Viracor-IBT Laboratories Research Articles

Diagnostic testing for galactose-alpha-1,3-galactose, United States, 2010 to 2018

BackgroundAlpha-gal syndrome (AGS) is an emerging immunoglobulin E (IgE)–mediated allergy to galactose-alpha-1,3-galactose (alpha-gal). The geographic distribution and burden of AGS in the United States are unknown. ObjectiveTo characterize alpha-gal IgE testing patterns and describe the trends and distribution from 2010 to 2018 in the United States. MethodsThis retrospective analysis included all persons tested for alpha-gal IgE antibodies by Viracor-IBT Laboratories (Lee’s Summit, Missouri), the primary site of testing in the United States. Data included age and sex of person tested, specimen state of origin, collection date, and result value; persons with at least 1 positive test result (≥0.1 kU/L) were compared with negatives. Proportions tested and with positive test results were calculated using the US Census population estimates. ResultsOverall, 122,068 specimens from 105,674 persons were tested for alpha-gal IgE during July 1, 2010, to December 31, 2018. Nearly one-third (34,256, 32.4%) had at least 1 positive result. The number of persons receiving positive test results increased 6-fold from 1110 in 2011 to 7798 in 2018. Of those receiving positive test results, mean [SD] age was 46.9 (19.8) years; men were more likely to test positive than women (43.3% vs 26.0%). Arkansas, Virginia, Kentucky, Oklahoma, and Missouri had the highest number of persons who were tested and had a positive result per 100,000 population. ConclusionMore than 34,000 persons, most presumably symptomatic, have received positive test results for IgE antibodies to alpha-gal, suggesting AGS is an increasingly recognized public health problem. The geographic distribution of persons who tested positive is consistent with exposure to Amblyomma americanum ticks.

ICOSL+ Plasmacytoid Dendritic Cells As Biomarker and Inducer of Graft-Versus-Host Disease

Acute graft-versus-host disease (GVHD) remains one of the leading causes of death post allogeneic hematopoietic cell transplantation (HCT). Gastrointestinal GVHD (GI-GVHD), the most fatal type of GVHD, would benefit from additional biomarkers that are therapeutic targets. Using state-of-the-art quantitative proteomics we previously identified and validated an increased CD4+CD146+ T cell population in GI-GVHD patients. This population expressed a Th1 and Th17 phenotype and was induced by Inducible COStimulator (ICOS), a critical costimulatory molecule for the development of pathogenic Th17 (Li W. et al, J. Clin. Invest. Insights, 2016).ICOS binds its ligand, ICOSL, which is expressed on dendritic cells (DCs) that prime naïve T cells to initiate immune responses. This prompted us to examine ICOSL expression on the two blood DCs subsets that can be identified in human peripheral blood: Lineage-HLADR+CD11c+ myeloid DCs (mDCs) and Lineage-HLADR+CD123+ plasmacytoid DCs (pDCs). Using the same cohort of patients aforementioned, the frequency of ICOSL was significantly higher on pDCs in 64 GI-GVHD patients when compared to 22 non-GVHD enteritis patients, 35 skin GVHD patients, and 39 patients without GVHD (Figure 1). The numbers and frequencies of total DCs, mDCs and pDCs were similar between groups.The growth factor fms-related tyrosine kinase 3 ligand (Flt3l) is necessary for the development and differentiation of pDCs, and the transcription factor, Stat3, is required for Flt3l-dependent dendritic cell differentiation in mice. The role of pDCs in acute GVHD is still controversial (tolerogenic or initiator of GVHD depending on the murine model), and confirmatory studies about their functions are necessary before a therapeutic approach based on this mechanism can be contemplated. Based on the patients' data and previous knowledge, we hypothesized that absence of ICOSL signaling in donor DCs would protect against GVHD through Flt3l, Stat3, or both. We first found that knocking out (KO) ICOSL in the donor bone marrow (BM) extended survival compared to wild-type (WT) mice in the major mismatch (B6, H-2b à BALB/c, H-2d) experimental HCT model, while recipients of Stat3KO BM did not show any difference in GVHD mortality (Figure 2A). We also found a significant decrease of Flt3l levels in plasma collected at day 3 from ICOSLKO BM recipients compared to WT mice (Figure 2B). We then analyzed the recipients' infiltrating intestinal immune cells at day 10 post-HCT for the infiltration of pDCs and pathogenic Th17 cells. We found significantly lower frequencies of intestinal pDCs (CD11b-CD11c+B220+CD103+) (Figure 2C), and intestinal T cells coexpressing interferon (IFN)g and IL-17 (Figure 2D) in recipients of ICOSLKO BM compared to recipients of WT BM. Absolute counts of these two populations followed the same trend (data not shown). To confirm these data were not strain-specific, we performed similar analyses in the haplo-identical (B6, H-2b à B6D2F1, H-2d) experimental model showing similar outcomes for pDCs and IFNγ+IL-17+ T cells frequencies and counts in recipients of ICOSLKO BM compared to recipients of WT BM. Importantly, and in contrast to human T cells, CD146 is not expressed on naïve murine T cells and thus cannot be measured in vivo in acute GVHD models. Transcriptome analyses by Nanostring technology (Immunology panels) comparing 14 days post-HCT of sorted pDCs from ICOSLKO BM versus WT haplo-identical recipients showed increased expression of key molecules required for development (Itgax, Nos2, Socs1, Tcf4 and Bst2), costimulation (Cd80, Cd48, Cd74 and Cd86), and function (Tyrobp, Ikbkg, Nod2 and Irf7) of pDCs (Figure 3A). Lastly, we found increased T cell activation markers including Prf1, Il17a, eomes in sorted CD4+ T cells from ICOSLKO BM compared to WT recipients (Figure 3B).We conclude that early quantification of ICOSL+ pDCs frequency may allow identification of patients at risk of GI-GVHD development. Targeting ICOSL may represent a new avenue to treat acute GVHD. [Display omitted] DisclosuresPaczesny:Viracor IBT Laboratories: Patents & Royalties.

Outcomes of Pediatric Patients with Acute Leukemia Following Adult Unrelated Donor Transplant: The Impact of Donor KIR Gene Content and KIR Ligand Matching

Abstract In patients with acute leukemia, relapse remains one of the most frequent causes of treatment failure after allogeneic transplantation (allo-HCT). Early after transplant, natural killer (NK) cells predominate and thus, they have been implicated in graft vs. leukemia (GVL) reactions after allo-HCT. Killer Ig Receptors (KIR) are a family of 15 distinct genes that both positively and negatively regulate NK cell function. Individuals vary in the number of genes that they possess within their genome and thus, may vary in their GVL capacity. Accordingly, numerous studies have investigated the association of donor KIR gene content (B/x vs. A/A), KIR B gene score (neutral, better, best), KIR composite score (taking into account CenB/x and TelA/A gene status) and KIR ligand mismatching in the setting of allo-HCT. These studies have largely focused on adult transplant recipients, yielding variable outcomes. However, pediatric patients may differ from adult transplant recipients in a variety of ways including differential sensitivity of leukemic blasts to NK cytotoxicity, distinct cytogenetic changes, the higher propensity to receive myeloablative conditioning and an earlier adoption of minimal residual disease-driven timing of transplantation. The purpose of this analysis was to investigate the impact of KIR gene content and KIR ligand mismatching on outcomes of children transplanted with acute lymphoblastic and acute myeloid leukemia (ALL and AML, respectively), reported to CIBMTR. The study cohort included pediatric patients (0-<19 years, n=716), who received a first HLA 7-8/8 matched, unmanipulated allo-HCT from an adult donor for either B precursor ALL (n=372) or AML (n=344), where donor KIR typing was available. The time period studied was from 1995-2016. For ALL and AML patients, most (95% and 86%) received myeloablative conditioning, without in vivo T cell depletion (79% and 70%). GVHD prophylaxis was with calcineurin inhibitor + MTX+/- serotherapy (79% and 76%) or calcineurin inhibitor + MMF +/- serotherapy (13% and 17%). Median follow up was 59 and 54 months for ALL and AML, respectively. The cumulative incidence of relapse for ALL and AML at 3 years was 22% (18-26%) and 33% (28-39%) and the disease free survival 58% (53-63%) and 48% (43-54%), respectively. The primary endpoints of the analysis were the impact of KIR gene content on relapse and disease free survival (DFS). Other secondary endpoints included acute GVHD, treatment related mortality and overall survival. Considering the various KIR models (Bx vs AA, KIR B gene content, and the KIR composite score (i.e., Tel A/A, Cen B) and KIR ligand matching, there was no significant association with the primary (relapse or DFS) or secondary endpoints in multivariate analysis when taking into account relevant covariates. Likewise, similar results were obtained when considering AML and ALL separately. Lastly, a subgroup analysis considering just patients who received in vivo T cell depletion also showed a lack of correlation between KIR genes and transplant outcomes. Collectively, these studies do not support the selection of an unmanipulated adult URD based on KIR gene content in pediatric AML or ALL transplant recipients. Disclosures Fleischhauer: GENDX: Research Funding. Fleischhauer:GENDX: Research Funding. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Paczesny:Viracor IBT Laboratories: Patents & Royalties. Lee:Kadmon: Research Funding; Amgen: Consultancy, Research Funding; Incyte: Consultancy; Mallinckrodt: Honoraria; Pfizer: Consultancy; Takeda: Research Funding; Onyx: Research Funding.

The Role of HLA-E Polymorphism in Acute Leukemia Patients Receiving a 10/10 HLA Matched Unrelated HSCT

Introduction: HLA-E is a class I HLA molecule that is involved in NK and CTL functions. It has limited polymorphism but can present unconventional peptides if it is aberrantly expressed under stress conditions. Donor-recipient (D/R) mismatching may potentiate NK-mediated alloreactivity. The functional role of HLA-E in HSCT is uncertain. We used CIBMTR data and samples to investigate the effect of HLA-E polymorphism on HSCT outcome in a 10/10 HLA matched unrelated acute leukemia setting by addressing the following questions: 1) Does D/R HLA-E match status affect HSCT outcome? 2) Does a specific D/R HLA-E genotype correlate with outcome?Methods: The study included 1840 adult AML/ALL patients in complete remission who received first T-cell replete, 10/10 high resolution HLA matched transplants from unrelated donors between 2000 to 2015. Cohort characteristics are summarized in Table 1. Both D/R were HLA-E genotyped by NGS using a validated protocol on an Illumina Miseq platform. Overall survival (OS), leukemia free survival (LFS), relapse (RI), transplant related mortality (TRM), acute GvHD (aGvHD) and chronic GvHD (cGvHD) were evaluated; p< 0.01 was considered significant. Factors violating the proportional hazards assumption were adjusted via stratification, while a stepwise model building approach was used to select variables related to a given outcome with a threshold of 0.05 for both entry and retention in the model. HLA-E genotype effects in D/R were tested separately by comparing 0, 1 or 2 copies of a particular HLA-E allele (i.e. HLA-E*01:03). Transplant pairs with HLA-E genotypes including other HLA-E alleles (i.e. other than HLA-E*01:01 or *01:03) were excluded from this analysis. The joint effect of D/R HLA-E genotype was also explored through a 4-level analysis of D/R HLA-E*01:03 homozygous vs other.Results: HLA-E*01:01 and *01:03 frequencies in both D/R as well as the rate of HLA-E discrepant transplant pairs (32.7%) were in accordance with previously reported data. Distribution of HLA-E matched vs HLA-E mismatched cases was balanced for all significant clinical predictors, while no significant interactions between the HLA-E matching status variable and the adjusted covariates were detected. HLA-E mismatch had no significant association with any of the outcome endpoints in both univariate and multivariate models (data not shown). With respect to the HLA-E genotype effect on outcome, 1827/1840 transplant pairs were considered for analysis, as 13 transplant pairs were excluded due to new or rare HLA-E genotypes. When tested separately, both D/R HLA-E*01:03/01:03 genotypes were associated with significantly lower LFS (HR=1.28, p=0.0027 and HR=1.31, p=0.0017 respectively). While the impact of HLA-E genotype on other outcome endpoints was not statistically significant, relapse rates were higher in cases with D/R HLA-E*01:03/01:03 genotype. Joint D/R HLA-E genotype analysis suggest that donor HLA-E*01:03/01:03 is driving this effect. The results of LFS and RI multivariate models are summarized in Table 2. No significant interactions were observed between the HLA-E genotype variable and the adjusted covariates in the multivariate models.Conclusion: This is the largest study investigating the effect of HLA-E polymorphism on HSCT outcome. We found no apparent effect of HLA-E mismatch on HSCT outcome in AML/ALL patients in complete remission, while donor HLA-E*01:03/01:03 genotype was associated with lower LFS, contrary to some previous reports. Differential efficiency of the two HLA allelic forms in inducing the CD94/NKG2A inhibitory pathway may account for their divergent effect on leukemia control, although this hypothesis is yet to be confirmed by functional assays. Our results suggest that avoidance of HLA-E*01:03/01:03 donors may improve the outcome of HSCT from 10/10 matched unrelated grafts.This project was funded by the Else Kröner-Fresenius-Foundation and the National Marrow Donor Program® (NMDP). [Display omitted] DisclosuresLee:Kadmon: Research Funding; Amgen: Consultancy, Research Funding; Mallinckrodt: Honoraria; Incyte: Consultancy; Pfizer: Consultancy; Onyx: Research Funding; Takeda: Research Funding. Fleischhauer:GENDX: Research Funding. Paczesny:Viracor IBT Laboratories: Patents & Royalties. Schrezenmeier:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding.

Multiple Functional Donor Polymorphisms in IL1RL1 region Associate with Death Due to GvHD or Infection after Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation (HCT) for AML and MDS

The last two authors contributed equallyElevated soluble Stimulation-2 (sST2), the decoy IL-33 receptor, in plasma/serum post-HSCT is a biomarker for death due to GvHD. ST2 is the product of IL1RL1 (2q12.1) and this ~.5Mb region contains >300 single nucleotide polymorphisms (SNPs) significantly associated (P<5x10-8) with plasma levels of sST2 in healthy participants from the Framingham Heart Study (FHS). Many of these SNPs are genome-wide associated with infection-related phenotypes, including asthma, Crohn's disease, ulcerative colitis and celiac disease. Given these relationships, we analyzed the association of ST2 SNPs with death due to GvHD or infection within one year after HLA-matched unrelated donor HCT for AML or MDS to identify functional SNPs that could potentially aid in donor selection.We measured plasma/serum sST2 levels in pre-HCT samples in a subset of AML and MDS patients (n=759) and their donors (n=757) from DISCOVeRY-BMT, a GWAS of >3,000 recipient-unrelated donor pairs reported to the CIBMTR between 2000-2011. After quality control (info>.8, MAF>.005), 3613 SNPs in the IL1RL1 region were tested for association with sST2 levels; 1541 donor SNPs associated with sST2 levels (P<.05), which validated 99% of the FHS sST2 SNP associations. To assess the contribution of these donor variants to post-HCT survival, we constructed competing risk models with clinical variables using stepwise Akaike Information Criterion (AIC), then tested each of the 1541 donor SNPs for association with each outcome in the two DISCOVeRY-BMT cohorts. Causes of death were previously adjudicated by a multi-member panel. Meta-analyses of the two cohorts identified 13 GvHD-death associated SNPs and 118 infection-death associated SNPs. Sensitivity analyses show 91/118 SNPs are still significant after excluding patients with a history of acute GvHD grade III-IV (Figure 1). When taking correlations into account, we found 10 and three independent SNPs (R2<.6) associate with death due to infection and GvHD, respectively. There were no overlapping SNP associations between GvHD-death or infection-death (Figure 1). For GvHD-death associated SNPs, alleles associated with higher sST2 levels associate with increased risk for GvHD-death, while for infection-death SNPs, alleles associated with higher sST2 levels reduced risk of infection-death.AIC multivariable models for GvHD-death included AML diagnosis, recipient obesity (>30 mg/kg2), peripheral blood cell source, donor age, rs1558645 (Pmeta=.001), and rs2310241(Pmeta=.0003), for which the risk alleles at each SNP increased risk of GvHD death ~1.5 fold. The model for infection-death included advanced disease at HCT, recipient/donor CMV status, peripheral blood cell source, rs13019803 (Pmeta=1.1 x 10-6), rs13015714 (Pmeta=5 x 10-4) and rs4851601 (Pmeta=2 x 10-6), with risk alleles at each SNP increasing risk of infection-death ~2-fold. To capture the total contribution of these donor variants to each outcome we created multi-allele models for GvHD-death (0-4 alleles) and infection-death (0-6 alleles).The multi-allele GvHD models showed a 1.5 fold increased risk of GvHD-death with each additional allele (Pmeta=3.4 x 10-6 ) (Figure 2). Individuals whose donors are homozygous for both risk alleles at each SNP have a ~3 fold, and ~2 fold higher risk of dying of GvHD versus those with zero, and 1-2 risk allele(s), respectively. The multi-allele infection models (0-6 alleles) show a ~2 fold increased risk of infection-death (Pmeta= 1.22 x 10-10) with each risk allele (Figure 2).sST2 is one of the most reproducible GvHD biomarkers to date. As hypothesized, specific alleles correlated with high sST2 levels in donors and with GvHD-death. Our novel finding is that some ST2 alleles associated with low sST2 levels correlated with infection-death. These results are not without precedent, for example the low-sST2 associated allele in rs13019803 (T) associates with higher mortality in the CHARGE consortium. The risk allele in rs13015714 (T) is significantly associated with lower IL1RL1 and IL18R1 (P= 5 x 10-10) gene expression in lung tissue, indicating biochemical functions for these SNPs. Importantly the non-risk allele in all 5 variants are common (>20%) across races and ethnicities, which means there is an opportunity to select donors without combinations of these variants and perhaps without the requirement of measuring the protein level pre-transplant. [Display omitted] DisclosuresMcCarthy:Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria. Lee:Kadmon: Research Funding; Amgen: Consultancy, Research Funding; Mallinckrodt: Honoraria; Incyte: Consultancy; Pfizer: Consultancy; Onyx: Research Funding; Takeda: Research Funding. Paczesny:Viracor IBT Laboratories: Patents & Royalties.

ST2 and ROR-γ-t Roles in Intestinal Regulatory T Cells

Allogeneic hematopoietic cell transplantation (allo-HCT) is an essential therapeutic modality for patients with hematological malignancies and benign blood disorders. Unfortunately, acute graft-versus-host disease (aGVHD) remains the major complication of allo-HCT and is associated with high mortality. Soluble STimulation-2 (sST2) is increased during aGVHD while regulatory T cells (Tregs) that express membrane-bound ST2 prevent GVHD through unknown mechanisms. Herein, we studied nonlymphoid tissue ST2+ Tregs in murine models of allo-HCT. Transplantation of Foxp3- T cells and Tregs sorted from different Foxp3 reporter mice indicated that ST2+Tregs isolated from aGVHD mice were of donor origin, thymus-derived, and predominantly intestine localized (data not shown). To further understand the mechanism, we performed a transcriptome analysis of sorted Foxp3GFP Tregs from aGVHD mice receiving wild type (WT) CD25-depleted total T cells with either WT or ST2-/- Foxp3GFP Tregs in both major (B6, H-2bàBALB/c, H-2d, Mhc) and minor (B6, H-2bàC3H.SW, H-2b, miH) mismatched aGVHD models. ST2-/- Foxp3GFP Tregs showed decreased expression of key molecules needed for Treg function such as Foxp3, Il2ra, Icos, Il27, Ctla4, and Ahr. Strikingly, transcription of RAR-related-orphan-receptor-c (Rorc), essential for proinflammatory IL-17+ T cells development, was the most upregulated transcript in ST2-/- Foxp3GFP Treg isolated from the gut in both models (Figure 1A). The transcriptome finding led us to investigate the frequency and function of ST2+Tregs in the gut of naive mice deficient for Rorc. Isolation and staining of intestinal T cells showed a significant increase of ST2+Treg frequency in the gut of Rorc-/- mice compared to WT mice: 55% vs. 12%, respectively (Figure 1B). Rorc-/- T cells transplantation enhanced the day 10 post-HCT frequency of intestinal ST2+Tregs (Figure 1C, left) that co-expressed TIGIT and Helios expression (data not shown), and decreased aGVHD score and mortality (Figure 1C, right). Previous studies have shown that IL-33 signaling in Tregs elicits their amphiregulin expression and increases their ability to suppress effector T cells (Teffs). IL-23, a key proinflammatory cytokine in the maintenance and amplification of Th17/Tc17, can restrain Treg responses by inhibiting IL-33 responsiveness. Using ex vivo polyclonal Tregs from donor B6 cultured with IL-2 (control Tregs) vs IL-2+IL-33 (TregIL-33) vs IL-2+IL-33+IL-23+IL-17, we found that adding IL-33 doubled the frequencies of ST2+ Tregs compared to control Tregs, and adding IL-23+IL-17 reversed this activity (Figure 2A). Markers of Tregs activation (KLRG1) (Figure 2B) and function (Helios, LAG3) (data not shown) were augmented in TregIL-33 compared to other Treg conditions. TregIL-33 also expressed more amphiregulin than Tregs cultured in the two other conditions (Figure 2C). Next, we evaluated their function in vitro and in vivo. TregIL-33 displayed better immunosuppression than other Tregs on CD4+ T cells at a physiological Tregs:Teffs ratio of 1:12; differences were not seen at higher ratios or on suppression of CD8+ T cells (Figure 3A). Then we examined the in vivo capacity of donor TregIL-33 to protect from aGVHD intestinal damage by co-adoptive transfer of Tregs cultured in the three aforementioned conditions with CD25-depleted WT T cells in Mhc and miH mismatch aGVHD models. We used a physiological ratio of 1 donor Treg per 20 donor Teffs (5% of total T cells). Mice receiving TregIL-33 showed significantly less severe aGVHD and better survival compared to those cultured either without IL-33 or with IL-33 and IL23/IL17 in both Mhc (Figure 3B) and miH (data not shown) mismatched aGVHD models. Analysis of intestinal T cells of these mice showed decreased total lymphocytes infiltration in the gut at day 10 after HCT, and decreased intestinal Tbet and RORγt expressing Teffs in both Mhc (Figure 3C) and miH (not shown) mismatched aGVHD models. Altogether, we conclude that within GVHD target organs, ST2+Tregs are predominantly intestine localized, and inversely correlated with RORgt in steady state and inflammatory conditions. Triggering the ST2/IL-33 pathway in Tregs prevents severe aGVHD, possibly through a dual mechanism: upregulation of amphiregulin and immunosuppression on effector T cells. Thus, TregIL-33 is a potential cellular therapy avenue for preventing or treating aGVHD. DisclosuresBlazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Paczesny:Viracor IBT Laboratories: Patents & Royalties.

Small Molecule ST2 Inhibitors Cause Reduction of Soluble ST2 and Improve Gvhd and Survival In Vivo

Activation of the membrane-bound Suppression of Tumorigenecity 2 (mST2) by Interleukin-33 (IL-33) in T cells leads to type-2 (Th2) and Foxp3+ regulatory (Tregs) immune responses. The mST2/IL-33 axis is not engaged when a ST2 splice isoform containing only the ST2 ectodomain and acting as a decoy receptor, called soluble ST2 (sST2), sequesters IL-33. Clinically, elevated plasma sST2 has been reported and used as a prognostic biomarker in cardiac allo-rejection, inflammatory bowel disease, and graft-versus-host disease (GVHD). We hypothesized that releasing IL-33 from circulating sST2 may augment the mST2/IL-33 axis activation to modulate the Th1 and Th2/Tregs responses for treating immune related diseases. This is supported by our study of a ST2 antibody in the GVHD models (Zhang et. al., Sci. Trans. Med. 2015). Advantages of small-molecule therapies over biologics include easier administration especially by oral, superior tissue penetration, modifiable pharmacokinetic properties including half-life, and lower manufacturing cost. Here, we present the proof-of-concept of this approach using three small molecule ST2 inhibitors we discovered recently. Our data show two inhibitors cause reduction of sST2, alleviate GVHD and improve survival in two in vivo GVHD models.Our high throughput screening (HTS) using the AlphaLISA assay and computational analysis led to discover three classes of small molecules inhibiting ST2 binding to IL-33 which were confirmed by the functional cell-based HEK-Blue assay. Seven compounds showing low toxicity at 66 uM to peripheral blood mononuclear cells (PBMC) were selected for the dose escalation toxicity study in mice. Six of them showed no toxicity up to 20 mg/kg in 13 days while five of them can be tolerated at 40 mg/kg. The HTS workflow is summarized in Fig. 1.Three compounds (named iST2-1-3) were evaluated in the in vivo GVHD disease models. The first model is the transplantation of human PBMC to the NOD/SCID/IL2rgnull (NSG) mice one day after the mice received 300 cGy total body irradiation (TBI). Compounds were injected via the intraperitoneal route to the mice the day before transplantation and continued daily at their respective IC50 dosages for 21 days. At Day 14, both iST2-1 and iST2-2 caused reduction of IFNg+ CD4+ T cells and increase of Foxp3+CD4+ Tregs population in the human CD45+CD4+ cells extracted from the gastrointestinal (GI), the main GVHD target organ, compared with the DMSO control (Fig. 2). More profound effect exerted by iST2-1 was observed at Day 21. Plasma concentrations of human IFNg and sST2, indicators of Th1 response, showed the highest reduction in the iST2-1 treatment group. While the hsST2 plasma concentration is maintained at < 30 pg/ml throughout 28 days in the iST2-1 treatment group, escalation of hsST2 up to 180 pg/ml was found in the DMSO control and iST2-3 treated group. Plasma hIFNg was at least 50% lower in the iST2-1 treated group at day 14 and 21. The GVHD scores and survival of NSG mouse models paralleled the changes of GI T cell populations and systemic cytokines data; iST2-1 treatment showed decreased GVHD score to 3 from 6 in the DMSO control and improvement of survival rate to 45 % on day 35 whereas all mice in the DMSO control group were dead.The second model is the minor mismatched model of allogeneic hematopoietic cell transplantation (HCT) from B6 to C3H.SW mice. We observed similar trends of IFNg+ reduction and Foxp3+ increase in CD4+ Tcells from the GI (Fig. 3). Plasma sST2 and IFNg concentrations were greatly reduced on day 14 in the iST2-1 treated group compared with DMSO control (50 versus 10 ng/ml for sST2 and 350 versus 100 pg/ml for IFNg).Finally, we have collected the Small Angle X-ray Scattering data to construct structural models between iST2-1 and sST2 to guide the inhibitor optimization. The CRISP-Cas9 technology was also used to obtain sST2 deficient B6 mice. Allo-HCT using these donor sST2 deficient T cells will delineate the specific mode of action by our ST2 inhibitors on mST2 and sST2. Our findings reveal iST2-1 is an attractive small molecule ST2 inhibitor that can be used to study the mST2/IL-33 axis in disease models and further developed as therapeutics to treat GVHD. [Display omitted] [Display omitted] [Display omitted] DisclosuresPaczesny:Viracor-IBT Laboratories: Patents & Royalties.

Infection Burden of Double Stranded DNA (dsDNA) Viruses after CD34+ Selected, T-Cell Depleted (TCD) Hematopoietic Cell Transplantation (HCT) for Myeloid Malignancies at Memorial Sloan Kettering Cancer Center (MSK)

Background: Ex vivo T-cell depletion (TCD) by CD34+ selection has been used successfully for graft-versus-host disease prevention alleviating the need for additional pharmacologic immunosuppressants. TCD has been associated with higher rates of viral infections and cytomegalovirus (CMV) disease in particular compared to conventional (CONV) HCT. Modern methods of TCD achieve up to 5 log reduction of T-cells. At MSK, TCD HCT recipients (R) are routinely monitored for CMV, adenovirus (ADV), Epstein-Barr virus (EBV), and human herpes virus 6 (HHV6) in the blood by quantitative polymerase chain reaction (qPCR) assays. We report the incidence of viremia and end organ disease (EOD) by these viruses in TCD HCT recipients.Methods: Retrospective review of adults with acute leukemia (AL) and myelodysplastic syndrome (MDS) patients (pts) who received TCD HCT at MSK from June 2010 -December 2014. TCD was performed by the FDA approved CliniMACS CD34+ reagent system (Miltenyi Biotec, Gladbach, Germany). CMV was monitored at least weekly from day 14-100 and at least every 14 days until day 180. EBV was monitored through day 180. Monitoring for CMV and EBV was performed by the Clinical Microbiology Laboratory at MSK using Roche analyte specific reagents, and since March 2013, the Cobas Ampliprep/Cobas Taqman (CAP/CTM) CMV qPCR in plasma. Monitoring for ADV/HHV6 was performed by Viracor-IBT laboratories (Lenexa, KS). Preemptive treatment for CMV was started for ≥2 consecutive PCR >500copies/ml in whole blood or >300 IU/ml in plasma. Treatment of ADV/HHV6 viremia was at clinician's discretion. EBV post-transplant lymphoproliferative disorder (PTLD) was treated with rituximab as primary therapy. During the study, 11 pts were enrolled in CMV prevention trials of brincidofovir (BCV), and 5 pts received BCV for treatment of ADV. EOD was scored by standard criteria. Follow up was through June 2015. The cumulative incidence (CI) was estimated for viremia at 180 days and EOD at 365 days for CMV, ADV, HHV6, and EBV. Kaplan-Meier method and log-rank test were used to compare CI between groups. P <0.05 was deemed statistically significant.Results: Of239 pts (median age 56.3 years, range 19.6-73.3), 161 (67%) pts had AL and 78 (33%) pts had MDS. Donors were matched-related 76 (32%), matched unrelated 116 (48%), or mismatched (related or unrelated) 47(20%); CMV R+ 148 (62%) or R- 91 (38%). CMV infection had the earliest onset post HCT at a median of 28 days (d) (IQR: 25-33), followed by HHV6 33d (25-52), ADV 71d (44-89), and EBV 85d (58-107). 188 (79%) pts had ≥1 dsDNA viral infection with 182 (76%) pts experiencing infection prior to d100. The CIs of viremia were: CMV 42% (68% in R+ vs 0% in R-, P <0.0001), ADV 7% (10% in R+ vs 5% in R-, P =0.02), EBV 19%, and HHV6 61%. The CI of EBV and HHV6 viremia were similar between R+ and R- (P =NS). Among 100 pts with CMV viremia, 62 (62%) had dsDNA viral coinfections; including 40 (40%) with 1 coinfection, 19 (19%) with 2 coinfections, and 3 (3%) with 3 coinfections. Antiviral treatment was given to 99 pts for CMV (99% of CMV viremic pts, 67% of R+, 41% of total pts), 9pts for ADV (56% of ADV viremic pts; 4% of total), and 19 for HHV6 (16% of HHV6 viremic pts; 8% of total). In total, 42 (18%) pts developed EOD by dsDNA viruses. The most common EOD was EBV PTLD (20 pts, 9%), followed by CMV (14 pts, 7%; but 11% among CMV R+ pts), ADV (8 pts, 4%), and HHV6 (2 pts, 1%). Among 14 pts with CMV disease, 6 had pneumonitis, 5 colitis, 3 duodenitis, and 3 retinitis. Three pts had 2 sites of CMV EOD. Among 8 pts with ADV disease, 3 had colitis, 3 pneumonitis, 1 nephritis, and 1 hepatitis/colitis. Two pts had HHV6 pneumonitis.Conclusions: 1) Viremias by dsDNA viruses occurred frequently and early post TCD HCT: 189 (79%) pts had at least 1 dsDNA viral infection, with onset of first infection before d100 in 183 (77%) pts. Around 58% of pts with CMV had coinfections with additional dsDNA virus(es). 2) Two thirds (67%) of CMV R+ and 41% of total pts received CMV antiviral treatment. 3) Overall, 18% of patients developed end organ disease by dsDNA viruses. 4) Viremias were managed effectively with intense monitoring and antiviral treatment, and rates of EOD were comparable to those of CONV HCT. Our data highlights the need for effective strategies to reduce the total burden of dsDNA viruses after TCD HCT. [Display omitted] [Display omitted] [Display omitted] DisclosuresPerales:Merck: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Giralt:TAKEDA: Consultancy, Honoraria, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; AMGEN: Consultancy, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding.

Improved HLA typing by next-generation DNA sequencing

Aim High-resolution HLA typing by DNA sequencing has enabled accurate matching of donors and recipients in allogeneic hematopoietic stem cell transplantation (HSCT). Most laboratories perform high-resolution HLA typing by Sanger-based DNA sequencing methodology. Sequence-based typing (SBT) using this methodology has significant limitations preventing unambiguous HLA typing, often requiring additional testing to provide clinically-useful information. Methods This study examined the potential benefit of next-generation DNA sequencing (NGS) for accurate HLA typing. DNA samples extracted from peripheral blood leukocytes from potential HSCT donors and recipients being tested by SBT for high-resolution HLA typing (n = 95) were tested in parallel using Illumina TruSight HLA sequencing reagents. In brief, amplicons for HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 were generated by long-range PCR, fragmented and indexed using Nextera XT v2 reagents, and sequenced using a MiSeq Reagent kit v2 (250 cycle paired-end). Data were analyzed blinded using Conexio Genomics Assign software. Results NGS generated unambiguous 2-field high-resolution HLA typing results for 94.2% of alleles typed (Table 1). Ambiguities were clustered, with HLA-B∗44:02/19N, -DPB1∗04:01/105:01 with -DPB1∗04:02/126:01, and HLA-DRB1∗03, -DRB1∗12, and -DRB1∗15 representing 69.4% of all ambiguities. NGS results were 99.8% concordant with SBT. Importantly, NGS resolved ambiguous typings from SBT. Improved resolution was derived from the nearly full-gene sequencing and phased sequence analysis enabled by NGS. Conclusions NGS presents a significant technical advantage for accurate high-resolution HLA typing. The nearly full-gene sequencing combined with phased stranded sequencing is useful for resolving common ambiguities observed using current SBT methodology. N. Baird: Employee; Company/Organization; Illumina, Inc. B. Baas: Employee; Company/Organization; Illumina, Inc. A. Crawford: Employee; Company/Organization; Illumina, Inc. M. Won: Employee; Company/Organization; Illumina, Inc. N. Kim: Employee; Company/Organization; Illumina, Inc. D. Goodridge: Employee; Company/Organization; Conexio Genomics. A. Lindell: Employee; Company/Organization; Illumina, Inc. G.P. Morris: Grant/Research Support; Company/Organization; Illumina. 2. Consultant; Company/Organization; Viracor-IBT Laboratories. 4. Scientific/Medical Advisor; Company/Organization; Neuralstem, Inc. Download : Download full-size image

Alpha-Gal IgE Sensitization in the United States; Surveillance Update

S A T U R D A Y 114 Alpha-Gal IgE Sensitization in the United States; Surveillance Update Michelle L. Altrich, PhD, Sharlene P. Blum, Shannon M. Foster; Viracor-IBT Laboratories, Lee’s Summitt, MO, Viracor-IBT Laboratories. RATIONALE: Galactose-alpha-1,3-galactose (alpha-gal), is a crossreactive carbohydrate moiety found in red meats such as beef, pork and lamb and is associated with a delayed IgE response. A link to lone star tick bites was established by researchers at the University of Virginia and although the mechanism of sensitization is not clearly elucidated, it remains the primary suspect. METHODS: A retrospective review of national laboratory data was conducted for alpha-gal IgE testing performed in 2012 and 2013, utilizing data with de-identified patient health information only when the source location could be identified. This data was mapped according to 6 geographical regions and compared to the July 2011 CDC published map of lone star tick populations. RESULTS: For the total data set, 37% displayed positive results for alphagal IgE. In the subset of samples that could be tied to a source location, the positive rate declined to 33%. The regions with the highest prevalence of positive samples continue to overlap with the location of the lone star tick with a positive rate as high as 46% in TN andAR. In other non-lone star tick regions the positive rates varied from 8% to 17%. CONCLUSIONS: The overall positive rate of samples tested in 20122013 decreased compared to 2009-2011, and the total number of tests increased, suggesting there is more screening of patients potentially exposed to the lone star tick. The overall regional prevalence rates continue to support the hypothesis that lone star tick bites trigger the development of the alpha gal allergy.

Current Insights: Use of Immuknow in Heart Transplant Recipients

Despite the advances in medical management of congenital and acquired cardiac disease, heart transplant remains the only curative option for certain patients. Transplant physicians aim to maintain a fine balance between too much and too little immunosuppression, so as to prevent complications such as infections, malignant growths, and toxic effects of drugs on one hand and acute or chronic rejection of the graft on the other hand. The ImmuKnow assay (by Cylex, recently acquired by Viracor-IBT Laboratories, Inc) was first introduced in 2002 by the Food and Drug Administration for detecting cell-mediated global immunity, thus providing an additional tool to help identify patients at risk for infection and rejection. All studies done to date are reviewed to examine the use of ImmuKnow in heart transplant recipients, both adults and children. Advantages and disadvantages are described, as well as areas in need of further investigation and improvement.

Auto-antibodies in Chronic Idiopathic Urticaria (CIU) and Non-urticarial Systemic Autoimmune Disorders

T U E S D A Y 841 Auto-antibodies in Chronic Idiopathic Urticaria (CIU) and Non-urticarial Systemic Autoimmune Disorders S. A. Stutes, C. B. Cho, M. Altrich, S. P. Ardoin, P. U. Ogbogu; The Ohio State University Medical Center, Columbus, OH, Viracor-IBT Laboratories, Lee’s Summit, MO. RATIONALE: 30-50% of patients with CIU have ‘‘functional’’ autoantibodies to FceRI or IgE asmeasured by basophil histamine release assay or basophil activation markers. It is unknown if patients with systemic autoimmune diseases have a similar prevalence of these auto-antibodies. METHODS: Adult patients, 26 with CIU, 27 with Rheumatoid Arthritis (RA), and 26 with Systemic Lupus Erythematosus (SLE), were recruited from an allergy clinic and a rheumatology clinic. 20 healthy controls were compared. The patients with RA and SLE did not have chronic urticaria. Atopy history and disease activity were obtained. The CU Index Panel (basophil histamine release assay), anti-IgE, and anti-thyroid antibodies were performed by Viracor-IBT laboratories. RESULTS: CU Index values were significantly higher in the CIU group (mean 15.6) as compared to the RA group (mean 2.31, p50.007), but not in the SLE group (mean 7.6, p50.079). In contrast to patients in the RA and control groups, 11% of SLE and 7.6% of CIU patients had elevated antiIgE antibody levels (>188 ng/mL). Elevated anti-thyroid peroxidase (TPO) levels did not correlate with a positive CU Index. Elevated CU Index and anti-IgE values in the RA and SLE groups were not associated with disease severity. CONCLUSIONS: The CU Index values differentiated CIU patients from RA patients, but not from SLE patients. Interestingly, SLE patients were found to have ‘‘functional’’ auto-antibodies without associated urticaria. The presence of auto-antibodies did not correlate with disease activity or having thyroid antibodies. Functional auto-antibodies may not be useful in distinguishing autoimmune chronic urticaria from non-urticarial autoimmune disorders.

Association of anti-IgA antibodies with adverse reactions to γ-globulin infusion

Association of anti-IgA antibodies with adverse reactions to γ-globulin infusion

Statistical Analysis of Correlates of Pneumococcal Antibody Level and Avidity in Patients with Recurrent Infections

S U N D A Y 510 Statistical Analysis of Correlates of Pneumococcal Antibody Level and Avidity in Patients with Recurrent Infections A. J. Fried, M. L. Altrich, J. F. Halsey, F. A. Bonilla; Children’s Hospital Boston, Boston, MA, Viracor-IBT Laboratories, Lenexa, KS. RATIONALE: We correlated subject characteristics with the levels and avidities of antibodies to 12 pneumococcal serotypes. METHODS: We measured concentration and avidity of antibodies to 12 pneumococcal serotypes in 78 patients aged 0.6-18 years evaluated for recurrent infections, and in 80 individuals evaluated for peanut allergy ages 0.4-15 years. Non-parametric statistics were used to correlate gender, age, serum immunoglobulin levels, and immunization history with responses and to compare patients and ‘‘controls’’. RESULTS: Level and avidity were highly correlated only for two types among patients. There was no consistent correlation of age and level in either group; there was a significant negative correlation of age and avidity for approximately half of the types in both groups. There was inconsistent correlation of total IgG or IgG subclasses with either levels or avidity. Distributions of levels and avidity for each pneumococcal type varied significantly from each other. For serotypes in the conjugate vaccine, there was a general association of avidity (but not level) with the total number of vaccine doses. There was significant association of levels (but not avidity) with vaccination history for the non-conjugate types. There was inconsistent correlation of level and avidity with time from immunization for all types. CONCLUSION: Overall, concentration and avidity correlate independently with immunologic characteristics and exposure history and are markedly different between pneumococcal types.