RELT, a new member of the tumor necrosis factor receptor superfamily, is selectively expressed in hematopoietic tissues and activates transcription factor NF-κB by Gabriel L. Sica, Gefeng Zhu, Koji Tamada, Ding Liu, Jian Ni, and Lieping Chen Blood Volume 97(9):2702-2707 May 1, 2001 ©2001 by American Society of Hematology

Sequence analysis of RELT. (A) Amino acid sequence of RELT Sequence analysis of RELT.(A) Amino acid sequence of RELT. Single underline is signal peptide, double underline is location of transmembrane region as determined by hydrophilicity analysis. Sequence analysis of RELT.(A) Amino acid sequence of RELT. Single underline is signal peptide, double underline is location of transmembrane region as determined by hydrophilicity analysis. Arrow indicates potential proteolytic cleavage site. Asterisk indicates potential N-glycosylation site. Sequence analysis was performed with MacVector 6.5. (B) Amino acid alignment of the cysteine-rich extracellular domains of RELT with the cysteine-rich domain of other members of the TNFR superfamily. Dark gray indicates identical amino acid, and light gray indicates similar amino acid homologies. Amino acid alignment was performed with ClustaW. (C) Alignment of human RELT with the putative rat RELT derived from a partial EST sequence and full-length macaque sequence. GenBank Accession numbers AI071067, AB046039, and AF319553 are for rat, macaque, and human sequences, respectively. Gabriel L. Sica et al. Blood 2001;97:2702-2707 ©2001 by American Society of Hematology

RELT-hFc fusion protein and its expression RELT-hFc fusion protein and its expression.(A) Diagram of RELT-hFc fusion protein construct. RELT-hFc fusion protein and its expression.(A) Diagram of RELT-hFc fusion protein construct. ECD is the extracellular domain of RELT that is linked to the Fc portion of hIgG1. ARR/GV is the amino acid sequence of the proposed cleavage site for the truncated form of the soluble protein. (B) Western blot analysis of recombinant RELT-hFc fusion protein from transfected 293 supernatant. After SDS-PAGE and transfer to nitrocellulose filter paper, fusion protein was detected with a goat antihuman Fc specific HRP-conjugated polyclonal antibody. Gabriel L. Sica et al. Blood 2001;97:2702-2707 ©2001 by American Society of Hematology

Tissue distribution of RELT mRNA Tissue distribution of RELT mRNA.Northern blot analysis of RELT mRNA distribution in (A) normal human tissues and (B) transformed human cell lines. Tissue distribution of RELT mRNA.Northern blot analysis of RELT mRNA distribution in (A) normal human tissues and (B) transformed human cell lines. Gabriel L. Sica et al. Blood 2001;97:2702-2707 ©2001 by American Society of Hematology

RELT is able to activate NF-κB and binds TRAF1 RELT is able to activate NF-κB and binds TRAF1.(A) Activation of NF-κB by RELT. The 293 cells were transfected with increasing doses of RELT, starting at 0.1 μg, 0.2 μg, 0.5 μg, 1.0 μg, and 2.0 μg of DNA. Vector and RELT cytoplasmic domain deletion mutant (... RELT is able to activate NF-κB and binds TRAF1.(A) Activation of NF-κB by RELT. The 293 cells were transfected with increasing doses of RELT, starting at 0.1 μg, 0.2 μg, 0.5 μg, 1.0 μg, and 2.0 μg of DNA. Vector and RELT cytoplasmic domain deletion mutant (RELT-cyt) were used at a concentration of 2.0 μg. All wells were transfected with 0.5 μg κB-Luc and 0.5 μg TK-βgal. The total amount of DNA transfected in all wells was 3 μg. All values are normalized to β-gal activity. The experiment is representative of 3 experiments. (B) RELT binds TRAF1. The 293 cells were transfected with 5 μg RELT-HA and 5 μg of either FLAG TRAF 1, 2, 3, 5, or 6. Cell lysates were immunoprecipitated (I.P.) with anti-FLAG beads, separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted (I.B.) with anti-HA HRP. Blots were then stripped and re-probed with anti-FLAG M2 to detect the presence of tagged proteins. (C) Activation of NF-κB by RELT is not inhibited by a dominant-negative form of TRAF2. The 293T cells were transfected with a total of 3 μg DNA. All wells were transfected with 0.25 μg κB-luc and 0.25 μg TK-βgal. Cells were also transfected with 2 μg of either vector, RELT, or HVEM and 0.5 μg TRAF2DN or vector. Transfections were performed in duplicates, and this experiment is representative of 2 experiments. Gabriel L. Sica et al. Blood 2001;97:2702-2707 ©2001 by American Society of Hematology

Effect of RELT-hFc fusion protein in T-cell responses Effect of RELT-hFc fusion protein in T-cell responses.(A) RELT-hFc binds PMA and ionomycin-activated T cells. Effect of RELT-hFc fusion protein in T-cell responses.(A) RELT-hFc binds PMA and ionomycin-activated T cells. Nylon wool–purified T cells were activated for 18 hours in 200 ng/mL ionomycin and 20 ng/mL PMA and then double stained with RELT-hFc biotin and anti-CD3 phycoerythrin. Histogram shows CD3 gated cells. Filled histogram is hIgG biotin control, and open histogram is RELT-hFc biotin. (B) RELT-hFc costimulates T-cell proliferation. For proliferation assays, 96-well plates were coated with the indicated concentration of anti-CD3, washed with PBS, and coated with 10 μg/mL of the indicated protein. After coating with fusion protein, wells were washed again with PBS, and T cells were added. T cells were incubated for 72 hours and pulsed with 1 μCi 3H thymidine for the last 18 hours before being harvested. All CPM values are from triplicate wells. This experiment is representative of 3 experiments. (C) Addition of soluble RELT-hFc selectively inhibits RELT-hFc costimulation of T-cell proliferation. Proliferation assays were performed as in (A). Soluble RELT-hFc was added at a dose of 10 μg/mL, and the dose of anti-CD3 is 6.25 ng/mL. (D) RELT-hFc does not inhibit the one-way mixed lymphocyte reaction. Nylon wool–purified T cells and stimulator PBMCs that had been irradiated for 3000 rads were incubated at various responder-to-stimulator ratios with 10 μg/mL hIgG or RELT-hFc. The mixed cells were incubated for 5 days and pulsed with 1 μCi 3H thymidine for the last 18 hours. All CPM values are from triplicate wells. This graph is representative of 3 experiments. Gabriel L. Sica et al. Blood 2001;97:2702-2707 ©2001 by American Society of Hematology