Phenotype and function of intestinal CD103+ dendritic cells

Mucosal tissues such as the intestine present an enormous surface area to the outside environment and are continually exposed to foreign antigens in food and microflora. Thus, the intestinal mucosa must must remain tolerant to these innocuous antigens while at same time be able to mount effective immune responses to pathogens. Intestinal dendritic cells (DCs) and macrophages, found throughout the villous lamina propria and intestinal lymphoid tissue in both mice and humans are thought to play key roles in this process. DCs at these sites constantly sample and process both luminal and self-antigens and are key players in presenting these antigens to T cells. Recently, intestinal CD103+ DCs have been shown to promote induction of both gut homing receptors on responding T cells and differentiation of FoxP3+ regulatory T cells via the vitamin A metabolite retinoic acid (RA). Another intestinal DC/macrophage subset, expressing the chemokine receptor CX3CR1+ has been suggested to take up intestinal luminal antigens via transepithelial dendrites. The focus of this thesis has been to phenotypically and functionally characterize intestinal CD103+ DCs and to investigate the mechanisms that underlie their enhanced ability to generate gut homing T cells. In this thesis we demonstrate that CD103+ DCs present in the murine small intestinal mucosa and draining mesenteric lymph nodes (MLN) have an enhanced ability to deliver RA signals and induce gut homing receptors on responding T cells in vitro compared to CD103- DCs and CD103+ DCs outside the intestine. Importantly we show that that CD103+ DCs also are present in human MLN and have a similar ability to induce RA dependent gut tropism on T cells. We demonstrate that CD103+ DCs and CX3CR1+ cells are non- overlapping populations of intestinal cells that differ both in phenotype and in function. Our results suggest that CD103+ DCs are the major migratory DC population in the intestinal mucosa and play a central role in initiating immune responses to luminal antigen in draining MLNs. In contrast we find that CX3CR1+ cells resemble tissue macrophages, are non-migratory and inefficient at activating naïve T cells. We further show that both populations have different growth factor requirements and turnover rates in the intestinal mucosa. We have also examined potential mechanisms underlying the selective ability of intestinal CD103+ DCs to metabolize vitamin A. Results from these studies suggest that the Vitamin A metabolite RA itself induces retinol-metabolizing activity in CD103+ DCs in vivo, and that bile is an important source of these retinoids. Collectively, these studies have contributed to our knowledge of the mechanisms regulating intestinal immune responses and will hopefully lead to novel strategies for treatments for intestinal inflammatory diseases and mucosal vaccine design

The diverse ontogeny and function of murine small intestinal dendritic cell/macrophage subsets.

Intestinal dendritic cell and macrophage subsets are believed to play key roles in maintaining intestinal homeostasis in the steady state and in driving protective immune responses in the setting of intestinal infection. This mini-review focuses on recent progress regarding the ontogeny and function of small intestinal lamina propria dendritic cell/macrophage subsets. In particular we discuss recent findings suggesting that small intestinal CD103(+) dendritic cells and Cx3cr1(+) cells derive from distinct precursor populations and that CD103(+) dendritic cells represent the major migratory population of cells with a key role in initiating adaptive immune responses in the draining mesenteric lymph node. In contrast, Cx3cr1(+) cells appear to represent a tissue resident population, phenotypically indistinguishable from tissue resident macrophages. These latter observations suggest an important division of labour between dendritic cell/macrophage subsets in the regulation of intestinal immune responses in the steady state

Bile retinoids imprint intestinal CD103(+) dendritic cells with the ability to generate gut-tropic T cells.

Small intestinal lamina propria (SI-LP) CD103(+) dendritic cells (DCs) are imprinted with an ability to metabolize vitamin A (retinol), a property underlying their enhanced capacity to induce the gut-homing receptors CC chemokine receptor-9 and α4β7 on responding T cells. In this study, we demonstrate that imprinting of CD103(+) DCs is itself critically dependent on vitamin A and occurs locally within the small intestine (SI). The major vitamin A metabolite retinoic acid (RA) induced retinol-metabolizing activity in DCs both in vitro and in vivo, suggesting a direct role for RA in this process. Consistent with this, SI-LP CD103(+) DCs constitutively received RA signals in vivo at significantly higher levels than did colonic CD103(+) DCs. Remarkably, SI CD103(+) DCs remained imprinted in mice depleted of dietary but not of systemic retinol. We found that bile contained high levels of retinol, induced RA receptor-dependent retinol-metabolizing activity in bone marrow-derived DCs, and imprinted these cells with the ability to generate gut-tropic T cells. Taken together, these results suggest a novel and unexpected role for bile in SI-LP CD103(+) DC imprinting.Mucosal Immunology advance online publication 2 February 2011. doi:10.1038/mi.2010.91

Retinoic acid receptor signaling levels and antigen dose regulate gut homing receptor expression on CD8(+) T cells

Recent studies have highlighted a central role for intestinal dendritic cells (DCs) and vitamin A metabolite retinoic acid (RA) in the generation of alpha 4 beta 7(+) CCR9(+) "gut tropic" effector T cells. Here, using RA-responsive element reporter mice, we demonstrate that both splenic and mesenteric lymph node (MLN) DCs enhanced retinoic acid receptor (RAR) signaling in CD8(+) T cells; however, only a subset of MLN DCs, expressing the integrin alpha-chain CD103, induced an early RAR signal that is required for efficient CCR9 induction. MLN-primed CD8(+) T cells also received enhanced RAR-dependent signals compared with splenic-primed CD8(+) T cells in vivo. Further DC-mediated induction of gut homing receptors was inhibited at a high antigen dose without influencing RAR signaling events, and resulted in less efficient CD8(+) T-cell entry into the small intestinal mucosa. These results highlight a complex interplay between antigen dose and DC subset-induced RAR signaling events in the generation of tissue tropic effector T-cell subsets

Cellular Barcoding Links B-1a B Cell Potential to a Fetal Hematopoietic Stem Cell State at the Single-Cell Level

Hematopoietic stem cells (HSCs) undergo a functional switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Here, we investigated whether the developmental attenuation of B-1a cell output is a consequence of a shift in stem cell state during ontogeny. Using cellular barcoding for in vivo single-cell fate analyses, we found that fetal liver definitive HSCs gave rise to both B-1a and B-2 cells. Whereas B-1a potential diminished in all HSCs with time, B-2 output was maintained. B-1a and B-2 plasticity could be reinitiated in a subset of adult HSCs by ectopic expression of the RNA binding protein LIN28B, a key regulator of fetal hematopoiesis, and this coincided with the clonal reversal to fetal-like elevated self-renewal and repopulation potential. These results anchor the attenuation of B-1a cell output to fetal HSC behavior and demonstrate that the developmental decline in regenerative potential represents a reversible HSC state

PTIP chromatin regulator controls development and activation of B cell subsets to license humoral immunity in mice

B cell receptor signaling and downstream NF-κB activity are crucial for the maturation and functionality of all major B cell subsets, yet the molecular players in these signaling events are not fully understood. Here we use several genetically modified mouse models to demonstrate that expression of the multifunctional BRCT (BRCA1 C-terminal) domain-containing PTIP (Pax transactivation domain-interacting protein) chromatin regulator is controlled by B cell activation and potentiates steady-state and postimmune antibody production in vivo. By examining the effects of PTIP deficiency in mice at various ages during ontogeny, we demonstrate that PTIP promotes bone marrow B cell development as well as the neonatal establishment and subsequent long-term maintenance of self-reactive B-1 B cells. Furthermore, we find that PTIP is required for B cell receptor- and T:B interaction-induced proliferation, differentiation of follicular B cells during germinal center formation, and normal signaling through the classical NF-κB pathway. Together with the previously identified role for PTIP in promoting sterile transcription at the Igh locus, the present results establish PTIP as a licensing factor for humoral immunity that acts at several junctures of B lineage maturation and effector cell differentiation by controlling B cell activation

Lin28b controls a neonatal to adult switch in B cell positive selection

The ability of B-1 cells to become positively selected into the mature B cell pool, despite being weakly self-reactive, has puzzled the field since its initial discovery. Here, we explore changes in B cell positive selection as a function of developmental time by exploiting a link between CD5 surface levels and the natural occurrence of self-reactive B cell receptors (BCRs) in BCR wild-type mice. We show that the heterochronic RNA binding protein Lin28b potentiates a neonatal mode of B cell selection characterized by enhanced overall positive selection in general and the developmental progression of CD5+ immature B cells in particular. Lin28b achieves this by amplifying the CD19/PI3K/c-Myc positive feedback loop, and ectopic Lin28b expression restores both positive selection and mature B cell numbers in CD19-/- adult mice. Thus, the temporally restricted expression of Lin28b relaxes the rules for B cell selection during ontogeny by modulating tonic signaling. We propose that this neonatal mode of B cell selection represents a cell-intrinsic cue to accelerate the de novo establishment of the adaptive immune system and incorporate a layer of natural antibody-mediated immunity throughout life