Fig 2: IL-32 induces a systemic CD8+ T cell–mediated tumor-specific immune response.(A) Experimental setup for in vivo tumor treatments. MC38 and B16F10 were inoculated in C57BL/6J mice, and 4T1 tumors in BALB/c mice (A–O). (B) Growth curves of IL-32–, cGAMP-, or PBS-treated primary and (C) contralateral, nontreated B16F10 melanomas. *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative of 4 independent experiments, with n = 6 mice per group. (D) Kaplan-Meier survival curves of B16F10-bearing mice treated with IL-32 (n = 6) or PBS (n = 8). (E) Representative growth curves of IL-32–treated and untreated MC38 colon adenocarcinoma (n = 6) and (F) orthotopic 4T1 breast tumors (n = 10). (G–O) On day 14, the primary treated tumors and spleens were harvested for flow cytometric analyses, IHC or TCRβ chain sequencing. (G) Representative flow cytometry plots displaying frequencies of CD45+ immune cells for each treatment group and (H) their quantification shown as relative frequencies (n = 18). (I) Relative frequencies of CD8+ and CD4+ T cells as a percentage of live cells (n = 18). (J) Representative immunohistochemical staining and (K) morphometric enumeration as cells/mm2 of CD8+ T cells (n = 3). Scale bar: 20 μm. (L) Relative frequencies of IFN-γ+ cells as percentage of CD8+ T cells (n = 12). (M) Frequencies of Nur77-GFP+ cells of CD8+ T cells, as determined by flow cytometry in B16F10-inoculated and PBS- (n = 14) and IL-32–treat (n = 13) Nur77 mice. (N) TCR clonality in tumors (left, PBS, n = 7; IL-32, n = 5) and spleens (right, PBS, n = 7; IL-32, n = 6), representative of 2 independent experiments. (O) Relative frequencies of TRP-2 tetramer–positive cells, as a proportion of CD8+ T cells in tumors (left, n = 6) and spleens (right, n = 18). (P) Growth curves of IL-32–treated and untreated B16F10 primary tumors (PBS, IL-32, PBS + aCD8, n = 18; IL-32 + aCD8, n = 16) and (Q) untreated contralateral tumors with or without CD8 depletion (n = 16–18). *P = 0.0171; **P = 0.003; ****P < 0.0001. (R) Growth curves of IL-32–treated and untreated B16F10 primary tumors with or without CD4 depletion (n = 11) or (S) NK depletion (PBS, n = 12; IL-32, n = 18). (B, C, E, F, and P–S) Growth curves are shown as mean ± SEM, using 2-way ANOVA followed by Šidák’s multiple comparisons test. (D) Comparison of survival curves was performed using a log-rank (Mantel-Cox) test. (H, I, K–M, and O) Data are displayed as mean ± SEM. Statistical significance was determined by unpaired, 2-tailed Student’s t test. ****P < 0.0001.

Fig 3: Samples contaminated with different non-adipose tissues can affect experimental data(A) Tissue samples that sink in Wash/Digestion buffer are most likely not adipose tissue. When acquiring adipose tissue (AT) from mice, if testes (middle) or lymph nodes (LN) (right) are accidentally acquired, ensure they are removed before digestion.(B) Tissue contaminants can distort different cell populations. As shown via flow cytometry analysis, when lymph nodes from a mouse are present during digestion, samples have increased CD45+ cells. This can compromise studies investigating T cell (CD45+ CD3+) abundance (right).

Fig 4: rhIL-7-hyFc enhances mCART19 expansion and persistence in immunocompetent mice.a C57BL/6J mice were injected with 5 × 104 syngeneic tumor cells (9523, a myeloid cell line to which murine CD19 expression was added) followed by cyclophosphamide (Cytoxan) conditioning one day prior to receiving 6 × 106 UTD or murine CART19 (mCART19), then given serial injections of rhIL-7-hyFc on days +1, +15, and +29 (n = 4/group). b Overall survival of mice in each group. p Values were calculated using two-sided Wilcoxon test (mCART19 + rhIL-7-hyFc vs. mCART19, p = 0.32, mCART19+rhIL-7-hyFc vs. UTD + rhIL-7-hyFc, p = 0.044). c Peripheral blood total WBC counts over time. d Absolute numbers of mCART19 cells over time. e Total WBC counts were fractionated by flow cytometry into percent CD45.1+ (adoptively transferred T cells) and CD45.2+ (endogenous cells); CD45.2 cells are subdivided into CD19+ B cells, CD3+ T cells, and Gr-1+ myeloid cells at the indicated timepoints. f Absolute numbers of CD45.2+ B cells, T cells, and myeloid cells over time, calculated by multiplying the total WBC count by the fraction of each cell determined by flow cytometry. All data represent median ± range. Dotted lines represent rhIL-7-hyFc injection time points. Source data are provided as a Source data file. ns: not significant, *p ≤ 0.05.

Fig 5: rhIL-7-hyFc reduces the number of CAR T cells required for the biological effect.a–e Balb/c mice were first given 1 × 106 A20 tumor cells, followed by cyclophosphamide (Cytoxan) conditioning 1 day prior to receiving mCART19 (5 × 104, 2.5 × 105, or 1.25 × 106 cells per mouse), then given serial injections of rhIL-7-hyFc on days +1, +15, and +29 (n = 4/group). b Overall survival of mice for all groups. c Peripheral blood CAR T cell expansion was measured over time by quantification of CD45.1+GFP+ cells. Each line represents values from one mouse. d B cell numbers were measured over time by quantification CD45.2+ CD19+ cells. Data represented as median ± range. Note that 5 × 104 cells (left panel) are depicted on a different scale. e B cell numbers on day +101 after T cell injection. f NSG mice were injected with 5 × 105 RamosCBR-GFP followed by UCART19 treatment (5 × 104, 2.5 × 105, or 1.25 × 106 cells per mouse) with serial injections of rhIL-7-hyFc on days +1, +15, and +29 (n = 5 mice per group). The top panels represent overall survival, while the bottom panels show serial tumor burden measurements (each line represents one mouse). p Values were calculated using two-sided Wilcoxon test (UCART19 1.25 × 106 + rhIL-7-hyFc vs. UCART19 1.25 × 106 only, p = 0.0039). Source data are provided as a Source data file. **p = 0.01.