T-cell Frequencies Research Articles

Non-uniform in vivo Expansion of Epstein-Barr Virus-Specific T-Cells Following Donor Lymphocyte Infusion for Post-transplant Lymphoproliferative Disease.

Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) is a life-threatening complication of T-lymphocyte deplete allogeneic hematopoietic stem cell transplantation (allo-HSCT). For patients with PTLD refractory to Rituximab, donor lymphocyte infusion (DLI) is established as a successful option for salvage therapy. However, although in vivo lymphocyte expansion has been correlated with good clinical outcome following DLI, the specificity and functional characteristics of EBV-specific T-cell responses remain poorly characterized. Here we describe two patients with Rituximab-refractory PTLD complicating T-cell deplete allo-HSCT, both of whom were successfully rescued with 1 × 106/Kg unselected stem cell donor-derived DLI. Prospective analyses revealed that complete clinical and radiological responses were associated with in vivo expansion of T and NK cells. Furthermore, EBV MHC tetramer, and interferon gamma analyses revealed a marked increase in EBV-specific T-cell frequency from 4 weeks after DLI. Reactivity was demonstrated against a range of EBV latent and lytic antigens, including those detected in tumor biopsy material. The immunodominant EBV-specific T cell response expanding in vivo following infusion matched the dominant response present in the DLI preparations prior to administration. Furthermore, differences in the repertoire of subdominant antigen-specific T-cells were also detected, suggesting that antigen-encounter in vivo can shape the immune response. These results demonstrate the value of prospectively studying in vivo T-cell responses, by facilitating the identification of important specificities required for clinical efficacy. Applying this approach on a larger scale promises to yield data which may be essential for the optimization of future adoptive immunotherapeutic strategies for PTLD.

CD154-expressing CMV-specific T cells associate with freedom from DNAemia and may be protective in seronegative recipients after liver or intestine transplantation.

Cell-mediated immunity to CMV, if known, could improve antiviral drug therapy in at-risk children and young adults with LT and IT. Host immunity has been measured with CMV-specific T cells, which express IFNγ, but not those which express CD154, a possible substitute for IFNγ. CMV-specific CD154+ T cells and their subsets were measured with flow cytometry after stimulating PBL from recipient blood samples with an overlapping peptide mix of CMV-pp65 antigen for up to 6hours. CMV-specific CD154+ T cells co-expressed IFNγ in PBL from three healthy adults and averaged 3.8% (95% CI 3.2%-4.4%) in 40 healthy adults. CMV-specific T cells were significantly lower in 19 CMV DNAemic LT or IT recipients, compared with 126 non-DNAemic recipients, 1.3% (95% CI 0.8-1.7) vs 4.1 (95% CI 3.6-4.6, P<.001). All T-cell subsets demonstrated similar between-group differences. In logistic regression analysis of 46 training set samples, 12 with DNAemia, all obtained between days 0 and 60 from transplant, CMV-specific T-cell frequencies ≥1.7% predicted freedom from DNAemia with NPV of 93%. Sensitivity, specificity, and PPV were 83%, 74%, and 53%, respectively. Test performance was replicated in 99 validation samples. In 32 of 46 training set samples, all from seronegative recipients, one of 19 recipients with CMV-specific T-cell frequencies ≥1.7% experienced DNAemia, compared with 8 of 13 recipients with frequencies <1.7% (P=.001). CMV-specific CD154+ T cells are associated with freedom from DNAemia after LT and IT. Among seronegative recipients, CMV-specific T cells may protect against the development of CMV DNAemia.

Disseminated cytomegalovirus disease after bendamustine: a case report and analysis of circulating B- and T-cell subsets

BackgroundBendamustine, used for the treatment of indolent B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia, is known to cause prolonged myelosuppression and lymphocytopenia and has been associated with the risk of developing serious and fatal infections. While reports of localized CMV infections in asymptomatic patients exist, disseminated CMV disease has not been described.Case presentationWe report the first case of disseminated CMV infection in a 75-year-old male diagnosed with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia with massive bone marrow infiltration. Despite 6-cycle R-bendamustine chemotherapy resulted in a good partial response, the patient developed persistent fever and severe weight loss. Analysis of cerebrospinal fluid and peripheral blood revealed the presence of CMV-DNA, while the fundus oculi examination revealed bilateral CMV retinitis. Treatment with induction and maintenance drugs was complicated by neutropenia and deterioration of renal function with electrolyte imbalance. From an immunological standpoint, we observed a profound imbalances in phenotype and function of B- and T-cell subsets, with a high proportion of circulating total, activated CD69+ and CD80+ B-cells, a low γ/δ T-cell frequency with a high proportion of CD69- and CD38-expressing cells, and hyperactivated/exhausted CD4+ and CD8+ T-cell phenotypes unable to face CMV challenge.ConclusionsWe hereby describe a severe form of disseminated CMV disease after R-bendamustine treatment. Our observations strongly support the careful clinical monitoring of CMV reactivation/infection in oncologic patients undergoing this therapeutic regimen.

Icaritin promotes tumor T-cell infiltration and induces antitumor immunity in mice.

Icaritin, a hydrolytic product of icariin isolated from traditional Chinese herbal medicine genus Epimedium, has many pharmacological and biological activities. Here, we show that icaritin can effectively decrease tumor burden of murine B16F10 melanoma and MC38 colorectal tumors in a T-cell dependent manner. The treatment effects are associated with increased CD8 T-cell infiltration and increased effector memory T-cell frequency. In vivo depletion of CD8 Tcell using an anti-CD8 monoclonal antibody abolished the antitumor effect, which supports the critical role of CD8 Tcells during icaritin treatment. By analyzing immune cells in the tumor tissue, we found reduced frequency of CD11b+ Gr1+ myeloid-derived suppression cells (MDSCs) infiltration and downregulation of PD-L1 expression on MDSCs after icaritin treatment. This was not limited to MDSCs, as icaritin also decreased the expression of PD-L1 on neutrophils. Importantly, the combination of anti-PD-1/CTLA-4 and icaritin significantly enhances antitumor ability and increases the efficacy of either treatment alone. Our findings reveal that icaritin induces antitumor immunity in a CD8 T-cell-dependent way and justify further investigation of combining immune checkpoint therapy to icaritin-based antitumor therapy.

CD8+CD28- T cells: key cytotoxic players impacting disease pathogenesis in chronic HBV infection.

During chronic hepatitis B (CHB), CD8+ T cells down-regulate CD28, the primary co-stimulation molecule for T-cell activation. Diverse functional attributes of CD8+CD28- T cells are suggested in various disease contexts. The present study aimed to characterize CD8+CD28- T cells in different phases of chronic Hepatitis B virus (HBV) infection (CHI)- Immune-tolerance (IT), Hepatitis B e-antigen-positive CHB (EP-CHB), Inactive carriers (IC) and Hepatitis B e-antigen-negative CHB (EN-CHB), to appraise their contribution in HBV-related disease pathophysiology. Flow cytometry analysis of T cells in peripheral blood of study subjects revealed enhanced CD8+CD28- T-cell accumulation in EP-/EN-CHB, compared with IT/IC and they expanded equivalently in HBV-specific and non-specific CD8+ T-cell compartments. Profound increase in CD8+CD28- T cells expressing perforin/granzyme-B/CD57/IFN-γ/TNF-α and markers of terminal differentiation were observed exclusively in EP-/EN-CHB. Further, activation with anti-NKG2D resulted in heightened IFN-γ/TNF-α production selectively from CD8+CD28- T cells, suggesting NKG2D-mediated alternative co-stimulation. CD8+CD28- T cells sorted from CHB patients induced enhanced apoptosis of peripheral blood mononuclear cells (PBMC), including CD4+ T cells. However, NKG2D-ligand (major histocompatibility complex class I chain-related molecule A/B (MICA/B)) was preferentially expressed by HBV-specific CD4+ T cells of CHB patients, making these cells a potential target to NKG2D-dependent CD8+CD28- T-cell killing. Both CD28+ and CD28- T cells in CHB expressedCXCR3 at similar levels and thus capable of homing to the liver. A positive correlation was seen between CD8+CD28- T-cell frequency and serum-alanine transaminase (ALT) levels and CHB-derived CD8+CD28- T cells caused pronounced cell death in HBV-transfected Huh7 cells. Immunofluorescence staining identified greater intrahepatic incidence of CD8+CD28- T cells but decline in CD4+ T cells in CHB than IC. Collectively, CD8+CD28- T cells demonstrated differential distribution and phenotypic/functional skewing in different CHI phases and contribute to disease progression by Perforin-Granzyme- or IFN-γ-TNF-α-mediated cytotoxicity while restraining antiviral immunity through NKG2D-dependent HBV-specific CD4+ T-cell depletion.

Systematic Assessment of Immune Marker Variation in Type 1 Diabetes: A Prospective Longitudinal Study.

Immune analytes have been widely tested in efforts to understand the heterogeneity of disease progression, risk, and therapeutic responses in type 1 diabetes (T1D). The future clinical utility of such analytes as biomarkers depends on their technical and biological variability, as well as their correlation with clinical outcomes. To assess the variability of a panel of 91 immune analytes, we conducted a prospective study of adults with T1D (<3 years from diagnosis), at 9–10 visits over 1 year. Autoantibodies and frequencies of T-cell, natural killer cell, and myeloid subsets were evaluated; autoreactive T-cell frequencies and function were also measured. We calculated an intraclass correlation coefficient (ICC) for each marker, which is a relative measure of between- and within-subject variability. Of the 91 analytes tested, we identified 35 with high between- and low within-subject variability, indicating their potential ability to be used to stratify subjects. We also provide extensive data regarding technical variability for 64 of the 91 analytes. To pilot the concept that ICC can be used to identify analytes that reflect biological outcomes, the association between each immune analyte and C-peptide was also evaluated using partial least squares modeling. CD8 effector memory T-cell (CD8 EM) frequency exhibited a high ICC and a positive correlation with C-peptide, which was also seen in an independent dataset of recent-onset T1D subjects. More work is needed to better understand the mechanisms underlying this relationship. Here we find that there are a limited number of technically reproducible immune analytes that also have a high ICC. We propose the use of ICC to define within- and between-subject variability and measurement of technical variability for future biomarker identification studies. Employing such a method is critical for selection of analytes to be tested in the context of future clinical trials aiming to understand heterogeneity in disease progression and response to therapy.

Partner HIV Serostatus Impacts Viral Load, Genital HIV Shedding, and Immune Activation in HIV-Infected Individuals.

Studies of seronegative individuals in HIV discordant relationships provide important insights into the effects of HIV exposure on the seronegative partner, but few have examined the impact of partner serostatus on disease progression in seropositive individuals. We investigated the impact of HIV serostatus on clinical and biological factors influencing HIV disease progression in 337 HIV-infected heterosexual individuals in stable long-term HIV-seroconcordant or HIV-serodiscordant relationships. Seroconcordant individuals had significantly higher plasma viral loads (pVLs) than HIV-infected partners in serodiscordant partnerships [4.4 log10 copies RNA/mL (interquartile range 3.7-5.0) versus 3.9 (3.3-4.5), P < 0.0001], irrespective of gender. pVLs correlated inversely with CD4 T-cell counts, although CD4 counts did not differ significantly between seroconcordant and serodiscordant individuals. HIV+ seroconcordant individuals had higher frequencies of CCR5 CD4 and CD8 T cells (P = 0.03 and P = 0.02, respectively) than HIV+ individuals in serodiscordant relationships and higher concentrations of plasma IL-1β (P = 0.04), TNF-α (P = 0.02), and IL-10 (P = 0.02). Activated CD4 T-cell frequencies and TNF-α were the most influential in determining variation in pVLs, independently of CD4 counts. In addition, HIV+ seroconcordant women had significantly higher genital VLs (gVLs) than HIV+ women in serodiscordant relationships (P < 0.001), with pVLs correlating significantly with gVLs (Rho = 0.65, P < 0.0001). Cervical and blood T-cell activation tended to correlate positively, although partner seroconcordance did not influence genital T-cell activation. We conclude that HIV+ seroconcordant individuals have higher frequencies of activated, CCR5-expressing T cells in blood and higher pVLs and gVLs than their HIV+ counterparts in discordant relationships, which could translate to faster disease progression or larger viral reservoir.

A Randomized Comparison of Antiretroviral Therapy Alone Versus Antiretroviral Therapy with a 'Kick-and-Kill' Approach, on Measures of the HIV Reservoir Amongst Participants with Recent HIV Infection: The RIVER Trial

Background: Antiretroviral therapy (ART) alone cannot cure HIV infection because of a persistent reservoir of latently infected cells. Approaches that force HIV transcription from latently infected cells, making them susceptible to targeted killing - termed "kick and kill" -have the capacity to eliminate cells that comprise the reservoir, and have been explored as a strategy towards an HIV cure. RIVER is the first randomized trial to determine the impact of ART alone versus ART plus 'kick-and-kill' on markers of the HIV reservoir. Methods: RIVER was an open-label, multicenter, 1:1 randomized controlled trial of ART-only (control) versus ART plus the histone deacetylase inhibitor vorinostat (the 'kick') and replication-deficient viral vector vaccines encoding conserved HIV sequences ChAdV63.HIVconsv-prime, MVA.HIVconsv-boost T-cell vaccination (the 'kill') (ART+V+V; intervention) in HIV-positive adults treated in recent HIV-infection. The primary endpoint was total HIV DNA in peripheral blood CD4+ T-cells at weeks 16 and 18 post-randomization. Secondary endpoints included safety, quantitative viral outgrowth, HIV-specific T-cell frequencies, CD8+ T-cell mediated viral inhibition, and histone acetylation. Findings: Sixty HIV-positive male participants were randomized and completed the study interventions, with no loss-to-follow-up. There were no intervention-related serious adverse events. Histone acetylation increased 3.2-fold two hours post-vorinostat dosing (p<0.001). Significantly higher HIV-specific T-cell responses were observed in the ART+V+V than the ART-only arm. Overall mean total HIV DNA was 3.04 log10 copies HIV DNA/106 CD4+ T-cells with no statistically significant difference between the arms in the primary endpoint (mean difference (intervention - control) of 0.04 (95%CI -0.03, 0.11) log10 total HIV DNA copies/106 CD4+ T-cells (p=0.256)), or in the proportion of participants with undetectable viral outgrowth (Odds Ratio 0.41 (95%CI 0.13,1.35) (p=0.145)). Interpretations: Despite significantly enhanced vaccine-induced HIV-specific T-cell immunity and vorinostat-induced histone acetylation, this 'kick-and-kill' approach conferred no significant benefit compared to ART alone on measures of the HIV reservoir. Trial Registration: NCT02336074 Funding Statement: The trial was funded by grant MR/L00528X/1 MRC DPFS; The vaccine GMP manufacture was jointly funded by the UK Medical Research Council and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreements (MRC G0701669) by award to Tomas Hanke (TH) and Lucy Dorrell (LD), and by EC FP7 award 305632 to LD, TH and others. TH and LD are the Jenner Institute, University of Oxford Investigators. Sarah Fidler is partly funded through the Imperial College NIHR BRC. John Frater is funded by the Medical Research Council (MR/L006588/1). The MRC Clinical Trials Unit at University College London is supported by grants from the MRC (MC-UU-12023/23 and MC-UU-12023/26).This work is supported by the CHERUB Collaboration. Declaration of Interests: No competing interests to disclose. Ethics Approval Statement: Ethics committee approval was obtained from all participating centres, (14/SC/1372) in accordance with the principles of the Declaration of Helsinki.

Defining remission in rheumatoid arthritis: does it matter to the patient? A comparison of multi-dimensional remission criteria and patient reported outcomes.

In a cross-sectional study, we evaluated the prevalence of 'multi-dimensional remission' (MDR) and its component parameters, assessed using objective measures in a cohort of RA patients in treatment-induced DAS28-remission, and their relationship with patient-reported outcome measures. We sought to confirm the feasibility and face validity of the MDR construct, providing a platform for future longitudinal studies in which its clinical utility might be further established. 605 patients were selected from an inflammatory arthritis register using DAS28(CRP)<2.6. Demographic, clinical and patients reported outcomes (PRO) data were collected. Ultrasound power doppler synovitis (n = 364) and T-cell subsets (n = 297) were also measured. Remission using clinical parameters was defined as: tender and swollen joint count (TJC/SJC) and CRP all ⩽1; ultrasound remission: total power doppler = 0 and T cell remission: positive normalized naïve T-cell frequency. MDR was defined as the achievement of all three dimensions. Overall, only 53% (321/605) of the patients achieved clinical parameters, failures being mainly due to raised CRP (52%), TJC (28)>1 (37%) or SJC (28)>1 (16%). 211/364 (58%) of patients achieved ultrasound remission and 193/297 (65%) patients showed T-cell remission. Complete data were available for 231 patients. MDR was observed in only 35% and was associated with the best (lower) PRO scores (all P ⩽ 0.05 vs non-MDR) when compared with the other definitions of remission assessed. The MDR rate was similar in early and established RA patients on b-DMARDs; however, it was lower in established RA patients who received multiple cs-DMARDs (P = 0.011). In this study, MDR, which may represent a state closer to normality, was found to occur in about a third of DAS28-remission patients and was associated with better patient-reported outcome measures. MDR could be a novel optimal treatment target, notably from a patient's perspective. The relevance of these findings needs further assessment.

Peripheral PD-1+CD56+ T-cell frequencies correlate with outcome in stage IV melanoma under PD-1 blockade

Immune checkpoint blockade with anti-PD-1 antibodies is showing great promise for patients with metastatic melanoma and other malignancies, but despite good responses by some patients who achieve partial or complete regression, many others still do not respond. Here, we sought peripheral blood T-cell biomarker candidates predicting treatment outcome in 75 stage IV melanoma patients treated with anti-PD-1 antibodies. We investigated associations with clinical response, progression-free survival (PFS) and overall survival (OS). Univariate analysis of potential biological confounders and known biomarkers, and a multivariate model, was used to determine statistical independence of associations between candidate biomarkers and clinical outcomes. We found that a lower than median frequency of peripheral PD-1+CD56+ T-cells was associated with longer OS (p = 0.004), PFS (p = 0.041) and superior clinical benefit (p = 0.009). However, neither frequencies of CD56-CD4+ nor CD56-CD8+ T-cells, nor of the PD-1+ fraction within the CD4 or CD8 subsets was associated with clinical outcome. In a multivariate model with known confounders and biomarkers only the M-category (HR, 3.11; p = 0.007) and the frequency of PD-1+CD56+ T-cells (HR, 2.39; p = 0.028) were identified as independent predictive factors for clinical outcome under PD-1 blockade. Thus, a lower than median frequency of peripheral blood PD-1+CD56+ T-cells prior to starting anti-PD-1 checkpoint blockade is associated with superior clinical response, longer PFS and OS of stage IV melanoma patients.

Effect of homeostatic T-cell proliferation in the vaccine responsiveness against influenza in elderly people

BackgroundSeasonal influenza virus infection is a significant cause of morbimortality in the elderly. However, there is poor vaccine efficacy in this population due to immunosenescence. We aimed to explore several homeostatic parameters in the elderly that could impact influenza vaccine responsiveness.MethodsSubjects (> 60 years old) who were vaccinated against influenza virus were included, and the vaccine response was measured by a haemagglutination inhibition (HAI) test. At baseline, peripheral CD4 and CD8 T-cells were phenotypically characterized. Thymic function and the levels of different inflammation-related biomarkers, including Lipopolysaccharide Binding Protein (LBP) and anti-cytomegalovirus (CMV) IgG antibodies, were also measured.ResultsInfluenza vaccine non-responders showed a tendency of higher frequency of regulatory T-cells (Tregs) before vaccination than responders (1.49 [1.08–1.85] vs. 1.12 [0.94–1.63], respectively, p = 0.061), as well as higher expression of the proliferation marker Ki67 in Tregs and different CD4 and CD8 T-cell maturational subsets. The levels of inflammation-related biomarkers correlated with the frequencies of different proliferating T-cell subsets and with thymic function (e.g., thymic function with D-dimers, r = − 0.442, p = 0.001).ConclusionsAge-related homeostatic dysregulation involving the proliferation of CD4 and CD8 T-cell subsets, including Tregs, was related to a limited responsiveness to influenza vaccination and a higher inflammatory status in a cohort of elderly people.

Abstract 4049: Cellular phenotypes of spleen cells in cancer patients targeted for adoptive cellular therapy

Abstract The spleen is a rich resource of large numbers of lymphocytes, with potential for use in adoptive cellular therapy (ACT). This is of particular relevance to patients with a distal pancreatic ductal adenocarcinoma (PDAC) who frequently undergo a pancreatectomy that required a splenectomy to clear the nodal basin. In this study we compared the phenotypes of eight paired sets of peripheral blood (PB) and spleen cell populations to assess the frequency of myeloid derived suppressor cells (MDSC) and lymphocyte subpopulations. Because a positive clinical response to ACT requires a low frequency of T-regs and MDSC’s, both systemically and in the tumor microenvironment these cells were one of the foci for our studies. A high frequency of these cells negatively correlate with survival and positively with tumor burden. Indeed, in this study we observed a significant decrease in the frequency of granulocytic, monocytic and immature MDSC’s in the spleen vs PB (4.8±1.6 vs. 54.4±6.1; 0.2±0.1 vs. 0.9±0.4; and 0.6±0.1 vs. 2.3±0.8 percent respectively) but not T-regulatory (T-reg) cells (0.8+0.5 vs. 0.1+0.04 percent) supporting the potential superiority of spleen cells relative to PB as a cellular source for ACT. In addition to a significantly higher number of spleen cells as compared to the PB for expansion/infusion, spleens have a higher frequency of T-cells as compared to the PB including CD3+ (35+5 vs. 16+4%), CD4+ (19+2 vs. 10+2%) and CD8+ (10+1 vs. 5+2%) T-cells. The frequency of transitional memory (Ttm) T-cells, which have a high proliferative potential were also significantly increased in the spleen vs. PB (37.5±6 vs. 26±3.0%) as was the frequency of PD1+CD8+ T-cells (8+2 vs 1+0.5%), which have been shown to have tumor specific cytotoxicity in cancer patients. In summary the increased frequency of PD-1+CD8+ cells, a Ttm memory phenotype and low levels of an exhaustion phenotype (TIM3 and Lag3 expression) in spleen cells vs the PB is supportive of the potential utility of these cells for expansion and therapeutic utility for these cells. Citation Format: Kathryn Cole, Quan Ly, Jesse L. Cox, Michael A. Hollingsworth, James C. Padussis, Jason M. Foster, Luciano M. Vargas, James E. Talmadge. Cellular phenotypes of spleen cells in cancer patients targeted for adoptive cellular therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4049.

Lung Injury on Antiretroviral Therapy in Adults With Human Immunodeficiency Virus/Tuberculosis.

Immune restoration on antiretroviral therapy (ART) can drive inflammation in people living with human immunodeficiency virus (HIV) who have pulmonary tuberculosis (TB), but its effects on the lungs have not been assessed. We evaluated associations between pulmonary inflammation, recovery of pathogen-specific CD4 T-cell function, and lung injury prior to and after ART initiation in adults with HIV and pulmonary TB. This was a prospective cohort study in South Africa, following adults with HIV and pulmonary TB prior to and up to 48 weeks after ART initiation. Pulmonary-specific inflammation was defined as total glycolytic activity (TGA) on [18]F-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT) at baseline and 4 weeks after ART initiation. Spirometry, respiratory symptom tests, and flow cytometry were performed at the same times to assess lung involvement and the frequency of mycobacteria-specific CD4 T-cells. In addition, we evaluated lung function longitudinally up to 48 weeks after ART initiation. Greater lung TGA on FDG PET-CT was associated with worse lung function and respiratory symptoms prior to ART initiation, and nearly half of subjects experienced worsening lung inflammation and lung function at Week 4 of ART. Worsening Week 4 lung inflammation and pulmonary function were both associated with greater increases in pathogen-specific functional CD4 T-cell responses on ART, and early decreases in lung function were independently associated with persistently lower lung function months after TB treatment completion. Increases in pulmonary inflammation and decreases in lung function are common on ART, relate to greater ART-mediated CD4 T-cell restoration, and are associated with the persistent impairment of lung function in individuals with HIV/TB.

Alteration of liver-infiltrated and peripheral blood double-negative T-cells in primary biliary cholangitis.

Double-negative (DN) T-cell is a unique regulatory T-cell, which is essential for maintaining immune system homoeostasis. However, the role of DN T-cells in the pathogenesis of primary biliary cholangitis (PBC) is still unknown. We investigated the number and function of DN T-cells in peripheral blood and liver biopsy specimens of PBC patients. The number and frequency of DN T-cells significantly decreased in peripheral blood and liver tissue of PBC patients. Furthermore, the frequency of DN T-cells in PBC was negatively correlated with disease severity and positively correlated with ursodeoxycholic acid response. In vitro assays showed that perforin expression and the suppressive capability of DN T-cells on the proliferation of CD4+ and CD8+ T-cells were impaired in PBC. Finally, lithocholic acid, the most hydrophobic acid, could downregulate the proliferation and perforin expression of DN T-cells. Decreased quantity and function of DN T-cells in PBC may result in the loss of immune regulations on effector CD4+ and cytotoxic CD8+ T-cells, and thereby may break the immune tolerance and promote the pathogenesis of PBC.

Dynamic Changes in Ex Vivo T-Cell Function After Viral Clearance in Chronic HCV Infection

Direct-acting antiviral (DAA) agents can successfully treat chronic hepatitis C virus (HCV) infection. However, the ex vivo HCV-specific T-cell function following viral clearance remains unknown. We investigated functional alterations and phenotypic changes in ex vivo HCV-specific CD8+ T cells with a longitudinal analysis of 41 patients with chronic HCV infection who were undergoing DAA treatment. A patient subset exhibited a significantly increased T-cell response (mainly CD8+ T cells) at week 4 of treatment. However, this increased T-cell response diminished in later weeks. Relative to pretreatment levels, the ex vivo HCV-specific CD8+ T-cell frequency decreased at 12 weeks after the end of treatment, along with a decreased antigen-experienced CD8+ T-cell population. DAA treatment increased the proliferative capacity of HCV-specific CD8+ T cells, but this change was not correlated with ex vivo function. Patients experiencing viral breakthrough or relapse exhibited defective restoration of T-cell function. Our present results indicated that DAA-mediated viral clearance only transiently restored ex vivo T-cell function, suggesting a need to enhance T-cell function in DAA-treated patients.

Immune profiling of tumor-infiltrating T cells using mass cytometry.

2607 Background: Immunotherapy recent successes have opened new avenues for the treatment of cancer and the presence of tumor-specific CD8+ T cells in tumor-bearing individuals offer a promising therapeutic target. However, the detection and profiling of such T cells are challenging due to the need to detect rare antigen-specific T cell subpopulations in patient samples that are limited in size thus making it difficult to exploit these parameters for predictive signatures of clinical response. Moreover, the identification and analysis of neoantigen-specific CD8+ T-cells in tumor-bearing individuals is challenging due to the small pool of such cells. Methods: In order to identify therapy-relevant tumor antigens and to facilitate a concurrent in-depth characterization of cells directed towards these targets, immunoSCAPE leverages the high-dimensional immune profiling capabilities of cytometry by time of flight (CyTOF) combined with a unique technology allowing the identification rare antigen-specific T-cell subsets. Results: We applied this technology to patient tumor-infiltrating lymphocytes from human cancer samples and tumor-derived neoantigens recognized by T-cells were identified and characterized. Interestingly, the majority of patient-derived tumor infiltrates consisted of tumor-unrelated T-cells characterized by a diverse phenotype. Strikingly, the expression of CD39 was absent from these bystander cells, suggesting that CD39 could be a useful biomarker for the identification of putative tumor-reactive T cells. Conclusions: Simultaneous immune profiling revealed that tumor-unrelated, bystander CD8+ T-cells are phenotypically different in human tumor infiltrates and identified CD39 as a putative marker of neoantigen-specific T-cells. By providing insights into the nature, frequency and phenotype of antigen-specific T-cells, immunoSCAPE’s unique target discovery and high-dimensional immune profiling platform is a valuable tool for the development of novel diagnostic and therapeutic strategies in immunotherapy.

NK- and T-cell subsets in malignant mesothelioma patients: Baseline pattern and changes in the context of anti-CTLA-4 therapy.

Malignant mesothelioma (MM) is a highly aggressive form of cancer with limited treatment options. Although the role of NK cells has been studied in many solid tumors, the pattern of NK-cell subsets and their recognition of mesothelioma cells remain to be explored. We used RNA expression data of MM biopsies derived from the cancer genome atlas to evaluate the immune cell infiltrates. We characterized the phenotype of circulating NK and T cells of 27 MM patients before and after treatment with an anti-CTLA-4 antibody (tremelimumab). These immune cell profiles were compared to healthy controls. The RNA expression data of the MM biopsies indicated the presence of NK cells in a subgroup of patients. We demonstrated that NK cells recognize MM cell lines and that IL-15 stimulation improved NK cell-mediated lysis in vitro. Using multivariate projection models, we found that MM patients had a perturbed ratio of CD56bright and CD56dim NK subsets and increased serum concentrations of the cytokines IL-10, IL-8 and TNF-α. After tremelimumab treatment, the ratio between the CD56bright and CD56dim subsets shifted back towards physiological levels. Furthermore, the improved overall survival was correlated with low TIM-3+ CD8+ T-cell frequency, high DNAM-1+ CD56dim NK-cell frequency and high expression levels of NKp46 on the CD56dim NK cells before and after immune checkpoint blockade. Together, our observations suggest that NK cells infiltrate MM and that they can recognize and kill mesothelioma cells. The disease is associated with distinct lymphocytes patterns, some of which correlate with prognosis or are affected by treatment with tremelimumab.

Frequency of Myeloid Derived Suppressor Cells in Spleen and Peripheral Blood from Cancer Patients

Abstract The peripheral blood (PB) of cancer patients is frequently neutrophilic and lymphopenic with few studies focused on tissue immunity. In this study we examined eight paired sets of PB and spleen populations to assess the frequency and sub-populations of myeloid derived suppressor- (MDSC) and T-cells. These studies observed a significant decrease in the frequency of granulocytic (G), monocytic (M) and immature MDSC’s in the spleen vs PB (4.8±1.6 vs. 54.4±6.1; 0.2±0.1 vs. 0.9±0.4; and 0.6±0.1 vs. 2.3±0.8 % respectively) but not T-regulatory cells (0.8±0.5 vs. 0.1±0.04 percent). The difference in the G vs M MDSC was consistent with arginase and nitric oxide synthase expression. In contrast, the % of splenic T-cells was increased relative to the PB including CD3+ (35±5 vs. 16±4%), CD4+ (19±2 vs. 10±2%) and CD8+ (10±1 vs. 5±2%). The frequency of transitional memory T-cells (Ttm) was significantly higher in the spleen vs. PB (37.5±6 vs. 26±3.0%) as was the frequency of PD1+CD8+ T-cells (8±2 vs 1±0.5% respectively). The increase in Ttm with their heightened proliferation, effector potential and tumor specific cytotoxicity of PD1+CD8+ cells support the superiority of spleen vs PB cells for adoptive cellular therapy. In support of this both the PD1+ cells were LAG3− and TIM3−, and thus not exhausted. In summary, the spleen cells of cancer patients have an increase in tumor specific effector cells with proliferative potential supporting their utility in therapeutic protocols.

Myelin oligodendrocyte glycoprotein revisited—sensitive detection of MOG-specific T-cells in multiple sclerosis

Autoreactive CD4+ T-cells are believed to be a main driver of multiple sclerosis (MS). Myelin oligodendrocyte glycoprotein (MOG) is considered an autoantigen, yet doubted in recent years. The reason is in part due to low frequency and titers of MOG autoantibodies and the challenge to detect MOG-specific T-cells. In this study we aimed to analyze T-cell reactivity and frequency utilizing a novel method for detection of antigen-specific T-cells with bead-bound MOG as stimulant.Peripheral blood mononuclear cells (PBMCs) from natalizumab treated persons with MS (n = 52) and healthy controls (HCs) (n = 24) were analyzed by IFNγ/IL-22/IL-17A FluoroSpot. A higher number of IFNγ (P = 0.001), IL-22 (P = 0.003), IL-17A (P < 0.0001) as well as double and triple cytokine producing MOG-specific T-cells were detected in persons with MS compared to HCs. Of the patients, 46.2–59.6% displayed MOG-reactivity. Depletion of CD4+ T-cells or monocytes or blocking HLA-DR completely eliminated the MOG specific response. Anti-MOG antibodies did not correlate with T-cell MOG-responses.In conclusion, we present a sensitive method to detect circulating autoreactive CD4+ T-cells producing IFNγ, IL-22 or IL-17A using MOG as a model antigen. Further, we demonstrate that MOG-specific T-cells are present in approximately half of persons with MS.

Characterization of a Subset of Patients With Rheumatoid Arthritis for Whom Current Management Strategies are Inadequate.

ObjectiveA subset of patients with seropositive rheumatoid arthritis (RA) do not mount a C‐reactive protein (CRP) response during flares. We hypothesize that these patients are more likely to experience poor clinical care and less likely to respond to traditional therapy. This study questioned whether this presentation was associated with worse disease outcome and distinct immunological features.MethodsUsing Power Doppler ultrasound, 48 RA patients with active synovitis were recruited; 30 had normal (n)CRP (5 mg/L or less) and 18 had high (h)CRP (more than 5 mg/L) levels. All had equivalent disease burden assessed by other clinical and laboratory parameters.ResultsTime to diagnosis and time to first disease‐modifying antirheumatic drug were significantly longer in nCRP compared with hCRP patients (P < 0.05). Significantly more nCRP patients needed escalation to biologics after 2‐year follow‐up (P = 0.01). The inflammatory milieu was also different between the two subgroups. Synergy between inflammatory cytokines observed in hCRP patients was lost in nCRP patients, and nCRP patients had significantly increased regulatory T‐cell (Treg) frequencies that correlated positively with predictors of poor disease outcome. Conversely, hCRP but not nCRP patients demonstrated a significant upregulation of alternative complement pathway factors that correlated negatively with Treg frequency.ConclusionPatients with nCRP during flares of RA had an altered immunological profile compared with hCRP patients and experienced diagnostic delays and responded less favorably to conventional treatment.