(E)-4-Hydroxy-3-methyl-but-2 enyl pyrophosphate-stimulated Vγ9Vδ2 T cells possess T helper type 1-promoting adjuvant activity for human monocyte-derived dendritic cells

Vγ9Vδ2 T cells respond to pyrophosphate antigens and display potent antitumour activity in vitro. We have investigated the potential of the most potent phosphoantigen known to activate Vγ9Vδ2 T cells, (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP), as an adjuvant for dendritic cell (DC)-based vaccines. A single stimulation of peripheral blood mononuclear cells with HMB-PP and IL-2 was sufficient to generate lines of effector memory Vγ9Vδ2 T cells that retained their cytolytic and cytokine secretion activities. These cells induced differentiation of DC into semi-mature antigen-presenting cells expressing CD86, CD11c, CD54, HLA-DR, CD83 and CD40, which secreted low levels of bioactive IL-12 but no IL-10. Vγ9Vδ2 T cells also strongly costimulated IL-12 release but inhibited IL-10 production by lipopolysaccharide (LPS)-stimulated DC. When substituted for Vγ9Vδ2 T cells, IFN-γ did not induce full DC maturation but it augmented IL-12 and inhibited IL-10 release by LPS-stimulated DC, in a manner similar to HMB-PP-activated Vγ9Vδ2 T cells. Our findings indicate that Vγ9Vδ2 T cells, stimulated with nanomolar concentrations of HMB-PP, strongly promote T helper type 1 (Th1) responses through their ability to induce DC maturation and IL-12 secretion. This adjuvant activity may prove useful in DC-based cancer therapies.     

Addition of a prominent epitope affects influenza A virus-specific CD8+ T cell immunodominance hierarchies when antigen is limiting

A reverse genetics strategy was used to insert the OVA peptide (amino acid sequence SIINFEKL; OVA(257-264)) into the neuraminidase stalk of both the A/PR8 (H1N1) and A/HKx31 (H3N2) influenza A viruses. Initial characterization determined that K(b)OVA257 is presented on targets infected with PR8-OVA and HK-OVA without significantly altering D(b) nucleoprotein (NP)366 presentation. There were similar levels of K(b)OVA257- and D(b)NP366-specific CTL expansion following both primary and secondary intranasal challenge. Interestingly, while variable, the presence of the immunodominant K(b)OVA257-specific response resulted in diminished D(b) acidic polymerase224- and K(b) basic polymerase subunit 1(703)-, but not D(b)NP366-specific responses and didn't alter endogenous influenza A virus-specific immunodominance hierarchies. However, challenging PR8-OVA-primed mice with HK-OVA via the i.p. route, and thereby limiting Ag dose, led to a reduction in the magnitude of all the influenza A virus-specific responses measured. A similar reduction in CTL response to native epitopes was also seen following primary respiratory HK-OVA infection of mice that received substantial numbers of K(b)OVA257-specific TCR transgenic T cells. Thus, during the course of infection, the generation of individual virus-specific CTL responses is independently regulated. However, in cases in which Ag is limiting, or high precursor frequency, the presence of immunodominant CTL responses can impact on the magnitude of other specific populations. Therefore, depending on both the size of the T cell precursor pool and the mode of Ag presentation, the addition of a major epitope can diminish the size of endogenous, influenza-specific CD8+ T cell responses, although never to the point that these are totally compromised.     

Combination of cytosine deaminase with uracil phosphoribosyl transferase leads to local and distant bystander effects against RM1 prostate cancer in mice

BACKGROUND: We aimed to evaluate the efficacy of gene-directed enzyme-prodrug therapy (GDEPT) using cytosine deaminase in combination with uracil phosphoribosyl transferase (CDUPRT) against intraprostatic mouse androgen-refractory prostate (RM1) tumors in immunocompetent mice. The product of the fusion gene, CDUPRT, converts the prodrug, 5-fluorocytosine (5FC), into 5-fluorouracil (5FU) and other cytotoxic metabolites that kill both CDUPRT-expressing and surrounding cells, via a 'bystander effect'. METHODS: Stably transformed andogen-independent mouse prostate cancer (PC) cells, RM1-CDUPRT, -GFP or GFP/LacZ cells were used. To assess the local bystander effects of CDUPRT-GDEPT, immunocompetent C57BL/6 mice implanted with cell mixtures of RM1-GFP/CDUPRT and RM1-GFP cells in different proportions intraprostatically were treated with 5FC. Pseudo-metastases in the lungs were established by a tail vein injection of untransfected RM1 cells. At necropsy, prostate weight/volume and lung colony counts were assessed. Tumors, lymph nodes, spleens and lungs were frozen or fixed for immunohistochemistry. RESULTS: CDUPRT expression in RM1-GFP/CDUPRT cells or tumors was confirmed by enzymic conversion of 5FC into 5FU, using HPLC. Treatment of mice bearing intraprostatic RM1-GFP/CDUPRT tumors with 5FC resulted in complete regression of the tumors. A 'local bystander effect' was seen, even though only 20% of the cells expressed CDUPRT. More importantly a significant reduction in pseudo-metastases of RM1 cells in lungs indicated a 'distant bystander effect'. Immunohistochemical evaluation of the treated tumors showed increased necrosis and apoptosis, with decreased tumor vascularity. There was also a significant increase in tumour-infiltration by macrophages, CD4+ T and natural killer cells. CONCLUSIONS: We conclude that CDUPRT-GDEPT significantly suppressed the aggressive growth of RM1 prostate tumors and lung pseudo-metastases via immune mechanisms involving necrosis and apoptosis.     

Survival estimates for Royal Terns in southern California

ABSTRACT.   Once the most abundant large tern in California, populations of Royal Terns ( Thalasseus maximus ) are now greatly reduced statewide. However, these terns are still common in southern California where the population appears to be increasing. We estimated survival of this species, for which no robust estimate of survival is available, using mark-resighting data. Apparent survival of birds >3 yr of age was estimated to be ∼0.95; first-year survival was lower at 0.86. The lower survival estimate for first-year birds probably reflects increased mortality, but could also be a function of permanent emigration by individuals in this age class. The adult survival probability is the highest reported among terns and suggests that survival is playing an important role in the recovery of this tern population and that the current population growth is not solely due to immigration. Improvements in resighting protocols, marking technology, and mark-recapture methods are recommended to better use live resighting, dead recovery, and multistate data.     

Structural determinants of T-cell receptor bias in immunity

Antigen-specific T-cell responses induced by infection, transplantation, autoimmunity or hypersensitivity are characterized by cells expressing biased profiles of T-cell receptors (TCRs) that are selected from a diverse, naive repertoire. Here, we review the evidence for these TCR biases, focusing on crystallographic analysis of the structural constraints that determine the binding of a TCR to its ligand and the persistence of certain TCRs in an immune repertoire. We discuss the ways in which diversity in a selected TCR repertoire can contribute to protective immunity and the implications of this for vaccine design and immunotherapy.     

Alternaria induces STAT6-dependent acute airway eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness

The fungal allergen, Alternaria, is specifically associated with severe asthma, including life-threatening exacerbations. To better understand the acute innate airway response to Alternaria, naive wild-type (WT) mice were challenged once intranasally with Alternaria. Naive WT mice developed significant bronchoalveolar lavage eosinophilia following Alternaria challenge when analyzed 24 h later. In contrast to Alternaria, neither Aspergillus nor Candida induced bronchoalveolar lavage eosinophilia. Gene microarray analysis of airway epithelial cell brushings demonstrated that Alternaria-challenged naive WT mice had a >20-fold increase in the level of expression of found in inflammatory zone 1 (FIZZ1/Retnla), a resistin-like molecule. Lung immunostaining confirmed strong airway epithelial FIZZ1 expression as early as 3 h after a single Alternaria challenge that persisted for ≥5 d and was significantly reduced in STAT6-deficient, but not protease-activated receptor 2-deficient mice. Bone marrow chimera studies revealed that STAT6 expressed in lung cells was required for epithelial FIZZ1 expression, whereas STAT6 present in bone marrow-derived cells contributed to airway eosinophilia. Studies investigating which cells in the nonchallenged lung bind FIZZ1 demonstrated that CD45(+)CD11c(+) cells (macrophages and dendritic cells), as well as collagen-1-producing CD45(-) cells (fibroblasts), can bind to FIZZ1. Importantly, direct administration of recombinant FIZZ1 to naive WT mice led to airway eosinophilia, peribronchial fibrosis, and increased thickness of the airway epithelium. Thus, Alternaria induces STAT6-dependent acute airway eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness. This may provide some insight into the uniquely pathogenic aspects of Alternaria-associated asthma.     

Chondrocalcinosis is common in the absence of knee involvement

Introduction

We aimed to describe the distribution of radiographic chondrocalcinosis (CC) and to examine whether metacarpophalangeal joint (MCPJ) calcification and CC at other joints occurs in the absence of knee involvement.

Methods

This was a cross-sectional study embedded in the Genetics of Osteoarthritis and Lifestyle study (GOAL). All participants (n = 3,170) had radiographs of the knees, hands, and pelvis. These were scored for radiographic changes of osteoarthritis (OA), for CC at knees, hips, symphysis pubis, and wrists, and for MCPJ calcification. The prevalence of MCPJ calcification and CC overall, at each joint, and in the presence or absence of knee involvement, was calculated.

Results

The knee was the commonest site of CC, followed by wrists, hips, and symphysis pubis. CC was more likely to be bilateral at knees and wrists but unilateral at hips. MCPJ calcification was usually bilateral, and less common than CC at knees, hips, wrists, and symphysis pubis. Unlike that previously reported, CC commonly occurred without any knee involvement; 44.4% of wrist CC, 45.9% of hip CC, 45.5% of symphysis pubis CC, and 31.3% of MCPJ calcification occurred in patients without knee CC. Those with meniscal or hyaline articular cartilage CC had comparable ages (P = 0.21), and neither preferentially associated with fibrocartilage CC at distant joints.

Conclusions

CC visualized on a plain radiograph commonly occurs at other joints in the absence of radiographic knee CC. Therefore, knee radiographs alone are an insufficient screening test for CC. This has significant implications for clinical practice, for epidemiologic and genetic studies of CC, and for the definition of OA patients with coexistent crystal deposition.     

Early Priming Minimizes the Age-Related Immune Compromise of CD8+ T Cell Diversity and Function

The elderly are particularly susceptible to influenza A virus infections, with increased occurrence, disease severity and reduced vaccine efficacy attributed to declining immunity. Experimentally, the age-dependent decline in influenza-specific CD8⁺ T cell responsiveness reflects both functional compromise and the emergence of ‘repertoire holes’ arising from the loss of low frequency clonotypes. In this study, we asked whether early priming limits the time-related attrition of immune competence. Though primary responses in aged mice were compromised, animals vaccinated at 6 weeks then challenged >20 months later had T-cell responses that were normal in magnitude. Both functional quality and the persistence of ‘preferred’ TCR clonotypes that expand in a characteristic immunodominance hierarchy were maintained following early priming. Similar to the early priming, vaccination at 22 months followed by challenge retained a response magnitude equivalent to young mice. However, late priming resulted in reduced TCRβ diversity in comparison with vaccination earlier in life. Thus, early priming was critical to maintaining individual and population-wide TCRβ diversity. In summary, early exposure leads to the long-term maintenance of memory T cells and thus preserves optimal, influenza-specific CD8⁺ T-cell responsiveness and protects against the age-related attrition of naïve T-cell precursors. Our study supports development of vaccines that prime CD8⁺ T-cells early in life to elicit the broadest possible spectrum of CD8⁺ T-cell memory and preserve the magnitude, functionality and TCR usage of responding populations. In addition, our study provides the most comprehensive analysis of the aged (primary, secondary primed-early and secondary primed-late) TCR repertoires published to date.