Joan Glick Bieler

Recruitment of latent pools of high-avidity CD8+ T cells to the antitumor immune response

A major barrier to successful antitumor vaccination is tolerance of high-avidity T cells specific to tumor antigens. In keeping with this notion, HER-2/neu (neu)-targeted vaccines, which raise strong CD8+ T cell responses to a dominant peptide (RNEU420-429) in WT FVB/N mice and protect them from a neu-expressing tumor challenge, fail to do so in MMTV-neu (neu-N) transgenic mice. However, treatment of neu-N mice with vaccine and cyclophosphamide-containing chemotherapy resulted in tumor protection in a proportion of mice. This effect was specifically abrogated by the transfer of neu-N–derived CD4+CD25+ T cells. RNEU420-429-specific CD8+ T cells were identified only in neu-N mice given vaccine and cyclophosphamide chemotherapy which rejected tumor challenge. Tetramer-binding studies demonstrated that cyclophosphamide pretreatment allowed the activation of high-avidity RNEU420-429-specific CD8+ T cells comparable to those generated from vaccinated FVB/N mice. Cyclophosphamide seemed to inhibit regulatory T (T reg) cells by selectively depleting the cycling population of CD4+CD25+ T cells in neu-N mice. These findings demonstrate that neu-N mice possess latent pools of high-avidity neu-specific CD8+ T cells that can be recruited to produce an effective antitumor response if T reg cells are blocked or removed by using approaches such as administration of cyclophosphamide before vaccination.

Increased TCR Avidity after T Cell Activation: A Mechanism for Sensing Low-Density Antigen

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.

Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

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.

Nanoscale artificial antigen presenting cells for T cell immunotherapy

UNLABELLED 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.

Probing T cell membrane organization using dimeric MHC-Ig complexes

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.

Enrichment and Expansion with Nanoscale Artificial Antigen Presenting Cells for Adoptive Immunotherapy.

Adoptive immunotherapy (AIT) can mediate durable regression of cancer, but widespread adoption of AIT is limited by the cost and complexity of generating tumor-specific T cells. Here we develop an Enrichment + Expansion strategy using paramagnetic, nanoscale artificial antigen presenting cells (aAPC) to rapidly expand tumor-specific T cells from rare naïve precursors and predicted neo-epitope responses. Nano-aAPC are capable of enriching rare tumor-specific T cells in a magnetic column and subsequently activating them to induce proliferation. Enrichment + Expansion resulted in greater than 1000-fold expansion of both mouse and human tumor-specific T cells in 1 week, with nano-aAPC based enrichment conferring a proliferation advantage during both in vitro culture and after adoptive transfer in vivo. Robust T cell responses were seen not only for shared tumor antigens, but also for computationally predicted neo-epitopes. Streamlining the rapid generation of large numbers of tumor-specific T cells in a cost-effective fashion through Enrichment + Expansion can be a powerful tool for immunotherapy.

Quantum Dot Fluorescence Characterizes the Nanoscale Organization of T Cell Receptors for Antigen

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.

Streptococcus pneumoniae Serotype 1 Capsular Polysaccharide Induces CD8+CD28− Regulatory T Lymphocytes by TCR Crosslinking

Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an α-helix configuration. In this paper we look at the immune response of CD8+ T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8+CD28− T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8+CD28− T lymphocytes. The Sp1-induced CD8+CD28− T lymphocytes are CD122lowCTLA-4+CD39+. They synthesize IL-10 and TGF-β. The Sp1-induced CD8+CD28− T cells exhibit immunosuppressive properties on CD4+ T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8+ T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8+ T cell activation by enhancing crosslinking of TCR. The expansion of CD8+CD28− T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8+CD28− T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-κB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8+CD28− population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8+CD28− T lymphocytes in vivo and in vitro. The underlying mechanism of CD8+ T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system.

Ex vivo induction and expansion of natural killer T cells by CD1d1-Ig coated artificial antigen presenting cells

Natural killer T (NKT) cells play a pivotal role in maintaining immune homostasis. They recognize lipid antigen in the context of CD1d molecules and subsequently produce cytokines that activate cells of both the innate and adaptive immune responses. Many studies examining patients with autoimmune disease or cancer have shown that there is a reduction in both NKT cell number and function. Due to the complexities of manipulating NKT cells in vivo, ex vivo expanded effector NKT cells would be an excellent therapeutic modality. To date, immunotherapy utilizing the NKT/CD1d system has been dependent on the use of autologous DC in the presence or absence of a synthetic glycolipid, alpha-galactocylceramide. Here we report a novel technique that facilitates the growth and analysis of NKT cells through the use of CD1d-expressing aAPC. CD1d-based aAPC can effectively propagate both canonical (iNKT cells) and noncanonical (Valpha14(-)) NKT cells. Importantly, CD1d-Ig aAPC can expand NKT cells from cancer patients. Thus, CD1d-expressing aAPC will enhance our knowledge of NKT cell biology and could potentially be used as a novel tool in adoptive immunotherapeutic strategies.

Modulation of MHC binding by lateral association of TCR and coreceptor.

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 K(b)-SIY than cells activated by a low dose of MHC/peptide. In contrast, both cell populations bound a CD8-independent ligand L(d)-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.