Immunosuppression causes dynamic changes in expression QTLs in psoriatic skin.

Psoriasis is a chronic, systemic inflammatory condition primarily affecting skin. While the role of the immune compartment (e.g., T cells) is well established, the changes in the skin compartment are more poorly understood. Using longitudinal skin biopsies (n = 375) from the "Psoriasis Treatment with Abatacept and Ustekinumab: A Study of Efficacy"(PAUSE) clinical trial (n = 101), we report 953 expression quantitative trait loci (eQTLs). Of those, 116 eQTLs have effect sizes that were modulated by local skin inflammation (eQTL interactions). By examining these eQTL genes (eGenes), we find that most are expressed in the skin tissue compartment, and a subset overlap with the NRF2 pathway. Indeed, the strongest eQTL interaction signal - rs1491377616-LCE3C - links a psoriasis risk locus with a gene specifically expressed in the epidermis. This eQTL study highlights the potential to use biospecimens from clinical trials to discover in vivo eQTL interactions with therapeutically relevant environmental variables.

Autoreactive Plasmablasts After B Cell Depletion With Rituximab and Relapses in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: Autoreactive B cells are responsible for antineutrophil cytoplasmic antibody (ANCA) production in ANCA-associated vasculitis (AAV). Rituximab (RTX) depletes circulating B cells, including autoreactive B cells. We aimed to evaluate changes and associations with relapse of the circulating autoreactive B cell pool following therapeutic B cell depletion in AAV. METHODS: Sequential flow cytometry was performed on 148 samples of peripheral blood mononuclear cells from 23 patients with proteinase 3 (PR3)-ANCA-positive AAV who were treated with RTX for remission induction and monitored after stopping therapy during long-term follow-up in a prospective clinical trial. PR3 was used as a ligand to target autoreactive PR3-specific (PR3+) B cells. B cell recurrence was considered as the first blood sample with ≥10 B cells/µl after RTX treatment. RESULTS: At B cell recurrence, PR3+ B cell frequency among B cells was higher than baseline (P < 0.01). Within both PR3+ and total B cells, frequencies of transitional and naive subsets were higher at B cell recurrence than at baseline, while memory subsets were lower (P < 0.001 for all comparisons). At B cell recurrence, frequencies of B cells and subsets did not differ between patients who experienced relapse and patients who remained in remission. In contrast, the plasmablast frequency within the PR3+ B cell pool was higher in patients who experienced relapse and associated with a shorter time to relapse. Frequencies of PR3+ plasmablasts higher than baseline were more likely to be found in patients who experienced relapse within the following 12 months compared to those in sustained remission (P < 0.05). CONCLUSION: The composition of the autoreactive B cell pool varies significantly following RTX treatment in AAV, and early plasmablast enrichment within the autoreactive pool is associated with future relapses.

Stabilization of leukocytes from cerebrospinal fluid for central immunophenotypic evaluation in multicenter clinical trials.

Analysis of cerebrospinal fluid (CSF) represents a valuable window into the pathogenesis of neuroinflammatory diseases, such as multiple sclerosis (MS). However, analysis of the cellular fraction of CSF is often neglected because CSF cells die rapidly ex vivo. Immunophenotyping of CSF cells in multicenter clinical trials requires sample preservation and shipping to a centralized lab. Yet, there is no consensus on the best method to preserve intact CSF cells and no detailed evaluation of subset-specific cell loss. We used flow cytometry to compare major leukocyte populations in fresh CSF (processed within 2 h) to cells fixed for 48 h with TransFix-EDTA® or cryopreserved and thawed after 96 h. We observed a statistically significant loss of total mononuclear cells, total T cells, CD3+ CD8- T cells, and CD3+ CD8+ T cells after cryopreservation compared to fresh or fixed (p < 0.001), with no significant difference between fresh and fixed. Thus, our results demonstrate that TransFix-EDTA® was superior to cryopreservation for preserving intact CSF T cells. Surprisingly, neither cryopreservation nor fixation had a significant effect on recovery of low frequency cell subsets in CSF, including B cells, NK cells, NKT-like cells, CD14+ monocytes, or CD123+ DCs, versus fresh CSF. To determine the effect of prolonged fixation on cell recovery, we analyzed major CSF cell subsets by flow cytometry after 24, 48, or 72 h of fixation with TransFix-EDTA®. We observed a consistent and progressive loss in the absolute counts of all subsets over time, although this effect was not statistically significant. We conclude that for immunophenotyping of major CSF cell subsets by flow cytometry, fixation with TransFix-EDTA®, shipment to a central lab, and analysis within 48 h is a feasible method to ensure stability of both absolute cell number and relative frequency. This method is a valuable alternative to fresh CSF analysis and can be implemented in multicenter clinical trials.

A phase 1b open-label dose-finding study of ustekinumab in young adults with type 1 diabetes.

OBJECTIVES: We assessed the safety of ustekinumab (a monoclonal antibody used in psoriasis to target the IL-12 and IL-23 pathways) in a small cohort of recent-onset (<100 days of diagnosis) adults with type 1 diabetes (T1D) by conducting a pilot open-label dose-finding and mechanistic study (NCT02117765) at the University of British Columbia. METHODS: We sequentially enrolled 20 participants into four subcutaneous dosing cohorts: (i) 45 mg loading weeks 0/4/16, (ii) 45 mg maintenance weeks 0/4/16/28/40, (iii) 90 mg loading weeks 0/4/16, and (iv) 90 mg maintenance weeks 0/4/16/28/40. The primary endpoint was safety as assessed by an independent data and safety monitoring board (DSMB) but we also measured mixed meal tolerance test C-peptide, insulin use/kg, and HbA1c. Immunophenotyping was performed to assess immune cell subsets and islet antigen-specific T cell responses. RESULTS: Although several adverse events were reported, only two (bacterial vaginosis and hallucinations) were thought to be possibly related to drug administration by the study investigators. At 1 year, the 90 mg maintenance dosing cohort had the smallest mean decline in C-peptide area under the curve (AUC) (0.1 pmol/ml). Immunophenotyping showed that ustekinumab reduced the percentage of circulating Th17, Th1, and Th17.1 cells and proinsulin-specific T cells that secreted IFN-γ and IL-17A. CONCLUSION: Ustekinumab was deemed safe to progress to efficacy studies by the DSMB at doses used to treat psoriasis in adults with T1D. A 90 mg maintenance dosing schedule reduced proinsulin-specific IFN-γ and IL-17A-producing T cells. Further studies are warranted to determine if ustekinumab can prevent C-peptide AUC decline and induce a clinical response.

Effect of Costimulatory Blockade With Abatacept After Ustekinumab Withdrawal in Patients With Moderate to Severe Plaque Psoriasis: The PAUSE Randomized Clinical Trial.

IMPORTANCE: Psoriasis relapse may involve compensatory T-cell activation pathways in the presence of CD28-CD80/CD86 blockade with abatacept. OBJECTIVE: To determine whether costimulatory signaling blockade with abatacept prevents psoriasis relapse after ustekinumab withdrawal. DESIGN, SETTING, AND PARTICIPANTS: Psoriasis Treatment with Abatacept and Ustekinumab: a Study of Efficacy (PAUSE), a parallel-design, double-blind, placebo-controlled randomized clinical trial, was conducted at 10 sites in the US and Canada. Participant enrollment opened on March 19, 2014, and concluded on April 11, 2016. Participants were adults with moderate to severe plaque psoriasis and received ustekinumab in a lead-in phase. Those who responded to ustekinumab at week 12 were randomized 1:1 to either the continued with ustekinumab group (ustekinumab group) or the switched to abatacept group (abatacept group). Treatment was discontinued at week 39, and participants were followed up for psoriasis relapse until week 88. Statistical analyses were performed in the intention-to-treat (ITT) and safety samples from May 3, 2018, to July 6, 2021. INTERVENTIONS: Participants received subcutaneous ustekinumab at weeks 0 and 4 (45 mg per dose for those ≤100 kg; 90 mg per dose for those >100 kg). Participants randomized to the abatacept group at week 12 received subcutaneous abatacept, 125 mg weekly, from weeks 12 to 39 and ustekinumab placebo at weeks 16 and 28. Participants randomized to the ustekinumab group received ustekinumab at weeks 16 and 28 and abatacept placebo weekly from weeks 12 to 39. MAIN OUTCOMES AND MEASURES: The primary end point was the proportion of participants with psoriasis relapse (loss of ≥50% of the initial Psoriasis Area and Severity Index improvement) between weeks 12 and 88. Secondary end points included time to psoriasis relapse, proportion of participants with psoriasis relapse between weeks 12 and 40, and adverse events. The psoriasis transcriptome and serum cytokines were evaluated. RESULTS: A total of 108 participants (mean [SD] age, 46.1 [12.1] years; 73 [67.6%] men) were treated with open-label ustekinumab; 91 were randomized to blinded treatment. Similar proportions of participants in the abatacept group and the ustekinumab group relapsed between weeks 12 and 88 (41 of 45 [91.1%] vs 40 of 46 [87.0%]; P = .41). Median time to relapse from the last dose of ustekinumab was similar between groups as well: 36 weeks (95% CI, 36-48 weeks) in the abatacept group vs 32 weeks (95% CI, 28-40 weeks) in the ustekinumab group. Similar numbers and rates of adverse events occurred. Abatacept did not maintain suppression of the pathogenic IL-23-mediated psoriasis molecular signature in lesions after ustekinumab withdrawal, and serum IL-19 levels increased. CONCLUSIONS AND RELEVANCE: This parallel-design, double-blind randomized clinical trial found that abatacept did not prevent psoriasis relapse that occurred after ustekinumab withdrawal because it did not completely block the pathogenic psoriasis molecular pathways that led to relapse. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01999868.

Circulating autoreactive proteinase 3+ B cells and tolerance checkpoints in ANCA-associated vasculitis.

BACKGROUNDLittle is known about the autoreactive B cells in antineutrophil cytoplasmic antibody-associated (ANCA-associated) vasculitis (AAV). We aimed to investigate tolerance checkpoints of circulating antigen-specific proteinase 3-reactive (PR3+) B cells.METHODSMulticolor flow cytometry in combination with bioinformatics and functional in vitro studies were performed on baseline samples of PBMCs from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-AAV and myeloperoxidase-AAV (MPO-AAV) and 27 healthy controls (HCs). Clinical data and outcomes from the trial were correlated with PR3+ B cells (total and subsets).RESULTSThe frequency of PR3+ B cells among circulating B cells was higher in participants with PR3-AAV (4.77% median [IQR, 3.98%-6.01%]) than in participants with MPO-AAV (3.16% median [IQR, 2.51%-5.22%]) and participants with AAV compared with HCs (1.67% median [IQR, 1.27%-2.16%], P < 0.001 for all comparisons), implying a defective central tolerance checkpoint in patients with AAV. Only PBMCs from participants with PR3-AAV contained PR3+ B cells capable of secreting PR3-ANCA IgG in vitro, proving they were functionally distinct from those of participants with MPO-AAV and HCs. Unsupervised clustering identified subtle subsets of atypical autoreactive PR3+ memory B cells accumulating through the maturation process in patients with PR3-AAV. PR3+ B cells were enriched in the memory B cell compartment of participants with PR3-AAV and were associated with higher serum CXCL13 levels, suggesting an increased germinal center activity. PR3+ B cells correlated with systemic inflammation (C-reactive protein and erythrocyte sedimentation rate, P < 0.05) and complete remission (P < 0.001).CONCLUSIONThis study suggests the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients with PR3-AAV, elucidating the selection process of autoreactive B cells.Trial registrationClinicalTrials.gov NCT00104299.FundingThe Vasculitis Foundation, the National Institute of Allergy and Infectious Diseases of the NIH, and the Mayo Foundation for Education and Research.

Differential induction of allergen-specific IgA responses following timothy grass subcutaneous and sublingual immunotherapy.

INTRODUCTION: There is no detailed comparison of allergen-specific immunoglobulin responses following sublingual immunotherapy (SLIT) and subcutaneous immunotherapy (SCIT). OBJECTIVE: We sought to compare nasal and systemic timothy grass pollen (TGP)-specific antibody responses during 2 years of SCIT and SLIT and 1 year after treatment discontinuation in a double-blind, double-dummy, placebo-controlled trial. METHODS: Nasal fluid and serum were obtained yearly (per-protocol population, n = 84). TGP-specific IgA1, IgA2, IgG4, IgG, and IgE were measured in nasal fluids by ELISA. TGP-specific IgA1, IgA2, and Phleum pratense (Phl p)1, 2, 4, 5b, 6, 7, 11, and 12 IgE and IgG4 were measured in sera by ELISA and ImmunoCAP, respectively. RESULTS: At years 2 and 3, TGP-IgA1/2 levels in nasal fluid were elevated in SLIT compared with SCIT (4.2- and 3.0-fold for IgA1, 2.0- and 1.8-fold for IgA2, respectively; all P < .01). TGP-IgA1 level in serum was elevated in SLIT compared with SCIT at years 1, 2, and 3 (4.6-, 5.1-, and 4.7-fold, respectively; all P < .001). Serum TGP-IgG level was higher in SCIT compared with SLIT (2.8-fold) at year 2. Serum TGP-IgG4 level was higher in SCIT compared with SLIT at years 1, 2, and 3 (10.4-, 27.4-, and 5.1-fold, respectively; all P < .01). Serum IgG4 levels to Phl p1, 2, 5b, and 6 were increased at years 1, 2, and 3 in SCIT and SLIT compared with placebo (Phl p1: 11.8- and 3.9-fold; Phl p2: 31.6- and 4.4-fold; Phl p5b: 135.5- and 5.3-fold; Phl p6: 145.4- and 14.7-fold, respectively, all at year 2 when levels peaked; P < .05). IgE to TGP in nasal fluid increased in the SLIT group at year 2 but not at year 3 compared with SCIT (2.8-fold; P = .04) and placebo (3.1-fold; P = .02). IgA to TGP and IgE and IgG4 to TGP components stratified participants according to treatment group and clinical response. CONCLUSIONS: The observed induction of IgA1/2 in SLIT and IgG4 in SCIT suggest key differences in the mechanisms of action.

A CD8+ NK cell transcriptomic signature associated with clinical outcome in relapsing remitting multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) with the majority of cases characterised by relapsing/remitting (RRMS) attacks of neurologic dysfunction followed by variable resolution. Improving clinical outcomes in RRMS requires both a better understanding of the immunological mechanisms driving recurrent demyelination and better means of predicting future disease course to facilitate early targeted therapy. Here, we apply hypothesis-generating network transcriptomics to CD8+ cells isolated from patients in RRMS, identifying a signature reflecting expansion of a subset of CD8+ natural killer cells (NK8+) associated with favourable outcome. NK8+ are capable of regulating CD4+ T cell activation and proliferation in vitro, with reduced expression of HLA-G binding inhibitory receptors and consequent reduced sensitivity to HLA-G-mediated suppression. We identify surrogate markers of the NK8+ signature in peripheral blood leucocytes and validate their association with clinical outcome in an independent cohort, suggesting their measurement may facilitate early, targeted therapy in RRMS.

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.

Biomarkers of severity and threshold of allergic reactions during oral peanut challenges.

BACKGROUND: Oral food challenge (OFC) is the criterion standard to assess peanut allergy (PA), but it involves a risk of allergic reactions of unpredictable severity. OBJECTIVE: Our aim was to identify biomarkers for risk of severe reactions or low dose threshold during OFC to peanut. METHODS: We assessed Learning Early about Peanut Allergy study, Persistance of Oral Tolerance to Peanut study, and Peanut Allergy Sensitization study participants by administering the basophil activation test (BAT) and the skin prick test (SPT) and measuring the levels of peanut-specific IgE, Arachis hypogaea 2-specific IgE, and peanut-specific IgG4, and we analyzed the utility of the different biomarkers in relation to PA status, severity, and threshold dose of allergic reactions to peanut during OFC. RESULTS: When a previously defined optimal cutoff was used, the BAT diagnosed PA with 98% specificity and 75% sensitivity. The BAT identified severe reactions with 97% specificity and 100% sensitivity. The SPT, level of Arachis hypogaea 2-specific IgE, level of peanut-specific IgE, and IgG4/IgE ratio also had 100% sensitivity but slightly lower specificity (92%, 93%, 90%, and 88%, respectively) to predict severity. Participants with lower thresholds of reactivity had higher basophil activation to peanut in vitro. The SPT and the BAT were the best individual predictors of threshold. Multivariate models were superior to individual biomarkers and were used to generate nomograms to calculate the probability of serious adverse events during OFC for individual patients. CONCLUSIONS: The BAT diagnosed PA with high specificity and identified severe reactors and low threshold with high specificity and high sensitivity. The BAT was the best biomarker for severity, surpassed only by the SPT in predicting threshold. Nomograms can help estimate the likelihood of severe reactions and reactions to a low dose of allergen in individual patients with PA.

Extensive intrathecal T cell renewal following hematopoietic transplantation for multiple sclerosis.

A recent study of autologous hematopoietic stem cell transplantation (AHSCT) for active relapsing-remitting multiple sclerosis (RRMS) showed efficacy in preventing disease worsening. However, the immunologic basis for efficacy remains poorly defined. Multiple sclerosis pathology is known to be driven by inflammatory T cells that infiltrate the CNS. Therefore, we hypothesized that the preexisting T cell repertoire in the intrathecal compartment of active RRMS participants was ablated and replaced with new clones following AHSCT. T cell repertoires were assessed using high-throughput TCRß chain sequencing in paired cerebrospinal fluid (CSF) and peripheral blood CD4+ and CD8+ T cells from participants that underwent AHSCT, before and up to 4 years following transplantation. More than 90% of the preexisting CSF repertoire in participants with active RRMS was removed following AHSCT and replaced with clonotypes predominantly generated from engrafted autologous stem cells. Of the preexisting clones in CSF, approximately 60% were also detected in blood before therapy, and concordant treatment effects were observed for clonotypes in both compartments following AHSCT. These results indicate that replacement of the preexisting TCR repertoire in active RRMS is a mechanism for AHSCT efficacy and suggest that peripheral blood could serve as a surrogate for CSF to define mechanisms associated with efficacy in future studies of AHSCT.

Elicited and pre-existing anti-Neu5Gc antibodies differentially affect human endothelial cells transcriptome.

Humans cannot synthesize N-glycolylneuraminic acid (Neu5Gc) but dietary Neu5Gc can be absorbed and deposited on endothelial cells (ECs) and diet-induced anti-Neu5Gc antibodies (Abs) develop early in human life. While the interaction of Neu5Gc and diet-induced anti-Neu5Gc Abs occurs in all normal individuals, endothelium activation by elicited anti-Neu5Gc Abs following a challenge with animal-derived materials, such as following xenotransplantation, had been postulated. Ten primary human EC preparations were cultured with affinity-purified anti-Neu5Gc Abs from human sera obtained before or after exposure to Neu5Gc-glycosylated rabbit IgGs (elicited Abs). RNAs of each EC preparation stimulated in various conditions by purified Abs were exhaustively sequenced. EC transcriptomic patterns induced by elicited anti-Neu5Gc Abs, compared with pre-existing ones, were analyzed. qPCR, cytokines/chemokines release, and apoptosis were tested on some EC preparations. The data showed that anti-Neu5Gc Abs induced 967 differentially expressed (DE) genes. Most DE genes are shared following EC activation by pre-existing or anti-human T-cell globulin (ATG)-elicited anti-Neu5Gc Abs. Compared with pre-existing anti-Neu5Gc Abs, which are normal component of ECs environment, elicited anti-Neu5Gc Abs down-regulated 66 genes, including master genes of EC function. Furthermore, elicited anti-Neu5Gc Abs combined with complement-containing serum down-regulated most transcripts mobilized by serum alone. Both types of anti-Neu5Gc Abs-induced a dose- and complement-dependent release of selected cytokines and chemokines. Altogether, these data show that, compared with pre-existing anti-Neu5Gc Abs, ATG-elicited anti-Neu5Gc Abs specifically modulate genes related to cytokine responses, MAPkinase cascades, chemotaxis, and integrins and do not skew the EC transcriptome toward a pro-inflammatory profile in vitro.

Abatacept Targets T Follicular Helper and Regulatory T Cells, Disrupting Molecular Pathways That Regulate Their Proliferation and Maintenance.

Abatacept is a CTLA-4-Ig fusion protein that binds to the costimulatory ligands CD80 and CD86 and blocks their interaction with the CD28 and CTLA-4 receptors expressed by T cells, therefore inhibiting T cell activation and function. Abatacept has shown clinical efficacy in treating some autoimmune diseases but has failed to show clinical benefit in other autoimmune conditions. The reasons for these disparate results are not clear and warrant further investigation of abatacept's mode of action. Longitudinal specimens from the Immune Tolerance Network's A Cooperative Clinical Study of Abatacept in Multiple Sclerosis trial were used to examine the effects of abatacept treatment on the frequency and transcriptional profile of specific T cell populations in peripheral blood. We found that the relative abundance of CD4+ T follicular helper (Tfh) cells and regulatory T cells was selectively decreased in participants following abatacept treatment. Within both cell types, abatacept reduced the proportion of activated cells expressing CD38 and ICOS and was associated with decreased expression of genes that regulate cell-cycle and chromatin dynamics during cell proliferation, thereby linking changes in costimulatory signaling to impaired activation, proliferation, and decreased abundance. All cellular and molecular changes were reversed following termination of abatacept treatment. These data expand upon the mechanism of action of abatacept reported in other autoimmune diseases and identify new transcriptional targets of CD28-mediated costimulatory signaling in human regulatory T and Tfh cells, further informing on its potential use in diseases associated with dysregulated Tfh activity.

Treatment of type 1 diabetes with teplizumab: clinical and immunological follow-up after 7 years from diagnosis.

AIMS/HYPOTHESIS: The long-term effects of successful immune therapies for treatment of type 1 diabetes have not been well studied. The Autoimmunity-Blocking Antibody for Tolerance (AbATE) trial evaluated teplizumab, an Fc receptor non-binding humanised anti-CD3 monoclonal antibody in individuals with new-onset type 1 diabetes, and ended in 2011. Clinical drug-treated responders showed an increased frequency of 'partially exhausted' CD8+ T cells. We studied the clinical, immunological and metabolic status of participants after an average follow-up of 7 years. METHODS: Participants with detectable C-peptide at year 2 of AbATE returned for follow-up. C-peptide responses were assessed by 4 h mixed-meal tolerance test. Autoantibodies and HbA1c levels were measured and average daily insulin use was obtained from patient logs. Peripheral blood mononuclear cells were analysed by flow cytometry and cytokine release. RESULTS: Fifty-six per cent of the original participants returned. Three of the original control group who did not return had lost all detectable C-peptide by the end of the 2 year trial. The C-peptide responses to a mixed-meal tolerance test were similar overall in the drug vs control group of participants but were significantly improved, with less loss of C-peptide, in drug-treated responders identified at 1 year. However, the improvements in C-peptide response were not associated with lower HbA1c levels or insulin use. Drug-treated responders showed a significantly increased frequency of programmed cell death protein 1-positive central memory and anergic CD8+ T cells at follow-up. CONCLUSIONS/INTERPRETATION: These findings suggest there is reduced decline in C-peptide and persistent immunological responses up to 7 years after diagnosis of diabetes in individuals who respond to teplizumab. TRIAL REGISTRATION: ClinicalTrials.gov NCT02067923; the protocol is available at www.immunetolerance.org (ITN027AI).

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.

Challenges and Opportunities for Biomarkers of Clinical Response to AHSCT in Autoimmunity.

Autoimmunity represents a broad category of diseases that involve a variety of organ targets and distinct autoantigens. For patients with autoimmune diseases who fail to respond to approved disease-modifying treatments, autologous hematopoietic stem cell transplantation (AHSCT) after high-dose immunosuppressive therapy provides an alternative strategy. Although more than 100 studies have been published on AHSCT efficacy in autoimmunity, the mechanisms that confer long-term disease remission as opposed to continued deterioration or disease reactivation remain to be determined. In a phase II clinical trial, high-dose immunosuppressive therapy combined with autologous CD34+ hematopoietic stem cell transplant in treatment-resistant, relapsing-remitting multiple sclerosis (RRMS) resulted in 69.2% of participants achieving long-term remission through 60 months follow-up. Flow cytometry data from the 24 transplanted participants in the high-dose immunosuppression and autologous stem cell transplantation for poor prognosis multiple sclerosis (HALT-MS) trial are presented to illustrate immune reconstitution out to 36 months in patients with aggressive RRMS treated with AHSCT and to highlight experimental challenges inherent in identifying biomarkers for relapse and long-term remission through 60 months follow-up. AHSCT induced changes in numbers of CD4 T cells and in the composition of CD4 and CD8 T cells that persisted through 36 months in participants who maintained disease remission through 60 months. However, changes in T cell phenotypes studied were unable to clearly discriminate durable remission from disease reactivation after AHSCT, possibly due to the small sample size, limited phenotypes evaluated in this real-time assay, and other limitations of the HALT-MS study population. Strategies and future opportunities for identifying biomarkers of clinical outcome to AHSCT in autoimmunity are also discussed.

Synchronous immune alterations mirror clinical response during allergen immunotherapy.

BACKGROUND: Three years of treatment with either sublingual or subcutaneous allergen immunotherapy has been shown to be effective and to induce long-term tolerance. The Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy (GRASS) trial demonstrated that 2 years of treatment through either route was effective in suppressing the response to nasal allergen challenge, although it was insufficient for inhibition 1 year after discontinuation. OBJECTIVE: We sought to examine in the GRASS trial the time course of immunologic changes during 2 years of sublingual and subcutaneous immunotherapy and for 1 year after treatment discontinuation. METHODS: We performed multimodal immunomonitoring to assess allergen-specific CD4 T-cell properties in parallel with analysis of local mucosal cytokine responses induced by nasal allergen exposure and humoral immune responses that included IgE-dependent basophil activation and measurement of serum inhibitory activity for allergen-IgE binding to B cells (IgE-facilitated allergen binding). RESULTS: All 3 of these distinct arms of the immune response displayed significant and coordinate alterations during 2 years of allergen desensitization, followed by reversal at 3 years, reflecting a lack of a durable immunologic effect. Although frequencies of antigen-specific TH2 cells in peripheral blood determined by using HLA class II tetramer analysis most closely paralleled clinical outcomes, IgE antibody-dependent functional assays remained inhibited in part 1 year after discontinuation. CONCLUSION: Two years of allergen immunotherapy were effective but insufficient for long-term tolerance. Allergen-specific TH2 cells most closely paralleled the transient clinical outcome, and it is likely that recurrence of the T-cell drivers of allergic immunity abrogated the potential for durable tolerance. On the other hand, the persistence of IgE blocking antibody 1 year after discontinuation might be an early indicator of a protolerogenic mechanism.

Remodeling T cell compartments during anti-CD3 immunotherapy of type 1 diabetes.

The immunological mechanism(s) of action whereby teplizumab preserves C-peptide levels in the progression of patients with recent onset type 1 diabetes (T1D) is still not well understood. In the present study, we evaluated the kinetics of T cell modulation in peripheral blood following two 14-day courses of teplizumab therapy one year apart in recent onset T1D participants in the AbATE clinical trial. Transient rises in PD-1+Foxp3+ Treg and potentially anergic (CD57-KLRG1-PD-1+) cells in the circulating CD4 T cell compartment were paralleled by more profound increases in circulating CD8 T cells with traits of exhaustion (CD57-KLRG1+PD-1+, TIGIT+KLRG1+, and persistent down-modulation of CD127). The observed phenotypic changes across cell types were associated with favorable response to treatment in the subgroup of study participants that did not develop anti-drug antibodies after the first course of therapy. These findings provide new insights on the duration and complexity of T cell modulation with teplizumab therapy in recent onset T1D, and in addition, suggest that coordinated immune mechanisms of tolerance that favor CD4 Treg function and restrain CD4 non-Treg and CD8 T cell activation may contribute to treatment success.

Anti-Gal and Anti-Neu5Gc Responses in Nonimmunosuppressed Patients After Treatment With Rabbit Antithymocyte Polyclonal IgGs.

BACKGROUND: Polyclonal antihuman thymocyte rabbit IgGs (antithymocyte globulin [ATG]) are popular immunosuppressive drugs used to prevent or treat organ or bone-marrow allograft rejection, graft versus host disease, and autoimmune diseases. However, animal-derived glycoproteins are also strongly immunogenic and rabbit ATG induces serum sickness disease in almost all patients without additional immunosuppressive drugs, as seen in the Study of Thymoglobulin to arrest Type 1 Diabetes (START) trial of ATG therapy in new-onset type 1 diabetes. METHODS: Using enzyme-linked immunosorbent assay, we analyzed serial sera from the START study to decipher the various anti-ATG specificities developed by the patients in this study: antitotal ATG, but also antigalactose-α1-3-galactose (Gal) and anti-Neu5Gc antibodies, 2 xenocarbohydrate epitopes present on rabbit IgG glycans and lacking in humans. RESULTS: We show that diabetic patients have substantial levels of preexisting antibodies of the 3 specificities, before infusion, but of similar levels as healthy individuals. ATG treatment resulted in highly significant increases of both IgM (for anti-ATG and anti-Neu5Gc) and IgG (for anti-ATG, -Gal, and -Neu5Gc), peaking at 1 month and still detectable 1 year postinfusion. CONCLUSIONS: Treatment with rabbit polyclonal IgGs in the absence of additional immunosuppression results in a vigorous response against Gal and Neu5Gc epitopes, contributing to an inflammatory environment that may compromise the efficacy of ATG therapy. The results also suggest using IgGs lacking these major xenoantigens may improve safety and efficacy of ATG treatment.

Effect of 2 Years of Treatment With Sublingual Grass Pollen Immunotherapy on Nasal Response to Allergen Challenge at 3 Years Among Patients With Moderate to Severe Seasonal Allergic Rhinitis: The GRASS Randomized Clinical Trial.

Importance: Sublingual immunotherapy and subcutaneous immunotherapy are effective in seasonal allergic rhinitis. Three years of continuous treatment with subcutaneous immunotherapy and sublingual immunotherapy has been shown to improve symptoms for at least 2 years following discontinuation of treatment. Objective: To assess whether 2 years of treatment with grass pollen sublingual immunotherapy, compared with placebo, provides improved nasal response to allergen challenge at 3-year follow-up. Design, Setting, and Participants: A randomized double-blind, placebo-controlled, 3-parallel-group study performed in a single academic center, Imperial College London, of adult patients with moderate to severe seasonal allergic rhinitis (interfering with usual daily activities or sleep). First enrollment was March 2011, last follow-up was February 2015. Interventions: Thirty-six participants received 2 years of sublingual immunotherapy (daily tablets containing 15 µg of major allergen Phleum p 5 and monthly placebo injections), 36 received subcutaneous immunotherapy (monthly injections containing 20 µg of Phleum p 5 and daily placebo tablets) and 34 received matched double-placebo. Nasal allergen challenge was performed before treatment, at 1 and 2 years of treatment, and at 3 years (1 year after treatment discontinuation). Main Outcomes and Measures: Total nasal symptom scores (TNSS; range; 0 [best] to 12 [worst]) were recorded between 0 and 10 hours after challenge. The minimum clinically important difference for change in TNSS within an individual is 1.08. The primary outcome was TNSS comparing sublingual immunotherapy vs placebo at year 3. Subcutaneous immunotherapy was included as a positive control. The study was not powered to compare sublingual immunotherapy with subcutaneous immunotherapy. Results: Among 106 randomized participants (mean age, 33.5 years; 34 women [32.1%]), 92 completed the study at 3 years. In the intent-to-treat population, mean TNSS score for the sublingual immunotherapy group was 6.36 (95% CI, 5.76 to 6.96) at pretreatment and 4.73 (95% CI, 3.97 to 5.48) at 3 years, and for the placebo group, the score was 6.06 (95% CI, 5.23 to 6.88) at pretreatment and 4.81 (95% CI, 3.97 to 5.65) at 3 years. The between-group difference (adjusted for baseline) was -0.18 (95% CI, -1.25 to 0.90; [P = .75]). Conclusions and Relevance: Among patients with moderate to severe seasonal allergic rhinitis, 2 years of sublingual grass pollen immunotherapy was not significantly different from placebo in improving the nasal response to allergen challenge at 3-year follow-up. Trial Registration: clinicaltrials.gov Identifier: NCT01335139; EudraCT Number: 2010-023536-16.