Quantitative biochemistry and cell biology assays revealed that PD-L1:CD80 cisheterodimerization inhibited both PD-L1:PD-1 and CD80:CTLA-4 interactions through distinct mechanisms but preserved the ability of CD80 to activate the T cell co-stimulatory receptor CD28. Furthermore, PD-L1 expression on antigen-presenting cells (APCs) prevented CTLA-4-mediated trans-endocytosis of CD80. Atezolizumab (anti-PD-L1), but not anti-PD-1, reduced cell surface expression of CD80 on APCs, and this effect was negated by co-blockade of CTLA-4 with ipilimumab (anti-CTLA-4). Thus, PD-L1 exerts an immunostimulatory effect by repressing the CTLA-4 axis; this has implications to the synergy of anti-PD-L1 and anti-CTLA-4 combination therapy.

SUMMARYThe PD-1 pathway, consisting of the co-inhibitory receptor PD-1 on T cells and its ligand (PD-L1) on antigen-presenting cells (APCs), is a major mechanism of tumor immune evasion. PD-1 and PD-L1 blockade antibodies have produced remarkable clinical activities against a subset of cancers. Binding between T cell-intrinsic PD-1 and APC-intrinsic PD-L1 triggers inhibitory signaling to attenuate the T cell response. Here, we report that PD-1 is co-expressed with PD-L1 on tumor cells and tumor-infiltrating APCs. Using reconstitution and cell culture assays, we demonstrate that the co-expressed PD-1 binds to PD-L1 in cis. Such interaction inhibits the ability of PD-L1 to bind T cell-intrinsic PD-1 in trans and, in turn, represses canonical PD-L1/PD-1 inhibitory signaling. Selective blockade of tumor-intrinsic PD-1 frees up tumor-intrinsic PD-L1 to inhibit T cell signaling and cytotoxicity. Our study uncovers another dimension of PD-1 regulation, with important therapeutic implications.

Blockade antibodies of the immunoinhibitory receptor PD-1 can stimulate the anti-tumor activity of T cells, but clinical benefit is limited to a fraction of patients. Evidence suggests that BTLA, a receptor structurally related to PD-1, may contribute to resistance to PD-1 targeted therapy, but how BTLA and PD-1 differ in their mechanisms is debated. Here, we compared the abilities of BTLA and PD-1 to recruit effector molecules and to regulate T cell signaling. While PD-1 selectively recruited SHP2 over the stronger phosphatase SHP1, BTLA preferentially recruited SHP1 to more efficiently suppress T cell signaling. Contrary to the dominant view that PD-1 and BTLA signal exclusively through SHP1/2, we found that in SHP1/2 double-deficient primary T cells, PD-1 and BTLA still potently inhibited cell proliferation and cytokine production, albeit more transiently than in wild type T cells. Thus, PD-1 and BTLA can suppress T cell signaling through a mechanism independent of both SHP1 and SHP2.

Cancer stem-like cells (CSC) are thought to drive tumor initiation, metastasis, relapse, and therapeutic resistance, but their specific pathogenic characters in many cancers, including non-small cell lung cancer (NSCLC), have yet to be well defined. Here, we develop findings that the growth factor HGF promotes CSC sphere formation in NSCLC cell populations. In patient-derived sphere-forming assays (PD-SFA) with HGF, CD49f and CD104 were defined as novel markers of lung CSC (LCSC). In particular, we isolated a subpopulation of CD166CD49fCD104Lin LCSC present in all human specimens of NSCLC examined, regardless of their histologic subtypes or genetic driver mutations. This specific cell population was tumorigenic and capable of self-renewal, giving rise to tumor spheres and orthotopic lung tumors in immune-compromised mice. Mechanistic investigations established that was preferentially expressed in this cell subpopulation and required for self-renewal via the transcription factor HES1. Through a distinct HES1-independent pathway, NOTCH1 also protected LCSCs from cisplatin-induced cell death. Notably, treatment with a γ-secretase inhibitor that blunts NOTCH1 function ablated self-renewing LCSC activity and restored platinum sensitivity and Overall, our results define the pathogenic characters of a cancer stem-like subpopulation in lung cancer, the targeting of which may relieve platinum resistance in this disease. .