Fig 2: B7-H3.CAR-T cells co-expressing CCR2b have superior antitumor activity against the A549 tumor in the brain.a Schematic of the A549 brain tumor model in which NSG mice were implanted intracranially (i.c.) with FFluc-A549 cells (5 × 104 cells) into the right brain hemisphere, and then treated with CD19.28, B7-H3.28, or CCR2b.B7-H3.28 CAR-T cells (10 × 106) inoculated i.v. 18 days later. b, c Representative tumor BLI images (b) and BLI kinetics (c) in the model shown in (a). Days indicated in (b) represent the days post T cell infusion. d Kaplan–Meier survival curves of mice in (b). ***p = 0.0008 (CCR2b.B7-H3.28 vs. B7-H3.28), ?2 test. N = 4 mice in CD19.28 group, and n = 9 mice in B7-H3.28 and CCR2b.B7-H3.28 in (c, d). e In the separate experiment, mice were euthanized 8 days after CAR-T cell infusion, and human CD45+CD3+ T cells were enumerated in blood, spleen, and the right brain hemisphere by flow cytometry. Bar graph summary are shown, data are presented as mean values + SD (n = 6 mice in B7-H3.28, n = 7 mice in CCR2b.B7-H3.28), *p = 0.0464, unpaired t test with Welch’s correction and two-tailed p value calculation. f In another experiment, the same procedure as the experiment in (e), when the mice were euthanized 8 days after CAR-T cell infusion, human CD45+CD3+ T cells in both the left brain hemisphere (without tumor) and right brain hemisphere (with tumor) were enumerated by flow cytometry, besides blood and spleen. Bar graph summary are shown, data are presented as mean values + SD (n = 5 mice/group), **p = 0.0062, ****p < 0.0001, two-way ANOVA with Sidak’s multiple comparisons test. Source data for (c, d, e, f) are provided as a Source Data file.

Fig 3: CCR2b co-expressing B7-H3.CAR-T cells have comparable antitumor activity when coculture with NSCLC cell lines in vitro.a Representative flow plots of CARs expression in CD19.28, B7-H3.28, B7-H3.BB, CCR2b.B7-H3.28 and CCR2b.B7-H3.BB CAR-T cells as assessed by flow cytometry. b, c Summary of the percentage (b) and MFI (c) of CAR expression in B7-H3.28, B7-H3.BB, CCR2b.B7-H3.28 and CCR2b.B7-H3.BB CAR-T cells (n = 10 independent experiments using CAR-T cells generated from ten different donors, *p = 0.0219 for CD19.28 vs. B7-H3.28, *p = 0.0240 for CD19.28 vs. B7-H3.BB, *p = 0.0295 for B7-H3.BB vs. CCR2b.B7-H3.28, *p = 0.0217 for B7-H3.BB vs. CCR2b.B7-H3.BB, one-way ANOVA). Data are presented as mean values ± SD. d, e GFP-labeled NSCLC cell lines were cocultured with CD19.28, B7-H3.28, B7-H3.BB, CCR2b.B7-H3.28 or CCR2b.B7-H3.BB CAR-T cells at the CAR-T cell to tumor cell ratio of 1:5. On day 5, NSCLC cells (GFP+) and CAR-T cells (CD3+) were enumerated by flow cytometry. Representative flow-cytometry plots (d) and quantification of residual tumor cells (e) are illustrated. Data are presented as mean values + SD. f, g Summary of IFN? (f) and IL2 (g) released by CAR-T cells in the culture supernatant after 24 h of coculture with the indicated cell lines as measured by ELISA, n = 4 independent experiments using CAR-T cells generated from four different donors in (d–g). Data are presented as mean values + SD. Source data for (b, c, e, f, g) are provided as a Source Data file.

Fig 4: Coexpression of CCR2b in B7-H3.CAR-T cells improves the migration toward CCL2.a Mean gene expression distributions of CCL and CXCL family members (41 genes) in lung cancer adenocarcinoma (LUAD) (n = 517) and lung cancer squamous cell carcinoma (LUSC) (n = 501) cohorts from TCGA data set. The solid curve lines indicate the distribution of the quantiles of CCL2 in all 41 chemokines genes at each expression level, and the dotted line indicates the mean expression of CCL2 in LUAD and LUSC. b CCL2 measured in the culture supernatant of NSCLC cell lines (1 × 105/mL cells cultured in 24-well plates for 24 h) (n = 4 independent samples). Data are presented as mean values + SD. c Mean gene expression distributions of CCL and CXCL family members (39 genes) from 37 NSCLC paired with brain metastasis. The solid curve lines indicate the distribution of the quantiles of CCL2 in all the 39 genes at each expression level and the dotted line indicates the mean expression of CCL2 in lung cancer and brain metastasis. CCL2 expression is in the top 15 and 8% quantiles of the 39 CCL and CXCL genes expression in primary lung cancer tissue and brain metastasis, respectively. d Schematic of the vectors encoding B7-H3.CAR and CCR2b. e Representative flow plots showing CCR2 expression in control non-transduced T cells (NT), B7-H3.28, B7-H3.BB, CCR2b.B7-H3.28 and CCR2b.B7-H3.BB CAR-T cells. f Summary of CCR2 expression percentage (top) and MFI (bottom) in NT, B7-H3.28, B7-H3.BB, CCR2b.B7-H3.28 and CCR2b.B7-H3.BB CAR-T cells (n = 4 independent experiments using CAR-T cells generated from four different donors). Data are presented as mean values ± SD. g Cell numbers of NT, B7-H3.28, B7-H3.BB, CCR2b.B7-H3.28 and CCR2b.B7-H3.BB CAR-T cells migrated to the bottom of the transwell chambers with 5 µm pores that were induced by the culture supernatant of NSCLC cell lines and human CCL2 recombinant protein (10 ng/mL), (n = 3 independent experiments using CAR-T cells generated from three different donors). Data are presented as mean values + SD. Source data for (b, f, g) are provided as a Source Data file. Source data for (a, c) are provided as Supplementary Table 1.

Fig 5: The characteristics of PD-1highCXCR5– Tph cells(A) Representative data of PD-1highCXCR5– Tph cells in each group (left), the proportion of these T cells among healthcare workers (HCs) (n = 55), non-ICU patients with COVID-19 (non-ICU) (n = 56), and ICU patients (ICU) (n = 36). One-way ANOVA with Dunn’s multiple comparisons tests were performed (right). Data are represented as mean ± SEM.(B) Correlation between PD-1highCXCR5– Tph cells and plasmablasts in patients (both non-ICU and ICU, n = 51). Linear regression is shown with 95% confidence interval (gray area). Correlation statistics is two-tailed Spearman’s rank correlation test.(C) Principal-component analysis (PCA) of RNA-seq transcriptomes (n = 3, patients with COVID-19). Based on the expressions of PD-1 and CXCR5, six subsets were evaluated.(D) Heatmap of Tfh-related genes.21(E) Clustered heatmap of 100 genes that were differentially expressed (left column) in PD-1highCXCR5– Tph cells compared with cTfh cells (PD-1highCXCR5+ Tfh cells) (|log2FC| > 1, FDR < 0.05). The right column shows the log2FC.(F) Representative data of CCR5 and CCR2 expression on PD-1highCXCR5– Tph cells compared with PD-1highCXCR5+ Tfh cells.See also Figures S1–S3.