Cloning and Expression of DR6
To investigate the physiological function of DR6, a knockout strategy was devised, the first step of which was cloning a murine cDNA encoding DR6. Murine and human DR6 displayed 88% protein sequence identity ( A). Examination of mouse tissues by Northern blot analysis demonstrated the presence of a 4-kb DR6 transcript in adult lymphoid tissues including PBLs, thymus, and spleen. The transcript could also be detected at varying levels in every adult or fetal tissue analyzed ( B). To further characterize the expression pattern of DR6 in various lymphoid cell populations, we performed real time quantitative RT-PCR by Taqman™ assay using total RNA extracted from various cell populations. Our analysis indicated that DR6 was present on CD4+, CD8+, CD19+, and CD14+ cells, suggesting that DR6 is specifically expressed on T cells, B cells, and monocytes (data not shown). DR6 expression was also determined in Th0 and differentiated Th1 and Th2 cells. DR6 mRNA was found in Th0 cells as well as in Th1 and Th2 cells; however, levels of DR6 transcript were twofold higher in Th2 cells ( C). These results reinforced the notion that DR6 may be important in immune function and potentially regulate Th cell responses.
Figure 1. Amino acid sequence and tissue distribution of DR6. (A) Amino acid sequence of mouse and human DR6. (B) Northern blot analysis of human DR6. Human multiple tissue Northern blots (CLONTECH) were probed with a cDNA fragment corresponding to the cytoplasmic (more ...)
Generation of DR6-deficient Mice
To create DR6-deficient mice, a gene-targeting vector was constructed such that homologous recombination would delete the entire extracellular and transmembrane domain of the endogenous mouse DR6 gene. The 3.9-kb deletion included exons 2 and 3, resulting in a mutant DR6 allele lacking amino acid residues Q33 to G415 ( A). Three targeted ES cell lines were injected into C57BL/6 blastocysts. Founder mice with germline transmission were interbred to produce homozygous DR6-deficient mice, which were born at the expected Mendelian ratio. Inactivation of the DR6 gene locus was verified by Southern blot analysis ( B) and loss of gene expression was demonstrated by RT-PCR analysis of total liver RNA ( C). DR6-deficient mice were viable, fertile, and detailed histological analysis did not reveal any obvious abnormalities (data not shown).
Figure 2. Targeting of the DR6 gene by homologous recombination. (A) Structure of the mouse DR6 gene and gene targeting vector. Exons 2 and 3 corresponding to the extracelluar and transmembrane regions of mDR6 were replaced by a neomycin resistance gene under the (more ...)
Lymphocyte Populations in DR6-deficient Mice
Initial profiling of immune cells revealed a slight, though statistically significant (P < 0.05) increase in the percentage of CD3+ T cells in the thymus and PBL compartments of the knockout mice ( A). We further measured thymic weight and compared the total number of thymocytes in DR6−/− mice to control wild-type littermates. There was no significant change in the size of the thymus or in the number of thymocytes (data not shown). Additional analysis showed that both CD4+ and CD8+ T cells were almost elevated twofold in the PBL compartment ( B and C). Flow cytometry analysis of cells stained with mAbs to B220, Gr.1, NK1.1, F4/80, and CD14 did not indicate any gross differences in B cells, neutrophils, NK cells, or monocyte/macrophages in lymphoid organs of DR6-deficient mice when compared with age- and sex-matched controls (data not shown).
Figure 3. Effect of DR6 on T cell development. Total T cells and CD8+ T subpopulations were determined in thymus (Thy), blood (PBL), spleen (Sp), and LNs from DR6 knockout (black bars) and wild-type (white bars) mice. Organ-specific (A) total CD3+ T cells, (B) (more ...)
In Vitro T Cell Responses
Given the significant increase in T cell numbers in DR6-deficient mice, this phenotype was further characterized by measuring their proliferative capacity. Purified CD4+ T cells from DR6-deficient and control wild-type littermates were stimulated with Con A, a polyclonal T cell stimulator or with α-CD3 mAb alone or α-CD3 plus α-CD28. Subsequently, proliferative response and IL-2 production were measured. T cells from DR6-deficient mice hyperproliferated and produced increased amounts of IL-2 as compared with wild-type cells regardless of the nature of the proliferative stimulus (). These data indicated that CD4+ T cells lacking DR6 were intrinsically hyperresponsive to mitogenic, TCR, and costimulatory signals consistent with the notion that DR6 normally functions to suppress T cell activation and cytokine production.
Figure 4. In vitro activation of T cells. For in vitro responses of T cells, purified CD4+ T cells from DR6−/− mice (black squares) or wild-type littermates (black circles) were cultured in the presence of different concentrations of ConA or anti-CD3 (more ...)
In Vivo T Cell Responses
To determine the influence of DR6 on in vivo T cell responses, wild-type and DR6-deficient mice were immunized with KLH and 9 d later draining LNs (DLNs) were harvested. After in vitro stimulation of DLN cells with KLH, cytokine production and recall proliferative ability of these cells was assessed. T cells from primed DR6-deficient mice had increased proliferative responses with production of higher levels of IL-2 and IFN-γ ( A–C). Additionally, production of IL-4 was markedly enhanced in DLN cells from DR6-deficient mice, levels being almost twofold higher ( D). These results indicate that DR6-deficient mice are hyperresponsive to in vivo antigenic challenge. This is consistent with our in vitro data reinforcing the notion that DR6 functions to suppress T cell responsiveness in vivo. Although T cells from DR6-deficient mice produced somewhat increased levels of various cytokines, the level of IL-4 was significantly elevated, underscoring a role for DR6 in the Th2 response. The in vivo KLH/CFA challenge experiments were designed to examine the influence of antigenic challenge on general T cell responses and not Th differentiation which typically requires immunization with KLH in alum. However, even under these sub-optimal conditions, we did see some preference for Th2 differentiation.
Figure 5. Antigen-induced in vivo responses in DR6-deficient mice. Cytokine production and proliferative response of lymph node cells from wild-type (white circles) and DR6-deficient (white squares) mice immunized with KLH in CFA. Lymph node cells were collected (more ...)
In Vivo Humoral Responses
Since Th2 cells regulate humoral immunity and Ig production, particularly IgG1 and IgE antibodies while the Th1 response contributes to the production of IgG2a and, to a lesser extent, IgG3 antibodies, serum Ig levels were determined in DR6-deficient mice. Naive DR6-deficient mice had Ig levels comparable to wild-type littermates ( A). However, upon in vivo challenge with TNP-conjugated KLH, DR6-deficient mice developed markedly increased titers of TNP-specific IgG1 and IgE ( B), whereas levels of IgM and IgG2a were only slightly elevated. The levels of IgG1 were increased by ~200% in DR6-deficient mice, consistent with preferential Th2 cell differentiation and hyperproliferation. This marked polarization toward a Th2 response was also substantiated by the significant increase in IL-4 production in KLH-primed DR6−/− T cells ( D).
Figure 6. Effect of DR6 on Ig subclasses. Serum was collected from wild-type (black bars) and DR6-deficient mice (white bars) and subclass concentrations determined by ELISA (A). Serum was obtained from wild-type and DR6-deficient mice immunized with TNP-KLH in (more ...)
In Vitro Differentiation of T Cells
Since DR6-deficient mice exhibited an enhanced Th2 response in vivo, we investigated the role of DR6 in mediating Th differentiation in vitro. Purified naive CD4+ T cells from wild-type and DR6-deficient mice were differentiated into either Th1 cells by activating them with α-CD3 plus α-CD28 in the presence of antibodies to IL-12 and IL-4 or into Th2 cells in the presence of IL-4 and antibody to IFN-γ. When differentiated into Th1 cells, DR6-deficient lymphocytes produced levels of IFN-γ equivalent to wild-type controls (). In contrast, DR6-deficient lymphocytes grown in the presence of IL-4 and anti–IFN-γ produced markedly enhanced Th2 cytokines such as IL-4 itself. Cytokine production was also determined by staining the T cells for intracellular IFN-γ and IL-4. A significantly higher number of T cells produced IL-4 (data not shown), indicating that the IL-4 producing T cells pool was expanded in DR6−/− mice. Taken together, our results indicate that DR6 plays a major role in Th cell differentiation both in vivo and in vitro.
Figure 7. In vitro induction of Th cell differentiation and effect of DR6 on cell death. T cells purified from spleens of wild-type (black bars) or DR6-deficient (white bars) mice were differentiated into Th1 or Th2 cells with anti-CD3 plus anti-CD28. Production (more ...)
DR6 Does Not Regulate T Cell Differentiation through Apoptosis
Since DR6 possesses an intracellular DD, this raised the possibility that it may regulate T cell activation and differentiation by modulating apoptosis. To address this, AICD of thymocytes and splenic T cells from DR6-deficient mice was measured in response to various apoptotic stimuli. No appreciable difference was observed in susceptibility of splenocytes or thymocytes to AICD or death induced by other proapoptotic stimuli ( A and B). Therefore, it was unlikely that decreased apoptosis accounts for the increased proliferative potential of DR6-deficient T cells. In sum, our data are consistent with an intrinsic, cell autonomous growth inhibitory role for DR6, removal of which, as in the knockout cells leads to a hyperproliferative response.
Figure 8. Effects of DR6 on thymocyte apoptosis. (A) Apoptosis was induced in thymocytes from wild-type (black bars) and DR6-deficient (white bars) mice with soluble FLAG-tagged FasL and α-FLAG antibody, plate-bound α-CD3 plus α-CD28, etoposide, (more ...)
DR6 Regulates Differentiation of T Cells by Activation of the JNK Pathway
Given the role of the JNK-1 pathway in Th cell differentiation and the polarized Th2 response observed in both JNK-1(12) and DR6 null mice (this study), we asked if JNK activity was correspondingly reduced in DR6-deficient T cells. Purified CD4+
T cells from wild-type or DR6-deficient mice were stimulated with α-CD3 plus α-CD-28 and JNK activity measured using recombinant c-Jun as substrate. In DR6-deficient T cells, JNK activity was significantly reduced ( C), consistent with the notion that attenuation of this MAP kinase contributes to the polarized Th cell differentiation observed in JNK-deficient mice (12