The efficacy of chimeric antigen receptor T cells (CART) in solid tumors is limited by immune inhibition. In our study, we observed that effector cytokines mediated the upregulation of the PD-L1 immune checkpoint in primary glioblastoma. To offset the PD-L1 inhibitory signal, we engineered PD-1 checkpoint reversal receptors (CPR) with a CD28 or 41BB costimulatory endodomain and coexpressed them with a first-generation or a CD28-containing second-generation HER2-specific CAR (CPR/CART) using bicistronic vectors. We found that bipartite T-cell activation, by CAR-generated signal 1 and CPR costimulation (signal 2), fine-tuned proinflammatory cytokine release and sustained antitumor activity. Whereas both CPR28 and CPR41BB effectively counteracted the PD-1 signal in vitro, CPR41BB, when coexpressed with a first-generation CAR (CARζ/CPR41BB), promoted central memory differentiation following repeat antigenic stimulation. CARζ/CPR41BB T cells formed a robust immune synapse with tumor targets, similar to a 41BB-containing second-generation CART, maintained the favorable metabolic parameters associated with 41BB costimulation, and demonstrated superior antitumor function after adoptive transfer in xenograft models of gioblastoma and metastatic osteosarcoma. Thus, a CPR molecule with 41BB costimulation that curtails PD-1 inhibition and complements CAR signaling to optimize T-cell activation could enhance CART efficacy against solid tumors.
Enhancing CART function and persistence while balancing immune effector-mediated inflammation is crucial. Using our clinically relevant HER2-CAR platform, we demonstrate that tumor-intrinsic signals like the PD-1/PD-L1 immune checkpoint can be leveraged in CART design to modulate immune synapse and metabolic parameters, improving antitumor function without increasing cytokine production.
©2025 The Authors; Published by the American Association for Cancer Research.

Purpose: BRAF and MEK inhibitors (BRAF/MEKi) favor melanoma-infiltrating lymphocytes, providing the rationale for current combinatorial trials with anti-PD-1 antibody. A portion of melanoma cells may express PD-1, and anti-PD-1 antibody could have a direct antitumor effect. Here, we explore whether BRAF/MEKi modulate rates of PD-1+ melanoma cells, supporting an additional-lymphocyte-independent-basis for their therapeutic combination with anti-PD-1 antibody.Experimental Design: With data mining and flow cytometry, we assessed PD-1, PD-L1/2 expression on melanoma cell lines (CCLE, N = 61; validation cell lines, N = 7) and melanoma tumors (TCGA, N = 214). We explored in vitro how BRAF/MEKi affect rates of PD-1+, PD-L1/2+ melanoma cells, and characterized the proliferative and putative stemness features of PD-1+ melanoma cells. We tested the functional lymphocyte-independent effect of anti-PD-1 antibody alone and in combination with BRAF/MEKi in vitro and in an in vivo immunodeficient murine model.Results: PD-1 is consistently expressed on a small subset of melanoma cells, but PD-1+ cells increase to relevant rates during BRAF/MEKi treatment [7.3% (5.6-14.2) vs. 1.5% (0.7-3.2), P = 0.0156; N = 7], together with PD-L2+ melanoma cells [8.5% (0.0-63.0) vs. 1.5% (0.2-43.3), P = 0.0312; N = 7]. PD-1+ cells proliferate less than PD-1- cells (avg. 65% less; t = 7 days) and are preferentially endowed with stemness features. In vivo, the direct anti-melanoma activity of PD-1 blockage as monotherapy was negligible, but its association with BRAF/MEKi significantly delayed the development of drug resistance and tumor relapse.Conclusions: BRAF/MEKi increase the rates of PD-1+ melanoma cells that may sustain tumor relapse, providing a lymphocyte-independent rationale to explore combinatory strategies with anti-PD-1 antibody. Clin Cancer Res; 24(14); 3377-85. ©2018 AACR.
©2018 American Association for Cancer Research.

Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor-ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4+ and CD8+ T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus.

Vitamin D modulates inflammation in Crohn's disease (CD). Programmed death (PD)-1 receptor contributes to the maintenance of immune tolerance. Vitamin D might modulate PD-1 signalling in CD.
To investigate PD-1 expression on T cell subsets in CD patients treated with vitamin D or placebo.
We included 40 CD patients who received 1200 IU vitamin D3 for 26 weeks or placebo and eight healthy controls. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated at baseline and week 26. The expressions of PD-1, PD-L1, and surface activation markers were analysed by flow cytometry. Soluble PD-1 plasma levels were measured by ELISA.
PD-1 expression upon T cell stimulation was increased in CD4+CD25+int T cells in vitamin D treated CD patients from 19% (range 10 - 39%) to 29% (11 - 79%)(p = 0.03) compared with placebo-treated patients. Vitamin D treatment, but not placebo, decreased the expression of the T cell activation marker CD69 from 42% (31 - 62%) to 33% (19 - 54%)(p = 0.01). Soluble PD-1 levels were not influenced by vitamin D treatment.
Vitamin D treatment increases CD4+CD25+int T cells ability to up-regulate PD-1 in response to activation and reduces the CD69 expression in CD patients.