblast and adipocyte differentiation. Myofibroblast differentiation was confirmed using immunohistochemistry and collagen gel contraction assay. Replicative senescence was examined by β-gal staining. PDLSCs changed their morphology from spindle to flat and wide during ex vivo expansion. After the morphological change, PDLSCs showed several features of myofibroblast including extensive stress fiber formation, contraction activity, and myofibroblast marker expression. Upon the morphological change, osteoblastic and adipocyte differentiation capacity were reduced and expression of stem cell-related genes were decreased. β-Gal staining was not always correlated with the morphological change of PDLSCs. Moreover, exogenous addition of bFGF and PDGF-BB served to maintain spindle shape and osteoblastic differentiation potential of PDLSCs. This study demonstrates that spontaneous differentiation of PDLSCs during ex vivo expansion and may provide the important information of cell culture condition of PDLSCs for clinical use.     

Dendritic cells: driving the differentiation programme of T cells in viral infections

Protective immunity against viral pathogens depends on the generation and maintenance of a small population of memory CD8(+) T cells. Successful memory cell generation begins with early interactions between na?ve T cell and dendritic cells (DCs) within the inflammatory milieu of the secondary lymphoid tissues. Recent insights into the role of different populations of DCs, and kinetics of antigen presentation, during viral infections have helped to understand how DCs can shape the immune response. Here, we review the recent progress that has been made towards defining how specific DC subsets drive effector CD8(+) T-cell expansion and differentiation into memory cells. Further, we endeavour to examine how the molecular signals imparted by DCs coordinate to generate protective CD8(+) T-cell immunity.     

Cancer immunotherapy using cells modified with cytokine genes

The ability of various cytokines to hamper tumor growth or to induce anti-tumor immune response has resulted in their study as antitumor agents in gene therapy approaches. In this review we will concentrate on the costimulation of antitumor immune responses using modification of various cell types by cytokine genes. Several strategies have emerged such as (i). modification of tumor cells with cytokine genes ex vivo (whole tumor cell vaccines), (ii). ex vivo modification of other cell types for cytokine gene delivery, (iii). delivery of cytokine genes into tumor microenvironment in vivo, (iv). modification of dendritic cells with cytokine genes ex vivo. Originally single cytokine genes were used. Subsequently, multiple cytokine genes were applied simultaneously, or in combination with other factors such as chemokines, membrane bound co-stimulatory molecules, or tumor associated antigens. In this review we discuss these strategies and their use in cancer treatment as well as the promises and limitations of cytokine based cancer gene therapy. Clinical trials, including our own experience, employing the above strategies are discussed.