CD8 + T cells are specialized cells of the adaptive immune system capable of finding and eliminating pathogen-infected cells. To date it has not been possible to observe the destruction of any pathogen by CD8 + T cells in vivo. Here we demonstrate a technique for imaging the killing of liver-stage malaria parasites by CD8 + T cells bearing a transgenic T cell receptor specific for a parasite epitope. We report several features that have not been described by in vitro analysis of the process, chiefly the formation of large clusters of effector CD8 + T cells around infected hepatocytes. The formation of clusters requires antigen-specific CD8 + T cells and signaling by G protein-coupled receptors, although CD8 + T cells of unrelated specificity are also recruited to clusters. By combining mathematical modeling and data analysis, we suggest that formation of clusters is mainly driven by enhanced recruitment of T cells into larger clusters. We further show various death phenotypes of the parasite, which typically follow prolonged interactions between infected hepatocytes and CD8 + T cells. These findings stress the need for intravital imaging for dissecting the fine mechanisms of pathogen recognition and killing by CD8 + T cells.Plasmodium | immunity | lymphocytes

Malaria, a disease caused by parasites of the Plasmodium genus, begins when Plasmodium-infected mosquitoes inject malaria sporozoites while searching for blood. Sporozoites migrate from the skin via blood to the liver, infect hepatocytes, and form liver stages which in mice 48 h later escape into blood and cause clinical malaria. Vaccine-induced activated or memory CD8 T cells are capable of locating and eliminating all liver stages in 48 h, thus preventing the blood-stage disease. However, the rules of how CD8 T cells are able to locate all liver stages within a relatively short time period remains poorly understood. We recently reported formation of clusters consisting of variable numbers of activated CD8 T cells around Plasmodium yoelii (Py)-infected hepatocytes. Using a combination of experimental data and mathematical models we now provide additional insights into mechanisms of formation of these clusters. First, we show that a model in which cluster formation is driven exclusively by T-cell-extrinsic factors, such as variability in “attractiveness” of different liver stages, cannot explain distribution of cluster sizes in different experimental conditions. In contrast, the model in which cluster formation is driven by the positive feedback loop (i.e., larger clusters attract more CD8 T cells) can accurately explain the available data. Second, while both Py-specific CD8 T cells and T cells of irrelevant specificity (non-specific CD8 T cells) are attracted to the clusters, we found no evidence that non-specific CD8 T cells play a role in cluster formation. Third and finally, mathematical modeling suggested that formation of clusters occurs rapidly, within few hours after adoptive transfer of CD8 T cells, thus illustrating high efficiency of CD8 T cells in locating their targets in complex peripheral organs, such as the liver. Taken together, our analysis provides novel insights into and attempts to discriminate between alternative mechanisms driving the formation of clusters of antigen-specific CD8 T cells in the liver.

The t-haplotype, a mouse meiotic driver found on chromosome 17, has been a model for autosomal segregation distortion for close to a century, but several questions remain regarding its biology and evolutionary history. A recently published set of population genomics resources for wild mice includes several individuals heterozygous for the t-haplotype, which we use to characterize this selfish element at the genomic and transcriptomic level. Our results show that large sections of the t-haplotype have been replaced by standard homologous sequences, possibly due to occasional events of recombination, and that this complicates the inference of its history. As expected for a long genomic segment of very low recombination, the t-haplotype carries an excess of fixed nonsynonymous mutations compared to the standard chromosome. This excess is stronger for regions that have not undergone recent recombination, suggesting that occasional gene flow between the t and the standard chromosome may provide a mechanism to regenerate coding sequences that have accumulated deleterious mutations. Finally, we find that t-complex genes with altered expression largely overlap with deleted or amplified regions, and that carrying a t-haplotype alters the testis expression of genes outside of the t-complex, providing new leads into the pathways involved in the biology of this segregation distorter.KEYWORDS meiotic driver; t-haplotype; genome evolution M EIOTIC drivers (also known as segregation distorters) are selfish alleles or chromosome variants that can transmit themselves to over 50% of the progeny of heterozygous individuals (Burt and Trivers 2009;Lindholm et al. 2016), often by killing or inactivating gametes that carry the nondriver allele. This requires the combined action of at least one distorter gene, which attacks gametes, and a responder gene, which protects gametes carrying the driver [reviewed in Lindholm et al. (2016)]. Linkage between the distorter and responder genes is required for the survival of the driver, and successful drivers often arise in regions of low recombination (Schwander et al. 2014). Conversely, the presence of drivers can select for reduced recombination around the driving and responding loci (Charlesworth and Hartl 1978).Autosomal drivers usually have no detectable phenotypic effects, and much of what is known about them comes primarily from studies of two model systems: Segregation Distorter in Drosophila melanogaster (Larracuente and Presgraves 2012) and the t-haplotype of the domestic mouse Mus musculus.The t-haplotype is a 40-Mb variant of the proximal portion of chromosome 17 (Burt and Trivers 2009; Herrmann and Bauer 2012), which shows suppressed recombination with the standard chromosome due to the accumulation of several inversions (three on the t-haplotype and one on the standard chromosome; Artzt et al. 1982;Herrmann et al. 1986;Burt and Trivers 2009).When present in females, it is transmitted to 50% of the progeny, but . 90% of the progeny of t-carrying males inherit it (Chesley an...

The t -haplotype of mice is a classical model for autosomal transmission distortion. A largely non-recombining variant of the proximal region of chromosome 17, it is transmitted to more than 90% of the progeny of heterozygous males through the disabling of sperm carrying a standard chromosome. While extensive genetic and functional work has shed light on individual genes involved in drive, much less is known about the evolution and function of the rest of its hundreds of genes. Here, we characterize the sequence and expression of dozens of t -specific transcripts and of their chromosome 17 homologues. Many genes showed reduced expression of the t -allele, but an equal number of genes showed increased expression of their t -copy, consistent with increased activity or a newly evolved function. Genes on the t -haplotype had a significantly higher non-synonymous substitution rate than their homologues on the standard chromosome, with several genes harbouring dN/dS ratios above 1. Finally, the t -haplotype has acquired at least two genes from other chromosomes, which show high and tissue-specific expression. These results provide a first overview of the gene content of this selfish element, and support a more dynamic evolutionary scenario than expected of a large genomic region with almost no recombination.

Using a unique combination of visual, statistical, and data mining methods, we tested the hypothesis that an immune cell's movement pattern can convey key information about the cell's function, antigen specificity, and environment. We applied clustering, statistical tests, and a support vector machine (SVM) to assess our ability to classify different datasets of imaged flouresently labelled T cells in mouse liver. We additionally saw clusters of different movement patterns of T cells of identical antigenic specificity. We found that the movement patterns of T cells specific and non-specific for malaria parasites are differentiable with 72% accuracy, and that specific cells have a higher tendency to move towards the parasite than non-specific cells. Movements of antigen-specific T cells in uninfected mice vs. infected mice were differentiable with 69.8% accuracy. We additionally saw clusters of different movement patterns of T cells of identical antigenic specificity. We concluded that our combination of methods has the potential to advance the understanding of cell movements in vivo.