After infection, many factors coordinate the population expansion and differentiation of CD8+ effector and memory T cells. Using data of unparalleled breadth from the Immunological Genome Project, we analyzed the CD8+ T cell transcriptome throughout infection to establish gene-expression signatures and identify putative transcriptional regulators. Notably, we found that the expression of key gene signatures can be used to predict the memory-precursor potential of CD8+ effector cells. Long-lived memory CD8+ cells ultimately expressed a small subset of genes shared by natural killer T and γδ T cells. Although distinct inflammatory milieu and T cell precursor frequencies influenced the differentiation of CD8+ effector and memory populations, core transcriptional signatures were regulated similarly, whether polyclonal or transgenic, and whether responding to bacterial or viral model pathogens. Our results provide insights into the transcriptional regulation that influence memory formation and CD8+ T cell immunity.

Protein phosphorylation events during T cell receptor (TCR) signaling control the formation of complexes among proteins proximal to the TCR, the activation of kinase cascades, and the activation of transcription factors; however, the mode and extent of the influence of phosphorylation in coordinating the diverse phenomena associated with T cell activation are unclear. Therefore, we used the human Jurkat T cell leukemia cell line as a model system and performed large-scale quantitative phosphoproteomic analyses of TCR signaling. We identified 10,665 unique phosphorylation sites, of which 696 showed TCR-responsive changes. In addition, we analyzed broad trends in phosphorylation data sets to uncover underlying mechanisms associated with T cell activation. We found that, upon stimulation of the TCR, phosphorylation events extensively targeted protein modules involved in all of the salient phenomena associated with T cell activation: patterning of surface proteins, endocytosis of the TCR, formation of the F-actin cup, inside-out activation of integrins, polarization of microtubules, production of cytokines, and alternative splicing of messenger RNA. Further, case-by-case analysis of TCR-responsive phosphorylation sites on proteins belonging to relevant functional modules together with network analysis allowed us to deduce that serine-threonine (S-T) phosphorylation modulated protein-protein interactions (PPIs) in a system-wide fashion. We also provide experimental support for this inference by showing that phosphorylation of tubulin on six distinct serine residues abrogated PPIs during the assembly of microtubules. We propose that modulation of PPIs by stimulus-dependent changes in S-T phosphorylation state is a widespread phenomenon applicable to many other signaling systems.

Elucidating the rules for receptor triggering in cell-cell and cell-matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This is comprised of monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer (SLB). The TCR ligand could be co-planar with the SLB (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engineering for immunotherapy.

T cells constitute a crucial arm of the adaptive immune system and their optimal function is required for a healthy immune response. After the initial step of T cell-receptor (TCR) triggering by antigenic peptide complexes on antigen presenting cell (APC), the T cell exhibits extensive cytoskeletal remodeling. This cytoskeletal remodeling leads to formation of an “immunological synapse” [1] characterized by regulated clustering, segregation and movement of receptors at the interface. Synapse formation regulates T cell activation and response to antigenic peptides and proceeds via feedback between actin cytoskeleton and TCR signaling. Actin polymerization participates in various events during the synapse formation, maturation, and eventually its disassembly. There is increasing knowledge about the actin effectors that couple TCR activation to actin rearrangements [2, 3], and how defects in these effectors translate into impairment of T cell activation. In this review we aim to summarize and integrate parts of what is currently known about this feedback process. In addition, in light of recent advancements in our understanding of TCR triggering and translocation at the synapse, we speculate on the organizational and functional diversity of microfilament architecture in the T cell.

Successful treatment of multiple cancer types requires early detection and identification of reliable biomarkers present in specific cancer tissues. To test the feasibility of identifying proteins from archival cancer tissues, we have developed a methodology, termed direct tissue proteomics (DTP), which can be used to identify proteins directly from formalin-fixed paraffin-embedded prostate cancer tissue samples. Using minute prostate biopsy sections, we demonstrate the identification of 428 prostate-expressed proteins using the shotgun method. Because the DTP method is not quantitative, we employed the absolute quantification method and demonstrate picogram level quantification of prostate-specific antigen. In depth bioinformatics analysis of these expressed proteins affords the categorization of metabolic pathways that may be important for distinct stages of prostate carcinogenesis. Furthermore, we validate Wnt-3 as an upregulated protein in cancerous prostate cells by immunohistochemistry. We propose that this general strategy provides a roadmap for successful identification of critical molecular targets of multiple cancer types.

Multisite phosphorylation is an important mechanism for achieving intricate regulation of protein function. Here we extended the absolute quantification of abundance (AQUA) methodology and validated its applicability to quantitatively study multisite phosphorylation. As a test case, we chose the conserved inhibitory site of the cyclindependent kinases (CDKs), Cdk1, Cdk2, and Cdk3, which are important regulators of cell cycle transitions and apoptosis. Inhibitory phosphorylation at Thr 14 and Tyr 15 of the CDKs is modulated by complex regulatory mechanisms involving multiple kinases and phosphatases. Yet the resulting quantitative dynamics among the four possible phosphorylated and non-phosphorylated versions of CDKs (T14p-Y15p, T14p-Y15, T14-Y15p, and T14-Y15) has not been investigated to date. Hence we used the heavy isotope-labeled tryptic peptides spanning the inhibitory site as internal standards and quantified all four versions by LC-selected reaction monitoring. Quantification of the phosphorylation status of the inhibitory site in the cell extracts provided novel quantitative insights. 1) The transition to mitotic phase was dominated by the conversion of "T14p-Y15p" to the "T14-Y15" form, whereas the two monophosphorylated forms were considerably lower in abundance.2) The amount of all four forms decreased during the progression of apoptosis but with differing kinetics. Analysis of immunoprecipitated Cdk1 and Cdk2 revealed that the inhibitory site phosphorylation state of both kinases at different stages of the cell cycle followed the same trend. Quantitative immunoblotting using antibodies to Cdk1 and Cdk2 and to the T14-Y15p form suggested that quantification by AQUA was reliable and accurate. These results highlight the utility of internal standard peptides to achieve accurate quantification of multisite phosphorylation status.

The CD2-CD58 recognition system promotes adhesion and signaling and counters exhaustion in human T cells. We found that CD2 localized to the outer edge of the mature immunological synapse (IS), with cellular or artificial APC, in a pattern we refer to as a "CD2 corolla". The corolla captured engaged CD28, ICOS, CD226 and SLAM-F1 costimulators. The corolla amplified active phosphorylated Src-family kinases (pSFK), LAT and PLC-γ over T cell receptor (TCR) alone. CD2-CD58 interactions in the corolla boosted signaling by 77% compared to central CD2-CD58 interactions. Engaged PD-1 invaded the CD2 corolla and buffered CD2 mediated amplification of TCR signaling. CD2 numbers and motifs in its cytoplasmic tail controlled corolla formation. CD8 + tumor infiltrating lymphocytes displayed low expression of CD2 in the majority of colorectal, endometrial and ovarian cancer patients. CD2 down-regulation may attenuate anti-tumor T cell responses with implications for checkpoint immunotherapies.

Global understanding of tissue-specific differences in mitochondrial signal transduction requires comprehensive mitochondrial protein identification from multiple cell and tissue types. Here, we explore the feasibility and efficiency of protein identification using the one-dimensional gel electrophoresis in combination with the nano liquidchromatography tandem mass spectrometry (GeLC-MS/ MS). The use of only 40 g of purified mitochondrial proteins and data analysis using stringent scoring criteria and the molecular mass validation of the gel slices enables the identification of 227 known mitochondrial proteins (membrane and soluble) and 453 additional proteins likely to be associated with mitochondria. Replicate analyses of 60 g of mitochondrial proteins on the faster scanning LTQ mass spectrometer validate all the previously identified proteins and most of the single hit proteins except the 81 single hit proteins. Among the identified proteins, 466 proteins are known to functionally participate in various processes such as respiration, tricarboxylic acid cycle (TCA cycle), amino acid and nucleotide metabolism, glycolysis, protection against oxidative stress, mitochondrial assembly, molecular transport, protein biosynthesis, cell cycle control, and many known cellular processes. The distribution of identified proteins in terms of size, pI, and hydrophobicity reveal that the present analytical strategy is largely unbiased and very efficient. Thus, we conclude that this approach is suitable for characterizing subcellular proteomes form multiple cells and tissues.