Cell-mediated immunity stems from the proliferation of naive T lymphocytes expressing T cell antigen receptors (TCRs) specific for foreign peptides bound to host major histocompatibility complex (MHC) molecules. Because of the tremendous diversity of the T cell repertoire, naive T cells specific for any one peptide:MHC complex (pMHC) are extremely rare. Thus, it is not known how many naive T cells of any given pMHC specificity exist in the body or how that number influences the immune response. By using soluble pMHC class II (pMHCII) tetramers and magnetic bead enrichment, we found that three different pMHCII-specific naive CD4(+) T cell populations vary in frequency from 20 to 200 cells per mouse. Moreover, naive population size predicted the size and TCR diversity of the primary CD4(+) T cell response after immunization with relevant peptide. Thus, variation in naive T cell frequencies can explain why some peptides are stronger immunogens than others.

Plasmonic photothermal therapy (PPTT) is a minimally-invasive oncological treatment strategy in which photon energy is selectively administered and converted into heat sufficient to induce cellular hyperthermia. The present work demonstrates the feasibility of in vivo PPTT treatment of deep-tissue malignancies using easily-prepared plasmonic gold nanorods and a small, portable, inexpensive near-infrared (NIR) laser. Dramatic size decreases in squamous cell carcinoma xenografts were observed for direct (P<0.0001) and intravenous (P<0.0008) administration of pegylated gold nanorods in nu/nu mice. Inhibition of average tumor growth for both delivery methods was observed over a 13-day period, with resorption of >57% of the directly-injected tumors and 25% of the intravenously-treated tumors.

Appropriate development of regulatory T (Treg) cells is necessary to prevent autoimmunity. Neonatal mice, unlike adults, lack factors required for Treg cell development. It is unclear what these missing factors are. However, signals emanating from the T cell receptor (TCR), the costimulatory receptor CD28, and the family of gammac-dependent cytokine receptors are required for Treg cell development. Herein we demonstrate that expression of a constitutively active Stat5b transgene (Stat5b-CA) allowed for Treg cell development in neonatal mice and restored Treg cell numbers in Cd28(-/-) mice. Sequence analysis of TCR genes in Stat5b-CA Treg cells indicated that ectopic STAT5 activation resulted in a TCR repertoire that more closely resembled that of naive T cells. Using MHCII tetramers to identify antigen-specific T cells, we showed that STAT5 signals diverted thymocytes normally destined to become naive T cells into the Treg cell lineage. Our data support a two-step model of Treg cell differentiation in which TCR and CD28 signals induce cytokine responsiveness and STAT5-inducing cytokines then complete the program of Treg cell differentiation.

Unraveling the complexity of the adaptive immune system requires the study of T cells in vivo. This protocol describes how populations of T cells specific for a given peptide: Major Histocompatibility Complex (pMHC) epitope can be identified in mice and tracked throughout the course of an immune response. The methodology involves the adoptive transfer of T-cell receptor (TCR) transgenic T cells with defined epitope specificity into histocompatible mice and the subsequent detection of these cells through the use of congenic or clonotypic markers. Alternatively, endogenous epitope-specific T cells can be tracked directly through the use of pMHC tetramers. Using magnetic bead-based enrichment and advanced multi-parameter flow cytometry, populations as small as 5 epitope-specific T cells can be detected from the peripheral lymphoid organs of a mouse. The adoptive transfer procedure can be completed within 3 h, while analysis of epitope-specific cells from mice can be completed within 6 h.

Millions of T cells are produced in the thymus, each expressing a unique alpha/beta T cell receptor (TCR) capable of binding to a foreign peptide in the binding groove of a host major histocompatibility complex (MHC) molecule. T cell-mediated immunity to infection is due to the proliferation and differentiation of rare clones in the preimmune repertoire that by chance express TCRs specific for peptide-MHC (pMHC) ligands derived from the microorganism. Here we review recent findings that have altered our understanding of how the preimmune repertoire is established. Recent structural studies indicate that a germline-encoded tendency of TCRs to bind MHC molecules contributes to the MHC bias of T cell repertoires. It has also become clear that the preimmune repertoire contains functionally heterogeneous subsets including recent thymic emigrants, mature naive phenotype cells, memory phenotype cells, and natural regulatory T cells. In addition, sensitive new detection methods have revealed that the repertoire of naive phenotype T cells consists of distinct pMHC-specific populations that consistently vary in size in different individuals. The implications of these new findings for the clonal selection theory, self-tolerance, and immunodominance are discussed.

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Enhancing the maturity of the newly formed blood vessels is critical for the success of therapeutic angiogenesis. The maturation of vasculature relies on active participation of mural cells to stabilize endothelium and a basal level of relevant growth factors. We set out to design and successfully achieved robust angiogenesis using an injectable polyvalent coacervate of a polycation, heparin, and fibroblast growth factor-2 (FGF2). FGF2 was loaded into the coacervate at nearly 100% efficiency. In vitro assays demonstrated that the matrix protected FGF2 from proteolytic degradations. FGF2 released from the coacervate was more effective in the differentiation of endothelial cells and chemotaxis of pericytes than free FGF2. One injection of 500 ng of FGF2 in the coacervate elicited comprehensive angiogenesis in vivo. The number of endothelial and mural cells increased significantly, and the local tissue contained more and larger blood vessels with increased circulation. Mural cells actively participated during the whole angiogenic process: Within 7 d of the injection, pericytes were recruited to close proximity of the endothelial cells. Mature vasculature stabilized by vascular smooth muscle cells persisted till at least 4 wk. On the other hand, bolus injection of an identical amount of free FGF2 induced weak angiogenic responses. These results demonstrate the potential of polyvalent coacervate as a new controlled delivery platform.A ngiogenesis is a physiological process involving the formation of nascent vasculature from existing blood vessels. The complex interactions between endothelial cells and mural cells including vascular smooth muscle cells and pericytes are highly coordinated by various signals (1, 2). Therapeutic angiogenesis is promising in treating many human diseases, especially coronary and peripheral ischemia (3, 4). Among various approaches to therapeutic angiogenesis, delivery of growth factors is the most simple and direct because it does not need viral vectors in gene therapy or cells in cell therapy. Direct injection of free growth factors failed to demonstrate efficacy in clinical trials (5). Therefore, appropriate controlled delivery strategy for growth factor is highly desirable and extensively studied. However, loading capacity and long-term efficacy still present significant challenges to growth factor delivery.In the human body, most secretory growth factors are associated with extracellular matrix usually through interactions with glycosaminoglycans. Glycosaminoglycans are negatively charged linear polysaccharides that can have different composition, function, and distribution in the body (6). Together with other extracellular matrix molecules, glycosaminoglycans provide a substratum for cell attachment (7). Furthermore, their interaction with growth factors is critical in many biological processes such as development (8, 9) and cancer progression (10, 11). Heparin and heparan sulfate are well-studied glycosaminoglycans for their high affinity to a variety of growth factors including hep...

Therapeutic angiogenesis aims at treating ischemic diseases by generating new blood vessels from existing vasculature. It relies on delivery of exogenous factors to stimulate neovasculature formation. Current strategies using genes, proteins and cells have demonstrated efficacy in animal models. However, clinical translation of any of the three approaches has proved to be challenging for various reasons. Administration of angiogenic factors is generally considered safe, according to accumulated trials, and offers off-the-shelf availability. However, many hurdles must be overcome before therapeutic angiogenesis can become a true human therapy. This article will highlight protein-based therapeutic angiogenesis, concisely review recent progress and examine critical challenges. We will discuss growth factors that have been widely utilized in promoting angiogenesis and compare their targets and functions. Lastly, since bolus injection of free proteins usually result in poor outcomes, we will focus on controlled release of proteins.Blood vessels that carry oxygen, nutrients, cells and signals are critical in both developmental and adult physiology. Without sufficient blood supply, tissues and organs cannot maintain regular activities. On the other hand, induction of neovasculature provides a potential strategy to treat many ischemic illnesses, especially cardiovascular diseases (CVDs) including coronary and peripheral arterial diseases. The morbidity, mortality and cost of CVDs are highlighted by the latest statistics from the American Heart Association [1]: an estimated 82,600,000 American adults (≥20 years old) have one or more types of CVDs; CVDs caused 813,804 of all 2,243,712 deaths (33.6%) or one of every 2.9 deaths in 2007, and coronary heart disease caused approximately one of every six deaths. The direct and indirect cost of CVD was estimated to be US$286 billion in 2007. The amount is higher than the $228 billion spent on cancer and benign neoplasms. As a potential therapy to revascularize ischemic tissues, including ischemic heart, therapeutic angiogenesis has drawn much attention in the last 20 years.Neovasculature can be obtained by three approaches based on different mechanisms: promoting expression of angiogenic genes, supplying potent angiogenic factors and delivering progenitor or stem cells. No matter which approach is used, angiogenic activity has to be precisely controlled in order to achieve stable vascularization. For cell delivery, the