P. Rod Dunbar

Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141+ DC subset. CD141+ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-β, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c+ DC subset. Polyinosine-polycytidylic acid (poly I:C)–activated CD141+ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8+ cytotoxic T lymphocytes than poly I:C–activated CD1c+ DCs. Importantly, CD141+ DCs, but not CD1c+ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141+ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8α+ DC subset. The data demonstrate a role for CD141+ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.

Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells that initiate protective T-cell immunity in mice. To study the immunogenicity of DCs in humans, we injected 9 healthy subjects subcutaneously with a control injection of autologous monocyte-derived, mature DCs, followed 4-6 weeks later by DCs pulsed with keyhole limpet hemocyanin (KLH), HLA-A*0201-positive restricted influenza matrix peptide (MP), and tetanus toxoid (TT). Four more subjects received these antigens without DCs. Injection of unpulsed DCs, or antigens alone, failed to immunize. Priming of CD4(+) T cells to KLH was observed in all 9 subjects injected with KLH-pulsed DCs, and boosting of TT-specific T-cell immunity was seen in 5 of 6 subjects injected with TT-pulsed DCs. Injection of antigen-pulsed DCs led to a severalfold increase in freshly isolated MP-specific, IFN-gamma-secreting CD8(+) T cells in all 6 HLA-A*0201-positive subjects, as early as 7 days after injection. When T cells were boosted in culture, there was an increase in MHC tetramer-binding cells and cytotoxic T cells after DC vaccination. These data provide the first controlled evidence of the immunogenicity of DCs in humans, and demonstrate that a single injection of mature DCs rapidly expands T-cell immunity.

Structural and Kinetic Basis for Heightened Immunogenicity of T Cell Vaccines

Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR–peptide–MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)–A2 tumor epitope NY–ESO-1157–165–SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine–tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA–A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials.

Human adipose‐derived stem cells: isolation, characterization and applications in surgery

The ideal stem cell for use in functional tissue engineering needs to be abundantly available, harvested with minimal morbidity, differentiated reliably down various pathways and able to be transplanted safely and efficaciously. Adult human adipose tissue contains a population of mesenchymal stem cells, termed ‘adipose‐derived stem cells’ (ASC), which seem to fulfil most, if not all, of these criteria. ASC can be harvested readily, safely, and in relative abundance by modern liposuction techniques. They are capable of differentiating into other mesenchymal tissue types, including adipocytes, chondrocytes, myocytes and osteoblasts. They also show angiogenic properties, with recent evidence of a potential role in healing radiotherapy‐damaged tissue, possibly due to their secretion of vascular endothelial growth factor. Similarly, they may have a role in healing chronic wounds, and as such are being investigated in phase 1 trials for their ability to aid healing of recurrent Crohn’s fistulae. Subsequently they have a wide range of potential clinical uses in all fields of surgery. This article reviews the current and potential clinical applications of ASC in relation to surgery, as well as methods for their isolation, differentiation and molecular characterization.

A Shift in the Phenotype of Melan-A-Specific CTL Identifies Melanoma Patients with an Active Tumor-Specific Immune Response

In a significant proportion of melanoma patients, CTL specific for the melan-A26/7–35 epitope can be detected in peripheral blood using HLA-A2/peptide tetramers. However, the functional capacity of these CTL has been controversial, since although they prove to be effective killers after in vitro expansion, in some patients they have blunted activation responses ex vivo. We used phenotypic markers to characterize melan-A tetramer+ cells in both normal individuals and melanoma patients, and correlated these markers with ex vivo assays of CTL function. Melanoma patients with detectable melan-A tetramer+ cells in peripheral blood fell into two groups. Seven of thirteen patients had a CCR7+ CD45R0− CD45RA+ phenotype, the same as that found in some healthy controls, and this phenotype was associated with a lack of response to melan-A peptide ex vivo. In the remaining six patients, melan-A tetramer+ cells were shifted toward a CCR7− CD45R0+ CD45RA− phenotype, and responses to melan-A peptide could be readily demonstrated ex vivo. When lymph nodes infiltrated by melan-A-expressing melanoma cells were examined, a similar dichotomy emerged. These findings demonstrate that activation of melan-A-specific CTL occurs in only some patients with malignant melanoma, and that only patients with such active immune responses are capable of responding to Ag in ex vivo assays.

Concise review: human adipose-derived stem cells: separating promise from clinical need

Human adipose‐derived stem cells (ASCs) have become an increasing interest to both stem cell biologists and clinicians because of their potential to differentiate into adipogenic, osteogenic, chondrogenic, and other mesenchymal lineages, as well as other clinically useful properties attributed to them, such as stimulation of angiogenesis and suppression of inflammation. ASCs have already been used in a number of clinical trials, and some successful outcomes have been reported, especially in tissue reconstruction. However, a critical review of the literature reveals considerable uncertainty about the true clinical potential of human ASC. First, the surgical needs that ASC might answer remain relatively few, given the current difficulties in scaling up ASC‐based tissue engineering to a clinically useful volume. Second, the differentiation of ASC into cell lineages apart from adipocytes has not been conclusively demonstrated in many studies due to the use of rather simplistic approaches to the confirmation of differentiation, such as the use of nonspecific histological dyes, or a small number of molecular markers of uncertain significance. Third, the ASC prepared from human lipoaspirate for different studies differ in purity and molecular phenotype, with many studies using cell preparations that are likely to contain heterogeneous populations of cells, making it uncertain whether ASC themselves are responsible for effects observed. Hence, while one clinical application already looks convincing, the full clinical potential of ASC awaits much deeper investigation of their fundamental biology. STEM CELLS 2011,29:404–411

A homozygous diploid subset of commercial wine yeast strains

Genetic analysis was performed on 45 commercial yeasts which are used in winemaking because of their superior fermentation properties. Genome sizes were estimated by propidium iodide fluorescence and flow cytometry. Forty strains had genome sizes consistent with their being diploid, while five had a range of aneuploid genome sizes that ranged from 1.2 to 1.8 times larger. The diploid strains are all Saccharomyces cerevisiae, based on genetic analysis of microsatellite and minisatellite markers and on DNA sequence analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA of four strains. Four of the five aneuploid strains appeared to be interspecific hybrids between Saccharomyces kudriavzevii and Saccharomyces cerevisiae, with the fifth a hybrid between two S. cerevisiae strains. An identification fingerprint was constructed for the commercial yeast strains using 17 molecular markers. These included six published trinucleotide microsatellites, seven new dinucleotide microsatellites, and four published minisatellite markers. The markers provided unambiguous identification of the majority of strains; however, several had identical or similar patterns, and likely represent the same strain or mutants derived from it. The combined use of all 17 polymorphic loci allowed us to identify a set of eleven commercial wine yeast strains that appear to be genetically homozygous. These strains are presumed to have undergone inbreeding to maintain their homozygosity, a process referred to previously as ‘genome renewal’.

Cutting Edge: CD1a+ Antigen-Presenting Cells in Human Dermis Respond Rapidly to CCR7 Ligands

Recent data from murine models have confirmed that Langerhans cells are not the only population of APCs in the skin involved in initiating immune responses. In healthy human skin, we identify CD1a+ dermal APCs located close to the lymphatic vessels in the upper layers of the dermis that are unequivocally distinct from migrating Langerhans cells but exhibit both potent allostimulatory capacity and a chemotactic response to CCR7 ligands. In contrast, CD14+ dermal APCs are distributed throughout the dermis and lack a chemotactic response to CCR7 ligands. CD1a+ dermal APCs therefore represent an APC population distinct from Langerhans cells that are capable of migrating to lymph nodes and stimulating naive T cells. In humans, CD1a+ dermal APCs may fulfill some of the roles previously ascribed to Langerhans cells.

Cancer/testis antigens can be immunological targets in clonogenic CD133+ melanoma cells

Abstract“Cancer stem cells” that resist conventional treatments may be a cause of therapeutic failure in melanoma. We report a subpopulation of clonogenic melanoma cells that are characterized by high prominin-1/CD133 expression in melanoma and melanoma cell lines. These cells have enhanced clonogenicity and self-renewal in vitro, and serve as a limited in vitro model for melanoma stem cells. In some cases clonogenic CD133+ melanoma cells show increased expression of some cancer/testis (CT) antigens. The expression of NY-ESO-1 in an HLA-A2 expressing cell line allowed CD133+ clonogenic melanoma cells to be targeted for killing in vitro by NY-ESO-1-specific CD8+ T-lymphocytes. Our in vitro findings raise the hypothesis that if melanoma stem cells express CT antigens in vivo that immune targeting of these antigens may be a viable clinical strategy for the adjuvant treatment of melanoma.

Three-colour fluorescence immunohistochemistry reveals the diversity of cells staining for macrophage markers in murine spleen and liver

Macrophages have traditionally been identified in murine tissues using a small range of markers, typically F4/80, CD68 and CD11b. However many studies have suggested that substantial heterogeneity exists in macrophage populations, and no single marker, nor even pair of markers, can necessarily identify all the populations. Further, many of the key monoclonal antibodies have been raised in the same species, making it difficult to combine them in histochemical studies. Here we have optimised a triple colour immunofluorescent staining protocol, utilising an anti-FITC technique, to allow antibodies to macrophage markers to be used simultaneously. We highlight the substantial heterogeneity of cells in both normal liver and spleen that stain for F4/80, CD68, CD11b, and CD11c. Using diet-induced steatohepatitis as a model of liver inflammation, we show that CD11b is expressed by newly migrating macrophage precursors, but is an unreliable marker for macrophage precursors when used alone because it is also expressed by migrating neutrophils. In healthy livers CD11c expression is a unique feature of a population of cells immediately surrounding the sinusoids. However, during hepatic inflammation CD11c can also be co-expressed by other cells, including both infiltrating cells and F4/80+ cells within the liver parenchyma. While no one marker alone is sufficient to account for all macrophage populations, we confirm that F4/80 marks the majority of the tissue-resident macrophages in both the liver and the spleen, although F4/80- populations that are positive for CD68, CD11b, or CD11c also exist. Distinguishing between tissue macrophages and dendritic cells with these markers remains problematic.