Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-1.

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.

Activated group 3 innate lymphoid cells promote T-cell-mediated immune responses.

Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1β stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.

Trunk neuromuscular responses to a single whole-body vibration session in patients with chronic low back pain: a cross-sectional study.

OBJECTIVE: Whole-body vibration (WBV) exercise is progressively adopted as an alternative therapeutic modality for enhancing muscle force and muscle activity via neurogenic potentiation. So far, possible changes in the recruitment patterns of the trunk musculature after WBV remain undetermined. The main objective of this study was to evaluate the short-term effects of a single WBV session on trunk neuromuscular responses in patients with chronic low back pain (cLBP) and healthy participants. METHODS: Twenty patients with cLBP and 21 healthy participants performed 10 trunk flexion-extensions before and after a single WBV session consisting of five 1-minute vibration sets. Surface electromyography (EMG) of erector spinae at L2-L3 and L4-L5 and lumbopelvic kinematic variables were collected during the trials. Data were analyzed using 2-way mixed analysis of variance models. RESULTS: The WBV session led to increased lumbar EMG activity during the flexion and extension phases but yielded no change in the quiet standing and fully flexed phases. Kinematic data showed a decreased contribution to the movement of the lumbar region in the second extension quartile. These effects were not different between patients with cLBP and healthy participants. CONCLUSIONS: Increased lumbar EMG activity after a single WBV session most probably results from potentiation effects of WBV on lumbar muscles reflex responses. Decreased EMG activity in full trunk flexion, usually observed in healthy individuals, was still present after WBV, suggesting that the ability of the spine stabilizing mechanisms to transfer the extension torque from muscles to passive structures was not affected.

The influence of environmental conditions on immune responses, morphology and recapture probability of nestling house martins (Delichon urbica)

Nestling birds produced later in the season are hypothesized to be of poor quality with a low probability of survival and recruitment. In a Spanish population of house martins (Delichon urbica), we first compared reproductive success, immune responses and morphological traits between the first and the second broods. Second, we investigated the effects of an ectoparasite treatment and breeding date on the recapture rate the following year. Due probably to a reverse situation in weather conditions during the experiment, with more rain during rearing of the first brood, nestlings reared during the second brood were in better condition and had stronger immune responses compared with nestlings from the first brood. Contrary to other findings on house martins, we found a similar recapture rate for chicks reared during the first and the second brood. Furthermore, ectoparasitic house martin bugs had no significant effect on the recapture rate. Recaptured birds had similar morphology but higher immunoglobulin levels when nestlings compared with non-recaptured birds. This result implies that a measure of immune function is a better predictor of survival than body condition per se.

Differential Recognition of Old World and New World Arenavirus Envelope Glycoproteins by Subtilisin Kexin Isozyme 1 (SKI-1)/Site 1 Protease (S1P).

The arenaviruses are an important family of emerging viruses that includes several causative agents of severe hemorrhagic fevers in humans that represent serious public health problems. A crucial step of the arenavirus life cycle is maturation of the envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). Comparison of the currently known sequences of arenavirus GPCs revealed the presence of a highly conserved aromatic residue at position P7 relative to the SKI-1/S1P cleavage side in Old World and clade C New World arenaviruses but not in New World viruses of clades A and B or cellular substrates of SKI-1/S1P. Using a combination of molecular modeling and structure-function analysis, we found that residueY285 of SKI-1/S1P, distal from the catalytic triad, is implicated in the molecular recognition of the aromatic "signature residue" at P7 in the GPC of Old World Lassa virus. Using a quantitative biochemical approach, we show that Y285 of SKI-1/S1P is crucial for the efficient processing of peptides derived from Old World and clade C New World arenavirus GPCs but not of those from clade A and B New World arenavirus GPCs. The data suggest that during coevolution with their mammalian hosts, GPCs of Old World and clade C New World viruses expanded the molecular contacts with SKI-1/S1P beyond the classical four-amino-acid recognition sequences and currently occupy an extended binding pocket.

Assessment of speed of human locomotion using a differential satellite global positioning system.

PURPOSE: The objective was to explore whether a satellite-based navigation system, global positioning system used in differential mode (DGPS), could accurately assess the speed of running in humans. METHODS: A subject was equipped with a portable GPS receptor coupled to a receiver for differential corrections, while running outdoors on a straight asphalt road at 27 different speeds. Actual speed (reference method) was assessed by chronometry. RESULTS: The accuracy of speed prediction had a standard deviation (SD) of 0.08 km x h(-1) for walking, 0.11 km x h(-1) for running, yielding a coefficient of variation (SD/mean) of 1.38% and 0.82%, respectively. There was a highly significant linear relationship between actual and DGPS speed assessment (r2 = 0.999) with little bias in the prediction equation, because the slope of the regression line was close to unity (0.997). CONCLUSION: the DGPS technique appears to be a valid and inconspicuous tool for "on line" monitoring of the speed of displacement of individuals located on any field on earth, for prolonged periods of time and unlimited distance, but only in specific environmental conditions ("open sky"). Furthermore, the accuracy of speed assessment using the differential GPS mode was improved by a factor of 10 as compared to non-differential GPS.

Expression of apoptosis and survival genes in human memory T cell populations

RESUME Introduction: Les cellules T mémoires humaines sont classées en trois sous-populations sur la base de l'expression d'un marqueur de surface cellulaire, CD45RA, et du récepteur aux chimiokines, CCR7. Ces sous-populations, nommées cellules mémoires centrales (TcM), mémoires effectrices (TEM) et mémoires effectrices terminales (ITEM), ont des rôles fonctionnels distincts, ainsi que des capacités de prolifération et de régénération différentes. Cependant, la génération de ces différences reste encore mal comprise et on ignore les mécanismes moléculaires impliqués. Matériaux et Méthodes: Des cellules mononucléaires humaines du sang périphérique ont été séparées par cytométrie de flux selon leur expression de CD4, CD8, CD45RA et CCR7 en sous-populations de cellules CD4+ ou CD8+ naïves, TcM, TEM ou ITEM. Dans chacune de ces sous-populations, 14 gènes impliqués dans l'apoptose, la survie ou la capacité proliférative des cellules T ont été quantifiés par RT-PCR en temps réel, relativement à l'expression d'un gène de référence endogène. L'ARN provenant de 450 cellules T a été utilisé par gène et par sous-population. Les gènes analysés (cibles) comprenaient des gènes de survie (BAFF, APRIL, BAFF-R, BCMA, TACI, IL-15Rα, IL-7Rα), des gènes anti-apoptotiques (Bcl-2, BclxL, FLIP), des gènes pro-apoptotiques (Bad, Bax, Fast) et le gène anti-prolifératif, Tob. A l'aide de la méthode comparative delta-delta-CT, le taux d'expression des gènes cibles de chaque sous-population des cellules T mémoires CD4+ et CD8+, à été comparée à leur taux d'expression dans les cellules T naïves CD4+ et CD8+. Résultats: Dans les cellules CD8+, les gènes pro-apoptotiques Bax et Fast étaient surexprimés dans toutes les sous-populations mémoires, tandis que l'expression des facteurs anti-apoptotiques et de survie comme Bcl-2, APRIL et BAFF-R, étaient diminués. Ces deux tendances étaient particulièrement accentuées dans les sous-groupes des cellules mémoires TEM et TTEM. A noter que malgré le fait que leur expression était également diminuée dans les autres cellules mémoires, le facteur de survie IL-7Ra, était sélectivement surexprimé dans la sous-population de cellules TcM et l'expression d'IL-15Ra était sélectivement augmentée dans les TEM. Dans les cellules CD4+, le taux d'expression des gènes analysés était plus variable entre les sujets étudiés que dans les cellules CD8+, ne permettant pas de définir un profil d'expression spécifique. L'expression du gène de survie BAFF par contre, a été significativement augmentée dans toutes les sous-populations mémoire CD4+. Il en va de même pour l'expression d' APRIL et de BAFF-R, bien que dans moindre degré. A remarquer que l'expression du facteur anti-apoptotique Fast a été observée uniquement dans la souspopulation des TTEM. Discussion et Conclusions: Cette étude montre une nette différence entre les cellules CD8+ et CD4+, en ce qui concerne les profils d'expression des gènes impliqués dans la survie et l'apoptose des cellules T mémoires. Ceci pourrait impliquer une régulation cellulaire homéostatique distincte dans ces deux compartiments de cellules T mémoires. Dans les cellules CD8+ l'expression d'un nombre de gènes impliqués dans la survie et la protection de l'apoptose semblerait être diminuée dans les populations TEM et TTEM en comparaison à celle des sous-populations naïves et TEM, tandis que l'expression des gènes pro-apoptotiques semblerait être augmentée. Comme ceci paraît être plus accentué dans les TTEM, cela pourrait indiquer une plus grande disposition à l'apopotose dans les populations CCR7- (effectrices) et une perte de survie parallèlement à l'acquisition de capacités effectrices. Ceci parlerait en faveur d'un modèle de différentiation linéaire dans les cellules CD8+. De plus, l'augmentation sélective de l'expression d'IL-7Ra observée dans le sous-groupe de cellules mémoires TEM, et d'IL-15Ra dans celui des TEM, pourrait indiquer un moyen de sélection pour des réponses immunitaires mémoires à long terme par une réponse distincte à ces cytokines. Dans les cellules CD4+ par contre, aucun profil d'expression n'a pu être déterminé; les résultats suggèrent même une résistance relative à l'apoptose de la part des cellules mémoires. Ceci pourrait favoriser l'existence d'un modèle de différentiation plus flexible avec des possibilités d'interaction multiples. Ainsi, la surexpression sélective de BAFF, APRIL et BAFF-R dans les sous-populations individuelles des cellules mémoires pourrait être un indice de l'interaction de ces sous-groupes avec des cellules B. ABSTRACT Introduction: Based on their surface expression of the CD45 isoform and of the CCR7 chemokine receptor, memory T cells have been divided into the following three subsets: central memory (TAM), effector memory (TEM) and terminal effector memory (ITEM). Distinct functional roles and different proliferative and regenerative capacities have been attributed to each one of these subpopulations. The molecular mechanisms underlying these differences; however, remain poorly understood. Materials and Methods: According to their expression of CD4, CD8, CD45RA and CCR7, human peripheral blood mononuclear cells were sorted by flow-cytometry into CD4+ or CD8+ naïve, TAM, TEM and ITEM subsets. Using real-time PCR, the expression of 14 genes known to be involved in apoptotis, survival or proliferation of T cells was quantified separately in each individual subset, relative to an endogenous reference gene. The RNA equivalent of 450 T cells was used for each gene and subset. The target gene panel included the survival genes BAFF, APRIL, BAFF-R, BCMA, TACI, IL-15Rα and IL-7Rα, the anti-apoptotic genes Bcl2, Bcl-xL and FLIP, the pro-apoptotic genes Bad, Bax and Fast, as well as the antiproliferative gene Tob. Using the comparative CT-method, the expression of the target genes in the three memory T cell subsets of both CD4+ and CD8+ T cell populations was compared to their expression in the naïve T cells. Results: In CD8+ cells, the pro-apoptotic factors Bax and Fast were found to be upregulated in all memory T cell subsets, whereas the survival and anti-apoptotic factors Bcl-2, APRIL and BAFF-R were downregulated. These tendencies were most accentuated in TEM and TTEM subsets. Even though the survival factor IL-7Rα was also downregulated in these subsets, interestingly, it was selectively upregulated in the CD8+ TAM subset. Similarly, IL-15Rαexpression was shown to be selectively upregulated in the CD8+ TEM subset. In CD4+ cells, the expression levels of the analyzed genes showed a greater inter-individual variability than in CD8+ cells, thus suggesting the absence of any particular expression pattern for CD4+ memory T cells. However, the survival factor BAFF was found to be significantly upregulated in all CD4+ memory T cell subsets, as was also the expression of APRIL and BAFF-R, although to a lesser extent. Furthermore, it was noted that the pro-apoptotic gene Fast was only expressed in the TTEM CD4+ subset. Discussion and Conclusions: Genes involved in apoptosis and survival in human memory T cells have been shown to be expressed differently in CD8+ cells as compared to CD4+ cells, suggesting a distinct regulation of cell homeostasis in these two memory T cell compartments. The present study suggests that, in CD8+ T cells, the expression of various survival and antiapoptotic genes is downregulated in TEM and TTEM subsets, while the expression of proapoptotic genes is upregulated in comparison to the naïve and the TAM populations. These characteristics, potentially translating to a greater susceptibility to apoptosis in the CCR7- (effector) memory populations, are accentuated in the TTEM population, suggesting a loss of survival in parallel to the acquisition of effector capacities. This speaks in favour of a linear differentiation model in CD8+ T memory cells. Moreover, the observed selectively increased expression of IL-7Rα in CD8+ TAM cells - as that of IL-15Rα in CD8+ TEM cells -suggest that differential responsiveness to cytokines could confer a selection bias for distinct long-term memory cell responses. Relative to the results for CD8+ T cells, those for CD4+ T cells seem to indicate a certain resistance of the memory subsets to apoptosis, suggesting the possibility of a more flexible differentiation model with multiple checkpoints and potential interaction of CD4+ memory cells with other cells. Thus, the selective upregulation of BAFF, APRIL and BAFF-R in individual memory subsets could imply an interaction of these subsets with B cells.

Chronic wound healing by fetal cell therapy may be explained by differential gene profiling observed in fetal versus old skin cells.

Engineering of fetal tissue has a high potential for the treatment of acute and chronic wounds of the skin in humans as these cells have high expansion capacity under simple culture conditions and one organ donation can produce Master Cell Banks which can fabricate over 900 million biological bandages (9 x 12cm). In a Phase 1 clinical safety study, cases are presented for the treatment of therapy resistant leg ulcers. All eight patients, representing 13 ulcers, tolerated multiple treatments with fetal biological bandages showing no negative secondary effects and repair processes similar to that seen in 3rd degree burns. Differential gene profiling using Affymetrix gene chips (analyzing 12,500 genes) were accomplished on these banked fetal dermal skin cells compared to banked dermal skin cells of an aged donor in order to point to potential indicators of wound healing. Families of genes involved in cell adhesion and extracellular matrix, cell cycle, cellular signaling, development and immune response show significant differences in regulation between banked fetal and those from banked old skin cells: with approximately 47.0% of genes over-expressed in fetal fibroblasts. It is perhaps these differences which contribute to efficient tissue repair seen in the clinic with fetal cell therapy.

Assessment of vaccine-induced CD4 T cell responses to the 119-143 immunodominant region of the tumor-specific antigen NY-ESO-1 using DRB1*0101 tetramers.

Abstract: Purpose: NY-ESO-1 (ESO), a tumor-specific antigen of the cancer/testis group, is presently viewed as an important model antigen for the development of generic anticancer vaccines. The ESO119-143 region is immunodominant following immunization with a recombinant ESO vaccine. In this study, we generated DRB1*0101/ESO119-143 tetramers and used them to assess CD4 T-cell responses in vaccinated patients expressing DRB1*0101 (DR1). Experimental Design: We generated tetramers of DRB1*0101 incorporating peptide ESO119-143 using a previously described strategy. We assessed ESO119-143-specific CD4 T cells in peptide-stimulated post-vaccine cultures using the tetramers. We isolated DR1/ESO119-143 tetramer(+) cells by cell sorting and characterized them functionally. We assessed vaccine-induced CD4(+) DR1/ESO119-143 tetramer(+) T cells ex vivo and characterized them phenotypically. Results: Staining of cultures from vaccinated patients with DR1/ESO119-143 tetramers identified vaccine-induced CD4 T cells. Tetramer(+) cells isolated by cell sorting were of T(H)1 type and efficiently recognized full-length ESO. We identified ESO123-137 as the minimal optimal epitope recognized by DR1-restricted ESO-specific CD4 T cells. By assessing DR1/ESO119-143 tetramer(+) cells using T cell receptor (TCR) beta chain variable region (V beta)-specific antibodies, we identified several frequently used V beta. Finally, direct ex vivo staining of patients' CD4 T cells with tetramers allowed the direct quantification and phenotyping of vaccine-induced ESO-specific CD4 T cells. Conclusions: The development of DR1/ESO119-143 tetramers, allowing the direct visualization, isolation, and characterization of ESO-specific CD4 T cells, will be instrumental for the evaluation of spontaneous and vaccine-induced immune responses to this important tumor antigen in DR1-expressing patients

Phenotype and function of protective T cell immune responses in HIV.

PURPOSE OF REVIEW: Definition of T cell immune correlates in HIV infection remains a lofty goal towards our understanding of the HIV-specific immune response. This review will focus upon recent developments and controversies in our understanding of protective T cell responses against HIV. RECENT FINDINGS: It has become clear that multiple functions and phenotypic markers of T cells must be assessed to accurately characterize the complexity of CD4 and CD8 T cell responses. While evidence indicates that a hallmark of protective immune responses in HIV infection is the presence of 'polyfunctional' T cell responses, a disconnect remains between the function and phenotype of effective HIV-specific T cells. Moreover, there may be inherent differences in the ability of specific human leukocyte antigen class I families to promote CD8 T cell effector versus polyfunctional responses. It remains to be determined how polyfunctional responses arise in HIV infection, which functions are important for control, and whether surface phenotype markers provide an indication of protective capacity. SUMMARY: Polyfunctional and phenotypic assessment of T cell responses have clearly advanced our understanding of HIV specific immune responses. Critical questions remain, however, especially whether polyfunctional T cell responses control, or are controlled by, HIV replication.

Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii.

ABSTRACT: BACKGROUND: Sphingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. Thus far, however, little is known about the adaptive strategies used by Sphingomonas bacteria to respond to changes in water potential. To improve our understanding, strain RW1 was perturbed with either the cell-permeating solute sodium chloride or the non-permeating solute polyethylene glycol with a molecular weight of 8000 (PEG8000). These solutes are assumed to simulate the solute and matric components of the total water potential, respectively. The responses to these perturbations were then assessed and compared using a combination of growth assays, transcriptome profiling, and membrane fatty acid analyses. RESULTS: Under conditions producing a similar decrease in water potential but without effect on growth rate, there was only a limited shared response to perturbation with sodium chloride or PEG8000. This shared response included the increased expression of genes involved with trehalose and exopolysaccharide biosynthesis and the reduced expression of genes involved with flagella biosynthesis. Mostly, the responses to perturbation with sodium chloride or PEG8000 were very different. Only sodium chloride triggered the increased expression of two ECF-type RNA polymerase sigma factors and the differential expression of many genes involved with outer membrane and amino acid metabolism. In contrast, only PEG8000 triggered the increased expression of a heat shock-type RNA polymerase sigma factor along with many genes involved with protein turnover and repair. Membrane fatty acid analyses further corroborated these differences. The degree of saturation of membrane fatty acids increased after perturbation with sodium chloride but had the opposite effect and decreased after perturbation with PEG8000. CONCLUSIONS: A combination of growth assays, transcriptome profiling, and membrane fatty acid analyses revealed that permeating and non-permeating solutes trigger different adaptive responses in strain RW1, suggesting these solutes affect cells in fundamentally different ways. Future work is now needed that connects these responses with the responses observed in more realistic scenarios of soil desiccation.

Allergen-specific antibody and cytokine responses, mast cell reactivity and intestinal permeability upon oral challenge of sensitized and tolerized mice.

BACKGROUND: Food allergy has reached an epidemic level in westernized countries and although central mechanisms have been described, the variability associated with genetic diversity underscores the still unresolved complexity of these disorders. OBJECTIVE: To develop models of food allergy and oral tolerance, both strictly induced by the intestinal route, and to compare antigen-specific responses. METHODS: BALB/c mice were mucosally sensitized to ovalbumin (OVA) in the presence of the mucosal adjuvant cholera toxin, or tolerized by intra-gastric administrations of OVA alone. Antibody titres and cytokines were determined by ELISA, and allergic status was determined through several physiologic parameters including decline in temperature, diarrhoea, mast cell degranulation and intestinal permeability. RESULTS: OVA-specific antibodies (IgE, IgGs and IgA in serum and feces) were produced in sensitized mice exclusively. Upon intra-gastric challenge with OVA, sensitized mice developed anaphylactic reactions associated with a decline of temperature, diarrhoea, degranulation of mast cells, which were only moderately recruited in the small intestine, and increased intestinal permeability. Cytokines produced by immune cells from sensitized mice included T-helper type 2 cytokines (IL-5, IL-13), but also IL-10, IFN-gamma and IL-17. In contrast, all markers of allergy were totally absent in tolerized animals, and yet the latter were protected from subsequent sensitization, demonstrating that oral tolerance took place efficiently. CONCLUSION: This work allows for the first time an appropriate comparison between sensitized and tolerized BALB/c mice towards OVA. It highlights important differences from other models of allergy, and thus questions some of the generally accepted notions of allergic reactions, such as the protective role of IFN-gamma, the importance of antigen-specific secretory IgA and the role of mucosal mast cells in intestinal anaphylaxis. In addition, it suggests that IL-17 might be an effector cytokine in food allergy. Finally, it demonstrates that intestinal permeability towards the allergen is increased during challenge.

RHAMM-R3 peptide vaccination in patients with acute myeloid leukemia, myelodysplastic syndrome, and multiple myeloma elicits immunologic and clinical responses.

The receptor for hyaluronic acid-mediated motility (RHAMM) is an antigen eliciting both humoral and cellular immune responses in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and multiple myeloma (MM). We initiated a phase 1 clinical trial vaccinating 10 patients with R3 (ILSLELMKL), a highly immunogenic CD8(+) T-cell epitope peptide derived from RHAMM. In 7 of 10 patients, we detected an increase of CD8(+)/HLA-A2/RHAMM R3 tetramer(+)/CD45RA(+)/CCR7(-)/CD27(-)/CD28(-) effector T cells in accordance with an increase of R3-specific CD8(+) T cells in enzyme linked immunospot (ELISpot) assays. In chromium release assays, a specific lysis of RHAMM-positive leukemic blasts was shown. Three of 6 patients with myeloid disorders (1/3 AML, 2/3 MDS) achieved clinical responses: one patient with AML and one with MDS showed a significant reduction of blasts in the bone marrow after the last vaccination. One patient with MDS no longer needed erythrocyte transfusions after 4 vaccinations. Two of 4 patients with MM showed a reduction of free light chain serum levels. Taken together, RHAMM-R3 peptide vaccination induced both immunologic and clinical responses, and therefore RHAMM constitutes a promising target for further immunotherapeutic approaches. This study is registered at http://ISRCTN.org as ISRCTN32763606 and is registered with EudraCT as 2005-001706-37.