Immunosuppression withdrawal in living-donor renal transplant recipients following induction with antithymocyte globulin and rituximab: Results of a prospective clinical trial.

Durable tolerance in kidney transplant recipients remains an important but elusive goal. We hypothesized that adding B cell depletion to T cell depletion would generate an immune milieu postreconstitution dominated by immature transitional B cells, favoring tolerance. The Immune Tolerance Network ITN039ST Research Study of ATG and Rituximab in Renal Transplantation was a prospective multicenter pilot study of live donor kidney transplant recipients who received induction with rabbit antithymocyte globulin and rituximab and initiated immunosuppression (IS) withdrawal (ISW) at 26 weeks. The primary endpoint was freedom from rejection at 52 weeks post-ISW. Six of the 10 subjects successfully completed ISW. Of these 6 subjects, 4 restarted immunosuppressive medications due to acute rejection or recurrent disease, 1 remains IS-free for over 9 years, and 1 was lost to follow-up after being IS-free for 42 weeks. There were no cases of patient or graft loss. CD19+ B cell frequencies returned to predepletion levels by 26 weeks posttransplant; immunoglobulin D+CD27--naïve B cells predominated. In contrast, memory cells dominated the repopulation of the T cell compartment. A regimen of combined B and T cell depletion did not generate the tolerogenic B cell profile observed in preclinical studies and did not lead to durable tolerance in the majority of kidney transplant recipients.

Efficacy and Safety of Immunosuppression Withdrawal in Pediatric Liver Transplant Recipients: Moving Toward Personalized Management.

BACKGROUND AND AIMS: Tolerance is transplantation's holy grail, as it denotes allograft health without immunosuppression and its toxicities. Our aim was to determine, among stable long-term pediatric liver transplant recipients, the efficacy and safety of immunosuppression withdrawal to identify operational tolerance. APPROACH AND RESULTS: We conducted a multicenter, single-arm trial of immunosuppression withdrawal over 36-48 weeks. Liver tests were monitored biweekly (year 1), monthly (year 2), and bimonthly (years 3-4). For-cause biopsies were done at investigators' discretion but mandated when alanine aminotransferase or gamma glutamyltransferase exceeded 100 U/L. All subjects underwent final liver biopsy at trial end. The primary efficacy endpoint was operational tolerance, defined by strict biochemical and histological criteria 1 year after stopping immunosuppression. Among 88 subjects (median age 11 years; 39 boys; 57 deceased donor grafts), 33 (37.5%; 95% confidence interval [CI] 27.4%, 48.5%) were operationally tolerant, 16 were nontolerant by histology (met biochemical but failed histological criteria), and 39 were nontolerant by rejection. Rejection, predicted by subtle liver inflammation in trial entry biopsies, typically (n = 32) occurred at ≤32% of the trial-entry immunosuppression dose and was treated with corticosteroids (n = 32) and/or tacrolimus (n = 38) with resolution (liver tests within 1.5 times the baseline) for all but 1 subject. No death, graft loss, or chronic, severe, or refractory rejection occurred. Neither fibrosis stage nor the expression level of a rejection gene set increased over 4 years for either tolerant or nontolerant subjects. CONCLUSIONS: Immunosuppression withdrawal showed that 37.5% of selected pediatric liver-transplant recipients were operationally tolerant. Allograft histology did not deteriorate for either tolerant or nontolerant subjects. The timing and reversibility of failed withdrawal justifies future trials exploring the efficacy, safety, and potential benefits of immunosuppression minimization.

Immunosuppression Withdrawal in Liver Transplant Recipients on Sirolimus.

BACKGROUND AND AIMS: As conversion from calcineurin inhibitor to sirolimus (SRL), a mechanistic target of rapamycin inhibitor (mTOR-I), has been shown to enhance immunoregulatory profiles in liver transplant (LT) recipients (LTRs), mTOR-I therapy might allow for increased success of immunosuppression (IS) withdrawal. Our aim was to determine if operational tolerance could be observed in LTRs withdrawn from SRL and if blood/graft tolerance biomarkers were predictive of successful withdrawal. APPROACH AND RESULTS: We performed a prospective trial of SRL monotherapy withdrawal in nonimmune, nonviremic LTRs > 3 years post-LT. SRL was weaned over ~6 months, and biopsies were performed 12 months postweaning or at concern for acute rejection. Twenty-one LTRs consented; 6 were excluded due to subclinical acute rejection on baseline biopsy or other reasons, and 15 underwent weaning (age 61.3 ± 8.8 years; LT to SRL weaning 6.7 ± 3 years). Eight (53%) achieved operational tolerance (TOL). Of the 7 who were nontolerant (non-TOL), 6 had mild acute rejection on biopsy near the end of weaning or at study end; 1 was removed from the trial due to liver cancer recurrence. At baseline preweaning, there were statistically increased blood tolerogenic dendritic cells and cell phenotypes correlating with chronic antigen presentation in the TOL versus non-TOL groups. A previously identified biopsy gene signature accurately predicted TOL versus non-TOL in 12/14 LTRs before weaning. At study end, biopsy staining revealed statistically significant increases in antigen-presenting cell:leukocyte pairings, FOXP3+ /CD4+ T cells, Tbet+ /CD8+ T cells, and lobular dendritic cells in the non-TOL group. CONCLUSIONS: This study evaluated IS withdrawal directly from mTOR-I therapy in LTRs and achieved > 50% operational tolerance. Preweaning gene expression and peripheral blood mononuclear cell profiling may be useful as predictors of successful mTOR-I therapy withdrawal. NCT02062944.

IgG4 donor-specific HLA antibody profile is associated with subclinical rejection in stable pediatric liver recipients.

The impact of donor-specific HLA antibody (DSA) following liver transplantation remains controversial. We hypothesized DSA IgG subclass characteristics, compared to total DSA IgG, might correlate with specific histopathological phenotype(s) of subclinical graft injury. We therefore studied 129 stable, arguably "clinically ideal," pediatric liver recipients at the time of a screening biopsy to enter an immunosuppression withdrawal trial. Sixty-five (50%) subjects tested positive for class II DSA. IgG subclass profile was characterized by mean fluorescence intensity (MFI) and normalized subclass composition (>5%). A prominent IgG4 DSA profile was strongly correlated with greater HLA mismatch, a histopathological phenotype characterized by the presence of interface activity (with variable degrees of fibrosis), and a transcriptional profile of attenuated T cell-mediated rejection. Specifically, compared to those without class II DSA, those with IgG4 class II DSA MFI sum >2000 exhibited an odds ratio (OR) of 20.79 (95% confidence interval [CI] 4.38-98.69) and IgG4 subclass composition >5% exhibited an OR of 8.99 (95% CI 2.70-29.9). Our data suggest that IgG4 DSA may serve as a useful biomarker to identify, among clinically and biochemically stable liver transplant recipients, a subset with histological and transcriptional features indicative of an active, suboptimally controlled alloimmune response.

Evidence of Chronic Allograft Injury in Liver Biopsies From Long-term Pediatric Recipients of Liver Transplants.

BACKGROUND & AIMS: A substantial proportion of pediatric liver transplant recipients develop subclinical chronic allograft injury. We studied whether there are distinct patterns of injury based on histopathologic features and identified associated immunologic profiles. METHODS: We conducted a cross-sectional study of 157 stable, long-term pediatric recipients of transplanted livers (70 boys; > 6 years old at time of transplantation; mean, 8.9 ± 3.46 years after liver transplantation) who underwent liver biopsy analysis from August 13, 2012, through May 1, 2014. Participants had received livers from a living or deceased donor and had consistently normal results from liver tests. Liver biopsy specimens were scored by a central pathologist; an unsupervised hierarchical cluster analysis of histologic features was used to sort biopsy samples into 3 clusters. We conducted transcriptional and cytometric analyses of liver tissue samples and performed a systems biology analysis that incorporated clinical, serologic, histologic, and transcriptional data. RESULTS: The mean level of alanine aminotransferase in participants was 27.6 ± 14.57 U/L, and the mean level of γ-glutamyl transferase was 17.4 ± 7.93 U/L. Cluster 1 was characterized by interface activity (n = 34), cluster 2 was characterized by periportal or perivenular fibrosis without interface activity (n = 45), and cluster 3 had neither feature (n = 78). We identified a module of genes whose expression correlated with levels of alanine aminotransferase, class II donor-specific antibody, portal inflammation, interface activity, perivenular inflammation, portal and perivenular fibrosis, and cluster assignment. The module was enriched in genes that regulate T-cell-mediated rejection (TCMR) of liver and other transplanted organs. Functional pathway analysis showed overrepresentation of TCMR gene sets for cluster 1 but not clusters 2 or 3. CONCLUSION: In an analysis of biopsies from an apparently homogeneous group of stable, long-term pediatric liver transplant recipients with consistently normal liver test results, we found evidence of chronic graft injury (inflammation and/or fibrosis). Biopsy samples with interface activity had a gene expression pattern associated with TCMR.

Five-year histological and serological follow-up of operationally tolerant pediatric liver transplant recipients enrolled in WISP-R.

Pediatric liver transplant recipients arguably have the most to gain and the most to lose from discontinuing immunosuppression (IS). Whereas IS undoubtedly exerts a cumulative toll, there is concern that insufficient or no IS may contribute to allograft deterioration. Twelve pediatric recipients of parental living donor liver grafts, identified as operationally tolerant through complete IS withdrawal (WISP-R; NCT00320606), were followed for a total of 5 years (1 year of IS withdrawal and 4 years off IS) with serial liver tests and autoantibody and alloantibody assessments. Liver biopsies were performed 2 and 4 years off IS, and, at these time points, immunoglobulin G (IgG) subclass and C1q binding activity for donor-specific antibodies (DSAs) were determined. There were no cases of chronic rejection, graft loss, or death. Allografts did not exhibit progressive increase in inflammation or fibrosis. Smooth-muscle actin expression by stellate cells and CD34 expression by liver sinusoidal endothelial cells remained stable, consistent with the absence of progressive graft injury. Three subjects never exhibited DSA. However, 3 subjects showed intermittent de novo class I DSA, 4 subjects showed persistent de novo class II DSA, and 5 subjects showed persistent preexisting class II DSA. Class II DSA was predominantly against donor DQ antigens, often of high mean fluorescence intensity, rarely of the IgG3 subclass, and often capable of binding C1q. CONCLUSION: Operationally tolerant pediatric liver transplant recipients maintain generally stable allograft histology in spite of apparently active humoral allo-immune responses. The absence of increased inflammation or progressive fibrosis suggests that a subset of liver allografts seem resistant to the chronic injury that is characteristic of antibody-mediated damage. (Hepatology 2017;65:647-660).

Alpha-1 antitrypsin treatment of new-onset type 1 diabetes: An open-label, phase I clinical trial (RETAIN) to assess safety and pharmacokinetics.

OBJECTIVE: To determine the safety and pharmacokinetics of alpha-1 antitrypsin (AAT) in adults and children. RESEARCH DESIGN AND METHODS: Short-term AAT treatment restores euglycemia in the non-obese mouse model of type 1 diabetes. A phase I multicenter study in 16 subjects with new-onset type 1 diabetes studied the safety and pharmacokinetics of Aralast NP (AAT). This open-label, dose-escalation study enrolled 8 adults aged 16 to 35 years and 8 children aged 8 to 15 years within 100 days of diagnosis, to receive 12 infusions of AAT: a low dose of 45 mg/kg weekly for 6 weeks, followed by a higher dose of 90 mg/kg for 6 weeks. RESULTS: C-peptide secretion during a mixed meal, hemoglobin A1c (HbA1c), and insulin usage remained relatively stable during the treatment period. At 72 hours after infusion of 90 mg/kg, mean levels of AAT fell below 2.0 g/L for 7 of 15 subjects. To identify a plasma level of AAT likely to be therapeutic, pharmacodynamic ex vivo assays were performed on fresh whole blood from adult subjects. Polymerase chain reaction (PCR) analyses were performed on inhibitor of IKBKE, NOD1, TLR1, and TRAD gene expression, which are important for activation of nuclear factor-κB (NF-κB) and apoptosis pathways. AAT suppressed expression dose-dependently; 50% inhibition was achieved in the 2.5 to 5.0 mg/mL range. CONCLUSIONS: AAT was well tolerated and safe in subjects with new-onset type 1 diabetes. Weekly doses of AAT greater than 90 mg/kg may be necessary for an optimal therapeutic effect.

A composite score associated with spontaneous operational tolerance in kidney transplant recipients.

New challenges in renal transplantation include using biological information to devise a useful clinical test for discerning high- and low-risk patients for individual therapy and ascertaining the best combination and appropriate dosages of drugs. Based on a 20-gene signature from a microarray meta-analysis performed on 46 operationally tolerant patients and 266 renal transplant recipients with stable function, we applied the sparse Bolasso methodology to identify a minimal and robust combination of six genes and two demographic parameters associated with operational tolerance. This composite score of operational tolerance discriminated operationally tolerant patients with an area under the curve of 0.97 (95% confidence interval 0.94-1.00). The score was not influenced by immunosuppressive treatment, center of origin, donor type, or post-transplant lymphoproliferative disorder history of the patients. This composite score of operational tolerance was significantly associated with both de novo anti-HLA antibodies and tolerance loss. It was validated by quantitative polymerase chain reaction using independent samples and demonstrated specificity toward a model of tolerance induction. Thus, our score would allow clinicians to improve follow-up of patients, paving the way for individual therapy.

Changes in T-cell subsets identify responders to FcR-nonbinding anti-CD3 mAb (teplizumab) in patients with type 1 diabetes.

The mechanisms whereby immune therapies affect progression of type 1 diabetes (T1D) are not well understood. Teplizumab, an FcR nonbinding anti-CD3 mAb, has shown efficacy in multiple randomized clinical trials. We previously reported an increase in the frequency of circulating CD8(+) central memory (CD8CM) T cells in clinical responders, but the generalizability of this finding and the molecular effects of teplizumab on these T cells have not been evaluated. We analyzed data from two randomized clinical studies of teplizumab in patients with new- and recent-onset T1D. At the conclusion of therapy, clinical responders showed a significant reduction in circulating CD4(+) effector memory T cells. Afterward, there was an increase in the frequency and absolute number of CD8CM T cells. In vitro, teplizumab expanded CD8CM T cells by proliferation and conversion of non-CM T cells. Nanostring analysis of gene expression of CD8CM T cells from responders and nonresponders versus placebo-treated control subjects identified decreases in expression of genes associated with immune activation and increases in expression of genes associated with T-cell differentiation and regulation. We conclude that CD8CM T cells with decreased activation and regulatory gene expression are associated with clinical responses to teplizumab in patients with T1D.

Antithymocyte globulin therapy for patients with recent-onset type 1 diabetes: 2 year results of a randomised trial.

AIMS/HYPOTHESIS: Type 1 diabetes results from T cell mediated destruction of beta cells. We conducted a trial of antithymocyte globulin (ATG) in new-onset type 1 diabetes (the Study of Thymoglobulin to ARrest T1D [START] trial). Our goal was to evaluate the longer-term safety and efficacy of ATG in preserving islet function at 2 years. METHODS: A multicentre, randomised, double-blind, placebo-controlled trial of 6.5 mg/kg ATG (Thymoglobulin) vs placebo in patients with new-onset type 1 diabetes was conducted at seven university medical centres and one Children's Hospital in the USA. The site-stratified randomisation scheme was computer generated at the data coordinating centre using permuted-blocks of size 3 or 6. Eligible participants were between the ages of 12 and 35, and enrolled within 100 days from diagnosis. Subjects were randomised to 6.5 mg/kg ATG (thymoglobulin) vs placebo in a 2:1 ratio. Participants were blinded, and the study design included two sequential patient-care teams: an unblinded study-drug administration team (for the first 8 weeks), and a blinded diabetes management team (for the remainder of the study). Endpoints assessed at 24 months included meal-stimulated C-peptide AUC, safety and immunological responses. RESULTS: Fifty-eight patients were enrolled; at 2 years, 35 assigned to ATG and 16 to placebo completed the study. The pre-specified endpoints were not met. In post hoc analyses, older patients (age 22-35 years) in the ATG group had significantly greater C-peptide AUCs at 24 months than placebo patients. Using complete preservation of baseline C-peptide at 24 months as threshold, nine of 35 ATG-treated participants (vs 2/16 placebo participants) were classified as responders; nine of 11 responders (67%) were older. All participants reported at least one adverse event (AE), with 1,148 events in the 38 ATG participants vs 415 in the 20 placebo participants; a comparable number of infections were noted in the ATG and placebo groups, with no opportunistic infections nor difficulty clearing infections in either group. Circulating T cell subsets depleted by ATG partially reconstituted, but regulatory, naive and central memory subsets remained significantly depleted at 24 months. Beta cell autoantibodies did not change over the 24 months in the ATG-treated or placebo participants. At 12 months, ATG-treated participants had similar humoral immune responses to tetanus and HepA vaccines as placebo-treated participants, and no increased infections. CONCLUSIONS/INTERPRETATION: A brief course of ATG substantially depleted T cell subsets, including regulatory cells, but did not preserve islet function 24 months later in the majority of patients with new-onset type 1 diabetes. ATG preserved C-peptide secretion in older participants, which may warrant further study. TRIAL REGISTRATION: ClinicalTrials.gov NCT00515099 PUBLIC DATA REPOSITORY: START datasets are available in TrialShare www.itntrialshare.org FUNDING: National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH). The trial was conducted by the Immune Tolerance Network (ITN).

Prediction and detection of emotional tone in online social media mental disorder groups using regression and recurrent neural networks.

Online social media networks have become a significant platform for persons with mental illnesses to discuss their struggles and obtain emotional and informational assistance in recent years. One such platform is Reddit, where sub-groups called 'subreddits' exist, based on a variety of topics including mental illnesses such as anxiety or depression. We analyse the user's interactions to calculate the mental health status by formulating and using a parameter called 'emotional tone' representing the user's emotional state. VADER sentiment analysis and TextBlob are used to categorise emotional tone and find distribution of emotional polarity and subjectivity of comments. For final tone prediction, RNN and State-Of-The-Art word embedding techniques are used to develop a predictive model. The resultant model provides end-to-end categorization and prediction of emotional tone. We obtain results with respect to Weighted L1 Loss that deals with extreme responses. The MODEL transcends all the baselines by at least 12.1% and the final emotional status of the authors is positive.

Phase II Randomized Trial of Rituximab Plus Cyclophosphamide Followed by Belimumab for the Treatment of Lupus Nephritis.

OBJECTIVE: To assess the safety, mechanism of action, and preliminary efficacy of rituximab followed by belimumab in the treatment of refractory lupus nephritis (LN). METHODS: In a multicenter, randomized, open-label clinical trial, 43 patients with recurrent or refractory LN were treated with rituximab, cyclophosphamide (CYC), and glucocorticoids followed by weekly belimumab infusions until week 48 (RCB group), or treated with rituximab and CYC but no belimumab infusions (RC group). Patients were followed up until week 96. Percentages of total and autoreactive B cell subsets in the patients' peripheral blood were analyzed by flow cytometry. RESULTS: Treatment with belimumab did not increase the incidence of adverse events in patients with refractory LN. At week 48, a complete or partial renal response occurred in 11 (52%) of 21 patients receiving belimumab, compared to 9 (41%) of 22 patients in the RC group who did not receive belimumab (P = 0.452). Lack of improvement in or worsening of LN was the major reason for treatment failure. B cell depletion occurred in both groups, but the percentage of B cells remained lower in those receiving belimumab (geometric mean number of B cells at week 60, 53 cells/µl in the RCB group versus 11 cells/µl in the RC group; P = 0.0012). Percentages of total and autoreactive transitional B cells increased from baseline to week 48 in both groups. However, percentages of total and autoreactive naive B cells decreased from baseline to week 48 in the belimumab group compared to the no belimumab RC group (P = 0.0349), a finding that is consistent with the observed impaired maturation of naive B cells and enhanced censoring of autoreactive B cells. CONCLUSION: The addition of belimumab to a treatment regimen with rituximab and CYC was safe in patients with refractory LN. This regimen diminished maturation of transitional to naive B cells during B cell reconstitution, and enhanced the negative selection of autoreactive B cells. Clinical efficacy was not improved with rituximab and CYC in combination with belimumab when compared to a therapeutic strategy of B cell depletion alone in patients with LN.

IL-6 receptor blockade does not slow ß cell loss in new-onset type 1 diabetes.

BackgroundIL-6 receptor (IL-6R) signaling drives development of T cell populations important to type 1 diabetes pathogenesis. We evaluated whether blockade of IL-6R with monoclonal antibody tocilizumab would slow loss of residual ß cell function in newly diagnosed type 1 diabetes patients.MethodsWe conducted a multicenter, randomized, placebo-controlled, double-blind trial with tocilizumab in new-onset type 1 diabetes. Participants were screened within 100 days of diagnosis. Eligible participants were randomized 2:1 to receive 7 monthly doses of tocilizumab or placebo. The primary outcome was the change from screening in the mean AUC of C-peptide collected during the first 2 hours of a mixed meal tolerance test at week 52 in pediatric participants (ages 6-17 years).ResultsThere was no statistical difference in the primary outcome between tocilizumab and placebo. Immunophenotyping showed reductions in downstream signaling of the IL-6R in T cells but no changes in CD4 memory subsets, Th17 cells, Tregs, or CD4+ T effector cell resistance to Treg suppression. A DC subset decreased during therapy but regressed to baseline once therapy stopped. Tocilizumab was well tolerated.ConclusionTocilizumab reduced T cell IL-6R signaling but did not modulate CD4+ T cell phenotypes or slow loss of residual ß cell function in newly diagnosed individuals with type 1 diabetes.Trial RegistrationClinicalTrials.gov NCT02293837.FundingNIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and National Institute of Allergy and Infectious Diseases (NIAID) UM1AI109565, UL1TR000004 from NIH/National Center for Research Resources (NCRR) Clinical and Translational Science Award (CTSA), NIH/NIDDK P30DK036836, NIH/NIDDK U01DK103266, NIH/NIDDK U01DK103266, 1UL1TR000064 from NIH/NCRR CTSA, NIH/National Center for Advancing Translational Sciences (NCATS) UL1TR001878, UL1TR002537 from NIH/CTSA; National Health and Medical Research Council Practitioner Fellowship (APP1136735), NIH/NIDDK U01-DK085476, NIH/CTSA UL1-TR002494, Indiana Clinical and Translational Science Institute Award UL1TR002529, Vanderbilt Institute for Clinical and Translational Research UL1TR000445. NIH/NCATS UL1TR003142, NIH/CTSA program UL1-TR002494, Veteran Affairs Administration, and 1R01AI132774.

Alefacept provides sustained clinical and immunological effects in new-onset type 1 diabetes patients.

BACKGROUND: Type 1 diabetes (T1D) results from destruction of pancreatic ß cells by autoreactive effector T cells. We hypothesized that the immunomodulatory drug alefacept would result in targeted quantitative and qualitative changes in effector T cells and prolonged preservation of endogenous insulin secretion by the remaining ß cells in patients with newly diagnosed T1D. METHODS: In a multicenter, randomized, double-blind, placebo-controlled trial, we compared alefacept (two 12-week courses of 15 mg/wk i.m., separated by a 12-week pause) with placebo in patients with recent onset of T1D. Endpoints were assessed at 24 months and included meal-stimulated C-peptide AUC, insulin use, hypoglycemic events, and immunologic responses. RESULTS: A total of 49 patients were enrolled. At 24 months, or 15 months after the last dose of alefacept, both the 4-hour and the 2-hour C-peptide AUCs were significantly greater in the treatment group than in the control group (P = 0.002 and 0.015, respectively). Exogenous insulin requirements were lower (P = 0.002) and rates of major hypoglycemic events were about 50% reduced (P < 0.001) in the alefacept group compared with placebo at 24 months. There was no apparent between-group difference in glycemic control or adverse events. Alefacept treatment depleted CD4+ and CD8+ central memory T cells (Tcm) and effector memory T cells (Tem) (P < 0.01), preserved Tregs, increased the ratios of Treg to Tem and Tcm (P < 0.01), and increased the percentage of PD-1+CD4+ Tem and Tcm (P < 0.01). CONCLUSIONS: In patients with newly diagnosed T1D, two 12-week courses of alefacept preserved C-peptide secretion, reduced insulin use and hypoglycemic events, and induced favorable immunologic profiles at 24 months, well over 1 year after cessation of therapy. TRIAL REGISTRATION: https://clinicaltrials.gov/ NCT00965458. FUNDING: NIH and Astellas.