SUMMARY Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (Prf1) transcription, and repressed re-expression of memory CTL markers Bcl6 and IL-7Rα. Binding of Eomes and STAT5 to Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Rα and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Rα repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Rα-deficient effector CD8+ T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1+ and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.

Activation of naive CD8 + T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs

Stimulation of immune cells triggers Ca2؉ entry through store-operated Ca 2؉ release-activated Ca 2؉ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca 2؉ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca 2؉ release-activated Ca 2؉ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca 2؉ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca 2؉ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells. Ca2ϩ is a key second messenger in intracellular signaling pathways. In lymphocytes, specialized store-operated Ca 2ϩ channels known as CRAC 5 channels are required for sustained Ca 2ϩ influx across the plasma membrane (1). The resulting prolonged elevation of intracellular free Ca 2ϩ entry is essential for sustained nuclear translocation of the transcription factor NFAT, a small family of proteins whose activation is critical for a productive immune response (2). NFAT proteins reside in the cytoplasm of resting lymphocytes in a highly phosphorylated form and translocate to the nucleus upon dephosphorylation by the Ca 2ϩ /calmodulin-dependent serine/threonine phosphatase calcineurin (2, 3). In the nucleus, NFAT proteins bind to promoters and regulatory regions of a large number of cytokine genes and other activation-associated genes, thereby mediating the activation, proliferation, and differentiation of T cells, B cells, and other immune system cells.Although the notion of Ca 2ϩ influx through "store-operated" Ca 2ϩ channels was first proposed in 1986 (4, 5), the molecular identity of the proteins involved in this process remained unknown until the advent of large-scale RNAi-based screens. The first components of the pathway to be identified were Drosophila Stim (dStim) and its human homologues STIM1 and STIM2 through large-scale (albeit not genome-wide) RNAibased screens ...

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs)1–3, and LSC deficiency is a major cause of blindness worldwide4. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts5, a gene allowing for prospective LSC enrichment has not been identified so far5. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5)6,7 marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs2 in mice and p63α-positive LSCs8 in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

Age-related macular degeneration (AMD) is a major cause of blindness in the developed world. Oxidative stress and inflammation are implicated in AMD, but precise mechanisms remain poorly defined. Carboxyethylpyrrole (CEP) is an AMD-associated lipid peroxidation product. We previously demonstrated that mice immunized with CEP-modified albumin developed AMD-like degenerative changes in the outer retina. Here, we examined the kinetics of lesion development in immunized mice and the presence of macrophages within the interphotoreceptor matrix (IPM), between the retinal pigment epithelium and photoreceptor outer segments. We observed a significant and time-dependent increase in the number of macrophages in immunized mice relative to young age-matched controls prior to overt pathology. These changes were more pronounced in BALB/c mice than in C57BL/6 mice. Importantly, IPM-infiltrating macrophages were polarized toward the M1 phenotype but only in immunized mice. Moreover, when Ccr2-deficient mice were immunized, macrophages were not present in the IPM and no retinal lesions were observed, suggesting a deleterious role for these cells in our model. This work provides mechanistic evidence linking immune responses against oxidative damage with the presence of proinflammatory macrophages at sites of future AMD and experimentally demonstrates that manipulating immunity may be a target for modulating the development of AMD.

Age-related macular degeneration (AMD) is a major disease affecting central vision, but the pathogenic mechanisms are not fully understood. Using a mouse model, we examined the relationship of two factors implicated in AMD development: oxidative stress and the immune system. Carboxyethylpyrrole (CEP) is a lipid peroxidation product associated with AMD in humans and AMD-like pathology in mice. Previously, we demonstrated that CEP immunization leads to retinal infiltration of pro-inflammatory M1 macrophages before overt retinal degeneration. Here, we provide direct and indirect mechanisms for the effect of CEP on macrophages, and show for the first time that antigen-specific T cells play a leading role in AMD pathogenesis. In vitro, CEP directly induced M1 macrophage polarization and production of M1-related factors by retinal pigment epithelial (RPE) cells. In vivo, CEP eye injections in mice induced acute pro-inflammatory gene expression in the retina and human AMD eyes showed distinctively diffuse CEP immunolabeling within RPE cells. Importantly, interferon-gamma (IFN-γ) and interleukin-17 (IL-17)-producing CEP-specific T cells were identified ex vivo after CEP immunization and promoted M1 polarization in co-culture experiments. Finally, T cell immunosuppressive therapy inhibited CEP-mediated pathology. These data indicate that T cells and M1 macrophages activated by oxidative damage cooperate in AMD pathogenesis.

Perforin gene (PRF1) transcription regulates perforin expression in NK cells and CTL. Here we identified the locus-wide ensemble of cis-acting sequences that drives PRF1 transcription physiologically. By using chromosome transfer, we revealed that de novo activation of a silent PRF1 locus was controlled by a 150 kb domain comprised of 16 DNase I hypersensitive sites (DHSs). These cis-acting sequences included a locus control region (LCR) and conferred developmentally appropriate and lineage-specific expression of human perforin from BAC transgenes. The LCR included four distal DHSs that were required for perforin expression from its natural locus, and their engineered deletion from the PRF1 BAC transgene abolished LCR function and led to rapid gene silencing. Thus, LCR function is central for regulating the developmental and activation-specific PRF1 promoter activity characteristic of NK cells and CTL.

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