The discovery of immune checkpoints provided a breakthrough for cancer therapy. Immune checkpoints are inhibitory receptors that are up-regulated on chronically stimulated lymphocytes and have been shown to hinder immune responses to cancer. Monoclonal antibodies against the checkpoint molecules PD-1 and CTLA-4 have shown early clinical success against melanoma and are now approved to treat various cancers. Since then, the list of potential candidates for immune checkpoint blockade has dramatically increased. The current paradigm stipulates that immune checkpoint blockade therapy unleashes pre-existing T cell responses. However, there is accumulating evidence that some of these immune checkpoint molecules are also expressed on Natural Killer (NK) cells. In this review, we summarize our latest knowledge about targetable NK cell inhibitory receptors. We discuss the HLA-binding receptors KIRS and NKG2A, receptors binding to nectin and nectin-like molecules including TIGIT, CD96, and CD112R, and immune checkpoints commonly associated with T cells such as PD-1, TIM-3, and LAG-3. We also discuss newly discovered pathways such as IL-1R8 and often overlooked receptors such as CD161 and Siglecs. We detail how these inhibitory receptors might regulate NK cell responses to cancer, and, where relevant, we discuss their implications for therapeutic intervention.

Ag-specific tolerizing immunotherapy is considered the optimal strategy to control type 1 diabetes, a childhood disease involving autoimmunity toward multiple islet antigenic peptides. To understand whether tolerizing immunotherapy with a single peptide could control diabetes driven by multiple Ags, we coencapsulated the high-affinity CD4 + mimotope (BDC2.5 mim ) of islet autoantigen chromogranin A (ChgA) with or without calcitriol (1a,25-dihydroxyvitamin D3) into liposomes. After liposome administration, we followed the endogenous ChgA-specific immune response with specific tetramers. Liposome administration s.c., but not i.v., induced ChgA-specific Foxp3 + and Foxp3 2 PD1 + CD73 + ICOS + IL-10 + peripheral regulatory T cells in prediabetic mice, and liposome administration at the onset of hyperglycemia significantly delayed diabetes progression. After BDC2.5 mim /calcitriol liposome administration, adoptive transfer of CD4 + T cells suppressed the development of diabetes in NOD severe combined immunodeficiency mice receiving diabetogenic splenocytes. After BDC2.5 mim /calcitriol liposome treatment and expansion of ChgA-specific peripheral regulatory T cells. IFN-g production and expansion of islet-specific glucose-6-phosphatase catalytic subunit-related protein-specific CD8 + T cells were also suppressed in pancreatic draining lymph node, demonstrating bystander tolerance at the site of Ag presentation. Thus, liposomes encapsulating the single CD4 + peptide, BDC2.5 mim , and calcitriol induce ChgA-specific CD4 + T cells that regulate CD4 + and CD8 + self-antigen specificities and autoimmune diabetes in NOD mice.

Enhancement of regulatory T cell (Treg cell) frequency and function is the goal of many therapeutic strategies aimed at treating type 1 diabetes (T1D). The interleukin-2 (IL-2) pathway, which has been strongly implicated in T1D susceptibility in both humans and mice, is a master regulator of Treg cell homeostasis and function. We investigated how IL-2 pathway defects impact Treg cells in T1D-susceptible nonobese diabetic (NOD) mice in comparison with protected C57BL/6 and NOD congenic mice. NOD Treg cells were reduced in frequency specifically in the lymph nodes and expressed lower levels of CD25 and CD39/CD73 immunosuppressive molecules. In the spleen and blood, Treg cell frequency was preserved through expansion of CD25(low), effector phenotype Treg cells. Reduced CD25 expression led to decreased IL-2 signaling in NOD Treg cells. In vivo, treatment with IL-2-anti-IL-2 antibody complexes led to effective upregulation of suppressive molecules on NOD Treg cells in the spleen and blood, but had reduced efficacy on lymph node Treg cells. In contrast, NOD CD8(+) and CD4(+) effector T cells were not impaired in their response to IL-2 therapy. We conclude that NOD Treg cells have an impaired responsiveness to IL-2 that reduces their ability to compete for a limited supply of IL-2.

The autoimmune disease type 1 diabetes is predominantly mediated by CD8+ cytotoxic T‐cell destruction of islet beta cells, of which islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP)206–214 is a dominant target antigen specificity. Previously, we found that a liposome‐based antigen‐specific immunotherapy encapsulating the CD4+ T‐cell islet epitope 2.5mim together with the nuclear factor‐κB inhibitor calcitriol induced regulatory T cells and protected from diabetes in NOD mice. Here we investigated whether the same system delivering IGRP206–214 could induce antigen‐specific CD8+ T‐cell‐targeted immune regulation and delay diabetes. Subcutaneous administration of IGRP206–214/calcitriol liposomes transiently activated and expanded IGRP‐specific T‐cell receptor transgenic 8.3 CD8+ T cells. Liposomal co‐delivery of calcitriol was required to optimally suppress endogenous IGRP‐specific CD8+ T‐cell interferon‐γ production and cytotoxicity. Concordantly, a short course of IGRP206–214/calcitriol liposomes delayed diabetes progression and reduced insulitis. However, when IGRP206–214/calcitriol liposomes were delivered together with 2.5mim/calcitriol liposomes, disease protection was not observed and the regulatory effect of 2.5mim/calcitriol liposomes was abrogated. Thus, tolerogenic liposomes that target either a dominant CD8+ or a CD4+ T‐cell islet epitope can delay diabetes progression but combining multiple epitopes does not enhance protection.

No abstract

Growing interest surrounds adoptive cellular therapies utilizing Natural Killer (NK) cells, which can be obtained from various sources, including umbilical cord blood (UCB) and adult peripheral blood (APB). Understanding NK cell receptor expression and diversity in such cellular sources will guide future therapeutic designs. We used a 20‐color flow cytometry panel to compare unstimulated and cytokine‐activated UCB and APB NK cells. Our analysis showed that UCB NK cells express slightly higher levels of the immune checkpoints PD‐1, TIGIT, and CD96 compared to their APB counterparts. Unsupervised hierarchical clustering and dimensionality reduction analyses revealed enrichment in CD56neg as well as mature NKp46neg and CD56+CD16+ NK cell populations in UCB whereas CD57+ terminally differentiated NK cells with variable expression of KIRs and CD16 were found in APB. These populations were conserved following stimulation with IL‐12, IL‐15, and IL‐18. Cytokine stimulation was associated with the downregulation of TIGIT and CD16 on multiple NK cell subsets in UCB and APB. Among UCB CD16− NK cell populations, TIGIT+ NK cells produced more IFN‐γ than their TIGIT− counterparts. Our data demonstrate higher immune checkpoint expression on UCB NK cells compared to APB. However, the expression of TIGIT immune checkpoint is not indicative of NK cell exhaustion.

Type 1 diabetes (T1D) is an autoimmune disease in which the β-cells of the pancreas are attacked by the host’s immune system, ultimately resulting in hyperglycemia. It is a complex multifactorial disease postulated to result from a combination of genetic and environmental factors. In parallel with increasing prevalence of T1D in genetically stable populations, highlighting an environmental component, consumption of advanced glycation end products (AGEs) commonly found in in Western diets has increased significantly over the past decades. AGEs can bind to cell surface receptors including the receptor for advanced glycation end products (RAGE). RAGE has proinflammatory roles including in host–pathogen defense, thereby influencing immune cell behavior and can activate and cause proliferation of immune cells such as islet infiltrating CD8+ and CD4+ T cells and suppress the activity of T regulatory cells, contributing to β-cell injury and hyperglycemia. Insights from studies of individuals at risk of T1D have demonstrated that progression to symptomatic onset and diagnosis can vary, ranging from months to years, providing a window of opportunity for prevention strategies. Interaction between AGEs and RAGE is believed to be a major environmental risk factor for T1D and targeting the AGE-RAGE axis may act as a potential therapeutic strategy for T1D prevention.

Adoptive cellular therapies using Natural Killer (NK) cells are of growing interest. NK cells can be obtained from various sources, including umbilical cord blood (UCB) and adult peripheral blood (APB). Understanding the diversity of NK cell populations and their receptor expression in both UCB and APB will guide future therapeutic designs. In this study, we used a 20-colour flow cytometry panel to compare unstimulated and cytokine-activated UCB and APB NK cells. Our analysis showed that UCB NK cells express slightly higher levels of the immune checkpoints PD-1, TIGIT and CD96 compared to their APB counterparts. Unsupervised hierarchical clustering and principal component analyses revealed previously unappreciated differences in NK cell populations from UCB and APB. UCB was characterised by an enrichment in CD56neg as well as mature NKp46neg and CD56+CD16+ NK cell populations whereas CD57+ terminally differentiated NK cells with variable expression of KIRs and CD16 were found in APB. These populations were conserved following two-days of IL-15 culture as well as overnight stimulation with IL-12, IL-15, and IL-18. Interestingly, cytokine stimulation was associated with the up-regulation of LAG-3 and DNAM-1 together with the downregulation of NKG2D, TIGIT and CD16 on multiple NK cell subsets in both UCB and APB. TIM-3 was also up-regulated with activation, but only in UCB. Overall, our data indicate that NK cells in UCB have a more immature phenotype than APB NK cells and UCB NK cells might be more amenable to immune checkpoint therapy.