Fig 1: DuoBody-CD40×4-1BB enhances TIL expansion. Tumor tissues resected from patients were cut into pieces of 1–2 mm3 and cultured in the presence of IL-2 (10 U/mL) and DuoBody-CD40×4-1BB (0.0008, 0.003, 0.0125, 0.05 or 0.2 µg/mL), a non-humanized variant of DuoBody-CD40×4-1BB (0.01–1 µg/mL), control antibodies (0.2 µg/mL) or IL-2 only for 10–15 days. (A–D) Cell numbers after expansion were determined by flow cytometry, and normalized to reference beads. Total TIL, CD8+, CD4+ T-cell and NK-cell numbers are shown for a patient with colon cancer (A) and three patients with NSCLC (B–D) *p<0.05. One-way ANOVA with Dunnett’s multiple comparisons test. (E) TCR repertoire analysis was performed by TRB RNA sequencing of TIL expanded in (D), in the presence of a non-humanized variant of DuoBody-CD40×4-1BB (0.1 µg/mL) or IL-2 only. Cumulative frequency of shared clonotypes, the 20 most abundant clonotypes in the DuoBody-CD40×4-1BB-treated cultures are shown. ANOVA, analysis of variance; NK, natural killer; NSCLC, non-small cell lung cancer; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte.
Fig 2: Inhibition of DYRK1B function suppresses Th1 and Th17, but enhances Treg differentiation. (A–C) Human naïve CD4+ T cells were stimulated by anti-CD3 and anti-CD28 and then differentiated under Th1-, Th17- and Th2-polarizing conditions, respectively, in the absence or presence of a selective DYRK1B inhibitor at 4 different concentrations for 96 h. The scatter dot plots represent data from triplicate sample and analyzed by flow cytometry for CD4+IFN-γ+, CD4+IL17A+, and CD4+IL4+, respectively. (D) Human naïve CD4+ T cells were stimulated by anti-CD3 and anti-CD28 and then differentiated in the presence of TGF-β1 and IL-2 with or without a selective DYRK1B inhibitor at 4 different concentrations for 96 h. The scatter dot plot represents data from triplicate samples as analyzed by flow cytometry for iTreg (CD4+CD25+FOXP3+) population. The results are summarized in the bar graphs, and data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001), ns non-significant).
Fig 3: Gut microbial functional modules with altered abundance in the ATLL patient group.(A) Comparison of KO abundances between patients with ATLL and healthy controls. X-axis, fold change (log2-transformed) in the mean relative abundance of each bacterium in each group; Y-axis, -log10-transformed P values from pairwise comparisons. The horizontal line represents P = 0.05. P values were calculated by the two-tailed Brunner–Munzel test. The plot size shows the KO prevalence (number of subjects) in the ATLL patient group.(B) Enrichment analysis of KOs that were significantly more abundant in patients with ATLL. P values are transformed by -log10. The vertical line represents P = 0.05. ∗, q < 0.05.(C) SSA synthesis module. Abbreviations: HPC, homoprotocatechuate; CHMS, 5-carboxymethyl-2-hydroxymuconic-semialdehyde; CHM, 5-carboxymethyl-2-hydroxymuconate; OPET, 5-oxo-pent-3-ene-1,2,5-tricarboxylate; HHDD, 2-hydroxyhepta-2,4-diene-1,7-dioate; OHED, 2-oxo-hept-3-ene-1,7-dioate; HHED, 2,4-dihydroxy-hept-2-ene-1,7-dioate; SSA, succinic semialdehyde.(D) Association of KO abundance and genus Klebsiella with the SSA synthesis module. The box plot represents the relative abundance of the KOs (left) and ortholog genes assigned to the KO from Klebsiella (right). The bar plot represents the bacterial composition of the KOs of each subject (right). The top two genera with the highest average relative abundance in at least one KO are displayed.(E) (F) Cell growth rate. (E) IL-2-dependent ILT-Mat cells and (F) IL-2-independent MT-1 cells treated with SSA. The values represent the means ± s.d. ∗P < 0.05; unpaired two-tailed Student's t-test.
Fig 4: Oxidized-desialylated LDL downregulates cytotoxicity receptor CD56 and upregulates the CD3 receptor. Activated and expanded LAK cells were cultured in serum free X-VIVO 10 media in a V-bottom 96 well plate with IL-2 in the absence or presence of native LDL or oxidized-desialylated LDL at 50 µg/ml for 72 hours. Then LAK cells were washed three times with X-VIVO 10 serum free media, reconstituted in PBS, and labeled with anti-CD56 antibodies, as well as with the dead staining dye SytoxBlue. A) Live cells were gated and plotted against CD3 and CD56. B) Oxidized-desialylated LDL decreased the number of CD3-CD56+ cells. C) The number of NKT cells (CD3+ CD56+) also decreased significantly. D) The number of CD3 positive cells increased significantly upon oxidized-desialylated LDL treatment of LAK cells. n = 5 per group. Error bars represent standard deviation. ** indicates statically significant differences (p<0.0001), determined using one-way ANOVA with Tukey posthoc test.
Fig 5: Sample inclusion flow chart and timeline. (A) All samples were initially assessed with at least one of the assays (K562- or HMBPP-induced expression of CD107a on NK cells or Vγ9Vδ2 T cells, respectively). Assays with fewer than 250 flow cytometry events were excluded (n=14). The sample donors were subsequently categorized as fHLH patients or controls after genetic testing or clinical follow-up: Blue: No degranulation deficiency; 190 samples from 170 individuals; with 18 of these being IL-2 prestimulated. Red: Genotype consistent with a degranulation deficiency; 23 samples from 12 patients with primary HLH [including homozygous or compound heterozygous variants in UNC13D (n=10), STXBP2 (n=1), or RAB27A (n=1)]. Three of these samples were IL-2 prestimulated. (B) The NK cell degranulation assay was introduced in May 2014, while Vγ9Vδ2 T cell-based assay was introduced in April 2021. Blood samples were collected between May 2014 and November 2022, with healthy control samples collected from July 2019 onwards.
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