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. Author manuscript; available in PMC: 2019 Mar 11.
Published in final edited form as: Nat Med. 2018 May 28;24(6):731–738. doi: 10.1038/s41591-018-0041-7

Figure 3: Modulation of macrophage function determines CRS severity.

Figure 3:

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 (Supplementary 7b). (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.