Multiparameter Flow Cytometry Analysis Research Articles

Relevance of Antibody Validation for Flow Cytometry.

Antibody reagents are the key components of multiparametric flow cytometry analysis. Their quality performance is an absolute requirement for reproducible flow cytometry experiments. While there is an enormous body of antibody reagents available, there is still a lack of consensus about which criteria should be evaluated to select antibody reagents with the proper performance, how to validate antibody reagents for flow cytometry, and how to interpret the validation results. The achievements of cytometry moved the field to a higher number of measured parameters, large data sets, and computational data analysis approaches. These advancements pose an increased demand for antibody reagent performance quality. This review summarizes the codevelopment of cytometry, antibody development, and validation strategies. It discusses the diverse issues of the specificity, cross-reactivity, epitope, titration, and reproducibility features of antibody reagents, and this review discusses the validation principles and methods that are currently available and those that are emerging. We argue that significant efforts should be invested by antibody users, developers, manufacturers, and publishers to increase the quality and reproducibility of published studies. More validation data should be presented by all stakeholders; however, the data should be presented in sufficient experimental detail to foster reproducibility, and community effort shall lead to the public availability of large data sets that can serve as a benchmark for antibody performance. © 2019 International Society for Advancement of Cytometry.

Abstract 473: Higher levels of genomic complexity correlates with an advanced plasma cell differentiation status in newly diagnosed multiple myeloma patients

Abstract Introduction: plasticity is a hallmark of Multiple Myeloma (MM) clone(s), where both quiescent MM cells, acting as tumor-initiating cells, and proliferative MM cells, able to invade and disseminate, might co-exist. Aim of the study is to stratify patients (pts) according to both the level of chromosomal instability (CIN) and their plasma cells (PCs) differentiation stages, and to evaluate the impact of this stratification on the disease outcome. Patients & Methods: 145 newly diagnosed MM pts were included in the study. Whole-genome copy number alterations (CNAs) were analysed by SNP array both in the CD138+PCs and CD19+B-cells. In each pts, both the CD138+/CD38high PCs and CD19+B-cells compartments were characterized by 6-color multi-parameter flow cytometry analysis, combining CD138-PE, CD38-PE-Cy7, CD20-APC, CD19-APC-Cy7, CD27-FITC, CD45-FITC, CD28-APC, CD44-FITC, CD54-APC, CD81-PerCP-Cy5.5, CD56-APC. Results: According to the CD138+ PCs’ CIN, as described both by the total amount of CNAs and by the portion of genome changed (GC), three major pts subgroups were identified: the most representative, including 21/64 pts, was characterized by a higher CIN (median CNAs: 550 %GC ≥ 25) as compared to the others (intermediate and low CIN, median CNAs: 220 10≤ GC%≥25). Hyperdyploidy, but also high-risk features [i.e. 17p del (TP53)] mainly characterized CD138+ PCs with high CIN. On the contrary, in the same pts, the CD19+B-cells display a quite simple karyotype with very few microalterations (>50kb), mostly involved in the signal transduction pathway (loss on KRAS, chr12p12.1 and on SIRPB1, chr20p13). According to the the co-expression of CD19/CD81, describing the MM clone(s) differentiation status, PCs with a high level of CIN resulted more mature CD19-/CD81-. Both the high expression of CD28 and CD44 and the reduced expression of CD20, CD27 and CD45 confirmed the advanced differentiation status. Finally, although baseline clinical features of pts with more mature, genomically instable PCs, are associated to bad prognosis (e.g. PET lesions, k/l ratio, ISS III, β2-microglobulin; p<.05), they are more likely to obtain high quality response rates (≥CR) to PI induction therapy. Conclusion: High level of genomic complexity correlates with advanced PCs differentiation stages, and this is lastly associated with a prevalence of poor prognosis features. Both CIN and phenotypic pliancy represent important, yet poorly defined, mechanisms by which MM clone(s) accelerate their own evolution and survival. Acknowledgements: AIRC, AIL, HARMONY, Berlucchi Citation Format: Marina Martello, Rosalinda Termini, Enrica Borsi, Vincenza Solli, Andrea Poletti, Lucia Pantani, Serena Rocchi, Katia Mancuso, Elena Zamagni, Paola Tacchetti, Mario Arpinati, Gabriella Chirumbolo, Nicoletta Testoni, Giulia Marzocchi, Giovanni Martinelli, Michele Cavo, Carolina Terragna. Higher levels of genomic complexity correlates with an advanced plasma cell differentiation status in newly diagnosed multiple myeloma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 473.

PB2112 COEXISTENCE OF MONOCLONAL B‐ CELL POPULATION IN PATIENTS WITH NEWLY DIAGNOSED MULTIPLE MYELOMA; INCIDENCE, IMMUNOPHENOTYPING AND CLINICAL CHARACTERIZATION

Background:Clustering of B‐cell malignancies in families, and recently described genetic correlations between multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) (Went M et al; Blood Cancer Journal 2018) suggest shared biological pathways influencing the development of these malignancies. Since we began applying multiparameter flow cytometry (FACS) analysis of 8‐color panel for plasma cell dyscrasia (PCD), we observed several cases of newly diagnosed multiple myeloma (NDMM) with a co‐existing monoclonal B‐cell clone. Herein, we retrospectively review the incidence of this phenomenon and describe phenotypic and clinical characteristics.Aims:To investigate patients with NDMM and a co‐existing monoclonal B‐cell population.MethodsHospital records of all consecutive patients who underwent FACS analysis of bone marrow aspirates between January 2016 and September 2018 were reviewed and NDMM, active or smoldering, according to IMWG criteria, were identified. Cases of Waldenstrom's macroglobulinemia and lymphoplasmacytic lymphoma were excluded. FACS analysis included a PCD panel comprising CD45, CD138, CD38, CD27, CD56, CD19, and CytoKappa/Lambda. Cases that were found to have a monoclonal B‐cell population were further evaluated by a lymphoid screening tube (LST) and a B‐cell chronic lymphoproliferative disorders (BCLD) panel as proposed by EuroFlow™. Demographics, clinical characteristics, and outcomes of patients were documented from the hospital records.Results:Out of 160 consecutive FACS analyses of patients with NDMM, 13 cases (8.1%) had a coexisting monoclonal B‐cell population. In most cases, the monoclonal B‐cells had a CLL‐like phenotype (n = 8, 61.5%), whereas in the rest they were CD5‐negative (n = 5, 38.5%), with mean percentage of the monoclonal B‐cells of 5.7% (±1.7) out of all leukocytes. Patients with MM and co‐existing monoclonal B‐cells had a mean age of 72 years (range: 46‐86), 6 had smoldering and 7 had active myeloma and none of them had lymphadenopathy or splenomegaly. Involved immunoglobulin was IgG (n = 7), IgA (n = 3), IgM (n = 1) and undetected/unavailable (n = 2). PCD involved light chain was Kappa in 9 and Lambda in 4 patients. Involved free light chain level median was 332 mg/L (range 34‐9310) and M‐SPIKE median was 2.3 g/dL (range 0‐8.1). Plasma cells light chain subtype was concordant with that of the monoclonal B‐cells in 8 patients (61.5%). Hemoglobin median was 11.8 g/dL (range: 7.4‐14), absolute lymphocyte counts median was 1.7 × 109/L (1.1‐4.3). CRAB symptoms included lytic or focal lesions (n = 5), anemia (n = 5), renal failure (n = 1). Five of the 6 patients who received anti‐myeloma therapy responded. At a median follow‐up time of 9 months, none of the patients developed expansion of their B‐cell clone or progressed to an overt clinical lymphoproliferative disorder and 11 (80%) patients remain alive.Summary/Conclusion:We describe for the first time presence of a B‐cell monoclonal population in 8.1% of patients with NDMM. The clone is mostly with a CLL‐like phenotype, and did not expand or require therapy. While this incidence is slightly higher than expected, it may still be merely a coincidence reflecting prevalence of monoclonal‐B cell lymphocytosis in older population; alternatively, it may be caused by shared genetic drivers. Further investigations, including longer follow‐up and correlative molecular studies, are required.

PS1234 APPLICATION OF FLOW SOM TO CLASSICAL FLOW CYTOMETRY: UNSUPERVISED ANALYSIS OF HEMATOPOIESIS AND HEMATOLOGICAL MALIGNANCIES

Background:Multiparameter flow cytometry (MFC) is an essential tool for the diagnosis and follow‐up of hematological malignancies. Numerous panels have been devised that best allow to achieve these goals. However, analysis of MFC graphs still heavily relies on the expertise of biologists and on specifically well adapted cascade‐gating strategies. Because of the development of mass cytometry, which generates data in a multidimensional environment of up to 50 dimensions, new strategies have been developed, that could be applied to classical MFC.Aims:In this study, the capacities of a rapid new tool for multidimensional analysis, FlowSOM (self‐organizing maps) have been combined to a classical MFC analysis tool, Kaluza®, for the identification of normal and diseased human bone marrow (BM) cell subpopulations.Methods:Normal BM samples collected during bone surgery were analyzed in MFC with four antibody panels adapted to the exploration of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) respectively. The listmode files obtained were merged and submitted to FlowSOM analysis, using R packages, which yielded trees of 100 nodes defining normal BM cell subsets. FlowSOM parameters were then transferred to Kaluza® for a bidimensional representation of the trees within which selected navigation could be performed with Kaluza® tools. In a second part of the study, the same stainings were performed on myelodysplastic or leukemic (AML or ALL) samples (diagnosis or follow‐up), which were submitted to FlowSOM together with the reference merged BM.Results:Node‐by‐node selection allowed for a precise definition of each of the unsupervised subsets generated by the software. This disclosed a selective segregation of delicate subsets such as non‐classical monocytes, hematogones or stem cells. Direct simultaneous comparison of diseased samples and normal BM allowed for a precise definition of abnormal cells (blasts) at diagnosis, and their immediate recognition as minimal residual disease in follow‐up samples.Summary/Conclusion:Flow SOM appears as a new and efficient tool for a rapid identification of BM cell subsets, highlighting differences from normal BM in dysplastic of leukemic samples. The node‐by‐node approach facilitates the definition of abnormal cells and their quantification, supervision intervening only after the generation of subsets delineated by artificial intelligence.

PS1235 RED BLOOD CELL GENERATION FROM HUMAN INDUCED PLURIPOTENT STEM CELLS

Background: Multiparameter flow cytometry (MFC) is an essential tool for the diagnosis and follow-up of hematological malignancies. Numerous panels have been devised that best allow to achieve these goals. However, analysis of MFC graphs still heavily relies on the expertise of biologists and on specifically well adapted cascade-gating strategies. Because of the development of mass cytometry, which generates data in a multidimensional environment of up to 50 dimensions, new strategies have been developed, that could be applied to classical MFC. Aims: In this study, the capacities of a rapid new tool for multidimensional analysis, FlowSOM (self-organizing maps) have been combined to a classical MFC analysis tool, Kaluza®, for the identification of normal and diseased human bone marrow (BM) cell subpopulations. Methods: Normal BM samples collected during bone surgery were analyzed in MFC with four antibody panels adapted to the exploration of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) respectively. The listmode files obtained were merged and submitted to FlowSOM analysis, using R packages, which yielded trees of 100 nodes defining normal BM cell subsets. FlowSOM parameters were then transferred to Kaluza® for a bidimensional representation of the trees within which selected navigation could be performed with Kaluza® tools. In a second part of the study, the same stainings were performed on myelodysplastic or leukemic (AML or ALL) samples (diagnosis or follow-up), which were submitted to FlowSOM together with the reference merged BM. Results: Node-by-node selection allowed for a precise definition of each of the unsupervised subsets generated by the software. This disclosed a selective segregation of delicate subsets such as non-classical monocytes, hematogones or stem cells. Direct simultaneous comparison of diseased samples and normal BM allowed for a precise definition of abnormal cells (blasts) at diagnosis, and their immediate recognition as minimal residual disease in follow-up samples. Summary/Conclusion: Flow SOM appears as a new and efficient tool for a rapid identification of BM cell subsets, highlighting differences from normal BM in dysplastic of leukemic samples. The node-by-node approach facilitates the definition of abnormal cells and their quantification, supervision intervening only after the generation of subsets delineated by artificial intelligence.

Myelodysplasia as assessed by multiparameter flow cytometry refines prognostic stratification provided by genotypic risk in systemic mastocytosis.

Systemic mastocytosis (SM) is characterized by extreme heterogeneity of manifestations and prognosis. Several disease-related biomarkers, including clinical, hematological and molecular variables, have been correlated with prognosis. Although relevant, the mutation profile closely reflects the WHO classification that has per se prognostic value. High-risk mutations (HRM) are largely confined to advanced forms, and thus fail in providing information regarding progression and outcome in the not-advanced variants. In this work, we studied hematopoietic cells by multi-parameter flow cytometry (MFC) in order to highlight dysplastic traits that might provide insights into outcome. A score previously validated for myelodysplastic syndromes, with high reproducibility in standard diagnostics, was used. The application of an MFC score to a cohort of 71 SM cases, concurrently genotyped for configuring a HRM category, resulted in the identification of two separate patients' categories (MFC+ and MFC-) characterized by significantly different clinical and laboratory features at presentation. The extent of dysplasia by MFC tended to parallel WHO-category and genotype-related stratification. MFC+ patients had shorter survival compared to MFC- ones, for whom the incidence of progression and/or death was virtually null. Of note, MFC score remained prognostically informative in unadvanced subsets. Furthermore, the integration of MFC and HRM was an independent predictor for outcome, also overcoming WHO-categories in multivariate analysis for EFS. Our results support the use of MFC analysis in the evaluation of patients with SM, alone and in combination with HRM, for refinement of prognosis assessment.

BKV Clearance Time Correlates With Exhaustion State and T-Cell Receptor Repertoire Shape of BKV-Specific T-Cells in Renal Transplant Patients.

Reactivation of the BK polyomavirus is known to lead to severe complications in kidney transplant patients. The current treatment strategy relies on decreasing the immunosuppression to allow the immune system to clear the virus. Recently, we demonstrated a clear association between the resolution of BKV reactivation and reconstitution of BKV-specific CD4+ T-cells. However, which factors determine the duration of viral infection clearance remains so far unclear. Here we apply a combination of in-depth multi-parametric flow cytometry and NGS-based CDR3 beta chain receptor repertoire analysis of BKV-specific T-cells to a cohort of 7 kidney transplant patients during the clinical course of BKV reactivation. This way we followed TCR repertoires at single clone levels and functional activity of BKV-specific T-cells during the resolution of BKV infection. The duration of BKV clearance did not depend on the number of peripheral blood BKV-specific T-cells nor on a few immunodominant BKV-specific T-cell clones. Rather, the T-cell receptor repertoire diversity and exhaustion status of BKV-specific T-cells affected the duration of viral clearance: high clonotype diversity and lack of PD1 and TIM3 exhaustion markers on BKV-specific T-cells was associated with short clearance time. Our data thus demonstrate how the diversity and the exhaustion state of the T-cells can determine the clinical course of BKV infection.

MTOR Inhibitor Everolimus in Regulatory T Cell Expansion for Clinical Application in Transplantation.

Experimental and preclinical evidence suggest that adoptive transfer of regulatory T (Treg) cells could be an appropriate therapeutic strategy to induce tolerance and improve graft survival in transplanted patients. The University of Kentucky Transplant Service Line is developing a novel phase I/II clinical trial with ex vivo expanded autologous Treg cells as an adoptive cellular therapy in renal transplant recipients who are using everolimus (EVR)-based immunosuppressive regimen. The aim of this study was to determine the mechanisms of action and efficacy of EVR for the development of functionally competent Treg cell-based adoptive immunotherapy in transplantation to integrate a common EVR-based regimen in vivo (in the patient) and ex vivo (in the expansion of autologous Treg cells). CD25 Treg cells were selected from leukapheresis product with a GMP-compliant cell separation system and placed in 5-day (short) or 21-day (long) culture with EVR or rapamycin (RAPA). Multi-parametric flow cytometry analyses were used to monitor the expansion rates, phenotype, autophagic flux, and suppressor function of the cells. phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway profiles of treated cells were analyzed by Western blot and cell bioenergetic parameters by extracellular flux analysis. EVR-treated cells showed temporary slower growth, lower metabolic rates, and reduced phosphorylation of protein kinase B compared with RAPA-treated cells. In spite of these differences, the expansion rates, phenotype, and suppressor function of long-term Treg cells in culture with EVR were similar to those with RAPA. Our results support the feasibility of EVR to expand functionally competent Treg cells for their clinical use.

A Multiparameter Flow Cytometry Analysis Panel to Assess CD163 mRNA and Protein in Monocyte and Macrophage Populations in Hyperinflammatory Diseases.

CD163 facilitates regulation and resolution of inflammation and removal of free hemoglobin and is highly expressed in myeloid cells from patients with inflammatory disorders, such as systemic juvenile idiopathic arthritis (SJIA) and macrophage activation syndrome (MAS). Our recent studies indicate that regulation of CD163 mRNA expression is a key functional property of polarized monocytes and macrophages and is mediated at the transcriptional and posttranscriptional level, including via microRNAs. The goal of the current study is to develop a multiparameter flow cytometry panel incorporating detection of CD163 mRNA for polarized monocyte and macrophage populations in disorders such as SJIA and MAS. THP-1 cells and CD14+ human monocytes were stained using fluorochrome-conjugated Abs to myeloid surface markers, along with CD163 mRNA. Staining for mRNA could reliably detect CD163 expression while simultaneously detecting different macrophage populations using Abs targeting CD14, CD64, CD80, CD163, and CD209. This approach was found to be highly sensitive for increased mRNA expression when macrophages were polarized with IL-10 [M(IL-10)], with a strong signal over a broad range of IL-10 concentrations, and showed distinct kinetics of CD163 mRNA and protein induction upon IL-10 stimulation. Finally, this panel demonstrated clear changes in polarization markers in unstimulated monocytes from patients with SJIA and MAS, including upregulated CD163 mRNA and increased CD64 expression. This approach represents a robust and sensitive system for RNA flow cytometry, useful for studying CD163 expression as part of a multimarker panel for human monocytes and macrophages, with broad applicability to the pathogenesis of hyperinflammatory diseases.

IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation

IL-17E (IL-25) is a member of the IL-17 cytokine family involved in the promotion of type 2 immune responses. Recently, IL-17E has been reported to be up-regulated in distinct skin inflammatory diseases such as psoriasis and atopic and contact dermatitis. We assessed the role played by IL-17E in skin inflammation. Here, we show that IL-17E induces skin inflammation invivo, characterized by the expression of innate immune response genes and the recruitment of innate immune cells, particularly neutrophils. Genetic deletion or IL-17E neutralization ameliorated skin inflammation induced by imiquimod application or tape stripping, with reductions in neutrophil and macrophage infiltration as assessed by t-distributed stochastic neighbor embedding-guided multiparameter flow cytometry analysis, in mice. In humans, IL-17E promotes the recruitment of neutrophils via activation of macrophages in a p38-dependent mechanism. In addition, IL-17E is up-regulated in neutrophil-rich inflammatory skin diseases, such as pyoderma gangrenosum and acute generalized exanthematous pustulosis. Our data show a role for IL-17E in skin inflammation that is unrelated to the development of type 2 immune reactions. We propose that IL-17E is an important common denominator of chronic skin inflammation, promoting innate immune cell recruitment and activation.

Haploidentical Transplantation with Regulatory and Conventional T Cells Improves Outcome of Patients Affected By Acute Myeloid Leukemia with Complex Karyotype and/or Monosomy 7/Del(7q)

▪Acute myeloid leukemias (AML) with complex karyotype (≥ 3 abnormalities) and/or monosomy 7/del(7q) are a subset of AML with extremely poor prognosis. Hematopoietic stem cell transplantation (HSCT) is the only potentially curative treatment, but relapse rate is high (most often above 50%) and negatively impacts on patients' survival. (Ciurea et al. Cancer 2018) Such poor results are even questioning the role of HSCT and rise the need of new transplant procedures for these patients. To this end, we are performing a clinical trial of haploidentical HSCT with adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) in the absence of post-transplant pharmacologic immune suppression (Treg-Tcon haplo-HSCT). Such approach allows for an extremely low relapse rate in patients with high-risk acute leukemias transplanted in complete remission. (Martelli et al. Blood 2014)In this study we analyzed the outcome of all the patients that were referred to our center from January 1st 2007 to May 31st 2018 with a diagnosis of AML with complex karyotype and/or monosomy 7/del(7q) and that were fit for receiving an induction treatment.49 patients were included. Median age at diagnosis was 52; 22 were female, 27 were male. 20/49 had chromosome 7 abnormalities (isolated in 12 patients; in a complex karyotype in 8 patients). 11 patients received a hypomethylating agent as first line treatment, while the other 38 patients received high-dose chemotherapy. We performed 30 HSCT procedures in 25 patients; 5 patients received a second transplant (4 after disease relapse and 1 after rejection). 6 underwent a HLA-matched HSCT, 6 a T-depleted haploidentical HSCT, and 18 a Treg-Tcon haplo-HSCT. Median age at transplant was 42 with a median follow up of 40 months.Regarding disease status at transplant, 26 HSCT were performed in patients that were in 1st or 2nd hematological remission and 4 in patients with refractory disease. Cytogenetic analysis (karyotype and/or FISH analysis) revealed the presence of abnormal clones in 11 patients and multiparametric flow cytometry analysis detected minimal residual disease in 16 patients. Thus, most of the patients (17/30, 57%) were transplanted with detectable disease.15/49 patients were disease-free and alive at the time of the analysis, all of them after receiving HSCT (HSCT vs Non-HSCT, p<0.01, Fig. A). Treg-Tcon haplo-HSCT allowed for a markedly better disease free survival as compared to the other transplant procedures (p=0.01, Fig. B). Such survival advantage was only due to a reduced rate of disease relapse (31% vs 79%, p=0.02, Fig. C), as transplant related mortality was similar between the 2 groups (9% vs 9%). 17/18 Treg-Tcon haplo-HSCT patients engrafted and only 2/17 developed acute GvHD grade ≥ 2. 4/17 patients relapsed and none of them received a transplant from a NK alloreactive donor. (Ruggeri et al. Science 2002) Multivariate analysis that included chromosome 7 involvement, presence of disease at multiparametric flow cytometry and/or cytogenetic analysis, and blast count at transplant confirmed that Treg-Tcon haplo-HSCT was the only predictor of a better survival.In conclusion, our results not only confirm that HSCT is the only potentially curative treatment for AML with complex karyotype and/or chromosome 7 involvement, but also demonstrate that Treg-Tcon haplo-HSCT allows for a strong antileukemic effect, the only needed for a long term control of such unfavorable AMLs with high disease burden at transplant. While it is necessary to confirm these results in a larger cohort of patients, Treg-Tcon haplo-HSCT could represent the best transplant option for this subset of very high-risk AML patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Distinct Alterations in the Composition of Mucosal Innate Lymphoid Cells in Newly Diagnosed and Established Crohn's Disease and Ulcerative Colitis.

Innate lymphoid cells [ILC] have been suggested to play a role in inflammatory bowel disease [IBD]. Here, we investigated the ILC compartment in intestinal biopsies and blood from distinct patient groups with Crohn's disease [CD] and ulcerative colitis [UC], either newly diagnosed or with disease established for at least 1 year. This approach allowed us to simultaneously investigate temporal, disease-specific, and tissue-specific changes in ILC composition in IBD. ILC subset frequencies, phenotype, and transcription factor profile in blood and intestinal biopsies were investigated by multi-parameter flow cytometry analysis. Endoscopic disease severity was judged using the ulcerative colitis endoscopic index of severity and the simple endoscopic score for Crohn's disease. The frequency of NKp44+ILC3 was decreased in inflamed tissue, both in patients with CD and those with UC, already at the time of diagnosis, and correlated with disease severity. Simultaneously, the frequency of ILC1 was increased in patients with CD, whereas the frequency of ILC2 was increased in patients with UC. However, in patients with established UC or CD, both ILC1 and ILC2 were increased. In contrast to the ILC composition in inflamed tissue, ILC in non-inflamed tissue or blood were unchanged compared with non-IBD controls. Finally, in patients undergoing treatment with an anti-α4β7 antibody the frequencies of ILC in peripheral blood remained unchanged. We report both shared and distinct changes in ILC composition depending on diagnosis and disease duration. The alterations in ILC composition in IBD occur selectively at inflamed sites in the gut.

IMMU-70. GLOBAL IMMUNE FINGERPRINTING IN GLIOBLASTOMA REVEALS IMMUNE-SUPPRESSION SIGNATURES ASSOCIATED WITH PROGNOSIS

Glioblastoma (GBM) remains uniformly lethal, and, despite a large accumulation of immune cells in the microenvironment, there is limited anti-tumor immune response, even with newly developed immune checkpoint therapies. To overcome these challenges and enhance the efficacy of immunotherapies, a comprehensive understanding of the immune system in GBM and changes during disease progression is required. Here, we integrated multi-parameter flow cytometry and mass cytometry time of flight (CyTOF) analysis of patient blood to determine changes in the immune system among tumor types and over disease progression. Utilizing multi-parameter flow cytometry analysis in a cohort of over 250 patients with brain tumors ranging from benign to malignant primary and metastatic, we found that GBM patients had a significant elevation in myeloid-derived suppressor cells (MDSCs) in blood, but not immunosuppressive T regulatory cells. We validated these findings in GBM patient tissue and found that increased numbers of MDSCs in recurrent GBM portended poor prognosis. CyTOF analysis of peripheral blood from a cohort of newly diagnosed GBM patients revealed that reduction in MDSC frequency over time is accompanied by a concomitant increase in dendritic cells and natural killer cells. This reduced MDSC profile was present in GBM patients with extended survival and was similar to that of low-grade glioma (LGG) patients. Our findings provide a rationale for developing strategies to target MDSCs, which are elevated in GBM patients and predict poor prognosis, either by directly targeting or by shifting the immune profile to induce differentiation toward the immune profile of LGGs.

Bilinear Staphylococcus aureus detection based on suspension immunoassay

A novelty bilinear suspension immunoassay biosensor is developed for Staphylococcus aureus specific detection. In the present study, a dual-color-based sandwich immunosensor was constructed for sensitive and selective detection of this bacterium. In this bioassay system, the monoclonal antibodies of Staphylococcus aureus were immobilized on carboxyl-modified fluorescent microspheres (PSA-R6G) which acted as a capture probe. A secondary fluorescein isothiocyanate (FITC)-labelled antibody of Staphylococcus aureus acted as a sensitive reporter antibody. After dual-labelling with R6G and FITC, the enriched Staphylococcus aureus bacteria were observed by using the multiparameter flow cytometry analysis. In this method, two regression equations were obtained with IFITC = 1.56lgC + 4.50 and IR6G = 1.54lgC + 2.78, respectively. It can be noted that the two slopes were very similar, which indicated the false positives decrease significantly. For general applications, the quantificational measurements of Staphylococcus aureus in milk and water samples were also carried out. The suspension immunoassay exhibited an excellent specificity to Staphylococcus aureus in contrast to conventional culture-based method.

Abstract 5616: Rituximab chemotherapy induces a partial recovery from T-cell exhaustion in follicular lymphoma patients

Abstract For many years, the clinical responses observed in cancer patients treated with antitumor antibodies have been viewed only as a consequence of the recruitment and activation of innate immune cells. Several preclinical studies have indicated that anti-tumor antibodies can also induce a potent anti-tumor adaptive T-cell response capable of protecting animals from tumor challenge. Our studies have shown that CD4+ Th1 cells play a central role in the response to anti-CD20 tumor therapy by opposing Treg expansion and that CD20-specific memory T cells are responsible for anti-tumor protection observed in surviving animals subjected to tumor rechallenge. Whether an adaptive T-cell response is elicited in patients with Follicular Lymphoma (FL) receiving anti-CD20-based treatment is not known. In the present work, we analyzed peripheral T cells before and during anti-CD20-chemotherapy treatment of FL patients (R-CHOP) with high tumor burden. We first examined in ELISPOT assays the IFN-γ production by PBMC from 20 patients against human CD20-derived peptides previously selected by combining in silico and in vivo screening using transgenic human HLA-DR mice. Both FL patients before treatment and patients undergoing R-CHOP treatment exhibited peripheral T-cell responses to the selected CD20-derived peptides. However, these responses were markedly lower than those observed in healthy donors. Similar results were also obtained when T cell responses against viruses commonly infecting large numbers of individuals (CMV, EBV, influenza) were assessed, suggesting that high tumor burden FL patients exhibit an immune exhaustion of T cells. Multiparametric flow cytometry analyses suggested that their poor ability to respond to viral and CD20-derived peptides is related to the presence of an immunocompromised and exhausted blood T-cell peripheral compartment with higher percentages of TIGIT- and PD-1-expressing T cells, of peripheral Treg, and terminally differentiated CD8+ TEMRA. R-CHOP therapy induced a shift of CD4+ and CD8+ T cells toward a central memory phenotype (TCM), an additional shift of CD8+ T cells to a naive phenotype, and a decrease in the number of PD-1+ and TIGIT+ T cells, making these cells evolving toward a lesser degree of exhaustion. However, it did not restore a level of specific responses similar to those observed in healthy donors. These results support the use of anti-PD-1 and anti-TIGIT antibodies in combination with anti-CD20 in FL patients with high-tumor burden. Citation Format: Benoit Milcent, Pauline Brice, Catherine Thieblemont, Jean-Luc Teillaud, Sophie Siberil. Rituximab chemotherapy induces a partial recovery from T-cell exhaustion in follicular lymphoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5616.

Bi-specific ligand-controlled chimeric antigen receptor T-cell therapy for non-small cell lung cancer.

Our goal is to develop a switch-controlled approach to enable better control of reactivity and safety of chimeric antigen receptor (CAR)-T therapy for non-small-cell lung cancer (NSCLC). Lentiviral transduction was performed to generate anti-FITC CAR-T cells and target cells stably expressing either isoform of the folate receptor. Colorimetric-based cytotoxic assay, enzyme-linked immunosorbent assay, and multiparametric flow cytometry analysis were used to evaluate the specificity and activity of CAR-T cells in vitro. Human primary T cells stably expressing the fully human anti-FITC CAR were generated. Anti-FITC CAR-T cells displayed antigen-specific and folate-FTIC dependent reactivity against engineered A549-FRα and THP-1-FRβ. The selective activation and proliferation of anti-FITC CAR-T cells in vitro stringently relied on the co-existence of folate-FITC and FR- expressing target cells and was dose-titratable with the folate-FITC switch. The excellent in vitro efficacy and specificity of an adaptor-controlled CAR-T therapy to target both tumor cells and tumor-associated macrophages in NSCLCs were validated.

Residual Peripheral Blood CD26+ Leukemic Stem Cells in Chronic Myeloid Leukemia Patients During TKI Therapy and During Treatment-Free Remission.

Chronic myeloid leukemia (CML) patients in sustained “deep molecular response” may stop TKI treatment without disease recurrence; however, half of them lose molecular response shortly after TKI withdrawing. Well-defined eligibility criteria to predict a safe discontinuation up-front are still missing. Relapse is probably due to residual quiescent TKI-resistant leukemic stem cells (LSCs) supposedly transcriptionally low/silent and not easily detectable by BCR-ABL1 qRT-PCR. Bone marrow Ph+ CML CD34+/CD38− LSCs were found to specifically co-express CD26 (dipeptidylpeptidase-IV). We explored feasibility of detecting and quantifying CD26+ LSCs by flow cytometry in peripheral blood (PB). Over 400 CML patients (at diagnosis and during/after therapy) entered this cross-sectional study in which CD26 expression was evaluated by a standardized multiparametric flow cytometry analysis on PB CD45+/CD34+/CD38− stem cell population. All 120 CP-CML patients at diagnosis showed measurable PB CD26+ LSCs (median 19.20/μL, range 0.27–698.6). PB CD26+ LSCs were also detectable in 169/236 (71.6%) CP-CML patients in first-line TKI treatment (median 0.014 cells/μL; range 0.0012–0.66) and in 74/112 (66%), additional patients studied on treatment-free remission (TFR) (median 0.015/μL; range 0.006–0.76). Notably, no correlation between BCR-ABL/ABLIS ratio and number of residual LSCs was found both in patients on or off TKIs. This is the first evidence that “circulating” CML LSCs persist in the majority of CML patients in molecular response while on TKI treatment and even after TKI discontinuation. Prospective studies evaluating the dynamics of PB CD26+ LSCs during TKI treatment and the role of a “stem cell response” threshold to achieve and maintain TFR are ongoing.

Maturation of cord blood natural killer (NK) cells differs among neonates

Abstract The source, timing of colonization, and stability of the neonatal microbiome is largely unknown. We and other groups recently demonstrated that infant colonization may begin in utero, challenging the notion that neonates are born sterile. However, neonates are particularly vulnerable to viral infection partially due to dampened responses from NK cells and delayed maturation of adaptive immunity. Given the degree of variability in neonatal vulnerability to infection, we hypothesized that intrauterine exposure to microbes enables a greater diversity of NK cells in the neonates, relative to the adult, to allow for immune tolerance. Here, we performed multi-parametric flow cytometry analyses to dissect the normal range and expression of human NK cell developmental markers from umbilical cord (UC) blood (n=37). NK cells were identified as CD45+ CD3− CD56+ using FlowJo X and Cytobank. UC blood had a lower frequency of mature NK cells than adult peripheral blood. Furthermore, examination of NK cell maturation via spanning analysis of density events (SPADE) highlighted inter-individual diversity of neonatal NK cell subsets. Diversity of NK cells subsets between neonates and adults was significantly different with higher diversity in neonates (Simpson’s Diversity Index, p = 0.008). Our findings suggest diversity in stages of NK cell development between individual neonates that may reflect early immune exposure. Given ours and others recent work demonstrating that the intrauterine environment is not sterile, we speculate that microbial exposures during gestation may be a driving difference in the diversification NK cell populations in human infants and hence enable immune tolerance at the risk of neonatal viral vulnerability.

The dinoponeratoxin peptides from the giant ant Dinoponera quadriceps display in vitro antitrypanosomal activity.

The crude venom of the giant ant Dinoponera quadriceps is a cocktail of polypeptides and organic compounds that shows antiparasitic effects against Trypanosoma cruzi, the causative agent of Chagas disease. In order to investigate the venom-derived components responsible for such antitrypanosomal activity, four dinoponeratoxins (DnTxs) were identified, namely M-PONTX-Dq3a, -Dq3b, -Dq3c and -Dq4e, that are diverse in size, net charge, hydrophobicity and propensity to interact with eukaryote cell membranes. These peptides were tested against epimastigote, trypomastigote and amastigote forms of benznidazole (Bz)-resistant Y strain of T. cruzi and in mammalian host cells. The M-PONTX-Dq3a and -Dq4e inhibited all developmental forms of T. cruzi, including amastigotes, the responsible form for the maintenance of infection on chronic phase of the disease. The M-PONTX-Dq3a showed the highest selectivity index (SI) (80) and caused morphological alterations in T. cruzi, as observed by scanning electron microscopy (SEM), and induced cell death through necrosis, as seen by multiparametric flow cytometry analysis with specific biochemical markers. Altogether, the D. quadriceps venom appears as a source for the prospection of trypanocidal peptides and the M-PONTX-Dq3a arises as a candidate among the dinoponeratoxin-related peptides in the development of compounds against Chagas disease.

284: Maturation of cord blood natural killer (NK) cells differs among neonates

The source, timing of colonization, and stability of the neonatal microbiome is largely unknown, and it remains unclear how microbes influence early immunity. We and others have recently shown that infant colonization may begin in utero, challenging the notion that neonates are born sterile. However, some neonates are particularly vulnerable to viral infection, which is thought to be due to dampened responses from NK cells and delayed maturation of adaptive immunity. Given the degree of variability in neonatal vulnerability to infection, we hypothesized that intrauterine exposure to microbes in the more common non-sterile environment actually enables a greater diversity of NK cells in the neonates relative to the adult to allow for immune tolerance. Here, we performed the largest multi-parametric flow cytometry analyses to dissect the normal range and expression of human NK cell developmental markers from umbilical cord (UC) blood. UC blood was collected at delivery in a large number of neonates (n=35). Whole blood was stained with NK cell maturation markers and data was acquired by flow cytometry. Data was analyzed using FlowJo X and Cytobank. Spanning analysis density of events (SPADE) analysis was applied to identify NK cell developmental subsets by individual. Diversity between neonates and adults was calculated using Simpson’s Diversity Index. NK cells were identified in UC blood as CD3−CD45+CD56+. UC blood had a lower frequency of mature NK cells than adult peripheral blood (Fig. A) Furthermore, examination of NK cell maturation via SPADE analysis identified highlighted inter-individual diversity of neonatal NK cell subsets (Fig. B). Diversity of NK cells subsets between neonates and adults was significantly different with higher diversity in neonates (Fig. C, p = 0.008). Our findings suggest diversity in stages of NK cell development between individual neonates that may reflect early immune exposure and harken viral susceptibility. Given ours and others recent work demonstrates that the intrauterine environment is not sterile, we speculate that microbial exposures during gestation may be a driving difference in the diversification NK cell populations in human infants and hence enable immune tolerance at the risk of neonatal viral vulnerability.