Mixed-model of ANOVA for measurement reproducibility in proteomics

This work is a statistical analysis of reproducibility of a MALDI-TOF mass spectrometry experiment. Its aim is to evaluate measurement variability and compare peak intensities from two types of MALDI-TOF platforms. We compared and commented on the abilities of Principal Component Analysis and mixed-model analysis of variance to evaluate the biological variability and the technical variability of peak intensities in different patients. The properties and hypotheses of both methods are summarized and applied to spectra from plasma of patients with Hodgkin lymphoma. Principal Component Analysis checks rapidly the balance between the two variabilities; however, a mixed-model analysis of variance is necessary to quantify the biological and technical components of the experimental variance as well as their interactions and to split the total variance into between-subjects and within-subject components. The latter method helped to assess the reproducibility of measurements from two MALDI-TOF platforms and to decompose the technical variability according to the experimental design.     

Leadership in Moving Human Groups

How is movement of individuals coordinated as a group? This is a fundamental question of social behaviour, encompassing phenomena such as bird flocking, fish schooling, and the innumerable activities in human groups that require people to synchronise their actions. We have developed an experimental paradigm, the HoneyComb computer-based multi-client game, to empirically investigate human movement coordination and leadership. Using economic games as a model, we set monetary incentives to motivate players on a virtual playfield to reach goals via players'' movements. We asked whether (I) humans coordinate their movements when information is limited to an individual group member''s observation of adjacent group member motion, (II) whether an informed group minority can lead an uninformed group majority to the minority''s goal, and if so, (III) how this minority exerts its influence. We showed that in a human group – on the basis of movement alone – a minority can successfully lead a majority. Minorities lead successfully when (a) their members choose similar initial steps towards their goal field and (b) they are among the first in the whole group to make a move. Using our approach, we empirically demonstrate that the rules of swarming behaviour apply to humans. Even complex human behaviour, such as leadership and directed group movement, follow simple rules that are based on visual perception of local movement.     

Dendritic cells: driving the differentiation programme of T cells in viral infections

Protective immunity against viral pathogens depends on the generation and maintenance of a small population of memory CD8(+) T cells. Successful memory cell generation begins with early interactions between na?ve T cell and dendritic cells (DCs) within the inflammatory milieu of the secondary lymphoid tissues. Recent insights into the role of different populations of DCs, and kinetics of antigen presentation, during viral infections have helped to understand how DCs can shape the immune response. Here, we review the recent progress that has been made towards defining how specific DC subsets drive effector CD8(+) T-cell expansion and differentiation into memory cells. Further, we endeavour to examine how the molecular signals imparted by DCs coordinate to generate protective CD8(+) T-cell immunity.     

Compromised influenza virus-specific CD8(+)-T-cell memory in CD4(+)-T-cell-deficient mice

The primary influenza A virus-specific CD8(+)-T-cell responses measured by tetramer staining of spleen, lymph node, and bronchoalveolar lavage (BAL) lymphocyte populations were similar in magnitude for conventional I-A(b+/+) and CD4(+)-T-cell-deficient I-A(b-/-) mice. Comparable levels of virus-specific cytotoxic-T-lymphocyte activity were detected in the inflammatory exudate recovered by BAL following challenge. However, both the size of the memory T-cell pool and the magnitude of the recall response in the lymphoid tissues (but not the BAL specimens) were significantly diminished in mice lacking the CD4(+) subset. Also, the rate of virus elimination from the infected respiratory tract slowed at low virus loads following challenge of na?ve and previously immunized I-A(b-/-) mice. Thus, though the capacity to mediate the CD8(+)-T-cell effector function is broadly preserved in the absence of concurrent CD4(+)-T-cell help, both the maintenance and recall of memory are compromised and the clearance of residual virus is delayed. These findings are consistent with mathematical models that predict virus-host dynamics in this, and other, models of infection.     

Contemporary analysis of MHC-related immunodominance hierarchies in the CD8+ T cell response to influenza A viruses

Early studies of influenza virus-specific CD8+ T cell-mediated immunity indicated that the level of CTL activity associated with H2Db is greatly diminished in mice that also express H2Kk. Such MHC-related immunodominance hierarchies are of some interest, as they could lead to variable outcomes for peptide-based vaccination protocols in human populations. The influence of H2Kk on the H2Db-restricted response profile has thus been looked at again using a contemporary, quantitative, IFN-gamma-based flow cytometric assay. The depressive effect of H2Kk was very apparent for the influenza DbPA224 epitope and was also reproduced when CTL activity was measured for H2Db-expressing targets pulsed with the immunodominant NP366 peptide. The secondary CD8+IFN-gamma+ DbNP366-specific response was much greater in parental H2b than in H2kxbF1 mice, but the sizes of the CD8+ sets specific for KkNP50 and DbNP366 were essentially equivalent in the F1 animals. Thus, although the immunodominance profile associated with DbNP366 is lost when H2Kk is also present, the response is still substantial. A further, MHC-related effect was also identified for the KkNS1152 epitope, which was consistently associated with a greater CD8+IFN-gamma+ response in H2KkDb recombinant than in (H2KkDk x H2KbDb)F1 mice. The diminished DbPA224 response in H2kxbF1 mice was characterized by loss of a prominent Vbeta7 TCR responder phenotype, supporting the idea that TCR deletion during ontogeny shapes the available repertoire. The overall conclusion is that these MHC-related immunodominance hierarchies are more subtle than the early CTL assays suggested and, although inherently unpredictable, are unlikely to cause a problem for peptide-based vaccine strategies.     

DCs as targets for vaccine design

The increasingly stringent requirements laid down by regulatory authorities have brought to an end the largely empirical design of vaccines. Vaccines must now be designed rationally, in order that appropriate immune responses are elicited with few or no side effects. The DC plays a pivotal role in determining the type of immune response that ensues following exposure of the host to an Ag. In this review, we identify some of the features and properties of DCs, and how these properties can be exploited in the design of smart vaccines.     

The role of antigen in the localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza pneumonia.

The role of Ag in the recruitment and localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza infection was explored using TCR-transgenic (Tg) mice. Naive, Thy1.2(+)CD8(+) OT-I TCR-Tg cells were primed and recruited to the lung after transfer into congenic Thy1.1(+) recipients challenged with a genetically engineered influenza virus (influenza A/WSN/33 (WSN)-OVA(I)) containing the K(b) restricted OVA(257-264) epitope (siinfekl) in the viral neuraminidase stalk. However, if the transferred animals were infected with a similar influenza virus that expressed an irrelevant K(b) epitope (WSN-PEPII), no TCR-Tg T cells were detectable in the lung, although they were easily visible in the lymphoid organs. Conversely, there were substantial numbers of OT-I cells found in the lungs of WSN-PEPII-infected mice when the animals had been previously, or were concurrently, infected with a recombinant vaccinia virus expressing OVA. Similar results were obtained with nontransgenic populations of memory CD8(+) T cells reactive to a murine gamma-herpesvirus-68 Ag. Interestingly, the primary host response to the immunodominant influenza nucleoprotein epitope was not affected by the presence of memory or recently activated OT-I T cells. Thus, although Ag is required to activate the T cells, the subsequent localization of T cells to the lung during a virus infection is a property of recently activated and memory T cells and is not necessarily driven by Ag in the lung.     

Cutting edge: precursor frequency affects the helper dependence of cytotoxic T cells.

Generation of CTL immunity often depends on the availability of CD4 T cell help. In this report, we show that CTL responses induced by cross-priming can be converted from CD4-dependent to CD4-independent by increasing the frequency of CTL precursors. In the absence of CD4 T cells, high numbers of CTL precursors were able to expand in number and become effector CTL. The ability of high frequencies of CD8 T cells to override help was not due to their ability to signal CD40 via expression of CD154. These findings suggest that when precursor frequencies are high, priming of CD8 T cell responses may not require CD4 T cell help.     

Dendritic cells present lytic antigens and maintain function throughout persistent gamma-herpesvirus infection

The activation and maintenance of Ag-specific CD8(+) T cells is central to the long-term control of persistent infections. These killer T cells act to continuously scan and remove reservoirs of pathogen that have eluded the acute immune response. Acutely cleared viral infections depend almost exclusively on dendritic cells (DC) to present Ags to, and to activate, the CD8(+) T cell response. Paradoxically, persistent pathogens often infect professional APCs such as DC, in addition to infecting a broad range of nonprofessional APC, raising the possibility that many cell types could present viral Ags and activate T cells. We addressed whether in persistent viral infection with murine gammaherpesviruses, DC or non-DC, such as B cells and macrophages, were required to maintain the continued activation of Ag-specific CD8(+) T cells. We found that presentation of the surrogate Ag, OVA, expressed under a lytic promoter to CD8(+) T cells during persistent infection was largely restricted to DC, with little contribution from other lymphoid resident cells, such as B cells. This is despite the fact that B cells harbor a very large reservoir of latent virus. Our results support that, during persistent viral infection, continual presentation of lytic Ags by DC leads to T cell activation critical for maintaining CD8(+) T cells capable of limiting persistent viral infection.