Solid tumour-induced systemic immunosuppression involves dichotomous myeloid-B cell interactions

Abstract

Solid tumours induce systemic immunosuppression that involves myeloid and T cells. B cell-related mechanisms remain relatively understudied. Here we discover two distinct patterns of tumour-induced B cell abnormality (TiBA; TiBA-1 and TiBA-2), both associated with abnormal myelopoiesis in the bone marrow. TiBA-1 probably results from the niche competition between pre-progenitor-B cells and myeloid progenitors, leading to a global reduction in downstream B cells. TiBA-2 is characterized by systemic accumulation of a unique early B cell population, driven by interaction with excessive neutrophils. Importantly, TiBA-2-associated early B cells foster the systemic accumulation of exhaustion-like T cells. Myeloid and B cells from the peripheral blood of patients with triple-negative breast cancer recapitulate the TiBA subtypes, and the distinct TiBA profile correlates with pathologic complete responses to standard-of-care immunotherapy. This study underscores the inter-patient diversity of tumour-induced systemic changes and emphasizes the need for treatments tailored to different B and myeloid cell abnormalities.

Extended Data Fig. 1.. Remote tumors disrupt early B cell development in two different patterns

a, Representative flow cytometry gating of mouse B cell subsets in the BM of TiBA-1 tumor-bearing (LLC), TiBA-2 tumor-bearing (PyMT-N) and Sham mice. b, The percentages of BM B cell subsets among total B cells in tumor-bearing and Sham mice were analyzed by flow cytometry. B6 background (LLC, PyMT-N, E0771), BALB/c background (4T1, 2208L, T12, T11). BM was analyzed by flow cytometry when tumor models had similar tumor sizes (around 1cm3). Sham-B6 (n=14 mice), PyMT-N (n=13 mice), LLC (n=10 mice), 2208L (n=7 mice), and other groups (n=5 mice). Two-way ANOVA with Dunnett’s multiple comparisons test compared to Sham. c,d, The absolute cell numbers of BM B cell subsets in tumor-bearing and Sham mice were analyzed by flow cytometry. BM was analyzed by flow cytometry when tumor models had similar tumor sizes (around 1cm3). Sham-B6 (n=14 mice), PyMT-N (n=13 mice), LLC (n=10 mice), 2208L (n=7 mice), and other groups (n=5 mice). One-way ANOVA (Brown-Forsythe and Welch ANOVA) with Dunnett’s T3 multiple comparisons test compared to Sham. e, Flow cytometry analyzed B220+ B cell numbers in the BM across different tumor models (similar tumor sizes). Sham-B6 (n=27 mice), PyMT-N (n=24 mice), LLC (n=15 mice) and other groups (n=5 mice). One-way ANOVA with Dunnett’s multiple comparisons test compared to Sham. f, Diagram summarizing TiBA phenotypic characteristics in the BM. For all boxplots (b-e), the line inside the box is the median value, and the bottom/top bars of the box indicate min to max values.

Extended Data Fig. 2.. scRNA-seq of BM cells in naïve mice and different TiBA tumor-bearing models

a, Diagram showing FACS-sorting of BM cells from Sham and TiBA tumor-bearing mice for scRNA-seq. b, Heatmap of top 5 markers expression of BM cell clusters in the BALB/c dataset. c, Heatmap of top 5 markers expression of BM cell clusters in the C57BL/6 dataset. d, Feature plots show the expression of early B cell marker genes across BM cell clusters. e, Diagram showing the firefly-luciferase/GFP-labeled 4T1 cells were transplanted into the mammary fat pad (MFP). f, Representative image showing negative luc-signals at different bones. g, Heatmaps of log10 fold change in the percentage of various B cell subsets in BM of tumor-bearing mice compared with Sham. n=3 mice/group.

Extended Data Fig. 3.. TiBA-1 and TiBA-2 drive distinct changes to systemic B cell profiles

a, Representative flow cytometry gating of mouse B cell subsets in the PB of Sham and 4T1 tumor-bearing mice. b, Quantification of Tie-B cell numbers in the PB and spleen in different tumor models by flow cytometry, Sham-B6 (n=11 mice), PyMT-N (n=6 mice) and other groups (n=5 mice). One-way ANOVA with Dunnett’s multiple comparisons test compared to Sham. c,d, Absolute cell numbers of various B cell subsets in the PB (c) and spleen (d) of tumor-bearing and Sham mice. PB was analyzed by flow cytometry when tumor models had similar tumor sizes (around 1cm³). n=6 mice (for Sham-B6 or PyMT-N) and n=5 mice (for other groups) mice, respectively. One-way ANOVA (Brown-Forsythe and Welch ANOVA) with Dunnett’s T3 multiple comparisons test compared to Sham. e, Heatmap of top 5 markers expression of PB cell clusters. f, Feature plots show the expression of Breg cell marker genes across PB cell clusters. g,h, Flow cytometry analysis of IL10 expression on different cell subsets in TiBA-2 tumor-bearing mice. MFI, mean fluorescent intensity. n=5 mice/group. One-way ANOVA with Dunnett’s multiple comparisons test compared to NC, mean ± s.d.. i-k, Flow cytometry analyzed tumor-infiltrating B cell subsets in different TiBA tumors (tumor sizes around 1cm³). TiBA-1 (n=11 mice), TiBA-2 (n=14 mice), TiBA-0 (n=14 mice). One-way ANOVA with Dunnett’s multiple comparisons test compared to TiBA-2, mean ± s.d.. l, Flow cytometry analyzed tumor-infiltrating plasma cells in TiBA tumors. n=5. One-way ANOVA with Tukey’s multiple comparisons test compared to TiBA-2, mean ± s.d.. m, Flow cytometry analyzed Tie-B cell numbers in tumors (tumor sizes around 1.5 cm³), blood and spleen of TiBA-2 models. n=8 mice. One-way ANOVA with Dunnett’s multiple comparisons test, mean ± s.d.. For all boxplots (b-d), the line inside the box is the median value, and the bottom/top bars of the box indicate min to max values.

Extended Data Fig. 4.. Tie-B cells impair anti-tumor immunity and ICB efficacy

a, Representative flow cytometry images of B cells in the BM and PB. b, Flow cytometry analyzed B cells in 2208L tumor-bearing mice after B cell depletion (day 27). n=5 mice/group. Unpaired two-tailed Student’s t-test. c, Flow cytometry analyzed total B cells or Tie-B cells in PyMT-N tumor-bearing mice after B cell depletion (day 27). n=5 mice/group. Unpaired two-tailed Student’s t-test. d,e, Adoptive transfer of B220⁺IgM⁻CD93⁻ or B220⁺IgM⁺ non-Tie-B cells into μMT mice bearing PyMT-N tumors and quantification of tumor growth curves. n=5 mice/group. f, E0771 tumor growth curves during IgG or anti-PD-1&CTLA-4 treatment. n=5 mice/group, unpaired two-tailed Student’s t-test on day 22. g, ICB responsive E0771 cells were orthotopically transplanted into μMT mice, 5 days later, adoptively transfer of PBS, B220⁺IgM⁻CD93⁻ or B220⁺IgM⁺ cells every 5 days. Meanwhile, anti-PD1&CTLA4 were treated every 3 days. h, E0771 tumor growth curves in mice from the experiment (g). n=5 mice/group. i, Tie-B cells were stained with CFSE and injected into WT mice. j, Flow cytometry analysis of CFSE⁺ Tie-B cell numbers across different organs 18h after injection. n=3 mice per group. One-way ANOVA with Dunnett’s multiple comparisons test compared to the spleen group, mean ± s.d.. k, Tie-B cells were isolated from TiBA-2 tumor-bearing CD45.1 mice, then intravenously injected into different TiBA tumor mice. l-n, Flow cytometry analysis of CD45.1⁺ Tie-B cell numbers in the spleen (l), BM (m) and tumor (n) of different TiBA models 5 days after injection. n=4 mice/group. One-way ANOVA with Dunnett’s multiple comparisons test compared to TiBA-2. o-t, Flow cytometry analyzed the percentages of indicated B cell subsets in total CD45.1⁺B220⁺ cells in the spleen or BM of different recipients. n=4 mice/group. One-way ANOVA with Dunnett’s multiple comparisons test compared to TiBA-2. For all boxplots (b,c,l-t), the line inside the box is the median value, and the bottom/top bars of the box indicate min to max values.

Extended Data Fig. 5.. Tie-B promotes the systemic accumulation of exhaustion-like T cells

a,b, Tumor cell numbers four days after cocultured with Tie-B cells. n=3 independent repeats. Unpaired two-tailed Student’s t-test, mean ± s.d.. c, PyMT-M tumor growth curves during T-cell transfer and ICB treatment. Unpaired two-tailed Student’s t-test on day 20. d, PD-1⁺CTLA-4⁺ T cells in 2208L-bearing mice after B cell depletion. n=5 mice per group. One-way ANOVA with Tukey’s multiple comparisons test (left panel). The line inside the box is the median value, and the bottom/top bars of the box indicate min to max values. e, B220⁺ cells and PD-1⁺CTLA-4⁺ T cells in naïve mice after B cell depletion. n=5 mice. Unpaired two-tailed Student’s t-test, mean ± s.d.. f, Mean fluorescent intensity (MFI) of ID2 protein in T cells from naïve or TiBA-2 tumor-bearing mice. n=4 mice. Unpaired two-tailed Student’s t-test, mean ± s.d.. g, PD1⁺CTLA4⁺ proportions after CD8T cells were co-cultured with Tie-B cells. n=3 biological replicates. Unpaired two-tailed Student’s t-test, mean ± s.d.. h, The expression level of MHC-II genes in B cell clusters based on the scRNA-seq data. i,j, MFI of MHC-II protein in B cell subsets in TiBA-2 tumor-bearing mice. n=10 mice. Two-way ANOVA with Dunnet’s multiple comparisons test (i) and one-way ANOVA with Tukey’s multiple comparisons test (j), mean ± s.d.. k,l, Tie-B cells were stimulated with ovalbumin^323-339 peptides, co-cultured with OT-II CD4⁺ T cells, then detected with MHC-II-OVA-tetramer antibodies. n=3 biological replicates, unpaired two-tailed Student’s t-test, mean ± s.d.. DC, dendritic cells (positive control) m, Number of migrated T cells. n=3 biological replicates, unpaired two-tailed Student’s t-test, mean ± s.d.. n, Representative IF staining images of Tie-B cells (B220⁺CD93⁺) and CD8 T cells in spleen. Yellow arrows indicate aggregations. Representative of three independent experiments. o, Establishment of tumor-bearing Cd79a-Cre^ER;Hmgb1^fl/fl mice for tamoxifen treatment. p, PD1⁺CTLA4⁺ proportions after co-culture of CD3T and Tie-B cells. n=3 biological replicates. One-way ANOVA with Dunnet’s multiple comparisons test, mean ± s.d.. q, MFI of ID2 protein in T cells after co-culturing with Tie-B. n=3 biological replicates. One-way ANOVA with Dunnet’s multiple comparisons test, mean ± s.d..

Extended Data Fig. 6.. TiBA1/2 cannot be induced solely by direct interactions between cancer cells and early B cells

a, Flow cytometry analysis of myeloid cell numbers in E0771 tumors from the experiment in Fig. 4t-u. n=5 mice/group. b-e, Flow cytometry analysis of myeloid cell subsets in the PB, spleen, BM and tumors of PyMT-N tumor-bearing Cd79a-Cre^ER or Cd79a-Cre^ER;Hmgb1^fl/fl mice after tamoxifen treatment. n=4 mice/group. Unpaired two-tailed Student’s t-test, mean ± s.d.. f-h, Flow cytometry analysis of B cell subsets in the PB, spleen and BM of PyMT-N tumor-bearing Cd79a-Cre^ER or Cd79a-Cre^ER;Hmgb1^fl/fl mice after tamoxifen treatment. n=4 mice/group. Unpaired two-tailed Student’s t-test, mean ± s.d.. i, Diagram showing co-culture of TiBA-1 or TiBA-2 tumor cells with early B cells in a non-direct contact manner to mimic the remote effect of cancer cells. j,k, Flow cytometry analysis of the absolute cell numbers of early B cell subsets 2 days after co-culture with TiBA-1 (LLC) or TiBA-2 (PyMT-N) tumor cells. n=3 (B cells were isolated from 3 biological replicate mice). Two-way ANOVA with Dunnett’s multiple comparisons test compared to “B+NC” group, mean ± s.d.. l, Diagram illustrates the 2208L (TiBA-1) tumor-bearing CD11b-DTR or WT mice that received DT treatment. m, Flow cytometry analysis of absolute cell numbers of mature B cells (Fr. F) in the BM of 2208L tumor-bearing WT or CD11b-DTR mice after DT treatment (day 21). n=4 mice/group. The line inside the box is the median value, and the bottom/top bars of the box indicate min to max values. n, Feature plot showing the CD11b expression in BM cell clusters. o, Representative flow cytometry images of CD11b and B220 expression in the BM of 2208L tumor-bearing mice.

Extended Data Fig. 7.. Development of TiBA-2 involves abnormal neutrophil-early B cell crosstalk

a, Flow cytometry quantification of monocytes (CD45⁺CD11b⁺Ly6C^hiLy6G⁻) numbers in PyMT-N tumor-bearing mice after anti-Ly6C depletion. n=5 mice/group. Unpaired two-tailed Student’s t-test, mean ± s.d.. b-d, Flow cytometry analysis of indicated B cell subsets in the PB, spleen and BM of PyMT-N tumor-bearing mice after IgG or anti-Ly6C treatment. n=5 mice/group, mean ± s.d.. e, Diagram showing early-B cells were co-cultured with neutrophils (isolated from E0771 models) in the presence of E0771 tumor cells. The numbers of pre-B cell subsets were analyzed by flow cytometry 3 days after co-culture. Mean ± s.d. f, Dot plot showing the expression level of predicted target genes in pro-B (Fr. B-C) and pre-B (Fr. C’-D) clusters. g,h, Volcano plots show the differential gene expression of Fr. B/C and Fr. D cell clusters between PyMT-N tumor-bearing and Sham mice. Wilcoxon rank-sum test. i, Feature plot showing the Tnfsf13b and Il15 expression distribution in BM cell clusters. j, GSEA shows down- and up-regulated gene sets in the BM neutrophil cluster of TiBA-1 tumor-bearing mice compared to Sham. Hypergeometric p-values were adjusted for multiple testing using the Benjamini-Hochberg method. k,l, Feature plots and Violin plots show the Itgb2 expression in the BM neutrophil clusters between PyMT-N tumor-bearing and Sham mice. m, Diagram showing early-B cells and neutrophils cocultured and treated with anti-ITGB2 blocking antibodies. n, Dot plot showing the expression levels of ITGB2 receptors in all BM B cell clusters. o, Diagram showing early-B cells and neutrophils cocultured and treated with anti-ICAM2 blocking antibodies. p, Flow cytometry analysis of the numbers of pre-B cells 4 days after co-culture in the experiment (o). n=3 biological replicates. One-way ANOVA with Tukey’s multiple comparisons test, mean ± s.d..

Extended Data Fig. 8.. Chemotherapy plus ICB treatment on TiBA-2 models and human PB cell flow cytometry analysis

a, Diagram showing PyMT-N tumor-bearing mice (or Sham mice) treated with vehicle or paclitaxel in combination with ICB (anti-PD-1&CTLA-4). b-d, Flow cytometry analysis of transitional B cells in the PB of PyMT-N tumor-bearing mice after six doses of treatment as described in experiment (a). TiBA-2 groups (n=5 mice), Sham (n=4 mice). One-way ANOVA with Dunnett’s multiple comparisons test compared to the “TiBA-2+Vec” group, mean ± s.d.. e-g, Flow cytometry analysis of BM Fr. D subsets and PB Tie-B cells in PyMT-N tumor-bearing mice after six doses of treatment as described in experiment (a). TiBA-2 groups (n=5 mice), Sham (n=4 mice). One-way ANOVA with Dunnett’s multiple comparisons test compared to the “TiBA-2+Vec” group, mean ± s.d.. h, PyMT-N tumor growth curves during paclitaxel in combination with anti-PD-1&CTLA4 therapy. n=5 mice per group. i, Representative flow cytometry gating of human B cell subsets and cell counting beads. j, The age of healthy females and different TiBA patients. Healthy (N=34), TiBA-1 TNBC patients (N=18), TiBA-2 TNBC patients (N=9), TiBA-0 TNBC patients (N=15). Mean ± s.d.. k, Human T cells were co-cultured with TiBA-2 patient CD19⁺IgM⁺IgD⁺ cells. Flow cytometry analysis of PD1⁺CTLA4⁺ T cell numbers 3 days after co-culture. n=3 biologically replicates, mean ± s.d.. l, Working model of distinct TiBA phenotypes.

Fig. 1.. Remote tumours disrupt early B cell development in two different patterns

a,b, Heatmaps of log10 fold change in the percentage (a) and absolute cell numbers (b) of B cell subsets in BM of tumour-bearing mice compared with Sham (tumour-free) mice. BM was analyzed by flow cytometry when tumour models had similar tumour sizes (around 1 cm³). n=10 mice (for LLC), n=7 mice (for 2208L), n=13 mice (for PyMT-N), and n=5 mice (for T12, E0771 or B16-F10). c, UMAP clustering of BM cells from BALB/c models with color annotations of cell identity. d, UMAP plots indicate the origin of BM cells from different tumour-bearing or sham BALB/c mice. BM cells from n=5 mice (per group) were pooled into one sample for scRNA-seq. e, UMAP clustering of BM cells from B6 models with color annotations of cell identity. f, UMAP plot indicating the origin of BM cells from PyMT-N-bearing (Cyan) and sham (Red) mice. BM cells from n=5 mice (per group) were pooled into one sample for scRNA-seq. g,h, The proportion of BM B cell subsets among total B cells in BALB/c models (g) or B6 models (h) based on scRNA-seq analysis. The numbers indicate absolute cell counts.

Fig. 2.. TiBA-1 and TiBA-2 drive distinct changes to systemic B cell profiles

a, Representative flow cytometry gating of mouse transitional B cells and Tie-B cells in the PB of tumour-bearing and Sham mice. b,c, Heatmaps of log10 fold change in cell numbers of various B cell subsets in the PB (b) and spleen (c) of tumour-bearing mice compared with Sham (tumour-free) mice. PB or spleen was analyzed by flow cytometry when tumour models had similar tumour sizes (around 1 cm³). n=6 mice (for PyMT-N) and n=5 mice (for other groups). d, UMAP clustering of PB cells with color annotations of cell identity. e, UMAP plot indicating the origin of PB cells from PyMT-N-bearing (Cyan) and sham (Red) mice. PB cells from n=5 biologically independent mice (per group) were pooled into one sample for scRNA-seq. f, Percentage of B cell subsets among total PB B cells in PyMT-N-bearing and Sham mice based on scRNA-seq analysis. g, Expression distribution of indicated early B cell marker genes in PB cells. h, UMAP plot of scRNA-seq data of PyMT-N tumour-infiltrating B cells. i, Expression distribution of indicated early B cell marker genes in PyMT-N tumour-infiltrating B cells. j, Representative in situ immunofluorescence (IF) staining of Tie-B cells (yellow arrows, B220⁺CD93⁺) in TiBA-2 tumours. This result is representative of three independent experiments. k, The frequency of B cell subsets within tumour-infiltrating B cells was determined by flow cytometry and then transformed into Z-scores. The analyzed tumours had similar tumour sizes (around 1cm³). TiBA-1 (n=11 mice), TiBA-2 or TiBA-0 (n=14 mice). l, Quantification of tumour-infiltrating total B cells (CD45⁺B220⁺) by flow cytometry. Tumour volumes were around 1cm³. n=22 mice (for TiBA-1 or TiBA-2 tumours) and n=12 mice (for TiBA-0 tumours). One-way ANOVA with Dunnett’s multiple comparisons test compared to TiBA-1. The line inside the box is the median value, and the bottom/top bars of the box indicate min to max values.

Fig. 3.. TiBA-2 impairs anti-tumour immunity and ICB efficacy

a, Diagram shows transplantation of TiBA-1 or TiBA-2 tumour cells into WT or μMT B6 mice. b,c, Tumour growth curves of TiBA-1 or TiBA-2 tumours in WT and μMT mice. n=5 mice/group. Multiple unpaired t tests (two-tailed). d, The diagram shows tumour-bearing mice treated with B cell-depleting antibodies. e,f, Tumour growth curves of TiBA-1 or TiBA-2 tumours in WT mice treated with IgG or B cell-depleting antibodies. n=5 mice/group. Multiple unpaired t-tests (two-tailed). g, The experimental design shows the adoptive transfer of total B cells (isolated from WT mice) following TiBA-1 tumour implantation into μMT mice. h, TiBA-1 Tumour growth curves in μMT mice during B cell adoptive transfer. n=5 mice/group. Multiple unpaired t tests (two-tailed). i, The experimental design shows the adoptive transfer of Tie-B cells (purified from PyMT-N-bearing mice) following TiBA-2 tumour implantation into μMT mice. j, TiBA-2 Tumour growth curves in μMT mice during Tie-B cell adoptive transfer. n=5 mice/group. Multiple unpaired t tests (two-tailed). k, Diagram illustrates the experimental design. ICB responsive E0771 cells were orthotopically transplanted into μMT mice, 5 days later, adoptively transfer (AT) of WT B cells (Group-0), PBS (Group-1) or Tie-B cells (Group-2) every 5 days. In the meantime, ICB (anti-PD-1 plus anti-CTLA-4) was treated every 3 days. l, Tumour growth curves show responses of E0771 tumours in mice from the experiment (k) to ICB therapy. n=6 mice/group. One-way ANOVA with Dunnett’s multiple comparisons test (compared to Group-2) on day 25.

Fig. 4.. Tie-B cells promote the systemic accumulation of exhaustion-like T cells

a,b, Adoptive cell transfer and ICB treatment in E0771 tumour-bearing Rag1-KO mice and tumour growth curves. n=10 (T+ICB group), n=5 (other groups). One-way ANOVA with Tukey’s multiple comparisons test on day 19, mean ± s.d.. c,d, PD1⁺CTLA4⁺ T cell levels in Sham and PyMT-N models (treated with B cell depleting antibodies). n=5. One-way ANOVA with Tukey’s multiple comparisons test. The line inside the box is the median value, and the bottom/top bars of the box indicate min to max values. e, PD1⁺CTLA4⁺ T cells in tumours. n=8. Unpaired two-tailed Student’s t-test, mean ± s.d.. f,g, The expression of PD-1 and CTLA-4 in T cells after co-cultured with Tie-B cells. n=3 biological replicates. One-way ANOVA with Dunnett’s multiple comparisons test compared to “T+NC” (f), mean ±  s.d.. h, The expression of TIM3 and LAG3 in T cells from TiBA-2 tumour models. n=5. i, Cell numbers four days post-proliferation. n=3 biological replicates. Unpaired two-tailed Student’s t-test, mean ± s.d.. j,k, OT-I CD8⁺ T cells were cocultured with Tie-B or non-Tie-B (B220⁺IgM⁺) cells, followed by incubating with tumour cells for one day and quantification of tumour cell numbers. n=3 biological replicates. One-way ANOVA with Tukey’s multiple comparisons test. l,m, NicheNet analysis of early B cells (sender) and T cells (receiver) based on the scRNA-seq of PyMT-N model. Ligands expression in early B cells (l); Ligand–target matrix denotes the regulatory potential between sender-derived ligands and targets from the receiver (m). n,o, Ligand expression in Tie-B (n) and Hmgb1 expression in all B cells (o). p,q, HMGB1 expression in Tie-B cells of PyMT-N models. n=3 (Cd79a-cre^ER) and n=4 (Cd79a-cre^ER;Hmgb1^fl/fl). Unpaired two-tailed Student’s t-test, mean ± s.d.. r, PD1⁺CTLA4⁺ T cell levels in PyMT-N models. n=5. Unpaired two-tailed Student’s t-test, mean ± s.d.. s, OT-I CD8⁺ T cells were cocultured with Tie-B for four days, followed by incubation with ovalbumin^257-264-tumour cells for one day and quantification of tumour cell numbers. n=3 biological replicates. Unpaired two-tailed Student’s t-test, mean ± s.d.. t,u, Adoptive cell transfer and ICB treatment in E0771-bearing Rag1-KO mice and tumour growth curves. n=5. Unpaired two-tailed Student’s t-test on day 19.

Fig. 5. Both TiBA-1 and TiBA-2 are associated with abnormal myelopoiesis in the BM.

a, Flow cytometry analyzed absolute cell numbers of CD11b⁺ myeloid cells in the BM of tumour-bearing or Sham (naïve) mice. n=10 mice (for Sham-B6, PyMT-N, LLC) and n=5 mice (for other groups) per group. One-way ANOVA with Dunnett’s multiple comparisons test compared to Sham. b-d, Absolute cell numbers of myeloid cells and B220⁺ B cells in the BM or PB of 2208L tumour-bearing WT or CD11b-DTR mice after DT treatment (day 21), analyzed by flow cytometry. n=4 mice/group. Two-way ANOVA with Sidak’s multiple comparisons test (b), Unpaired two-tailed Student’s t-test (c,d). e, Absolute cell numbers of GMPs (Lin⁻Sca-1⁻c-Kit⁺CD34⁺CD16/32⁺) in the BM of 2208L tumour-bearing WT or CD11b-DTR mice after DT treatment (day 21), analyzed by flow cytometry. n=4 mice/group. Unpaired two-tailed Student’s t-test. f, Correlation between B cell numbers and GMP numbers in the BM of TiBA-1 tumour-bearing mice. n=39 mice. Pearson correlation coefficients and two-sided t-tests, error bands indicate 95% confidence intervals. g, IF staining of pre-pro-B cells (yellow arrows, B220⁺CD19⁻) and GMPs (White, Lin⁻Sca-1⁻CD41⁻CD115⁻c-Kit⁺) in the BM of LLC tumour-bearing mice. The dashed line (endosteum), vasculature (green). Representative images from 3 biologically independent animals. h, Diagram illustrates the TiBA-2 tumour-bearing CD11b-DTR or WT mice that received DT or PBS treatment. i, Heatmaps of log10 fold change in absolute cell numbers of various cell subsets in PB, BM and spleen of PyMT-N or 4T1 tumour-bearing mice after myeloid depletion (day 21), analyzed by flow cytometry. n=5 mice/group. Multiple t tests (two-tailed). j,k, Representative flow cytometry images of mouse B cell subsets in the BM (j) or Tie-B cells in the spleen (k) of TiBA-2 tumour-bearing mice after myeloid depletion (day 21). l, Diagram illustrates that CD11b⁺ cells (isolated from TiBA-2 tumour-bearing mice) were added to the co-culture of cancer cells and with WT early B cells. m, Heatmaps of log10 fold change in absolute cell numbers of early B cell subsets 2 days after co-culture. n=4 biological replicates. For all boxplots (a-e), the line inside the box is the median value, and the bottom/top bars of the box indicate min to max values.

Fig. 6.. Development of TiBA-2 involves abnormal neutrophil-early B crosstalk

a, In vivo anti-Ly6G or anti-Ly6C depletion strategy. b-d, Neutrophil (b) and pre-B subsets (d) in the BM of PyMT-N tumour-bearing mice after neutrophil depletion (day 21). IgG group (n=5 mice), anti-Ly6G group (n=4 mice). Unpaired two-tailed Student’s t-test. e, Tie-B cell numbers in PyMT-N tumour-bearing mice after neutrophil depletion (day 21). IgG group (n=5 mice), anti-Ly6G group (n=4 mice). Unpaired two-tailed Student’s t-test. f,g, Early-B cells were co-cultured with neutrophils in direct or non-direct contact (DC or NDC) in the presence of tumour cells (f), then analyzed by flow cytometry 3 days after co-culture (g). n=4 biological replicates. Two-way ANOVA with Dunnett’s multiple comparisons test compared to “B (NC)” group, mean ± s.d.. h, Representative IF staining of pre-B cells (B220⁺IgM⁻CD43⁻) and neutrophils (Ly6G⁺) in the femur of PyMT-N tumour-bearing mice. Aggregated pre-B and neutrophils (Blue dashed circles). The results are representative of three independent experiments. i,j, Intercellular communication analysis between neutrophils (sender) and early-B cells (receiver) based on the scRNA-seq of PyMT-N model. ligand expression in neutrophil clusters (i); Ligand–target matrix denotes the regulatory potential between sender-derived ligands and targets from the receiver (j). k, GSEA showing upregulated gene sets in BM neutrophil cluster of PyMT-N tumour-bearing mice. Hypergeometric p-values were adjusted for multiple testing using the Benjamini-Hochberg method. l, Flow cytometry analyzed absolute cell numbers of pre-B subsets 3 days after co-culture. n=3 biological replicates. Two-way ANOVA with Tukey’s multiple comparisons test, mean ± s.d.. m, Mice treated with anti-ITGB2 blocking antibodies. n, The absolute cell numbers of BM pre-B subsets after treatment (day 21). n=5 mice/group. Two-way ANOVA with Sidaks’s multiple comparisons test. o, Flow cytometry analysis of Tie-B cells in tumour-bearing mice during treatment. n=5 mice/group. Two-way ANOVA with Sidaks’s multiple comparisons test. p, Tumour growth curves in TiBA-2 models. n=5 mice/group. One-way ANOVA with Dunnett’s multiple comparisons test compared to IgG (on day 25). For all boxplots (b, d, e, n, o), the line inside the box is the median value, and the bottom/top bars of the box indicate min to max values.

Fig. 7.. Distinct TiBA patterns in human TNBC patients correlate with myeloid cells and clinical outcomes

a, Diagram illustrates the collection of PB sample and therapeutic outcome from human TNBC patients. b, Unsupervised hierarchical clustering dendrograms of log2 fold change (normalized to the mean of healthy females) in cell numbers for indicated cell subsets in PB of human TNBC patients compared with healthy females. Healthy (N=34), TNBC patients (N=42). c, Proportion of residual cancer burden (RCB) scores in different TiBA patients following chemotherapy combined with pembrolizumab. Fisher’s Exact Test (two-sided). d, Flow cytometry of PD1⁺CTLA4⁺ T cells in the PB of healthy females and human TNBC patients according to TiBA classification in (b). Healthy (N=31), TiBA-1 (N=16), TiBA-2 (N=9), TiBA-0 (N=15). One-way ANOVA with Dunnett’s multiple comparisons test compared to Healthy, mean ± s.d.. e, Expression signatures of indicated genes in TCGA TNBC patient datasets. f, Diagram showing co-culture of human T cells with human Tie-B cells (CD19⁺IgM⁻IgD⁻CD10⁺CD38⁺ isolated from TiBA-2 patients) and anti-HMGB1 neutralizing antibody treatment. g, Flow cytometry analysis of PD1⁺CTLA4⁺ population in T cells 3 days after co-culture. N=4 biologically replicates. One-way ANOVA with Tukey’s multiple comparisons test, mean ± s.d.. h,i, Flow cytometry analysis of HMGB1 expression in different B cell subsets in the PB of TiBA-2 TNBC patients. N=9 patients. One-way ANOVA with Dunnett’s multiple comparisons test compared to Early-B, mean ± s.d.. j, Diagram illustrates the sampling process for co-culturing human tumour cells with human BM B cells and neutrophils. k, Flow cytometry analyzed absolute cell numbers of human pre-B cells 3 days after co-culture. n=3 independent repeats. One-way ANOVA with Fisher’s LSD test compared to NC, mean ± s.d.. l,m, Diagram showing co-cultured human pre-B cells and neutrophils were treated with anti-ITGB2 blocking antibodies (l), followed by flow cytometry analysis 3 days after co-culture (m). Unpaired two-tailed Student’s t-test, mean ± s.d..