Azathioprine therapy selectively ablates human Vδ2⁺ T cells in Crohn's disease.

Tumor-derived and bacterial phosphoantigens are recognized by unconventional lymphocytes that express a Vγ9Vδ2 T cell receptor (Vδ2 T cells) and mediate host protection against microbial infections and malignancies. Vδ2 T cells are absent in rodents but readily populate the human intestine, where their function is largely unknown. Here, we assessed Vδ2 T cell phenotype and function by flow cytometry in blood and intestinal tissue from Crohn's disease patients (CD patients) and healthy controls. Blood from CD patients included an increased percentage of gut-tropic integrin ß7-expressing Vδ2 T cells, while "Th1-committed" CD27-expressing Vδ2 T cells were selectively depleted. A corresponding population of CD27+ Vδ2 T cells was present in mucosal biopsies from CD patients and produced elevated levels of TNFα compared with controls. In colonic mucosa from CD patients, Vδ2 T cell production of TNFα was reduced by pharmacological blockade of retinoic acid receptor-α (RARα) signaling, indicating that dietary vitamin metabolites can influence Vδ2 T cell function in inflamed intestine. Vδ2 T cells were ablated in blood and tissue from CD patients receiving azathioprine (AZA) therapy, and posttreatment Vδ2 T cell recovery correlated with time since drug withdrawal and inversely correlated with patient age. These results indicate that human Vδ2 T cells exert proinflammatory effects in CD that are modified by dietary vitamin metabolites and ablated by AZA therapy, which may help resolve intestinal inflammation but could increase malignancy risk by impairing systemic tumor surveillance.

Prenatally acquired vitamin A deficiency alters innate immune responses to human rotavirus in a gnotobiotic pig model.

We examined how prenatally acquired vitamin A deficiency (VAD) modulates innate immune responses and human rotavirus (HRV) vaccine efficacy in a gnotobiotic (Gn) piglet model of HRV diarrhea. The VAD and vitamin A-sufficient (VAS) Gn pigs were vaccinated with attenuated HRV (AttHRV) with or without concurrent oral vitamin A supplementation (100,000 IU) and challenged with virulent HRV (VirHRV). Regardless of vaccination status, the numbers of conventional and plasmacytoid dendritic cells (cDCs and pDCs) were higher in VAD piglets prechallenge, but decreased substantially postchallenge as compared with VAS pigs. We observed significantly higher frequency of CD103 (integrin αEß7) expressing DCs in VAS versus VAD piglets postchallenge, indicating that VAD may interfere with homing (including intestinal) phenotype acquisition. Post-VirHRV challenge, we observed longer and more pronounced diarrhea and higher VirHRV fecal titers in nonvaccinated VAD piglets. Consistent with higher VirHRV shedding titers, higher IFN-α levels were induced in control VAD versus VAS piglet sera at postchallenge day 2. Ex vivo HRV-stimulated mononuclear cells (MNCs) isolated from spleen and blood of VAD pigs prechallenge also produced more IFN-α. In contrast, at postchallenge day 10, we observed reduced IFN-α levels in VAD pigs that coincided with decreased TLR3(+) MNC frequencies. Numbers of necrotic MNCs were higher in VAD pigs in spleen (coincident with splenomegaly in other VAD animals) prechallenge and intestinal tissues (coincident with higher VirHRV induced intestinal damage) postchallenge. Thus, prenatal VAD caused an imbalance in innate immune responses and exacerbated VirHRV infection, whereas vitamin A supplementation failed to compensate for these VAD effects.

Commitment toward the natural T (iNKT) cell lineage occurs at the CD4+8+ stage of thymic ontogeny.

T lineage commitment occurs in a discrete, stage-specific manner during thymic ontogeny. Intrathymic precursor transfer experiments and the identification of CD4(+)8+ double-positive (DP), V alpha 14J alpha 18 natural T (iNKT) cells suggest that commitment to this lineage might occur at the DP stage. Nevertheless, this matter remains contentious because others failed to detect V alpha 14J alpha 18-positive iNKT cells that are CD4(+)8+. In resolution to this issue, we demonstrate that retinoic acid receptor-related orphan receptor gamma (ROR gamma)0/0 thymi, which accumulate immature single-positive (ISP) thymocytes that precede the DP stage, do not rearrange V alpha 14-to-J alpha 18 gene segments, suggesting that this event occurs at a post-ISP stage. Mixed radiation bone marrow chimeras revealed that RORgamma functions in an iNKT cell lineage-specific manner. Further, introgression of a Bcl-x(L) transgene into ROR gamma(0/0) mice, which promotes survival and permits secondary rearrangements of distal V alpha and J alpha gene segments at the DP stage, rescues V alpha 14-to-J alpha 18 recombination. Similarly, introgression of a rearranged V alpha 14J alpha 18 transgene into ROR gamma(0/0) mice results in functional iNKT cells. Thus, our data support the "T cell receptor-instructive (mainstream precursor) model" of iNKT cell lineage specification where V alpha 14-to-J alpha 18 rearrangement, positive selection, and iNKT cell lineage commitment occur at or after the DP stage of ontogeny.

Expression of retinoid X receptors and COUP-TFI in a human salivary gland adenocarcinoma cell line.

The growth of the adenocarcinoma cell line derived from human salivary gland (HSG) is regulated by all-trans-retinoic acid (t-RA), which binds to its specific receptor, retinoic acid receptors (RARs), located in the nucleus, and thereby transactivates target genes. In this study, we examined the binding characteristics of the nuclear extract of HSG cells to the retinoic acid response element (RARE) compared with those of in vitro translated RAR alpha and retinoid X receptor alpha (RXR alpha), a heterodimeric partner of RAR alpha. Gel shift analysis using anti-RAR alpha and anti-RXR alpha antibodies revealed that the translated RAR alpha bound to RARE as a heterodimer with RXR alpha. In contrast, the binding of the nuclear extract of HSG cells to RARE showed a heterogeneous pattern, suggesting the existence of several species of RXRs as well as RARs in the nuclei of HSG cells. We therefore tried to clone these putative RXRs by the polymerase chain reaction using degenerated oligonucleotide primers conserved across the RXR family. The DNA sequencing of the recombinant clones revealed the expression of RXR alpha and RXR beta. In addition, chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI), which is also an RXR family member, was cloned. To evaluate the transcriptional activity of RARs and RXRs endogenously expressed in HSG cells, we performed a transient transfection analysis. When HSG cells were transfected with a luciferase reporter plasmid containing two repeats of either the RARE of the RAR beta gene or that of cellular retinol-binding protein II gene, positioned upstream of a thymidine kinase promoter fused to the luciferase sequence, a 2-3-fold induction of luciferase activity was observed in both cases. These results suggest that RARs and RXRs endogenously expressed in HSG cells were transcriptionally active in vivo. Thus, our findings showed that RXR alpha, RXR beta, and COUP-TFI are expressed in HSG cells and suggest that these molecules function as heterodimeric partners of RARs and (or) competitive repressors for RAREs and are involved in cellular responses mediated by retinoids.

Aberrant metabolism of retinoid X receptor proteins in human hepatocellular carcinoma.

A polyclonal antibody was raised against a recombinant ligand binding domain construct of the human retinoid X receptor (RXR) alpha. This antibody reacted with an endogenous 54 kDa nuclear protein from human hepatoma-derived HuH7 cells in immunoblot analyses. Immunoblotting of nuclear proteins from human hepatocellular carcinomas (HCCs) and their surrounding tissues revealed the presence of a 44 kDa RXR distinct from the 54 kDa RXR and a dramatic decrease in the relative amounts of 44 kDa RXR to 54 kDa RXR in all HCCs compared with normal tissue. In vitro shift and intracellular conversion from 54 kDa RXR to 44 kDa species were observed with the nuclear extracts of HuH7 cells. Furthermore, transfection of hRXR alpha cDNA into HuH7 cells resulted in the increase of 54 kDa RXR, whereas transfected mouse hepatocytes accumulated 44 kDa RXR. These results strongly indicated that 44 kDa RXR was a physiological proteolytic fragment of 54 kDa RXR and that post-translational metabolism of RXR was impaired in HCC and the HuH7 hepatoma-derived cell line.