MiRNome and transcriptome aided pathway analysis in human regulatory T cells.

Owing to their manifold immune regulatory functions, regulatory T cells (Treg) have received tremendous interest as targets for therapeutic intervention of diverse immunological pathologies or cancer. Directed manipulation of Treg will only be achievable with extensive knowledge about the intrinsic programs that define their regulatory function. We simultaneously analyzed miR and mRNA transcript levels in resting and activated human Treg cells in comparison with non-regulatory conventional T cells (Tcon). Based on experimentally validated miR-target information, both transcript levels were integrated into a comprehensive pathway analysis. This strategy revealed characteristic signal transduction pathways involved in Treg biology such as T-cell receptor-, Toll-like receptor-, transforming growth factor-ß-, JAK/STAT (Janus kinase/signal transducers and activators of transcription)- and mammalian target of rapamycin signaling, and allowed for the prediction of specific pathway activities on the basis of miR and mRNA transcript levels in a probabilistic manner. These data encourage new concepts for targeted control of Treg cell effector functions.

Tracking cell proliferation using the far red fluorescent dye SNARF-1.

BACKGROUND: The [(3)H]thymidine incorporation assay and staining of living cells with fluorescent dyes like carboxyfluorescein diacetate, succinimidyl ester (CFSE) have evolved as valuable methods for studying T cell responses. To assess proliferation of cells already labeled by FITC, CFSE, GFP, or other "green" molecules or to simultaneously track two otherwise indistinguishable cell populations in mixed cell cultures, it would be desirable to have a dye with distinct fluorescent properties for this application. METHODS: We analyzed the dilution of the far red fluorescent dye SNARF-1 in proliferating cells by flow cytometric analysis. The results were compared with the CFSE dilution technique as well as the [(3)H]thymidine incorporation assay. RESULTS: Staining of primary human lymphocytes revealed that SNARF-1 labeling was equivalent to CFSE for estimating proportions of proliferating cells in stimulated cell cultures and yielded results comparable to [(3)H]thymidine incorporation. We showed that SNARF-1 offers the possibility to simultaneously analyze the proliferation of phenotypically indistinguishable subsets of hematopoietic cells and can also be used to track uniformly proliferating, non hematopoietic cells like HEK293. CONCLUSIONS: In summary, we have demonstrated that labeling of cells with SNARF-1 allows for estimating cell proliferation of cells of hematopoietic and non-hematopoietic origin.

A human immunodeficiency syndrome caused by mutations in CARMIL2.

Human T-cell function is dependent on T-cell antigen receptor (TCR) and co-signalling as evidenced by immunodeficiencies affecting TCR-dependent signalling pathways. Here, we show four human patients with EBV+ disseminated smooth muscle tumours that carry two homozygous loss-of-function mutations in the CARMIL2 (RLTPR) gene encoding the capping protein regulator and myosin 1 linker 2. These patients lack regulatory T cells without evidence of organ-specific autoimmunity, and have defective CD28 co-signalling associated with impaired T-cell activation, differentiation and function, as well as perturbed cytoskeletal organization associated with T-cell polarity and migration disorders. Human CARMIL2-deficiency is therefore an autosomal recessive primary immunodeficiency disorder associated with defective CD28-mediated TCR co-signalling and impaired cytoskeletal dynamics.

Stable nonviral gene transfer into primary human T cells.

Effective techniques for the stable genetic modification of peripheral T cells would facilitate functional gene studies and the development of gene therapeutic approaches. However, many approaches to genetically modify T cells are hampered by low transfection efficiency, direct cell toxicity, and the need for specialized laboratory space. In this study we investigated the Amaxa Nucleofector platform, a nonviral technique to transfect primary human T cells. A plasmid equipped with two different promoters enabled concomitant expression of a gene of interest and of a cell surface marker allowing for immunomagnetic cell enrichment. This resulted in highly purified populations of gene-modified T cells and, after repeated enrichment steps, provided stably and homogeneously transfected, fully functional human T cells. In summary, this study provides proof of principle that human T cells can be altered to homogeneously and stably express a gene of interest by a nonviral technique. This should enable further studies on T cell physiology and ultimately facilitate the translation of treatment approaches either for diseases that are caused by defective gene function in T cells or for diseases that require genetically designed T cell therapy.