Evaluation of Sézary cell marker expression and cell death behaviour upon in vitro treatment by flow cytometry in Sézary syndrome patients.

The diagnosis of Sézary syndrome (SS) relies on the identification of blood Sézary cells (SC) by different markers via flow cytometry. Treatment of SS is challenging since its pathogenesis is characterized by cell death resistance rather than hyperproliferation. In this study, we establish an integrated approach that considers both the expression of SC markers and sensitivity to cell death both spontaneously and upon in vitro treatment. Peripheral blood mononuclear cells were isolated from 20 SS patients and analysed for the SC markers CD7 and CD26 loss as well as CD158k and PD1 gain. The cells were then treated with different established and experimental therapies in vitro and cell death was measured. Spontaneous and therapeutically induced cell death were measured and correlated to cellular marker profiles. In the marker-positive cells, spontaneous cell death sensitivity was reduced. Different treatments in vitro managed to specifically induce cell death in the putative CTCL cell populations. Interestingly, a repeated analysis after 3 months of treatment revealed the CTCL cell death sensitivity to be restored by therapy. We propose this novel integrated approach comprising the evaluation of SC marker expression and analysis of cell death sensitivity upon treatment that can also enable a better therapy stratification.

Phase 1 study of CAR-37 T cells in patients with relapsed or refractory CD37+ lymphoid malignancies.

ABSTRACT: We report a first-in-human clinical trial using chimeric antigen receptor (CAR) T cells targeting CD37, an antigen highly expressed in B- and T-cell malignancies. Five patients with relapsed or refractory CD37+ lymphoid malignancies were enrolled and infused with autologous CAR-37 T cells. CAR-37 T cells expanded in the peripheral blood of all patients and, at peak, comprised >94% of the total lymphocytes in 4 of 5 patients. Tumor responses were observed in 4 of 5 patients with 3 complete responses, 1 mixed response, and 1 patient whose disease progressed rapidly and with relative loss of CD37 expression. Three patients experienced prolonged and severe pancytopenia, and in 2 of these patients, efforts to ablate CAR-37 T cells, which were engineered to coexpress truncated epidermal growth factor receptor, with cetuximab were unsuccessful. Hematopoiesis was restored in these 2 patients after allogeneic hematopoietic stem cell transplantation. No other severe, nonhematopoietic toxicities occurred. We investigated the mechanisms of profound pancytopenia and did not observe activation of CAR-37 T cells in response to hematopoietic stem cells in vitro or hematotoxicity in humanized models. Patients with pancytopenia had sustained high levels of interleukin-18 (IL-18) with low levels of IL-18 binding protein in their peripheral blood. IL-18 levels were significantly higher in CAR-37-treated patients than in both cytopenic and noncytopenic cohorts of CAR-19-treated patients. In conclusion, CAR-37 T cells exhibited antitumor activity, with significant CAR expansion and cytokine production. CAR-37 T cells may be an effective therapy in hematologic malignancies as a bridge to hematopoietic stem cell transplant. This trial was registered at www.ClinicalTrials.gov as #NCT04136275.

Flow cytometric profiles with CD7 and CADM1 in CD4+ T cells are promising indicators for prognosis of aggressive ATL.

ABSTRACT: Adult T-cell leukemia/lymphoma (ATL) is a poor prognosis hematological malignancy originating from human T-cell leukemia virus 1 (HTLV-1)-infected CD4+ T cells. Flow cytometric plots of CADM1 and CD7 in CD4+ T cells are useful for separating HTLV-1-uninfected T cells and ATL cells. They are indicators of clonal evolution of HTLV-1-infected cells and disease progression of asymptomatic carriers or indolent ATL. However, the impacts of the plots on the clinical course or prognosis of ATL, especially in aggressive ATL, remain unclear. We focused on the N fraction (CD4+ CADM1+ CD7-) reflecting ATL cells and analyzed the flow cytometric profiles and clinical course of 497 samples from 92 HTLV-1-infected patients who were mainly aggressive ATL. The parameters based on N fractions showed significant correlations with known indicators of ATL disease status (soluble interleukin-2 receptor, lactate dehydrogenase, abnormal lymphocytes, etc.) and sensitively reflected the treatment response of aggressive ATL. The parameters based on N fractions significantly stratified the prognosis of aggressive ATL at 4 different time points: before treatment, after 1 course of chemotherapy, at the best response after chemotherapy, and before allogeneic hematopoietic cell transplantation. Even after mogamulizumab administration, which shows potent effects for peripheral blood lesions, the N fraction was still a useful indicator for prognostic estimation. In summary, this report shows that CADM1 vs CD7 plots in CD4+ T cells are useful indicators of the clinical course and prognosis of aggressive ATL. Therefore, this CADM1 and CD7 profile is suggested to be a useful prognostic indicator consistently from HTLV-1 carriers to aggressive ATL.

Characteristics and outcomes of acute myeloid leukaemia patients with baseline CD7 expression.

Targeted therapy development for acute myeloid leukaemia (AML) requires an understanding of specific expression profiles. We collected flow cytometry data on 901 AML patients and recorded aberrant CD7 expression on leukaemic blasts. 263 (29.2%) had blasts positive for CD7. CD7+ AML was more likely to be adverse risk (64.6% vs. 55.6%, p = 0.0074) and less likely to be favourable risk (15.2% vs. 24.1%, p = 0.0074) by European LeukemiaNet 2022 criteria. Overall survival was inferior (11.9 [95% CI, 9.7-15.9] vs. 19.0 months [95% CI, 16.1-23.0], p = 0.0174). At relapse, 30.4% lost and 19.0% gained CD7, suggesting moderate instability over time.

CD7 activation regulates cytotoxicity-driven pathology in systemic sclerosis, yielding a target for selective cell depletion.

OBJECTIVES: Cytotoxic T cells and natural killer (NK) cells are central effector cells in cancer and infections. Their effector response is regulated by activating and inhibitory receptors. The regulation of these cells in systemic autoimmune diseases such as systemic sclerosis (SSc) is less defined. METHODS: We conducted ex vivo analysis of affected skin and blood samples from 4 SSc patient cohorts (a total of 165 SSc vs 80 healthy individuals) using single-cell transcriptomics, flow cytometry and multiplex immunofluorescence staining. We further analysed the effects of costimulatory modulation in functional assays, and in a severely affected SSc patient who was treated on compassionate use with a novel anti-CD3/CD7 immunotoxin treatment. RESULTS: Here, we show that SSc-affected skin contains elevated numbers of proliferating T cells, cytotoxic T cells and NK cells. These cells selectively express the costimulatory molecule CD7 in association with cytotoxic, proinflammatory and profibrotic genes, especially in recent-onset and severe disease. We demonstrate that CD7 regulates the cytolytic activity of T cells and NK cells and that selective depletion of CD7+ cells prevents cytotoxic cell-induced fibroblast contraction and inhibits their profibrotic phenotype. Finally, anti-CD3/CD7 directed depletive treatment eliminated CD7+ skin cells and stabilised disease manifestations in a severely affected SSc patient. CONCLUSION: Together, the findings imply costimulatory molecules as key regulators of cytotoxicity-driven pathology in systemic autoimmune disease, yielding CD7 as a novel target for selective depletion of pathogenic cells.

Naturally selected CD7 CAR-T therapy without genetic editing demonstrates significant antitumour efficacy against relapsed and refractory acute myeloid leukaemia (R/R-AML).

BACKGROUND: The survival rate for patients with relapsed and refractory acute myeloid leukaemia (R/R-AML) remains poor, and treatment is challenging. Chimeric antigen receptor T cells (CAR-T cells) have been widely used for haematologic malignancies. Current CAR-T therapies for acute myeloid leukaemia mostly target myeloid-lineage antigens, such as CD123 and CD33, which may be associated with potential haematopoietic toxicity. As a lineage-specific receptor, CD7 is expressed in acute myeloid leukaemia cells and T cells but is not expressed in myeloid cells. Therefore, the use of CD7 CAR-T cells for R/R-AML needs to be further explored. METHODS: In this report, immunohistochemistry and flow cytometry were used to analyse CD7 expression in clinical samples from R/R-AML patients and healthy donors (HDs). We designed naturally selected CD7 CAR-T cells to analyse various functions and in vitro antileukaemic efficacy based on flow cytometry, and xenograft models were used to validate in vivo tumour dynamics. RESULTS: We calculated the percentage of cells with CD7 expression in R/R-AML patients with minimal residual disease (MRD) (5/16, 31.25%) from our institution and assessed CD7 expression in myeloid and lymphoid lineage cells of R/R-AML patients, concluding that CD7 is expressed in T cells but not in myeloid cells. Subsequently, we designed and constructed naturally selected CD7 CAR-T cells (CD7 CAR). We did not perform CD7 antigen knockdown on CD7 CAR-T cells because CD7 molecule expression is naturally eliminated at Day 12 post transduction. We then evaluated the ability to target and kill CD7+ acute myeloid leukaemia cells in vitro and in vivo. Naturally selected CD7 CAR-T cells efficiently killed CD7+ acute myeloid leukaemia cells and CD7+ primary blasts of R/R-AML patients in vitro and significantly inhibited leukaemia cell growth in a xenograft mouse model. CONCLUSION: Naturally selected CD7 CAR-T cells represent an effective treatment strategy for relapsed and refractory acute myeloid leukaemia patients in preclinical studies.

Prognostic value of lymphoid marker CD7 expression in acute myeloid leukemia patients undergoing allogeneic hematopoietic cell transplantation in first morphological complete remission.

Although defined as a lymphoid surface marker, CD7 is aberrantly expressed on a subtype of acute myeloid leukemia cells and appears to be associated with an inferior response to chemotherapy. Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative modality but no data has been reported in CD7-positive AML patients. We performed a retrospective analysis involving 141 AML patients who underwent allo-HCT in first morphological complete remission (CR1). The results showed that CD7-positive AML patients had a poor 2-year overall survival (64.5% vs 82.0%, P = 0.040), relapse-free survival (RFS) (56.5% vs 79.4%, P = 0.005), and higher cumulative incidence of relapse (27.0% vs 9.7%, P = 0.003) post-HCT. In addition, expression of CD7 was related to RAS and RUNX1 mutation, and high residual disease level pre-HCT. Multivariate analyses showed CD7 expression at diagnosis was an independent risk factor for RFS (P = 0.016, HR = 0.418) and relapse (P = 0.014, HR = 0.307). We concluded that for AML patients in CR1, CD7 is a negative predictor for allo-transplant outcomes.

An antibody-drug conjugate with intracellular drug release properties showing specific cytotoxicity against CD7-positive cells.

Refractory T cell acute leukaemias that no longer respond to treatment would benefit from new modalities that target T cell-specific surface proteins. T cell associated surface proteins (the surfaceome) offer possible therapy targets to reduce tumour burden but also target the leukaemia-initiating cells from which tumours recur. Recent studies of the T cell leukaemia surfaceome confirmed that CD7 is highly expressed in overt disease. We have used an anti-CD7 antibody drug conjugate (ADC) to show that the binding of antibody to surface CD7 protein results in rapid internalization of the antigen together with the ADC. As a consequence, cell killing was observed via induction of apoptosis and was dependent on cell surface CD7. The in vitro cytotoxic activity (EC50) of the anti-CD7 ADC on T cell acute leukaemia (T-ALL) cells Jurkat and KOPT-K1 was found to be in the range of 5-8 ng/mL. In a pre-clinical xenograft model of human tumour growth expressing CD7 antigen, growth was curtailed by a single dose of ADC. The data indicate that CD7 targeting ADCs may be developed into an important second stage therapy for T cell acute leukaemia, for refractory CD7-positive leukaemias and for subsets of acute myeloid leukaemia (AML) expressing CD7.

Chimeric antigen receptor T cells targeting CD7 in a child with high-risk T-cell acute lymphoblastic leukemia.

Effective systemic treatments for relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) are limited. Recent clinical application of chimeric antigen receptor (CAR) immunotherapy has demonstrated successful control of B-cell malignancies by CAR-T cells; however, designing CARs for T-ALL remains a challenge. CD7 overexpression in T-cell malignancies may be an attractive target for immunotherapy in T-ALL. This study aimed to describe the safe and effective use of autologous CD7-CAR T cells (4SCAR7) for the treatment of T-ALL with induction failure in an 11-year-old patient. Based on The Chinese Children's Cancer Group-ALL (CCCG-ALL) study protocol, minimal residual disease (MRD) by flow cytometry (FC) analysis was detected on days 19 and 46 of remission induction. At the end of remission-induction chemotherapy, the patient achieved morphologic complete remission, though with MRD 16.13% and RT-PCR of KMT2A-MLLT1 fusion positive, which indicated induction failure. The cerebrospinal fluid (CSF) was negative for blasts at diagnosed. CAR-T therapy and allogeneic transplant were recommended as the next treatment options. CD3+ lymphocytes were collected from the patient 18 days after the high-dose MTX chemotherapy through leukapheresis. The 4SCAR7 CD7-targeting CAR-T cells were generated thereafter. The patient received lymphodepleting chemotherapy prior to 4SCAR7 infusion. Oral administration of itraconazole and sulfamethoxazole was performed from day 0 after CAR-T cell infusion. The patient did not have hypotension, hypoxia, or serious biochemical change or abnormality, but had fever on day 9. Although grade 1 cytokine-release syndrome (CRS) was diagnosed, it was successfully treated with ibuprofen. Anti-CD7 CAR transgene copy numbers in peripheral blood were determined by qPCR, which showed effective expansion initially, then dropped quickly, and persisted at a low level. Although experienced cytopenia from days 14 to 21, the patient achieved remission on day 17. After complete remission, the patient received hematopoietic stem cell transplantation (HSCT) and has recovered well to thisdate. Overall, this report suggested that 4SCAR7 could be a safe and effective strategy for the treatment of pediatric patients with high-risk T-cell malignancies.

Base-edited CAR T cells for combinational therapy against T cell malignancies.

Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by 'T v T' fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in 'self-enrichment' yielding populations 99.6% TCR-/CD3-/CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic 'off-target' activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

CD34 is not Expressed by Blasts in a Third of B-ALL Patients and its Negativity is associated with Aberrant Marker Expression: A Retrospective Analysis.

BACKGROUND: CD34 antigen is expressed by early hematopoietic progenitor cells and acute leukemia cells. Its expression is associated with good prognosis in acute myeloid leukemia. Literature is sparse on its prognostic significance in B- acute lymphoblastic leukemia (B-ALL) especially from India. Hence the present study was undertaken to analyse the frequency of CD34 expression in B-ALL in Indian patients and determine its prognostic significance by associating with other prognostic markers and aberrant antigen expression. METHODS: Seventy-five B-ALL patients diagnosed by flow cytometry over a period of 3½ year were studied. Correlation of CD34 expression was studied with gender, age, total leucocyte count (TLC), French-American-British (FAB) morphological type, immuno-phenotypic markers, cytogenetics and minimal residual disease. Differences between groups were evaluated using Student's T-test for quantitative data and Chi-square test/Fishers exact T-test for qualitative variables. P value.

Diagnostic significance of flow cytometry scales in diagnostics of myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) can present a challenge for clinicians. Multicolor flow cytometry (MFC) can aid in establishing a diagnosis. The aim of this study was to determine the optimal MFC approach for MDS. METHODS: The study included 102 MDS (39 low-grade MDS), 83 cytopenic patients without myeloid neoplastic disorders (control group), and 35 healthy donors. Bone marrow was analyzed using a six-color MFC. Analysis was conducted according to the "Ogata score," "Wells score," and the integrated flow cytometry (iFC) score. RESULTS: The respective sensitivity and specificity values were 77.5% and 90.4% for the Ogata score, 79.4% and 81.9% for the Wells score, and 87.3% and 87.6% for the iFC score. Specificity was not 100% due to deviations of MFC parameters in the control group. Patients with paroxysmal nocturnal hemoglobinuria (PNH) had higher levels of CD34+ CD7+ myeloid cells than donors. Aplastic anemia and PNH were characterized by a high proportion of CD56+ cells among CD34+ precursors and neutrophils. The proportion of MDS-related features increased with the progression of MDS. The highest number of CD34+ blasts was found in MDS with excess blasts. MDS with isolated del(5q) was characterized by a high proportion of CD34+ CD7+ cells and low granularity of neutrophils. In 39 low-grade MDS, the sensitivities were 53.8%, 61.5%, and 71.8% for Ogata score, Wells score, and iFC, respectively. CONCLUSION: The results support iFC as a useful diagnostic tool in MDS.

[Expression of CD7 and its correlation with prognosis in patients with NK/T-cell lymphoma].

Objective: To analysis the expression of CD7 in NK/T-cell lymphoma as well as study the correlations between CD7 and clinical survival and prognosis. Methods: The clinical and pathological indicators of 112 NKTCL patients who were admitted to or consulted at the First Affiliated Hospital of Zhengzhou University between May 2008 and December 2019 were analyzed retrospectively. The CD7 expression in the tumor tissues was detected using immunohistochemistry staining, and the influence of CD7 expression on the survival and prognosis in the patients was analyzed. Results: The CD7 expression rate was 84.82% in 112 NKTCL patients, and its expression was not influenced by sex, age, and the primary site. An analysis of the complete clinical data of 72 patients showed that the CD7 expression was significantly correlated with the PINK score, tumor metastasis, and peripheral blood EBV-DNA level. However, the Ann Arbor stage, bone marrow involvement, B symptoms, IPI/aaIPI score, Ki-67, EBER, TIA-1, Granzyme B, LDH, and ß(2)-MG were not associated with the CD7 expression. The 1-year, 3-year, and 5-year overall survival (OS) rates of the 72 patients were 81.2%, 61.8%, and 58.8%, respectively, and the progression-free survival (PFS) rates were 53.5%, 29.4%, and 24.0%, respectively. The median overall survival (median-OS, mOS) was 81 mon, and the median progression-free survival (median-PFS, mPFS) was 14 mon. The 3-year OS rates in the CD7-positive group and the CD7-negative group were 58.1% and 83.9%, respectively, (P>0.05) . The 3-year PFS rates were 21.7% and 51.9%, respectively (P<0.05) . The univariate analysis showed that age, primary tumor site, Ann Arbor stage, IPI/aaIPI score, PINK score, LDH, ß(2)-microglobulin, EBV-DNA, Ki-67, and CD7 influenced patient prognosis. The multivariate analysis showed that Ann Arbor stage and CD7 were independent prognostic factors for PFS, while PINK score and Ki-67 were independent prognostic factors for OS. Conclusions: The expression rate of CD7 in NKTCL was high and was closely related to poor patient prognosis. The patients with high levels of EBV-DNA, metastatic disease, or high PINK score were more likely to express CD7.

Clinical and hematological correlates of aberrant immunophenotypic profiles in adult and pediatric acute myeloid leukemia at presentation.

BACKGROUND: Aberrant phenotypes in acute leukemia have been reported with varying frequencies in independent studies and their association with prognostic factors is still a matter of debate. AIM: This study aims to identify the frequency of aberrant immunophenotypes in de novo acute myeloid leukemia (AML) and to evaluate their association with initial clinical and hematological features. MATERIALS AND METHODS: A total of 181 patients of de novo AML were included during the time (July 2010-June 2012). The immunophenotype of all cases of AML was studied by using flow cytometry. RESULTS: Aberrant lymphoid antigen expression was seen in 43.1% cases. Most frequent aberrant lymphoid antigen was CD7, seen in 26.5% cases. All French-American-British (FAB) subtypes except AML-M3 expressed aberrant lymphoid antigens. The expression was most common in AML-M4 in the current study. CD34 expression in AMLs was significantly associated with the expression of aberrant lymphoid antigens. Lymphoid antigen expression in adult AML was significantly associated with higher white blood cell (WBC) count (>50,000/mm3) and higher number of peripheral blasts (>70%). CONCLUSION: In summary, CD7 is the most common aberrant lymphoid antigen expressed in AML. FAB subtype AML-M3 is usually not associated with aberrant lymphoid antigen expression. AML cases with CD34 positivity are more likely to express aberrant lymphoid markers. The current study also supports that aberrant lymphoid antigen expression in adult AML is associated with adverse presenting hematological features (WBC count >50,000/mm3, peripheral blasts >70%). Pediatric Ly + AML cases are not associated with adverse presenting clinical and biological features.

FLT3-ITD DNA and mRNA levels in AML do not correlate with CD7, CD33 and CD123 expression.

INTRODUCTION: FLT3 internal tandem duplication (ITD) mutations are found in around 25% of all acute myeloid leukaemia (AML) cases and is associated with shorter disease-free and overall survival. Previous reports have shown that FLT3-ITD induces a specific phenotype in leukemic blasts, which is characterized by high levels of CD33 and CD123, and that expression of CD33 and CD123 is directly influenced by the DNA FLT3-ITD/wild-type FLT3 allelic ratio (AR). METHODS: A total of 42 FLT3-ITD and 104 FLT3-ITD-negative AML patients were analysed. Immunophenotyping data were used to calculate antigen expression levels as the ratio between the geometric mean fluorescence intensities (MFIs) of leukemic blasts and MFIs of negative lymphocyte populations. FLT3-ITD-DNA and RNA analysis was performed, under the same conditions, by capillary electrophoresis. RESULTS: Compared with the control group, the FLT3-ITD cohort presented significantly higher CD7, CD33 and CD123 levels. In order to assess the impact of FLT3-ITD abundance on antigen expression, the patients were grouped for each parameter into two cohorts using the following threshold values: (a) 0.5 for the AR, according to current AML guidelines; (b) 0.7 for the FLT3-ITD/FLT3-WT mRNA ratio (RR); and (c) 1.3 for the FLT3-ITD RR/AR ratio. We found higher values of CD33 for RR/AR ≥1.3, and no other statistical differences between CD7, CD33 and CD123 levels of the other FLT3-ITD groups. In terms of correlations between MFI values and FLT3-ITD parameters, we only observed a moderate interdependence between CD33 MFI and the RR/AR ratio, and a weak negative correlation between CD123 MFI and AR. CONCLUSION: FLT3-ITD mutations induce a specific antigen profile in AML blasts, and our data do not onfirm previous reports of FLT3-ITD AR influencing both CD33 and CD123 expression.

Glycodelin regulates the numbers and function of peripheral natural killer cells.

Natural killer (NK) cells comprise of ∼70% of the immune cell population in the maternal decidua and ∼15% of the mononuclear cells in the peripheral blood. The decidual NK cells capable of producing high levels of cytokines are functionally distinct from the peripheral NK cells that exhibit high cytotoxicity. The numbers of peripheral NK cells and their cytotoxicity potential have been correlated with pregnancy outcome. In the same context, glycodelin, an immunomodulatory protein, has been recognized to be essential for the establishment and maintenance of pregnancy, and its' reduced levels are associated with recurrent spontaneous abortions. We investigated the effect of glycodelin on the peripheral NK cells. Our results reveal that glycodelin suppresses the cytotoxicity of peripheral NK cells via downregulating perforin, granzyme B and IFNγ. Glycodelin also induces caspase-dependent death in only activated peripheral NK cells, the effect suggested to be mediated by glycodelin upon engaging with the CD7 cell surface receptor. Thus, during pregnancy, glycodelin modulates the function and the number of cytotoxic NK cells that pose a deleterious effect on the fetus, a semi-allograft. This study provides insights into the mechanism of the regulatory effect of glycodelin on NK cells and could possibly be exploited for the management of miscarriages.

UM171 expands distinct types of myeloid and NK progenitors from human pluripotent stem cells.

Scaling up blood cell production from hPSCs is critical to advancing hPSC technologies for blood transfusion, immunotherapy, and transplantation. Here we explored the potential of the HSC agonist pyrimido-indole derivative UM171, to expand hematopoietic progenitors (HPs) derived from hPSCs in chemically defined conditions. We revealed that culture of hPSC-HPs in HSC expansion conditions (SFEM with added TPO, SCF, FLT3L, IL3 and IL6) in the presence of UM171 predominantly expanded HPs with a unique CD34+CD41aloCD45+ phenotype that were enriched in granulocytic progenitors (G-CFCs). In contrast, in lymphoid cultures on OP9-DLL4, in the presence of SCF, FLT3L, and IL7, UM171 selectively expanded CD34+CD45+CD7+ lymphoid progenitors with NK cell potential, and increased NK cell output up to 10-fold. These studies should improve our understanding of the effect of UM171 on de novo generated HPs, and facilitate development of protocols for robust granulocyte and lymphoid cell production from hPSCs, for adoptive immunotherapies.