PURPOSE: Immunotherapy targeting the PD-1/PD-L1 pathway has changed the treatment landscape of non-small-cell lung carcinoma (NSCLC). We demonstrated that HHLA2, a newly identified immune inhibitory molecule, was widely expressed in NSCLC. We now compared the expression and function of PD-L1 with alternative immune checkpoints, B7x and HHLA2. EXPERIMENTAL DESIGN: Expression was examined in tissue microarrays consisting of 392 resected NSCLC tumors. Effects of PD-L1, B7x and HHLA2 on human T cell proliferation and cytokine production were investigated. RESULTS: PD-L1 expression was identified in 25% and 31% of tumors in the discovery and validation cohorts, and was associated with higher stage and lymph node involvement. The multivariate analysis showed that stage, TIL status and lymph node involvement were independently associated with PD-L1 expression. B7x was expressed in 69% and 68%, while HHLA2 was positive in 61% and 64% of tumors in the two sets. The co-expression of PD-L1 with B7x or HHLA2 was infrequent, 6% and 3%. The majority (78%) of PD-L1 negative cases expressed B7x, HHLA2 or both. The triple positive group had more TIL infiltration than the triple negative group. B7x-Ig and HHLA2-Ig inhibited TCR-mediated proliferation of CD4 and CD8 T cells more robustly than PD-L1-Ig. All three significantly suppressed cytokine productions by T cells. CONCLUSION: The majority of PD-L1 negative lung cancers express alternative immune checkpoints. The roles of the B7x and HHLA2 pathway in mediating immune evasion in PD-L1 negative tumors deserves to be explored to provide the rationale for an effective immunotherapy strategy in these tumors.