PMC

a. Weight change of tumour bearing mice after 1928z CAR T cell transfer. Weight per mouse is normalised to starting weight pre-CAR transfer (Tumour only n=8 mice, 1928z-LNGFR n=7 mice, 1928z-mCD40L n=5 mice). Results from one experiment. (Two-way ANOVA).
b. Percent survival of tumour bearing mice treated with 1928z-LNGFR or 1928z-mCD40L CAR T cells. (1928z-LNGFR n=16 mice, 1928z-mCD40L n=13 mice). Results pooled from two independent experiments. (Log-rank Mantel-Cox test).
c. Representative flow cytometric plot showing Total Peritoneal Macrophages within the gated population (Resident Peritoneal Macrophages and CRS-Associated Macrophages). Cells were obtained by peritoneal lavage. Analysis of peritoneal macrophages for levels of F4/80 and Ly6C in the presence of the 1928z-mCD40L CAR are representative of one experiment of cells obtained from Tumour only n=8 mice, 1928z-LNGFR n=7 mice, 1928z-mCD40L n=5 mice. Experiment was performed once.
d. Percent of CD40+ total peritoneal macrophages, obtained by peritoneal lavage at 61 hours post 1928z-LNGFR or 1928z-mCD40L CAR T cell transfer, analyzed by flow cytometry (Tumour only n=8 mice, 1928z-LNGFR n=7 mice, 1928z-mCD40L n=5 mice). Analysis of peritoneal macrophages for surface expression of CD40 in the presence of the 1928z-mCD40L CAR was performed once. (Unpaired two sample t-test, two-tailed).
e. Serum levels of murine cytokines at 18 hours post CAR T cell transfer. Cytokine levels were measured by Cytokine Bead Array (CBA). (Tumour only n=8 mice, 1928z-LNGFR n=7 mice, 1928z-mCD40L n=7 mice). Serum cytokine measurements when using the 1928z-mCD40L CAR were performed once. (Unpaired two sample t-test, two-tailed).
f. Percent of myeloid populations from peritoneum, spleen and bone marrow expressing iNOS protein in tumour only mice and tumour + CAR mice. iNOS expression was determined by intracellular flow cytometry. (For peritoneum n=14 mice per group, for spleen and bone marrow n=10 mice per group). For peritoneum results are shown from three pooled independent experiments. For spleen and bone marrow results are shown from two pooled independent experiments. (Unpaired two sample t-test, two-tailed).
g. Weight change of tumour bearing mice after 1928z CAR T cell transfer. Weight per mouse is normalised to starting weight pre-CAR transfer. Mice were treated with L-NIL or vehicle (PBS). (Tumour only n=7 mice, CAR + L-NIL n=7 mice, CAR + Vehicle n=8 mice). Weight measurement under conditions of CRS was performed once with L-NIL and once with 1400W (). (Two-way ANOVA).
h. Percent survival of tumour bearing mice after 1928z CAR T cell transfer receiving 1400W or Vehicle (PBS). (Vehicle n=20 mice, 1400W n=19 mice). Results pooled from three independent experiments. (Log-rank Mantel-Cox test).
All data are means ± s.e.m.

a. Schematic of mouse model. Raji tumour cells are intraperitoneally injected in mice and allowed to grow for three weeks, where they eventually grow into vascularized solid tumour masses. Thirty million CAR T cells are transferred and mice are monitored over the following days. Mice are sacrificed and cells are obtained for analysis through peritoneal lavage and tissue harvesting for further analysis
b. Weight change of tumour bearing mice after 1928z CAR T cell transfer. Weight per mouse is normalised to starting weight pre-CAR transfer (Tumour only n=12 mice, Tumour + CAR n=18 mice). Experiments monitoring weight under CRS conditions were performed in at least 20 independent experiments. (Two-way ANOVA).
c. Percent survival of mice after 1928z CAR T cell transfer (Tumour only n=12 mice, Tumour + CAR n=18 mice). Experiments monitoring survival under CRS conditions were performed in at least 20 independent experiments. (Log-rank Mantel-Cox test).
d. Serum levels of murine SAA3 at 42 hours post 1928z CAR T cell transfer as measured by ELISA (No Tumor no CAR n=5 mice, tumour only n=5 mice, CAR only n=5 mice, tumour + CAR n=7 mice). SAA3 levels under CRS conditions were measured at least in two independent experiments. (Unpaired two sample t-test, two-tailed).
e. Cytokine levels 4.5 hours before (pre-car) and 24 hours post 1928z CAR T cell transfer (CRS or Severe CRS). Mice that eventually died from CRS were grouped under Severe CRS while mice that survived but suffered greater than 10% weight loss were grouped under CRS. “m” prefix denotes murine origin while “h” prefix denotes human origin. Cytokine levels were measured by Cytokine Bead Array (CBA). (Severe CRS n=5 mice, CRS n=10 mice, pre-CAR n=16 mice). Stratification of cytokines based on survival were performed at least in three independent experiments. (Unpaired two sample t-test, two-tailed).
f. Species of origin of pro-inflammatory cytokines. (Human cytokines from n=15 mice, Mouse cytokines from n=15 mice).
g. Percent survival of tumour bearing mice treated with 1928z CAR T cells that received anti-murine IL-6R blocking antibody or isotype (vehicle). (Vehicle n=6 mice, anti-mIL-6R n=5 mice). Experiments measuring survival after blocking murine IL-6R were performed once. (Log-rank Mantel-Cox test).
All data are means ± s.e.m.

a. Immunohistochemical staining for Mac2 of sections from 3-week Raji tumour explants. Mice had not received CAR T cells. Immunohistochemical staining of tumor explants for Mac 2 was performed once.
b. Absolute counts of myeloid cell populations obtained by peritoneal lavage 68 hours after 1928z CAR T cell transfer. Phenotypes were analyzed by flow cytometry and absolute quantification was performed by the addition of counting beads. (No tumor no CAR n=5 mice, CAR only n=5 mice, Tumour only n=6 mice, Tumour + CAR n=7 mice). Enumeration of myeloid cells under these conditions was performed at least 10 times. (Unpaired two sample t-test, two-tailed).
c. Representative flow cytometric plot showing Total Peritoneal Macrophages within the gated population (Resident Peritoneal Macrophages F4/80^hi Ly6C^neg-lo and CRS-Associated Macrophages F4/80^int-lo Ly6C^int-hi). Cells were obtained from peritoneal lavage. Analysis of peritoneal macrophages for levels of F4/80 and Ly6C was performed at least 10 times.
d. Absolute counts of myeloid cell populations obtained from multiple organs 18 hours after 1928z CAR T cell transfer (Tumour only n=4 mice, Tumour + CAR n=4 mice). Enumeration of cells retrieved from peritoneum, spleen and bone marrow under CRS conditions was performed at least 3 times. (Unpaired two sample t-test, two-tailed).
e and f. Fold change of pro-inflammatory cytokine gene expression in myeloid populations as determined by RNAseq analysis. Fold change was determined by comparing each population under tumour only and tumour + CAR conditions. Significant downregulation (green bars), significant upregulation (red bars), no significant change (gray bars). Gene expression levels were determined from cells obtained from Tumour only n=9 mice and Tumour + CAR n=9 mice. Cells from three mice of the same group were randomly pooled to create one biological replicate group to the end of obtaining three biological replicate groups of Tumour only mice and three biological replicate groups of Tumor + CAR mice. (Binomial test, FDR-adjusted p-values).
All data are means ± s.e.m. except in (d) which are means ± s.d.

a and b. Fold change of IL-1 signaling component gene expression in myeloid populations as determined by RNAseq analysis. Fold change was determined by comparing each population under tumour only and tumour + CAR conditions. Significant downregulation (green bars), significant upregulation (red bars), no significant change (grey bars). Gene expression levels were determined from cells obtained from Tumour only n=9 SSCID-beige mice and Tumour + CAR n=9 SCID-beige mice. Cells from three mice of the same group were randomly pooled to create one biological replicate group to the end of obtaining three biological replicate groups of Tumour only mice and three biological replicate groups of Tumor + CAR mice. (Binomial test, FDR-adjusted p-values).
c. Percent survival of tumour bearing SCID-beige mice after 1928z CAR T cell transfer receiving Anakinra or Vehicle (PBS). (Anakinra n=11 mice, Vehicle n=10 mice). Results pooled from two independent experiments. (Log-rank Mantel-Cox test).
d. Percent of peritoneal macrophages expressing iNOS at 18 hours post CAR T cell transfer. SCID-beige mice were treated with isotype, murine IL-6 blocking antibody, Anakinra or murine IL-6 blocking antibody + Anakinra. (Tumour only n=4 mice, Isotype n=3 mice, Anti-mIL-6 n=3 mice, Anakinra n=3 mice, Anti-mIL-6 + Anakinra n=4 mice). Measurement of %iNOS+ macrophages with isotype or anti-mIL-6 blockade was performed in two independent experiments. Measurement of %iNOS+ macrophages with isotype, Anakinra or Anakinra + anti-mIL-6 blocking antibody was performed once. (One-way ANOVA).
e. Levels of murine IL-1Ra in supernatants of 1928z-LNGFR and 1928z-mIL1Ra transduced CAR T cells after 48 hours in culture as determined by ELISA. One sample analyzed in duplicate for each CAR construct. Measurement of IL-1Ra in supernatants of 1928z-mIL1Ra CAR T cells was performed once.
f. Percent survival of tumour bearing SCID-beige mice after 1928z-LNGFR or 1928z-mIL1Ra CAR T cell transfer. (1928z-LNGFR n=22 mice, 1928z-mIL1Ra n=18 mice). Results from three pooled independent experiments. (Log-rank Mantel-Cox test).
g. Serum levels of murine cytokines from SCID-beige mice surviving CRS at 18 hours post CAR T cell transfer. Tumour bearing mice received 1928z-LNGFR or 1928z-mIL1Ra CAR T cells. Cytokine levels were measured by Cytokine Bead Array (CBA) (1928z-LNGFR n=3 mice, 1928z-mIL1Ra n=3 mice). Results representative of three independent experiments. (Unpaired two sample t-test, two-tailed).
h. Percent tumour free survival of NSG mice receiving 0.2e6 or 0.5e6 1928z-LNGFR or 1928z-mIL1Ra CAR T cells. Tumours were injected intravenously on Day −4 and CAR T cells on Day 0. (Tumour only n=4 mice, 0.2e6 1928z-LNGFR n=7 mice, 0.2e6 1928z-mIL-1Ra n=7 mice, 0.5e6 1928z-LNGFR n=11 mice, 0.5e6 1928z-mIL-1Ra n=11 mice). Experiment was performed once. (Log-rank Mantel-Cox test).
All data are means ± s.e.m.