Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy.

Based on the memory for the re-expression of certain cytokine genes, different subsets of Th cells have been defined. In Th type 1 (Th1) and Th2 memory lymphocytes, the genes for the cytokines interferon-c and interleukin (IL)-4 are imprinted for expression upon restimulation by the expression of the transcription factors T-bet and GATA-3, respectively, and epigenetic modification of the cytokine genes. In Th17 cells, IL-17 expression is dependent on the transcription factors RORct and RORa. Here, we analyze the stability and plasticity of IL-17 memory in Th17 cells. We have developed a cytometric IL-17 secretion assay for the isolation of viable Th cells secreting IL-17. For Th17 cells generated in vitro, IL-17 expression itself is dependent on continued TGF-b/IL-6 or IL-23 signaling and is blocked by interferon-c and IL-4 signaling. In response to IL-12 and IL-4, in vitro generated Th17 cells are converted into Th1 or Th2 cells, respectively. Th17 cells isolated ex vivo, however, maintain their IL-17 memory upon subsequent in vitro culture, even in the absence of IL-23. Their cytokine memory is not regulated by IL-12 or IL-4. Th17 cells generated in vivo are a stable and distinct lineage of Th cell differentiation.Key words: Cytokine memory . Interleukin-17 . T-cell differentiation Supporting Information available online IntroductionTh memory lymphocytes are imprinted for the re-expression of distinct cytokine genes upon restimulation. Originally, two types of Th effector memory cells had been defined: T helper type 1 (Th1) cells re-expressing interferon-g (IFN-g), and Th2 cells, reexpressing interleukin (IL)-4, -5 and -13 [1]. Recently, a third lineage of Th effector memory cells has been described, characterized by the re-expression of IL-17A, ). Th17 cells can induce autoimmune inflammation [3] and are protective in response to fungal infection [4]. In vitro, naïve murine Th cells can be induced to differentiate into Th17 cells by combined TGF-b and IL-6 signalling [5,6]. IL-23 promotes survival and proliferation of Th17 cells [6]. IL-21 can induce IL-17 independent of IL-6 and is expressed by Th17 cells themselves, as part of a positive regulatory feedback loop for IL-17 re-expression [7,8]. In human Th cells, similar signals are required for the differentiation of IL-17 re-expressing Th memory cells [9][10][11]. STAT3 is involved as a signal transducer and and the retinoic acid receptor-related orphan 2654Frontline receptors RORgt [13] and RORa [14] as transcription factors controlling lineage development. Ectopic over-expression of RORgt and RORa in naïve Th cells is sufficient to induce IL-17 expression [14].As part of their functional memory, the capacity of effector memory Th cells to stably re-express particular cytokines has been demonstrated for Th1 cells and IFN-g expression and for Th2 cells and their IL-4 and IL-10 expression [15,16]. This memory cytokine expression depends on TcR signals, but does not require the original instructive signals. It even occurs in the presence of adverse instruc...

T helper (Th) lymphocytes, when reactivated, recall expression of those cytokines they had been instructed to express in earlier activations, even in the absence of specific cytokine-inducing factors. In cells that memorize their expression, the cytokine genes are modified by chromatin rearrangement and demethylation, suggesting that they have been somatically imprinted. Here we show, by using inhibitors blocking the cell cycle in various stages, that for the instruction of a Th cell to express interleukin (IL)-4 or IL-10 upon restimulation, entry of the cell into the S phase of the first cell cycle after initial activation is required. Separation of the IL-4 receptor (IL-4R) and T cell antigen receptor (TCR) signals in time, demonstrates that this instruction is dependent on concomitant signaling from both receptors. In Th cells, inhibited to progress into the first S phase after activation, the IL-4R and TCR signals can be memorized for at least 1 d, priming the T cell to become instructed for expression of IL-4 upon restimulation, when entering the S phase after release of the cell cycle block. The requirement of the initial S phase of T cell activation, for instruction of Th cells to express IL-4 or IL-10 upon restimulation points to the decisive role of epigenetic modification of cytokine genes as a molecular correlate of the memory to express particular cytokines.

Th1 and Th17 cells are distinct lineages of effector/memory cells, imprinted for reexpression of , by upregulated expression of T-bet and retinoic acid-related orphan receptor ct (RORct) , respectively. Apparently, Th1 and Th17 cells share tasks in the control of inflammatory immune responses. Th cells coexpressing IFN-c and IL-17 have been observed in vivo, but it remained elusive, how these cells had been generated and whether they represent a distinct lineage of Th differentiation. It has been shown that ex vivo isolated Th1 and Th17 cells are not interconvertable by TGF-b/IL-6 and IL-12, respectively. Here, we show that ex vivo isolated Th17 cells can be converted into Th1/Th17 cells by combined IFN-c and IL-12 signaling. IFN-c is required to upregulate expression of the IL-12Rb2 chain, and IL-12 for Th1 polarization. These Th1/Th17 cells stably coexpress RORct and T-bet at the single-cell level. Our results suggest a molecular pathway for the generation of Th1/Th17 cells in vivo, which combine the pro-inflammatory potential of Th1 and Th17 cells. IntroductionTh1 cells, with a memory for IFN-g expression and determined by the master transcription factor T-bet, are considered to be essential for protection against intracellular pathogens, and had been viewed as the major pathogenic drivers of chronic autoimmune inflammation, e.g. EAE [1][2][3], uveitis [4] or colitis [5]. Recently, Th17 cells, with a memory for expression of IL-17 and determined by the transcription factor retinoic acid related orphan receptor gt (RORgt), have been identified as another pathogenic Th-cell lineage driving pathogenesis in these autoimmune models [6,7]. Th17 cells contribute to inflammation through the recruitment of neutrophils and the induction of secretion of pro-inflammatory mediators such as IL-6, IL-8, TNF-a, IL-1b, CXCL1, CXCL10 and matrix metalloproteinases from tissue cells (reviewed in [8]). Th1 cells contribute to inflammation by activation of macrophages [9]. The concerted action of IFN-g and IL-17 has been shown to be essential in the effective induction and maintenance of autoimmunity [10,11], e.g. Th1 cells being required for the recruitment of Th17 cells into the central nervous system in EAE.In inflamed tissue of autoimmune patients, Th cells coexpressing IFN-g and IL-17 have been identified [12][13][14].Ã These authors have contributed equally to this study. ResultsIn vivo, Th17 cells do not express IL-12Rb2 and do not respond to IL-12Although IL-17-expressing cells isolated from cultures stimulated in vitro respond to subsequent stimulation with IL-12 with gain of IFN-g expression and loss of IL-17 expression (Fig. 1A, upper panel), IL-17 expressing cells directly isolated ex vivo maintained IL-17 expression and could not be induced to express IFN-g in the presence of IL-12 (Fig. 1A, lower panel) [17,19]. To identify the molecular mechanism of refraction of in vivo generated Th17 cells to conversion by IL-12, we here compared the expression of genes relevant for IL-12 signaling by Th17 cells generated in vit...

Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50–100 nm in diameter) and avidin-coated quantum dot nanocrystals, (~30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo.