Physiological levels of 25-hydroxyvitamin D3 induce a suppressive CD4+ T cell phenotype not reflected in the epigenetic landscape.

1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), the active metabolite of vitamin D3 has a strong impact on the differentiation and function of immune cells. Here we analysed the influence of its precursor 25-hydroxyvitamin D3 (25(OH)D3 ) on the differentiation of human CD4+ T cells applying physiological concentrations in vitro. Our data show that 25(OH)D3 is converted to its active form 1,25(OH)2 D3 by T cells, which in turn supports FOXP3, CD25 and CTLA-4 expression and inhibits IFN-γ production. These changes were not reflected in the demethylation of the respective promoters. Furthermore, we investigated the impact of vitamin D3 metabolites under induced Treg (iTreg) polarization conditions using TGF-ß. Surprisingly, no additive effect but a decreased percentage of FOXP3 expressing cells was observed. However, the combination of 25(OH)D3 or 1,25(OH)2 D3 together with TGF-ß further upregulated CD25 and CTLA-4 and significantly increased soluble CTLA-4 and IL-10 secretion whereas IFN-γ expression of iTreg was decreased. Our data suggest that physiological levels of 25(OH)D3 act as potent modulator of human CD4+ T cells and autocrine or paracrine production of 1,25(OH)2 D3 by T cells might be crucial for the local regulation of an adaptive immune response. However, since no epigenetic changes are detected by 25(OH)D3 a rather transient phenotype is induced.

Transcription and enhancer profiling in human monocyte subsets.

Human blood monocytes comprise at least 3 subpopulations that differ in phenotype and function. Here, we present the first in-depth regulome analysis of human classical (CD14(++)CD16(-)), intermediate (CD14(+)CD16(+)), and nonclassical (CD14(dim)CD16(+)) monocytes. Cap analysis of gene expression adapted to Helicos single-molecule sequencing was used to map transcription start sites throughout the genome in all 3 subsets. In addition, global maps of H3K4me1 and H3K27ac deposition were generated for classical and nonclassical monocytes defining enhanceosomes of the 2 major subsets. We identified differential regulatory elements (including promoters and putative enhancers) that were associated with subset-specific motif signatures corresponding to different transcription factor activities and exemplarily validated novel downstream enhancer elements at the CD14 locus. In addition to known subset-specific features, pathway analysis revealed marked differences in metabolic gene signatures. Whereas classical monocytes expressed higher levels of genes involved in carbohydrate metabolism, priming them for anaerobic energy production, nonclassical monocytes expressed higher levels of oxidative pathway components and showed a higher mitochondrial routine activity. Our findings describe promoter/enhancer landscapes and provide novel insights into the specific biology of human monocyte subsets.

Polyclonal expansion of human CD4(+)CD25(+) regulatory T cells.

Based on results from experimental animal models, the adoptive transfer of CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) is expected to be efficacious in treating autoimmune and inflammatory diseases, as well as in preventing alloresponses after solid organ or stem-cell transplantation. For potential clinical applications, large numbers of Treg cells in maximum purity will be required to avoid the risk of disease exacerbation by contaminating effector T cells. We have recently described methods for the efficient in vitro expansion of human Treg cells and identified CD4(+)CD25(high)CD45RA(+) T cells as the ideal starting population for the generation of homogeneous and stable Treg cell products. Here, we provide detailed instructions for their identification, isolation, expansion, and functional characterization.

Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation.

The adoptive transfer of CD4(+)CD25(+) natural regulatory T cells (Treg) is a promising strategy for the treatment of autoimmune diseases and the prevention of alloresponses after transplantation. Clinical trials exploring this strategy require efficient in vitro expansion of this rare cell population. Protocols developed thus far rely on high-grade purification of Treg prior to culture initiation, a process still hampered by the lack of Treg cell-specific surface markers. Depletion of CD127(+) cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3(+) cells with all functional and molecular characteristics of natural Treg. Here, we demonstrate that upon in vitro expansion, CpG methylation in a conserved region within the FOXP3 gene locus increased in CD4(+)CD25(+)CD127(low) Treg, correlating with loss of FOXP3 expression and emergence of pro-inflammatory cytokines. Further analysis identified CD45RA(-)FOXP3(+) memory-type Treg as the main source of converting cells, whereas CD45RA(+)FOXP3(+) Treg from the same donors showed no conversion within 3 wk of in vitro expansion. Thus, Treg cell lineage differentiation does not seem to represent a final fate decision, as natural Treg can lose their cell-type-specific characteristics after repetitive TCR stimulation.

Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion.

Thymus-derived CD4+ CD25+ regulatory T cells suppress autoreactive CD4+ and CD8+ T cells and thereby protect from autoimmunity. In animal models, adoptive transfer of CD4+ CD25+ regulatory T cells has been shown to prevent and even cure autoimmune diseases as well as pathogenic alloresponses after solid organ and stem-cell transplantations. We recently described methods for the efficient in vitro expansion of human regulatory T cells for clinical applications. We now demonstrate that only CCR7- and L-selectin (CD62L)-coexpressing cells within expanded CD4+ CD25high T cells maintain phenotypic and functional characteristics of regulatory T cells. Further analysis revealed that these cells originate from CD45RA+ naive cells within the CD4+ CD25high T-cell compartment, as only this subpopulation homogeneously expressed CD62L, CCR7, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and forkhead box P3 (FOXP3), produced no inflammatory cytokines and maintained robust suppressive activity after expansion. In contrast, cell lines derived from CD45RA- memory-type CD4+ CD25high T cells lost expression of lymph node homing receptors CCR7 and CD62L, contained interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) as well as IL-10-secreting cells, showed only moderate suppression and, most importantly, did not maintain FOXP3 expression. Based on these unexpected findings, we suggest that isolation and expansion of CD45RA+ naive CD4+ CD25high T cells is the best strategy for adoptive regulatory T (Treg)-cell therapies.

Isolation of CD4+CD25+ regulatory T cells for clinical trials.

The adoptive transfer of donor CD4+CD25+ regulatory T cells has been shown to protect from lethal graft-versus-host disease after allogeneic bone marrow transplantation in murine disease models. Efficient isolation strategies that comply with good manufacturing practice (GMP) guidelines are prerequisites for the clinical application of human CD4+CD25+ regulatory T cells. Here we describe the isolation of CD4+CD25+ T cells with regulatory function from standard leukapheresis products by using a 2-step magnetic cell-separation protocol performed under GMP conditions. The generated cell products contained on average 49.5% CD4+CD25high T cells that phenotypically and functionally represented natural CD4+CD25+ regulatory T cells and showed a suppressive activity comparable to that of CD4+CD25+ regulatory T-cell preparations purified by non-GMP-approved fluorescence-activated cell sorting.

Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells.

CD4(+)CD25+ regulatory T (Treg) cells are pivotal for the maintenance of self-tolerance, and their adoptive transfer gives protection from autoimmune diseases and pathogenic alloresponses after solid organ or bone marrow transplantation in murine model systems. In vitro, human CD4(+)CD25+ Treg cells display phenotypic and functional characteristics similar to those of murine CD4(+)CD25+ Treg cells: namely, hyporesponsiveness to T-cell receptor (TCR) stimulation and suppression of CD25- T cells. Thus far, the detailed characterization and potential clinical application of human CD4(+)CD25+ Treg cells have been hampered by their paucity in peripheral blood and the lack of appropriate expansion protocols. Here we describe the up to 40 000-fold expansion of highly purified human CD4(+)CD25high T cells in vitro through the use of artificial antigen-presenting cells for repeated stimulation via CD3 and CD28 in the presence of high-dose interleukin 2 (IL-2). Expanded CD4(+)CD25high T cells were polyclonal, maintained their phenotype, exceeded the suppressive activity of freshly isolated CD4(+)CD25high T cells, and maintained expression of the lymph node homing receptors L-selectin (CD62L) and CCR7. The ability to rapidly expand human CD4(+)CD25high Treg cells on a large scale will not only facilitate their further exploration but also accelerate their potential clinical application in T cell-mediated diseases and transplantation medicine.