Fig 2: Phenotypic Characterization of ?miR-124 versus WT Cells(A) Immunostainings of indicated neuronal markers at 4 dpi and 0 dpi (hiPSC control).(B) Loss of pluripotency (TRA-1-60) and neural lineage commitment (NCAM1) 0–4 dpi measured by flow cytometry using the surface markers indicated.(C) Representative images of WT and ?miR-124 neurons at 4 dpi with differing morphology.(D) Quantification of bipolar morphology for WT and ?miR-124 neurons at 4 dpi. n, number of independent samples.(E) Quantification of neurite outgrowth velocities of WT and ?miR-124 neurons. n, number of neurites measured.(F) Quantification of bipolarity of ?miR-124 with and without miR-124 overexpression at 4 dpi. The overexpression of miR-124 rescued the typical bipolar morphology (see also STAR Methods). n, number of independent samples.(G) Quantification of neurite outgrowth velocities of ?miR-124 neurons at 5 dpi with and without miR-124 overexpression. The impairment was rescued by miR-124 overexpression. n, number of neurites measured.(H) Example traces and action potential (AP) quantification for 7 and 14 dpi of WT and ?miR-124. APs were induced by current injection.(I) Quantification of cell capacitance of WT and ?miR-124 neurons at 7 and 14 dpi, indicating similar cell sizes for each time point.(J) Quantification of membrane voltage of WT and ?miR-124 at 7 and 14 dpi, an indicator for potassium and proton channel activity.(K–M) Quantification of peak and steady currents with example traces (K) at 7 and 14 dpi. Peak currents are sodium-channel dominated, whereas steady currents are potassium-channel dominated.Scale bars, 100 µm. Sample number, n, refers to the number of biological samples (B, D, and F), number of analyzed neurites (E and G), and number of recorded cells as stated in (H) (H–M). Statistical analysis, unpaired Student’s t tests for two-group comparisons and Holm-Sidak correction for multiple comparisons with *p = 0.05, **p = 0.01, ***p = 0.001. Data are represented as mean ± SEM.See also Figure S2.

Fig 3: BAFF-R expressing B cells at different maturational stages are present in decidua.To identify lymphocytes, in the first panel to the far left, live cells were gated using a viability dye that marks all dead cells and the lymphocyte gate was then set within CD45-expressing singlet leukocytes. (a–c) Within lymphocytes, CD56+ NK cells, CD3+ CD56neg T cells, CD3+ CD56+ NK-T-like cells and CD19+ CD20+ B cells were identified (second and third panels). B cell maturational stages were distinguished based on CD24 and CD38 expression (fourth panel). The BAFF-R expression on the different lymphocyte populations is depicted to the far right. (d) The proportion of different cell subsets with in the lymphocyte gate and (e) the proportion of B cells at different maturational stages isolated from decidua basalis, decidua parietalis and cord blood. (f) The mean fluorescence intensity of BAFF on B cells at different maturational stages. Floating bars show minimum to maximum values, and horizontal bar indicates the median (n = 3). Approximately 40.000 cells were collected in the lymphocyte gate for d. basalis and CBMC and 20.000 for d. parietalis.

Fig 4: In vivo activation of lymphocytes, monocytes and neutrophils after CHMI. In vivo leucocyte activation was determined by direct staining of fresh whole blood with fluorescent antibodies every 2 days post-challenge. Lymphocytes were defined based on forward scatter and sideward scatter characteristics, and duplet events were excluded. a NK cell activation was defined as the percentage of CD3-CD56dimCD16+ live cells expressing CD69. b ?dT cell activation was defined as the percentage of CD3+?dTCR+ live cells expressing CD69. c NKT cell activation was defined as the percentage of CD3+?dTCR-CD56+ live cells expressing CD69. d aßT cell activation was defined the as percentage of CD3+?dTCR-CD56- live cells expressing CD69. e Monocytes were defined based on forward and side scatter characteristics, and the as HLA-DR+CD14+. Within the monocyte population, cells were then divided into CD16- and CD16+ monocytes. f–g Within the CD16- monocyte population, the relative change in mean fluorescent intensity of HLA-DR and CD86 compared to pre-malaria challenge values was determined. h Neutrophils were defined based on forward and side scatter characteristics, and then defined as HLA-DR-CD14-CD16+CD11b+. Activated neutrophils were defined as CD62LdimCD11bhigh. i–j IFN-? and granzyme B were measured by Luminex assay in citrate plasma taken ever 2 days. Circulating cytokine levels are corrected for baseline levels (pre-BCG vaccination time point) at each time point. In all graphs the grey dots represent non-BCG vaccinated control group volunteers (n = 10), and each coloured dot shows an individual BCG vaccinated volunteer (n = 9). Statistical analysis are between BCG vaccinated and control volunteers at a single time point, and p-values are the results of Mann–Whitney U-test. *p < 0.05. k Circulating CRP levels were measured in citrate plasma are shown for each BCG vaccinated volunteer (colours consistently represent the same volunteers across each graph)

Fig 5: Gemcitabine does not negatively impact HPC-NK cell phenotype and functionality. A-K. Phenotype of HPC-NK cells treated with gemcitabine for 48 h. Percentage positivity corrected for background staining in the corresponding control sample is shown. Control samples did not contain the antibodies of interest. Protein expression of (a) NKG2A on viable CD56+CD3- cells, or (b) CD158b, (c) CD158e1, (d) NKp30, (e) NKp44, (f) NKp46, (g) DNAM-1, (h) NKG2D, (i) CD69 (all n = 3) on viable CD56+CD3-NKG2A+ cells, or (j) FasL, (k) TRAIL (both n = 3) on living CD56+ cells is shown. L-N. Functionality of HPC-NK cells treated with gemcitabine for 48 h, after 4 h stimulation with (white) or without (black) K562 (all n = 3). Percentage positivity as protein expression of (l) perforin on viable CD56+ cells, (m) CD107a (degranulation) and (n) IFN? on viable CD56+ perforin+ HPC-NK cells is shown. Graphs show individual donors (NK1-6). Statistical significance was tested by repeated-measures One-way ANOVA for A-K and repeated-measures Two-way ANOVA for L-N, all ANOVAs with Bonferroni correction