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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. From the Clinical Editor Artifical antigen presenting cells could revolutionize the field of cancer-directed immunotherapy. This team of investigators have manufactured two types of nanoscale particle platform-based aAPCs and demonstrates that both iron-dextran particles and quantum dot nanocrystals enhance tumor rejection in a melanoma model, providing the first description of nanoscale aAPCs that lead to effective T cell stimulation and inhibition of tumor growth.

The structure of a T cell receptor (TCR) and its affinity for cognate antigen are fixed, but T cells regulate binding sensitivity through changes in lateral membrane organization. TCR microclusters formed upon antigen engagement participate in downstream signaling. Microclusters are also found 3–4 days after activation, leading to enhanced antigen binding upon rechallenge. However, others have found an almost complete loss of antigen binding four days after T cell activation, when TCR clusters are present. To resolve these contradictory results, we compared binding of soluble MHC-Ig dimers by transgenic T cells stimulated with a high (100 μM) or low (100 fM) dose of cognate antigen. Cells activated by a high dose of peptide bound sixfold lower amounts of CD8-dependent ligand Kb-SIY than cells activated by a low dose of MHC/peptide. In contrast, both cell populations bound a CD8-independent ligand Ld-QL9 equally well. Consistent with the differences between binding of CD8-dependent and CD8-independent peptide/MHC, Förster resonance energy transfer (FRET) measurements of molecular proximity reported little nanoscale association of TCR with CD8 (16 FRET units) compared to their association on cells stimulated by low antigen dose (62 FRET units). Loss of binding induced by changes in lateral organization of TCR and CD8 may serve as a regulatory mechanism to avoid excessive inflammation and immunopathology in response to aggressive infection.

In this report, we review a novel method for probing the membrane organization of T cells using dimeric major histocompatibility complexes (MHC), MHC-Ig. MHC-Ig complexes are useful reagents for quantitative analysis of binding data since their valency is controlled. These complexes can be easily labeled and loaded with a variety of peptides. A binding assay using these dimers and quantitative analysis of the MHC-Ig dimer-T cell binding curves is described in detail. Using this approach, we show that the organization of TCR on activated T cells is different from TCR organization on nai;ve T cells. The implications of these findings are discussed with regards to current models of T cell recognition. This analysis offers insight into how T cell controls their biological range of responsiveness. Specifically, these findings reveal the biophysical basis of the ability of activated T cells to recognize low amounts of antigen independent of costimulation.

Changes in the clustering of surface receptors modulate cell responses to ligands. Hence, global measures of receptor clustering can be useful for characterizing cell states. Using T cell receptor for antigen as an example, we show that k-space image correlation spectroscopy of quantum dots blinking detects T cell receptor clusters on a scale of tens of nanometers and reports changes in clustering after T cell activation. Our results offer a general approach to the global analysis of lateral organization and receptor clustering in single cells, and can thus be applied when the cell type of interest is rare.

While activated T cells are known to have enhanced biological responses to antigen stimulation, the biophysical basis of this increased sensitivity remains unknown. Here, we show that, on activated T cells, the TCR avidity for peptide-MHC complexes is 20- to 50-fold higher than the TCR avidity of naive T cells. This increased avidity for peptide-MHC depends on TCR reorganization and is sensitive to the cholesterol content of the T cell membrane. Analysis of the binding data indicates the enhanced avidity is due to increases in cross-linking of TCR on activated T cells. Activation-induced membrane (AIM) changes in TCR avidity represent a previously unrecognized means of increasing the sensitivity of activated T cells to small amounts of antigen in the periphery.

Background:Harnessing the cytotoxic properties of T cells to destroy tumor cells has been the central goal of anti-cancer immunotherapy. One approach to achieve this goal has been the use adoptive cell transfer (ACT). This method involves the isolation, in vitro manipulation and re-infusion of antigen-specific T cells into cancer patients. This approach has been used to mediate durable responses in a number of malignancies. Despite its significant therapeutic potential, the routine use of ACT has been limited. This has been due, in large part, to the high cost, intricate and labor-intensive methods required to successfully generate autologous antigen-specific T cells. There is a clear need for less expensive more efficient and streamlined approaches for generation of antigen-specific T cells for use in ACT. To this end, we have developed a T cell Enrichment+Expansion strategy using paramagnetic, Nano scale artificial Antigen Presenting Cells (nano-aAPCs), which are capable of enriching rare tumor-specific T cells in a magnetic column and activating them. Nano-aAPCs, are not only more biocompatible than traditional aAPC, but by manipulating paramagnetic nanoparticle based aAPC with magnetic fields, we propose a method to quickly generate large numbers of high frequency tumor-specific T cells. Our hypothesis is that magnetic enrichment and expansion with nano-aAPC can reduce time in culture and increase frequency of antigen-specific cells, two factors that we predict will improve T cell expansion and persistence after adoptive transfer. Methods:HLA-Ig dimer was loaded with tumor antigen peptides for MART1, NY-ESO and WT1. To manufacture nano-APCs, magnetic nanoparticles (size 50-100nm) were decorated with loaded HLA-Ig (signal 1) and anti-CD28 antibody (signal 2). Peripheral blood mononuclear cells (PBMCs) were collected from normal donors and CD8+ T cells were isolated. CD8+ T cells were subsequently incubated with nano-APCs. The T cell/nano-APCs mixtures were then pass through a magnetic column. The relevant T cells were bound to the nano-APCs and therefore were attached to the column while the irrelevant T cells passed through the column. Relevant T cells were then washed off the column and cultured using standard T cell culture techniques for 7 days. Cell number and specificity were evaluated on Day 0 and day 7. Results: CD8+ T cells specific for the tumor antigens MART1, NYESO and WT1 were successfully expanded using our nano-APCs. After one week, we saw fold expansions of >100-fold for all 3 antigens studied. T cell specificity increased from Conclusions: The initial data presented here represent proof of principle evidence of the feasibility and efficacy of our proposed streamlined and cost effective enrichment and expansion approach for the generation of antigen specific T cells. Here we expanded significant numbers of CD8+ T cells targeting the tumor antigens MART1 NY-ESO and WT1 in just 7 days using our nano-APCs. While MART1 was used a model antigen to develop or system, both NY-ESO and WT1 are tumor antigens that are relevant for several hematological malignancies. Streamlining the generation of large numbers of high-frequency tumor-specific T cells in a cost effective, reproducible fashion through Enrichment+Expansion could be a powerful addition to current tumor immunotherapy protocols. Disclosures Oelke: NextImmune: Equity Ownership. Schneck:NextImmune: Equity Ownership.

Meeting abstracts Adoptive T cell therapy can mediate durable regression of cancer [[1][1]]. While pre-existing anti-tumor responses can only be cultured from a minority of cancer patients [[2][2]], T cells specific for a wide variety of tumor antigens can be generated by stimulation of naive

Changes in distribution of membrane receptor organization are used by cells to modulate the dynamic range of their responses to environmental cues. Consequently, it is important to develop experimental methods that can accurately measure receptor transport and aggregation. We used quantum dot (QD) labeling of T cell receptors (TCR) and a recently developed technique, k-space image correlation spectroscopy (kICS) to characterize TCR state as a function of cell differentiation. We developed kICS to measure transport coefficients of fluorescently labeled membrane proteins while taking into account nanoparticle emission blinking. We use kICS to measure T cell receptor (TCR) aggregation in live cells by characterizing quantum dot (QD) blinking and distribution on the cell surface. 2C TCR transgenic cells in culture were observed from the naive state to 12 days after activation by antigen. Cells were labeled with biotin-MHC/peptide to bind the TCR with relatively high avidity, followed by streptavidin-quantum dots. They were then imaged on an emCCD-equipped microscope and kICS analysis was applied. Spatial intensity fluctuations in an image measured the clustering of receptors on the 100s of nm length scale. Changes in the intensity correlation function of the blinking QDs characterized clustering on the 10s of nm length scale. We also used kICS to measure changes in TCR diffusive transport. When T cells exhibited maximum activity 3-4 days after exposure to antigen, the degree of their TCR aggregation on both length scales was significantly higher than that of naive cells, while TCR diffusion was a minimum. This new technology has powerful applications as it can be applied to just a few cells and we will show that it is able to detect changes in receptor organization of cells in vivo.