The Immunological Genome Project combines immunology and computational biology laboratories in an effort to establish a complete 'road map' of gene-expression and regulatory networks in all immune cells.

N atural killer (NK) cells express a variety of inhibitory killer Ig-like receptors (KIR) that inhibit cytotoxicity upon recognition of class I MHC proteins (1). In this manner NK cells detect diseased cells through their loss of expression of self-MHC protein rather than by directly detecting foreign antigen, a conjecture known as the missing self-hypothesis (2). Inhibitory NK receptors containing two Ig domains, denoted KIR1 (or KIR2DL1) and KIR2 (or KIR2DL2), recognize the class I MHC proteins, HLA-Cw4 or -Cw6 and HLA-Cw3 or -Cw7, respectively (3, 4). Crystal structures of both class I MHC and KIR extracellular domains have been determined (5-9), and the appropriate binding sites have been mapped by site-directed mutagenesis (10-15). Although the binding kinetics between soluble KIR͞MHC proteins, determined by surface plasmon resonance, are extremely fast (16-18), video microscopy of NK cell immunosurveillance shows that intercellular contacts last for minutes (not shown). Thus, we set out to delineate the molecular mechanisms of NK cell recognition that occur over this time frame.An enhanced variant of green fluorescent protein (EGFP) (19), originally discovered and cloned from Aequorea victoria jellyfish (20, 21), was used to mark the location of HLA-C. Plasmids encoding EGFP attached to the intracellular C terminus of class I MHC protein were transfected into 721.221, a B cell line derived by mutagenesis that does not express class I MHC protein (22,23). These transfectants then were incubated with various NK cell lines for 20 min at 37°C, after which time many NK cell͞target cell conjugates were formed. Conjugates of living NK and target cells were imaged by laser-scanning confocal fluorescence microscopy. This methodology advances previous imaging of mouse T cell͞target cell intercellular contacts that used paraformaldehyde-fixed cells (24) or live T cells interacting with MHC protein-rich lipid bilayers (25). Here, immune synapses are shown to exist at the contact between two living human cells. Materials and MethodsCell Lines and Transfectants. Plasmids encoding EGFP attached to the C terminus of HLA-Cw3 or -Cw4 were prepared by PCR of the appropriate HLA-C allele to remove the stop codon and add an EcoRI restriction site at the 5Ј end and a NotI site at the 3Ј end. EGFP was prepared by PCR from the plasmid pEGFP (CLONTECH) to contain a NotI site at the 5Ј end and a BamHI site at the 3Ј end. These PCR products then were joined and amplified together by PCR by using primers at the 5Ј end of HLA-C and the 3Ј end of EGFP. The product was cloned first into pBABE and then into pcDNA3 (Invitrogen). Plasmids encoding other GFP-linked proteins were prepared by PCR of the appropriate HLA-C allele to remove the stop codon and cloned as KpnI͞NotI fragments into the vector initially encoding HLA-Cw3-GFP. All primers were purchased from Life Technologies (Gaithersburg, MD) and all plasmid inserts were sequenced by the Core Facilities, Dana-Farber Cancer Institute (Boston, MA). 721.221 cells were transfected with 10...

SUMMARY Perturbations in the transcriptional programs specifying epidermal differentiation cause diverse skin pathologies ranging from impaired barrier function to inflammatory skin disease. However, the global scope and organization of this complex cellular program remain undefined. Here we report single-cell RNA sequencing profiles of 92,889 human epidermal cells from 9 normal and 3 inflamed skin samples. Transcriptomics-derived keratinocyte subpopulations reflect classic epidermal strata but also sharply compartmentalize epithelial functions such as cell-cell communication, inflammation, and WNT pathway modulation. In keratinocytes, ~12% of assessed transcript expression varies in coordinate patterns, revealing undescribed gene expression programs governing epidermal homeostasis. We also identify molecular fingerprints of inflammatory skin states, including S100 activation in the interfollicular epidermis of normal scalp, enrichment of a CD1C+CD301A+ myeloid dendritic cell population in psoriatic epidermis, and IL1βhi CCL3hiCD14+ monocyte-derived macrophages enriched in foreskin. This compendium of RNA profiles provides a critical step toward elucidating epidermal diseases of development, differentiation, and inflammation.

The transcription factor FoxP3 partakes dominantly in the specification and function of FoxP3+CD4+ T regulatory cells (Tregs), but is neither strictly necessary nor sufficient to determine the characteristic Treg signature. Computational network inference and experimental testing assessed the contribution of other transcription factors (TF). Enforced expression of Helios or Xbp1 elicited specific signatures, but Eos, Irf4, Satb1, Lef1 and Gata1 elicited exactly the same outcome, synergizing with FoxP3 to activate most of the Treg signature, including key TFs, and enhancing FoxP3 occupancy at its genomic targets. Conversely, the Treg signature was robust to inactivation of any single cofactor. A redundant genetic switch thus locks-in the Treg phenotype, a model which accounts for several aspects of Treg physiology, differentiation and stability.