Identification of tribbles-1 as a novel binding partner of Foxp3 in regulatory T cells.

In a previous study, we identified TRIB1, a serine-threonine kinase-like molecule, as a biomarker of chronic antibody-mediated rejection of human kidneys when measured in peripheral blood mononuclear cells. Here, we focused our analysis on a specific subset of peripheral blood mononuclear cells that play a dominant role in regulating immune responses in health and disease, so-called CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). We isolated both human and murine Treg and non-Treg counterparts and analyzed TRIB1 and Foxp3 mRNA expression by quantitative PCR on the freshly isolated cells or following 24 h of activation. Physical interaction between the human TRIB1 and Foxp3 proteins was analyzed in live cell lines by protein complementation assay using both flow cytometry and microscopy and confirmed in primary freshly isolated human CD4(+)CD25(hi)CD127(-) Tregs by co-immunoprecipitation. Both TRIB1 and Foxp3 were expressed at significantly higher levels in Tregs than in their CD4(+)CD25(-) counterparts (p < 0.001). Moreover, TRIB1 and Foxp3 mRNA levels correlated tightly in Tregs (Spearman r = 1.0; p < 0.001, n = 7), but not in CD4(+)CD25(-) T cells. The protein complementation assay revealed a direct physical interaction between TRIB1 and Foxp3 in live cells. This interaction was impaired upon deletion of the TRIB1 N-terminal but not the C-terminal domain, suggesting an interaction in the nucleus. This direct interaction within the nucleus was confirmed in primary human Tregs by co-immunoprecipitation. These data show a direct relationship between TRIB1 and Foxp3 in terms of their expression and physical interaction and highlight Tribbles-1 as a novel binding partner of Foxp3 in Tregs.

Frequency of circulating autoreactive T cells committed to myelin determinants in relapsing-remitting multiple sclerosis patients.

Multiple sclerosis (MS) is considered as an autoimmune disease in which T cell reactivity to self-antigens expressed in the brain, particularly myelin antigens, plays a pivotal role. Various myelin-derived peptides, including peptides of myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) have been studied as putative target in MS. However, CD4(+) and CD8(+) T cells recognizing autoantigens from brain have been detected in the blood of MS patients as well as the blood of normal individuals. Here we review and discuss studies focused on the assessment of the frequency of autoreactive T cells responding to a given antigen using different assays including LDA, IFNγ-ELISPOT and TRAP (T cell Recognition of Antigen Presenting Cells by Protein transfer) in MS.

Pattern of Clinical Progression Until Metastatic Castration-Resistant Prostate Cancer: An Epidemiological Study from the European Prostate Cancer Registry.

BACKGROUND: Prostate cancer (PCa) is the most frequently diagnosed cancer in men in Europe. The impact of PCa natural history and therapeutic management on the outcomes of castration-resistant prostate cancer patients with metastasis (mCRPC) remains unclear. OBJECTIVE: The objective of this study was to describe retrospectively patterns of clinical progression through diagnosis sequences before the mCRPC stage and to assess how these sequences impacted patients' disease progression and overall survival at mCRPC stage. PATIENTS AND METHODS: Patients with mCRPC were identified from the Prostate Cancer Registry (PCR), an observational study in a real-world setting in 16 countries between 2013 and 2016. Patients were grouped in diagnosis sequences before mCRPC and defined by date of PCa diagnosis, first metastasis, and castration resistance. Distribution of time-to-event variables were estimated using Kaplan-Meier product-limit survival curves for overall survival (OS) and progression-free survival (PFS). Non-adjusted Cox models were conducted for efficacy endpoints (OS, PFS) to estimate hazard ratios between diagnosis sequences. RESULTS: At the end of study, 2859 mCRPC patients were included in this analysis. Among mCRPC four diagnosis sequences were identified: 35% developed metastases (mHSPC) before becoming castration resistant (sequence 1, metachronous mHSPC), 10% developed castration resistance (nmCRPC) before metastases (sequence 2), 27% developed metastases and castration resistance within 4 months (sequence 3) and 28% of patients were de novo mHSPC (sequence 4). Median OS was 17.7 months (interquartile range (IQR): 8.8-29.9) and PFS was 6.4 months (IQR: 3.2-12.0). The univariate analyses showed no correlation between mCRPC patients' OS or PFS and the diagnosis sequence. CONCLUSION: This large European study describe four different patterns of prostate cancer progression to mCRPC stage. Our results indicate that patient survival becomes comparable after progression to mCRPC, regardless of the diagnosis sequence. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02236637; registered September 2014.

Patients with relapsing-remitting multiple sclerosis have normal Treg function when cells expressing IL-7 receptor alpha-chain are excluded from the analysis.

Multiple sclerosis (MS) is a chronic inflammatory disease that results in demyelination in the central nervous system, and a defect in the regulatory function of CD4+CD25high T cells has been implicated in the pathogenesis of the disease. Here, we reanalyzed the function of this T cell subset in patients with MS, but we depleted cells expressing IL-7 receptor alpha-chain (CD127), a marker recently described as present on activated T cells but not Tregs. Similar to other studies, we observed a marked defect in the suppressive function of unseparated CD4+CD25high T cells isolated from MS patients. However, when CD127(high) cells were removed from the CD4+CD25high population, patient and control cells inhibited T cell proliferation and cytokine production equally. Likewise, when the CD25 gate used to sort the cells was stringent enough to eliminate CD127high cells, CD4+CD25high T cells from patients with MS and healthy individuals had similar regulatory function. Additional analysis indicated that the CD127high cells within the CD4+CD25high T cell population from patients with MS appeared more proliferative and secreted more IFN-gamma and IL-2 than the same cells from healthy individuals. Taken together, we conclude that CD4+CD25highCD127low Tregs from MS patients and healthy individuals exhibit similar suppressive functions. The decreased inhibitory function of unfractioned CD4+CD25high cells previously observed might be due to abnormal activation of CD127high T cells in patients with MS.

T cell recognition of self-antigen presenting cells by protein transfer assay reveals a high frequency of anti-myelin T cells in multiple sclerosis.

Although peripheral blood myelin-autoreactive T cells are thought to play a key role in multiple sclerosis, they are generally considered to have qualitative differences rather than quantitative ones when compared to those found in healthy individuals. Here, we revisited the assessment of myelin-autoreactive T cells in a new approach based on their combined ability to acquire membrane proteins from autologous antigen presenting cells, and to respond to whole myelin extract as the stimulating autoantigen. Using this approach, the myelin-autoreactive T cell frequency in patients with multiple sclerosis was found to be unexpectedly high (n = 22, subtracted values median 2.08%, range 0-6%; background median 1%, range 0-4%) and to exceed that of age/gender-matched healthy individuals significantly (n = 18, subtracted values median 0.1%, range 0-5.3%, P < 0.0001; background median 1.45%, range 0.1-4%). Higher anti-myelin autoreactivity was stable in patients with multiple sclerosis after several months. These data correlated with whole myelin-induced gamma interferon-enzyme-linked immunosorbent spot assay performed under the same conditions, although the values obtained with enzyme-linked immunosorbent spot assay under all conditions were 58 times lower than with this new method. The myelin-autoreactive T cells were memory T cells expressing CD40L with a CD62(low) phenotype, suggesting their ability for homing to tissues. Collectively, these new data show a higher frequency of autoreactive T cells during multiple sclerosis than in age/gender-matched healthy individuals, and support an autoimmune aetiology in multiple sclerosis.

Patients with drug-free long-term graft function display increased numbers of peripheral B cells with a memory and inhibitory phenotype.

Several transplant patients maintain stable kidney graft function in the absence of immunosuppression. Here we compared the characteristics of their peripheral B cells to that of others who had stable graft function but were under pharmacologic immunosuppression, to patients with chronic rejection and to healthy volunteers. In drug-free long-term graft function (DF) there was a significant increase in both absolute cell number and frequency of total B cells; particularly activated, memory and early memory B cells. These increased B-cell numbers were associated with a significantly enriched transcriptional B-cell profile. Costimulatory/migratory molecules (B7-2/CD80, CD40, and CD62L) were upregulated in B cells; particularly in memory CD19(+)IgD(-)CD38(+/-)CD27(+) B cells in these patients. Their purified B cells, however, responded normally to a polyclonal stimulation and did not have cytokine polarization. This phenotype was associated with the following specific characteristics which include an inhibitory signal (decreased FcgammaRIIA/FcgammaRIIB ratio); a preventive signal of hyperactive B-cell response (an increase in BANK1, which negatively modulates CD40-mediated AKT activation); an increased number of B cells expressing CD1d and CD5; an increased BAFF-R/BAFF ratio that could explain why these patients have more peripheral B cells; and a specific autoantibody profile. Thus, our findings show that patients with DF have a particular blood B-cell phenotype that may contribute to the maintenance of long-term graft function.

Failure of glatiramer acetate to modify the peripheral T cell repertoire of relapsing-remitting multiple sclerosis patients.

Glatiramer acetate (GA) is a random copolymer used as an immunomodulatory treatment in relapsing-remitting multiple sclerosis (RR-MS). Its mechanisms of action are poorly understood, and several hypotheses have been put forward, the majority of which rely on in vitro studies. It has been hypothesised that further to processing by APC, GA could provide a large number of different epitopes with a possible sequence similarity to auto-antigens, which are able to stimulate a large proportion of T cells. Given that in a previous study we showed that the circulating T cells of MS patients present more alterations of the Vbeta T cell receptor (TCR) usage than normal individuals, we explored the possible effect of GA on the ex vivo T cell repertoire of MS patients. Here we used quantitative PCR and electrophoresis to longitudinally analyse (and without any ex vivo stimulation), the CDR3 length distribution (LD) and the amount of Vbeta TCR, as well as various cytokines, in the blood T cells of 10 RR-MS patients before and after 3 months and 2 years of GA treatment. In addition, we also determined the status of responder and non-responder patients after 24 months of GA treatment based on clinical and radiological criteria. We found no significant modification of cytokine production, Vbeta TCR mRNA accumulation or CDR3-LD in the patients after short-term and long-term treatment. In addition, we did not observe any difference in CDR3-LD in the GA responder patients (n=6) compared to non-responder patients (n=4). Focusing our study on responder patients, we performed TCR repertoire analysis in the CD4+ and CD8+ compartment. Alterations of CDR3-LD were predominantly found in the CD8+ compartment, without any significant influence of GA treatment. Finally, the T cell repertoire variations in MS patients treated with GA and healthy controls were equivalent. Collectively, our data suggest that GA therapy does not induce significant variations in cytokine production or TCR usage in MS patients.

A network meta-analysis to compare simeprevir with boceprevir and telaprevir in combination with peginterferon-α and ribavirin in patients infected with genotype 1 Hepatitis C virus.

OBJECTIVE: To conduct a network meta-analysis (NMA) to assess the relative efficacy and safety of simeprevir, a second generation oral protease inhibitor (PI), compared to telaprevir and boceprevir in combination with pegylated interferon-α and ribavirin (PR) in patients with chronic hepatitis C. METHODS: A systematic literature review and NMA of randomized controlled trials involving anti-virals added to PR were conducted. Electronic database searches and hand searches were conducted to identify relevant publications. Outcomes of interest included sustained virologic response (SVR), incidence of adverse events (AEs), and discontinuation due to AEs. Networks were based on treatment-, dose-, and duration-specific nodes. Sub-group analyses were conducted to investigate heterogeneity, based on Metavir scores, sub-genotypes 1a/1b, and prior response. RESULTS: A total of 15 publications were considered for the base case of the meta-analysis. Simeprevir was associated with higher SVR rates than PR alone. Compared to telaprevir and boceprevir, SVR rates tended to be higher for simeprevir, with odds ratios ranging from 1.27 [0.81-2.00] to 2.61 [1.44-4.74] in treatment-naïve and from 1.04 [0.78-1.38] to 1.74 [0.84-3.61] in treatment-experienced patients, respectively. In terms of safety, the risks of anemia and discontinuations due to AEs were lower for simeprevir compared to PR alone, telaprevir, and boceprevir. The risk of rash was lower for simeprevir compared to telaprevir, and similar compared to PR alone and boceprevir. CONCLUSION: This NMA in genotype 1 HCV patients suggests a similar or better efficacy and tolerability profile for simeprevir compared to telaprevir and boceprevir.

The involvement of SMILE/TMTC3 in endoplasmic reticulum stress response.

BACKGROUND: The state of operational tolerance has been detected sporadically in some renal transplanted patients that stopped immunosuppressive drugs, demonstrating that allograft tolerance might exist in humans. Several years ago, a study by Brouard et al. identified a molecular signature of several genes that were significantly differentially expressed in the blood of such patients compared with patients with other clinical situations. The aim of the present study is to analyze the role of one of these molecules over-expressed in the blood of operationally tolerant patients, SMILE or TMTC3, a protein whose function is still unknown. METHODOLOGY/PRINCIPAL FINDINGS: We first confirmed that SMILE mRNA is differentially expressed in the blood of operationally tolerant patients with drug-free long term graft function compared to stable and rejecting patients. Using a yeast two-hybrid approach and a colocalization study by confocal microscopy we furthermore report an interaction of SMILE with PDIA3, a molecule resident in the endoplasmic reticulum (ER). In accordance with this observation, SMILE silencing in HeLa cells correlated with the modulation of several transcripts involved in proteolysis and a decrease in proteasome activity. Finally, SMILE silencing increased HeLa cell sensitivity to the proteasome inhibitor Bortezomib, a drug that induces ER stress via protein overload, and increased transcript expression of a stress response protein, XBP-1, in HeLa cells and keratinocytes. CONCLUSION/SIGNIFICANCE: In this study we showed that SMILE is involved in the endoplasmic reticulum stress response, by modulating proteasome activity and XBP-1 transcript expression. This function of SMILE may influence immune cell behavior in the context of transplantation, and the analysis of endoplasmic reticulum stress in transplantation may reveal new pathways of regulation in long-term graft acceptance thereby increasing our understanding of tolerance.

Peripheral blood CD4+ T lymphocytes from multiple sclerosis patients are characterized by higher PSGL-1 expression and transmigration capacity across a human blood-brain barrier-derived endothelial cell line.

Mechanisms of T lymphocyte trafficking in the brain remain unclear in MS. We hypothesized that MS is associated with increased CD4+ and CD8+ T lymphocyte trafficking across the BBB. To test this hypothesis, we calculated the frequency of PSGL-1+/CD4+ and PSGL-1+CD8+ or LFA-1+/CD4+/CD8+ T cells in the PBMC of 27 patients with a RR-MS (21 untreated and six IFN-beta-treated) and 18 HI. Next, we measured their ex vivo TR across resting and TNF-alpha-activated human BBB-derived hCMEC/D3 endothelial layers under static conditions. The frequency of PSGL-1+CD4+ T lymphocytes was significantly higher in treated or untreated MS patients than HI. Furthermore, resting hCMEC/D3 TR of CD4+ lymphocytes (purified or in PBMC) from treated or untreated MS patients were significantly higher than those of HI and associated with significant enrichments of CD4+PSGL+ or CD4+PSGL-1+CD45RO+ T cells in their transmigrating fractions. The TR of CD4+ and CD8+ from MS patients across TNF-alpha-activated hCMEC/D3 were also significantly higher than that observed in HI. Resting hCMEC/D3 transmigration was blocked significantly by anti-PSGL-1/anti-LFA-1 in all groups, and anti-VLA-4 inhibited transmigration of MS T cells specifically. Purified PSGL-1-negative CD4+ lymphocytes transmigrated resting hCMEC/D3 with <10% of transmigrating cells re-expressing PSGL-1, suggesting PSGL-1-independent transmigration mechanisms. The frequency of PSGL-1 was unchanged in CD8+ cells from MS patients, whereas CD8+LFA-1(high) were reduced significantly in IFN-beta-treated patients specifically. Collectively, MS is associated with an expanding pool of PSGL-1+CD4+ T lymphocytes able to transmigrate the BBB endothelium in vitro and possibly contributing to brain pathology.

Blood CD8+ T cell responses against myelin determinants in multiple sclerosis and healthy individuals.

Patients with multiple sclerosis (MS) display significant peripheral blood CD8(+) T cell receptor biases, suggesting clonal selection. Our objective was to identify relevant myelin-derived peptides capable of eliciting responses of fresh blood CD8+ T cells in MS patients. We focused our analysis on the HLA supertypes (HLA-A3, -A2, -B7, -B27, -B44) predominant in a patient cohort. Three myelin protein (MBP, PLP and MOG) sequences were screened for HLA binding motifs and peptides were tested for their binding to HLA molecules. The cellular responses of 27 MS patients and 19 age- and sex-matched healthy controls (HC) were tested in IFN-gamma ELISPOT assays only detecting pre-committed CD8+ T cells. Sixty-nine new epitopes elicited positive responses, with MOG-derived peptides being the most immunogenic and peptides binding to HLA-A3 being the most frequent. However, MS patients and HC displayed the same frequency of autoreactive cells. The epitopes inducing the strongest responses were not those with the highest HLA binding, suggesting an effective thymic selection in MS patients. Our data extend the concept that the frequency of myelin-reactive T cells in MS patient blood is not increased compared to HC. The description of this set of myelin-derived peptides (MHC class I restricted, recognized by CD8+ T cells) offers new tools to explore the CD8+ cell role in MS.