γδ T cells are an important innate source of interleukin-17 (IL-17). In contrast to T helper 17 (Th17) cell differentiation, which occurs in the periphery, IL-17-producing γδ T cells (γδT17 cells) are probably committed during thymic development. To study when γδT17 cells arise during ontogeny, we used TcrdH2BeGFP reporter mice to monitor T cell receptor (TCR) rearrangement and IL-17 production in the embryonic thymus. We observed that several populations such as innate lymphoid cells and early T cell precursors were able to produce IL-17 prior to (and thus independent of) TCR recombination. γδT17 cells were absent after transplantation of IL-17-sufficient bone marrow into mice lacking both Il17a and Il17f. Also, γδT17 cells were not generated after genetic restoration of defective Rag1 function in adult mice. Together, these data suggested that these cells developed exclusively before birth and subsequently persisted in adult mice as self-renewing, long-lived cells.

Stable expression of Foxp3 in regulatory T cells (Tregs) depends on DNA demethylation at the Treg-specific demethylated region (TSDR), a conserved, CpG-rich region within the Foxp3 locus. The TSDR is selectively demethylated in ex vivo Tregs purified from secondary lymphoid organs, but it is unclear at which stage of Treg development demethylation takes place. In this study, we show that commitment to a stable lineage occurred during early stages of murine thymic Treg development by engraving of lineage-specific epigenetic marks in parallel with establishment of a Treg-specific gene expression profile. TSDR demethylation was achieved through an active mechanism and involved enzymes of the ten-eleven-translocation family and hydroxylation of methylated cytosines, a modification that is implicated as an initiating step of mitosis-independent DNA demethylation pathways and has not yet been observed at specific loci during immune cell differentiation. Together, our results demonstrate that initiating TSDR demethylation during early stages of thymic Treg development commences stabilization of Foxp3 expression and guarantees full functionality and long-term lineage stability of Tregs.

To study B-cell development from bone marrow (BM), we generated recombination-activating gene 1 (

Siglec-G is a negative regulator of BCR-mediated signaling in B1a cells. This population of B cells is highly increased in Siglec-G–deficient mice, but the mechanism of this expansion is not known so far. In this study, we demonstrate that Siglecg−/− B1a cells show a lower level of spontaneous apoptosis and a prolonged life span. Mechanistically, the lower apoptosis could result from higher expression levels of the transcription factor NFATc1 in Siglec-G–deficient B1a cells. Interestingly, Siglecg−/− B1a cells display an altered BCR repertoire compared with wild-type B1a cells. As the BCR repertoire and the VDJ composition of Igs of Siglecg−/− B1a cells resembles more the Abs produced by adult bone marrow-derived B cells rather than canonical fetal liver-derived B1a cells, this suggest that the selection into the B1a cell population is altered in Siglec-G–deficient mice.

AimsIn the present study we have investigated the comparative switching propensity of murine peritoneal and splenic B cell subpopulations to IgA in presence of retinoic acid (RA) and TGF-β.Methods and ResultsTo study the influence of RA and TGF-β on switching of B cell subpopulations to IgA, peritoneal (B1a, B1b and B2 cells) and splenic (B1a, marginal zone, and B2) B cells from normal BALB/c mice were FACS purified, cultured for 4 days in presence of RA and TGF-β and the number of IgA producing cells was determined by ELISPOT assay or FACS analysis. In presence of TGF-β, peritoneal B1b cells switched to IgA more potently than other peritoneal B cell subpopulations. When TGF-β was combined with retinoic acid (RA), switching to IgA was even more pronounced. Under these conditions, “innate” B cells like peritoneal and splenic B1 cells and MZ B cells produced IgA more readily than B2 cells. Additionally, high frequency of nucleotide exchanges indicating somatic hypermutation in VH regions was observed. Besides IgA induction, RA treatment of sorted PEC and splenic B cells led to expression of gut homing molecules - α4β7 and CCR9. Intraperitoneal transfer of RA-treated B1 cells into Rag1-/- recipients resulted in IgA in serum and gut lavage, most efficiently amongst B1b cell recipients.ConclusionPresent study demonstrates the differential and synergistic effect of RA and TGF-β on switching of different B cell subpopulations to IgA and establishes the prominence of peritoneal B1b cells in switching to IgA under the influence of these two factors. Our study extends our knowledge about the existing differences among B cell subpopulations with regards to IgA production and indicates towards their differential contribution to gut associated humoral immunity.

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The role of Foxp3-expressing regulatory T (Treg) cells in tolerance and autoimmunity is well-established. However, although of considerable clinical interest, the role of Treg cells in the regulation of hematopoietic homeostasis remains poorly understood. Thus, we analysed B and T lymphopoiesis in the scurfy (Sf) mouse model of Treg cell deficiency. In these experiments, the near-complete block of B lymphopoiesis in the BM of adolescent Sf mice was attributed to autoimmune T cells. We could exclude a constitutive lympho-hematopoietic defect or a B cell-intrinsic function of Foxp3. Efficient B cell development in the BM early in ontogeny and pronounced extramedullary B lymphopoietic activity resulted in a peripheral pool of mature B cells in adolescent Sf mice. However, marginal zone B and B-1a cells were absent throughout ontogeny. Developmental B lymphopoietic defects largely correlated with defective thymopoiesis. Importantly, neonatal adoptive Treg cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12. Our observations on autoimmune-mediated incapacitation of the BM environment in Foxp3-deficient mice will have direct implications for the rational design of BM transplantation protocols for patients with severe genetic deficiencies in functional Foxp3+ Treg cells.

B‐1a cells are found mainly in the peritoneal cavity of mice but are also present in the spleen. Gene expression profiling defined many genes differentially expressed in B‐1a cells from these two sites. To see whether this gene expression pattern was imprinted by the particular microenvironment, peritoneal or spleen cells from recombinant L2 mice mainly consisting of B‐1a cells were adoptively transferred into Rag1–/– mice. Re‐isolated peritoneal and splenic B‐1a cells were analyzed for expression of three indicator genes – vcam‐1, adamdec1 and spi‐c. The expression of these genes was up‐regulated in splenic and down‐regulated in peritoneal cells. This particular pattern was observed for peritoneal or splenic donor cells transferred either intraperitoneally or intravenously. Similar results were obtained when levels of surface IgM or frequencies of Mac‐1+ B‐1 cells were compared after transfer. This suggests that the environment induces the particular genetic program of B‐1a cells and argues against an independent ontogeny.