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Detection of hallmark genomic aberrations in acute myeloid leukemia (AML) is essential for diagnostic subtyping, prognosis, and patient management. However, cytogenetic/cytogenomic techniques used to identify those aberrations, such as karyotyping, fluorescence in situ hybridization (FISH), or chromosomal microarray analysis (CMA), are limited by the need for skilled personnel as well as significant time, cost, and labor. Optical genome mapping (OGM) provides a single, cost-effective assay with a significantly higher resolution than karyotyping and with a comprehensive genome-wide analysis comparable with CMA and the added unique ability to detect balanced structural variants (SVs). Here, we report in a real-world setting the performance of OGM in a cohort of 100 AML cases that were previously characterized by karyotype alone or karyotype and FISH or CMA. OGM identified all clinically relevant SVs and copy number variants (CNVs) reported by these standard cytogenetic methods when representative clones were present in >5% allelic fraction. Importantly, OGM identified clinically relevant information in 13% of cases that had been missed by the routine methods. Three cases reported with normal karyotypes were shown to have cryptic translocations involving gene fusions. In 4% of cases, OGM findings would have altered recommended clinical management, and in an additional 8% of cases, OGM would have rendered the cases potentially eligible for clinical trials. The results from this multi-institutional study indicate that OGM effectively recovers clinically relevant SVs and CNVs found by standard-of-care methods and reveals additional SVs that are not reported. Furthermore, OGM minimizes the need for labor-intensive multiple cytogenetic tests while concomitantly maximizing diagnostic detection through a standardized workflow.

The SARS-CoV-2 pandemic poses a great threat to global health, particularly in solid organ transplant recipients (SOTRs). Although a 3-dose mRNA vaccination protocol has been implemented for the majority of SOTRs, its effectiveness was still largely unknown. We analyzed 113 vaccinated SOTRs, and 30 healthy controls (HCs), some of whom had recovered from COVID, for their immune responses against the original vaccine strain and variants of concern (VOC), including the highly mutated-omicron variant. Here, we report that 3 doses of the mRNA vaccine had only a modest effect in eliciting anti-viral responses against all viral strains in the fully vaccinated SOTRs who did not contract the virus. Only 34.0% (16/47) of this group of patients demonstrated both detectable anti-RBD IgG and neutralization activities against alpha, beta, and delta variants, and only 8.5% (4/47) of them showed additional omicron-neutralizing capacities. In contrast, 79.5% (35/44) of the vaccinated recovered-SOTRs demonstrated both higher anti-RBD IgG levels and neutralizing activities against all VOC, including omicron. These findings illustrate a significant impact of previous infection on the development of anti-COVID immune responses in vaccinated SOTRs and highlight the need for alternative strategies to protect a subset of a lesser-vaccine responsive population.

Summary Background Refractory coeliac disease type 2 is a rare subtype of coeliac disease with high mortality rates; interleukin 15 (IL-15) is strongly implicated in its pathophysiology. This trial aimed to investigate the effects of AMG 714, an anti-IL-15 monoclonal antibody, on the activity and symptoms of refractory coeliac disease type 2. Methods This was a randomised, double-blind, placebo-controlled, phase 2a study of adults with a confirmed diagnosis of refractory coeliac disease type 2. Patients were randomly assigned at a 2:1 ratio to receive seven intravenous doses over 10 weeks of AMG 714 (8 mg/kg) or matching placebo. Biopsy samples were obtained at baseline and week 12 for cellular analysis and histology. The change in the proportion of aberrant intraepithelial lymphocytes from baseline to week 12 with respect to all intraepithelial lymphocytes was the primary endpoint and was quantified using flow cytometry. Secondary endpoints were the change in aberrant intraepithelial lymphocytes with respect to intestinal epithelial cells; intestinal histological scores (villous height-to-crypt depth ratio; VHCD); intraepithelial lymphocyte counts; Marsh score; and patient-reported symptom measures, including the Bristol stool form scale (BSFS) and gastrointestinal symptom rating scale (GSRS). Main analyses were done in the per-protocol population of patients who received their assigned treatment, provided evaluable biopsy samples, and did not have major protocol deviations; only patients with non-atypical disease were included in the analyses of aberrant intraepithelial lymphocytes, including the primary analysis. Safety was assessed in all patients who received at least one dose of study drug. This study is registered at ClinicalTrials.gov (NCT02633020) and EudraCT (2015-004063-36). Findings From April 13, 2016, to Jan 19, 2017, 28 patients were enrolled and randomly assigned to AMG 714 (n=19) and placebo (n=9). Six patients were not included in the primary analysis because of protocol deviation (one in the AMG 714 group), insufficient biopsy samples (one in the AMG 714 group), and atypical intraepithelial lymphocytes (three in the AMG 714 group and one in the placebo group). At 12 weeks, the least square mean difference between AMG 714 and placebo in the relative change from baseline in aberrant intraepithelial lymphocyte percentage was −4·85% (90% CI −30·26 to 20·56; p=0·75). The difference between the AMG 714 and placebo groups in aberrant intraepithelial lymphocytes with respect to epithelial cells at 12 weeks was −38·22% (90% CI −95·73 to 19·29; nominal p=0·18); the difference in change in Marsh score from baseline was 0·09% (95% CI −1·60–1·90; nominal p=0·92); the difference in VHCD ratio was 10·67% (95% CI −38·97 to 60·31; nominal p=0·66); and the difference in change in total intraepithelial lymphocyte count was −12·73% (95% CI −77·57–52·12); nominal p=0·69). Regarding symptoms, the proportion of patients with diarrhoea per the BSFS score decreased from ten (53%) of 19 at baseline to seven (37%) of 19 at week 12 in the AMG 714 group and increased from two (22%) of nine at baseline to four (44%) of nine at week 12 in the placebo group (nominal p=0·0008); and the difference between the groups in change in GSRS score was −0·14 (SE 0·19; nominal p=0·48). Eight (89%) patients in the placebo group and 17 (89%) in the AMG 714 group had treatment-emergent adverse events, including one (11%) patient in the placebo group and five (26%) in the AMG 714 group who had serious adverse events. The most common adverse event in the AMG 714 group was nasopharyngitis (eight [42%] patients vs one [11%] in the placebo group). Interpretation In patients with refractory coeliac disease type 2 who were treated with AMG 714 or placebo for 10 weeks, there was no difference between the groups in terms of the primary endpoint of aberrant intraepithelial lymphocyte reduction from baseline. Effects on symptoms and other endpoints suggest that further research of AMG 714 may be warranted in patients with refractory coeliac disease type 2. Funding Celimmune and Amgen.

Plasma cell neoplasms (PCNs), comprising plasma cell myelomas (PCMs) and plasmacytomas, occurring after solid organ transplantation, represent rare subtypes of monomorphic post-transplant lymphoproliferative disorders (M-PTLDs). Data regarding the clinical and pathological features of post-transplant (PT)-PCMs are limited. To gain a better understanding of disease biology, we performed comprehensive immunophenotypic analysis, reviewed cytogenetic analysis results and evaluated clinical outcomes of PT-PCMs diagnosed and treated at our institution. Fifteen PT-PCM (M: F - 4:1) and 2 PT-MGUS (2 males) cases were identified. The median age of PT-PCM patients was 68yrs (29-79 yrs) and PCMs presented at a median of 9.7yrs (0.5-24.7 yrs) after transplantation. PT-PCMs accounted for 11.6% of all M-PTLDs and the period prevalence was 9/3108 (0.29%), 3/1071 (0.28%), 2/1345 (0.15%) and 1/878 (0.11%) post kidney, heart, liver and lung transplantation. Lytic bone disease was observed in 1/11 (9%) patients. Marrow plasma cell infiltration ranged from 10%-70% (median 20%), with 10/15 (67%) and 5/15 (33%) cases manifesting immature and plasmablastic morphology. The immunophenotype of all cases and cytogenetic abnormalities, identified in 60% of cases, were similar to multiple myeloma (MM) of immunocompetent individuals. All PT-PCMs were EBER-. Ten of 11 (91%) patients with active MM were treated, all with proteasome inhibitor-based therapy. Treatment response and 5-year overall survival (54.5%) was comparable to MM of immunocompetent individuals. However, the survival of patients with plasmablastic PCMs was inferior to those with immature PCMs. 0ur findings indicate PT-PCMs to be predominantly late onset PTLDs that have similar clinicopathologic characteristics as conventional MM. This article is protected by copyright. All rights reserved.

Mastitis is one of the most frequently encountered diseases in dairy cattle, negatively affecting animal welfare and milk production. For this reason, contributions to understanding its genomic architecture are of great interest. Genome-wide association studies (GWAS) have identified multiple loci associated with somatic cell score (SCS) and mastitis in cattle. However, most of the studies have been conducted in different parts of the world on various breeds, and none of the investigations have studied the genetic architecture of mastitis in Romanian dairy cattle breeds up to this point in time. In this study, we report the first GWAS for SCS in dairy cattle breeds from Romania. For GWAS, we used an Axiom Bovine v3 SNP-chip (&gt, 63,000 Single Nucleotide Polymorphism -SNPs) and 33,330 records from 690 cows belonging to Romanian Spotted (RS) and Romanian Brown (RB) cattle. The results found one SNP significantly associated with SCS in the RS breed and 40 suggestive SNPs with −log10 (p) from 4 to 4.9 for RS and from 4 to 5.4 in RB. From these, 14 markers were located near 12 known genes (AKAP8, CLHC1, MEGF10, SATB2, GATA6, SPATA6, COL12A1, EPS8, LUZP2, RAMAC, IL12A and ANKRD55) in RB cattle, 3 markers were close to ZDHHC19, DAPK1 and MMP7 genes, while one SNP overlapped the HERC3 gene in RS cattle. Four genes (HERC3, LUZP2, AKAP8 and MEGF10) associated with SCS in this study were previously reported in different studies. The most significant SNP (rs110749552) associated with SCS was located within the HERC3 gene. In both breeds, the SNPs and position of association signals were distinct among the three parities, denoting that mastitis is controlled by different genes that are dependent according to parity. The current results contribute to an expansion in the body of knowledge regarding the proportion of genetic variability explained by SNPs for SCS in dairy cattle.

Introduction The combination of the BCL-2 inhibitor venetoclax with an HMA (HMA/Ven) has improved outcomes in previously untreated patients with AML not eligible for intensive induction therapy. In a phase Ib study, 67% of patients achieved a complete remission (CR) or CR with incomplete recovery of blood counts (CRi) with a median overall survival (OS) of 17.5 months (DiNardo CD et al. Blood 2019; 133(1):7-17). HMA/Ven has also demonstrated efficacy in a heavily pretreated population with relapsed or refractory (R/R) AML, the majority of whom had prior HMA exposure (DiNardo CD et al. Am J Hematol 2018; 93(3):401-7). Measurable residual disease (MRD) is recognized as an independent prognostic indicator important for risk stratification and treatment planning (Schuurhuis GJ et al. Blood 2018; 131(12):1275-91). To date, however, there have been few reports on the effect of HMA/Ven on MRD. Methods This is a retrospective case series of patients with AML at a single-center tertiary-care institution. Patients ≥ 18 years of age who were treated with HMA/Ven between January 2017 and June 2020, either in the upfront or salvage setting, for AML were included. Outcomes included CR/CRi rate, MRD response, relapse free survival (RFS), and OS. MRD was assessed via multicolor flow cytometry with a sensitivity of 10-3 (0.1%). Results Nineteen patients were identified, 12 (63%) of whom were female. The median age at the time of HMA/Ven initiation was 71 years (range, 21 - 87 years). Ten (53%) patients had de novo AML and 9 had secondary or therapy-related AML. By 2017 ELN criteria, 3 (16%) patients had favorable-risk, 9 (47%) had intermediate-risk, and 7 (37%) had adverse-risk AML. Nine (47%) patients had R/R AML; 5 received HMA/Ven as first salvage therapy, and 4 as 2nd or greater salvage. Three (16%) patients had prior HMA exposure. No patient had prior venetoclax exposure. Median follow-up was 9.1 months (range, 1-21.1 months). Ten (53%) patients received azacitidine and 9 (47%) were given decitabine. Venetoclax doses ranged from 50 to 400 mg daily, depending on participation in a clinical trial and concomitant medications. Eight patients achieved a CR and 7 patients achieved a CRi for a combined CR/CRi rate of 79%. The CR/CRi rate was 90% (9/10) in the upfront setting, and 66% (6/9) in the salvage setting. The median time and number of cycles to best clinical response was 2.3 months (range, 0.9-3.9 months) and 2 (range, 1-3 cycles), respectively. Eleven (73%) of the 15 responders achieved MRD clearance after a median of 2 cycles (range, 1-3 cycles) (Table 1). Two of 4 (50%) MRD-positive patients relapsed, while 4 (36%) of 11 MRD-negative patients relapsed (Figure 1). Relapse occurred at a median of 2.0 months (range, 1.3-2.7 months) in the MRD positive group and 11.0 months (range, 2.8-14 months) in the MRD negative group. One patient died of infectious complications while MRD negative. Three patients, all of whom were treated for R/R disease, proceeded to an allogeneic stem cell transplant (HSCT). Two were MRD negative at the time of HSCT and all remained in remission. At the time of data cutoff, 7 (64%) of 11 MRD-negative patients were alive, and all 4 MRD-positive patients were alive. Causes of death in the MRD-negative group included disease relapse (3 patients) and infection (1 patient). Median overall survival in the entire cohort (range, 32 days-NR) was not reached. Conclusions HMA/Ven was highly effective as both upfront and salvage therapy. Surprisingly, the salvage CR/CRi rate in this series was 66%, allowing half of the responders to proceed to HSCT. The majority (73%) of responders achieved MRD negativity. While MRD status influenced RFS, 36% of MRD-negative patients relapsed. Additionally, the same percentage of MRD-negative patients died during follow-up, versus none of the patients with MRD-positivity. This indicates the need for more sensitive methods to assess MRD and for novel therapeutic strategies to eliminate MRD, thereby improving long-term outcomes. Larger prospective studies are needed to define the role of MRD assessment with venetoclax-containing regimens. Disclosures Jurcic: AbbVie:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding;Celgene:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding;Syros Pharmaceuticals:Research Funding;PTC Therapeutics:Research Funding;Arog Pharmaceuticals:Research Funding;Kura Oncology:Research Funding;Forma Therapeutics:Research Funding;Astellas:Research Funding;Genentech:Research Funding;Novartis:Consultancy, Membership on an entity's Board of Directors or advisory committees;Daiichi-Sankyo:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding;BMS:Consultancy, Research Funding.Campbell:AstraZeneca:Consultancy.Lee:Genentech:Research Funding;Sumitomo Dainippon Pharma Oncology, Inc.:Research Funding;AbbVie:Research Funding;Novartis:Research Funding;Bayer:Research Funding;Celgene:Consultancy;Forty Seven:Research Funding.Heaney:Blueprint Medicines Corporation:Research Funding;BMS:Research Funding;CTI Biopharma:Consultancy, Research Funding;Deciphera:Research Funding;Incyte:Research Funding;Novartis:Consultancy, Research Funding;Sierra Oncology:Research Funding;AbbVie:Consultancy;Partner Therapeutics:Consultancy.Lamanna:Janssen:Consultancy, Membership on an entity's Board of Directors or advisory committees;Octapharma:Research Funding;Juno:Other: Institutional research grants, Research Funding;Gilead:Consultancy, Membership on an entity's Board of Directors or advisory committees;Astra Zeneca:Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional research grants, Research Funding;Pharmacyclics: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, Other: Institutional research grants, Research Funding;Bei-Gene:Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional research grants, Research Funding;Abbvie:Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional research grants, Research Funding;Oncternal, Verastem, TG Therapeutics:Other: Institutional research grants, Research Funding;MingSight:Other: Institutional research grants, Research Funding;Loxo:Research Funding;Celgene:Consultancy, Membership on an entity's Board of Directors or advisory committees;Columbia University Medical Center:Current Employment.

ObjectivesWe investigated the ability of routine flow cytometry (FC) to detect nonhematologic neoplasms (non-HN) using antibody panels routinely used for the diagnosis of hematologic neoplasms.MethodsFC analyses of 4,000 various diagnostic samples were retrospectively reviewed to identify cases in which an aberrant, viable CD45-negative, nonhematologic neoplastic population was detected by FC panels designed to evaluate hematologic neoplasms.ResultsA total of 57 (1.4%) diverse non-HNs were identified, representing neuroendocrine tumors (33/57) and carcinomas (9/57), as well as other malignancies (15/57) such as sarcoma and melanoma. The majority of neoplasms were positive for at least one antibody, typically CD56 (43/51, 84.3%), followed by CD117 (15/34, 44.1%) and CD138 (6/33, 18.2%).ConclusionsOur findings highlight the importance of carefully inspecting CD45-negative events to identify non-HNs by routine FC analysis. This can help expedite further downstream immunophenotypic analysis of specimens and triage samples for appropriate genetic and molecular studies.

Refractory celiac disease type II (RCD II), also referred to as "cryptic" enteropathy-associated T-cell lymphoma (EATL) or "intraepithelial T-cell lymphoma," is a rare clonal lymphoproliferative disorder that arises from innate intraepithelial lymphocytes. RCD II has a poor prognosis and frequently evolves to EATL. The pathogenesis of RCD II is not well understood and data regarding the immunophenotypic spectrum of this disease and underlying genetic alterations are limited. To gain further biological insights, we performed comprehensive immunophenotypic, targeted next-generation sequencing, and chromosome microarray analyses of 11 RCD II cases: CD4-/CD8- (n=6), CD8+ (n=4), and CD4+ (n=1), and 2 of 3 ensuing EATLs. Genetic alterations were identified in 9/11 (82%) of the RCD II cases. All 9 displayed mutations in members of the JAK-STAT signaling pathway, including frequent, recurrent STAT3 (7/9, 78%) and JAK1 (4/9, 44%) mutations, and 9/10 evaluable cases expressed phospho-STAT3. The mutated cases also harbored recurrent alterations in epigenetic regulators (TET2, n=5 and KMT2D, n=5), nuclear factor-κB (TNFAIP3, n=4), DNA damage repair (POT1, n=3), and immune evasion (CD58, n=2) pathway genes. The CD4-/CD8- and other immunophenotypic subtypes of RCD II exhibited similar molecular features. Longitudinal genetic analyses of 4 RCD II cases revealed stable mutation profiles, however, additional mutations were detected in the EATLs, which occurred at extraintestinal sites and were clonally related to antecedent RCD II. Chromosome microarray analysis demonstrated copy number changes in 3/6 RCD II cases, and 1 transformed EATL with sufficient neoplastic burden for informative analysis. Our findings provide novel information about the immunophenotypic and genomic characteristics of RCD II, elucidate early genetic events in EATL pathogenesis, and reveal potential therapeutic targets.

Detection of hallmark genomic aberrations in acute myeloid leukemia (AML) is essential for prognosis and patient management. Clinical practice guidelines for identifying such structural variants (SVs), established by the World Health Organization (WHO), European Leukemia Net (ELN) and National Comprehensive Cancer Network (NCCN), rely substantially on cytogenetic/cytogenomic techniques such as karyotyping, fluorescence in situ hybridization (FISH) or chromosomal microarray analysis (CMA). However, these techniques are limited by the need for skilled personnel as well as significant time and labor, making them cost-prohibitive for some patients. Optical genome mapping (OGM) addresses these limitations and allows for the accurate identification of clinically significant SVs using a novel, high throughput, inexpensive methodology. In a single assay, OGM offers a significantly higher resolution than karyotyping with comprehensive genome-wide analysis comparable to CMA and the added unique ability to detect balanced SVs that are missed by microarray. Here, we report the performance of OGM in a cohort of 100 AML cases, which were previously characterized by karyotype alone or karyotype and FISH. CMA was performed as an additional test in some cases. OGM identified all the clinically relevant SVs and CNVs reported by these standard cytogenetic methods. Moreover, OGM identified clinically relevant SVs in 11% of cases that had been missed by the routine methods. In 24% of cases, OGM refined the underlying genomic structure reported by traditional cytogenomic testing (13%), identified additional clinically relevant variants (7%) or both (4%). Three of 48 (6.25%) cases reported with normal karyotypes were shown to have cryptic translocations involving gene fusions. Two of these cases included fusion between NSD1-NUP98. Based on the comprehensive genomic profiling of the AML patients in this multi-institutional study, we recommend that OGM be considered as a first-line test for detection and identification of clinically relevant SVs.

Multiple sclerosis (MS) is a chronic autoimmune disease of the CNS that is characterized by demyelination, axonal loss, gliosis, and inflammation. The murine model of MS is the experimental autoimmune encephalopathy (EAE) induced by immunization of mice with myelin oligodendrocyte glycoprotein (MOG)35–55. Ig-like transcript 3 (ILT3) is an inhibitory cell surface receptor expressed by tolerogenic human dendritic cells. In this study, we show that the recombinant human ILT3.Fc protein binds to murine immune cells and inhibits the release of proinflammatory cytokines that cause the neuroinflammatory process that result in paralysis. Administration of ILT3.Fc prevents the rapid evolution of the disease in C57BL/6 mice and is associated with a profound reduction of proliferation of MOG35–55–specific Th1 and Th17 cells. Inhibition of IFN-γ and IL-17A in mice treated with ILT3.Fc is associated with delayed time of onset of the disease and its evolution to a peak clinical score. Neuropathological analysis shows a reduction in inflammatory infiltrates and demyelinated areas in the brains and spinal cords of treated mice. These results indicate that inhibition of Th1 and Th17 development provides effective suppression of EAE and suggests the feasibility of a clinical approach based on the use of ILT3.Fc for treatment of MS. Furthermore, our results open the way to further studies on the effect of the human ILT3.Fc protein in murine experimental models of autoimmunity and cancer.

Introduction: Natural Killer cells (NK) play an important role in tumor immune-surveillance. The innate cell engager AFM13 is a CD30/CD16A targeting high affinity bispecific tetravalent antibody that engages and activates NK. This study evaluates AFM13's clinical and immunological activity. It examines the immunologic changes in the tumor and in peripheral blood (PB) as a function of the dose and method of administration of AFM13. Methods: Subjects with relapsed or refractory CD30 expressing lymphoma with cutaneous involvement were recruited into 4 cohorts: 1.5 mg/kg IV weekly, 7 mg/kg IV weekly, 7 mg/kg continuous intravenous infusion (CIVI) over 5 days weekly and 200mg IV weekly. Each cohort consisted of 3 subjects with 3 additional subjects in cohort 4. Subjects received 8 weeks of therapy for up to 2 cycles. Response assessment was performed on week 11 of each cycle by mSWAT, photography, PET imaging and PB flow cytometry. A second cycle was administered if there was no progression. Subjects underwent skin biopsies and PB immunologic studies as follows: pretreatment, day 5, week 4 and week 8 of therapy. Biopsies were analyzed and evaluated by a pathologist and IHC image analyzer. PB samples were analyzed by flow cytometry. Results: Fourteen subjects were accrued. The median number of prior therapies was 4 (1-11) and 4 subjects had progressed on brentuximab vedotin. The disease etiologies, treatment emergent toxicities and response by cohort are presented in Table 1. An objective response rate of 40% was observed in the study. In the PB, flow cytometry revealed a decrease in circulating NK (CD16/56+ CD3- CD19- lymphocytes) during therapy with post therapy recovery. The activation marker CD69 on NK increased in responders (R) compared to non-responders (NR). Similarly, tumor biopsies in R showed increased infiltration and activation of CD56+ NK as opposed to NR (Figure 1). NK cytotoxicity through the expression of Granzyme B was seen in R vs. NR. Flow quantitation of circulating CD4+ CD25+ T cells (Tregs) showed a decrease in R vs. NR. Conclusions: AFM13 demonstrated a high ORR of 40% among a population of heavily pretreated subjects with CD30 positive lymphoproliferative T-cell malignancies. AFM13 exhibited activity post brentuximab vedotin failure. In addition, biological changes in NK infiltration and activation in the PB and tissue biopsy correlated with response. These data are the first to demonstrate that innate cell engagers modulate NK and T cell populations in the peripheral blood and tumor from patients and to determine that such changes are associated with patient benefit. Disclosures Sawas: Roche: Current equity holder in publicly-traded company; Affimed: Research Funding; Daiichi Sankyo: Speakers Bureau; Seattle Genetics: Speakers Bureau; Gilead: Speakers Bureau; Flatiron Health: Current Employment. Chen:Affimed: Research Funding. Lipschitz:Affimed: Research Funding. Rodig:Bristol Myers Squibb: Research Funding; Merck: Research Funding; Affimed: Research Funding; KITE/Gilead: Research Funding; Immunitas: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

A patient diagnosed with Chronic Lymphocytic Leukemia (CLL) received HSC transplantation, and displayed stable donor engraftment. Three years after transplantation, the patient experienced a relapse in the form of Diffuse Large B cell Lymphoma (DLBCL). Total blood, bone marrow, lymph node (LN) and lineage specific Chimerism measurements were performed using AmpFlSTR+ (Applied Biosystems) kit. The patient showed full myeloid engraftment and mixed T and B Chimerism immediately after transplant. Within 6–12 weeks all lineages studied (T, B, NK, myeloid and erythroid) were fully donor (99–100%), which remained stable for over 3 years. A miniscule drop in the B cell lineage Chimerism was noted (97.3%) 42 months after transplantation. Concurrent clinical status (lymphadenopathy, abdominal pain) suggested relapse. Histopathology and flow cytometry studies showed that the patient was experiencing Richter’s transformation (RT) to DLBCL. Molecular oncology testing demonstrated that the DLBCL IgH rearrangement was identical to the original CLL disease. Leftover LN tissue which had been submitted for flow cytometry studies and demonstrated the presence of immunophenotypically aberrant monotypic B cells (>50% of cellularity) was processed for Chimerism and showed 21% donor cells. RT is defined as transformation from CLL into a more aggressive lymphoma, typically DLBCL, with a reported incidence ranging from 1 to 9%. The median time to RT is 1.9 years after diagnosis. Importantly, RT changes the nature of the hematological neoplasm from leukemia, residing mostly in the blood and bone marrow, to lymphoma, a disease which mainly affects secondary lymphoid organs. Therefore, this patient displayed complete or near complete donor engraftment in the peripheral blood (including in lymphoid lineages) by routine Chimerism testing. The reemergence of neoplastic cells of recipient origin was clearly evident when the test was applied to an affected lymph node. In conclusion RT from leukemia to lymphoma may represent a significant challenge to monitoring disease relapse by Chimerism.

One of the challenges facing the increased usage of EVs is the possibility to add energy to them, be it in the form of gas (hydrogen) or electricity. The standard is set by classic petrol and diesel fueling system. Motor vehicles powered by hydrogen have currently quite a low market penetration. However, hydrogen refueling infrastructure is starting to build-up in order to make larger-scale hydrogen-powered motor vehicle deployment possible. The European Directive on the deployment of alternative fuels infrastructure requires states to create and submit national policy frameworks in order to develop the widespread use of alternative fuels and the required infrastructure, charging points for Electric Vehicles (EVs) and hydrogen refueling points. In present the fueling hydrogen stations for FCEV is separate of recharging points for EVs as BEV and PHEV. The paper deals the dual electricity - hydrogen charge station in interaction with smart grid and renewable energy sources all controlled by Energy Management System (EMS). The refueling stations have options for all-EVs: as BEV and PHEV or FCEVs. The EMS has the ability to control the loading and unloading of a hydrogen storage system and electricity production by fuel cell, managing the energy flow in order to maintain grid stability and could be solved if charging is used on times of low electricity demand and/or high supply. The principal elements of the charging station are the EMS and controller, photovoltaic setting with Maximum Power Point Tracking (MPPT), energy storage unit in DC link, two DC/DC isolated for elecrolyzer and fuel cell, hydrogen storage and bidirectional inverter to grid connection. Because the refueling stations for FCEV and the recharging points for EVs are regulated by the international standards the novelty of this paper is use and connection of the elecrolyzer and the fuel cells of types Proton Exchange Membrane (PEM) for configuration of the EMS and controller.

In the context of kidney transplantation, genomic incompatibilities between donor and recipient may lead to allosensitization against new antigens. We hypothesized that recessive inheritance of gene-disrupting variants may represent a risk factor for allograft rejection.We performed a two-stage genetic association study of kidney allograft rejection. In the first stage, we performed a recessive association screen of 50 common gene-intersecting deletion polymorphisms in a cohort of kidney transplant recipients. In the second stage, we replicated our findings in three independent cohorts of donor-recipient pairs. We defined genomic collision as a specific donor-recipient genotype combination in which a recipient who was homozygous for a gene-intersecting deletion received a transplant from a nonhomozygous donor. Identification of alloantibodies was performed with the use of protein arrays, enzyme-linked immunosorbent assays, and Western blot analyses.In the discovery cohort, which included 705 recipients, we found a significant association with allograft rejection at theWe found that the

Flow Cytometry is a high-throughput cell analysis method has generated heavy reliance on it in the biomedical field, especially in Immunology and Hematopathology. Flow Cytometry has a wide variety of applications [1]; the present chapter will focus on various Flow Cytometry errors relating to its main clinical missions of malignant and non-malignant immunophenotyping, cell enumeration and flow crossmatching. Cell sorting, DNA assays, image stream, mass cytometry, microbial flow, Ca++ flux and other exciting or exotic applications are somewhat beyond the routine clinical applications of Flow and the scope of this chapter. Technical as well as human errors that may affect results are also addressed.

Background In the context of kidney transplantation, genomic incompatibilities between donor and recipient may lead to allosensitization against new antigens. We hypothesized that recessive inheritance of gene-disrupting variants may represent a risk factor for allograft rejection. Methods We performed a two-stage genetic association study of kidney allograft rejection. In the first stage, we performed a recessive association screen of 50 common gene-intersecting deletion polymorphisms in a cohort of kidney transplant recipients. In the second stage, we replicated our findings in three independent cohorts of donor-recipient pairs. We defined genomic collision as a specific donor-recipient genotype combination in which a recipient who was homozygous for a gene-intersecting deletion received a transplant from a nonhomozygous donor. Identification of alloantibodies was performed with the use of protein arrays, enzyme-linked immunosorbent assays, and Western blot analyses. Results In the discovery cohort, which included 705 recipients, we found a significant association with allograft rejection at the LIMS1 locus represented by rs893403 (hazard ratio with the risk genotype vs. nonrisk genotypes, 1.84; 95% confidence interval [CI], 1.35 to 2.50; P=9.8x10(-5)). This effect was replicated under the genomic-collision model in three independent cohorts involving a total of 2004 donor-recipient pairs (hazard ratio, 1.55; 95% CI, 1.25 to 1.93; P=6.5x10(-5)). In the combined analysis (discovery cohort plus replication cohorts), the risk genotype was associated with a higher risk of rejection than the nonrisk genotype (hazard ratio, 1.63; 95% CI, 1.37 to 1.95; P=4.7x10(-8)). We identified a specific antibody response against LIMS1, a kidney-expressed protein encoded within the collision locus. The response involved predominantly IgG2 and IgG3 antibody subclasses. Conclusions We found that the LIMS1 locus appeared to encode a minor histocompatibility antigen. Genomic collision at this locus was associated with rejection of the kidney allograft and with production of anti-LIMS1 IgG2 and IgG3.

This paper presents the energy savings in refurbishment of the buildings using Life Cycle Assessment at material level by comparing three insulation materials that offer higher thermal performance and greater environmental sustainability. The comparison is made on products having the same insulation performance (U-value). After the enumeration of intervention practices for energy saving in buildings, the Life Cycle Assessment methodology in refurbishment of the buildings is applied at material level using Life Cycle Inventory to transform material volume input into impact on the environment as output. The Life Cycle Assessment (LCA) was carried out according to the requirements of the Environmental Product Declaration (EPD). After assessing the building performance before and after isolation, the “embodied energy”, which comes from the materials manufacturing as phases of the refurbishment of the buildings, was determinate and compared with the operational energy of the building. This LCA methodology in refurbishment of the buildings at material level was evaluated for Expanded Polystyrene (EPS), Extruded Polystyrene (XPS) and Rigid polyurethane (PUR) insulation products. This methodology has been used on a building in Galati, Romania (School No. 9). A spider diagram was used to represent the criteria in a consistent, graphical way in relative units. The extreme values for a given criteria indicate the poor performance of the insulation material. Finally, the assessment of the building performance before and after using the insulation and the determination of the “embodied energy” compared to the operational energy of the building resulted in a 55% energy saving in the use stage of the building after refurbishment.

Just like other parts of Eurasia,* 1 the South Caucasus is facing a new breed of East-West geopolitical competition interwoven with three evolving challenges:2 l)a growing ideological gap between Russia and the West; 2) the chronic persistence of protracted conflicts; 3) the dilemma of the post-Soviet states: European vs. Eurasian integration.More specifically, the South Caucasus geopolitical landscape is shaped by:1. Geopolitical competition between Russia and the West in the wake of the ongoing Ukrainian crisis, which effectively brought the era of European cooperative security to an end.2. Growing Russian regional assertiveness, whereby the Eurasian Economic Union (EEU) is increasingly used as a vehicle to counter strides towards European integration, while OSCE-led conflict resolution is manipulated to create geopolitical leverage over the regional states.3. A tacit Russian-Turkish partnership of convenience, basically motivated by both parties' focus on different fronts: Russia is engaged in the geopolitical confrontation with the West over Ukraine, while Turkey has been absorbed by the fluid evolutions in the Middle East (particularly in Syria and Iraq).4. The inability of the EU to exert, or at least claim, a bolder regional role because of its own institutional constraints and lack of appetite for new CSDP missions in the aftermath of the Euro crisis.5. NATO's self-restrained regional role limited to soft security cooperation in the "28+1 " format, driven by its refocus on deterrence and defense of the territory of its Eastern members against a resurgent Russia and the fact that the region is less of a strategic priority in the wake of unwinding the ISAF operation in Afghanistan.Overall, the geopolitical competition between Russia and the West over Ukraine may have a negative impact on the South Caucasus: it may either freeze the current status quo, or it may push it into the whirlwind of instability around Ukraine. Two factors seem decisive for this analysis:1. Russian progress in ensuring geopolitical control of Ukraine may rather tend to support the first option. Otherwise, faced with a stalemate in Ukraine, Moscow might have to deal with a strategic dilemma: either expand its confrontation with the West in the South Caucasus or reinforce the status quo to avoid annoying Turkey and prevent it from taking action.2. Turkish tacit acceptance of Russian incursions in Ukraine may also favor the status quo in the South Caucasus, while Ankara's brazen reaction-via NATO or directly-may dramatically raise the risk of instability in the South Caucasus.Against this complex and deeply worrying regional background, where Russia and Turkey (re)emerge as the dominant regional powers, what strategic policy changes might Western decision makers envisage consolidating their position as a viable South Caucasus player? From a methodological perspective, I address these questions through the lens of the evolving challenges in Eurasia.Unresolved European Security Issues LingerThe geopolitical competition between Russia and the West became predictable after President Vladimir Putin stated in April 2005: "Above all, we should acknowledge that the collapse of the Soviet Union was a major geopolitical disaster of the century. As for the Russian nation, it became a genuine drama. Tens of millions of our co-citizens and compatriots found themselves outside Russian territory."3 The seeds of the new confrontation were planted into those words, while alluding to both the goal and the strategy of the new Russian resurgence.However, this statement came after two rounds of NATO enlargement (1997 and 2004) and after the Big Bang enlargement of the European Union (2004). Moreover, it came after the Rose and Orange revolutions in Georgia and Ukraine, respectively, and brought pro-Western leaders into top state positions seeking NATO and EU membership for their countries. …

The blockade of immune checkpoints by anti-receptor and/or anti-ligand mAb is one of the most promising approaches to cancer immunotherapy. The interaction between Ig-like transcript 3 (ILT3), a marker of tolerogenic dendritic cells, also known as LILRB4/LIR5/CD85k, and its still unidentified ligand on the surface of activated human T cells is potentially important for immune checkpoint blockade. To identify the ILT3 ligand, we generated mAb by immunizing mice with Jurkat acute T cell leukemia, which binds ILT3.Fc to its membrane. Flow cytometry, mass spectrometry, and Biacore studies demonstrated that the ILT3 ligand is a CD166/activated leukocyte cell adhesion molecule. Knockdown of CD166 in primary human T cells by nucleofection abolished the capacity of ILT3.Fc to inhibit CD4+ Th cell proliferation and to induce the generation of CD8+CD28− T suppressor cells. CD166 displays strong heterophilic interaction with CD6 and weaker homophilic CD166–CD166 cell adhesion interaction. ILT3.Fc inhibited the growth of CD166+ tumor cell lines (TCL) derived from lymphoid malignancies in vitro and in vivo. CRISPR-Cas9–based knockout of CD166 from TCL abrogated ILT3.Fc binding and its tumor-inhibitory effect. The mechanism underlying the effect of ILT3.Fc on tumor cell growth involves inhibition of the p70S6K signaling pathway. Blockade of CD166 by ILT3.Fc inhibited progression of human TCL in NOD.Cg-Prkdc Il-2rg/SzJ mice, suggesting its potential immunotherapeutic value.

Background Pre-transplant sensitization is a limiting factor in solid-organ transplantation. In heart transplants, ventricular assist device (VAD) implantation has been associated with sensitization to human leukocyte antigens (HLA). The effect of VAD on non-HLA antibodies is unclear. We have previously shown that polyreactive natural antibodies (Nabs) contribute to pre-sensitization in kidney allograft recipients. Here we assessed generation of Nabs after VAD implantation in pre-transplant sera and examined their contribution to cardiac allograft outcome. Methods IgM and IgG Nabs were tested in pre-transplant serum samples collected from 206 orthotopic heart transplant recipients, including 128 patients with VAD (VAD patients) and 78 patients without VAD (no-VAD patients). Nabs were assessed by testing serum reactivity to apoptotic cells by flow cytometry and to the generic oxidized epitope, malondialdehyde, by enzyme-linked immunosorbent assay. Results No difference was observed in serum levels of IgM Nabs between VAD and no-VAD patients. However, serum IgG Nabs levels were significantly increased in VAD compared with no-VAD patients. This increase was likely due to the presence of the VAD, as revealed by lower serum IgG Nabs levels before implantation. Elevated pre-transplant IgG Nabs level was associated with development of primary graft dysfunction (PGD). Conclusions Our study demonstrates that VAD support elicits IgG Nabs reactive to apoptotic cells and oxidized epitopes. These findings further support broad and non-specific B-cell activation by VAD, resulting in IgG sensitization. Moreover, the association of serum IgG Nabs levels with development of PGD suggests a possible role for these antibodies in the inflammatory reaction accompanying this complication.

Immune activation needs to be tightly regulated to control immune-mediated tissue damage. Inhibitory pathways serve to terminate an immune response and resolve inflammation. Persistent exposure to antigens can drive development of adaptive regulatory cells. Similarly exposure of activated T cells to the recombinant ILT3-Fc molecule during priming triggers the differentiation of CD8 T suppressor cells and the induction of CD4 T helper anergy. Ts express high levels of immunoregulatory signature genes together with low levels of microRNA which control their function. Analysis of microRNA contained by exosomes from cultures in which T cells were alloactivated in the presence or absence of ILT3.Fc, demonstrated that this agent inhibits the release of inflammatory microRNA. The source of such inflammatory microRNA was found to reside in alloactivated CD4 T cells, since exosomes from MLC primed CD4 T cells were shown to diminish the suppressive activity of ILT3-Fc-induced CD8(+) Ts at high effector to suppressor T cell ratios. This indicates that inflammatory exosomes can swing the balance between effector and regulatory T cells in favor of immunity. These data suggest that isolation and characterization of micro-RNA containing exosomes in patients' circulation may be of use for treatment, prevention and monitoring of immune activation.

Presensitization against a broad array of HLA is associated with prolonged waiting times and inferior kidney allogaft survival. Although the use of solid phase assay (SPA) for the detection and characterization of anti-HLA antibodies provides greater sensitivity than complement-dependent lymphocytotoxicity (CDC) assay, it often detects donor specific antibodies (DSA) which turn out to be clinically irrelevant. Our data reinforce the concept that these two types of assays should be used in parallel for pre-and post-transplantation monitoring of anti-HLA antibodies in recipients of solid organ allografts.

Alloantigen specific CD8 T suppressor cells can be generated in vitro either by multiple stimulations of CD3 T cells with allogeneic APC or by single stimulation in primary MLC containing recombinant ILT3.Fc protein. The aim of the present study was to determine whether multiple MLC stimulation induced in CD8(+) CD28(-) T suppressor cells molecular changes that are similar to those observed in CD8 T suppressor cells from primary MLC containing ILT3.Fc protein. Our study demonstrates that the characteristic signatures of CD8 T suppressor cells, generated by either of these methods are the same consisting of up-regulation of the BCL6 transcriptional repressor and down-regulation of inflammatory microRNAs, miR-21, miR-30b, miR-146a, and miR-155 expression. In conclusion microRNAs which are increased under inflammatory conditions in activated CD4 and CD8 T cells with helper or cytotoxic function show low levels of expression in CD8 T cells which have acquired antigen-specific suppressor activity.

Bidirectional interactions between dendritic cells (DC) and Ag-experienced T cells initiate either a tolerogenic or immunogenic pathway. The outcome of these interactions is of crucial importance in malignancy, transplantation, and autoimmune diseases. Blockade of co-stimulation results in the induction of T helper cell anergy and subsequent differentiation of antigen specific CD8+ T suppressor/regulatory cells (Ts). Ts. Primed in the presence of inhibitory signals exert their inhibitory function in an antigen specific manner, a feature with tremendous clinical potential. In transplantation or autoimmunity, antigen specific Ts can enforce tolerance to auto- or alloantigens, while otherwise leaving the immune response to pathogens uninhibited. Alternatively, blockade of inhibitory receptors results in the generation of cytolytic CD8+ T cells (CTL), which is vital towards defense against tumors and viral diseases. Because CD8 T cells are MHC Class I restricted, they are able to recognize HLA-bound antigenic peptides presented not only by APC, but also on parenchymal cells, thus eliciting or suppressing auto- or allo- immune reactions.

Antigen-specific CD8 suppressor T cells (CD8 + Ts) are adaptive regulatory T cells that are induced in vivo and in vitro by chronic antigenic stimulation of human T cells. CD8 + Ts induce the upregulation of the inhibitory receptors ILT3 and ILT4 on monocytes and dendritic cells rendering these antigen presenting cells (APCs) tolerogenic. Tolerogenic APCs induce CD4 + T helper anergy and elicit the differentiation of CD4 + and CD8 + T regulatory/suppressor cells. Overexpression of membrane ILT3 in APC results in inhibition of NF-κB activation, transcription of inflammatory cytokines and costimulatory molecules. Soluble ILT3-Fc which contains only the extracellular, Ig-like domain linked to mutated IgG1 Fc, is strongly immunosuppressive. ILT3-Fc, induces the differentiation of human CD8 + Ts which inhibit CD4 + Th and CD8 + CTL effector function both in vitro and in vivo. The acquisition of Ts' function by primed CD8 + T cells treated with ILT3-Fc was demonstrated to be the effect of the significant upregulation of BCL6, a transcriptional repressor of IL-2, IFN-gamma, IL-5 and granzyme B. The upregulated expression of BCL6, SOCS1 and DUSP10 is integral to the signature of ILT3-Fc-induced CD8 + Ts. These genes are known inhibitors of cytokine production and TCR signaling and are targeted by miRNAs which are suppressed by ILT3-Fc. ILT3-Fc induces tolerance to allogeneic human islets and reverses rejection after its onset in a humanized NOD/SCID mouse model. Based on these findings we postulate that ILT3-Fc may become an important new agent for treatment of autoimmunity and transplant rejection.

We have investigated the mechanism underlying the immunoregulatory function of membrane Ig-like transcript 3 (ILT3) and soluble ILT3Fc. microRNA (miRNA) expression profile identified genes that were downregulated in ILT3-induced human CD8+ T suppressor cells (Ts) while upregulated in T cells primed in the absence of ILT3. We found that miR-21, miR-30b, and miR-155 target the 3′-untranslated region of genes whose expression was strongly increased in ILT3Fc-induced Ts, such as dual specificity phosphatase 10, B cell CLL/lymphoma 6, and suppressor of cytokine signaling 1, respectively. Transfection of miRNA mimics or inhibitors and site-specific mutagenesis of their 3′-untranslated region binding sites indicated that B cell CLL/lymphoma 6, dual specificity phosphatase 10, and suppressor of cytokine signaling 1 are direct targets of miR-30b, miR-21, and miR-155. Primed CD8+ T cells transfected with miR-21&30b, miR-21&155, or miR-21&30b&155 inhibitors displayed suppressor activity when added to autologous CD3-triggered CD4 T cells. Luciferase reporter assays of miR-21 and miR-155 indicated that their transcription is highly dependent on AP-1. Analysis of activated T cells showed that ILT3Fc inhibited the translocation to the nucleus of the AP-1 subunits, FOSB and c-FOS, and the phosphorylation of ZAP70 and phospholipase C-γ 1. In conclusion, ILT3Fc inhibits T cell activation and induces the generation of Ts targeting multiple inflammatory miRNA pathways.

We have analyzed the impact of anti-HLA antibodies present in the patients' circulation prior and/or following heart transplantation in a population of 108 pediatric recipients. Anti-HLA class I and class II antibodies were monitored by traditional CDC using donor and panel T and B lymphocytes and by SPA for detection of DSA. There was a highly significant correlation between the development of AMR and presence of CDC- or SPA-detected DSA. However, the fraction of the transplant population which remained AMR-free was much higher among patients with SPA-detected compared to CDC-detected DSA. Furthermore, long-term graft survival was negatively affected only by cytotoxic, complement-fixing anti-HLA class I antibodies developing following transplantation. Anti-HLA class I or class II antibodies detected by SPA had no effect on long-term survival rates.