Tissue-resident mucosal-associated invariant T (MAIT) cells in the human kidney represent a functionally distinct subset.

Mucosal-associated invariant T (MAIT) cells are innate-like T-cells that recognize bacterial riboflavin metabolites. They are present in human blood but are abundant at barrier sites, including the liver, lungs, and kidneys, where they possess a CD69+ /CD103+/- tissue-resident phenotype. In renal tissue, MAIT cells likely defend against the ascending uropathogens responsible for urinary tract infections (UTIs), which are common, especially among renal transplant recipients (RTRs). Nevertheless, the functional role for MAIT cells in renal tissue and the influence of renal transplantation on MAIT cells remains unclear. Using multiparameter flow cytometry and the MR1-tetramer, we characterized MAIT cell phenotype and function in healthy renal tissue (n = 6), renal transplants explanted after allograft failure (n = 14) and in blood from healthy controls (n = 20) and RTRs before and 1-year after transplantation (n = 21). MAIT cells in renal tissue constitute a distinct CD69+ CD103+/- population that displays typical phenotypic features of tissue-resident T-cells and is skewed toward IL-2, GM-CSF, and IL-17A production upon stimulation. The circulating MAIT cell population was not decreased in number in RTRs pre- or post-transplantation. Tissue-resident MAIT cells in the kidney represent a functionally distinct population. This shows how MAIT cells in the kidney may be involved in the protection against microorganisms.

Circulating mucosal-associated invariant T cells in subjects with recurrent urinary tract infections are functionally impaired.

BACKGROUND: Urinary tract infection recurrence is common, particularly in women and immunocompromised patients, such as renal transplant recipients (RTRs). Mucosal-associated invariant T (MAIT) cells play a role in the antibacterial response by recognizing bacterial riboflavin metabolites produced by bacteria such as Escherichia coli. Here, we investigated whether MAIT cells are involved in the pathogenesis of recurrent urinary tract infections (RUTIs). METHODS: Using multichannel flow cytometry, we characterized the MAIT cell phenotype and function in blood from immunocompetent adults with (n = 13) and without RUTIs (n = 10) and in RTRs with (n = 9) and without RUTIs (n = 10). RESULTS: There were no differences in the numbers of MAIT cells between the study groups. MAIT cells in patients with RUTI expressed T-bet more often than those in controls. MAIT cells from immunocompetent RUTI participants required more antigen-presenting cells coincubated with E. coli to evoke a similar cytokine and degranulation response than those from controls. This effect was absent in the RTR with RUTI vs RTR control groups, where the overall percentage of MAIT cells that responded to stimulation was already reduced. CONCLUSION: Circulating MAIT cells in immunocompetent individuals with RUTIs respond to bacterial stimuli with reduced efficacy, which suggests that they are involved in the pathogenesis of RUTIs.

Butyrate production in patients with end-stage renal disease.

Background: Chronic kidney disease (CKD) is associated with a decreased intestinal barrier function, causing bacterial translocation over the intestinal wall and triggering a systemic inflammatory response. Butyrate, a short-chain fatty acid produced by certain bacterial strains, is considered instrumental to keep the intestinal barrier intact. There are indications that a decreased amount of these specific bacterial species is part of the cause of the decreased intestinal barrier function in CKD. The aim of this study is (i) to determine if Dutch patients with end-stage renal disease (ESRD) have a decreased amount of butyrate-producing species and butyrate-producing capacity and (ii) whether this correlates with systemic inflammation. Methods: We used qPCR to evaluate the most abundant butyrate-producing species F. prauznitzii, E. rectale and Roseburia spp. and the BCoAT gene, which reflects the butyrogenic capacity of the intestinal microbiota. Fecal samples were collected from healthy kidney donors (n=15), preemptive renal transplant recipients (n=4) and dialysis patients (n=31). Markers of inflammation (CRP and IL-6) and intestinal permeability (D-lactate) were measured in plasma. Results: Patients with ESRD did not have a significantly decreased amount F. prauznitzii, E. rectale and Roseburia spp. or the BCoAT gene. Neither was there a significant correlation with CRP, IL-6 or D-lactate. On the individual level, there were some patients with decreased BCoAT levels and increased levels of CRP, IL-6 and D-lactate. Conclusions: Patients with ESRD do not have a decreased amount of the most abundant butyrate-producing species nor a decreased butyrate-producing capacity.

Immune responses in DAA treated chronic hepatitis C patients with and without prior RG-101 dosing.

BACKGROUND&AIMS: With the introduction of DAA's, the majority of treated chronic hepatitis C patients (CHC) achieve a viral cure. The exact mechanisms by which the virus is cleared after successful therapy, is still unknown. The aim was to assess the role of the immune system and miRNA levels in acquiring a sustained virological response after DAA treatment in CHC patients with and without prior RG-101 (anti-miR-122) dosing. METHODS: In this multicenter, investigator-initiated study, 29 patients with hepatitis C virus (HCV) genotype 1 (n = 11), 3 (n = 17), or 4 (n = 1) infection were treated with sofosbuvir and daclatasvir ± ribavirin. 18 patients were previously treated with RG-101. IP-10 levels were measured by ELISA. Ex vivo HCV-specific T cell responses were quantified in IFN-γ-ELISpot assays. Plasma levels of miR-122 were measured by qPCR. RESULTS: All patients had an SVR12. IP-10 levels rapidly declined during treatment, but were still elevated 24 weeks after treatment as compared to healthy controls (median 53.82 and 39.4 pg/mL, p = 0.02). Functional IFN-γ HCV-specific T cell responses did not change by week 12 of follow-up (77.5 versus 125 SFU/106 PBMC, p = 0.46). At follow-up week 12, there was no difference in plasma miR-122 levels between healthy controls and patients with and without prior RG-101 dosing. CONCLUSIONS: Our data shows that successful treatment of CHC patients with and without prior RG-101 dosing results in reduction of broad immune activation, and normalisation of miR-122 levels (EudraCT: 2014-002808-25). TRIAL REGISTRATION: EudraCT: 2014-002808-25.

Human intrahepatic CD69 + CD8+ T cells have a tissue resident memory T cell phenotype with reduced cytolytic capacity.

Tissue resident memory T cells (TRM) have been identified in various tissues, however human liver TRM to date remain unidentified. TRM can be recognized by CD69 and/or CD103 expression and may play a role in the pathology of chronic hepatitis B (CHB) and hepatitis C virus infection (CHC). Liver and paired blood mononuclear cells from 17 patients (including 4 CHB and 6 CHC patients) were isolated and CD8+ T cells were comprehensively analysed by flowcytometry, immunohistochemistry and qPCR. The majority of intrahepatic CD8+ T cells expressed CD69, a marker used to identify TRM, of which a subset co-expressed CD103. CD69 + CD8+ T cells expressed low levels of S1PR1 and KLF2 and a large proportion (>90%) was CXCR6+, resembling liver TRM in mice and liver resident NK cells in human. Cytotoxic proteins were only expressed in a small fraction of liver CD69 + CD8+ T cells in patients without viral hepatitis, however, in livers from CHB patients more CD69 + CD8+ T cells were granzyme B+. In CHC patients, less intrahepatic CD69 + CD8+ T cells were Hobit+ as compared to CHB and control patients. Intrahepatic CD69 + CD8+ T cells likely TRM which have a reduced cytolytic potential. In patients with chronic viral hepatitis TRM have a distinct phenotype.

Immune phenotype and function of natural killer and T cells in chronic hepatitis C patients who received a single dose of anti-MicroRNA-122, RG-101.

MicroRNA-122 is an important host factor for the hepatitis C virus (HCV). Treatment with RG-101, an N-acetylgalactosamine-conjugated anti-microRNA-122 oligonucleotide, resulted in a significant viral load reduction in patients with chronic HCV infection. Here, we analyzed the effects of RG-101 therapy on antiviral immunity. Thirty-two chronic HCV patients infected with HCV genotypes 1, 3, and 4 received a single subcutaneous administration of RG-101 at 2 mg/kg (n = 14) or 4 mg/kg (n = 14) or received a placebo (n = 2/dosing group). Plasma and peripheral blood mononuclear cells were collected at multiple time points, and comprehensive immunological analyses were performed. Following RG-101 administration, HCV RNA declined in all patients (mean decline at week 2, 3.27 log10 IU/mL). At week 8 HCV RNA was undetectable in 15/28 patients. Plasma interferon-γ-induced protein 10 (IP-10) levels declined significantly upon dosing with RG-101. Furthermore, the frequency of natural killer (NK) cells increased, the proportion of NK cells expressing activating receptors normalized, and NK cell interferon-γ production decreased after RG-101 dosing. Functional HCV-specific interferon-γ T-cell responses did not significantly change in patients who had undetectable HCV RNA levels by week 8 post-RG-101 injection. No increase in the magnitude of HCV-specific T-cell responses was observed at later time points, including 3 patients who were HCV RNA-negative 76 weeks postdosing. CONCLUSION: Dosing with RG-101 is associated with a restoration of NK-cell proportions and a decrease of NK cells expressing activation receptors; however, the magnitude and functionality of ex vivo HCV-specific T-cell responses did not increase following RG-101 injection, suggesting that NK cells, but not HCV adaptive immunity, may contribute to HCV viral control following RG-101 therapy. (Hepatology 2017;66:57-68).

Dynamics of the Immune Response in Acute Hepatitis B Infection.

BACKGROUND: Acute hepatitis B virus infection in adults is generally self-limiting but may lead to chronicity in a minority of patients. METHODS: We included 9 patients with acute hepatitis B virus (HBV) infection and collected longitudinal follow-up samples. Natural killer (NK) cell characteristics were analyzed by flowcytometry. HBV-specific T-cell function was analyzed by in vitro stimulation with HBV peptide pools and intracellular cytokine staining. RESULTS: Median baseline HBV DNA load was 5.12 log IU/mL, and median ALT was 2652 U/mL. Of 9 patients, 8 cleared HBsAg within 6 months whereas 1 patient became chronically infected. Early time points after infection showed increased CD56bright NK cells and an increased proportion of cells expressing activation markers. Most of these had normalized at week 24, while the proportion of TRAIL-positive CD56bright NK cells remained high in the chronically infected patient. In patients who cleared HBV, functional HBV-specific CD8+ and CD4+ responses could be observed, whereas in the patient who developed chronic infection, only low HBV-specific T-cell responses were observed. CONCLUSIONS: NK cells are activated early in the course of acute HBV infection. Broad and multispecific T-cell responses are observed in patients who clear acute HBV infection, but not in a patient who became chronically infected.

Restoration of T cell function in chronic hepatitis B patients upon treatment with interferon based combination therapy.

BACKGROUND & AIMS: Chronic hepatitis B virus (HBV) infection is characterized by functional impairment of HBV-specific T cells. Understanding the mechanisms behind T cell dysfunction and restoration is important for the development of optimal treatment strategies. METHODS: In this study we have first analysed the phenotype and function of HBV-specific T cells in patients with low viral load (HBV DNA <20,000IU/ml) and spontaneous control over the virus. Subsequently, we assessed HBV-specific T cells in patients with high viral load (HBV DNA >17,182IU/ml) treated with peginterferon/adefovir combination therapy who had various treatment outcomes. RESULTS: HBV-specific T cells could be detected directly ex vivo in 7/22 patients with low viral load. These showed an early differentiated memory phenotype with reduced ability to produce IL-2 and cytotoxic molecules such as granzyme B and perforin, but with strong proliferative potential. In a cohort of 28 chronic hepatitis B patients with high viral load treated with peginterferon and adefovir, HBV-specific T cells could not be detected directly ex vivo. However, HBV-specific T cells could be selectively expanded in vitro in patients with therapy-induced HBsAg clearance (HBsAg loss n=7), but not in patients without HBsAg clearance (n=21). Further analysis of HBV-specific T cell function with peptide pools showed broad and efficient antiviral responses after therapy. CONCLUSIONS: Our results show that peginterferon based combination therapy can induce HBV-specific T cell restoration. These findings may help to develop novel therapeutic strategies to reconstitute antiviral functions and enhance viral clearance.

Natural Killer Cell Characteristics in Patients With Chronic Hepatitis B Virus (HBV) Infection Are Associated With HBV Surface Antigen Clearance After Combination Treatment With Pegylated Interferon Alfa-2a and Adefovir.

BACKGROUND: The role of natural killer (NK) cells in the process of hepatitis B virus (HBV) surface antigen (HBsAg) clearance and whether their phenotype is related to treatment outcome in patients with chronic hepatitis B are currently unknown. METHODS: Patients with chronic hepatitis B (HBV DNA load, >17 000 IU/mL) were treated with pegylated interferon alfa-2a and adefovir for 48 weeks. NK cell phenotype and function were analyzed in 7 responders (defined as individuals with HBsAg clearance by week 72; 3 HBV e antigen [HBeAg]-positive and 4 HBeAg-negative), 7 matched nonresponders, and 7 healthy controls. Subsequently, 34 baseline samples from HBeAg-positive patients with chronic hepatitis B were analyzed. RESULTS: During treatment, the percentage and absolute number of CD56(bright) NK cells increased significantly, whereas the percentage and absolute number of CD56(dim) NK cells decreased. At baseline, responders had a significantly lower expression of chemokine receptor CX3CR1 on CD56(bright) NK cells and inhibitory receptor NKG2A on CD56(dim) NK cells, compared with nonresponders. In addition, responders had higher CD56(bright) TRAIL expression and interferon γ production at end of treatment. These baseline differences were not found in HBeAg-positive patients who had HBeAg seroconversion without HBsAg clearance. CONCLUSIONS: Combination therapy significantly influences NK cell phenotype and function. Differences between patients with chronic hepatitis B with HBsAg clearance and nonresponders suggest that NK cells play a role in the clearance of HBsAg during interferon-based combination therapy.

HBsAg loss in patients treated with peginterferon alfa-2a and adefovir is associated with SLC16A9 gene variation and lower plasma carnitine levels.

BACKGROUND & AIMS: Achievement of HBsAg loss remains the hallmark of chronic hepatitis B treatment. In order to identify host factors contributing to treatment-induced HBsAg loss, we performed a genome-wide screen of single nucleotide polymorphisms (SNPs) and studied its immunological consequence. METHODS: Chronic hepatitis B patients (40 HBeAg-positive and 44 HBeAg-negative) treated with peginterferon alfa-2a and adefovir were genotyped for 999,091 SNPs, which were associated with HBsAg loss at week 96 (n = 9). Plasma carnitine levels were measured by tandem-mass spectrometry, and the effect of carnitine on the proliferative capacity of hepatitis B virus (HBV)-specific and non-specific CD8 T cells was studied in vitro. RESULTS: One polymorphism, rs12356193 located in the SLC16A9 gene, was genome-wide significantly associated with HBsAg loss at week 96 (p = 1.84 × 10(-8)). The previously reported association of rs12356193 with lower carnitine levels was confirmed in our cohort, and baseline carnitine levels were lower in patients with HBsAg loss compared to patients with HBsAg persistence (p = 0.02). Furthermore, we demonstrated that carnitine suppressed HBV-specific CD8 T cell proliferation. CONCLUSIONS: In chronic hepatitis B patients treated with peginterferon and adefovir, we identified strong associations of SLC16A9 gene variation and carnitine levels with HBsAg loss. Our results further suggest that a lower baseline plasma carnitine level increases the proliferative capacity of CD8 T cells, making patients more susceptible to the immunological effect of this treatment. These novel findings may provide new insight into factors involved in treatment-induced HBsAg loss, and play a role in the prediction of treatment outcome.

Induction of Staphylococcus aureus-specific IgA and agglutination potency in milk of cows by mucosal immunization.

Lactating cows were immunized with inactivated Staphylococcus aureus strains and concentrated culture supernatants. Application of a repeated mucosal immunization scheme resulted in significant levels of S. aureus-specific IgA in milk of dairy cows. Average IgA titers against whole cell S. aureus increased during the first 10 weeks of immunization after which a plateau level was reached and maintained during lactation. Immune whey agglutinated both bovine and human S. aureus strains including methicillin-resistant S. aureus (MRSA) strains and recognized extracted S. aureus proteins on Western blot. ELISAs to quantify milk IgA reactive with a number of S. aureus virulence proteins (e.g. enterotoxins, microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) and immune modulating proteins) and cell wall components, demonstrated the polyclonality of the IgA. Correlations observed between agglutination and specific IgA titers for whey and for purified IgA suggested functionality of the induced antibodies. Milk from immunized cows may provide a way of producing potentially therapeutic polyclonal antibodies against S. aureus colonization and infection.