Fig 2: Expression Pattern of Fc?Rs in hFc?R Mice and Human TumorsThe expression of hFc?Rs was analyzed by flow cytometry in leukocyte suspensions obtained from blood and MCA205 tumors in hFc?R mice and from metastatic deposits of human melanoma and paired blood samples.(A) Representative histograms demonstrating Fc?R expression on CD3+ T cells, CD19+ B cells, NK1.1+ NK cells, CD11b+NK1.1-Ly6G-CD11clow/- monocyte/macrophages (Mo/MF) and CD11b+Ly6G+ granulocytes isolated from hFc?R mice 10 days after s.c. tumor inoculation.(B) Percentage of expression of Fc?Rs in hFc?R mice from (A) (n = 3). Results are representative of three independent experiments.(C) Representative histograms demonstrating Fc?R expression on CD3+CD56- T cells, CD19+CD3- B cells, CD56+CD3- NK cells, CD11b+CD14+HLA-DR+ Mo/M? and CD11b+CD15+CD14- granulocytes isolated from melanoma patient samples.(D) Percentage expression of Fc?Rs in metastatic deposits of human melanoma from (B) (n = 10). Error bars show ±SEM.*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S2 and Table S2.

Fig 3: rhIL-7-hyFc enhances UCART19 expansion and function in vitro.a rhIL-7-hyFc is an engineered IL-7 homodimer fused to IgD and IgG4 elements (hyFc©), promoting in vivo stability and reducing complement activation. b UCART19 was generated by activating human T cells with anti-CD3/CD28 beads, followed by CRISPR/Cas9 deletion of the human T cell receptor alpha subunit (TRAC), and lentiviral transduction of a third-generation anti-CD19 CAR. c–e UCART19 was incubated with CD19+ RamosCBR-GFP at an effector to target (E:T) ratio of 2:1 in 10 ng/ml of rhIL-7 or the indicated concentrations of rhIL-7-hyFc. On day 7, UCART19 was replated with RamosCBR-GFP at an E:T of 2:1 with the addition of fresh cytokines. UCART19 numbers and phenotypes were determined by serial counting and flow cytometry. Bar graphs represent median values, with each data point representing a different T cell donor. d Representative growth plot for a single donor (left) and compiled fold-expansion of all donors on day 14 (right, n = 3 donors for rhIL-7, 4 donors for all other groups). p Values were calculated by one-way ANOVA with Tukey’s multiple comparisons test (rhIL-7 10 vs. 0, p = 0.0055, rhIL-7-hyFc 10 vs. 0, p = 0.0246, rhIL-7 10 vs. rhIL-7-hyFc, p = 0.82). e UCART19 viability (%propidium iodide exclusion, rhIL-7 10 vs. 0: p < 0.0001; rhIL-7-hyFc 10 vs. 0: p < 0.0001; rhIL-7 10 vs. rhIL-7-hyFc: p = 0.9999), apoptosis (%annexinV+, rhIL-7 10 vs. 0: p = 0.0005; rhIL-7-hyFc 10 vs. 0: p = 0.0002; rhIL-7 10 vs. rhIL-7-hyFc: p = 0.9615) and proliferation (%Ki-67+, rhIL-7 10 vs. 0: p < 0.0001; rhIL-7-hyFc 10 vs. 0: p < 0.0001; rhIL-7 10 vs. rhIL-7-hyFc: p = 0.9805) measured after rhIL-7 or rhIL-7-hyFc treatment in the presence of target cells after 14 days (n = 3 different donors). P-values were calculated by one-way ANOVA with Tukey’s multiple comparisons test. f UCART19 was incubated with Ramos at an E:T of 1:1 for 16 h with either vehicle or rhIL-7y-hyFc (100 ng/ml) prior to loading purified CAR T cells onto the IsoCode chip for single-cell multiplex cytokine analysis. Polyfunctional strength index (PSI), as defined by the percentage of cells secreting multiple cytokines multiplied by the mean fluorescence intensity of the secreted proteins, was calculated for UCART19 on day 0 (UCART19 input cells), day 14 with vehicle, or day 14 with rhIL-7-hyFc. Bar graphs represent mean ± standard error of the mean (n = 1 donor, 2 technical replicates for day 0, 3 technical replicates for day 14 vehicle/rhIL-7-hyFc). Further details regarding the cytokines assayed, polyfunctionality, and signal strength is depicted in Supplemental Fig. 2. g, h UCART19 cells serially expanded in the presence of rhIL-7-hyFc and CD19+ Ramos cells at a 2:1 E:T ratio as in c were evaluated by flow cytometry on day +14. Bar graphs represent mean ± SD of four technical replicates (n = 1 donor). g CAR expression analysis on day +14 in CD4+ and CD8+ T cells (left), with representative flow cytometry plots of UCART19 using CD34 to define CAR-T populations (right). h Memory phenotype of T cells as measured by expression of CCR7 and CD45RO (T-EFF effector T cells, T-EM effector memory T cells, T-CM central memory T cells, T-N naive T cells). Source data are provided as a Source data file. ns: not significant, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.

Fig 4: Representative Flow Cytometry Plots Showing the Percentage of CD3+, CD4+, and CD8+ T Cells Isolated from LRSCs Using the Magnetic Technique

Fig 5: Innate lymphocyte subtype analysis within TILs population.(A) Gating strategy for identification of ?dT cells subtypes after gating on CD3 and ?dTCR positive cells. This representative example is from a TIL sample following initial 14-day expansion. (B) ?dT cell numbers expressed as % of CD3+ cells in TILs following initial expansion (n = 31) and then after REP (n = 41): statistical analysis by unpaired t-test. (C) ?dT cell numbers expressed as % of CD3+ cells in 6 paired TIL samples following initial expansion and then after REP. (D) ?dT cell subset distributions in the TILs population defined as in (A) following initial expansion (n = 14) and then after REP (n = 12). Vd1+ cell proportions fell significantly (p = 0.006) and Vd1-/Vd2- proportions rose significantly after REP (p = <0.0001): statistical comparison by one-way ANOVA with Sidak’s correction for multiple comparisons. (E) ?dT cell subset distributions in 5 paired TILs populations defined as in (A) following initial expansion and then after REP.