Low programmed cell death‐1 (PD‐1) expression in peripheral CD4+ T cells in Japanese patients with autoimmune type 1 diabetes

Programmed cell death‐1 (PD‐1) is a co‐stimulatory molecule that inhibits T cell proliferation. We aimed to clarify PD‐1 expression in CD4⁺ T cells and the association between PD‐1 expression and the 7785C/T polymorphism of PDCD1, with a focus on the two subtypes of type 1 diabetes, type 1A diabetes (T1AD) and fulminant type 1 diabetes (FT1D), in the Japanese population. We examined 22 patients with T1AD, 15 with FT1D, 19 with type 2 diabetes (T2D) and 29 healthy control (HC) subjects. Fluorescence‐activated cell sorting (FACS) and real‐time PCR were utilized to analyse PD‐1 expression quantitatively. Genotyping of 7785C/T in PDCD1 was performed using the TaqMan method in a total of 63 subjects (21 with T1AD, 15 with FT1D and 27 HC). FACS revealed a significant reduction in PD‐1 expression in CD4⁺ T cells in patients with T1AD (mean: 4·2 vs. 6·0% in FT1D, P = 0·0450; vs. 5·8% in T2D, P = 0·0098; vs. 6·0% in HC, P = 0·0018). PD‐1 mRNA expression in CD4⁺ T cells was also significantly lower in patients with T1AD than in the HC subjects. Of the 63 subjects, PD‐1 expression was significantly lower in individuals with the 7785C/C genotype than in those with the C/T and T/T genotypes (mean: 4·1 vs. 5·9%, P = 0·0016). Our results indicate that lower PD‐1 expression in CD4⁺ T‐cells might contribute to the development of T1AD through T cell activation.     

Differential control of CD4+ T‐cell subsets by the PD‐1/PD‐L1 axis in a mouse model of allergic asthma

Studies examining the role of PD‐1 family members in allergic asthma have yielded conflicting results. Using a mouse model of allergic asthma, we demonstrate that blockade of PD‐1/PD‐L1 has distinct influences on different CD4⁺ T‐cell subsets. PD‐1/PD‐L1 blockade enhances airway hyperreactivity (AHR), not by altering the magnitude of the underlying Th2‐type immune response, but by allowing the development of a concomitant Th17‐type immune response. Supporting differential CD4⁺ T‐cell responsiveness to PD‐1‐mediated inhibition, naïve PD‐1^?/? mice displayed elevated Th1 and Th17 levels, but diminished Th2 cytokine levels, and ligation of PD‐1 in WT cells limited cytokine production by in vitro polarized Th1 and Th17 cells, but slightly enhanced cytokine production by in vitro polarized Th2 cells. Furthermore, PD‐1 ligation enhanced Th2 cytokine production by naïve T cells cultured under nonpolarizing conditions. These data demonstrate that different CD4⁺ T‐cell subsets respond differentially to PD‐1 ligation and may explain some of the variable results observed in control of allergic asthma by the PD‐1 family members. As the PD‐1/PD‐L1 axis limits asthma severity by constraining Th17 cell activity, this suggests that severe allergic asthma may be associated with a defective PD‐1/PD‐L1 regulatory axis in some individuals.     

Programmed death (PD)‐1 molecule and its ligand PD‐L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus‐1‐infected individuals

Human immunodeficiency virus (HIV)‐1 causes T cell anergy and affects T cell maturation. Various mechanisms are responsible for impaired anti‐HIV‐1‐specific responses: programmed death (PD)‐1 molecule and its ligand PD‐L1 are negative regulators of T cell activity and their expression is increased during HIV‐1 infection. This study examines correlations between T cell maturation, expression of PD‐1 and PD‐L1, and the effects of their blockade. Peripheral blood mononuclear cells (PBMC) from 24 HIV‐1⁺ and 17 uninfected individuals were phenotyped for PD‐1 and PD‐L1 expression on CD4⁺ and CD8⁺ T cell subsets. The effect of PD‐1 and PD‐L1 blockade on proliferation and interferon (IFN)‐γ production was tested on eight HIV‐1⁺ patients. Naive (CCR7⁺CD45RA⁺) CD8⁺ T cells were reduced in HIV‐1 aviraemic (P = 0·0065) and viraemic patients (P = 0·0130); CD8 T effector memory subsets [CCR7^‐CD45RA^–(T_EM)] were increased in HIV‐1⁺ aviraemic (P = 0·0122) and viraemic (P = 0·0023) individuals versus controls. PD‐1 expression was increased in CD4 naive (P = 0·0496), central memory [CCR7⁺CD45RA^– (T_CM); P = 0·0116], T_EM (P = 0·0037) and CD8 naive T cells (P = 0·0133) of aviraemic HIV‐1⁺versus controls. PD‐L1 was increased in CD4 T_EMRA (CCR7^‐CD45RA⁺, P = 0·0119), CD8 T_EM (P = 0·0494) and CD8 T_EMRA (P = 0·0282) of aviraemic HIV‐1⁺versus controls. PD‐1 blockade increased HIV‐1‐specific proliferative responses in one of eight patients, whereas PD‐L1 blockade restored responses in four of eight patients, but did not increase IFN‐γ‐production. Alteration of T cell subsets, accompanied by increased PD‐1 and PD‐L1 expression in HIV‐1 infection contributes to anergy and impaired anti‐HIV‐1‐specific responses which are not rescued when PD‐1 is blocked, in contrast to when PD‐L1 is blocked, due possibly to an ability to bind to receptors other than PD‐1.     

Recent Insight into the Role of the PD‐1/PD‐L1 Pathway in Feto‐Maternal Tolerance and Pregnancy

Pregnancy presents a great challenge to the maternal immune system. Given that maternal alloreactive lymphocytes are not depleted during pregnancy, local and/or systemic mechanisms have to serve a central function in altering the maternal immune responses. Regulatory T cells (Tregs) and the PD‐1/PD‐L1 pathway are both critical in controlling the immune responses. Recent studies have proved the critical function of the PD‐1/PD‐L1 pathway in regulating the T‐cell homeostasis and the peripheral tolerance through promoting the development and function of Tregs, and inhibiting the activation of effector T cells. The function of the PD‐1/PD‐L1 pathway in feto‐maternal interface and pregnancy has been investigated in human and animal models of pregnancy. In this review, we provide recent insight into the role of the PD‐1/PD‐L1 pathway in regulating T‐cell homeostasis, maternal tolerance, and pregnancy‐related complications as well as its possible applicability in clinical immunotherapy.     

PD‐1 regulates extrathymic regulatory T‐cell differentiation

Treg cells and the programed death‐1/programed death ligand‐1 (PD‐1/PD‐L1) pathway are both critical for maintaining peripheral tolerance to self‐Ags. A significant subset of Treg cells constitutively expresses PD‐1, which prompted an investigation into the role of PD‐1/PD‐L1 interactions in Treg‐cell development, function, and induction in vivo. The phenotype and abundance of Treg cells was not significantly altered in PD‐1‐deficient mice. The thymic development of polyclonal and monospecific Treg cells was not negatively impacted by PD‐1 deficiency. The suppressive function of PD‐1^?/? Treg cells was similar to their PD‐1^+/+ counterparts both in vitro and in vivo. However, in three different in vivo experimental settings, PD‐1^?/? conventional CD4⁺ T cells demonstrated a strikingly diminished tendency toward differentiation into peripherally induced Treg (pTreg) cells. Our results demonstrate that PD‐1 is dispensable for thymic Treg‐cell development and suppressive function, but is critical for the extrathymic differentiation of pTreg cells in vivo. These data suggest that Ab blockade of the PD‐1/PD‐L1 pathway may augment T‐cell responses by acting directly on conventional T cells, and also by suppressing the differentiation of pTreg cells.     

B7‐1/2, but not PD‐L1/2 molecules,are required on IL‐10‐treated tolerogenic DC and DC‐derived exosomes for in vivo function

Costimulatory molecules, such as B7‐1/2 and PD‐L1/2 play an important role in the function of APC. The regulation of the surface levels of costimulatory molecules is one mechanism by which APC maintain the balance between tolerance and immunity. We examined the contributions of B7‐1/2 and PD‐L1/2 to the function of IL‐10‐treated, immunosuppressive DC as well as therapeutic exosomes derived from these DC. IL‐10 treatment of DC significantly downregulated surface expression of MHC II, B7‐1, B7‐2, and decreased levels of MHC I and PD‐L2. IL‐10 treatment of DC resulted in a modified costimulatory profile of DC‐secreted exosomes with a reduction in B7‐1, PD‐L1 and PD‐L2. We further demonstrate that absence of B7‐1 or B7‐2 on donor DC results in a loss of ability of IL‐10‐treated DC and their exosomes to suppress the delayed‐type hypersensitivity response, whereas IL‐10‐treated DC deficient in PD‐L1/2 as well as their secreted exosomes retained the ability to suppress delayed‐type hypersensitivity responses. We conclude that B7‐1 and B7‐2, but not PD‐L1 and PD‐L2, on IL‐10‐treated DC and DC‐derived exosomes play a critical role in immunosuppressive functions of both DC and exosomes.     

Influence of PD‐L1 cross‐linking on cell death in PD‐L1‐expressing cell lines and bovine lymphocytes

Programmed death‐ligand 1 (PD‐L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death‐1 (PD‐1). However, the mechanism of PD‐L1‐mediated inhibitory signalling after PD‐L1 cross‐linking by anti‐PD‐L1 monoclonal antibody (mAb) or PD‐1–immunogloblin fusion protein (PD‐1‐Ig) is still unknown, although it may induce cell death of PD‐L1⁺ cells required for regular immune reactions. In this study, PD‐1‐Ig or anti‐PD‐L1 mAb treatment was tested in cell lines that expressed PD‐L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD‐L1‐mediated cell death. PD‐L1 cross‐linking by PD‐1‐Ig or anti‐PD‐L1 mAb primarily increased the number of dead cells in PD‐L1^high cells, but not in PD‐L1^low cells; these cells were prepared from Cos‐7 cells in which bovine PD‐L1 expression was induced by transfection. The PD‐L1‐mediated cell death also occurred in Cos‐7 and HeLa cells transfected with vectors only encoding the extracellular region of PD‐L1. In bovine lymphocytes, the anti‐PD‐L1 mAb treatment up‐regulated interferon‐γ (IFN‐γ) production, whereas PD‐1‐Ig treatment decreased this cytokine production and cell proliferation. The IFN‐γ production in B‐cell‐depleted peripheral blood mononuclear cells was not reduced by PD‐1‐Ig treatment and the percentages of dead cells in PD‐L1⁺ B cells were increased by PD‐1‐Ig treatment, indicating that PD‐1‐Ig‐induced immunosuppression in bovine lymphocytes could be caused by PD‐L1‐mediated B‐cell death. This study provides novel information for the understanding of signalling through PD‐L1.     

Increased PD‐L1 expression and PD‐L1/CD86 ratio on dendritic cells were associated with impaired dendritic cells function in HCV infection

Impaired hepatitis C virus (HCV)‐specific T cell immunity was associated with the persistence of HCV infection. Dysfunction of dentritic cells (DCs) was believed to be involved in T cell exhaustion, but the mechanisms were rarely understood. In this study, surface costimulatory marker (CD83, CD86, and CD40), coinhibitory marker (PD‐L1) expression and allostimulatory capacity of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were evaluated in HCV‐infected patients. Results showed that the expression of both costimulatory and coinhibitory markers was increased in HCV‐infected patients compared with healthy controls. PD‐L1/CD86 ratio was increased and positively correlated with PD‐L1 expression on DCs in HCV‐infected patients. Allostimulatory capacity of DCs was impaired and inversely correlated with PD‐L1 expression and PD‐L1/CD86 ratio. These findings suggested that the effect of inhibitory marker PD‐L1 overwhelmed the effect of costimulatory markers and down regulated DC‐T activation in HCV‐infected patients. The results will be helpful to understand the mechanism of dysfunction of DCs in HCV infection and shed light on the DC‐based immunotherapeutic strategy. J. Med. Virol. 82: 1152–1159, 2010. © 2010 Wiley‐Liss, Inc.