Objectives We previously reported that CCL17 gene-deficient mice are protected from developing pain-like behaviour and exhibit less disease in destabilization of medial meniscus (DMM)-induced OA, as well as in high-fat diet (HFD)-exacerbated DMM-induced OA. Here, we explored if therapeutic neutralization of CCL17, using increasing doses of a neutralizing monoclonal antibody (mAb), would lead to a dose-dependent benefit in these two models. Design DMM-induced OA was initiated in male mice either fed with a control diet (7% fat) or 8 weeks of a 60% HFD, followed by therapeutic intraperitoneal administration (i.e. when pain is evident) of an anti-CCL17 mAb (B293, 25mg/kg, 5mg/kg or 1mg/kg) or isotype control (BM4; 25mg/kg). Pain-like behaviour and arthritis were assessed by relative static weight distribution and histology, respectively. The effects of B293 (25mg/kg) on HFD-induced metabolic changes, namely oral glucose tolerance test, insulin tolerance test and liver triglyceride levels, were examined. Results Therapeutic administration of B293 results in a dramatic amelioration of DMM-induced OA pain-like behaviour and the inhibition of disease progression, compared to BM4 (isotype control) treatment. A similar therapeutic effect was observed in HFD-exacerbated OA pain-like behaviour and disease. B293 treatment did not alter the measured HFD-induced metabolic changes. Conclusions Based on the data presented, CCL17 could be a therapeutic target in OA patients with joint injury alone or with obesity.

Introduction:Hemophilia A (HA) patients (HAPs) with the human leukocyte antigen (HLA)-class-II (HLAII) haplotype DRB1*15:01/DQB1*06:02, and thus antigen presenting cells which express HLAII β-polypeptide chains that form heterodimers of DR15- and DQ6-serotypes, respectively, have an increased risk of developing factor (F)VIII inhibitors (FEIs)-neutralizing antibodies against the therapeutic-FVIII-proteins (tFVIIIs) infused to prevent/arrest bleeding. As DRB1*15:01 and DQB1*06:02 exist in strong linkage disequilibrium, association analysis cannot determine which is the actual risk allele. Methods:To establish the true risk allele of this haplotype, we analyzed the tFVIII-derived peptides (tFVIII-dPs) bound to either the DR or DQ molecules that comprise the individual HLAII repertoires expressed by monocyte-derived dendritic cells obtained from 25 normal blood donors and six HAPs, four without and two with FEIs. We performed log-linear mixed model analyses, where the dependent variable is the log of the measured peptide count. Under Model 1, we analyzed an HLAII allele predictor consisting of ten levels (four DRB1 and six DQB1 alleles) in the fixed effects and variables in the random effects to account for non-independence. Model 2-where the HLAII allele variable consisted of only DRB1*15:01 and DQB1*06:02-compares the HLAII alleles. Results:Relative to the Model 1 reference, DRB1*15:01 and DQB1*06:02 significantly increased tFVIII-derived peptide counts, and DRB1*15:01 contributed significantly more than DQB1*06:02. Reported as risk ratios (RRs) and their 95% confidence interval (CI) lower- (LB) and upper-bound (UB), we found a RR (95% CI-LB, -UB) of 14.16 (10.38, 19.33) and 1.76 (1.24, 2.50) for DRB1*15:01 and DQB1*06:02, respectively. Under Model 2, we found an RR for DRB1*15:01 against DQB1*06:02 of 7.00 (5.80, 8.44). Discussion/conclusion:Our results suggest that DRB1*15:01 is the offending HLAII allele and that DR15 allotypes underlie the increased FEI risk in HAPs.

ObjectiveTissue-resident memory cells (Trm) are a subset of T cells residing persistently and long-term within specific tissues that contribute to persistent inflammation and tissue damage. We characterised the phenotype and function of Trm and the role of CD103 in primary Sjogren’s syndrome (pSS).MethodsIn both pSS and non-pSS sicca syndrome patients, we examined Trm frequency, cytokine production in salivary glands (SG) and peripheral blood (PB). We also analysed Trm-related gene expression in SG biopsies through bulk and single-cell RNA sequencing (scRNAseq). Additionally, we investigated Trm properties in an immunisation-induced animal model of pSS (experimental SS, ESS) mouse model and assessed the effects of Trm inhibition via intraglandular anti-CD103 monoclonal antibody administration.ResultsTranscriptomic pSS SG showed an upregulation of genes associated with tissue recruitment and long-term survival of Trm cells, confirmed by a higher frequency of CD8+CD103+CD69+cells in pSS SG, compared with non-specific sialadenitis (nSS). In SG, CD8+CD103+Trm contributed to the secretion of granzyme-B and interferon-γ, CD8+Trm cells were localised within inflammatory infiltrates, where PD1+CD8+ T cells were also increased compared with nSS and MALT lymphoma. scRNAseq of PB and pSS SG T cells confirmed expression ofCD69, ITGAE, GZMB, GZMKandHLA-DRB1among CD3+CD8+SG T cells. In the SG of ESS, CD8+CD69+CD103+Trm producing Granzyme B progressively expanded. However, intraglandular blockade of CD103 in ESS reduced Trm, reduced glandular damage and improved salivary flow.ConclusionsCD103+CD8+Trm cells are expanded in the SG of pSS and ESS, participate in tissue inflammation and can be therapeutically targeted.

Supplemental Figure S1. BMP pathway gene expression in activated NK cells.

Humans have a minor lymphocyte population of gamma-delta (γδ) T cells. The majority of these express a recombined Vγ9Vδ2 T cell receptor (TCR) attractive to immunotherapy. This distinct TCR conveys reactivity to phosphorylated antigens (pAg) that derive from pathogens or accumulate inside tumour cells. Such T cell responses are regulated by butyrophilin (BTN) 3A1 and other membrane-related proteins present on antigen-presenting cells. However, the activation mechanism and direct molecular ligand recognised by the γδ TCR remain a crucial unresolved question. Herein, we used pAg-reactive TCR probes in a whole-genome screen to identify BTN2A1 as an essential ligand. In further investigation, we elucidated its functionality working in cis with BTN3A1. Also, a mutational analysis unveiled critical regions of the γδ TCR are positioned at opposite sides. We locate germ-line encoded residues of the Vγ9 chain were responsible for BTN2A1 binding, whereas two amino-acids of the Vδ2 chain were necessary for a complete response to pAg. In conclusion, we propose a dual-ligand complex model that senses pAg to evoke immune responses, wherein BTN2A1 sets the framework to develop new opportunities on γδ T cell-based immunotherapies.

BACKGROUND:To compare the clinical efficacy of New York Esophageal squamous cell carcinoma-1 (NY-ESO-1) vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in a randomized, double-blind phase II study in participants with fully resected melanoma at high risk of recurrence. METHODS:Participants with resected stage IIc, IIIb, IIIc and IV melanoma expressing NY-ESO-1 were randomized to treatment with three doses of NY-ESO-1/ISCOMATRIX or ISCOMATRIX adjuvant administered intramuscularly at 4-week intervals, followed by a further dose at 6 months. Primary endpoint was the proportion free of relapse at 18 months in the intention-to-treat (ITT) population and two per-protocol populations. Secondary endpoints included relapse-free survival (RFS) and overall survival (OS), safety and NY-ESO-1 immunity. RESULTS:The ITT population comprised 110 participants, with 56 randomized to NY-ESO-1/ISCOMATRIX and 54 to ISCOMATRIX alone. No significant toxicities were observed. There were no differences between the study arms in relapses at 18 months or for median time to relapse; 139 vs 176 days (p=0.296), or relapse rate, 27 (48.2%) vs 26 (48.1%) (HR 0.913; 95% CI 0.402 to 2.231), respectively. RFS and OS were similar between the study arms. Vaccine recipients developed strong positive antibody responses to NY-ESO-1 (p≤0.0001) and NY-ESO-1-specific CD4+ and CD8+ responses. Biopsies following relapse did not demonstrate differences in NY-ESO-1 expression between the study populations although an exploratory study demonstrated reduced (NY-ESO-1)+/Human Leukocyte Antigen (HLA) class I+ double-positive cells in biopsies from vaccine recipients performed on relapse in 19 participants. CONCLUSIONS:The vaccine was well tolerated, however, despite inducing antigen-specific immunity, it did not affect survival endpoints. Immune escape through the downregulation of NY-ESO-1 and/or HLA class I molecules on tumor may have contributed to relapse.

BACKGROUND:Plasma-derived (pd) or recombinant (r) therapeutic factor VIII proteins (FVIIIs) are infused to arrest/prevent bleeding in patients with hemophilia A (PWHA). However, FVIIIs are neutralized if anti-FVIII-antibodies (inhibitors) develop. Accumulating evidence suggests that pdFVIIIs with von Willebrand factor (VWF) are less immunogenic than rFVIIIs and that distinct rFVIIIs are differentially immunogenic. Since inhibitor development is T-helper-cell-dependent, human leukocyte antigen (HLA)-class-II (HLAcII) molecules constitute an important early determinant. OBJECTIVES:Use dendritic cell (DC)-protein processing/presentation assays with mass-spectrometric and peptide-proteomic analyses to quantify the DP-bound, DQ-bound, and DR-bound FVIII-derived peptides in individual HLAcII repertoires and compare the immunogenic potential of six distinct FVIIIs based on their measured peptide counts. PATIENTS/METHODS:Monocyte-derived DCs from normal donors and/or PWHA were cultured with either: Mix-rFVIII, a VWF-free equimolar mixture of a full-length (FL)-rFVIII [Advate® (Takeda)] and four distinct B-domain-deleted (BDD)-rFVIIIs [Xyntha® (Pfizer), NovoEight® (Novo-Nordisk), Nuwiq® (Octapharma), and Afstyla® (CSL Behring GmBH)]; a pdFVIII + pdVWF [Beriate® (CSL Behring GmBH)]; Advate ± pdVWF; Afstyla ± pdVWF; and Xyntha + pdVWF. RESULTS:We showed that (i) Beriate had a significantly lower immunogenic potential than Advate ± pdVWF, Afstyla - pdVWF, and Mix-rFVIII; (ii) distinct FVIIIs differed significantly in their immunogenic potential in that, in addition to (i), Afstyla + pdVWF had a significantly lower immunogenic potential than Beriate, while the immunogenic potential of Beriate was not significantly different from that of Xyntha + pdVWF; and (iii) rFVIIIs with pdVWF had significantly lower immunogenic potentials than the same rFVIIIs without pdVWF. CONCLUSIONS:Our results provide HLAcII peptidomic level explanations for several important clinical observations/issues including the differential immunogenicity of distinct FVIIIs and the role of HLAcII genetics in inhibitor development.

The role played by N-linked glycans (NLGs) in the immunogenicity of therapeutic Factor VIII (tFVIII) proteins is poorly understood. Our study addresses this question using peptidomic profiling data on HLA-class-II (HLAcII)-presented peptides from dendritic cell (DC)-protein processing and presentation assays (PPPAs) performed across three independent experiments reported in the literature (Sorvillo et al. (2016), Peyron et al. (2018), and Diego et al. (2020)). Assuming that the number of peptides presented on HLAcII molecules is directly proportional to immunogenicity potential (IP), we asked if the NLGs on tFVIII proteins provide some degree of protection from proteolytic processing within DCs for the amino acid (AA) residues in their immediate vicinity. If NLGs are protective, then we expect an attenuated IP, which would reflect in lower counts of associated AAs. We examined the effects of NLGs both pointwise (i.e., at the single glycated asparagine residue) and at the glycosylation umbrella (GUMB), a term we defined as being −5 to +5 AAs from the glycated asparagine residue of all NLGs. Our first step in addressing these effects was to construct 2×2 contingency tables of the number of AA residues in the HLAcII bound and unbound fractions, and the number of AA residues falling either within the set of NLGs or within the set of GUMBs. We statistically evaluated our question by using Fisher's Exact tests of the hypothesis of no association, and calculated the odds ratio (OR) and its 95% confidence interval (CI). Results from the pointwise tests of the effect of NLGs reported in Figure 1 suggest that overall NLGs exert a protective effect in that non-glycated AAs were at significantly greater risk of being in the fraction of peptides bound to and presented by HLAcII molecules. The results from these statistical analyses-listed as OR (95% CI LB, 95% CI UB)-were: 7.0 (2.6, 21.9) for Diego et al.; 5.6 (0.9, 230.6) for Sorvillo et al. with 25 nM tFVIII; 6.5 (1.0, 269.0) for Sorvillo et al. with 50 nM tFVIII; 3.7 (1.1, 19.8) for Peyron et al.; and 4.4 (2.3, 9.3) for all data combined. The data from Sorvillo et al. (2016; for their experiments using 50 nM tFVIII but not for their experiments using 25 nM tFVIII), Peyron et al. (2018), and Diego et al. (2020), as well as the combined data, all revealed significantly greater risk of non-glycated AAs being in the HLAcII bound/presented fraction relative to glycated AAs. While the results from Sorvillo et al. with 25 nM tFVIII was not statistically significant, this was most likely due to the effects on power of its "small sample size" as the data was trending. The results for the GUMB-level tests reported in Figure 2 are even more robust with the data suggesting a protective effect of the GUMBs in that the AAs not located in their immediate vicinity (as defined above) were at significantly greater risk of being in the bound fraction of peptides presented on HLAcII molecules in all experiments, as well as in the combined data. The results from these statistical analyses-listed as OR (95% CI LB, 95% CI UB)-were: 6.0 (4.5, 8.2) for Diego et al.; 19.9 (6.7, 97.6) for Sorvillo et al. with 25 nM tFVIII; 7.3 (3.9, 15.6) for Sorvillo et al. with 50 nM tFVIII; 2.5 (1.8, 3.6) for Peyron et al.; and 3.8 (3.2, 4.6) for all data combined. In conclusion, these results-from the tFVIII proteins tested using peptidomic profiling of HLAcII-presented peptides from DC-PPPAs performed across three independent experiments-demonstrate NLGs provided significant protection from either proteolytic processing in DCs or peptide binding to HLAcII molecules, or both, and thus lower IP. Our results support the conclusion that NLGs play a significant role in the immunogenicity of tFVIII proteins. Disclosures Luu: Haplogenics Corporation: Current Employment. Hofmann:CSL Behring: Current Employment. Dinh:Haplogenics Corporation: Current Employment. Powell:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees. Mead:CSL Behring: Current Employment. Escobar:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maraskovsky:CSL Behring: Current Employment. Howard:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees.

Cancer vaccine development has proven challenging with the exception of some virally induced cancers for which prophylactic vaccines exist. Currently, there is only one FDA approved vaccine for the treatment of prostate cancer and as such prostate cancer continues to present a significant unmet medical need. In this study, we examine the effectiveness of a therapeutic cancer vaccine that combines the ISCOMATRIX™ adjuvant (ISCOMATRIX) with the Toll-like receptor 3 agonist, polyinosinic–polycytidylic acid (Poly I:C), and Flt3L, FMS-like tyrosine kinase 3 ligand. We employed the TRAMP-C1 (transgenic adenocarcinoma of the mouse prostate) model of prostate cancer and the self-protein mPAP (prostatic acid phosphatase) as the tumor antigen. ISCOMATRIX™–mPAP–Poly I:C–Flt3L was delivered in a therapeutic prime-boost regime that was consistently able to achieve complete tumor regression in 60% of animals treated and these tumor-free animals were protected upon rechallenge. Investigations into the underlying immunological mechanisms contributing to the effectiveness of this vaccine identified that both innate and adaptive responses are elicited and required. NK cells, CD4+ T cells and interferon-γ were all found to be critical for tumor control while tumor infiltrating CD8+ T cells became disabled by an immunosuppressive microenvironment. There is potential for broader application of this cancer vaccine, as we have been able to demonstrate effectiveness in two additional cancer models; melanoma (B16-OVA) and a model of B cell lymphoma (Eµ-myc-GFP-OVA).

Gamma delta (γδ) T cells are essential to protective immunity. In humans, most γδ T cells express Vγ9Vδ2+ T cell receptors (TCRs) that respond to phosphoantigens (pAgs) produced by cellular pathogens and overexpressed by cancers. However, the molecular targets recognized by these γδTCRs are unknown. Here, we identify butyrophilin 2A1 (BTN2A1) as a key ligand that binds to the Vγ9+ TCR γ chain. BTN2A1 associates with another butyrophilin, BTN3A1, and these act together to initiate responses to pAg. Furthermore, binding of a second ligand, possibly BTN3A1, to a separate TCR domain incorporating Vδ2 is also required. This distinctive mode of Ag-dependent T cell activation advances our understanding of diseases involving pAg recognition and creates opportunities for the development of γδ T cell-based immunotherapies.

The HLA haplotype DRB1*15:01/DQB1*06:02 has been shown to be associated with the development of inhibitory antibodies ("inhibitors") against therapeutic FVIII (tFVIII) proteins in Hemophilia A (HA) patients. Moreover, this same haplotype has also been implicated with an increased risk for the development of multiple sclerosis, narcolepsy, and drug-induced-hypersensitivity and -liver-injury. Recent reports in the literature have indicated that the alleles of this haplotype may have differential effects on immunogenicity despite the fact that they are in strong linkage disequilibrium (LD). Thus, the question arises as to which of the two constituent alleles of this haplotype is the true HLA risk allele. To address this question we analyzed peptidomic profiling data on HLA-class-II (HLAcII)-presented peptides from dendritic cell (DC)-protein processing and presentation assays (PPPAs) performed in two independent experiments performed by Peyron et al. (2018) and Diego et al. (2020) which reported results on both the HLA-DQ and -DR isomers. The tFVIII in both experiments is full-length recombinant FVIII (FL-rFVIII). We performed log-linear mixed model analyses under two models, where the dependent variable in both models is the logarithm of the expected peptide count. Under Model 1, we analyzed in the fixed effect component of the model a single HLA allele predictor variable consisting of 10 levels represented by distinct DRB1 and DQB1 alleles (4 and 6 levels, respectively). Under this model, we analyzed in the random effects component of the model variables for individuals (8 and 9 levels from Peyron et al. and Diego et al., respectively), experiments (2 levels for Peyron et al. and Diego et al.), and HLA isomers (2 levels for DQ and DR). The random effects component of the model serves to reduce the error (or "noise") in our estimation of the predictive effect of HLA alleles for the peptide counts by accounting for the clustering due to individuals, experiments, and isomers. This approach therefore can be understood as maximizing the signal-to-noise ratio. Model 2 was more focused in that the single HLA allele predictor variable in the fixed effect component consisted of only DQB1*06:02 and DRB1*15:01. Model 2 was also simpler in that we only accounted for two random effect variables, namely for individuals (in this case 2 and 6 levels for Peyron et al. and Diego et al., respectively) and experiments. Thus, Model 2 is a direct head-to-head comparison of the two main HLA alleles of interest. The Model 1 results are reported in Figure 1A and B, where it can be seen that relative to the baseline reference allele provided by DQB1*02:01, both DQB1*06:02 and DRB1*15:01 are at significantly increased risk of contributing to the overall peptide count. Results are reported as risk ratios (RRs) and their associated 95% confidence interval lower and upper bounds (95% CI LB and 95% CI UB). For DQB1*06:02 and DRB1*15:01 respectively, we found a RR (95% CI LB, 95% CI UB) of 1.76 (1.24, 2.50) and 14.16 (10.38, 19.33). Because they are compared to the same baseline, the two RRs may also be directly compared, thus showing that DRB1*15:01 contributes significantly more to the overall peptide count than DQB1*06:02. Under Model 2 (the head-to-head comparison), the RR for the DRB1*15:01 allele against the baseline DQB1*06:02 allele is 7.00 (5.80, 8.44) (Figure 2A and B). These results contribute to the field of precision medicine because they demonstrate that even in the case of alleles in tight LD, their effects can be effectively disentangled by a DC-PPPA and a log-linear mixed model analysis. Our results support the conclusion that in regard to the well-known risk haplotype of DQB1*06:02/DRB1*15:01, the true HLA risk allele seems to be DRB1*15:01. Disclosures Luu: Haplogenics Corporation: Current Employment. Hofmann:CSL Behring: Current Employment. Powell:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees. Dinh:Haplogenics Corporation: Current Employment. Escobar:National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maraskovsky:CSL Behring: Current Employment. Howard:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees.

AbstractObjectivesPlasmacytoid dendritic cells (pDCs), through the production of type 1 interferons (IFNs) and other cytokines, are major contributors to systemic lupus erythematosus (SLE) pathogenesis. IL‐3 promotes pDC survival, but its role in SLE is not well characterised. This study investigated serum IL‐3 and IFN levels, and a whole blood ‘IL‐3 gene signature’, in human SLE.MethodsSerum cytokine levels were measured by ELISA in n = 42 SLE patients, and n = 44 healthy donors. IL‐3‐regulated genes were determined by RNASeq of healthy donor whole blood cells (WBCs) stimulated in vitro with IL‐3 for 6 or 24 h. Whole blood cell RNASeq analysis was undertaken in a separate cohort of n = 31 SLE patients, and n = 28 healthy donors.ResultsSerum IL‐3 levels correlated with IFNα (r = 0.612, 95% CI 0.455–0.733, P < 0.001) and type III IFN (r = 0.585, 95% CI 0.406–0.720, P < 0.0001). IL‐3 stimulation of WBC in vitro altered 794 genes (−1 ≥ logFC ≥ 1, FDR < 0.05), of which 35 overlapped with genes differentially expressed between SLE and healthy donors. These 35 genes were expressed in 27/31 SLE donors, revealing the presence of an ‘IL‐3 gene signature’. There was strong correlation between the IL‐3 signature and an IFN signature, as determined by hierarchical clustering of the 500 most variable genes in SLE donors (r = 0.939, 95% CI 0.898–0.964, P < 0.0001).ConclusionA dual IL‐3/IFN gene signature is a feature of SLE. An association between IL‐3 and IFN raises the possibility that dual blockade of IL‐3 and IFN may be especially useful for SLE patients with this dual cytokine gene signature.

Background: Patients with the X-linked bleeding disorder hemophilia A have impaired blood clotting due to deficient or absent factor VIII (FVIII) coagulant activity. Bleeding can be managed by infusions of any of several plasma-derived (pd) or recombinant (r) therapeutic FVIII proteins (tFVIIIs). The efficacy of tFVIIIs can be eliminated, however, if neutralizing anti-tFVIII-antibodies called "inhibitors" develop. Since the development of inhibitors is T-helper-cell dependent, human leukocyte antigen (HLA)-class-II (HLAcII) molecules comprise an important early determinant. Accumulating evidence suggests that the presence of the FVIII chaperon protein, von Willebrand factor (VWF), in either pdFVIII or rFVIII concentrates, decreases the immunogenicity of these tFVIIIs by reducing their uptake by antigen presenting cells, especially dendritic cells (DCs). Objectives: Use a native (i.e., non-engineered) full-length (FL) tFVIII without ((−)) or with ((+)) pdVWF in DC-protein processing and presentation assays (PPPAs) followed by mass-spectrometric and peptide-proteomic analyses to identify and quantify the DP-, DQ-, and DR-bound/tFVIII-derived-peptides in individual HLAcII repertoires. Compare the number of peptides in the subset from any of the five globular domains (A1, A2, A3, C1 and C2) or three acidic-residue-rich connecting segments (a1, a2 and a3), which we collectively refer to as the non-B-domain (NBD) portion of a tFVIII, with the number of peptides from its non-globular "outrigger like" B-domain (BD) that contains ~80% of the N-linked glycans of a FL-FVIII molecule despite containing only 908 amino acid residues, i.e. slightly less than 40% of its 2,332 total residues. Methods: DC-PPPAs were performed using monocyte-derived (Mo)DCs obtained from 12 healthy blood donors. The tFVIII tested was a FL-rFVIII (Advate®) used (−) or (+) pdVWF (i.e., FL-rFVIII − pdVWF and FL-rFVIII + pdVWF) and the resulting data consists of counts of tFVIII-derived peptides presented on and extracted from HLAcII molecules. Difference of proportion tests were used to compare the effect of pdVWF as well as the NBD- and BD-regions of the FL tFVIII on the peptide counts. Results: FL-rFVIII − pdVWF yielded significantly more peptides (p<0.05) than FL-rFVIII + pdVWF from the NBD portions but not the BD. Interestingly, for the FL-rFVIII − pdVWF preparation, the NBD portions yielded significantly more peptides (p<0.05) than the BD, but this pattern was reversed for the FL-rFVIII + pdVWF preparation in that the NBD portions yielded significantly less peptides (p<0.05) than the BD. Conclusions: The Outrigger Hypothesis posits that in the presence of VWF the heavily glycosylated BD acts as an "outrigger" and renders this portion of a FL tFVIII relatively more likely to be internalized, proteolytically processed and HLAcII-presented. However, in the absence of VWF, the N-linked glycans individually act to protect the amide bonds in the underlying peptide bond backbone of a tFVIII from proteolytic processing, as posited by the GUMB Hypothesis. Our results support both hypotheses as important determinants in the pathogenesis of inhibitor development. Disclosures Howard: Haplogenics Corporation: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Luu:Haplogenics Corporation: Employment. Hofmann:CSL Behring: Employment. Dinh:Haplogenics Corporation: Employment. Mead:CSL Behring: Employment. Powell:Haplogenics: Membership on an entity's Board of Directors or advisory committees. Escobar:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees. Eugene:CSL Behring: Employment.

The immunogenicity of protein therapeutics is an important safety and efficacy concern during drug development and regulation. Strategies to identify individuals and subpopulations at risk for an undesirable immune response represent an important unmet need. The major histocompatibility complex (MHC)-associated peptide proteomics (MAPPs) assay directly identifies the presence of peptides derived from a specific protein therapeutic on a donor's MHC class II (MHC-II) proteins. We applied this technique to address several questions related to the use of factor VIII (FVIII) replacement therapy in the treatment of hemophilia A (HA). Although >12 FVIII therapeutics are marketed, most fall into 3 categories: (i) human plasma-derived FVIII (pdFVIII), (ii) full-length (FL)-recombinant FVIII (rFVIII; FL-rFVIII), and (iii) B-domain-deleted rFVIII. Here, we investigated whether there are differences between the FVIII peptides found on the MHC-II proteins of the same individual when incubated with these 3 classes. Based on several observational studies and a prospective, randomized, clinical trial showing that the originally approved rFVIII products may be more immunogenic than the pdFVIII products containing von Willebrand factor (VWF) in molar excess, it has been hypothesized that the pdFVIII molecules yield/present fewer peptides (ie, potential T-cell epitopes). We have experimentally tested this hypothesis and found that dendritic cells from HA patients and healthy donors present fewer FVIII peptides when administered pdFVIII vs FL-rFVIII, despite both containing the same molar VWF excess. Our results support the hypothesis that synthesis of pdFVIII under physiological conditions could result in reduced heterogeneity and/or subtle differences in structure/conformation which, in turn, may result in reduced FVIII proteolytic processing relative to FL-rFVIII.

Objective Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease that is difficult to treat. There is currently no optimal stratification of patients with SLE, and thus responses to available treatments are unpredictable. Here, we developed a new stratification scheme for patients with SLE, based on the whole-blood transcriptomes of patients with SLE. Methods We applied machine learning approaches to RNA-sequencing (RNA-seq) datasets to stratify patients with SLE into four distinct clusters based on their gene expression profiles. A meta-analysis on two recently published whole-blood RNA-seq datasets was carried out and an additional similar dataset of 30 patients with SLE and 29 healthy donors was contributed in this research; 141 patients with SLE and 51 healthy donors were analysed in total. Results Examination of SLE clusters, as opposed to unstratified SLE patients, revealed underappreciated differences in the pattern of expression of disease-related genes relative to clinical presentation. Moreover, gene signatures correlated to flare activity were successfully identified. Conclusion Given that disease heterogeneity has confounded research studies and clinical trials, our approach addresses current unmet medical needs and provides a greater understanding of SLE heterogeneity in humans. Stratification of patients based on gene expression signatures may be a valuable strategy to harness disease heterogeneity and identify patient populations that may be at an increased risk of disease symptoms. Further, this approach can be used to understand the variability in responsiveness to therapeutics, thereby improving the design of clinical trials and advancing personalised therapy.

Plasmacytoid dendritic cells (pDCs), through the production of type 1 interferons (IFNs) and other cytokines, are major contributors to systemic lupus erythematosus (SLE) pathogenesis. IL-3 promotes pDC survival, but its role in SLE is not well characterised. This study investigated serum IL-3 and IFN levels, and a whole blood ‘IL-3 gene signature’, in human SLE. Serum cytokine levels were measured by ELISA in n  = 42 SLE patients, and n  = 44 healthy donors. IL-3-regulated genes were determined by RNASeq of healthy donor whole blood cells (WBCs) stimulated in vitro with IL-3 for 6 or 24 h. Whole blood cell RNASeq analysis was undertaken in a separate cohort of n  = 31 SLE patients, and n  = 28 healthy donors. Serum IL-3 levels correlated with IFNα ( r  = 0.612, 95% CI 0.455–0.733, P  < 0.001) and type III IFN ( r  = 0.585, 95% CI 0.406–0.720, P  < 0.0001). IL-3 stimulation of WBC in vitro altered 794 genes (−1 ≥ logFC ≥ 1, FDR < 0.05), of which 35 overlapped with genes differentially expressed between SLE and healthy donors. These 35 genes were expressed in 27/31 SLE donors, revealing the presence of an ‘IL-3 gene signature’. There was strong correlation between the IL-3 signature and an IFN signature, as determined by hierarchical clustering of the 500 most variable genes in SLE donors ( r  = 0.939, 95% CI 0.898–0.964, P  < 0.0001). A dual IL-3/IFN gene signature is a feature of SLE. An association between IL-3 and IFN raises the possibility that dual blockade of IL-3 and IFN may be especially useful for SLE patients with this dual cytokine gene signature.

T cells specific for tFVIIIs in HA patients can induce neutralizing FVIII antibodies called inhibitors. DCs that present tFVIII derived peptides in their HLAcII repertoire may activate T cells. We employed DC protein processing and presentation assays (DCAs) to quantify tFVIII peptides in HLAcII peptidomes and used these as measures of the immunogenic potential (IP) of tFVIIIs as a function of their associated HLAcII/tFVIII peptide density. We used DCs from 28 healthy donors and 5 HA patients [2 inhibitor (Inh) positive and 3 Inh negative] in 3 DCAs. In DCA 1, DCs from healthy donors 1–12 were given an equimolar mix of 5 recombinant (r) FVIIIs. In DCA 2, DCs from healthy donors 13–24 were given a plasma derived (pd) FVIII + vWF or 1 of 2 rFVIIIs (BDT or FL) ± vWF. In DCA 3, DCs from healthy donors 25–28 and HA patients 1–5 were incubated with pdFVIII or FL, BDD, or BDT rFVIII, all + vWF (Fig. 1A). After DC lysis and DR, DQ and DP isolation, we identified tFVIII peptides by LC-MS/MS. We performed a log linear analysis of the DR bound tFVIII peptides to identify determinants of Inh risk (Fig. 1B-E) with random effects to control for potential donor- and DCA-clustering. Space limits prevent description of the DQ and DP peptides. The IP of tFVIIIs was greater in HA patients than in healthy donors and in Inh positive than in Inh negative patients (Fig. 1B-E). We found that BDT + vWF and FL ± vWF had lesser and greater IP, respectively, than pdFVIII + vWF; the IP of BDD + vWF was comparable to that of pdFVIII + vWF. The rFVIII mix had the greatest IP. The higher IP in HA patients versus healthy donors and in Inh positive vs Inh negative patients reflect significant correlations of 0.91 and 0.52 with the presence/absence of the FVIII Inh risk allele DRB1*15:01.Download : Download high-res image (400KB)Download : Download full-size image DC protein processing and presentation is affected by HLAcII repertoires but not by HA patient or FVIII Inh status after accounting for correlation with DRB1*15:01. Relative to pdFVIII, rFVIIIs have different IP with BDT and FL being significantly less and greater, respectively. T.E. Howard: 4. Scientific/Medical Advisor; Company/Organization; Haplomics Biotechnology. M. Hofmann: 5. Employee; Company/Organization; CSL Behring. L.V. Dinh: 5. Employee; Company/Organization; Haplomics Biotechnology. J. Powell: 5. Employee; Company/Organization; CSL Behring. E. Maraskovsky: 5. Employee; Company/Organization; CSL Limited.

The development of neutralizing antibodies-termed “inhibitors”-to infused therapeutic (t) factor VIII proteins (tFVIIIs) is the most serious obstacle to effective treatment of bleeding in Hemophilia A (HA) patients. As clinically significant FVIII immune responses are only initiated if dendritic cell (DC) cII-HLAs can present foreign tFVIII-derived peptides to naïve FVIII-specific T cells, we posit the “Gate Keeper” hypothesis in which the limiting determinant of inhibitor formation are patients' cII-HLA repertoires with the majority being individually distinct and each contributing slightly to the vast population level diversity of cII-HLAs. While cII-HLAs are critical at the cellular level for initiating immune responses, conflicting results from population studies have led some to describe their encoding HLA-II structural genes as weak determinants of inhibitor causation. Our main objective here is to test a hypothesis that gets at the heart of this disconnect between molecular-based expectations and population-level data by analyzing cII-HLA peptidomic data from DC-protein processing and presentation assays (PPPAs). The chief variable of DC-PPPA data is the peptide count, which we assume to be directly proportional to immunogenic potential (IP). Our working model is that inhibitor formation requires at minimum, in its initial stages, a complex between cII-HLAs and specific tFVIII-derived peptides. A testable null hypothesis under this thinking posits that a given cII-HLA allotype will have the same IP when exposed to several tFVIIIs. To test this hypothesis, we first performed model selection to determine the best set of predictor allotypes. To analyze the data, we employed a log-linear model where the peptide count is the dependent variable and allotype is a categorical independent variable consisting of 29 levels for 29 allotypes (8 DP, 10 DQ, and 11 DR allotypes). We used elastic net regression (ENR) to select the best set of allotype levels thus giving the best overall model consisting now of only four DR allotypes (Table 1). We then performed interaction analysis under the best-selected allotypes model in which we introduced as additional predictor variables, a tFVIII categorical variable consisting of five levels for five different tFVIIIs, namely full length (FL)-recombinant (r) FVIII (FL-rFVIII) ± von Willebrand Factor (VWF), B domain truncated (BDT)-rFVIII ± VWF, and plasma derived (pd) FVIII (pdFVIII) + VWF, and 12 interaction terms for the (4 - 1) × (5 - 1) possible interactions between the cII-HLA allotype and tFVIII variables. We found significant cII-HLA allotype × tFVIII interactions (Table 2). To get at the specific null hypothesis of interest, we examined within-allotype risk ratios (RRs) and their appropriately adjusted confidence intervals (CIs).1-4 It can be shown that an 84% CI is sufficient to achieve a significance level of α = 0.05 for the CI difference.2-4 Although there are 12 total interaction terms, per allotype there are only three possible CI comparisons on using the interaction term with the highest RR as a fixed reference. On constructing the adjusted CIs and correcting for multiple hypothesis testing,2 we found that two comparisons in Table 2 corresponded to significantly different RRs. We determined statistical power to detect a CI difference.1,3 As seen in Table 2, our study was extremely underpowered, which may explain why only two significant differences were found. Thus, at least for the two comparisons showing significant difference, we have refuted the null hypothesis of no difference across tFVIIIs for a given allotype, and have affirmed our working model that specific combinations of cII-HLAs and tFVIII-derived peptides are the triggering factor in inhibitor development.Schenker N, Gentleman J. On judging the significance of differences by examining the overlap between confidence intervals. Am Statistician. 2001; 55(3): 182-6.Julious S. Using confidence intervals around individual means to assess statistical significance between two means. Pharmaceut Statist. 2004; 3: 217-22.Maghsoodloo S, Huang C-Y. Comparing the overlapping of two independent confidence intervals with a single confidence interval for two normal population parameters. J Statist Plan & Infer. 2010; 140: 3295-305.Knol M, Pestman W, Grobbee D. The (mis)use of overlap of confidence intervals to assess effect modification. Eur J Epidemiol. 2011; 26(4): 253-4.

Dendritic cell activation of CD4 T cells in the lymph node draining a site of infection or vaccination is widely considered the central event in initiating adaptive immunity. The accepted dogma is that this occurs by stimulating local activation and antigen acquisition by dendritic cells, with subsequent lymph node migration, however the generalizability of this mechanism is unclear. Here we show that in some circumstances antigen can bypass the injection site inflammatory response, draining freely and rapidly to the lymph nodes where it interacts with subcapsular sinus (SCS) macrophages resulting in their death. Debris from these dying SCS macrophages is internalized by monocytes recruited from the circulation. This coordinated response leads to antigen presentation by monocytes and interactions with naïve CD4 T cells that can drive the initiation of T cell and B cell responses. These studies demonstrate an entirely novel pathway leading to initiation of adaptive immune responses in vivo.

Aim: Pilot clinical trial of NY-ESO-1 (ESO) protein in ISCOMATRIX™ adjuvant pulsed onto peripheral blood dendritic cells (PBDC), to ascertain feasibility, evaluate toxicity and assess induction of ESO-specific immune responses. Patients & methods: Eligible participants had resected cancers expressing ESO or LAGE-1 and were at high risk of relapse. PBDC were produced using CliniMACS®plus, with initial depletion of CD1c+B cells followed by positive selection of CD1c+PBDC. Patients received three intradermal vaccinations of ESO/IMX-pulsed PBDC at 4-week intervals. Results: The process was feasible and safe. No vaccine-induced immune responses were detected. Assays of immunomodulatory cells did not correlate with outcomes. One patient had a long lasting complete remission. Conclusion: This method was feasible and safe but was minimally immunogenic.