Mean Fluorescence Intensity Ratio Research Articles

Midostaurin Reduces Bite® Mediated Cytotoxicity Against Acute Myeloid Leukemia

T-cell recruiting antibody constructs represent a promising approach to recruit T-cells to tumor cells independent of TCR-specificity. So far, CD33 has been the most commonly targeted antigen in AML. However, its ubiquitous expression pattern within the healthy myeloid hematopoietic system remains a major challenge. To reduce on-target off-leukemia toxicity, alternative AML-associated targets with a more restricted expression pattern are sought after. We evaluated CD135 (FLT3 ; FSM-like tyrosine kinase 3) as a target antigen for T-cell recruiting antibody based immunotherapy in AML. In 159/192 of AML patient samples (83%; MFI ratio of >1.5) a significant expression level was detected by flow cytometry with no differences in expression level at time of initial diagnosis versus relapse (Initial diagnosis: n=161, median MFI ratio 3.0 vs relapse: n=18, median MFI ratio 3.5). Expression level could neither be correlated to FLT3 mutational status (median MFI ratio FLT3-ITD vs FLT3 wild type (WT): 3.7 vs 4.0, n=52 and 173 respectively) nor to FLT3 allelic ratio (median MFI ratio allelic ratio high vs low: 4.1 vs 4.5, n=13 and 19 respectively). Importantly, CD34+/CD38- leukemia initiating cells (LICs) also expressed CD135 at a similar level compared to AML bulk cells (Median MFI ratio LIC: 2.4 vs bulk: 2.6, p=0.1, n=81). Bone marrow progenitor cells (CD45DIM/SSCLOW) cells and CD34+/CD38- expressing hematopoietic precursors from healthy bone marrow expressed CD135 to a significantly lower level compared to AML bulk cells and LICs, respectively (median MFI Ratio: AML bulk vs healthy progenitor cells: 3.2 vs 2.1, p<0.0001; LICs vs HSCs: 3.1 vs 0.7, p=0.0003). Next, we evaluated a CD135xCD3 BiTE® antibody construct (FLT3 BiTE®) for its ability to mediate cytotoxicity against AML cell lines and primary AML cells. Target antigen expression level was relevant for FLT3 BiTE® mediated cytotoxicity as demonstrated by preferential lysis of CD135bright (MM6, MFI ratio 15) vs CD135dim (OCI-AML3, MFI ratio 1.5) AML cell lines. Cytotoxicity against primary AML cells was tested in our long-term culture system using either allogeneic or autologous T cells (Krupka et al, Blood 2014). In the allogeneic set-up, with a fixed E:T ratio of 1:5, the FLT3 BiTE® antibody construct mediated efficient elimination of primary AML cells within 9 days of culture (median % specific lysis 75.9, n=6). This was accompanied by a strong T-cell proliferation (fold change CD2+ T cells: 7.9, n=6). In the autologous system, the E:T ratio was not fixed and influenced by the number of residual T-cells in the respective AML sample. Despite low E:T ratios (1:12-1:74) the FLT3 BiTE® induced high cytotoxicity and T-cell proliferation against primary AML cells within 14 days of culture (median specific lysis: 34.4%; fold change CD2 T cells: 6.6, n=7). We further investigated the influence of Midostaurin on FLT3 BiTE® mediated cytotoxicity against AML cells. Midostaurin (1 µM) drastically decreased FLT3 BiTE® mediated cytotoxicity in 7/8 patient samples (median % cytotoxicity FLT3 BiTE® vs +Midostaurin: 59.0 vs 39.9, p=0.02). This corresponded to a significant decrease in BiTE®- as well as CD3/CD28-mediated T-cell proliferation (median % CD3/CD28 mediated T-cell proliferation FLT3 BiTE® vs +Midostaurin: 56.4 vs 1.46, n=5). It has been reported that Midostaurin inhibits Akt phosphorylation (Kawai, J Bio Sci 2015), a major factor in T-cell proliferation, which might explain the decrease in T-cell proliferation and cytotoxicity. In summary, CD135 was shown to be expressed in the majority of AML patients on AML bulk cells as well as LICs with a restricted expression pattern in the healthy hematopoietic compartment. The FLT3 BiTE® antibody construct mediated strong cytotoxicity against primary AML cells. This correlated to profound T-cell activation and proliferation. The combinatorial approach with Midostaurin abrogated BiTE® mediated cytotoxicity due to a decrease in T-cell proliferation. As T-cell recruiting antibody construct mediated cytotoxicity relies on T-cell function, our data do not support the combinatorial approach with the FLT3 -TKI Midostaurin for targeted therapy of AML. DisclosuresLindl:AMGEN Inc: Research Funding. Krupka:Amgen, Inc: Research Funding; Amgen Research (Munich): Research Funding. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership. Kischel:AMGEN: Employment. Kufer:AMGEN Research Munich: Employment. Subklewe:Amgen, Inc.: Research Funding; Amgen Research (Munich): Research Funding.

CD123 Expression Patterns and Potential of IMGN632, a CD123-Targeted Antibody Drug Conjugate, in Acute Lymphoblastic Leukemia

Background: The interleukin (IL)-3 receptor α-chain, CD123, is the primary low-affinity subunit of the IL-3 receptor. CD123 is expressed at various levels in hematologic malignancies and has thus become an attractive target of therapy. While CD123 expression in precursor B-lymphoblastic leukemia/lymphoma (B-ALL) has been reported, only limited data in T-ALL exists. Furthermore, the potential of CD123-targeted therapies in ALL remains largely unexplored. In this study, we examine CD123 expression levels in a large cohort of ALL patients and assess the impact of IMGN632, a conjugate of a CD123-binding antibody with the novel DNA-alkylating IGN payload DGN549, on in vitro viability of patient ALL cells and cell lines.Methods: The study cohort (n=188) included 162 B-ALL and 26 T-ALL patients diagnosed and treated at the University of Texas MD Anderson Cancer Center between 12/2012 and 1/2017. Among B-ALL patients, 59 (36%) were Philadelphia chromosome-positive (Ph+). T-ALL patients were further subtyped into the following immunophenotypic categories: early, early T precursor (ETP), thymic, and mature. CD123 expression was evaluated using multiparameter flow cytometry (MFC). Blasts were defined by lineage markers as well as CD45dim blast gate, and CD123 expression was measured for the percentage of positivity and mean fluorescence intensity (MFI) ratio over controls. An arbitrary cutoff of 20% was used for expressing nominal MFC results. Median follow up duration was 21.1 months. CD123 expression was correlated with clinical, laboratory, and outcomes results. The number of binding sites for IMGN632 on ALL blast and cell lines was quantified by QuantiBRITE method. Cytotoxicity of IMGN632 was evaluated after continuous exposure up to five days by WST-8 assays on cell lines or by MFC on primary ALL patient samples.Results: Patients had a median age of 46 years (range, 16-88 years) and included 117 (62%) men. The median CD123 positivity on ALL blasts was 78% (range, 0.3-100) with a median MFI ratio of 11 (range, 0-203). For categorical analysis purposes, we used a standard cutoff of 20% to define positive CD123 expression. Prevalence of CD123 expression was overall higher in B-ALL compared to T-ALL [145 (90%) vs. 11 (42%); p<0.001]. Expression prevalence was highest in Ph+ B-ALL (57/59; 97%) followed by Ph- B-ALL (88/103; 85%) and T-ALL (11/26; 42%). In Ph- B-ALL, CD123 expression correlated with CD34 expression (p=0.002) and lower bone marrow blast count (p=0.003). In T-ALL, CD123 expression correlated with immunophenotypic subtypes (early) (p=0.006). To test the potential of targeting CD123 in ALL, B-ALL cell lines (n=12) which express CD123 at levels similar to AML patient samples were exposed to titrated IMGN632 levels. IMGN632 IC50 values ranged between 0.6 and 20 pM, and the conjugate cytotoxicity was highly CD123-specific, as masking CD123 with a competing anti-CD123 antibody reduced the potency more than 1000-fold. To test the anti-leukemia activity of IMGN632 on primary B-ALL cells, bone marrow cells from B-ALL patients were exposed to IMGN632 following which blasts and non-malignant cells were enumerated by MFC. Notably, IMGN632 eliminated more than 90% of B-ALL blasts in 6 out of 8 samples from newly-diagnosed as well as relapsed-refractory patients at low pico-molar concentrations. Normal cells were not affected by IMGN632 at 100-fold higher concentrations.Conclusions: CD123 expression is prevalent across ALL subtypes, including 90% of B-ALL and 42% of T-ALL. CD123 expression was associated with CD34 expression in B-ALL and immunophenotype in T-ALL. The CD123 targeted antibody-drug conjugate IMGN632 demonstrates promising activity and safety in pre-clinical models of B-ALL._ [Display omitted] DisclosuresDaver:Incyte Corporation: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Immunogen: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Pfizer Inc.: Consultancy, Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Kiromic: Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy. Zweidler-McKay:ImmunoGen: Employment. Kantarjian:ARIAD: Research Funding; Amgen: Research Funding; Delta-Fly Pharma: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; Novartis: Research Funding. Jabbour:Bristol-Myers Squibb: Consultancy. Khoury:Stemline Therapeutics: Research Funding; Kiromics: Research Funding; Pfizer: Research Funding; Angle: Research Funding.

Identification of Novel Targets for Antibody or Chimeric Antigen Receptor (CAR) Based Immunotherapy in AML: Characterizing the Surfaceome from Primary Patient Samples

Background: Despite considerable advances in the field of immunotherapy of acute myeloid leukemia, on-target off-leukemia toxicity remains a challenge. Thus, novel target antigens are sought after. The Cell Surface Capture (CSC) technology enables a discovery-driven approach to detect surface proteins. However, direct processing of primary samples has been limited by the substantial number of viable cells needed for the CSC-workflow. We therefore addressed this issue by improving the technique including the use of our ex vivo culture system. This allowed for an unbiased, direct assessment of primary AML patient samples and thus enabled us to interrogate the AML surfaceome in clinically relevant samples.Methods: Primary cells from patients with newly diagnosed or relapsed AML were cultured in our ex vivo co-culture system as previously described (Krupka et al. 2014) using MS-5 feeder cells. After 3 days, non-adherent cells were harvested and immediately subjected to the CSC-workflow. Glyco-CSC and its variants Cys-Glyco-CSC and Lys-CSC were initially performed as described by Wollscheid and colleagues (Wollscheid et al. 2009 and Bausch-Fluck et al. 2012) and subsequently modified to improve the yield on AML samples. CSC samples were analyzed by tandem mass spectrometry on an Orbitrap Elite instrument and modified peptides were identified using MaxQuant software.Results: To enhance the number of successfully identified surface proteins, we adapted the original protocol. These modifications doubled the yield in identified proteins from AML cell lines from initially 125 to 252 in the modified protocol. More importantly, the modifications increased the specificity of the assay significantly. In the original Glyco-CSC experiments, only 54% of all identified peptides displayed a mass shift (of 0.984 Da) associated with successful N-glycosylation and had a transmembrane domain or a signal peptide annotated in UniProt. After modification of the protocol, 80.4% of all peptides fulfilled these criteria. The modified protocol was therefore used for all primary samples. 5 representative primary patient samples from initial diagnosis and 2 samples from relapsed disease were analyzed. All samples yielded sufficient viable cell numbers after ex vivo culture and could successfully be subjected to the CSC workflow. We identified a total of 719 surface proteins fulfilling all filter rules. 22.9% of these proteins had CD annotations. Next, we only considered proteins that were detected by CSC in at least half of the primary patient samples tested. In addition, proteins were filtered to eliminate targets that are abundantly expressed on normal human hematopoietic stem and progenitor cells as well as relevant healthy tissue using publicly available transcriptome databases. 84 proteins were selected as potential candidates for manual screening. Of note, the expression of several antigens currently under investigation for AML immunotherapy (i.e. CD33, CD123, CD135, CLL-1) were detected by the method. We selected 6 promising novel candidate markers previously not described as relevant targets in AML. These were assessed by FACS analysis in independent patient samples. 3/6 of our novel targets showed uniform expression in all independent primary AML samples tested (defined as MFI ratio >1.5).Conclusion: In conclusion, improvements in the CSC-Workflow combined with our ex vivo culture system allowed for the successful identification of the AML surfaceome from primary patient samples without the necessity of xeno-amplification. We identified 6 novel targets, 3/6 were found to be uniformly expressed in independent primary AML samples. These candidates are now being evaluated further as potential targets for antibody and CAR based immunotherapy in AML. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of a FLT3 Bite® for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a disease with high unmet medical need. Standard induction chemotherapy can lead to complete remission in patients with a favorable genetic risk profile; however, about half of patients are ineligible for this intensive regimen. In those patients with a non-favorable genetic risk profile, the preferred treatment strategy is allogeneic stem cell transfer, but the majority of patients are ineligible due to age and other comorbidities.FLT3 is a receptor tyrosine kinase that induces cell growth in myeloid progenitor cells. Activating mutations in FLT3 have been identified in approximately 30% of AML patients. Expression of mutant FLT3 and overexpression of wild type FLT3 are associated with poor prognosis in AML, leading to clinical investigation of targeted FLT3 inhibitors, including midostaurin which was recently approved for patients with newly diagnosed FLT3-mutant AML. These inhibitors are likely to be effective only in certain patient subpopulations and may ultimately give rise to acquired resistance. To drive cures in AML, effective therapies for broad patient populations that can eliminate residual disease are needed.Bispecific T cell engaging antibody constructs (BiTE®) are a class of immunotherapy agents which redirect T lymphocytes to a specific antigen expressed on tumor cells. Blinatumomab, a BiTE® containing a single chain variable fragment (scFv) that binds CD19 on B lymphocytes and an scFv that binds CD3 on T lymphocytes, has shown clinical efficacy for treating CD19-positive hematological malignancies such as ALL. To extend the serum half-life, next generation BiTE® antibody constructs have been modified to contain Fc-moieties, allowing for weekly dosing.To evaluate FLT3 as a BiTE® target in AML, we assessed expression of FLT3 on primary AML patient blasts using flow cytometry. Over 83% of AML patients (n = 192) were FLT3-positive (MFI ratio > 1.5) with similar expression in bulk and leukemic stem cells. A half-life extended FLT3 BiTE® (FLT3 HLE BITE®) was generated and evaluated in vitro as well as in vivo in a mouse xenograft tumor model and non-human primates (NHP). FLT3 HLE BiTE® had subnanomolar binding affinity for human FLT3 and nanomolar binding affinity for human CD3 and NHP FLT3 and CD3. In cytotoxic assays targeting human AML cell lines, FLT3 HLE BiTE® exhibited picomolar efficacy using as effector cells human pan T cells in a 10:1 E:T ratio (range: 2 to 10 pM) or NHP PBMCs in a 5:1 E:T ratio (range: 30 to 80 pM). In mice injected with FLT3-positive MOLM13 cells and human CD3-positive T cells, FLT3 HLE BiTE® induced robust anti-tumor activity and extended survival ≥ 2-fold at all doses (n = 10 per group, p ≤ 0.0015). Following a single 5 µg/kg dose in NHP (n=2), FLT3 HLE BiTE® exhibited a reproducible pharmacokinetic profile with a 66 hour half-life. In this study, we observed hallmarks of BiTE® activation such as induction of IL-6 and MCP-1 and indication of a pharmacodynamic effect through increased soluble FLT3 ligand. Following repeat-dosing with FLT3 HLE BiTE®, digital droplet PCR was used to measure FLT3 transcript in NHP blood and bone marrow. Robust depletion of FLT3+ cells was observed in both NHP blood (≥ 95% reduction) and bone marrow (≥ 68% reduction) at all doses (5 µg/kg, 25 µg/kg, 75 µg/kg, n = 3/group). Taken together, FLT3 HLE BiTE® has potent in vitro and in vivo activity and may provide therapeutic benefit to AML patients. DisclosuresGoldstein:Amgen: Employment. Henn:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Archibeque:Amgen: Employment. Frank:Amgen Inc.: Employment, Equity Ownership. Balazs:Amgen Inc.: Employment, Equity Ownership. Dahlhoff:Amgen Research (Munich): Employment, Equity Ownership. Raum:Amgen Research (Munich): Employment. Li:Amgen, Inc.: Employment. Wahl:Amgen Research (Munich): Employment. Rock:Amgen, Inc.: Employment, Equity Ownership. Thomas:Amgen Research (Munich): Employment. Karbowski:Amgen, Inc.: Employment. Krupka:Amgen Research (Munich): Research Funding; Amgen, Inc: Research Funding. Subklewe:Amgen Research (Munich): Research Funding; Amgen, Inc.: Research Funding. Coxon:Amgen, Inc.: Employment. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership.

Diagnostic Value of Flow Cytometry Technique By European Leukemianet for Idiopathic Cytopenia of Undetermined Significance

Introduction: Myelodysplastic syndromes (MDS) are heterogeneous hematological disorders characterized by hematopoietic dysplasia and/or chromosomal aberrations. However, the diagnosis of dysplasia is subjective and chromosomal aberrations are not always present. Thus, the diagnosis of MDS is not always straightforward when lacking specific characteristics. To resolve this diagnostic problem, a novel flow cytometry technique standardized by the European LeukemiaNet (ELN) working group for MDS [Haematologica 94: 1124, 2009] was proposed. However, this ELN MDS index has not been validated for the diagnosis of idiopathic cytopenia of undetermined significance (ICUS).Purpose: To determine the diagnostic value of the ELN MDS index for ICUS, we analyzed the diagnostic accuracy of morphology, cytogenetics, and flow cytometry for cytopenia.Methods: We investigated 253 consecutive patients who underwent bone marrow aspiration to examine the cause of cytopenia at our institute between 2012 and 2015. We used the ELN MDS index verified by Ogata et al. for low-grade MDS [Haematologica 94: 1066, 2009]. The ELN MDS index consists of the following four parameters: (1) CD34+ myeloblasts on all nucleated cells, ≥2.4%; (2) CD34+ B cells on all CD34+ cells, ≤5.0%; (3) lymphocyte/myeloblast CD45 MFI ratio, ≤4.0 or ≥7.8; and (4) granulocyte/lymphocyte SCC peak ratio, ≤6.0. When two or more parameters met the criteria, the patient was diagnosed with MDS by cytometry. We evaluated the accuracy of diagnosis with cytometry by using the clinical diagnosis as a standard reference. The clinical diagnosis was determined by more than two hematologists/specialists. Patients with MDS and ICUS were included in this study. ICUS was defined as follows: bone marrow blast, <5.0%; single- or multi-lineage dysplasia, <10%; and no detection of clonal chromosomal abberation characteristic to MDS.Results: A total of 253 bone marrow samples were evaluated (117 females). The median patient age was 66 years (range, 1-92 years). The clinical diagnosis was MDS in 60 patients (24 females; median age, 69 years; range, 30-91 years) and ICUS in 17 patients (9 females; median age, 76 years; range, 54-88 years). Other patients were identified to have non-MDS/non-ICUS diseases and were used as a control group. MDS subcategories were as follows: RCUD (n = 14), RARS (n = 3), RCMD (n = 10), RAEB-1 (n = 9), RAEB-2 (n = 12), MDS-U (n = 8), and 5q− syndrome (n = 4). Two or more criteria of the ELN MDS index were met in 34 MDS (56.7%; 34/60) and 4 ICUS patients (23.5%; 4/17). Positivity with the ELN MDS index among the MDS subcategories varied as follows: 6 RCUD (42.9%; 6/14), 1 RARS (33.3%; 1/3), 5 RCMD (50.0%; 5/10), 7 RAEB-1 (77.8%; 7/9), 12 RAEB-2 (100%; 12/12), 2 MDS-U (25.0%; 2/8), and 1 5q− syndrome cases (25.0%; 1/4). When cutoff scores were set as the ELN MDS index of ≥2, sensitivities for diagnoses of MDS and ICUS were 56.7% (34/60) and 25.0% (4/16), specificities were 83.1% (147/177) and 83.1% (147/177), positive predictive values were 53.1% (34/64) and 11.8% (4/34), and negative predictive values were 85.0% (147/173) and 92.5% (147/159), respectively.Conclusion: Application of the ELN MDS index in MDS revealed sensitivity and specificity in line with those of previous studies, while 25.0% of ICUS patients were indicated to have MDS. This prevalence of MDS in ICUS may correspond to 27.5% (33/120) of patients with ICUS who had genetically clonal evidence identified by targeted sequencing of 22 MDS-associated genes [Kwok et al., Blood 2015; 126: 2355]. Our data suggest that the ELN MDS index is a practical tool to identify a subset of ICUS patients with MDS trait. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

At Last the Explanation for Increased Spontaneous Miscarriages in Thyroid Autoantibody Positive Pregnant Women

In women who are pregnant, the presence of thyroid autoantibodies is associated with an increased rate of miscarriage in the first trimester, with multiple reports averaging ~17% compared with ~8% in autoantibody negative women. During pregnancy immune tolerance is altered to allow implantation of the semi-allogeneic fetus and T-regulatory cells (Tregs), which play a major role in such tolerance, have been shown to increase during pregnancy, reaching a peak in the second trimester. A deficiency in this Treg response has been widely associated with spontaneous miscarriages. While it is known that the number of Tregs in patients with autoimmune thyroid disease (AITD) is decreased there are no data on pregnant women with AITD. In this study, we examined both the number and function of Tregs in pregnant women with (n=26) and without (n=41) thyroid autoantibodies (anti-Tg and/or anti-TPO) as well as healthy non pregnant women (n=25). Tregs were measured by flow cytometry of isolated CD4+, CD25+ and FoxP3+ T-cells. We found that the total number of CD4+CD25+FoxP3+ high Tregs was significantly increased in pregnancy consistent with previous reports (from 11% to 22%, p<0.024). Furthermore, this increase in Tregs was less in pregnant women with thyroid autoantibodies (mean of 12%). In addition, studies of phosphorylated signal transducer and activator of transcription-5a (pStat-5a) as a marker of Treg function, showed that while Tregs were activated in pregnancy, their activity per cell was diminished in the pregnant antibody positive women with a frequency:MFI ratio of 201 compared to 316 in the negative group. These data demonstrate that pregnant women with thyroid antibodies have a reduced Treg response to pregnancy, both in number and function, and offer a likely explanation for the increased miscarriage rate in such patients.

The contribution of CD200 to the diagnostic accuracy of Matutes score in the diagnosis of chronic lymphocytic leukemia in limited resources laboratories.

Flow cytometry immunophenotyping has an essential role in distinguishing chronic lymphocytic leukemia from other B-chronic lymphoproliferative disorders. Recently, CD200 is considered as a relatively consistent marker in chronic lymphocytic leukemia. We retrospectively assessed CD200 expression in 252 patients with B chronic lymphoproliferative disorders with four-color flow cytometry. CD200 expression estimation included the proportion of positive cells (≥30%) and the mean fluorescence intensity ratio. Additionally, we have incorporated CD200 into Matutes score, also replaced FMC7 and CD79b in an attempt to improve the score discriminative power. Of 252 patients enrolled, 199(79%) patients were classified as chronic lymphocytic leukemia and 53 (21%) as other B-chronic lymphoproliferative disorders. All chronic lymphocytic leukemia cases and 20 of 53 (37.7%) of other B-chronic lymphoproliferative disorders demonstrated high CD200 expression (≥30%). Further, CD200 (≥30%) revealed a higher accuracy in comparison to other markers in Matutes score (range: 51%–92.5%). Also, CD200 addition to the Matutes score has correctly recognized all 199 chronic lymphocytic leukemia cases including 10 atypical chronic lymphocytic leukemia cases. As for non-CLL cases, 20 of 53 attained a higher score, yet keeping the original diagnosis. Moreover, CD200 enhanced the diagnostic accuracy of Matutes score to 100%, and when included in a simplified 4-markers score, showed an accuracy of 99.8% compared to 99.4% of Matutes score. In conclusion, CD200 is an accurate diagnostic marker for chronic lymphocytic leukemia, and can refine the modified Matutes score accuracy when added with other markers.

Treatment-Free Intervals Mitigate T-Cell Exhaustion Induced By Continuous CD19xCD3-BiTE® Construct Stimulation in Vitro

The bispecific T-cell engager (BiTE®) blinatumomab is approved for treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and applied as continuous infusion over 28 days. The overall response rate to blinatumomab reported in clinical trials was 43 % and correlated to T-cell expansion (Zugmaier et al. 2015). In chronic viral infections, continuous antigen stimulation induces T-cell exhaustion, defined by phenotypic changes and functional impairment (Wherry 2011). Thus, we hypothesized that continuous BiTE® construct stimulation leads to T-cell exhaustion and that a treatment-free interval (TFI) reverses progressive T-cell dysfunction. To simulate continuous application of a BiTE® construct in vitro, T-cell long-term co-cultures were set up. Healthy donor T cells were stimulated in the presence of CD19+ OCI-Ly1 cells for 28 days with AMG 562, a half-life extended CD19 and CD3 specific BiTE® construct. T cells were harvested from the co-culture every 3-4 days between day 7 and 28 and assessed for markers of T-cell exhaustion: (1) AMG 562-mediated cytotoxicity of T cells was evaluated as specific lysis of CD19+ Ba/F3 target cells after 3 days, (2) T-cell expansion during the cytotoxicity assay was calculated as fold change (FC) of CD2+ counts, (3) Cytokine secretion of AMG 562-stimulated T cells was evaluated in co-culture supernatants by cytometric bead array (CBA) or after PMA/Ionomycine stimulation via intracellular cytokine staining (ICCS), (4) T-cell metabolic fitness was determined by Mito- and Glycolytic Stress Test using a Seahorse Analyzer, and (5) expression of the exhaustion-related transcription factor TOX was assessed by multiparameter flow cytometry. In order to assess the effect of a TFI on T-cell function, we cultured T cells and CD19+ OCI-Ly1 cells in the absence of AMG 562 from day 7-14 and 21-28 and compared their activity to T cells stimulated continuously with AMG 562. On day 7 of continuous (CONT) AMG 562 stimulation, we observed high cytotoxic and proliferative potential (% specific lysis=93±0.2, FC=2.9±0.2) as well as high IFN-g and TNF-a secretion analyzed by ICCS (% CD8+IFN-g+TNF-a+=23±6.7). However, cytotoxicity and proliferation decreased gradually until day 28 (% specific lysis=28±8.9; FC=0.6±0.1). CBA analysis confirmed decreasing secretion of IFN-g (day 3: 61113±12482, day 24: 3085±1351 pg/ml) and TNF-a (day 3: 1160±567, day 24: 43±7.6 pg/ml) as well as decreased IL-2 and granzyme B levels in culture supernatants. We furthermore observed highest mitochondrial fitness and basal glycolysis in T cells on day 7 of stimulation (basal OCR=2.2±0.6, maximal OCR=3.7±1.0, SRC=1.5±1.1 pmol/min/1000 cells, basal ECAR=2.0±0.4 mpH/min/1000 cells) which decreased until day 28 (basal OCR=0.4±0.2, maximal OCR=1.5±0.5, SRC=1.0±0.2 pmol/min/1000 cells, basal ECAR=0.5±0.2 mpH/min/1000 cells). In concordance, TOX increased during continuous stimulation (MFI ratio CD8+ day 7=6±0.8 to 12±0.8 on day 28). Strikingly, implementation of a TFI of 7 days led to superior cytotoxicity in T cells compared to continuously stimulated T cells (% specific lysis on day 14 CONT=34±4.2, TFI=99±2.2) and granzyme B production (CD8+; MFI ratio on day 14 CONT=124±11, TFI=303±34). Furthermore, increased proliferation during the cytotoxicity assay was observed in previously rested T cells (FC CONT=0.2±0.0, TFI=1.6±0.6). Although T cell function also decreased over time in TFI T cells, they maintained a strikingly higher cytotoxic potential (CONT=6±4.4, TFI=52±9.9) as well as higher granzyme B production (CONT=25±2, TFI=170±11) on day 28 compared to continuously stimulated T cells. In addition, TFI T cells showed increased IFN-g and TNF-a secretion after PMA/Ionomycine stimulation on day 28 (% CD8+IFN-g+TNF-a+ CONT=21±3.8, TFI=38±11.6). Our in vitro results demonstrate that continuous AMG 562 exposure negatively impacts T-cell function. Comprehensive analysis of T-cell activity in an array of functional assays suggests that continuous BiTE® construct exposure leads to T-cell exhaustion which can be mitigated through TFI. Currently, T cells from patients receiving blinatumomab are being analyzed to confirm the clinical relevance of our findings. Furthermore, RNA-Seq of continuously vs. intermittently AMG 562-exposed T cells will help us to understand underlying transcriptional mechanisms of BiTE® construct induced T-cell exhaustion. Disclosures Zieger: AMGEN Research Munich: Research Funding. Buecklein:Pfizer: Consultancy; Novartis: Research Funding; Celgene: Research Funding; Amgen: Consultancy; Gilead: Consultancy, Research Funding. Brauchle:AMGEN Inc.: Research Funding. Marcinek:AMGEN Research Munich: Research Funding. Kischel:AMGEN: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Subklewe:Gilead Sciences: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

Natural soluble human leukocyte antigen class I in donor serum neutralizes donor-specific HLA alloantibodies in recipient serum.

BackgroundHuman leukocyte antigen (HLA) molecules are cell-bound but can be identified in a soluble form. These soluble HLA (sHLA) molecules have an immunomodulatory function. We investigated whether natural sHLA in donor serum can neutralize donor-specific HLA alloantibodies (DSAs) in recipient serum.MethodsNeutralizing effects of donor serum on DSAs in recipient serum were measured using inhibition assay principle of flow cytometric crossmatch (FCXM), performed using sera from 143 kidney transplant recipients and their donors. The adding of donor serum to recipient serum yielded lower mean fluorescence intensity (MFI) ratios (test/control) than when diluent was added [Roswell Park Memorial Institute (RPMI) or third-party serum], which was presumed to be caused by the neutralizing effects of sHLA.ResultsIn the recipient group with class I DSAs alone (N=14), donor serum addition to recipient serum resulted in lower T cell MFI ratios [2.25 (1.31‒32.51)] than those observed on RPMI addition [3.04 (1.33‒125.39), P<0.05]. In the recipient group with class II DSAs alone (N=27), donor serum addition showed no significant difference in B cell MFI ratios [5.03 (1.41‒103.53)] compared to diluent addition: RPMI [4.50 (1.34‒145.98)] or third-party serum [5.08 (1.44‒138.47)], P>0.05 for both.ConclusionUsing inhibition FCXM, we verified that natural sHLA class I in donor serum neutralizes DSAs in recipient serum. However, no neutralizing effects of sHLA class II were revealed in this study. These potentially beneficial effects of sHLA infused via blood-derived products should be considered when desensitizing highly HLA-sensitized patients.

Induction of Truncating ASXL1 Mutations in Human CD34+ HSPCs Mimics Human ASXL1-Mutated Clonal Hematopoiesis and Progression to Myeloid Malignancies

Introduction: Mutations in additional sex combs like 1 (ASXL1) are recurrently found in myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), but can also be found in pre-malignant states such as clonal hematopoiesis of indeterminate potential (CHIP). However, the mechanisms by which these mutations contribute to disease initiation and progression remain unresolved. Initial observations using murine models suggested a loss of function of the ASXL1 protein as relevant to disease. However more recently, several studies have suggested that recurrent mutations of ASXL1 result in a change-of function caused by a truncated ASXL1 protein. Most of these studies have been performed in a murine system and have used over-expression of truncated ASXL1. Methods: To interrogate the functional relevance of recurrent mutations in ASXL1 in human HSPCs, we generated a novel CRISPR/Cas9-mediated model that allows for the introduction of truncations or complete knockout of ASXL1 into CD34+ cord blood cells. Importantly, this is the first model which efficiently introduces these mutations into the native ASXL1 locus in human cord-blood and bone-marrow derived HSPCs while simultaneously introducing a fluorescent marker corresponding to the genotype, so individual cells can be tracked in vitro and in vivo. Results: Using this system, we demonstrate that truncated, but not deleted, ASXL1 shows a proliferative advantage, decreased ability to differentiate along the megakaryocyte and erythroid lineages, as well as increased serial replating in vitro. Interestingly, truncation of ASXL1 alone did not result in a myeloid differentiation block in vitro, in line with observations of mutant HSPCs in CHIP. In vivo, CD34+ cells harboring truncation of ASXL1 exhibited myeloid skewing and a proliferative advantage in competitive engraftment studies using a xenograft NSGS mouse model (both p&amp;lt;0.05). Human HSPCs expressing truncated ASXL1 showed polyclonal engraftment without development of overt myeloid disease or impaired lifespan in the majority of the host animals. However, similar to humans with ASXL1 CHIP, some of the engrafted animals (2/30) developed clonal expansion of ASXL1-truncated human HSPCs resulting in a myeloid-biased, proliferative disease-like state with infiltration of non-hematopoietic organs and severe cytopenia which ultimately led to shortened survival of affected mice. Functionally, ASXL1-truncated HSPCs displayed dysregulated gene expression with depletion of RUNX1, TAL1, and GATA1 target genes (Benjamini-Hochberg adjusted p&amp;lt;0.0001). In line with increased activity of the Polycomb repressive deubiquitinase complex, truncation of ASXL1, but not deletion, led to decreased H2AK119Ub in our system (MFI ratio 0.7 for truncated ASXL1 vs. control, p&amp;lt;0.05). Orthogonally, we were able to confirm these observations in several isogenic cell lines engineered to contain truncating ASXL1 mutations as well as correcting the mutation in ASXL1 found in K562 cells (Y591*) using CRISPR/Cas9. Finally, we aimed to determine whether the introduction of additional mutations commonly observed to co-occur with ASXL1 could alter the behavior of human HSPCs and their progression from healthy hematopoiesis to myeloid disease. Indeed, introduction of mutations in the native RUNX1 locus on the background of truncated ASXL1 showed aggressive expansion of immature myeloid progenitors in our xenograft model. This system allows us now to determine the relevant genomic target regions of truncated ASXL1 in human HSPCs, investigate the mechanism of action directly in the relevant biological system and identify therapeutic strategies targeting mutant ASXL1. Conclusion: In summary, our work demonstrates the utilization of a novel CRISPR/Cas9 model of human ASXL1-mutant HSPCs that closely resembles the phenotype seen in individuals with ASXL1 mutant CHIP and patients with myeloid malignancies carrying mutant ASXL1. Additionally, our model allows us to directly compare the deletion of ASXL1 with the truncation, adding to the evidence that truncated ASXL1 phenocopies pre-malignant and malignant hematopoiesis with recurrent ASXL1 mutations. Finally, we provide the first model for the spontaneous progression of pre-malignant human CHIP to myeloid malignancy, which should enable further studies of leukemogenesis and potentially disease prevention. Disclosures Majeti: FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy.

Correlation between Changes in Granzyme B Expression and Time to Progression in Patients with Newly Diagnosed Multiple Myeloma Treated with Lenalidomide and Dexamethasone Therapy

【Background】Triplet and quadruplet regimens of novel agents have improved the survival of patients with multiple myeloma (MM) and elicited deeper responses to treatment. Minimal residual disease (MRD) negativity is now used as a surrogate marker for longer progression-free survival. However, triplet and quadruplet regimens can cause severe toxicity especially for elderly frail patients and financial toxicity is now an issue. On the other hand, long duration of response is sometimes experienced to low-dose lenalidomide and dexamethasone (Ld) therapy. However, the predictors of long duration of response is unknown. Sustained response without severe toxicity even if not achieving the MRD negativity is beneficial in the treatment choice for some populations like frail elderly patients. Therefore, predicting the long-duration of response could lead to individualized therapy, reducing overtreatment. Here, we hypothesized that the immunomodulatory effects of lenalidomide correlates with a long duration of response. 【Methods】We have previously conducted a prospective trial of Ld therapy in 40 patients with newly diagnosed transplant-ineligible MM. Among them, the time to progression (TTP) was evaluated in 29 patients, excluding patients who discontinued Ld therapy before disease progression. In this study, we evaluated the correlation between immune profile changes and TTP in this study set. Immune profile was evaluated by flow cytometry using peripheral blood mononuclear cells (PBMCs) obtained before and during the first or second cycles of Ld therapy. We evaluated the T cell subset (CD4+ T cells, CD8+ T cells, regulatory T cells) and NK cells and NKT cells and myeloid-derived suppressor cells. Exhaustion markers of PD-1, Tim-3, CTLA-4 and the activation markers of 4-1BB in each lymphocyte subset and memory subset of T cells were evaluated. The cytokine production of granzyme B, IFN-γ, TNF-α and IL-2, was detected by intracellular cytokine staining after 6-h stimulation with phorbol 12-myristate 13-acetate (25 ng/mL) and ionomycin (1 μg/mL) in the presence of the protein transport inhibitor, Golgi stop (BD Bioscience). 【Results】The median age was 75 years old (range, 61-86) at the initiation of Ld therapy. Median TTP was 34 months (range, 1-70). We divided the cases into two groups using the median TTP as a cut-off point (Figure 1). There were no significant differences in baseline characteristics, male to female ratio, performance status, M-protein subtype, creatinine clearance, international staging system (ISS), and high risk cytogenetic profiles between these two subgroups, except for age (median age was 77 years old in subgroups with TTP &lt; 34 months compared to 72 years old, p=0.04). Median initial dosage of lenalidomide was not statistically different (10 mg/day and 15 mg/day in subgroups with TTP &lt; 34months and TTP &gt; 34months, respectively) and dexamethasone dosage was 20 mg/day in both groups. The achievement of best response more than partial response was also comparable in both subgroups (71% and 93% in subgroups of TTP &lt; 34months and TTP &gt; 34months). We compared the immune profile changes in these subgroups. Consequently, we found that mean fluorescence intensity (MFI) ratio of granzyme B expression in NK and NKT cells (before and after Ld therapy) and ΔMFI of granzyme B expression in CD8+ T cells were significantly higher (p&lt;0.05) in longer TTP subgroups (Figure 2). 【Conclusions】Our data suggest that early immune responses during the first or second cycle of Ld therapy could serve as the predictive marker of longer TTP following Ld therapy in patients with NDMM. This could benefit some patients, particularly frail elderly patients by helping them avoid unnecessary intensification of treatment. Disclosures Ikeda: Nippon Shinyaku Research Grant: Research Funding. Nishikawa:Daiichi Sankyo: Research Funding; Zennyaku: Research Funding; Kyowa Hakko Kirin: Research Funding; Taihou Pharmaceuticals: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Ono Pharmaceutical: Research Funding, Speakers Bureau; Chugai Pharmaceuticals: Research Funding, Speakers Bureau; Sysmex: Research Funding; Asahikasei Pharma: Research Funding. Takahashi:Kyowa Hakko Kirin: Research Funding; Bristol-Myers Squibb: Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Ono Pharmaceutical: Research Funding; Asahi Kasei Pharma: Research Funding; Chug Pharmaceuticals: Research Funding; Eisai Pharmaceuticals: Research Funding; Astellas Pharma: Research Funding; Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Novartis Pharmaceuticals: Research Funding, Speakers Bureau.

Interferon-γ upregulates Δ42PD1 expression on human monocytes via the PI3K/AKT pathway

BackgroundWe recently identified a novel alternatively spliced isoform of human programmed cell death 1 (PD-1), named Δ42PD1, which contains a 42-base-pair in-frame deletion compared with the full-length PD-1. Δ42PD1 is likely constitutively expressed on human monocytes and down-regulated in patients infected with human immunodeficiency virus type 1 (HIV-1). The mechanism underlying the regulation of Δ42PD-1 expression in monocytes remains unknown. MethodsBy flow cytometry, we investigated the effect of Interferon-gamma (INF-γ) on the expression of Δ42PD1 in primary human monocytes as well as monocytic cell lines THP-1 and U937 cells. In addition, signaling pathway inhibitors and Δ42PD1-specific blocking antibody were used to explore the pathway involved in INF-γ-induced Δ42PD1 upregulation, and to elucidate the relationship between Δ42PD1 and TNF-α or IL-6 production by INF-γ primed monocytes in response to pre-fixed E. coli. Furthermore, we assessed T-cell proliferation, activation and cytokine production as enriched CD4+ T cells were co-cultured with THP-1 or U937 cells, with or without Δ42PD1-blocking antibody. ResultsTreatment of human peripheral blood mononuclear cells (PBMCs) with IFN-γ resulted in an approximately 4-fold increase in the expression of Δ42PD1 on monocytes. Similarly, IFN-γ upregulates Δ42PD1 expression on human monocytic cell lines THP-1 and U937, in a time- and dose-dependent manner. IFN-γ-induced Δ42PD1 upregulation was abolished by JAK inhibitors Ruxolitinib and Tasocitinib, PI3K inhibitor LY294002, and AKT inhibitor MK-2206, respectively, but not by STAT1 inhibitor and MAPK signaling pathway inhibitors. JAK, PI3K-AKT, and MAPK signaling inhibitors abolished effectively the production of TNF-α and IL-6 in INF-γ-primed monocytes in response to pre-fixed E. coli. In contrast, Δ42PD1-specific blocking antibody did not affect the IFN-γ-induced priming effect. Furthermore, the MFI ratio of Δ42PD1 to full-length PD-1 (PD-1 Δ/F ratio) was significantly and positively correlated with TNF-α (P = 0.0289, r = 0.6038) produced by circulating CD14+ monocytes in response to pre-fixed E. coli. Notably, Δ42PD1 blockage significantly inhibited CD4+ T-cells proliferation and cytokine production in the co-culture conditions. ConclusionsWe demonstrated that IFN-γ increases Δ42PD1 expression on human monocytes via activating the PI3K/AKT signaling pathway downstream of JAKs, and that the PD-1 Δ/F ratio is a potential biomarker to predict the functional state of monocytes. Notably, we revealed the Δ42PD1 play a role in T-cell regulation, providing a novel potential approach to manipulate adaptive immune response.

Measuring anti-HLA antibody active concentration and affinity by surface plasmon resonance: Comparison with the luminex single antigen flow beads and T-cell flow cytometry crossmatch results

BackgroundAlthough the Luminex single antigen flow beads (SAFB) and the flow cytometry crossmatch (FCXM) are the most sensitive assays used for anti-HLA antibodies characterization in transplant recipients, their semi-quantitative fluorescence read-out is not closely linked to graft outcome. MethodsSurface plasmon resonance (SPR) was implemented to determine truly quantitative parameters of five human monoclonal anti-class I HLA antibodies (mAbs): first the active concentration and then the binding constants. The results were compared to those obtained with SAFB and T-cell FCXM (T-FCXM). ResultsThe five mAbs displayed different rate and equilibrium constants for their cognate antigens. No correlation was evidenced between SAFB MFI or T-FCXM ratio and the binding parameters measured by SPR. Some mAbs amino acid substitutions within the epitope that influenced SAFB MFI resulted in affinity variations evidenced by SPR. ConclusionThe SAFB MFI and T-FCXM ratio, both semi-quantitative parameters, only partially reflected the subtlety of the anti-HLA antibody/antigen interaction as it can be analyzed by SPR. Future clinical studies using SPR for anti-HLA antibodies characterization could bring novel insights into the understanding of HLA/anti-HLA interaction and therefore anti-HLA antibodies pathogenicity.

Usefulness of MOG-antibody titres at first episode to predict the future clinical course in adults.

To analyze whether myelin oligodendrocyte glycoprotein antibody (MOG-Ab) titres at onset of the disease were different according to the clinical phenotype at presentation, and to investigate whether the titres were associated with risk of further relapses or predicted clinical outcome in adult patients. Finally, we assessed an alternative method to the classical measurement of MOG-Ab levels by serial dilutions. This is a retrospective study including 79 MOG-Ab-positive adult patients, whose samples were obtained at first episode. MOG-Ab were tested by cell-based assay. HEK293 cells were transfected (tHEK293) with human-MOG plasmid. Non-tHEK293 cells were used as negative controls. Assessment of antibody titres was performed by serial dilution, and delta mean fluorescence intensity ratio signal (MOG-ratio ΔMFI) by flow cytometry. MOG-ratio ΔMFI was calculated as follows: (MFI tHEK293cells- MFI non-tHEK293cells)/MFI non-tHEK293cells. MOG-ratio ΔMFI was calculated from the first serum dilution at 1:320. The association between MOG-Ab titres and risk of relapse was analyzed by Cox regression. The association between MOG-Ab titres and visual or motor disability at last follow-up was performed by binary logistic regression. Poor visual outcome was defined when patients displayed some degree of visual disability (visual acuity [VA] < 20/20) and poor motor outcome when patients displayed some degree of motor disability (Disability Status Scale [DSS] > 1). We also investigated correlations between MOG-Ab titres and MOG-ratio ΔMFI. MOG-Ab titres were higher in Caucasians than in those with other ethnicities, and in patients with a more severe VA (VA ≤ 20/100) or motor disability (DSS ≥ 3.0) at onset (p = 0.006, 0.034, and 0.058, respectively). MOG-Ab titres were not associated with risk of relapses or with the final clinical outcome. MOG-ratio ΔMFI correlated with MOG-Ab titres in the whole cohort (ρ = 0.90; p < 0.001), and when stratified by initial clinical phenotype. High MOG-Ab titres at onset are associated with a more severe presentation, but do not predict the future disease course. MOG-ratio ΔMFI is an alternative and straightforward method to determine MOG-Ab levels.

Intracellular Thiol Oxidation Is Linked with Loss of Δψm and Disease Progression in Acute Promyelocytic Leukaemia

Acute promyelocytic leukaemia (APL) is a type of myeloid malignancy defined by the chromosomal translocation t(15;17) and subsequent expression of the PML-RARα fusion protein. Long term remission in APL is achieved through a combination of high dose all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), the latter of which has potential to induce mitochondrial derived oxidative stress and initiate the intrinsic apoptosis pathway. ATO is particularly effective in the treatment of APL when compared with acute myeloid leukaemia (AML), suggesting that the mitochondrial membrane potential (Δψm) of APL cells is compromised. Here we show that loss of Δψm is associated with intracellular thiol oxidation and disease progression in APL.The mitochondrial probe 3,3'-diethyloxacrbocyanine iodide (DiOC2(3)) was used to evaluate MRP8-PML/RARA transgenic mice Δψm in bone marrow mononuclear cells (BMMC) at three disease stages: 1) pre-APL, 2) APL and 3) ATRA treated APL. Our data show that MRP8-PML/RARA BMMC cells during the APL stage accumulate significantly less DiOC2(3) compared with pre-APL cells (0.05±0.02 vs 0.24±0.05, p<0.05; data are normalised FL1/FL3 MFI ratio). The DiOC2(3) accumulation in BMMC from ATRA treated APL mice was equivalent to that of pre-APL cells (p>0.05). This finding indicates a loss of Δψm in APL. This may result in free radical leakage from the mitochondria, which in turn could oxidise intracellular thiols.To evaluate intracellular thiol oxidation, a previously optimised flow cytometric assay that incorporates the thiol reactive probe fluorescein-5 maleimide (F5M) was used. Our data show a significant decrease in F5M MFI for MRP8-PML/RARA BMMC cells during the APL stage, compared with pre-APL cells (219.01±47.67 vs 672.66±131.04, p<0.05; data are F5M MFI relative to unstained controls). This finding signifies that there is more thiol oxidation at the APL stage. ATRA treated APL mice showed an equivalent F5M MFI to that of pre-APL cells. Confocal microscopy confirmed that the F5M signal was intracellular. This observation shows that there is an increase in intracellular thiol oxidation in APL, which may be tracked during disease progression and treatment course. This finding may explain why APL cells are more sensitive to pro-oxidant treatments i.e. ATO.To understand these observations further, the APL cell line (NB4) was subjected to glucose oxidase (GOX) mediated oxidative stress in cell culture over a 4 hour time-course. F5M MFI signal was compared with the THP1 and Kasumi-1 AML cell lines subjected to the same treatment. All cell lines showed similar significant increases in F5M MFI after 1 hour GOX treatment relative to control (p<0.05), indicating an increase in ‘reduced’ cellular thiol i.e. reductive stress. However, after 4 hours NB4 cells showed a significant decrease in F5M MFI relative to control (p<0.05), indicating an increase in ‘oxidised’ cellular thiol i.e. oxidative stress. In contrast, THP1 cells showed no difference in MFI after 4 hours (p>0.05), whereas F5M MFI Kasumi-1 remained significantly increased after 4 hours (p<0.05) relative to control. This finding indicates differences in the sensitivity to oxidative stress between APL and AML cells.Taken together, this study shows that APL cells are more sensitive to oxidative stress compared with AML cells. Our F5M flow cytometric assay illustrates that an increase in cellular thiol oxidation is linked to disease progression in APL, with loss of Δψm associated with this process. Our F5M flow cytometric assay may therefore have clinical utility in monitoring treatment efficacy in APL. DisclosuresNo relevant conflicts of interest to declare.

P140Proper setting of cut-off value for the lymphocyte crossmatch by flow cytometry in kidney transplantation

Aim The flow cytometric lymphocyte crossmatch (FCXM) has become a routine test performed prior to kidney transplantation. Many studies have reported that a proportion of transplant recipients experienced early graft loss despite a negative complement-dependent cytotoxicity (CDC) crossmatch test. The FCXM is able to detect Human Leukocyte Antigen (HLA) antibodies of low titer or of a non-complement binding nature that may be responsible for graft loss. It is important to report positive and negative results using appropriate cut-off values. We reviewed the results of FCXM with CDC and panel reactive antibody (PRA) and attempted to set clinically useful cut-off values for FCXM. Methods From January 2015 to October 2017, 518 kidney recipients’ results of FCXM, CDC, and PRA (Screening test, Panel kit, and single antigen bead assay, LABScreen Luminex kits, One Lambda, Canoga Park, CA, USA) were analyzed. The PRA test was performed simultaneously from 403 patients, and 159 patients who were negative for HLA antibodies in the all PRA tests. Fifty-seven patients were positive for CDC test results and 37 patients were both positive in T cells and B cells. In 20 patients, only B cells CDC results were positive, and 13 patients were negative for FCXM test results. Results The final interpretation of FCXM result was decided through CDC, PRA result and existence of donor specific antibody (DSA). T cell FCXM result was interpreted as negative in 451 patients and positive in 67 patients according to DSA. The appropriate cut-off value of the Mean fluorescent intensity (MFI) ratio using the ROC curve for 518 patients was 1.86 (sensitivity 100%, specificity 98.2%, Youden’s index 0.982). However, patients who underwent desensitization treatment due to DSA or ABO blood type incompatibility were included. The cut-off value obtained using the ROC curve for 446 patients who have not experienced desensitization treatment was 2.45 (sensitivity 100%, specificity 99.3%, Youden’s index 0.993). Generally used, Median + 2 SD value of MFI ratio of patients judged as negative FCXM was 1.60. Conclusions Based on the statistical method used, various cut-off values could be obtained. It is important to determine the clinically significant cut-off value based on the data accumulated in each laboratory, not the simple median value from negative patients.

Study of microRNA Profile as a Molecular Biomarker in Egyptian Chronic Lymphocytic Leukemia.

MicroRNAs target mRNAs for cleavage or translational repression. They play a critical role in the progression of malignancies and leukemias including chronic lymphocytic leukemia (CLL). However, microRNA expression levels in Egyptian patients with CLL, and their prognostic value remain elusive. Our main aim was to assess the expression pattern of a panel of microRNAs in CLL patients to create an informative microRNA profile. The study subjects were 40 newly diagnosed CLL patients of both sexes and 40 age and sex matched controls. The expression levels of 12 microRNAs were evaluated by qRT-PCR, including miR-15a, 16, 23b, 24, 29a, 29c, 34a, 146a, 155, 181a, 195, and 221. Flow cytometry was used to determine the expression levels of BCL2, CD38, and ZAP-70 in CLL patients. We identified various degrees of upregulated miRNAs (miR-29a, miR-29c, miR-34a, miR-155, miR-146a, and miR-195) and down-regulated ones (miR-15a, miR-16, miR-23b, miR-24, miR-181a, and miR-221) in CLL patients relative to controls. The mean fluorescence intensity ratio (MFI-R) of BCL2 was recorded and was significantly upregulated in CLL patients compared with normal controls. In addition, inverse correlations were observed between microRNAs (miR-15a, miR-16, miR-155, and miR-195) and BCL2 MFI-R while positive correlations were observed between miR-29a and miR-29c, and BCL2 MFI-R. These findings suggest that these miRNAs regulate BCL2 levels. Moreover, we found that miR-15a, miR-16, miR-155, miR-181a, miR-195 and miR-221 were significantly upregulated, while miR-29a and miR-29c were significantly downregulated in ZAP-70 positive CLL patients. Various miRNAs may play an important role in the pathogenesis of CLL and have the potential to be used for the prognosis of patients with CLL.

PO-285 Immunomodulatory effects of lactobacillus strains: emphasis on identification of probiotic candidates with anti-tumour responses

IntroductionMucosal macrophages are essential for driving immune responses in mucosal membranes. In homeostatic environments, regulatory responses are predominated by the M2 subset, whereas inflammatory responses are mediated via their ability to switch to a pro-inflammatory M1 subset. Tumour-associated macrophages (TAM) have been found to play a fundamental role in tumour development. Therefore, the aim of this study was to identify macrophage subset-specific responses to a panel of Lactobacilli with the objective of determining candidates that elicit anti-tumour responses for future in vivo management of colorectal cancer.Material and methodsA panel of candidate strains; L. plantarum (C28, VD23, MS18) and L. salivarius (MS3, MS6, MS16) were grown in MRS broth and harvested at the end of the exponential phase. The human monocytic cell line THP-1 was differentiated into M1 and M2 macrophages. Pro-inflammatory cytokine profile (IL1β, IL18, IL23 and TNFα) was investigated upon macrophage culture with Lactobacilli and quantified by sandwich ELISA. Phagocytic properties were measured by culturing macrophage subsets with CFSE-loaded bacteria and analysed by flow cytometry.Results and discussionsMS3 strain was the biggest inducer of pro-inflammatory cytokine secretion. This strain was even more potent inducer of IL1β, IL18 and IL23 secretion in M2-like macrophages and would represent the most appropriate strain to re-programme M2-like TAMs. VD23 and C28 exhibited a TNFαlo, IL1βlo, IL18lo, IL23lo phenotype. The phagocytic response was generally greater for M2 macrophages, whereby the greatest phagocytosis was observed with VD23 strain (MFI ratio 4,06±0,67) and the lowest response with the MS6 strain (MFI ratio 1,38±0,08).The highest phagocytic properties in M1 macrophages were observed with MS6 (MFI ratio 3,36±0,21), whereas the lowest was with MS3 (MFI ratio 1,28±0,12).ConclusionSelection of potential anti-tumour strains would be dependent on tumour stage, facilitating an M2-like phenotype for earlier stages and an M1-like phenotype for later stages. VD23 would appear to be the best strain to programme anti-inflammatory activity in M1/M2s whereas MS3 would represent the most appropriate strain to re-programme M2-like TAMs. The findings of this study suggest new strategies to probiotic bacteria modulating immune responses, leading to the treatment of diseases by switching to favourable macrophage subsets.This work was supported by VEGA 1/0476/17.

Repeated Freezing and Thawing Does Not Affect HLA Antibody Titer

Introduction For deceased donor organ transplantations, we are not currently using virtual crossmatches (XMs) and preliminary negative T cell XMs are mandatory for kidney and pancreas allocation in Korea. Korean Network for Organ Sharing (KONOS) distributes transplant candidates’ sera to 21 OPO laboratories and KONOS preliminary XMs are performed in these laboratories using frozen stored sera. We are performing more than 40% of nationwide KONOS XMs in the Korea Organ Donation Agency (KODA) laboratory, and we had difficulties in keeping the frozen sera from more than 20,000 transplant candidates in small aliquots to avoid HLA antibody deterioration. We investigated the extent of change of antibody titer after repeated freezing and thawing. Materials and Methods Two different sera (serum A and B), which were tested positive for both class I and class II HLA antibodies were selected for the investigation. Sodium azide (0.1% in final concentration) was added to the sera as preservative, and freezing-thawing cycles were repeated for 20 times. A total of 7 serum samples (1, 3, 6, 9, 12, 15, and 20 times frozen-thawed; labeled #1, #3, #6, #9, #12, #15, and #20) were used for HLA antibody screening test (One Lambda, USA), and 3 samples (1, 9, and 20 times frozen-thawed) were used for HLA antibody single antigen assay (One Lambda, USA) and T cell flow cytometry XM (FCXM) test. To avoid prozone phenomenon, 1:1 (neat), 1:5, and 1:20 diluted sera were tested by screening assay and 1:20 sera for both serum A and B were used for the investigation. HLA antibody strength in mean fluorescence intensity (MFI) values and FCXM result in MFI ratios were compared. Test results of #20 sample compared to #1 sample were calculated as percentage values and variabilities of the test results among different samples of repeated freezing-thawing were calculated as coefficient of variation (CV) values. Results and Discussion The results are summarized in Table 1. There was little change in MFI values after 20 times of repeated freezing-thawing cycles: percentage of #20 vs # 1 sample as baseline (100%) was in the range of 99-110% for screening assay and 90-136% for single antigen assay. For serum A, change of MFI values of 5 class I and 5 class II antibodies of the #1, #9 and #20 samples are shown (Fig. 1 and Fig. 2). Variability of the test results among different samples of repeated freezing-thawing was low, and most of the CV values (7 out of 8) were <10% (2.2-6.9%). T cell FCXM results (3 XMs) also showed little change in MFI ratios (103-107%) and low CV values (4.2-6.7%). Unlike the notion that antibodies are deteriorated after repeated freezing-thawing, the strength of HLA antibodies was little affected after 20 times of freezing-thawing cycles.Conclusion HLA antibody strength is little affected by up to 20 times of repeated freezing and thawing. For preliminary HLA XM tests for deceased donor organ transplantation, serum samples from transplant candidates can be stored frozen without aliquoting.

Rapid Flow Cytometry-Based Test for the Diagnosis of Lipopolysaccharide Responsive Beige-Like Anchor (LRBA) Deficiency.

The diagnosis of lipopolysaccharide-responsive beige-like-anchor-protein (LRBA) deficiency currently relies on gene sequencing approaches that do not support a timely diagnosis and clinical management. We developed a rapid and sensitive test for clinical implementation based on the detection of LRBA protein by flow cytometry in peripheral blood cells after stimulation. LRBA protein was assessed in a prospective cohort of 54 healthy donors and 57 patients suspected of LRBA deficiency. Receiver operating characteristics analysis suggested an LRBA:MFI ratio cutoff point of 2.6 to identify LRBA-deficient patients by FACS with 94% sensitivity and 80% specificity and to discriminate them from patients with a similar clinical picture but other disease-causing mutations. This easy flow cytometry-based assay allows a fast screening of patients with suspicion of LRBA deficiency reducing therefore the number of patients requiring LRBA sequencing and accelerating the treatment implementation. Detection of biallelic mutations in LRBA is however required for a definitive diagnosis.