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A retinoic acid–dependent checkpoint in the development of CD4+ T cell–mediated immunity

Immune cell activation induces concurrent temporal and spatial retinoic acid signaling, and CD4+ T cell–specific loss of RA signals reduces effector function, migration, and polarity.


Brief Definitive Report
The importance of vitamin A in host resistance to infectious disease is irrefutable (Semba, 1999). Up to 10 million malnourished children are at increased risk of complications and death from measles and other infectious diseases as con sequences of vitamin A deficiencies (VADs). Al though recent studies have revealed how retinoic acid (RA) may control the development of protective immunity, findings reported herein show that RA plays a far more fundamental role in inflammation than previously anticipated.
RA signaling to T cells imprints their hom ing to the mesenteric LNs and gut through the upregulation of 47 and CCR9 (Iwata et al., 2004;Mora et al., 2008;Svensson et al., 2008;Wang et al., 2010) and contributes to B cell homing and isotype switching to IgA (Mora et al., 2008). Furthermore, RA at physiological concentrations has been shown to be critical for the development of Th17 (Uematsu et al., 2008;Cha et al., 2010;Wang et al., 2010). These findings provide a plausible explanation for the epidemiological findings of impaired immunity in vitamin A-deficient populations. At odds with its proinflammatory role in immunity, it has been shown that RA (at higher concentra tions) can effectively interfere with the gen eration of inflammatory Th17 cells as well as enhance regulatory CD4 + T cell (T reg cell) fre quencies and function (Mucida et al., 2007;Schambach et al., 2007). In conjunction with TGF, RA enhances the expression of the tran scription factor FoxP3 (Benson et al., 2007;Coombes et al., 2007;Sun et al., 2007), the master regulator for T reg cells, and facilitates the differen tiation of CD4 + effector T cells to stable, adaptive T reg cells (aT reg cells; Benson et al., 2007), likely by acting differentially in specific subsets of the CD4 + T cell compartment (memory vs. naive It is known that vitamin A and its metabolite, retinoic acid (RA), are essential for host defense. However, the mechanisms for how RA controls inflammation are incompletely understood. The findings presented in this study show that RA signaling occurs concurrent with the development of inflammation. In models of vaccination and allogeneic graft rejection, whole body imaging reveals that RA signaling is temporally and spatially restricted to the site of inflammation. Conditional ablation of RA signaling in T cells significantly interferes with CD4 + T cell effector function, migration, and polarity. These findings provide a new perspective of the role of RA as a mediator directly controlling CD4 + T cell differentiation and immunity.
Constitutive luciferase signal was routinely observed in the abdominal area of mice because of signaling emanating from the area of small and large intestines. Surprisingly, the intra dermal administration of CD40, CD3, CFA, and LPS elicited a robust, enhanced luciferase signal that was localized to the site of administration as early as 24 h after immuniza tion ( Fig. 1 A). In addition, after the i.p. administration of CFA, a locally and temporally restricted luciferase signal was first induced over controls in 24 h, but the signal intensi fied by the following days, with a gradual decrease over the ensuing 20 d (Fig. 1 B). A more indepth analysis of the tem poral and spatial induction of RA reporting and identity of the cells reporting was undertaken after the i.p. administration of CFA and CD3. In this case, the administration of CFA triggered RA signaling on CD4 + T cells ( Fig. 1 C), but to a lesser extent than T cell activation induced by CD3, where a heightened activity was observed when compared with IgG control (Fig. 1 D). The in vivo administration of a RA antago nist before T cell isolation ablated the RA reporting, estab lishing the possibility that the enhanced reporting was specific for RA (Fig. 1,C and D). Therefore, different immune activa tors stimulate RA signaling in a spatially and temporally restricted manner.
In addition, RA reporting by CD4 + T cells was studied in a model of allogeneic skin graft rejection. DR5Luciferase CD4 + T cells were adoptively transferred into RAG / mice before syngeneic or allogeneic skin graft transplantation. After transfer, WBI was used to assess RA reporting by the DR5 Luciferase T cells. As shown in Fig. 2 A, no signal was found at early time points (first 5 d after transplantation); however, be tween 7 and 21 d after transplant, enhanced RA signaling was detected in allogeneic grafts versus the syngeneic grafts. By week 3 after transplant, RA reporting declined to near base line levels in both groups. Signaling from the transplanted allograft was associated with the presence of the allograft because RA reporting disappeared when the graft was fully rejected. In Fig. 2 B, quantified photon flux is presented for both mice transplanted with syngeneic and allogeneic grafts. Therefore, alloreactive CD4 + T cells that enter an allograft are triggered by RA.
Our findings establish that RA signaling in T cells may be linked with the development of inflammation. The tempo rally restricted and regionally controlled signaling by RA sug gests that RA regulates immunity within the inflammatory site over a prescribed period of time. In this study, polyclonal T cell activation in vivo augments RA signaling (or sensing) by CD4 + T cells. Although gutresident DCs (Coombes et al., 2007;Sun et al., 2007) have been identified as the major source of immunederived RA, it is likely that RA produc tion by DCs, other APCs, and even nonhematopoietic cells may be responsible for the reporting that is observed in intact mice. These observations are supported by several studies, in which not only gutresident DCs are able to synthesize RA but also draining LN (dLN)-and skinresident DCs (Guilliams et al., 2010), as well as nonhematopoietic cells (Hammerschmidt et al., 2008;Molenaar et al., 2009). populations; Hill et al., 2008). Both of these latter activities provide compelling evidence that RA may exert antiinflam matory effects within the host. Under what circumstances RA plays a proinflammatory role or an antiinflammatory role remains to be determined.
The molecular basis for RA signaling to T cells and the cellular sources of RA within the immune system have begun to resolve. Of the three RA receptors (RARs; , , and ), RA has been shown to control the suppressive (T reg ) and in flammatory activities (Th17) of the CD4 + T cell compart ment by signaling through RAR (Mucida et al., 2007;Hill et al., 2008;Hall et al., 2011). Although it was originally be lieved that RA produced by hematopoietic cells may be lim ited to the gut, the production of RA by both hematopoietic and nonhematopoietic cells outside the gut has been repeat edly demonstrated (Hammerschmidt et al., 2008;Molenaar et al., 2009;Guilliams et al., 2010). The capacity of cells to produce RA is dependent on the expression of retinaldehyde dehydrogenase (RALDH) enzymes, the key family of en zymes which drive the irreversible conversion of retinal to RA (Duester, 2000). It has been shown that gutresident CD103 + DCs (Coombes et al., 2007;Sun et al., 2007), splenic DCs, and stromal cells (Hammerschmidt et al., 2008;Molenaar et al., 2009) produce RA. Within the gut, the op posing regulatory actions of RA on T reg cell (to mediate suppres sion) and Th17 cell differentiation (to suppress inflammation) have been implicated as critical actions in maintaining gut immune homeostasis (Mucida et al., 2007).
Whereas the role of RA in regulation of gut immunity has pictured RA as an important homeostatic regulator of in flammation, the findings presented in this study provide a fundamentally new perspective on the role of RA in the de velopment of cellmediated immunity. Using mice that re port the upregulation of luciferase as a consequence of RA signaling, this study shows that robust RA signaling occurs concurrent with the development of inflammation. In models of vaccination and allogeneic graft rejection, whole body im aging (WBI) revealed that RA signaling was temporally and spatially restricted with the site of inflammation. Conditional ablation of RA signaling in T cells arrested inflammation by altering T cell effector function, migration, and polarity. Our findings and others (Hall et al., 2011) establish that RA signaling to T cells is critical as an early mediator in the devel opment of CD4 + T cell-mediated immunity and help to ex plain the profound impairment of immunity in vitamin Adeficient populations.

RESULTS AND DISCUSSION Immune activation elicits RA reporting on CD4 + T cells
To determine whether RA production and signaling occurs during immune activation, mice were administered immune activators, and WBI was performed. The distribution and in tensity of RA signaling after immune activation were deter mined using a RARELuciferase reporter mouse (DR5 Luciferase), which transcriptionally upregulates luciferase expression upon signaling by RA (Svensson et al., 2008). WBI was performed 24 h after immunization using IVIS technology. Quantification of photon flux is shown in the plot (right). n = 1-3 mice per group, and data represent three pooled independent experiments. Bars indicate the mean. (B) DR5-Luciferase reporter mice were immunized with CFA by i.p. injection, and kinetics for RA reporting was tracked by WBI. Basal reporting is shown in the top left picture (Luc+) including a negative control littermate (Luc-). Quantification of photon flux is included in the plot (right). n = 3 mice. (C and D) CD4 + T cells were isolated from the spleens of DR5-Luciferase mice previously immunized by i.p. injection with CFA (C) or -CD3 (D). In some mice, the RA-specific antagonist (Antag; NRX194310; 50 µg per mouse) was injected i.p. at the time of stimulus. Luciferase activity was measured in vitro as described in Materials and methods. n = 2-3 mice per group. Pooled data of at least two independent experiments are shown. Error bars indicate SEM. ns, nonstatistical difference; *, P ≤ 0.01; **, P ≤ 0.05. These findings are representative of two independent experiments.
dnRARCD4 Cre -OTII-Ly5.2 cells by i.v. injec tion, and the next day, mice were transplanted with either syngeneic (B6) or allogeneic (actinOVA) grafts. After 7 d, skin grafts and dLNs were removed and analyzed for OTII-CD4 + T cell infiltration. Fig. 3 (A and B) shows that the alteration of RA signaling in CD4 + T cells impairs accumulation of T cells at the site of inflammation (skin graft). No differences in the expression of either activation markers (CD44, CD69, and CD62L) or chemo kine receptors that are upregulated upon inflam mation and are associated with migration to inflamed sites (CCR4, CCR5, CCR6, CXCR3, and CCR10) were observed (Fig. S4). Therefore, early TCRmediated events and expansion of graft specific T cells appeared intact in the absence of RA signaling, but tissue accumulation was impaired.
It appeared that clonal T cell proliferation was intact in the absence of RA signaling, and so addi tional experiments were designed to evaluate the impact of RA signaling on T cell differentiation. Because of the difficulty of recovering sufficient numbers of transferred CD4 + T cells when the host is on the WT C57BL/6 background, skingrafted RAG / mice were used as recipi ents to ensure a high recovery and purity of OTII-CD4 + T cells. dnRAR-OTII-Ly5.2 or dnRARCD4 Cre -OTII-Ly5.2 cells were transferred to RAG / recipient mice and grafted the next day. dLNresident CD4 + T cells were isolated after 7 d and cocultured with unpulsed or ISQ pulsed DCs. After 72 h, cytokine and chemokines were quan tified. As shown in Fig. 4 A, the production of Th1type cytokine IFN and IL17 was significantly reduced (60% for both, with P ≤ 0.022 [IFN] and P ≤ 0.028 [IL17]) when dLNresident dnRARCD4 Cre -OTII-Ly5.2 T cells were recalled with their cognate antigen, as compared with their counterpart dnRAR-OTII-Ly5.2 T cells. Chemo kine production by recalled dnRARCD4 Cre -OTII-Ly5.2 and dnRAR-OTII-Ly5.2 T cells was also evaluated. As depicted in Fig. 4 B, the interruption of RAR signaling in CD4 + T cells blocks the production of Th1linked chemo kines such as MIP1 (P ≤ 0.014) and MIP1 (P ≤ 0.008), including a downregulation in the production of RANTES ( Fig. S5; Cook, 1996). Moreover, we observed a tendency to a Th2skewed/suppressive phenotype characterized by the upregulation in the production of IL4 (P ≤ 0.037), IL9

Disruption of RA signaling in CD4 + T cell impairs T cell tissue accumulation and effector cell function
To analyze the functional impact of intrinsic RA signaling on CD4 + T cell population dynamics during graft rejection, RA signaling was selectively ablated in graftspecific T cells. A dominantnegative form of the RAR, RAR403 (hereafter denoted dnRAR; Rajaii et al., 2008), was overexpressed in T cells by interbreeding the dnRAR with CD4 Cre mice.
The potential impact of dnRAR- gene expression on T cell ontogeny in the dnRARCD4 Cre was addressed. Analysis of the T cell development indicated that overexpres sion of the dnRAR- gene impacted the frequencies of thy mic singlepositive CD8 + T cell populations, as previously described (Zhou et al., 2008), but had little effect on T cell populations in the periphery (Fig. S1). Overexpression of the dnRAR- did not affect CD3-induced T cell proliferation in vitro or in vivo ( Fig. S2) but, as anticipated, did inhibit RA induced TGF-dependent FoxP3 conversion ( Fig. S3; Benson et al., 2007;Coombes et al., 2007;Sun et al., 2007). To study the impact of RA signaling on graft rejection, the dnRAR- gene was also overexpressed in OVAspecific OTII TCR transgenic T cells, and mice were grafted with skin that ex presses OVA as a transplantation rejection antigen (Ehst et al., 2003). C57BL/6 mice received dnRAR-OTII-Ly5.2 or in the dnRARCD4 Cre mice was com pletely abolished. Therefore, experiments with an RAR panantagonist and over expression of the dnRAR- gene in T cells demonstrate that RA signaling is required for T cell-mediated graft rejection.
Our data demonstrate that RA sig naling to CD4 + T cells is essential for the inflammatory responses that mediate the rejection of allogeneic skin grafts. The ab sence of RA signaling in CD4 + T cells alters T cell polarity, resulting in a shift from a Th1/Th17 to a Th2 phenotype. The selective impact of RA on T cell dif ferentiation and not clonal expansion is consistent with the welldescribed role of RA as a differentiation factor in develop mental biology (Maden, 2007). With the present data considered, it is clear that RA exerts both proinflammatory and anti inflammatory effects on cellmediated immunity. Numerous studies have impli cated RA in facilitating immune suppres sion via the enhanced differentiation of aT reg cells (Benson et al., 2007;Coombes et al., 2007;Sun et al., 2007) and the suppression of Th17 cell differentiation (Mucida et al., 2007;Elias et al., 2008). In fact, it has been shown that RA agonists can suppress several murine auto immune diseases, including systemic lupus erythematosus and experimental autoimmune encephalomyelitis (Yamauchi et al., 2005). In the latter, reduced differentiation of Th17 was at tributed to the resolution in disease. Furthermore, a major emphasis for the role of RA in immunity has been placed on its role in gut homeostasis. Studies have shown that CD103 + DCs from the gut can potentiate the differentiation of aT reg cells through their capacity to express RALDH enzymes re quired for RA synthesis (Coombes et al., 2007;Sun et al., 2007). Although its role in the gut is no doubt critical, the findings reported herein provide a completely new perspec tive of the role of RA in immunity. In this regard, our prelimi nary results show that stimuli like T cell activation by CD3 antibody treatment increases the frequencies of DCs express ing RALDH. In the skin transplantation model, although we observed heightened RA signaling in CD4 + T cells during (P ≤ 0.026; Fig. 4 C), and IL10 (P ≤ 0.128), although for IL10 this difference was not statistically significant (Fig. S5). The impairment of Th1linked chemokines by graftspecific T cells may contribute to the transcriptional program re quired for Th1/Th17 cell differentiation and the develop ment of CD4 + T cell-mediated inflammation.
To evaluate the functional significance of RA signaling to graft rejection, T cell-mediated graft rejection was studied in mice in which RA signaling was ablated chemically and ge netically. Mice receiving syngeneic (B6) or allogeneic (actin OVA) skins were treated with a RAR panantagonist (NRX 194310). As shown in Fig. 5 A, the blockade of RAR signal ing resulted in complete acceptance of the skin allograft (open red circles). Experiments then evaluated the involvement of RA signaling in CD4 + T cells during graft rejection. WT, dnRAR, and dnRARCD4 Cre were grafted with syngeneic or allogeneic skin, and the kinetics of graft rejection was eval uated over time. As depicted in Fig. 5 B, dnRAR mice re ject the allograft following the same kinetics previously described for C57BL/6 (Ehst et al., 2003). However, rejection control T cell plasticity in vivo, and dnRARCD4 Cre may have impairments in lineage commitment. Studies are under way to evaluate these hypotheses.
In addition, our data show that the disruption of RA sig naling in the CD4 + T cell compartment impairs T cell infil tration of the skin graft. Although the expression of the major skin/inflammationrelated chemokine receptors was not compromised, the added effect of reduced Th1 cytokines and chemokines may cooperate for the poor recruitment of CD4 + T effector cells to the allograft. We observed that dnRAR CD4 Cre CD4 + T cells produce less Th1linked chemokines, which can explain in part the lack of CD4 + T cell accumula tion within the graft if chemokine migration by infiltrating T cells amplifies the recruitment of additional T cells to the inflammatory site.
A recent publication (Hall et al., 2011) has highlighted the role of RA signaling of T cells in the mucosal immune response to Toxoplasma gondii. These investigators have shown that VAD impairs IFN recall responses in immune mice. Furthermore, they show that T cells derived from an RAR / mouse display abnormalities in cell growth and Ca 2+ mobilization and produce less IFN in response to vaccina tion with OVA protein and Escherichia coli toxin. In contrast to these findings, we show that T cells that are unresponsive to RA via overexpression of a dnRAR- proliferate in response to antigen in vivo and in vitro. An apparent explanation to the differences is not clear at this time. It is possible that T cells the rejection process, we did not see a significant difference in the numbers of RALDH + DCs between the syngeneic and allogeneic groups (either dLNs or skin grafts; unpublished data). Thus, signaling of RA to CD4 + T cell may not be cor related with differential production from other cell types (APCs and stromal cells). It is known that RA signaling can be regulated by the expression of RA transport proteins ex pressed by the target cell. Alternatively, RA concentrations in vivo can be regulated by its catabolism by the catabolic en zyme, Cyp26. In this regard, future experiments involving Cyp26, the protein which degrades RA in the cytosol, will be pursued to understand its role in immunity. In contrast, the molecular mechanisms underlying the ability of RA to con trol the lineage commitment of T cells in vivo are unresolved. Our data show that ablation of RA signaling in vivo reduces the expression of Th1 and Th17associated cytokines and chemokines. This effect is not seen in vitro when dnRAR CD4 Cre T cells are stimulated with CD3 (unpublished data). The role of RA in eliciting Th1 and Th17 inflammatory T cells may be through its capacity to influence the expres sion of master transcription factors that regulate the fate of Th cells, such as Tbet or RORt. Alternatively, RA may  . RA signaling to CD4 + T cells is essential for allograft rejection. (A) C57BL/6 mice were transplanted with syngeneic (B6) or allogeneic (actin-OVA) skin grafts. RAR pan-antagonist (Antag; NRX 194310) was given three times per week at 50 µg/mouse, and skin graft survival was monitored. (B) C57BL/6, dnRAR-CD4 Cre , and dnRAR- were grafted with either B6 or actin-OVA skin grafts. Skin graft survival was monitored over time. The number of mice per group is indicated in each graph. Pooled data from two representative independent experiments are shown.

Br ief Definitive Repor t
Conversion assay in vitro. CD4 + T cells were isolated from the spleens of dnRAR or dnRARCD4 Cre mice and stimulated with 10 µg/ml plate bound CD3 (clone 2c11) and 1 µg/ml CD28 (clone PV1) plus the addition of 100 U/ml IL2, 10 ng/ml TGF, and different concentrations of RA (SigmaAldrich; as indicated). After 4 d in culture, the expression of FoxP3 was evaluated by intracellular staining (eBioscience) and analyzed by flow cytometry.
Quantification of luciferase activity in vitro. CD11c + and CD4 + T cells were isolated from the spleens of immunized DR5Luciferase reporter mice. 0.5 × 10 6 cells were resuspended in 100 µl DPBS, and the procedure was performed according to the manufacturer's instructions (SteadyLite kit; Roche).
Skin grafting. Skin grafting was performed as described previously (Quezada et al., 2005). For tracking RA signaling on CD4 + T cells, splenic CD4 + T cells were isolated from the DR5Luciferase reporter mouse, and 3 × 10 6 cells were transferred into RAG / recipients by i.v. injection. The next day, syngeneic or allogeneic skin grafts were transplanted. For the antag onist treatment regimen, when indicated, recipient mice were treated three times/week with control vehicle (DMSO) or 50 µg/mouse of the RAR panantagonist (NRX 194310;NuRx Pharmaceuticals).

Skin-infiltrating cell isolation.
Skin transplants were removed from animals at the indicated day in the figure legends. The transplants were incubated in HBSS media with 4 mg/ml DNase, 4 mg/ml Liberase, and 10 mg/ml Collagenase D for 3 h at 37°C (all from Roche). After this, the tissue was disintegrated using a mortar and pestle and filtered through a 100µm nylon strainer. Cell suspension was stained for flow cyto metric analysis.
Cytokine and chemokine secretion. Supernatants were collected 72 h after culture and analyzed using ELISA (mouse IL17A kit; BioLegend) or Luminex (BioRad Laboratories), as indicated. The Luminex assay was per formed by the Immune Monitoring Core (Dartmouth College).
Statistical analysis. Differences between the means of experimental groups were analyzed using the MannWhitney test. Data analysis was performed using Prism software (version 4.0; GraphPad Software). A pvalue ≤0.05 was considered significant.
Online supplemental material. Fig. S1 shows the T cell frequencies and phenotype from different organs in WT, dnRAR, and dnRARCD4 Cre mice. Fig. S2 shows that CD4 + T cells from the dnRARCD4 Cre mice do not convert to FoxP3 cells in vitro. Fig. S3 shows that the ablation of RA sig naling on CD4 + T cells does not affect the expression of chemokine recep tors or activation markers. Fig. S4 shows that the lack of RAR signaling promotes the generation of Th2/suppressive CD4 + T cells. Fig. S5 shows that derived from RAR / are developmentally impaired. Alternatively, it is possible that the signaling function of the RAR molecule may still be intact in the dnRAR-expressing T cells. Future studies with conditional deletion of each of the RAR will help resolve these issues and shed in sights into the role of other RAR in immunity. Nonetheless, collectively, the study by Hall et al. (2011) and our study on the role of RA in immunity provide new insights into the function of this mediator in inflammation.
With regard to cell differentiation and stability, data from experiments examining the role of RA in aT reg cell genera tion have implicated RA as an important differentiation fac tor for T cells, which may impact their plasticity. Data show that RAtreated natural T reg cells are lineage committed and virtually irreversible in their phenotype, even in the face of extremely proinflammatory conditions (Zhou et al., 2010). As such, RA controls the plasticity of aT reg cells (Benson et al., 2007;Nolting et al., 2009) and perhaps Th1 and Th17 cells. The enhanced aT reg cell differentiation by RA has been re cently resolved to show that RA enhances the association of the RAR/RXR receptor to the FoxP3 enhancer, leading to enhanced histone acetylation and enhanced binding of phos phorylated Smad3 (Xu et al., 2010). For nearly a century, it has been clear that VAD compromises immunity, and these new findings on the role of RA in controlling T cell-mediated differentiation provide a rational, scientific basis for the increased susceptibility to infectious disease in vitamin Adeficient individuals.

MATERIALS AND METHODS
Animals. C57BL/6, RAG / , CD4 Cre , and actinOVA (Ehst et al., 2003) mice were purchased from the Jackson Laboratory. The DR5Luciferase re porter (Svensson et al., 2008) and dnRAR (Rajaii et al., 2008) have been described previously. Animal experiments were approved by the Institutional Animal Care Use Committee of Dartmouth Medical School. All animals were maintained in a pathogenfree facility at Dartmouth Medical School.
Imaging. Mice received 3 mg dLuciferin (Gold Biotechnology) in 200 µl DPBS (Cellgro) by i.p. injection and were imaged in the IVIS 2000 (Xeno gen Corp.) according to the manufacturer's instructions. Analysis and images were obtained using the Living Image Software (version 2.6.1; Caliper Life Sciences).
T cell phenotyping. Thymus, peripheral LNs, spleen, and mesenteric LNs were removed from WT, dnRAR, or dnRARCD4 Cre mice. Cell sus pensions were prepared, and staining for CD4 and CD8 T cells was per formed and analyzed by flow cytometry.
Proliferation assay in vitro. CD4 + T cells were isolated from the spleens of dnRAR or dnRARCD4 Cre mice and labeled with 5 µM CFSE (In vitrogen). In parallel, T cell-depleted splenocytes (APC) were prepared, and 0.15 × 10 5 APCs were cocultured with 0.5 × 10 5 CD4 + T cells in the pres ence of soluble CD3 (clone 2C11). CFSE dilution was evaluated by flow cytometry at day 3.