The Role of Slp-76 Phosphotyrosines in TCR Signal Transduction and T Cell Differentiation

The cytosolic adapter protein src homology 2(SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) lacks enzymatic activity but nucleates a multi-molecular signaling complex that links early T cell receptor (TCR)-induced phosphorylation events into multiple downstream signaling pathways. The N-terminus of SLP-76 contains three tyrosines at residues 112,128 and 145 that are phosphorylated following TCR ligation and, although the mechanisms are not entirely clear, they are required for optimal TCR signal transduction. TCR signals are required for T cell proliferation, cytokine production, and effector and memory differentiation. The experiments described in this dissertation have first tested the biochemical mechanisms by which the SLP-76 tyrosines transmit signals and second tested how alterations in the TCR signals transmitted through SLP-76 tyrosines influence T cell differentiation and effector function. Experiments were performed using two genomic knock-in (KI) mice that express tyrosine to phenylalanine mutations at residue 145 (Y145F) or 112 and 128 together (Y112/128F). Using biochemistry-, flow cytometry- and microscopy-based approaches we show that mutations in the tyrosines of SLP-76 result in graded defects in TCR-induced signals and function depending on the tyrosine(s) affected. Surprisingly, localization of SH2 domain containing effector proteins to mutant SLP-76-nucleated signaling complexes was not lost and therefore could not account for the observed signaling defects. Infection of SLP-76 KI mice with lymphocytic choriomeningitis virus (LCMV) resulted in normal CD8 expansion but graded enhancement of memory differentiation consistent with a model in which weaker TCR signals preferentially promote memory versus effector differentiation. Furthermore CD8+ effector and memory KI T cells failed to produce appropriate cytokine upon antigen restimulation. Similarly, in vitro polarized KI Th17 and Th2 cells failed to produce IL17a and IL4, respectively, following TCR restimulation. Taken together our data show that SLP-76 tyrosines are essential for optimal TCR signal transduction and, moreover, TCR signals sufficient to promote T cell differentiation are different than those required to elicit inflammatory cytokine production

The requirements for natural Th17 cell development are distinct from those of conventional Th17 cells

A distinct population of Th17 cells develops in the thymus with innate immune cell characteristics, different selection requirements, and skewed TCR gene usage compared with peripheral Th17 cells

Implications of the Plastid Genome Sequence of Typha (Typhaceae, Poales) for Understanding Genome Evolution in Poaceae

Plastid genomes of the grasses (Poaceae) are unusual in their organization and rates of sequence evolution. There has been a recent surge in the availability of grass plastid genome sequences, but a comprehensive comparative analysis of genome evolution has not been performed that includes any related families in the Poales. We report on the plastid genome of Typha latifolia, the first non-grass Poales sequenced to date, and we present comparisons of genome organization and sequence evolution within Poales. Our results confirm that grass plastid genomes exhibit acceleration in both genomic rearrangements and nucleotide substitutions. Poaceae have multiple structural rearrangements, including three inversions, three genes losses (accD, ycf1, ycf2), intron losses in two genes (clpP, rpoC1), and expansion of the inverted repeat (IR) into both large and small single-copy regions. These rearrangements are restricted to the Poaceae, and IR expansion into the small single-copy region correlates with the phylogeny of the family. Comparisons of 73 protein-coding genes for 47 angiosperms including nine Poaceae genera confirm that the branch leading to Poaceae has significantly accelerated rates of change relative to other monocots and angiosperms. Furthermore, rates of sequence evolution within grasses are lower, indicating a deceleration during diversification of the family. Overall there is a strong correlation between accelerated rates of genomic rearrangements and nucleotide substitutions in Poaceae, a phenomenon that has been noted recently throughout angiosperms. The cause of the correlation is unknown, but faulty DNA repair has been suggested in other systems including bacterial and animal mitochondrial genomes

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

T-cell receptor signals direct the composition and function of the memory CD8+ T-cell pool

SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) nucleates a signaling complex critical for T-cell receptor (TCR) signal propagation. Mutations in the tyrosines of SLP-76 result in graded defects in TCR-induced signals depending on the tyrosine(s) affected. Here we use 2 strains of genomic knock-in mice expressing tyrosine to phenylalanine mutations to examine the role of TCR signals in the differentiation of effector and memory CD8+ T cells in response to infection in vivo. Our data support a model in which altered TCR signals can determine the rate of memory versus effector cell differentiation independent of initial T-cell expansion. Furthermore, we show that TCR signals sufficient to promote CD8+ T-cell differentiation are different from those required to elicit inflammatory cytokine production