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Targeting immune evasion via immune checkpoint pathways has changed the treatment paradigm in cancer. Since CTLA-4 antibody was first approved in 2011 for treatment of metastatic melanoma, eight immune checkpoint inhibitors (ICIs) centered on PD-1 pathway blockade are approved and currently administered to treat 18 different types of cancers. The first part of the review focuses on the history of CTLA-4 and PD-1 discovery and the preclinical experiments that demonstrated the possibility of anti-CTLA-4 and anti-PD-1 as anti-cancer therapeutics. The approval process of clinical trials and clinical utility of ICIs are described, specifically focusing on non-small cell lung cancer (NSCLC), in which immunotherapies are most actively applied. Additionally, this review covers the combination therapy and novel ICIs currently under investigation in NSCLC. Although ICIs are now key pivotal cancer therapy option in clinical settings, they show inconsistent therapeutic efficacy and limited responsiveness. Thus, newly proposed action mechanism to overcome the limitations of ICIs in a near future are also discussed.

Chemokines are key factors that influence the migration and maintenance of relevant immune cells into an infected tissue or a tumor microenvironment. Therefore, it is believed that the controlled administration of chemokines in the tumor microenvironment may be an effective immunotherapy against cancer. Previous studies have shown that CCL3, also known as macrophage inflammatory protein 1-alpha, facilitates the recruitment of dendritic cells (DCs) for the presentation of tumor Ags and promotes T cell activation. Here, we investigated the role of CCL3 in regulating the tumor microenvironment using a syngeneic mouse tumor model. We observed that MC38 tumors overexpressing CCL3 (CCL3-OE) showed rapid regression compared with the wild type MC38 tumors. Additionally, these CCL3-OE tumors showed an increase in the proliferative and functional tumor-infiltrating T cells. Furthermore, PD-1 immune checkpoint blockade accelerated tumor regression in the CCL3-OE tumor microenvironment. Next, we generated a modified CCL3 protein for pre-clinical use by fusing recombinant CCL3 (rCCL3) with a non-cytolytic hybrid Fc (HyFc). Administering a controlled dose of rCCL3-HyFc via subcutaneous injections near tumors was effective in tumor regression and improved survival along with activated myeloid cells and augmented T cell responses. Furthermore, combination therapy of rCCL3-HyFc with PD-1 blockade exhibited prominent effect to tumor regression. Collectively, our findings demonstrate that appropriate concentrations of CCL3 in the tumor microenvironment would be an effective adjuvant to promote anti-tumor immune responses, and suggest that administering a long-lasting form of CCL3 in combination with PD-1 blockers can have clinical applications in cancer immunotherapy.

Acute viral infection or vaccination generates highly functional memory CD8 T cells following the Ag resolution. In contrast, persistent antigenic stimulation in chronic viral infection and cancer leads to a state of T-cell dysfunction termed T-cell exhaustion. We and other have recently identified a novel subset of exhausted CD8 T cells that act as stem cells for maintaining virus-specific CD8 T cells in a mouse model of chronic lymphocytic choriomeningitis virus infection. This stem cell-like CD8 T-cell subset has been also observed in both mouse and human tumor models. Most importantly, in both chronic viral infection and tumor models, the proliferative burst of Ag-specific CD8 T cells driven by PD-1-directed immunotherapy comes exclusively from this stem cell-like CD8 T-cell subset. Therefore, a better understanding of the mechanisms how CD8 T-cell subsets are regulated during chronic viral infection and cancer is required to improve the current immunotherapies that restore the function of exhausted CD8 T cells. In this review, we discuss the differentiation of virus-specific CD8 T cells during chronic viral infection, the characteristics and function of CD8 T-cell subsets, and the therapeutic intervention of PD-1-directed immunotherapy in cancer.

Quantitative PCR and plaque assay are powerful virological techniques used to measure the load of defective or infectious virus in mouse and human. However, these methods display limitations such as cross contamination and long run-time. Here, we describe a novel technique termed as semi-functional quantitative flow cytometry (SFQF) for the accurate estimation of the quantity of infectious lymphocytic choriomeningitis virus (LCMV). LCMV titration method using flow cytometry was previously developed but has technical shortcomings, owing to the less optimized parameters such as cell overgrowth, plate scale, and detection threshold. Therefore, we first established optimized conditions for SFQF assay using LCMV nucleoprotein (NP)-specific antibody to evaluate the threshold of the virus detection range in the plaque assay. We subsequently demonstrated that the optimization of the method increased the sensitivity of virus detection. We revealed several new advantages of SFQF assay, which overcomes some of the previously contentious points, and established an upgraded version of the previously reported flow cytometric titration assay. This method extends the detection scale to the level of single cell, allowing extension of its application for in vivo detection of infected cells and their phenotypic analysis. Thus, SFQF assay may serve as an alternative analytical tool for ensuring the reliability of LCMV titration and can be used with other types of viruses using target-specific antibodies.

CD80 is mainly expressed on Ag-presenting cells (APCs) as a costimulatory molecule but is also detected on T cells. However, the origin and physiological role of CD80 on CD8+ T cells remain unclear. In the present study, we demonstrated that effector and memory CD8+ T cells, but not naïve CD8+ T cells, displayed CD80 molecules on their surfaces after acute lymphocytic choriomeningitis virus infection. Using adoptive transfer of CD80-knockout (KO) CD8+ T cells into a wild type or CD80-KO recipient, we demonstrated that the effector CD8+ T cells displayed CD80 by both intrinsic expression and extrinsic acquisition, while memory CD8+ T cells displayed CD80 only by extrinsic acquisition. Interestingly, the extrinsic acquisition of CD80 by CD8+ T cells was observed only in the lymphoid organs but not in the periphery, indicating the trogocytosis of CD80 molecules via interaction between CD8+ T cells and APCs. We compared the recall immune responses by memory CD8+ T cells that either extrinsically acquired CD80 or were deficient in CD80, and found that CD80, presented by memory CD8+ T cells, played a role in limiting their expansion and IL-2 production upon exposure to secondary challenge. Our study presents the in vivo dynamics of the extrinsic acquisition of CD80 by Ag-specific CD8+ T cells and its role in the regulation of recall immune responses in memory CD8+ T cells.

CD80 is mainly expressed on Ag-presenting cells (APCs) as a costimulatory molecule but is also detected on T cells. However, the origin and physiological role of CD80 on CD8+ T cells remain unclear. In the present study, we demonstrated that effector and memory CD8+ T cells, but not naive CD8+ T cells, displayed CD80 molecules on their surfaces after acute lymphocytic choriomeningitis virus infection. Using adoptive transfer of CD80-knockout (KO) CD8+ T cells into a wild type or CD80-KO recipient, we demonstrated that the effector CD8+ T cells displayed CD80 by both intrinsic expression and extrinsic acquisition, while memory CD8+ T cells displayed CD80 only by extrinsic acquisition. Interestingly, the extrinsic acquisition of CD80 by CD8+ T cells was observed only in the lymphoid organs but not in the periphery, indicating the trogocytosis of CD80 molecules via interaction between CD8+ T cells and APCs. We compared the recall immune responses by memory CD8+ T cells that either extrinsically acquired CD80 or were deficient in CD80, and found that CD80, presented by memory CD8+ T cells, played a role in limiting their expansion and IL-2 production upon exposure to secondary challenge. Our study presents the in vivo dynamics of the extrinsic acquisition of CD80 by Ag-specific CD8+ T cells and its role in the regulation of recall immune responses in memory CD8+ T cells.

Unlike conventional T cells, innate CD8 T cells develop a memory-like phenotype in the thymus and immediately respond upon antigen stimulation, similar to memory T cells. The development of innate CD8 T cells in the thymus is known to require IL-4, which upregulates Eomesodermin (Eomes). These features are similar to that of virtual memory CD8 T cells and IL-4-induced memory-like CD8 T cells generated in the peripheral tissues. However, the relationship between these cell types has not been clearly documented. In the present study, IL-4-induced memory-like CD8 T cells generated in the peripheral tissues were compared with innate CD8 T cells in terms of phenotype and function. When an IL-4/anti-IL-4 antibody complex (IL-4C) was injected into C57BL/6 mice daily for 7 days, the Eomes(hi)CXCR3 (+) CD8 T cell population was markedly increased in the peripheral lymphoid organs and blood. These cells were generated from naive CD8 T cells or accumulated via the expansion of pre-existing CD44(hi)CXCR3 (+) CD8 T cells. Initially, the majority of these CXCR3 (+) CD8 T cells expressed low levels of CD44, which was followed by the conversion to the CD44(hi) phenotype. This conversion was associated with the acquisition of enhanced effector function. After discontinuation of IL-4C treatment, Eomes expression levels gradually decreased in CXCR3 (+) CD8 T cells. Taken together, the results of this study demonstrate that IL-4-induced memory-like CD8 T cells generated in the peripheral lymphoid tissues are phenotypically and functionally similar to the innate CD8 T cells generated in the thymus.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that bridge innate and adaptive immune responses, thereby leading to immune activation. DCs have been known to recognize pathogen-associated molecular patterns such as lipopolysaccharides (LPS) and nucleic acids via their pattern recognition receptors, which trigger signaling of their maturation and effector functions. Furthermore, DCs take up and process antigens as a form of peptide loaded on the major histocompatibility complex (MHC) and present them to T cells, which are responsible for the adaptive immune response. Conversely, DCs can also play a role in inducing immune suppression under specific circumstances. From this perspective, the role of DCs is related to tolerance rather than immunity. Immunologists refer to these special DCs as tolerogenic DCs (tolDCs). However, the definition of tolDCs is controversial, and there is limited information on their development and characteristics. In this review, we discuss the current concept of tolDCs, cutting-edge methods for generating tolDCs in vitro, and future applications of tolDCs, including clinical use.

Chronic virus infection leads to the functional impairment of dendritic cells (DCs) as well as T cells, limiting the clinical usefulness of DC-based therapeutic vaccine against chronic virus infection. Meanwhile, B cells have been known to maintain the ability to differentiate plasma cells producing antibodies even during chronic virus infection. Previously, α-galactosylceramide (αGC) and cognate peptide-loaded B cells were comparable to DCs in priming peptide-specific CD8(+) T cells as antigen presenting cells (APCs). Here, we investigated whether B cells activated by αGC can improve virus-specific T cell immune responses instead of DCs during chronic virus infection. We found that comparable to B cells isolated from naïve mice, chronic B cells isolated from chronically infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) after αGC-loading could activate CD1d-restricted invariant natural killer T (iNKT) cells to produce effector cytokines and upregulate co-stimulatory molecules in both naïve and chronically infected mice. Similar to naïve B cells, chronic B cells efficiently primed LCMV glycoprotein (GP) 33-41-specific P14 CD8(+) T cells in vivo, thereby allowing the proliferation of functional CD8(+) T cells. Importantly, when αGC and cognate epitope-loaded chronic B cells were transferred into chronically infected mice, the mice showed a significant increase in the population of epitope-specific CD8(+) T cells and the accelerated control of viremia. Therefore, our studies demonstrate that reciprocal activation between αGC-loaded chronic B cells and iNKT cells can strengthen virus-specific T cell immune responses, providing an effective regimen of autologous B cell-based therapeutic vaccine to treat chronic virus infection.