Micah J. Benson

All-trans retinoic acid mediates enhanced T reg cell growth, differentiation, and gut homing in the face of high levels of co-stimulation

We demonstrate that all-trans retinoic acid (RA) induces FoxP3+ adaptive T regulatory cells (A-Tregs) to acquire a gut-homing phenotype (α4β7+ CC chemokine receptor 9+) and the capacity to home to the lamina propria of the small intestine. Under conditions that favor the differentiation of A-Tregs (transforming growth factor–β1 and interleukin 2) in vitro, the inclusion of RA induces nearly all activated CD4+ T cells to express FoxP3 and greatly increases the accumulation of these cells. In the absence of RA, A-Treg differentiation is abruptly impaired by proficient antigen presenting cells or through direct co-stimulation. In the presence of RA, A-Treg generation occurs even in the presence of high levels of co-stimulation, with RA attenuating co-stimulation from interfering from FoxP3 induction. The recognition that RA induces gut imprinting, together with our finding that it enhances A-Treg conversion, differentiation, and expansion, indicates that RA production in vivo may drive both the imprinting and A-Treg development in the face of overt inflammation.

Molecular mechanism and function of CD40/CD40L engagement in the immune system

Summary:  During the generation of a successful adaptive immune response, multiple molecular signals are required. A primary signal is the binding of cognate antigen to an antigen receptor expressed by T and B lymphocytes. Multiple secondary signals involve the engagement of costimulatory molecules expressed by T and B lymphocytes with their respective ligands. Because of its essential role in immunity, one of the best characterized of the costimulatory molecules is the receptor CD40. This receptor, a member of the tumor necrosis factor receptor family, is expressed by B cells, professional antigen‐presenting cells, as well as non‐immune cells and tumors. CD40 binds its ligand CD40L, which is transiently expressed on T cells and other non‐immune cells under inflammatory conditions. A wide spectrum of molecular and cellular processes is regulated by CD40 engagement including the initiation and progression of cellular and humoral adaptive immunity. In this review, we describe the downstream signaling pathways initiated by CD40 and overview how CD40 engagement or antagonism modulates humoral and cellular immunity. Lastly, we discuss the role of CD40 as a target in harnessing anti‐tumor immunity. This review underscores the essential role CD40 plays in adaptive immunity.

Cutting Edge: The Dependence of Plasma Cells and Independence of Memory B Cells on BAFF and APRIL

Memory B (BMEM) cells and long-lived bone marrow plasma cells (BM-PCs) persist within local environmental survival niches that afford cellular longevity. However, the factors supporting BMEM cell survival within the secondary lymphoid organs and allowing BM-PC persistence in the bone marrow remain poorly characterized. We report herein that long-lived BMEM cell survival and function are completely independent of BAFF (B cell-activating factor of the TNF family) or APRIL (a proliferation-inducing ligand). Thus, BMEM cells represent the only mature B2 lineage subset whose survival is independent of these ligands. We have previously shown that the TNFR family member receptor BCMA (B cell maturation Ag) is a critical survival receptor for BM-PC survival in vivo. We identify in this study the ligands critical for BM-PC survival and show that either BAFF or APRIL supports the survival of BM-PCs in vivo. These data define the BAFF/APRIL-dependent and -independent components of long-lived humoral immunity.

Retinoic Acid Can Directly Promote TGF-β-Mediated Foxp3+ Treg Cell Conversion of Naive T Cells

The article by Hill et al. (2008), published in the November 14, 2008 issue of Immunity, describes a mechanism by which retinoic acid (RA) enhances TGF-β-induced Foxp3 expression. The authors propose that RA does not act directly on naive T cells during activation in culture but rather indirectly via negative regulation of an accompanying population of effector or memory CD4+ CD44hi cells. They reasoned that the increased generation of Foxp3+ cells in response to RA in culture, as described previously (Coombes et al., 2007; Elias et al., 2008; Mucida et al., 2007; Sun et al., 2007; Xiao et al., 2008), represented the lifting by RA of inhibition imparted by accompanying CD4+CD44hi T cells, rather than by direct or indirect effects of RA on the Foxp3 expression of the primed naive T cells themselves. In order to assess the effects of RA on naive T cells in the absence of accompanying CD4+CD44hi T cells, we sorted (CD4+CD25−CD44low CD62L+) GFP− T cells (more than 99.7% purity) from Foxp3-eGFP reporter mice (Figure S1A available online) by flow cytometry. After 4 days of stimulation with anti-CD3 and anti-CD28, we stained CD4 cells with 7AAD to exclude dead cells; additionally, forward and side scatter (area versus width) was used to exclude doublets, and we evaluated Foxp3 expression via GFP staining. Addition of RA enhanced Foxp3 induction more than 50% by use of 1 or 10 ng/ml doses of TGF-β (Figure S1B). Because the sorting efficiency is not 100%, it is possible that extremely low numbers of “accompanying” memory or effector cells could still influence these results. To exclude this possibility, we used FACS-sorted CD4+CD25−CD44lo CD62L+ T cells, isolated from B7-1 and B7-2 double-deficient mice (Cd80−/− Cd86−/−), which even before sorting already contain less than 5% of memory or effector CD44hi cells (data not shown). RA also greatly enhanced Foxp3 induced by TGF-β in CD4+CD25−CD44lo CD62L+ naive T cells isolated from Cd80−/− Cd86−/− mice (Figure S1C). Moreover, we showed previously that RA is able to counterbalance the inhibitory effects of costimulation on TGF-β-mediated Foxp3 induction, with either CD4+CD25− or CD4+Foxp3− T cells (Benson et al., 2007). To confirm these results, we used OTII TCR transgenic CD4+CD25−CD44lo CD62L+ cells sorted by flow cytometry and tested the effects of RA by using increasing doses of anti-CD28 stimulation. We found that RA markedly enhanced TGF-β-mediated Foxp3 induction on pure naive CD4+ T cells that were stimulated with anti-CD3 and various doses of anti-CD28 (Figure S1D). The enhanced Foxp3 expression mediated by RA is more pronounced on naive monoclonal OTII TCR transgenic T cells as compared to polyclonal T cells, consistent with a lesser frequency of “contaminating” memory T cells. Finally, because we showed previously that RA-mediated enhanced expression of Foxp3 is greatly reduced in the absence of IL-2 (Mucida et al., 2007), we investigated the effects of RA on naive T cells with various doses of exogenous IL-2. Although IL-2-deficient mice develop inflammatory disorders, Il2−/−Cd80−/−Cd86−/− mice are healthy and, more importantly, they do not contain T regulatory cells. At steady state, ~99% of all CD4+ T cells isolated from Il2−/−Cd80−/−Cd86−/− mice are naive (data not shown). The CD4+ T cells were further sorted by flow cytometry so that highly purified naive CD4+CD25−CD44lo CD62L+ cells (more than 99.9% purity) were obtained. The sorted naive CD4+ Il2−/−Cd80−/−Cd86−/− T cells were tested for TGF-β-induced Foxp3 expression in the presence of increasing doses of IL-2 and anti-CD3 and anti-CD28 coated beads, with or without RA. The data showed that 1 nM RA distinctly enhanced TGF-β (1 ng/ml)-mediated Foxp3 induction in pure naive CD4+ T cells at all doses of IL-2 examined (Figures S1E and S1F). Strikingly, although the expression of Foxp3 was much reduced, RA enhanced TGF-β-mediated Foxp3 induction not only in the absence of memory or effector T cells but also in the absence of IL-2. These data demonstrate that RA mediates enhanced TGF-β-induced Foxp3 expression upon activation of pure naive T cells in the absence of accompanying CD4+CD44hi T cells. In addition, we confirmed, as Hill et al. (2008) proposed, that RA also efficiently counteracts inhibitory effects of CD44hi T cells on Foxp3 induction (data not shown), which indicates that RA is able to enhance Foxp3 expression both, via effects directly on the primed naive T cells as well as indirectly via inhibitory effects on accompanying CD4+CD44hi T cells. There is no doubt that the new findings by Hill et al. (2008) add an important new pathway by which RA can enhance Foxp3 induction, which had been suggested previously (Elias et al., 2008; Mucida et al., 2007; Xiao et al., 2008).Nevertheless, published data, together with the data presented here, disagree with the central statement proposed by Hill et al. (2008) that the enhanced expression of TGF-β driven Foxp3 mediated by RA is an indirect effect that requires suppression of accompanying CD4+CD44hi T cells rather than via direct or indirect effects on the primed T cells themselves. Under physiological conditions, naive T cells may be exposed to cytokines and effector or memory cells, and hence it is likely that during priming of naive T cells, both mechanisms of RA-mediated enhanced TGF-β driven Foxp3 expression in the primed T cells will synergize in vivo. Therefore, elucidating and understanding both processes by which RA affects naive and already differentiated T cells is important and may lead to the identification of possible targets for therapeutic interventions to treat various inflammatory and autoimmune diseases.

Mast cell degranulation breaks peripheral tolerance

Mast cells (MC) have been shown to mediate regulatory T‐cell (Treg)‐dependent, peripheral allograft tolerance in both skin and cardiac transplants. Furthermore, Treg have been implicated in mitigating IgE‐mediated MC degranulation, establishing a dynamic, reciprocal relationship between MC and Treg in controlling inflammation. In an allograft tolerance model, it is now shown that intragraft or systemic MC degranulation results in the transient loss of Treg suppressor activities with the acute, T‐cell dependent rejection of established, tolerant allografts. Upon degranulation, MC mediators can be found in the skin, Treg rapidly leave the graft, MC accumulate in the regional lymph node and the Treg are impaired in the expression of suppressor molecules. Such a dramatic reversal of Treg function and tissue distribution by MC degranulation underscores how allergy may causes the transient breakdown of peripheral tolerance and episodes of acute T‐cell inflammation.

Retinoic Acid in the Immune System

On occasion, emerging scientific fields intersect and great discoveries result. In the last decade, the discovery of regulatory T cells (Treg) in immunity has revolutionized our understanding of how the immune system is controlled. Intersecting the rapidly emerging field of Treg function, has been the discovery that retinoic acid (RA) controls both the homing and differentiation of Treg. Instantly, the wealth and breadth of knowledge of the molecular basis for RA action, its receptors, and how it controls cellular differentiation can and will be exploited to understand its profound effects on Treg. Historically, vitamin A deprivation and repletion and RA agonists have been shown to profoundly affect immunity. Now these findings can be interpreted in light of the revelations that RA controls leukocyte homing and Treg function.

Distinction of the memory B cell response to cognate antigen versus bystander inflammatory signals

The hypothesis that bystander inflammatory signals promote memory B cell (BMEM) self-renewal and differentiation in an antigen-independent manner is critically evaluated herein. To comprehensively address this hypothesis, a detailed analysis is presented examining the response profiles of B-2 lineage B220+IgG+ BMEM toward cognate protein antigen in comparison to bystander inflammatory signals. After in vivo antigen encounter, quiescent BMEM clonally expand. Surprisingly, proliferating BMEM do not acquire germinal center (GC) B cell markers before generating daughter BMEM and differentiating into plasma cells or form structurally identifiable GCs. In striking contrast to cognate antigen, inflammatory stimuli, including Toll-like receptor agonists or bystander T cell activation, fail to induce even low levels of BMEM proliferation or differentiation in vivo. Under the extreme conditions of adjuvanted protein vaccination or acute viral infection, no detectable bystander proliferation or differentiation of BMEM occurred. The absence of a BMEM response to nonspecific inflammatory signals clearly shows that BMEM proliferation and differentiation is a process tightly controlled by the availability of cognate antigen.

CCR6-Dependent Positioning of Memory B Cells Is Essential for Their Ability To Mount a Recall Response to Antigen

Chemokine-dependent localization of specific B cell subsets within the immune microarchitecture is essential to ensure successful cognate interactions. Although cognate interactions between T cells and memory B cells (Bmem) are essential for the secondary humoral immune responses, the chemokine response patterns of Bmem cells are largely unknown. In contrast to naive B cells, this study shows that Ag-specific Bmem cells have heightened expression of CCR6 and a selective chemotactic response to the CCR6 ligand, CCL20. Although CCR6 appears be nonessential for the initial clonal expansion and maintenance of Bmem, CCR6 is essential for the ability of Bmem to respond to a recall response to their cognate Ag. This dependency was deemed intrinsic by studies in CCR6-deficient mice and in bone marrow chimeric mice where CCR6 deficiency was limited to the B cell lineage. Finally, the mis-positioning of CCR6-deficient Bmem was revealed by immunohistological analysis with an altered distribution of CCR6-deficient Bmem from the marginal and perifollicular to the follicular/germinal center area.

B cell survival: an unexpected mechanism of lymphocyte vitality

Sustaining the vast peripheral B cell repertoire is critical for the immunological health of the host. B-cell activating factor of the TNF family (BAFF) is essential for the survival of mature B cells. A recent publication in Immunity by Robert Brink and coworkers has afforded important new insights into how interactions between BAFF and one of its receptors—BAFF receptor (BAFF-R)—function in regulating the development of the mature B cell repertoire.1