RORgamma t, a novel isoform of an orphan receptor, negatively regulates Fas ligand expression and IL-2 production in T cells

Abstract

We have identified RORgamma t, a novel, thymus-specific isoform of the orphan nuclear receptor RORgamma that is expressed predominantly in CD4+ CD8+ double-positive thymocytes. Ectopic expression of RORgamma t protects T cell hybridomas from activation-induced cell death by inhibiting the upregulation of Fas ligand. Following hybridoma stimulation, RORgamma t also inhibits IL-2 production but does not affect the induction of Nur-77 and Egr-3 nor the upregulation of CD69. Both the ligand-binding and DNA-binding domains of RORgamma t are required for this effect. We propose that the role of RORgamma t expression in immature thymocytes is to inhibit Fas ligand expression and cytokine secretion following engagement of their TCR during positive or negative selection.

Figure 1. RORγt Is a Novel Isoform of RORγ

(A) cDNA sequence of RORγt and its predicted amino acid sequence. The cDNA sequence of RORγt was derived from sequencing the rescued cDNA insert from DO11.10 clones that were resistant to PMA plus ionomycin-induced cell death. Several independent RT-PCR from thymocyte RNA yielded the same sequence. Sequences for the DBD (aa 10–75) and LBD (aa 305–460) are underlined. The 3′ untranslated region is identical to that of RORγ (Medvedev et al., 1997) and is not included. (B) Sequence comparison of RORγt and RORγ. Aligned are the first 114 nt and the predicted amino acid sequence for RORγt and RORγ. They share the same sequence beginning with the underlined nucleotides through the 3′ end of each cDNA.

Figure 2. Differential Expression of RORγt and RORγ

(A) Schematic representation of RORγt and RORγ cDNAs and the locations of the primers used in (B) and (C). The shaded region represents the distinct nucleotide sequences. Specific primers are 1 and 2. Common primers are 3, 4, and 5. Diagram is not to scale. (B) Expression of RORγt and RORγ. RT-PCR products of different tissues were blotted and probed with a full-length RORγt cDNA. HPRT serves as an internal control. Numbers in parentheses indicate the primers shown in (A) used in the PCR. (C) Expression of RORγt in thymocyte subpopulations. RT reactions of different thymocyte subpopulations were serially diluted at 1:3 and subjected to PCR with primers shown in (A). PCR products were blotted and probed as in (B). HPRT RT-PCR was performed from the same RT samples for cDNA template quantity control.

Figure 3. Expression of RORγt Protects T Cell Hybridomas from Activation-Induced Cell Death

(A) Vector control cells and DO11.10 or KMIs-8.3.5 clones expressing RORγt were cultured in either 2C11-coated plates or in PMA (10 ng/ml) plus various amounts of ionomycin and measured for their [³H]Thymidine incorporation. The [³H]Thymidine uptake of individual cell lines cultured in medium alone is calculated as 100%. Shown are the results from two individual hybridoma clones expressing RORγt (circles) or control cells (squares). (B) KMIs-8.3.5 vector control cells and a clone expressing RORγt were cultured in PMA (10 ng/ml) plus ionomycin (0.2 μg/ml) for 16 hr. Apoptotic cells with subdiploid DNA content were determined by propidium iodide uptake, and the percentage is indicated.

Figure 4. Protection of T Hybridomas from Activation-Induced Apopotosis by RORγt Deletion Mutants or RORγ

(A) Both the DBD and the LBD of RORγt are required to protect T cell hybridomas from activation-induced cell death. DO11.10 or KMIs-8.3.5 cells expressing full-length or truncated RORγt or hCD2 were cultured in 2C11-coated plates (125 ng/ml) and tested for their [³H]Thymidine incorporation. Incorporation of cells cultured in medium alone is calculated as 100%. Numbers indicate the amino acids contained in each RORγt deletion mutant. An initiation methionine was added to RORγt deletion mutant 113–495. (B) FACS analysis of hCD2 expression on KMIs-8.3.5 cells expressing RORγ or RORγt. Polyclonal KMIs-8.3.5 cell lines expressing RORγ or RORγt were generated by panning on anti-hCD2 MAb-coated plates, and adherent cells were stained with a FITC-anti-hCD2 as indicated. The profile at the far left represents parental KMIs-8.3.5 cells stained with anti-hCD2 MAb. (C) Comparison of the anti-apopototic effects by RORγ and RORγt. KMIs-8.3.5 cells expressing RORγ (diamonds), RORγt (circles), or control cells (squares) shown in (B) were cultured in 2C11-coated plates and tested as described in (A).

Figure 5. RORγt Inhibits Activation-Induced Fas Ligand Upregulation in KMIs-8.3.5 Cells

(A) FACS analysis of cell surface expression of Fas, Fas ligand, and CD69 in KMIs-8.3.5hCD2 control or KMIs-8.3.5RORγt cells after TCR-mediated activation. KMI-8.3.5 cells expressing RORγt and control cells were activated on 2C11-coated plates for 4.5 hr and the cell surface phenotype was examined by FACS (filled, before stimulation; unfilled, after stimulation). A similar pattern was observed when PMA (10 ng/ml) and ionomycin (0.2 μg/ml) were used as stimuli (data not shown). (B) Northern blot analysis of RNA expression of Fas ligand, Nur-77, and Egr-3 in KMIs-8.3.5hCD2 control, or KMIs-8.3.5RORγt cells after TCR-mediated activation. Cells were activated in 2C11-coated plates (125 ng/ml) for the indicated time, harvested, and extracted for total RNA. 15 μg total RNA was used for each sample. GAPDH serves as a loading control.

Figure 6. Expression of RORγt Inhibits IL-2 Production by T Cell Hybridomas

KMIs-8.3.5hCD2 control (squares) or KMIs-8.3.5RORγt cells (circles) were cultured for 20 hr in 96-well plates coated with the indicated amount of 2C11, and the supernatants were tested for IL-2 using a HT-2 bioassay. Shown are representative results from three similar experiments.

Figure 7. Expression of RORγt in T Cell Hybridoma Does Not Prevent Cell Death Induced by Fas Signaling or Other Stimuli

(A) RORγt does not inhibit Fas-induced killing in KMIs-8.3.5 cells. KMIs-8.3.5hCD2 control or KMIs-8.3.5RORγt cells were cocultured with L929 or L929FasL cells at a ratio of 1:3. Two days later, the percentage of hCD2 positive cells from cells cocultured with L929 (empty column) or L929FasL (filled column) were determined by FACS. (B) RORγt does not inhibit cell death induced by dexamethasone, ceramide, and staurosporine. KMIs-8.3.5hCD2 control or KMIs-8.3.5RORγt cells were cultured in the presence of indicated stimuli and [³H]Thymidine incorporation measured. [³H]Thymidine uptake of cells cultured in medium alone is calculated as 100%. Shown are results from two individual KMIs-8.3.5RORγt clones (circles) and KMIs-8.3.5hCD2 control cells (squares).