Exosomes in HNSCC plasma as surrogate markers of tumour progression and immune competence

Exosomes in plasma of head and neck squamous cell carcinoma (HNSCC) patients comprise subsets of vesicles derived from various cells. Recently, we separated CD3⁽⁺⁾ from CD3^(–) exosomes by immune capture. CD3^(–) exosomes were largely tumour‐derived (CD44v3⁺). Both subsets carried immunosuppressive proteins and inhibited functions of human immune cells. The role of these subsets in immune cell reprogramming by the tumour was investigated by focusing on the adenosine pathway components. Spontaneous adenosine production by CD3⁽⁺⁾ or CD3^(–) exosomes was measured by mass spectrometry, as was the production of adenosine by CD4⁺CD39⁺ regulatory T cells (T_reg) co‐incubated with these exosomes. The highest level of CD39/CD73 ectoenzymes and of adenosine production was found in CD3^(–) exosomes in patients with the stages III/IV HNSCCs). Also, the production of 5′‐AMP and purines was significantly higher in T_reg co‐incubated with CD3^(–) than CD3⁽⁺⁾ exosomes. Consistently, CD26 and adenosine deaminase (ADA) levels were higher in CD3⁽⁺⁾ than CD3^(–) exosomes. ADA and CD26 levels in CD3⁽⁺⁾ exosomes were significantly higher in patients with early (stages I/II) than advanced (stages III/IV) disease. HNSCC patients receiving and responding to photodynamic therapy had increased ADA levels in CD3⁽⁺⁾ exosomes with no increase in CD3^(–) exosomes. The opposite roles of CD3⁽⁺⁾ ADA⁺CD26⁺ and CD3^(–)CD44v3⁺ adenosine‐producing exosomes in early versus advanced HNSCC suggest that, like their parent cells, these exosomes serve as surrogates of immune suppression in cancer.     

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.     

RORγt antagonist suppresses M3 muscarinic acetylcholine receptor‐induced Sjögren's syndrome‐like sialadenitis

We showed recently that M3 muscarinic acetylcholine receptor (M3R)‐reactive CD3⁺ T cells play a pathogenic role in the development of murine autoimmune sialadenitis (MIS), which mimics Sjögren's syndrome (SS). The aim of this study was to determine the effectiveness and mechanism of action of retinoic acid‐related orphan receptor‐gamma t (RORγt) antagonist (A213) in MIS. Splenocytes from M3R knockout (M3R^–/–) mice immunized with murine M3R peptide mixture were inoculated into recombination‐activating gene 1 knockout (Rag‐1^–/–) mice (M3R^–/–→Rag‐1^–/–) with MIS. Immunized M3R^–/– mice (pretransfer treatment) and M3R^–/–→Rag‐1^–/– mice (post‐transfer treatment) were treated with A213 every 3 days. Salivary volume, severity of sialadenitis and cytokine production from M3R peptide‐stimulated splenocytes and lymph node cells were examined. Effects of A213 on cytokine production were analysed by enzyme‐linked immunosorbent assay (ELISA) and on T helper type 1 (Th1), Th17 and Th2 differentiation from CD4⁺ T cells by flow cytometry. Pretransfer A213 treatment maintained salivary volume, improved MIS and reduced interferon (IFN)‐γ and interleukin (IL)‐17 production significantly compared with phosphate‐buffered saline (PBS) (P < 0·05). These suppressive effects involved CD4⁺ T cells rather than CD11c⁺ cells. Post‐transfer treatment with A213 increased salivary volume (P < 0·05), suppressed MIS (P < 0·005) and reduced IFN‐γ and IL‐17 production (P < 0·05). In vitro, A213 suppressed IFN‐γ and IL‐17 production from M3R‐stimulated splenocytes and CD4⁺ T cells of immunized M3R^–/– mice (P < 0·05). In contrast with M3R specific responses, A213 suppressed only IL‐17 production from Th17 differentiated CD4⁺ T cells without any effect on Th1 and Th2 differentiation in vitro. Our findings suggested that RORγt antagonism is potentially suitable treatment strategy for SS‐like sialadenitis through suppression of IL‐17 and IFN‐γ production by M3R‐specific T cells.     

Comprehensive T‐cell immunophenotyping and next‐generation sequencing of human papillomavirus (HPV)‐positive and HPV‐negative head and neck squamous cell carcinomas

The success of programmed cell death 1 (PD‐1) inhibition in achieving a clinical response in a subset of head and neck squamous cell carcinoma (HNSCC) patients emphasizes the need to better understand the immunobiology of HNSCC. Immunophenotyping was performed for 30 HCSCC patients [16 human papillomavirus (HPV)‐positive; 14 HPV‐negative] on matched tissue from the primary tumour site, locally metastatic cervical lymph nodes (LNs), uninvolved local cervical LNs, and peripheral blood. CD4⁺ and CD8⁺ T‐cell lymphocytes obtained from tissue were analysed for expression levels of the inhibitory receptors PD‐1, TIM‐3 and CTLA‐4. Next‐generation sequencing of the T‐cell receptor (TCR) β chain was performed on patients (n = 9) to determine receptor repertoire diversity and for clonality analysis. HPV‐negative HNSCC patients, particularly those with stage IV disease, had significantly higher proportions of CD8⁺ T cells expressing CTLA‐4 in tumour tissue (P = 0.0013) and in peripheral blood (P = 0.0344) than HPV‐positive patients, as well as higher expression levels of TIM‐3⁺PD‐1⁺ CD8⁺ T cells (P = 0.0072) than controls. For all patients, PD‐1 expression on CD8⁺ T cells – particularly in HPV‐negative HNSCC cases – strongly correlated (r = 0.63, P = 0.013) with tumour size at the primary site. The top CD8⁺ TCR clones from tumour tissue significantly overlapped with circulating peripheral blood TCR clones (r = 0.946), and HPV‐positive patients had frequently expanded TCR clones that were more hydrophobic – and potentially more immunogenic – than those from HPV‐negative patients. Collectively, our findings demonstrate, for the first time, that high‐stage HPV‐negative HNSCC patients with primary tumours at different sites in the head and neck have elevated peripheral CTLA‐4⁺CD8⁺ T‐cell levels, that tumour‐familiar CD8⁺ T cells are detectable in peripheral blood from HNSCC patients, and that TCRs from HPV‐positive HNSCC patients potentially recognize distinctly immunogenic cognate antigens. However, our findings are preliminary, and need to be further confirmed in a larger patient cohort; also, how these factors affect patient response to immunotherapy needs to be determined. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Effect of activated human polymorphonuclear leucocytes on T lymphocyte proliferation and viability

Human polymorphonuclear leucocytes (PMN) are thought to be immunosuppressive. The suppressive mechanism(s) used by PMN are, however, not well defined and in this study they were analysed using T‐cell responses to CD3⁺ CD28 monoclonal antibodies (mAb) as a readout. We demonstrate that in vitro activated PMN (PMN^act) can, without any T‐cell interaction, induce apparent T‐cell suppression by inhibiting the stimulatory capacity of the CD3 mAb. However, a cell‐directed suppression of T‐cell proliferation was observed when PMN^act were added to pre‐activated T cells that are already committed to polyclonal proliferation. This suppression was partially reversed by catalase addition (P < 0·01) and largely reversed by addition of exogenous interleukin‐2 (P < 0·001) but was not significantly reduced by nitric oxide synthase inhibition, myeloperoxidase inhibition or addition of excess arginine. Following removal of PMN^act, suppressed T cells could respond normally to further stimulation. In addition to suppressing proliferation, co‐culture with PMN^act also induced a significant decrease in T‐cell viability that was reversed by catalase addition (P < 0·05). The addition of the arginase inhibitor N‐hydroxy‐nor‐l ‐arginine induced both a further significant, catalase‐sensitive, loss in T‐cell viability and increased nitrite release (P < 0·001). These data demonstrate that PMN, when activated, can both induce T‐cell death and reversibly inhibit proliferation of activated T cells. The mechanisms underlying these distinct processes and the effects of arginase inhibitors on PMN induced cytotoxicity merit further investigation.     

Tumour YAP1 and PTEN expression correlates with tumour‐associated myeloid suppressor cell expansion and reduced survival in colorectal cancer

The expansion of myeloid‐derived suppressor cells (MDSCs) correlates with tumorigenesis in colorectal cancer (CRC). Here, we found a significant association between CD33⁺ MDSC number and Yes‐associated protein 1 (YAP1) and phosphatase and tensin homologue (PTEN) levels in CRC patients (P < 0·05). Moreover, the CD33⁺ MDSCs, YAP1 and PTEN were identified as predictors for the prognosis of CRC patients (P < 0·05). Notably, CD33⁺ MDSCs were an independent survival predictor for CRC patients through a Cox model analysis. In vitro data determined that the expression levels of YAP1 and PTEN in CRC‐derived cell lines were associated with CRC‐derived MDSC induction, and the blockade of YAP1 and PTEN decreased CRC‐derived MDSC induction. A mechanistic analysis revealed that YAP1 promoted CRC‐derived MDSC induction by suppressing PTEN expression to up‐regulate COX‐2, P‐AKT and P‐p65 in CRC‐derived cells, leading to secretion of the cytokine granulocyte–macrophage colony‐stimulating factor. Our findings establish a novel mechanism of pro‐tumorigenic MDSC induction mediated by ectopic YAP1 and PTEN expression in CRC.     

1141420984Placental exosome suppression of T cell activation and differential target of T cell subsets

Problem: One immunoregulatory pathway that has received little attention is placental exosome release. In normal pregnancy, as factors linked with early immunomodulation decline, placental exosomes become key in modulating T‐cell activation, maintaining the absence of effector T‐cells by enhancing T cell apoptosis and loss of CD3‐z. Method of Study: Placental exosomes were isolated from the maternal peripheral circulation by a chromatographic/MACS procedure developed to specifically purify exosomes of placental origin. Exosomal FasL was identified by immunoelectron microscopy (IEM) and quantified by ELISA. Exosomal suppression of T cell signaling molecules and induction of apoptosis was determined by flow cytometry and DNA fragmentation, respectively. The role of FasL in these events was defined by use of FasL blocking antibody. The differential effect of exosomes on CD3‐z on T subsets was analyzed by western immunoblot (WB). Results: When T cells were co‐incubated with placental exosomes, CD3‐z was down‐regulated, with placental exosomes treated cells expressing 18,900 ± 7,000 MESF units of zeta, while control fraction‐treated cells expressed 49,000 ± 4,800 MESF units of zeta (P < 0.001). This down‐regulation of CD3‐z was partially reversed by pre‐incubating T cells with ZB4 antibody, suggesting a role for FasL. IEM revealed FasL‐antibody complexes located on the exterior of placental exosomes; however, only 20/27 preparations were FasL+. When placental exosomes were added to T cells, the level of CD3‐z expression on CD8⁺ cells was 7.1%, (8.7% vs 59.7%) inhibited 1.43‐fold more than in CD4⁺ cells (85.5 P < 0.01). On CD4⁺CD25⁺ cells, CD3‐z was not significantly inhibited. (P < 0.05). Conclusions: While all term placental exosome preparations tested induced apoptosis, exosomes obtained from 7/27 patients were negative for FasL but were able to induce DNA degradation. These results indicate that, while exosomal FasL is apoptogenic, additional exosome components are capable of inducing apoptosis, but these factors exhibit differential effect on T subsets.     

Increased natural killer cell subsets with inhibitory cytokines and inhibitory surface receptors in patients with recurrent miscarriage and decreased or normal subsets in kidney transplant recipients late post‐transplant

Patients with recurrent miscarriage (RM) show up‐regulated cytotoxic natural killer (NK) cells that are suspected to play a causal role in abortion. In the present study, we investigated counter‐regulating inhibitory mechanisms and compared the results in RM patients with those of healthy controls (HC), patients with end‐stage renal disease (ESRD) and kidney transplant recipients late post‐transplant (TX). NK, NK T and T cell subsets were analysed in the peripheral blood of 31 RM, 14 female ESRD and nine female TX patients as well as 21 female HC using eight‐colour fluorescence flow cytometry. Compared with HC, RM patients showed significantly higher absolute numbers of CD56⁺ NK cells co‐expressing the phenotype interferon (IFN)‐γR⁺, IL‐4⁺, transforming growth factor (TGF)‐β⁺, IL‐4⁺ human leucocyte antigen D‐related (HLA‐DR)⁺, TGF‐β⁺HLA‐DR⁺, IL‐4⁺TGF‐β⁺, IL‐4⁺TGF‐β^–, IFN‐γ⁺ and/or IL‐10^–IFN‐γ⁺ (all P ≤ 0·01), more IL‐17⁺CD56^bright (P = 0·028) NK cells and more CD56^dimCD16⁺ NK cells co‐expressing IFN‐γR, IFN‐γ, IL‐4 and/or TGF‐β (all P ≤ 0·01). When the same cell subsets were analysed in ESRD or TX patients, cytokine‐producing NK cell subsets were not significantly different from those of HC. RM patients showed significantly higher absolute numbers of CD158a⁺, CD158b⁺, CD158a^–CD158e⁺ (all P < 0·05), NKG2D⁺NKG2A⁺, NKG2D ⁺NKG2A^–, NKG2D⁺ and/or NKG2A⁺ (all P ≤ 0·01) CD56⁺ NK cells and higher CD158a⁺, CD158b⁺ (all P < 0·05), NKG2D⁺ and/or NKG2A⁺ (all P < 0·01) CD56^dim+CD16⁺ NK cells than HC. In contrast, ESRD patients had normal and TX recipients had lower CD158a⁺ and NKG2D⁺NKG2A^–CD56⁺ NK cells and lower CD158a⁺CD56^dim+CD16⁺ NK cells (all P < 0·05) than HC. RM patients have abnormally high circulating NK cells expressing inhibitory cytokines and inhibitory surface receptors which might contribute to the pathogenesis of RM.     

Macrophages play an essential role in antigen‐specific immune suppression mediated by T CD8+ cell‐derived exosomes

Murine contact sensitivity (CS) reaction could be antigen‐specifically regulated by T CD8⁺ suppressor (Ts) lymphocytes releasing microRNA‐150 in antibody light‐chain‐coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell‐exosome‐mediated antigen‐specific suppression as well as modulation of Mφ antigen‐presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) ‐specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome‐treated, antigen‐pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome‐treated, TNP‐conjugated Mφ. Additionally, exosome‐pulsed, TNP‐conjugated Mφ mediated suppression of CS in mice pre‐treated with a low‐dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome‐treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen‐specific immune suppression mediated by Ts cell‐derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation.     

Diminished CXCR5 expression in peripheral blood of patients with Sjögren's syndrome may relate to both genotype and salivary gland homing

Genetic investigations of Sjögren's syndrome (SS) have identified a susceptibility locus at p23.3 of chromosome 11, which contains the CXCR5 gene. C‐X‐C motif chemokine receptor 5 (CXCR5) is a chemokine receptor expressed on B and T cell subsets, and binds the chemotactic ligand C‐X‐C motif chemokine ligand 13 (CXCL13). In this study we aimed to link the genetic association with functional effects and explore the CXCR5/CXCL13 axis in SS. Expression quantitative trait loci analysis of the 11q23.3 locus was performed using B cell mRNA expression data from genotyped individuals. Lymphocyte surface markers were assessed by flow cytometry, and CXCL13 levels by a proximity extension assay. CXCR5⁺ and CXCL13⁺ cells in minor salivary glands were detected using immunohistochemistry. Our results demonstrated that SS‐associated genetic polymorphisms affected the expression of CXCR5 (P < 0·01). Notably, a decreased percentage of CXCR5⁺ cells, with lower CXCR5 expression, was observed for most circulating B and T cell subsets in SS patients, reaching statistical significance in CD19⁺CD27⁺immunoglobulin (Ig)D⁺ marginal zone (P < 0·001), CD19⁺CD27⁺IgD^– memory (P < 0·05) and CD27‐IgD double‐negative (P < 0·01) B cells and CD4⁺CXCR3^–CCR6⁺ Th17 cells (P < 0·05). CXCL13 levels were increased in patient plasma (P < 0·001), and immunohistochemical staining revealed expression of CXCL13 and higher numbers of CXCR5⁺ cells (P < 0·0001) within focal infiltrates and interstitially in salivary glands of SS patients. In conclusion, we link a genetic susceptibility allele for SS to a functional phenotype in terms of decreased CXCR5 expression. The decrease of CXCR5⁺ cells in circulation was also related to homing of B and T cells to the autoimmune target organ. Therapeutic drugs targeting the CXCR5/CXCL13 axis may be useful in SS.     

Evaluation of membrane‐bound and soluble forms of human leucocyte antigen‐G in systemic sclerosis

Systemic sclerosis (SSc) is a complex disease characterized by immune dysregulation, extensive vascular damage and widespread fibrosis. Human leucocyte antigen‐G (HLA‐G) is a non‐classic class I major histocompatibility complex (MHC) molecule characterized by complex immunomodulating properties. HLA‐G is expressed on the membrane of different cell lineages in both physiological and pathological conditions. HLA‐G is also detectable in soluble form (sHLA‐G) deriving from the shedding of surface isoforms (sHLA‐G1) or the secretion of soluble isoforms (HLA‐G5). Several immunosuppressive functions have been attributed to both membrane‐bound and soluble HLA‐G molecules. The plasma levels of sHLA‐G were higher in SSc patients (444·27 ± 304·84 U/ml) compared to controls (16·74 ± 20·58 U/ml) (P < 0·0001). The plasma levels of transforming growth factor (TGF)‐β were higher in SSc patients (18 937 ± 15 217 pg/ml) compared to controls (11 099 ± 6081 pg/ml; P = 0·003), and a significant correlation was found between TGF‐β and the plasma levels of total sHLA‐G (r = 0·65; P < 0·01), sHLA‐G1 (r = 0·60; P = 0·003) and HLA‐G5 (r = 0·47; P = 0·02). The percentage of HLA‐G‐positive monocytes (0·98 ± 1·72), CD4⁺ (0·37 ± 0·68), CD8⁺ (2·05 ± 3·74) and CD4⁺CD8⁺ double‐positive cells (14·53 ± 16·88) was higher in SSc patients than in controls (0·11 ± 0·08, 0·01 ± 0·01, 0·01 ± 0·01 and 0·39 ± 0·40, respectively) (P < 0·0001). These data indicate that in SSc the secretion and/or shedding of soluble HLA‐G molecules and the membrane expression of HLA‐G by peripheral blood mononuclear cells (PBMC) is clearly elevated, suggesting an involvement of HLA‐G molecules in the immune dysregulation of SSc.     

Human dominant‐negative class II transactivator transgenic pigs – effect on the human anti‐pig T‐cell immune response and immune status

Swine leucocyte antigen (SLA) class II molecules on porcine (p) cells play a crucial role in xenotransplantation as activators of recipient human CD4⁺ T cells. A human dominant‐negative mutant class II transactivator (CIITA‐DN) transgene under a CAG promoter with an endothelium‐specific Tie2 enhancer was constructed. CIITA‐DN transgenic pigs were produced by nuclear transfer/embryo transfer. CIITA‐DN pig cells were evaluated for expression of SLA class II with/without activation, and the human CD4⁺ T‐cell response to cells from CIITA‐DN and wild‐type (WT) pigs was compared. Lymphocyte subset numbers and T‐cell function in CIITA‐DN pigs were compared with those in WT pigs. The expression of SLA class II on antigen‐presenting cells from CIITA‐DN pigs was significantly reduced (40–50% reduction compared with WT; P < 0·01), and was completely suppressed on aortic endothelial cells (AECs) even after activation (100% suppression; P < 0·01). The human CD4⁺ T‐cell response to CIITA‐DN pAECs was significantly weaker than to WT pAECs (60–80% suppression; P < 0·01). Although there was a significantly lower frequency of CD4⁺ cells in the PBMCs from CIITA‐DN (20%) than from WT (30%) pigs (P < 0·01), T‐cell proliferation was similar, suggesting no significant immunological compromise. Organs and cells from CIITA‐DN pigs should be partially protected from the human cellular immune response.     

Co‐inhibitory profile and cytotoxicity of CD57+PD‐1– T cells in end‐stage renal disease patients

Blockade of the CD80/86‐CD28 pathway by belatacept after kidney transplantation is associated with an increased risk of rejection compared with standard, calcineurin inhibitor (CNI)‐based therapy. CD28^– T cells, which express CD57, are not susceptible to belatacept treatment. High numbers of CD4⁺CD57⁺programmed death 1 (PD‐1)^– T cells pretransplantation have been associated with a higher chance of rejection, although conflicting data have been reported. To investigate the working mechanism behind this possible higher chance of rejection, we studied the expression of co‐inhibitory molecules (CD223, CD244 and PD‐1), proliferative capacity and cytotoxic potential of fluorescence activated cell sorted (FACS) CD4⁺CD57⁺PD‐1^– and CD8⁺CD57⁺PD‐1^– T cells, and their CD57^– control populations, after alloantigen stimulation. The effect of belatacept on the cytotoxic capacity of pretransplantation peripheral blood mononuclear cells from 20 patients who received belatacept post‐transplantation was also tested. Expression of co‐inhibitory molecule CD223 increased by approximately 10‐fold after allogeneic stimulation in all four T cell subsets. Proliferation and up‐regulation of CD244 and PD‐1 was observed for CD4⁺CD57^‐PD‐1^– T cells after allogeneic stimulation, but no up‐regulation of these markers occurred on CD8⁺ T cells or CD4⁺CD57⁺PD‐1^– T cells. However, CD4⁺CD57⁺PD‐1^– T cells and, to a lesser extent, CD8⁺CD57⁺PD‐1^– T cells displayed higher cytotoxicity as indicated by granzyme B expression. Belatacept inhibited the cytotoxic potential of CD4⁺CD57⁺PD‐1^– T cells (median of inhibition 31%, P < 0·01) and CD8⁺CD57⁺PD‐1^– T cells (median of inhibition 10%, P < 0·05). In conclusion, alloantigen‐activated CD4⁺CD57⁺PD‐1^– T cells exhibited a less proliferative but more cytotoxic profile than their CD57^– counterparts. Their cytotoxic capacity can be inhibited partly by belatacept and was not associated with development of rejection after kidney transplantation.     

Decrease in the proportion of CD24hiCD38hi B cells and impairment of their regulatory capacity in type 1 diabetes patients

B10 cells restore immune balance by producing interleukin (IL)-10. Impaired B10 cell responses are related to numerous autoimmune diseases. However, the function of B10 cells in type 1 diabetes (T1D) patients is controversial. We hypothesized that there are numerical and functional defects of B10 cells in T1D. Sixty-two patients with T1D and 74 healthy volunteers were included in our study. We showed that B10 cells in human peripheral blood belong to a CD24^hiCD38^hi B cell subpopulation. CD24^hiCD38^hi B cells from healthy individuals possessed regulatory capacity, suppressed interferon (IFN)-γ, tumor necrosis factor (TNF)-α and IL-17A production and promoted IL-4 production and forkhead box protein 3 (FoxP3) expression in CD4⁺ T cells through an IL-10-dependent mechanism. Compared to healthy controls, B10 cell percentages in T1D were significantly lower (5·6 ± 3·5 versus 6·9 ± 3·3%; P < 0·05), produced less IL-10 (15·4 ± 4·3 versus 29·0 ± 4·5%; P < 0·001) and lacked regulatory capacity. In addition, Pearson’s correlation analysis showed that the frequency of circulating B10 cells was negatively correlated with the frequency of CD4⁺IFN-γ⁺ and CD4⁺TNF-α⁺ T cells (r = −0·248 and r = −0·283, P = 0·008 and P = 0·017, respectively), positively correlating with the frequency of CD4⁺CD25⁺FoxP3⁺ T cells (r = 0·247, P = 0·001). These data offer direct proof that there is a deficiency of circulating CD24^hiCD38^hi B cells in peripheral blood of patients with T1D, which participate in the T1D immune imbalance involved in the development of T1D.     

Exosomal miR‐24‐3p impedes T‐cell function by targeting FGF11 and serves as a potential prognostic biomarker for nasopharyngeal carcinoma

Recent studies have shown that extracellular microRNAs are not only potential biomarkers but are also involved in cell interactions to regulate the intercommunication between cancer cells and their microenvironments in various types of malignancies. In this study, we isolated exosomes from nasopharyngeal carcinoma (NPC) cell lines and patient sera (T‐EXOs), or control NP69 cells and healthy donor sera (HD‐EXOs). We found that miR‐24‐3p was markedly enriched in T‐EXOs as compared with HD‐EXOs; the serum exosomal miR‐24‐3p level was correlated with worse disease‐free survival of patients (p < 0.05). Knockdown of exosomal miR‐24‐3p (miR‐24‐3p‐sponge‐T‐EXOs) by a sponge RNA targeting miR‐24‐3p restored the T‐EXO‐mediated (control‐sponge‐T‐EXO) inhibition of T‐cell proliferation and Th1 and Th17 differentiation, and the induction of regulatory T cells (Tregs). Mechanistic analyses revealed that administration of exosomal miR‐24‐3p increased P‐ERK, P‐STAT1 and P‐STAT3 expression while decreasing P‐STAT5 expression during T‐cell proliferation and differentiation. Moreover, by in vivo and in vitro assessments, we found FGF11 to be a direct target of miR‐24‐3p. However, both miR‐24‐3p‐sponge‐T‐EXOs and T‐EXOs (control‐sponge‐T‐EXOs) impeded proliferation and Th1 and Th17 differentiation, but induced Treg differentiation, of lenti‐shFGF11‐transfected T cells. The levels of phosphorylated ERK and STAT proteins were different in lenti‐ScshRNA‐transfected T cells and lenti‐shFGF11‐transfected T cells following administration of miR‐24‐3p‐sponge‐T‐EXO. Interestingly, tumour FGF11 expression was positively correlated with the number of CD4⁺ and CD8⁺ T cells in vivo, and predicted favourable patient DFS (p < 0.05). Additionally, hypoxia increased cellular and exosomal miR‐24‐3p levels and enhanced the inhibitory effect of T‐EXO on T‐cell proliferation and differentiation. Collectively, our findings suggest that exosomal miR‐24‐3p is involved in tumour pathogenesis by mediating T‐cell suppression via repression of FGF11, and may serve as a potential prognostic biomarker in NPC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

CD14 expression is increased on monocytes in patients with anti‐neutrophil cytoplasm antibody (ANCA)‐associated vasculitis and correlates with the expression of ANCA autoantigens

Monocyte subsets with differing functional properties have been defined by their expression of CD14 and CD16. We investigated these subsets in anti‐neutrophil cytoplasm antibody (ANCA)‐associated vasculitis (AAV) and determined their surface expression of ANCA autoantigens. Flow cytometry was performed on blood from 14 patients with active AAV, 46 patients with AAV in remission and 21 controls. The proportion of classical (CD14^highCD16^neg/low), intermediate (CD14^highCD16^high) and non‐classical (CD14^lowCD16^high) monocytes and surface expression levels of CD14 and CD16 were determined, as well as surface expression of proteinase 3 (PR3) and myeloperoxidase (MPO) on monocyte subsets. There was no change in the proportion of monocytes in each subset in patients with AAV compared with healthy controls. The expression of CD14 on monocytes from patients with active AAV was increased, compared with patients in remission and healthy controls (P < 0·01). Patients with PR3‐ANCA disease in remission also had increased monocyte expression of CD14 compared with controls (P < 0·01); however, levels in patients with MPO‐ANCA disease in remission were lower than active MPO‐ANCA patients, and not significantly different from controls. There was a correlation between CD14 and both PR3 and MPO expression on classical monocytes in AAV patients (r = 0·79, P < 0·0001 and r = 0·42, P < 0·005, respectively). In conclusion, there was an increase in monocyte CD14 expression in active AAV and PR3‐ANCA disease in remission. The correlation of CD14 expression with ANCA autoantigen expression in AAV may reflect cell activation, and warrants further investigation into the potential for increased CD14 expression to trigger disease induction or relapse.