Deletion of PD-1 destabilizes the lineage identity and metabolic fitness of tumor-infiltrating regulatory T cells.

Regulatory T (Treg) cells have an immunosuppressive function and highly express the immune checkpoint receptor PD-1 in the tumor microenvironment; however, the function of PD-1 in tumor-infiltrating (TI) Treg cells remains controversial. Here, we showed that conditional deletion of PD-1 in Treg cells delayed tumor progression. In Pdcd1fl/flFoxp3eGFP-Cre-ERT2(+/-) mice, in which both PD-1-expressing and PD-1-deficient Treg cells coexisted in the same tissue environment, conditional deletion of PD-1 in Treg cells resulted in impairment of the proliferative and suppressive capacity of TI Treg cells. PD-1 antibody therapy reduced the TI Treg cell numbers, but did not directly restore the cytokine production of TI CD8+ T cells in TC-1 lung cancer. Single-cell analysis indicated that PD-1 signaling promoted lipid metabolism, proliferation and suppressive pathways in TI Treg cells. These results suggest that PD-1 ablation or inhibition can enhance antitumor immunity by weakening Treg cell lineage stability and metabolic fitness in the tumor microenvironment.

Viral coinfection promotes tuberculosis immunopathogenesis by type I IFN signaling-dependent impediment of Th1 cell pulmonary influx.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is often exacerbated upon coinfection, but the underlying immunological mechanisms remain unclear. Here, to elucidate these mechanisms, we use an Mtb and lymphocytic choriomeningitis virus coinfection model. Viral coinfection significantly suppresses Mtb-specific IFN-γ production, with elevated bacterial loads and hyperinflammation in the lungs. Type I IFN signaling blockade rescues the Mtb-specific IFN-γ response and ameliorates lung immunopathology. Single-cell sequencing, tissue immunofluorescence staining, and adoptive transfer experiments indicate that viral infection-induced type I IFN signaling could inhibit CXCL9/10 production in myeloid cells, ultimately impairing pulmonary migration of Mtb-specific CD4+ T cells. Thus, our study suggests that augmented and sustained type I IFNs by virus coinfection prior to the pulmonary localization of Mtb-specific Th1 cells exacerbates TB immunopathogenesis by impeding the Mtb-specific Th1 cell influx. Our study highlights a negative function of viral coinfection-induced type I IFN responses in delaying Mtb-specific Th1 responses in the lung.

Establishment of a mechanism-based in vitro coculture assay for evaluating the efficacy of immune checkpoint inhibitors.

Cancer immunotherapy, which blocks immune checkpoint molecules, is an effective therapeutic strategy for human cancer patients through restoration of tumor-infiltrating (TI) cell function. However, evaluating the efficacy of immune checkpoint inhibitors (ICIs) is difficult because no standard in vitro assay for ICI efficacy evaluation exists. Additionally, blocking a particular immune checkpoint receptor (ICR) is insufficient to restore T cell functionality, because other ICRs still transduce inhibitory signals. Therefore, limiting inhibitory signals transduced via other ICRs is needed to more accurately assess the efficacy of ICIs targeting a particular immune checkpoint. Here, we introduce a newly developed in vitro coculture assay using human peripheral blood mononuclear cells (hPBMCs) and engineered human cancer cell lines. We enriched CD8+ T cells from hPBMCs of healthy donors through low-dose T cell receptor stimulation and cytokine (human IL-2 and IL-7) addition. These enriched CD8+ T cells were functional and expressed multiple ICRs, especially TIM-3 and TIGIT. We also established immune checkpoint ligand (ICL) knockout (KO) cancer cell lines with the CRISPR-Cas9 system. Then, we optimized the in vitro coculture assay conditions to evaluate ICI efficacy. For example, we selected the most effective anti-TIM-3 antibody through coculture of TIM-3+CD8+ T cells with PD-L1-/-PVR-/- cancer cells. In summary, we developed a mechanism-based in vitro coculture assay with hPBMCs and ICL KO cancer cell lines, which could be a useful tool to identify promising ICIs by providing reliable ICI efficacy information.

Differential Role of PD-1 Expressed by Various Immune and Tumor Cells in the Tumor Immune Microenvironment: Expression, Function, Therapeutic Efficacy, and Resistance to Cancer Immunotherapy.

In the tumor immune microenvironment (TIME), tumor cells interact with various cells and operate various strategies to avoid antitumor immune responses. These immune escape strategies often make the TIME resistant to cancer immunotherapy. Neutralizing immune escape strategies is necessary to overcome resistance to cancer immunotherapy. Immune checkpoint receptors (ICRs) expressed in effector immune cells inhibit their effector function via direct interaction with immune checkpoint ligands (ICLs) expressed in tumor cells. Therefore, blocking ICRs or ICLs has been developed as a promising cancer immunotherapy by reinvigorating the function of effector immune cells. Among the ICRs, programmed cell death 1 (PD-1) has mainly been antagonized to enhance the survival of human patients with cancer by restoring the function of tumor-infiltrating (TI) CD8+ T cells. It has been demonstrated that PD-1 is expressed not only in TI CD8+ T cells, but also in other TI immune cells and even tumor cells. While PD-1 suppresses the function of TI CD8+ T cells, it is controversial whether PD-1 suppresses or amplifies the suppressive function of TI-suppressive immune cells (e.g., regulatory T cells, tumor-associated macrophages, and myeloid cells). There is also controversy regarding the role of tumor-expressing PD-1. Therefore, a precise understanding of the expression pattern and function of PD-1 in each cell subset is important for improving the efficacy of cancer immunotherapy. Here, we review the differential role of PD-1 expressed by various TI immune cells and tumor cells. We focused on how cell-type-specific ablation or blockade of PD-1 affects tumor growth in a murine tumor model. Furthermore, we will also describe how the blockade of PD-1 acts on TI immune cells in human patients with cancer.

Combination of PD-L1 and PVR determines sensitivity to PD-1 blockade.

Expression of immune checkpoint ligands (ICLs) is necessary to trigger the inhibitory signal via immune checkpoint receptors (ICRs) in exhausted T cells under tumor immune microenvironment. Nevertheless,to our knowledge, ICL expression profile in cancer patients has not been investigated. Using previously reported RNA-seq data sets, we found that expression of ICLs was patient specific but their coexpression can be patterned in non-small-cell lung cancers (NSCLCs). Since the expression of PD-L1 and poliovirus receptor (PVR) among various ICLs was independently regulated, we could stratify the patients who were treated with anti-PD-1 later into 4 groups according to the expression level of PD-L1 and PVR. Of interest, high PVR and low PVR expressions in PD-L1-expressing patients enriched nonresponders and responders to PD-1 blockade, respectively, helping in further selection of responders. Using a genetically engineered cancer model, we also found that PVR-deficient and PD-L1-sufficient tumor-bearing mice were highly sensitive to anti-PD-1 therapy, whereas PVR-sufficient and PD-L1-deficient tumor-bearing mice were resistant to anti-PD-1 therapy. Taken together, our study provides a concept that combinatorial expression patterns of PVR and PD-L1 are key determinants for PD-1 blockade and furthermore suggest a better therapeutic usage of immune checkpoint blockades (ICBs).

Sustained Type I Interferon Reinforces NK Cell-Mediated Cancer Immunosurveillance during Chronic Virus Infection.

The importance of natural killer (NK) cells in the early immune response to viral or bacterial infection is well known. However, the phenotype, function, and physiologic role of NK cells during the late stage of persistent viral infection have not been extensively studied. Here, we characterized NK cells in mice persistently infected with lymphocytic choriomeningitis virus clone 13 and showed that in contrast to NK cells from acutely infected or uninfected mice, NK cells from chronically infected mice expressed a terminally differentiated phenotype, stronger cytotoxicity, and reduced inhibitory receptor expression. In an in vivo tumor model, chronically infected mice exhibited significantly delayed tumor progression in an NK cell-dependent manner. NK cells from chronically infected mice also expressed high STAT1, and blocking the type I interferon (IFN) receptor revealed that type I IFN signaling directly regulated NK cell cytotoxicity. Our findings indicate that sustained type I IFN signaling during chronic viral infection potentiates the cytolytic function of NK cells and contributes to NK cell-dependent host immune surveillance.