Multiparameter Flow Cytometry Analysis Research Articles

Immunophenotypic characterization of leukemic stem cells in acute myeloid leukemia using single tube 10-colour panel by multiparametric flow cytometry: Deciphering the spectrum, complexity and immunophenotypic heterogeneity.

Despite extensive research, comprehensive characterization of leukaemic stem cells (LSC) and information on their immunophenotypic differences from normal haematopoietic stem cells (HSC) is lacking. Herein, we attempted to unravel the immunophenotypic (IPT) characteristics and heterogeneity of LSC using multiparametric flow cytometry (MFC) and single-cell sequencing. Bone marrow aspirate samples from patients with acute myeloid leukaemia (AML) were evaluated using MFC at diagnostic and post induction time points using a single tube-10-colour-panel containing LSC-associated antibodies CD123, CD45RA, CD44, CD33 and COMPOSITE (CLL-1, TIM-3, CD25, CD11b, CD22, CD7, CD56) with backbone markers that is, CD45, CD34, CD38, CD117, sCD3. Single-cell sequencing of the whole transcriptome was also done in a bone marrow sample. LSCs and HSCs were identified in 225/255 (88.2%) and 183/255 (71.6%) samples, respectively. Significantly higher expression was noted for COMPOSITE, CD45RA, CD123, CD33, and CD44 in LSCs than HSCs (p < 0.0001). On comparing the LSC specific antigen expressions between CD34+ (n = 184) and CD34- LSCs (n = 41), no difference was observed between the groups. More than one sub-population of LSC was demonstrated in 4.4% of cases, which further revealed high concordance between MFC and single cell transcriptomic analysis in one of the cases displaying three LSC subpopulations by both methods. A single tube-10-colour MFC panel is proposed as an easy and reproducible tool to identify and discriminate LSCs from HSCs. LSCs display both inter- and intra-sample heterogeneity in terms of antigen expressions, which opens the facets for single cell molecular analysis to elucidate the role of subpopulations of LSCs in AML progression.

A GC-MS-based untargeted metabolomics approach for comprehensive metabolic profiling of mycophenolate mofetil-induced toxicity in mice.

Background: Mycophenolate mofetil (MMF), the morpholinoethyl ester of mycophenolic acid, is widely used for maintenance immunosuppression in transplantation. The gastrointestinal toxicity of MMF has been widely uncovered. However, the comprehensive metabolic analysis of MMF-induced toxicity is lacking. This study is aimed to ascertain the metabolic changes after MMF administration in mice. Methods: A total of 700mg MMF was dissolved in 7mL dimethyl sulfoxide (DMSO), and then 0.5mL of mixture was diluted with 4.5mL of saline (100mg/kg). Mice in the treatment group (n = 9) were given MMF (0.1mL/10g) each day via intraperitoneal injection lasting for 2weeks, while those in the control group (n = 9) received the same amount of blank solvent (DMSO: saline = 1:9). Gas chromatography-mass spectrometry was utilized to identify the metabolic profiling in serum samples and multiple organ tissues of mice. The potential metabolites were identified using orthogonal partial least squares discrimination analysis. Meanwhile, we used the MetaboAnalyst 5.0 (http://www.metaboanalyst.ca) and Kyoto Encyclopedia of Genes and Genomes database (http://www.kegg.jp) to depict the metabolic pathways. The percentages of lymphocytes in spleens were assessed by multiparameter flow cytometry analysis. Results: Compared to the control group, we observed that MMF treatment induced differential expression of metabolites in the intestine, hippocampus, lung, liver, kidney, heart, serum, and cortex tissues. Subsequently, we demonstrated that multiple amino acids metabolism and fatty acids biosynthesis were disrupted following MMF treatment. Additionally, MMF challenge dramatically increased CD4+ T cell percentages but had no significant influences on other types of lymphocytes. Conclusion: MMF can affect the metabolism in various organs and serum in mice. These data may provide preliminary judgement for MMF-induced toxicity and understand the metabolic mechanism of MMF more comprehensively.

Comparison of Measurable Residual Disease in Pediatric B-Lymphoblastic Leukemia Using Multiparametric Flow Cytometry and Next-Generation Sequencing.

Measurable residual disease (MRD) testing, a standard procedure in B-lymphoblastic leukemia (B-ALL) diagnostics, is assessed using multiparametric flow cytometry (MFC) and next-generation sequencing (NGS) analysis of immunoglobulin gene rearrangements. We evaluated the concordance between eight-color, two-tube MFC-MRD the LymphoTrack NGS-MRD assays using 139 follow-up samples from 54 pediatric patients with B-ALL. We also assessed the effect of hemodilution in MFC-MRD assays. The MRD-concordance rate was 79.9% (N=111), with 25 (18.0%) and 3 (2.2%) samples testing positive only by NGS-MRD (MFC-NGS+MRD) and MFC-MRD (MFC+NGS-MRD), respectively. We found a significant correlation in MRD values from total nucleated cells between the two methods (r=0.736 [0.647-0.806], P<0.001). The median MRD value of MFC-NGS+MRD samples was estimated to be 0.0012% (0.0001%-0.0263%) using the NGS-MRD assays. Notably, 14.3% of MFC-NGS+MRD samples showed NGS-MRD values below the limit of detection in the MFC-MRD assays. The percentages of hematogones detected in MFC-MRD assays significantly differed between the discordant and concordant cases (P<0.001). MFC and NGS-MRD assays showed relatively high concordance and correlation in MRD assessment, whereas the NGS-MRD assay detected MRD more frequently than the MFC-MRD assay in pediatric B-ALL. Evaluating the hematogone percentages can aid in assessing the impact of sample hemodilution.

Lymphoma-Derived IL-10 Is a Key Immunomodulatory Factor at the Tumor Microenvironment of Activated B-Cell Diffuse Large B-Cell Lymphoma and Influences In Vivo Responses to Immunotherapy

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of aggressive non-Hodgkin's lymphoma with ~40% of patients experiencing refractory or relapsed disease to R-CHOP immunochemotherapy. These higher risk patients often exhibit gene expression profiles that resemble a late germinal center-derived activated B-cell (ABC) phenotype, where diverse specific mutations in genes along BCR and TLR signaling pathways converge into constitutive activation of the NF-κB pathway. Identification of factors that contribute to the survival of malignant ABC-DLBCL cells is crucial for designing specific interventions. In this setting, previous in vitro studies in DLBCL cell lines have evidenced that IL-10 is a direct target of NF-κB signaling that sustains lymphoma cell survival by an auto-stimulatory loop via STAT3 signaling. However, the action of IL-10 could be more complex within the lymphoma microenvironment, as IL-10 can have both stimulatory and inhibitory effects depending on the cellular source of IL-10, the timing of its secretion, and the type of immune cells that receives signals via IL-10 receptor (IL-10R). To explore in vivo the relevance of lymphoma-derived IL-10, we have generated a novel quintuple transgenic mouse model that we call pBIC10, after crossing our previous ABC-DLBCL-like pBIC mice carrying constitutive NF-κB signaling (Pascual et al., Blood 2019) to mice with a floxed Il10 gene for conditional knock-out of IL-10 specifically in malignant ABC-DLBCL cells. As expected, increased intracellular IL-10 signaling was detected in primary murine DLBCL cells from pBIC mice but not in IL-10 deficient pBIC10 mice, which promoted lymphoma cell survival ex vivo as revealed by perturbation studies of the IL-10/JAK1/STAT3 autocrine loop with blocking monoclonal antibodies or selected inhibitors. However, in vivo genetic deletion of lymphoma-derived IL-10 did not offer improved overall survival but, on the contrary, notably accelerated DLBCL progression in pBIC10 mice (Figure 1A, left). A comprehensive integration of multiparametric flow cytometry and transcriptomic analyses using bulk and single-cell RNA-seq of lymphoma cells and the tumor microenvironment coupled with BCR- and TCR-seq, identified lymphoma-derived IL-10 as a key immunomodulator of the DLBCL microenvironment, including unexpected protective paracrine functions in the progression of DLBCL. By comparing IL-10-proficient (pBIC) and -deficient (pBIC10) murine DLBCL models, we observed a multifaceted role of IL-10 produced by DLBCL cells, enabling immune-effector processes (facilitating immune chemotaxis, antigen presentation, and IFNγ responses) that yielded higher percentages of long-lived stem-like CD8 +TCF-1 +PD-1 lo/int T cells and dendritic cells, while restraining PD-1/LAG3-driven T-cell exhaustion and reducing immunosuppression by CD4 +CD25 +FOXP3 + regulatory T (Treg) cells. Thus, depletion of lymphoma-derived IL-10 decreased T-cell cytotoxicity and increased Treg-driven immunosuppression, which collectively accelerated lymphoma development. Consequently, T-cell targeted immune-checkpoint therapy (with anti-PD-1 antibodies) was effective only in IL-10-proficient DLBCL but not in IL-10-deficient pBIC10 mice (Figure 1A, right). Further comparative studies between pBIC and pBIC10 mice showed that tumor vascularization and expression of B-cell calcium channels, which have been previously associated with sensitivity to rituximab, were counteracted by lymphoma-derived IL-10. Accordingly, anti-CD20-based targeting of lymphoma cells (with rituximab surrogate antibodies) showed best in vivo responses in IL-10-deficient DLBCL mice with respect to pBIC mice (Figure 1A, right). Consistent with this preclinical data, analyses of transcriptional IL-10-associated hallmarks to stratify DLBCL patients (Schmitz et al., NEJM 2018) identified better clinical responses to R-CHOP in patients with pBIC10-like DLBCL phenotype (Figure 1B). Collectively, our study provides new functional and mechanistic insights into the role of lymphoma-derived IL-10 in the pathogenesis of DLBCL and the restraint of an immunosuppressed tumor microenvironment, which holds predictive potential as a biomarker for immunotherapy responses in DLBCL.

Unraveling Pathophysiology and Hematopoiesis of Vexas Syndrome By Multi-Omics Analyses and Targeted Gene Editing

Background and Rationale VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a newly described, treatment-refractory, severe, highly prevalent disease (~1:4,000 males aged &amp;gt;50 years) caused by somatic mutations in the UBA1 (ubiquitin-activating enzyme 1) gene of hematopoietic stem/progenitor cells (HSPCs). VEXAS is hallmarked by vacuoles in myeloid/erythroid precursors and presents with systemic inflammation. About 50% patients develop hematologic malignancies, including myelodysplasia, contributing to the poor prognosis. The pathophysiology and impact of VEXAS syndrome on human hematopoiesis remain elusive. Moreover, the lack of suitable disease models hampers investigation of disease development and preclinical drug testing. To fill these gaps, here we: i) integrated multiparametric flow cytometry and multi-omics analyses on VEXAS patients to achieve comprehensive molecular and phenotypic characterization of hematopoiesis; and ii) successfully developed in vitro and in vivo models of VEXAS syndrome by cutting-edge targeted base editing strategies. Methods Multiparametric immunophenotypic analyses and single-cell transcriptomics were performed on peripheral blood and bone marrow (BM) cells from six VEXAS patients (p.Met41&amp;gt;Thr; p.Met41&amp;gt;Val; p.Met41&amp;gt;Leu; c.118-1 G&amp;gt;C) recruited by our Unit. Circulating monocytes were analyzed by whole RNA-sequencing (RNA-seq) and metabolomics. UBA1 mutations were introduced by gene editing technologies in myeloid cell lines, primary T cells and healthy human HSPCs. Engineered HSPCs were used to reconstitute the hematopoietic compartment of immunodeficient mice to generate a humanized VEXAS model. Variant allele frequency of UBA1 mutant cells was quantified by targeted sequencing in isolated hematopoietic lineages and HSPCs. Results i) Multiparametric immunophenotypic analyses of BM cells showed unbalanced composition of the HSPC compartment in VEXAS patients compared to age-matched healthy individuals, with lower abundance of stem cells, multipotent and lymphoid progenitors and expanded myeloid progenitors. This was paralleled by a significant increase of circulating myeloid-biased HSPCs and immature myeloid cells. Transcriptomic analyses of patient-derived peripheral monocytes showed sustained VEXAS-associated inflammatory signatures and heightened glycolysis, which were confirmed by metabolomic profiling. Single-cell transcriptomics on the BM hematopoietic compartment of VEXAS patients harboring different UBA1 mutations displayed genotype-dependent gene expression patterns. Single-cell RNA-seq of peripheral blood mononuclear cells from VEXAS patients is ongoing. ii) In vitro and in vivo models of VEXAS, generated by UBA1 gene editing, faithfully recapitulated patient hematopoiesis and pathophysiology . Editing efficiency approached 90% in wild-type myeloid cell lines, primary human T cells and HSPCs. While UBA1-mutant T cells were counterselected over time in culture, myeloid ones were enriched. Colony forming unit assays of UBA1-edited HSPCs revealed an exclusive myeloid output in vitro and a significant loss of the erythroid HSPC differentiation potential compared to mock-edited controls. Transplantation of edited HSPCs in immunodeficient mice resulted in a 100-fold reduction in circulating B cells, while NK and myeloid compartments were preserved. Human BM HSPCs were 5-fold lower than in control mice, largely myeloid-biased, and displayed abnormal vacuolar morphology. Concordantly, while myeloid cells and HSPCs were mainly (&amp;gt;80%) UBA1 mutated, the lymphoid compartment showed predominance of wild-type alleles. Strikingly, the distribution of UBA1-mutant cells across lineages in humanized mice mirrored the composition of our VEXAS patient cohort, where UBA1-mutant cells accounted for 90% of total myeloid cells and HSPCs and for less than 5% of lymphoid lineages. Conclusions Our findingsprovide evidence thatmutations in UBA1 drive accumulation of myeloid-biased HSPCs and immature myeloid precursors. Mutant lymphoid cells are negatively selected and their peripheral myeloid counterpart displays inflammatory activation. Gene editing-based in vitro and in vivo models phenocopy most hematopoietic features of the disease and hold promise to enable preclinical testing and validation of novel therapeutics to treat VEXAS syndrome.

Evaluation of ICT01, a γ9δ2 T Cell-Activating Monoclonal Antibody, Combined with Venetoclax and Azacitidine in 1L AML (EVICTION Study)

Background: γ9δ2 T cells are emerging as a novel tumor-infiltrating effector cell that harbors strong cytolytic and pro-inflammatory activities, and whose intratumoral presence is associated with a favorable prognosis across solid and liquid cancer patients (Gentles et al., 2015; Tosolini et al., 2017). ICT01, a first-in-class anti-BTN3AmAb activating γ9δ2 T cells, completed Phase 1 testing in relapsed/refractory (r/r) solid tumors as monotherapy and in combination with pembrolizumab, and as monotherapy in r/r AML and lymphoma (EVICTION-NCT04243499). ICT01 activates circulating γ9δ2 T, CD8 T, and NK cells that leads to tumor infiltration and remodeling of the TME (De Gassart et al., 2021; Wermke et al., 2021) without any dose-limiting toxicities and a good safety profile. Encouraging clinical activity was observed in the hematologic cancer cohort with a 30% DCR on the 10 patients evaluable at week 8 or beyond (Garciaz, SITC 2023) supporting further clinical testing of ICT01 in combination with Venetoclax (VEN) and 5-Azacytidine (AZA), considered the standard of care for patients with newly diagnosed AML who are not candidates for intensive chemotherapy. Both ICT01 + γ9δ2 T cells and VEN/AZA induce killing of AML blasts, while VEN/AZA has been shown to enhance T-cell- and NK-cell-mediated cytotoxicity against AML blasts, suggesting that the combination of ICT01 plus VEN/AZA could lead to greater efficacy. We used preclinical models to demonstrate the ability of VEN/AZA to potentiate the anti-leukemic activity of ICT01-activated γ9δ2 T cells to support the clinical evaluation of ICT01 + VEN/AZA in 1L AML patients eligible to receive VEN/AZA. Results: Preclinical in vitro studies showed that ICT01-mediated activation of g9d2 T cells significantly increased survival against VEN toxicity by &amp;gt;50%, as compared to untreated control (p&amp;lt;0.01). Furthermore, killing assays performed by co-culture of AML blasts and HD-PBMC demonstrated that VEN/AZA enhances the blast killing capacity of ICT01-activated g9d2 T cells. These results were confirmed in vivo in NSG mice engrafted with human AML that were adoptively transferred with human g9d2 T cells and treated with VEN (40mg/kg/day OG)/AZA (2mg/kg/day IP), ICT01 (1mg/kg IV) or the combination. In this model the use of ICT01 together with VEN/AZA significantly delayed tumor growth (p&amp;lt;0.001) and significantly prolonged median survival to 42.5 days (vs 28 and 32.5 days for animals receiving ICT01 or VEN/AZA, respectively) (Figure 1). These results led to the design of the recently initiated Phase 2a arm of EVICTION in 1L AML where ICT01 is given at 10 mg or 75 mg IV Q4W on Day 1 of the 28-day approved regimen of VEN/AZA until toxicity or disease progression. Up to 25 patients will be treated at each dose level, following an interim analysis after 10 pts are treated at each dose. Clinical sites in France, Spain and the USA are participating. Biomarkers include multiparametric flow cytometry analysis of g9d2 T cells and other immune cells, and BTN3A expression in peripheral blood and in bone marrow aspirates at baseline and on Day 21-28. Circulating cytokine levels (including IFNγ, TNFa, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17a and MCP-1) are also assessed with MSD technology. Clinical response is evaluated per AML-specific standards (e.g., Cheson/IWG Criteria), with a target CR rate of &amp;gt;40%. Preliminary safety, biomarker and efficacy data from patients will be presented. Conclusion: Encouraging results obtained in the EVICTION Phase 1 trial and preclinical demonstration of the benefit of using ICT01 plus VEN/AZA allowed the initiation of a Phase 2a expansion cohort to evaluate the clinical benefit of this combination in 1L AML patients.

Partitioning of Circulating Monocyte Subsets in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an autoimmune disease characterized by accelerated platelet destruction with impaired platelet production leading to a platelet count decreased +/- bleeding manifestations, mostly caused by antiplatelet autoantibodies. The role of monocytes in ITP pathophysiology is not clearly established. Circulating monocyte subpopulations were identified phenotypically by flow cytometry and defined according to the surface expression of CD14 and of CD16 into classical (cMo), intermediate (iMo) and nonclassical (ncMo) monocytes. A new subpopulation, defined by the expression of the slan marker, has recently been described and represents a fraction of ncMo (ncMo slan +). Objectives The 1 st objective of the study was to analyze the partitioning of peripheral blood monocyte subsets by flow cytometry in ITP patients compared to a group of healthy blood donors (HD). Our second objective was to further characterize different phenotypic profiles of monocytes among ITP patients based on baseline characteristics, severity and treatment(s). Methods From April 2019 to January 2023, peripheral blood samples from ITP patients and HD were collected on EDTA and stained with the following antibodies: anti-CD45, CD2, CD56, CD24, CD14, CD16 and anti-slan. Acquisition analysis was performed either with a Navios or a DxFLEX cytometer. All files were analyzed with the previously described exclusion strategy of non-monocytes populations, using Kaluza™ analysis software, to characterize cMo, iMo, ncMo and ncMo slan + subsets. ITP patients were included regardless of their ITP status and/or phase of the disease. Results In total, 56 peripheral blood samples from 46 ITP patients and from 19 HD were analyzed. ITP patients had a median age of 50.5 years [19-91], 54.3% were females, whereas the median age of HD was 36 years [21-65] with 42.1% of females. ITP was defined as primary in 82.6% of the cases with a median disease duration of 3 years (&amp;lt;3 months or newly-diagnosed in 26.8% and &amp;gt;12 months or chronic ITP in 60.7%). Nine patients had undergone splenectomy (19.6%) at time of analysis. The platelet count was &amp;lt;50 x 10 9/L in 60.7% of ITP patients and absolute monocyte count ≥ 1 x 10 9/L was found in 21.4%. Regarding ITP treatment, 14 patients (25%) were receiving glucocorticoids (GC) at time of analysis whereas 9 (16.1%) had been exposed to GC within the previous month. Eleven patients (19.6%) had received intravenous immunoglobulin within a month prior to the analysis, 20 (35.7%) were on thrombopoietin receptor agonists, 9 (16.1%) had received rituximab in the previous year, 4 (7.1%) received another immunosuppressive therapy and 6 (10.7%) received another treatment. Eighteen patients (32.1%) were considered as “treatment-free” as they had not received any treatment for their ITP in the previous month or rituximab in the previous year. The latter group of “untreated” patients displayed a significant increase of the iMo relative fraction when compared to HD (4.92% vs 3.18%, p=0.018). There was no significant difference between the two groups regarding other monocytic subpopulations, including cMo, ncMo, ncMo slan+, the absolute and relative counts of monocytes. As for the treatment effect, the ongoing intake of GC therapy did not have a significant impact on monocytosis but was strongly associated with an increased proportion of cMo (94.2% vs 85.0%, p &amp;lt; 0.0001; Figure 1) and a decreased proportion of ncMo (2.31% vs 9.85%, p&amp;lt;0.0001) and ncMo slan+ (3.12% vs 7.46%, p&amp;lt;0.0001), mimicking the cMo accumulation characteristic of chronic myelomonocytic leukemia. Splenectomy and other treatments did not significantly interfere neither with monocyte count nor with the monocyte subpopulation partitioning. When excluding patients receiving GC, splenectomized patients had a higher monocyte count compared to non-splenectomized patients (1.0 vs 0.5G/L, p=0.008), with no difference in monocyte subset repartition. Conclusion Using multiparameter flow cytometry analysis, we demonstrated the expansion of iMo in untreated ITP patients. GC treatment led to the accumulation of cMo with reduced ncMo proportion, hence should be considered when interpreting the monocyte subset partitioning. These data suggest a potential role of monocytes in ITP pathogenesis and clinical response to GC therapy.

Mesenchymal Stromal Cells from Individuals with DNMT3A mut Clonal Hematopoiesis Instruct Young Healthy Hematopoietic Stem/Progenitor Cells Towards a Myeloid-Biased Aging Phenotype Resembling That of Patients with Clonal Cytopenia of Undetermined Significance (CCUS)

Clonal hematopoiesis of indeterminate potential (CHIP) describes the age-related acquisition of somatic mutations in hematopoietic stem/progenitor cells (HSPC) leading to clonal blood cell expansion and variable risk of progression to myeloid malignancy. DNMT3A is the most commonly mutated gene and while single mutations do not raise the likelihood of progression, high variant allele frequencies (VAF) and presence of co-mutations do. In myeloid diseases, the bone marrow (BM) microenvironment exhibits alterations promoting malignant transformation. However, it is unknown whether similar alterations exist in the pre-malignant niche. We set out to analyze whether human mesenchymal stromal cells (MSC) from CHIP individuals with a DNMT3A mutation ( DNMT3A mut) are altered and whether they are able to remodel the HSPC compartment in healthy hematopoiesis. From our published cohort of previously sequenced BM samples from hematologically healthy individuals undergoing hip replacement surgery, we selected samples with either an isolated DNMT3A mut at a low VAF (2.47-6.8%, n=4, median age 73.5y, termed low-risk CHIP), or DNMT3A mut samples with a high VAF (17.5-28.5%) and at least one co-mutation (n=5, median age 73y, termed high-risk CHIP) as well as 12 age-matched samples without any mutation (n=12, median age 72y). HSPC were isolated from 3 younger healthy donors (median age 50y). MSC were expanded by plastic adherence and subjected to multilevel characterization. Compared to age-matched healthy controls, MSC from DNMT3A mut BM samples (CHIP-MSC) exhibited higher proliferation rates, lower colony-forming-unit (CFU) fibroblast capacity, as well as reduced adipogenic and increased osteogenic differentiation potential. To understand how altered CHIP-MSC influence the hematopoietic compartment, younger healthy CD34+ HSPC were co-cultured with the different MSC subgroups for 42 hours in a low cytokine-containing medium, followed by bulk RNAseq on subsequently sorted cell populations (MSC: CD45-/CD90+; HSPC: CD45+/CD34+). We found 31 differentially expressed genes (DEG) (logFC±0.5, FDR&amp;lt;0.05) in CHIP-MSC compared to their age-matched counterparts. Functional analyses suggested an activation of communication towards HSPC via extracellular vesicles. Strikingly, differential gene expression of sorted young healthy HSPC after co-culture revealed 375 DEG (322 up-regulated, 53 down-regulated; logFC±0.5, FDR&amp;lt;0.05) in HSPC cultured with CHIP compared to non-CHIP age-matched MSC (Figure panel A). We then used gene set variation analysis (GSVA) to analyze HSPC compartment composition after co-culture. Based on enrichment scores, unsupervised clustering identified two distinctive profiles that correlated with HSPC cultured with either non-CHIP or high-risk CHIP-MSC, while those cultured with low-risk CHIP-MSC showed an intermediate phenotype (Figure panel B). Co-culture of HSPC with high-risk CHIP-MSC resulted in enrichment of hematopoietic stem cells (HSC) and multipotent progenitors (MPP), as well as myeloid skewing represented by higher scores for common-myeloid (CMP) and granulocyte-monocyte (GMP) progenitors. Conversely, in contrast to the control group, megakaryocyte-erythroid progenitors (MEP) were underrepresented. CFU capacity was increased in healthy HSPC cultured with CHIP-MSC and multiparameter flow cytometry analysis showed a trend towards increased CMP/GMP and decreased MEP numbers, consistent with the RNAseq data. Finally, we evaluated the BM HSPC compartment composition of a cohort of 49 CCUS patients with DNMT3A mut (VAF 1-49%, median age 76y) compared to 56 control BM samples using whole transcriptome sequencing data. Intriguingly, we observed the same profile with a myeloid shift in progenitor populations as in our healthy young HSPC exposed to high-risk CHIP-MSC. Taken together, our results indicate that MSC from individuals harboring a DNMT3Amutation are able to instruct young healthy HSPC and influence their fate, thereby promoting a differentiation bias that shifts the hematopoietic compartment toward that associated with an aged pre-malignant state, even in the absence of DNMT3A mut HSPC. These findings suggest that focusing on the interaction between MSC and HSPC is a promising therapeutic strategy to intercept disease progression. Functional analysis of identified deregulated pathways in DNMT3Amut CHIP is ongoing.

Potential biomarkers for fatal outcome prognosis in a cohort of hospitalized COVID-19 patients with pre-existing comorbidities.

The difficulty in predicting fatal outcomes in patients with coronavirus disease 2019 (COVID-19) impacts the general morbidity and mortality due to severe acute respiratory syndrome-coronavirus 2 infection, as it wears out the hospital services that care for these patients. Unfortunately, in several of the candidates for prognostic biomarkers proposed, the predictive power is compromised when patients have pre-existing comorbidities. A cohort of 147 patients hospitalized for severe COVID-19 was included in a descriptive, observational, single-center, and prospective study. Patients were recruited during the first COVID-19 pandemic wave (April-November 2020). Data were collected from the clinical history whereas immunophenotyping by multiparameter flow cytometry analysis allowed us to assess the expression of surface markers on peripheral leucocyte. Patients were grouped according to the outcome in survivors or non-survivors. The prognostic value of leucocyte, cytokines or HLA-DR, CD39, and CD73 was calculated. Hypertension and chronic renal failure but not obesity and diabetes were conditions more frequent among the deceased patient group. Mixed hypercytokinemia, including inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines, was more evident in deceased patients. In the deceased patient group, lymphopenia with a higher neutrophil-lymphocyte ratio (NLR) value was present. HLA-DR expression and the percentage of CD39+ cells were higher than non-COVID-19 patients but remained similar despite the outcome. Receiver operating characteristic analysis and cutoff value of NLR (69.6%, 9.4), percentage NLR (pNLR; 71.1%, 13.6), and IL-6 (79.7%, 135.2 pg/mL). The expression of HLA-DR, CD39, and CD73, as many serum cytokines (other than IL-6) and chemokines levels do not show prognostic potential, were compared to NLR and pNLR values.

906P Multi-parameter flow cytometry analysis of circulating immune cell populations in recurrent/metastatic adenoid cystic carcinoma

906P Multi-parameter flow cytometry analysis of circulating immune cell populations in recurrent/metastatic adenoid cystic carcinoma

Multiparametric Flow Cytometry for Determination of Viability, Caspase 3 and 7 Activity, and Lipid Peroxidation Adduct (4-Hydroxynonenal) in Equine Spermatozoa.

Flow cytometry is a powerful tool for the analysis of cell samples formed of multipopulations, such as spermatozoa. In recent years, multiparametric cytometers have evolved, allowing the study of different cellular characteristics, such as protein expression, DNA analysis, or mitochondrial activity. Whether using traditional fluorescent dyes or fluorophore-conjugated antibodies, each cell or cellular component is individually stained, the sample is analyzed at high velocities, and then is displayed and interpreted in a dot-plot. We hereby describe the procedure to perform a multiparametric flow cytometry analysis in equine spermatozoa using three sources of excitation and polychromatic flow cytometry for the detection of 4HNE, a lipid peroxidation adduct (by anti-4HNE antibody), apoptotic markers (by caspases 3 and 7 activity), and live/dead spermatozoa (by ethidium-homodimer) excluding the debris with Hoechst 33342 staining and gating. This multiparametric analysis allows the simultaneous detection of different spermatic parameters, providing useful information for the characterization of a seminal sample and fertility estimation. © 2023 Wiley Periodicals LLC. Basic Protocol: Determination of viability, caspase 3 and 7 activity, and 4-hydroxynonenal in equine spermatozoa by flow cytometry.

Leukemic Involvement Is a Common Feature in Waldenström Macroglobulinemia at Diagnosis.

Waldenström Macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with bone marrow (BM) involvement and IgM monoclonal gammopathy. To date, no studies have focused specifically on peripheral blood (PB) involvement. In this study, 100 patients diagnosed with WM according to the World Health Organization (WHO) criteria were included based on the demonstration of MYD88mut in BM and the availability of PB multiparametric flow cytometry (MFC) analysis. Leukemic involvement by MFC was detected in 50/100 patients. A low percentage of mature small lymphocytes in PB smears was observed in only 15 cases. MYD88mut by AS-qPCR was detected in PB in 65/100 cases. In cases with leukemic expression by MFC, MYD88mut was detected in all cases, and IGH was rearranged in 44/49 cases. In 21/50 patients without PB involvement by MFC, molecular data were consistent with circulating disease (MYD88mut by AS-qPCR 3/50, IGH rearranged 6/50, both 12/50). Therefore, PB involvement by standard techniques was detected in 71/100 patients. MYD88mut was detected in PB by dPCR in 9/29 triple negative cases. Overall, 80% of the patients presented PB involvement by any technique. Our findings support the role of PB MFC in the evaluation of patients with IgM monoclonal gammopathy and provide reliable information on correlation with molecular features. The development of a feasible MFC assay may stand as an objective tool in the classification of mature B cell neoplasms presenting with IgM monoclonal gammopathy.

The role of innate immune system in modulating CHK1 inhibitor (CHK1i) response in BRCA wild-type (BRCAwt), platinum-resistant high-grade serous ovarian cancer (PR-HGSOC): Exploratory analysis from a phase II study of CHK1i prexasertib.

5557 Background: Immune checkpoint blockade, e.g., anti-PD-1/PD-L1, demonstrated limited efficacy in HGSOC despite high levels of tumor-infiltrating lymphocytes, suggesting the innate, rather than adaptive, immune system may play a prominent role in anticancer response. Preclinical studies suggest CHK1i modulates the innate immune milieu. We previously showed clinical activity of prexasertib in BRCAwt, PR-HGSOC patients (pts). Here, we investigated the association of innate immunogenic features with CHK1i response. Methods: Prexasertib was given at 105 mg/m2 IV every 14 days in a 28-day cycle. Baseline (BL) fresh core biopsies were collected from pts enrolled in the biopsy (Bi) cohort for RNA sequencing. Computational immunogenomic analysis (CIBERSORT) was used for immune cell fractions quantification. Blood samples (BS) were obtained at BL and cycle 1 day 15 (C1D15) from pts enrolled in both Bi and non-Bi (nBi) cohorts for multiparametric flow cytometry (FC) analysis to evaluate dynamic changes in immune cell subsets. Mann-Whitney and Wilcoxon tests were used to compare unpaired and paired data, respectively. Results: 49 BRCAwt, PR-HGSOC pts were enrolled in the Bi cohort (n=25) and nBi cohort (n=24). 39 were evaluable per RECIST v1.1. 18/39 pts (46.2%) had a clinical benefit (CB), defined as progression-free survival≥6 months. Transcriptomic profiles of BL biopsies (n=15) met the quality control and assurance (Gene Similarity Index, Principal Component Analysis) for further in silico analysis. CIBERSORT analysis did not exhibit any differences in the immune cell fractions between CB and non-CB (NCB) groups at BL. FC analysis on paired BS (BL and C1D15) showed an increase of immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSCs, p&lt;0.001) and classical monocytes (CM, p=0.003) in the NCB group (18 paired BS). In the CB group (19 paired BS), CD141+ and CD1c+ dendritic cells (DCs) express higher rates of CD83, a marker of maturity and functionality (p&lt;0.001 and p&lt;0.001, respectively), despite no increase in the absolute number of DCs. At C1D15, a higher rate of M-MDSCs (median 7.8% vs. 5.52%; p=0.03) and CM (median 40.7% vs. 31.7%; p=0.03) were found in the NCB group (n=18) compared to the CB group (n=19); also, CM showed lower expression of HLA-DR in the NCB group (median 19.25% vs. 27.8%; p=0.02). Conclusions: Collectively, our data suggest the possible involvement of innate immunity in modulating CHK1i response. Increasing peripheral immunosuppressive cells and lower immunocompetence of the innate immune system may contribute to CHK1i resistance, whereas the functionality of DCs may be involved in the response. Enhancing innate immunity may represent a strategy to increase CHK1i response in this hard-to-treat population. Clinical trial information: NCT02203513 .

Oral prednisone regulates human skin T cells in delayed-type hypersensitivity reaction elicited by diphencyprone.

The potential benefit of inducing delayed-type hypersensitivity (DTH) reaction in healthy volunteers (HVs) as experimental models to study skin inflammatory disorders was recently reported using bulk molecular technologies. Immunophenotype of skin T cells, including cellular source of Type 1, 2, and 3 cytokines, in a local DTH reaction and their modulation by oral drugs remain to be investigated. Purified protein derivative (PPD), nickel, diphencyprone (DPCP), or house dust mite (HDM) was administered as sensitizer to 40 HVs. In addition, 20 HVs were randomized to receive oral prednisone or placebo before DPCP challenge. We characterized the immunophenotype and cytokine profile of CD3+ T cell infiltrate, and examined the modulation by oral prednisone at single cell level using multi-parameter flow cytometry and unsupervised analysis. PPD was biased towards a Th1 and Tc1 response, and HDM a Th2/Th17 and Tc2. Nickel and DPCP displayed a mixed Th1/Th2/Th17 and Tc1 response. CD4+ CD25+ FoxP3+ regulatory T cells (Tregs), the minor CD4+ CD25+ FoxP3- ICOS+ PD-1+ (activated PD-1+ Th), and CD103+ tissue resident memory (TRM) cells were detected in all groups. DPCP uniquely elicited rare CD8+ CD103+ CD25+ RoRγt+ PD-1+ ICOS+ IFNγ+ T cells (activated CD8+ IFNγ+ PD-1+ TRM). Oral prednisone decreased frequencies of activated PD-1+ Th and CD8+ IFNγ+ PD-1+ TRM subsets relative to placebo in DPCP reaction. The latter was positively correlated with improvement of clinical parameters with prednisone. DTH and skin CD3+ T cell profiles elicited by common sensitizers can be modulated by oral drugs. Corticosteroids reduce the frequencies of activated PD-1+ Th and CD8+ IFNγ+ PD-1+ TRM cells after DPCP exposure.

Specific immune response to mRNA vaccines against COVID-19 in patients receiving allogeneic stem cell transplantation for myeloid malignancy was altered by immunosuppressive therapy

BackgroundAllogeneic hematopoietic stem cell transplant (allo-HSCT) recipients are at high risk of complications associated with COVID-19 infection due to dysfunction of their immune system. Vaccination can protect from the adverse consequences of COVID-19. However, studies on the efficacy of COVID-19 vaccines in HSCT recipients with insufficient post-HSCT immune reconstitution are still scarce. In our study, we determined how immunosuppressive medication and the reconstitution of the cellular immune system influenced T cell responses specific for the surface glycoprotein of SARS-CoV-2 virus (S antigen) after two doses of mRNA vaccine against COVID-19 in patients with myeloid malignancies treated with HSCT. MethodsVaccination outcomes were followed in 18 (allo-HSCT) recipients and 8 healthy volunteers. The IgG antibodies against SARS-CoV-2 spike (S) and nucleocapsid (NCP) protein were determined in ELISA and S-specific T cells were detected using a sensitive ELISPOT-IFNγ based on in vitro expansion and restimulation of T cells in pre- and post-vaccination blood samples. Multiparametric flow cytometry analysis of peripheral blood leukocyte differentiation markers was employed for determination of reconstitution of the main subpopulations of T cells and NK cells at month 6 after HSCT. ResultsS- specific IgG antibody response detected in 72% of the patients was lower than in healthy vaccinees (100%). Vaccine-induced T-cell responses to S1 or S2 antigen were significantly reduced in HSCT recipients, which were treated with corticosteroids in dose 5 mg of prednisone- equivalents or higher during the vaccination period or in preceeding 100 days in comparison with recipients un-affected with corticosteroids. A significant positive correlation was found between the level of anti-SARS-Cov-2 spike protein IgG antibodies and the number of functional S antigen-specific T cells. Further analysis also showed that the specific response to vaccination was significantly influenced by the interval between administration of vaccine and transplantation. Vaccination outcomes were not related to age, sex, type of mRNA vaccine used, basic diagnosis, HLA match between HSC donor and recipient, and blood counts of lymphocytes, neutrophils, and monocytes at the time of vaccination. Multiparametric flow cytometry analysis of peripheral blood leukocyte differentiation markers showed that good humoral and cellular S-specific immune responses induced by vaccination were associated with well-reconstituted CD4+ T cells, mainly CD4+ effector memory subpopulation at six months after HSCT. ConclusionsThe results showed that both humoral and cellular adaptive immune responses of HSCT recipients to the SARS-CoV-2 vaccine were significantly suppressed by corticosteroid therapy. Specific response to the vaccine was significantly affected by the length of the interval between HSCT and vaccination. Vaccination as early as 5 months after HSCT can lead to a good response. Immune response to the vaccine is not related to age, gender, HLA match between HSC donor and recipient, or type of myeloid malignancy. Vaccine efficacy was dependent on well-reconstituted CD4+ T cells, at six months after HSCT.

Computational flow cytometry provides accurate assessment of measurable residual disease in chronic lymphocytic leukaemia.

Undetectable measurable residual disease (MRD) is associated with favourable clinical outcomes in chronic lymphocytic leukaemia (CLL). While assessment is commonly performed using multiparameter flow cytometry (MFC), this approach is associated with limitations including user bias and expertise that may not be widely available. Implementation of unsupervised clustering algorithms in the laboratory can address these limitations and have not been previously reported in a systematic quantitative manner. We developed a computational pipeline to assess CLL MRD using FlowSOM. In the training step, a self-organising map was generated with nodes representing the full breadth of normal immature and mature B cells along with disease immunophenotypes. This map was used to detect MRD in multiple validation cohorts containing a total of 456 samples. This included an evaluation of atypical CLL cases and samples collected from two different laboratories. Computational MRD showed high correlation with expert analysis (Pearson's r > 0.99 for typical CLL). Binary classification of typical CLL samples as either MRD positive or negative demonstrated high concordance (>98%). Interestingly, computational MRD detected disease in a small number of atypical CLL cases in which MRD was not detected by expert analysis. These results demonstrate the feasibility and value of automated MFC analysis in a diagnostic laboratory.

Abstract 3637: Extracellular matrix drives high-grade breast cancer immune suppression down-modulating PD-1 on Treg cells via the IL-23/SATB1 axis

Abstract The pathological grade defines tumor aggressiveness and, indeed, high-grade breast cancer (HGBC), regardless of molecular findings, is characterized by a very aggressive clinical course and a poor prognosis in spite of the availability of chemotherapy regimens and specific-targeted agents. Both the cellular and the extracellular compartments of the tumor microenvironment (TME) can contribute to the evolution of BC by immune escape and immune suppression processes. In this context, structural and extracellular components of the TME, namely the extracellular matrix (ECM), has been shown to contribute to many aspects of tumor progression, exerting important regulatory functions on tumor cells. The relevance of the ECM in cancer progression is strengthened by study showing that the ECM composition is a prognostic factor able to identify patients subgroups endowed with a different clinical outcome according to the distinct enrichment in ECM genes leading to four specific ECM signatures (ECM 1-4). Among the different ECM-related signatures, only the ECM3 signature - characterizing about 35 % of HGBC - identifies the most aggressive tumors with epithelial mesenchymal transition (EMT) features, poor prognosis, T-cell exclusion and an increased infiltration of myeloid-derived suppressor cells (MDSCs). To evaluate whether local immune suppressive features of ECM3+ tumors can be intercepted in the peripheral blood (PB), to represent a potential biomarker for this subset of tumors, we collected consecutive HGBC patients that included 22 ECM3+ and 30 ECM3- and performed a multiparametric flow cytometry analysis. We found PD-1neg Treg being correlated to ECM3 patients and through ad hoc mouse BC models, we provided the mechanisms through which SPARC, a key functional gene of the ECM3 signature, was responsible for the down-modulation of PD-1 on Treg, positively affecting their suppressive activity. By sustaining the release of IL-23, SPARC promotes SATB1 expression that repress PD-1. Notably, fluvastatin treatment in vivo decreased the expression of SATB1 in treated Treg cells, a result paralleled by an increased expression of PD-1 on Treg cells. Our data extend the regulatory activity of the extracellular matrix and SPARC, which are large characterized for myeloid cells, to regulatory T-cells also offering new possible targets for the treatment of high-grade breast tumors. Citation Format: Giovanna Talarico, Mara Lecchi, Massimo Costanza, Claudia Chiodoni, Vera Cappelletti, Paolo Verderio, Massimo Di Nicola, Francesco Bertolini, Mario Paolo Colombo, Sabina Sangaletti. Extracellular matrix drives high-grade breast cancer immune suppression down-modulating PD-1 on Treg cells via the IL-23/SATB1 axis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3637.

Pembrolizumab in Lymphopenic Metastatic Breast Cancer Patients Treated with Metronomic Cyclophosphamide: A Clinical and Translational Prospective Study.

Metastatic endocrine-resistant breast cancer (MBC) is a disease with poor prognosis and few treatment options. Low lymphocyte count is associated with limited overall survival. In a prospective cohort of lymphopenic patients with HER-2 negative MBC, we assessed the clinical and biological impact of pembrolizumab combined with metronomic cyclophosphamide. This multicenter Phase II study evaluated the safety and clinical activity of pembrolizumab (intravenous (IV), 200mg, every 3 weeks) combined with metronomic cyclophosphamide (50mg/day, per os) in lymphopenic adult patients with HER2-negative MBC previously treated by at least one line of chemotherapy in this setting according to a Simon's minimax two-stage design. Blood and tumor samples were collected to assess the impact of the combined treatment on circulating immune cells and the tumor immune microenvironment through multiparametric flow cytometry and multiplex immunofluorescence analyses. Primary endpoint was the clinical benefit rate at 6 months of treatment (CBR-6M). Secondary endpoints were objective response rate (ORR), duration of response, progression free survival (PFS), and overall survival (OS). Two out of the twenty treated patients presented clinical benefit (one Tumor Mutational Burden (TMB)-high patient with complete response (CR) and one patient with objective response (OR) per Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST V1.1) associated with a strong increase of cytokine-producing and proliferating CD4+ T cells and higher CD8+ T cells to macrophage ratios in the tumor. This impact on CD4+ and CD8+ T cell polyfunctionality was still observed more than one year for the patient with CR. A decreased in their absolute number of CD4+ and CD8+ memory T cells was observed in other patients. Pembrolizumab combined with metronomic cyclophosphamide was well tolerated, and displayed limited anti-tumoral activity in lymphopenic MBC. Correlative translational data of our trial advocates for additional studies with other chemotherapy combinations.

Deep Immunophenotyping of Human Whole Blood by Standardized Multi-parametric Flow Cytometry Analyses

Immunophenotyping is proving crucial to understanding the role of the immune system in health and disease. High-throughput flow cytometry has been used extensively to reveal changes in immune cell composition and function at the single-cell level. Here, we describe six optimized 11-color flow cytometry panels for deep immunophenotyping of human whole blood. A total of 51 surface antibodies, which are readily available and validated, were selected to identify the key immune cell populations and evaluate their functional state in a single assay. The gating strategies for effective flow cytometry data analysis are included in the protocol. To ensure data reproducibility, we provide detailed procedures in three parts, including (1) instrument characterization and detector gain optimization, (2) antibody titration and sample staining, and (3) data acquisition and quality checks. This standardized approach has been applied to a variety of donors for a better understanding of the complexity of the human immune system.

Immunophenotypic measurable residual disease monitoring in adult acute lymphoblastic leukemia patients undergoing allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) offers a survival benefit to adult patients affected by acute lymphoblastic leukemia (ALL). However, to avoid an overt disease relapse, patients with pre or post transplant persistence or occurrence of measurable residual disease (MRD) may require cellular or pharmacological interventions with eventual side effects. While the significance of multiparametric flow cytometry (MFC) in the guidance of ALL treatment in both adult and pediatric patients is undebated, fewer data are available regarding the impact of MRD monitoring, as assessed by MFC analysis, in the allo-HSCT settings. Aim of this article is to summarize and discuss currently available information on the role of MFC detection of MRD in adult ALL patients undergoing allo-HSCT. The significance of MFC-based MRD according to sensitivity level, timing, and in relation to molecular techniques of MRD and chimerism assessment will be also discussed.