• Allergy;
  • Nuclear receptor;
  • Retinoid X receptor α;
  • Vitamin A;
  • Th1/Th2


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

A viable hypomorphic allele of mouse retinoid X receptor α (Rxrα) was created by random germline mutagenesis. The mutation (I273N) alters the ligand binding and heterodimerization domain, and causes a 90% decline in ligand-inducible transactivation. Homozygotes develop progressive alopecia and dermal cysts, and progressive exaggeration of Th1 and loss of Th2 responses to antigen. Th1 skewing is directly caused by aberrant function of both antigen-presenting cells and naïve CD4 T cells; the predominant Th1 response to antigen is attributable to decreased suppression of regulatory T cells in mutant mouse. Dietary depletion of vitamin A in Th2-prone wild-type mice mimics the immune phenotype caused by the mutation. Hence, RXRα plays an important post-developmental role in the regulation of adaptive immune responses, and provides a plausible link between nutritional environment and the type of adaptive response that results from immunization.

See accompanying commentary


retinoic acid responsive element


retinoid X receptor α,β,γ


all-trans retinoic acid


CD4+CD25+ regulatory T cells


vitamin D receptor


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Nuclear receptors are a group of ligand-activated transcription factors that regulate the expression of target genes, affecting development, differentiation and metabolism 1, 2. Classical nuclear receptors mediates responses to lipophilic hormones and vitamins, including the steroids, retinoids, thyroid hormones and vitamin D 35. The retinoid X receptor (RXR) subfamily includes RXRα, -β, and -γ. RXRα serves as obligate heterodimerization partners for other nuclear receptors 6, and is required for a great number of hormone responses 5, 710. Targeted deletion of the Rxrα gene results in embryonic lethality between days 13.5 and 16.5 in mice 9, 11. Hence, the post-developmental and immunologic functions of RXRα have not been well studied.

T helper (Th) cell differentiation is the central process that defines the developing adaptive immune response 12. In part, Th1/Th2 balance is likely determined at the level of antigen-presenting cells (APC), which may elicit Th1 differentiation by producing IL-12 and IL-27 13, or Th2 differentiation by producing IL-10 14, 15. Th1 cells, in turn, produce IFN-γ and lymphotoxin, which maintain the Th1 phenotype, are important for protection against intracellular pathogens, and have also been associated with autoimmune pathologies. The Th2 subset produces IL-4, IL-5 and IL-13 and is responsible for eradicating helminth infections. However, the Th2 response also has allergic manifestations 16, 17. Because the incidence of allergic and atopic diseases such as asthma has undergone a dramatic increase in advanced societies 18, 19, it is evident that environmental factors influence Th1/Th2 balance. The “hygiene hypothesis” has been offered to explain this phenomenon, and holds that the key environmental difference is the low incidence of infections in advanced societies as compared to the developing world 20.

Much attention has focused on the molecular mechanisms that underlie Th1 and Th2 programming. The transcription factor T-bet has been identified as a primary determinant of Th1 differentiation 21, whereas primary determinants of Th2 differentiation include GATA3 and c-Maf 2225. However, the regulatory influences to which these transcription factors are subject have not been completely deciphered.

A number of reports have suggested that hormones might be involved in the regulation of Th1/Th2 differentiation. Vitamin A and vitamin D have been shown to directly enhance the development of Th2 cells in vitro2628. These data show that hormones and their RXRα-dependent nuclear receptors modulate the differentiation of T helper cells, probably by affecting the expression of pertinent transcription factors, cytokines and cytokine receptors. However, the embryonic lethal phenotype of Rxra9, 11 has prevented direct assessment of the influence of vitamins A and D on Th differentiation in vivo. A skin-specific deletion of the locus, also produced by gene targeting 29, 30, presumably has little or no effect on cells of the immune system.

In this report, we describe a viable hypomorphic allele of Rxrα (RxrαPke) induced by random germline mutagenesis using N-ethyl-N-nitrosourea (ENU). Initially identified as a visible recessive phenotype, the mutation causes a striking and progressive immunologic defect, in that Th2 differentiation is eroded and ultimately eliminated. The essential requirement for RXRα in Th2 differentiation is manifested both in T cells and in APC.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Identification of the pinkie phenotype and its cause

The pinkie phenotype is strictly recessive and entails premature graying, first apparent in hairs surrounding the snout as early as 5 weeks of age, with spreading to include truncal hair. Alopecia progresses from caudal ventral surface to involve the lower back, and then the entire body. Four-month-old mutant animals (C57BL/6 background) are typically hairless (Fig. 1A).

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Figure 1. Visible abnormality of pinkie mice. (A) Homozygous animals appear normal at 4 weeks of age (top left) but later develop premature graying (top middle) and progressive alopecia (top right). (B) Dermal cysts formed under the ventral skin with “black dots” on the top. The number and size of dermal cysts increases with age, and the severity of skin abnormality is generally worse for females than males. (C) A fraction of older mutant animals also develop kyphosis.

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Affected animals also develop cysts under the ventral skin (from 8 weeks for females), which increase in size and number with age, and resemble comedones. In general, males are less severely affected than females (Fig. 1B). By 1 year of age, some mutants develop a dorsal kyphosis (Fig. 1C). Late ocular findings include the appearance of “dry” eyes and corneal opacity (not shown). In several respects, pinkie homozygotes resemble mice with avitaminosis A and/or D 3133.

The pinkie locus was mapped to chromosome 2 based on genome-wide analysis of 27 meioses (Fig. 2A); progressive confinement of the mutation on 700 meioses permitted exclusion of 99.9% of the genome, yielding a 1.6 Mb critical region. Residing in the critical region, the Rxrα gene was considered a promising candidate since a conditional keratinocyte-specific knockout mutation of Rxrα produced a skin phenotype similar to that of Pinkie homozygotes 29, 30. A single nucleotide transversion (T[RIGHTWARDS ARROW]A) was identified in exon 6 of the Rxrα gene, causing the amino acid substitution I273N (Fig. 2B). This substitution is located in the third α-helix of the ligand binding domain of RXRα, which involves in the ligand-dependent allosteric change required for ligand-dependent transactivation 34, 35.

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Figure 2. The pinkie phenotype results from mutation of Rxrα gene. (A) Genetic mapping of the pinkie locus. On 27 meioses, pinkie locus is confined to proximal end of chromosome 2. (B) pinkie corresponds to a single nucleotide substitution of Rxrα gene. T[RIGHTWARDS ARROW]A transversion results in Ile replacement by Asn. The Consed display shows bi-directional sequencing of Rxrα cDNA isolated from homozygous pinkie skin.

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Rxrαpkecauses a 90% loss of RXRα activity in both retinoic acid and vitamin D signaling pathways, as measured in transfected HEK293 cells using a luciferase reporter gene with a promoter composed of either retinoic acid responsive element (RARE) or vitamin D responsive element (VDRE) (Fig. 3). The pinkie phenotype cannot be rescued by administration of large quantities of exogenous vitamin A or vitamin D (data not shown).

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Figure 3. Rxrαpke allele produces a receptor that is poorly responsive to retinoic acid and vitamin D. HEK293 cells were transfected with 1 μg pIRES vector or either Rxrpkeor wild-type Rxrα expression constructs together with 0.2 μg RARE-DR3-Luc reporter plasmid. Cells were stimulated with t-RA or vehicle for 12 h, and then the lysate was subject to luciferase assay (A); Alternatively, Vdr expression vector was co-transfected with either Rxrαpke or wild-type Rxrα expression construct into HEK293 cells, along with 24OHase-Luc reporter plasmid for VDR. Transfected cells were stimulated with 1α, 25-(OH)2D3 at a final concentration of 100 nM for 12 h (B). In both experiments, results are expressed as relative luciferase activity and represent the mean ± SEM of two independent assays.

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Rxrαpke/pke mice show progressive exaggeration of Th1 response to OVA immunization

The lymphocyte count and CD4+/CD8+ T cell ratio were identical in Rxrαpke/pke and control mice (data not shown). In view of the reported effect of Th polarization caused by retinoic acid 36, 37 and by vitamin D stimulation 38, 39, Rxrαpke/pke mice were immunized intraperitoneally with OVA mixed with alum, a strong promoter of Th2 responses, along with sex- and age-matched C57BL/6 control mice. Six-week-old Rxrapke homozygotes produced comparable amount of Th2-associated antigen-specific IgG1. However, 1-year-old Rxrapke homozygotes showed significant impairment of antigen-specific IgG1 production (p<0.05) (Fig. 4). Notably, an age-dependent decline in the Th2 associated response was also observed in normal mice, but a much greater decline was observed in Rxrapke homozygotes. The Th2 predominant BALB/c strain produced significantly higher levels of antigen-specific IgG1 as compared with C57BL/6 mice at all ages tested (data not shown).

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Figure 4. Increased Th1 response to OVA/alum immunization in pinkie mice. Two different age groups of C57BL/6 and pinkie mice were immunized intraperitoneally by OVA/alum for 14 days. The serum was collected after immunization and OVA-specific IgG1 production was analyzed (n=3). *p<0.05 compared to C57BL/6 mice. The results represent three independent experiments.

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In vitro, naïve CD4 T cells of Rxrapke homozygotes display Th1-predominant differentiation, and this effect is independent of the changes of expression of T-bet and GATA-3

Purified CD4+CD62Lhigh naïve T cells were exposed to plate-bound anti-CD3 and anti-CD28 in the presence of cytokines that promote Th1, Th2 or Th1/Th2 differentiation, respectively. Their Th differentiation status was assessed by intracellular staining of IFN-γ and IL-4 after 1 week of stimulation. Under conditions favoring exclusive Th1 differentiation, purified naïve CD4+ cells from Rxrapke homozygotes developed into highly polarized Th1 cells as compared to C57BL/6 controls, with an IFN-γ+/IL-4+ ratio of 309 vs. 108. A similar bias toward Th1 differentiation was observed under conditions favoring mixed Th1/Th2 differentiation. However, there was no difference between Rxrapke and wild-type cells under exclusive Th2-polarizing conditions. BALB/c cells were used as a Th2 polarization control (Fig. 5). These data suggest that RXRα-dependent signals normally suppress Th1 differentiation within naïve lymphoid cells. Once formed to excess in the absence of RXRα signaling, Th1 cells act to suppress Th2 differentiation. Hence, RXRα indirectly permits Th2 differentiation of naïve CD4+ cells, but is not required for Th2 differentiation per se.

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Figure 5. Skewed Th1 differentiation of naïve CD4 T cells in pinkie mice. Naïve CD4 T cells isolated from C57BL/6, pke/pke and BALB/c spleens, respectively, were cultured in the absence of APC and under indicated Th development condition for 1 week. The cells were then restimulated with PMA/ionomycin and cytokine profile was determined by intracellular staining. The results represent three independent experiments.

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In cells stimulated under mixed conditions favoring mixed Th1/Th2 differentiation, more T-bet mRNA and less GATA-3 mRNA were expressed in T cells homozygous for Rxrapke than in T cells from wild-type C57BL/6 mice. However, the difference was comparatively modest (Fig. 6), and we cannot conclude that this is the sole mechanism by which RXRα-dependent signals operate.

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Figure 6. Modest effect of the pinkie mutation on expression of T-bet and GATA-3 in developing Th cells. Quantitative real-time RT-PCR for T-bet and GATA-3 expression was performed with total RNA extracted from naïve CD4 T cells stimulated under Th1/Th2 condition. The expression level is presented with reference to mouse β-actin signal. The data are representative of two independent experiments.

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Rxrapke/pke DC produce more IL-12 upon stimulation by microbial inducers, and the effect is IL-10 independent

Rxrapke/pke DC consistently produce higher levels of IL-12 both in the presence and absence of lipopolysaccharide (LPS) at a concentration of 10 ng/mL as compared with control DC (p<0.05; Fig. 7A). However, as a major negative regulator of DC IL-12 expression 40, the production of IL-10 is comparable in Rxrapke/pke and control DC cultures (Fig. 7B). Hence, RXRα-dependent signals normally act to suppress IL-12 production in DC, both in the presence and in the absence of TLR4-dependent signals. However, this suppression does not involve IL-10 production.

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Figure 7. LPS-stimulated pinkie DC showed significantly increased IL-12 production. DC were stimulated with 10 ng/mL LPS for 24 h. Level of IL-12p40 (A) and IL-10 (B) in the supernatants were measured by ELISA. *p<0.05 compared to C57BL/6 mice.

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Rxrapke/pke regulatory T cells demonstrate progressive decreased suppression in vitro

There is accumulating evidence that regulatory CD4+CD25+ T cells (Treg) play a crucial role in the maintenance of immunologic self-tolerance and preventing autoimmune disease 4144. In view of the predominant Th1 response in pinkie mice, the natural Treg populations were examined. The ratio of CD4+CD25+/CD4+CD25 T cells is comparable between pinkie and wild-type C57BL/6 control mice, regardless of animal age (data not shown). In agreement with this, for both thymus and peripheral Treg, no significant difference in thymic or splenic expression of the lineage-specific marker foxp3 45 is observed between pinkie mice and control mice, although a modest reduction is witnessed in old mutant animals (Fig. 8A). However, the proliferation of naïve CD4+ T cells is consistently higher in the presence of Rxrapke/pke Treg as compared with Treg isolated from wild-type C57BL/6 mice at all ratios tested. The difference becomes most pronounced and is significant when cells from older animals are compared (p<0.05, Fig. 8B). The data imply that a functional RXRα is required to qualitatively maintain the suppressive function of CD4+CD25+ regulatory T cells in the periphery rather than regulate their thymic generation.

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Figure 8. Treg demonstrate decreased suppressive activity in pinkie mice. (A) Foxp3 expression level of Treg is not significantly altered in pinkie mice compared to control C57BL/6 mice. (B) CD25+CD4+ T cells were purified by FACS from spleens of 10-month-old pinkie and C57BL/6 mice, respectively. They were mixed at various ratios with normal CD25CD4+ T cells and cultured for 3 days with anti-CD3 mAb and mitomycin C-treated spleen cells as APC. CD4+ T cells proliferation was evaluated by thymidine uptake. The difference between pinkie mice and C57BL/6 is significant (p<0.05).

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Dietary vitamin A depletion favors a Th1 response to antigen in Th2-biased BALB/c mice

After dietary vitamin A depletion in utero and 9 weeks post weaning, the serum retinol level of BALB/c mice averaged 0.49 μM as compared to 1.43 μM in control mice, and showed no outward signs of vitamin A deficiency. Upon OVA/alum immunization, vitamin A-depleted BALB/c mice produce less antigen-specific IgG1 antibody than controls (Fig. 9A), and splenocytes isolated from vitamin A-depleted mice produce less Th2 cytokine IL-4 upon OVA re-stimulation in vitro (Fig. 9B). Although the difference is not quantitatively significant, the declining of Th2 response is apparent. While the genetic causes of Th2 predominance in BALB/c mice are unknown, a defined Rxrα-dependent ligand (retinoic acid) is required for Th2 polarization in this strain.

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Figure 9. Dietary depletion of vitamin A increased Th1 response to OVA/alum immunization in wild-type BALB/c mice. Two groups of BALB/c mice (dietary vitamin A depletion and control mice) were immunized intraperitoneally by OVA/alum for 14 days. (A) The serum was collected after immunization and OVA-specific IgG1 production was analyzed (n=8). (B) Splenocytes were isolated and restimulated with 100 μg/mL OVA for 96 h, and IL-4 production in the supernatant were measured by ELISA. The results represent two independent experiments.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The pinkie phenotype has not previously been reported in humans or in mice, and resulted from a viable hypomorphic mutation of Rxrα, a key component of several nuclear receptor complexes, including the receptors for vitamin A and vitamin D. The maximum ceiling of the mutant receptor response is approximately 10% that of the wild-type receptor. The viability of Rxrαpke homozygotes may partly be explained by compensatory overexpression of the mutant gene, which is expressed at about twice the level in homozygous mutant mice as in wild-type control animals according to quantitative real-time RT-PCR analysis (data not shown). In addition, redundant functions of RXRβ and RXRγ may offer redundancy. Phenotypic progression is most probably explained by the fact that expression of the Rxrα locus naturally decreases by 20–30% with senescence 46, 47, ultimately leading to decompensation.

As a member of nuclear receptor superfamily, RXRα not only serves as a receptor for retinoic acid, but also an obligatory heterodimerization partner for other nuclear receptors. RXRα is found predominantly in the skin 48. Further, the data from conditional knockout of RXRα in keratinocytes and vitamin D receptor (VDR) knockout mice have demonstrated the importance of RXRα/VDR in maintaining skin homeostasis postnatally, and in controlling hair-follicle cycling 29, 30, 49. Therefore, the skin lesions and alopecia seen in pinkie homozygotes are likely due to the defective RXRα/VDR signaling. The kyphosis often present in older mice provides further evidence for involvement of the VDR signaling pathway.

We have shown that RXRα deficiency causes failure of Th2 responses in vivo. This observation is broadly consistent with the previously reported opposite effects of hypervitaminoses A 50 and D 51, although these have never been examined in combination with one another, nor by means of a genetic approach. Because both vitamin A and vitamin D signals are RXRα dependent, an integrated syndrome resembling a mixed deficiency state is produced by the Rxrαpke mutation. Both an intrinsic defect in naïve CD4+ T cells and a defect in DC contribute to Th1 polarization in Rxrαpke/pke mice. In addition, age-dependent decrease of Treg suppression further exaggerates the Th1 predominance of Rxrαpke/pke mice upon antigen challenge.

DC from Rxrapke/pke animals produce significantly higher level of IL-12 under basal and activated condition, which indicates that Th1 skewing is at least partly due to the aberrant suppression of IL-12 production. IL-12 production is NF-κB dependent and is inhibited by negative regulators, including other cytokines (e.g., IL-10) and transcription factors (e.g., c-Maf) 52. In Rxrapke/pke mice, diminished IL-10 production by DC itself is not likely responsible for enhanced IL-12 production because identical levels of IL-10 are produced by DC from mutants and controls. It has been previously reported that both 1α, 25-(OH)2-vitamin D3 and retinoids significantly inhibit expression of the IL-12p40 subunit mRNA at a transcriptional level, and this transcriptional repression is achieved by down-regulation of NF-κB binding to an IL-12p40 promoter element 53, 54. These data collectively imply a direct role for nuclear receptors in the highly regulated process of IL-12 production. Since Rxrαpke results in defective heterodimers regardless of the partner, it is not surprising to see elevated IL-12 production stemming from a loss of negative regulation. This defect, manifested in APC of pinkie mice, creates a priming environment conductive to Th1 cell development.

In a system free of APC, differentiation of naïve CD4+ cells occurs identically in Rxrαpke/pke and control cultures under conditions that exclusively favor a Th2 phenotype. However, under conditions favoring Th1 or mixed Th1/Th2 differentiation, CD4+ T cells from Rxrαpke/pke mice show enhanced Th1 commitment. Rxrαpke/pke cells are thus Th2 competent, and the observed Th1/Th2 imbalance reflects an intrinsic defect in mechanisms that normally suppress Th1 commitment. When naïve CD4+ T cells from Rxrαpke homozygous mice were stimulated under mixed Th1/Th2 condition, the Th1-promoting factor T-bet is slightly overexpressed and the Th2-promoting factor GATA-3 is slightly under expressed. Analysis of the T-bet promoter sequence disclosed no nuclear receptor-responsive motifs. We surmise that the influence of RXRα is probably indirect, and in the case of GATA-3, may involve cross-suppression by T-bet 55. In the hierarchical network of cytokines and transcription factors that contribute to Th1 development, T-bet is readily induced by IFN-γ through Stat1, the expression of which up-regulates IL-12R expression on the naïve CD4 T cells 56, and IFN-γ production is induced by IL-12 through Stat4 signaling pathway 17. Whether negative regulation of Th1 differentiation by RXRα is solely exerted by inhibition of IL-12 production or whether it involves cross-regulation of several signaling pathways remains to be determined.

Although molecular basis of Treg suppressive function is not fully understood, it is likely that CD4+CD25+ cells suppress immunity by several distinct mechanisms 57. One entails directly suppression of other T cell functions by interference with TCR signaling upstream of transcription factors that induce responses by effector T cells; the other entails suppression of APC function, which is accomplished at least partly by inhibition of IL-12 production and co-stimulatory molecule expression on APC 5860. Through the latter mechanism, Treg suppress the default (Th1) response, offering resistance to helminth infection in vivo6163. Decreased Treg activity in Rxrαpke/pke mice releases this suppression, and may contribute to an exaggerated Th1 response. Intriguingly, without regard to the number of CD4+CD25+ cells, Treg of Rxrαpke/pke mice gradually lose their suppressive activity with increasing age. These data imply an essential role for RXRα signaling in the maintenance of Treg function in the periphery.

The rise in atopic disease frequency observed in developed countries cannot plausibly have a genetic cause, and therefore, an environmental change must be held culpable. An environmental change must be sensed by a biological system to exert an effect, and sensing usually implies the existence of a receptor. The hygiene hypothesis holds that the sensor is the immune system itself, and in recent times, has grown more specific in that Toll-like receptors (TLR) have been regarded as the key interface with the microbial world, and TLR-derived signals have been held to determine Th status. We have now adduced evidence of another environmental influence in that limitation of vitamin A and vitamin D sensing by a mutation in RXRα causes Th1 skewing. Moreover, dietary deficiency of the RXRα-dependent ligand vitamin A, substantially attenuates genetically programmed Th2 predominance. Conceivably, marginal consumption of vitamin A might be expected to prevent atopic diseases in the developing world, and is the normal state in many countries where overt malnutrition is problematic and clinical deficiency of vitamin A is prevalent 64. In the developed world, vitamin A consumption may tend to skew the Th balance toward a strong Th2 response, and environmental ligands yet unknown may possibly mimic the effect of retinoic acid as well.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements


C57BL/6 mice used in ENU mutagenesis were purchased from the Jackson Laboratories, and C3H/HeN mice used for outcrossing and mapping were purchased from Taconic. All studies involving animals were carried out in accordance with institutional regulations.


ENU, GM-CSF, PMA and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO). LPS was bought from Fluka. Antibodies and cytokines used in naïve CD4 T cell isolation and stimulation were purchased from eBiosciences and BD PharMingen (San Diego, CA). OVA was purified in house and was free of microbial contaminants.


Bone marrow cells were isolated from Rxrαpke homozygotes and C57BL/6 mice as described elsewhere 65, and cultured in the presence of GM-CSF and IL-4 to induce into DC differentiation. On day 8, cells were plated at a density of 2 × 105 cells/well in 24-well plate, and exposed to LPS at a concentration of 10 ng/mL for 24 h. The medium was then collected and subjected to IL-12 and IL-10 ELISA.

Splenocytes were prepared aseptically from C57BL/6 and Rxrαpke homozygotes and filtered through a 70-μm cell strainer to obtain a single-cell suspension. Contaminating red blood cells were then lysed by adding red cell lysis buffer (eBiosciences). Cells were centrifuged and resuspended in Iscove's modified Dulbecco's medium (IMDM). Naïve CD4 T cells and natural Treg were further purified by cell sorting. The purity of the sorted cells are comprised >95% of the cells in the final preparation.

ENU mutagenesis and genomic linkage analysis

As previously described 66, 6-week-old male C57BL/6 mice (G0) are treated with ENU administered in three weekly doses (100 mg/kg body weight) by intraperitoneal injection.

Homozygous pinkie mice were mated to wild-type C3H/HeN mice, their progeny were backcrossed to the homozygous mutant stock. The offspring of the F2 generation were scored for phenotype and genomic DNA was prepared from tail tips for genotyping. A total of 59 microsatellite markers were used for genome-wide linkage analysis.

Rxrα and Vdr expression constructs and transfection of HEK293 cells

Rxrα cDNA was amplified using primers: 5′-ATGGACACCAAACATTTCCTGCC-3′ and 5′-CTAGGTGGCTTGATGTGGTGCCTC-3′. Vdr cDNA was amplified using primers: 5′-ATGGAGGCAATGGCAGCCAGCACCTC-3′ and 5′-TCAGGAGATCTCATTGCCGAACACC-3′.

The cDNA were then cloned into pIRES expression vector (Clontech) between Nhe I and Xho I sites. The constructs were verified by DNA sequencing.

To evaluate the consequence of Rxrαpke in retinoic acid signaling pathway, 1 μg Rxrαpke or wild-type Rxrα expression construct was transfected into HEK293 cells together with 0.2 μg RARE-DR3-Luc reporter plasmid (kindly provided by Dr. Laszlo Tora in Strasbourg) using Lipofectamine™ 2000 (Invitrogen). The cellular response to all-trans retinoic acid (t-RA) stimulation was tested 24 h post-transfection. Prior to stimulation, transfected cells were serum-starved for 18 h, and stimulation with t-RA (1 μM final concentration) was carried out for 12 h at 37°C. The whole cell lysates were analyzed using the Luciferase Assay System (Promega). Similarly, to assess the vitamin D signaling pathway, Vdr expression vector was co-transfected with either Rxrαpke or wild-type Rxrα expression construct into HEK293 cells, along with 24OHase-Luc reporter plasmid for VDR (kindly provided by Dr. Hector F. DeLuca). The transfected cells were stimulated by 1α, 25-dihydroxyvitamin D3 at a final concentration of 100 nM.

In vivo immune response to OVA/alum immunization

Rxrαpke homozygotes and age-matched wild-type C57BL/6 mice were immunized with OVA/alum (100 μg OVA plus 2 mg alum) intraperitoneally for 14 days. Serum was collected before and after immunization. ELISA was performed to detect OVA-specific serum IgG1. Pooled serum from eight immunized C57BL/6 mice was used as a standard in ELISA. The standard was serially diluted in tenfold increments and results obtained for individual samples were expressed with reference to the standard (experimental units; EU).

In vitro stimulation of naïve CD4 T cells

Naïve CD4 T cells were suspended in IMDM supplemented with 10% heat-inactivated FBS (Invitrogen), 50 μM 2-mercaptoethanol, 2% penicillin/streptomycin (Invitrogen), and were plated at a density of 2 × 105/mL in 48-well culture plates (Falcon). The cells were stimulated with plate-bound anti-CD3 mAb (3 μg/mL, clone 145–2C11, BD PharMingen) and soluble anti-CD28 mAb (3 μg/mL, clone 37.51, BD PharMingen). Cytokines were added to wells at the initiation of cultures as indicated. To induce Th1 development, 4 ng/mL IL-12 and 5 μg/mL anti-IL-4 mAb were added; for Th2 development, 10 ng/mL IL-4 and 10 μg/mL anti-IFN-γ mAb were added; for mixed Th1/Th2 development, 4 ng/mL IL-12 and 10 ng/mL IL-4 were added. Cells were harvested on day 7, washed twice, counted and restimulated with PMA and ionomycin for intracellular cytokine staining 28.

In vitro T cells proliferation assay

Freshly isolated naïve CD4+ T cells were cultured in 96-well plate with 1.5 × 105/well mitomycin C-treated spleen cells as APC, along with Treg purified from wild-type C57BL/6 mice or Pinkie mice, respectively, at various ratios. The cells were stimulated by soluble anti-CD3 mAb (1 μg/mL) for 72 h at 37°C, and pulsed with 0.2 µCi [3H]thymidine for the last 12 h. The proliferation of naïve CD4+ T cells were evaluated by thymidine incorporation.

Dietary depletion of vitamin A

Pregnant BALB/c female mice were fed with a vitamin A-deficient diet (Harlan, TD88407) from the 2nd week of gestation until the pups were weaned at age 3 week. The weanlings were fed with the same vitamin A-deficient diet for another 6 weeks before being analyzed for immune response to antigen. The serum retinol level was evaluated by HPLC (Antech Diagnostics).

mRNA analysis

Total RNA was prepared using TRIzol® (Invitrogen). Analysis of gene expression was performed by quantitative real-time PCR on an ABI Prism 7900HT Sequence Detection System. Fluorogenic primers were designed for RXRα, GATA-3, T-bet and Foxp3 (non-fluorescent, SYBR® green for detection); β-actin was used as the endogenous control (purchased from Invitrogen).


Statistical analysis

The significance of mean values was determined by Student's t-test. Values of p<0.05 were considered significant.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The authors thank Dr. Terry Meehan (TSRI), Drs. Laurie Glimcher, Kerri Mowen, and Dallas Jones (Harvard University) and Dr. Edith Janssen (La Jolla Institute for Allergy and Immunology) for their helpful suggestions and discussions. This work was supported by NIH grant U54-AI54523.

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