Our data suggest that sTL1A is potentially a useful adjuvant that can be combined with vaccines aimed at eliciting human anti-tumor CD8+ T-cell responses. J558L plasmacytoma cells originally derived from BALB/c mice were obtained from the European Collection of Cell Cultures and J558L cells expressing TL1A were described previously 16. Soluble recombinant TL1A (sTL1A) was produced as a fusion protein with domains 3 and 4 of rat CD4 in CHO cells using previously described methods 17. Briefly, DNA encoding the extracellular domain of mouse TL1A

(amino acids 77–252) was cloned downstream of the sequence encoding the leader peptide and domains 3 and 4 of rat CD4 in the expression vector pEE14 17. Anti-TL1A mAb (TAN2-2) was described previously 16, and biotinylated anti-TNFRSF25 Ab was obtained from R&D Systems. Caspase inhibitor clinical trial PE-labeled KbOVA257–264 tetramer was produced by the Cancer Sciences Division Protein Expression Facility. Splenocytes from OT-I transgenic mice were depleted of CD4+ T cells (>98%) and cultured at 2×105 cells/well for 48 h (for the determination of IL-2) or 72 h (T-cell proliferation).

In some experiments, we isolated highly purified CD8+ T cells (≥95%) from WT or tnfrsf25 KO mice using a CD8 T-cell isolation kit (Miltenyi Biotec). Pure CD8+ T cells (1×105) were then stimulated with either plate-bound anti-CD3 or soluble anti-CD3 and irradiated (30 Gy) WT splenocytes as antigen-presenting cells. Where indicated sTL1A (2 μg/mL), neutralizing NVP-LDE225 anti-TL1A mAb (50 μg/mL) or anti-BCL1 Id control IgG (Mc39-16; 50 μg/mL) was added. RNA was extracted from splenocytes using the RNeasy mini kit (Qiagen) and cDNA generated with the Superscript III first-strand synthesis system (Invitrogen).

mRNA expression analyses were performed by qRT-PCR using TaqMan gene expression assays for granzyme B GPX6 (Mm00442834_m1), perforin (Mm00812512_m1) and IL-2 (Mm00434256_m1). Expression was normalized to that of CD3δ (Mm00442746_m1). For monitoring tumor growth, 5×106 tumor cells were injected s.c. into mice and tumor size (product of two perpendicular diameters) measured using calipers. CD4+ and CD8+ T-cell depletion was carried out by injection of anti-CD4 mAb (YTA3.1.2; 1 mg) or anti-CD8 mAb (YTS 169; 0.5 mg) on days –3, –1 and 3. Depletion was confirmed (days 6 and 15) by FACS analysis of blood samples. For adoptive transfer, 106 unlabeled or CFSE-labeled (10 μM; 10 min) OT-I cells were injected into C57BL/6 mice and OVA257–264 peptide (30 nmol) administered i.v. alone or with sTL1A (150 μg). Mice then received two additional injections of sTL1A on consecutive days. OVA-specific CD8+ T cells were enumerated by labeling with anti-CD8 mAb and KbOVA257–264 tetramer. Endotoxin levels of recombinant sTL1A were <1 ng/mg. In some groups, mice also received three consecutive doses of neutralizing anti-TL1A mAb (250 μg) to demonstrate the specificity of sTL1A.