Immunosuppressive plasma cells impede T-cell-dependent immunogenic chemotherapy

Abstract

Cancer-associated genetic alterations induce expression of tumour antigens that can activate CD8(+) cytotoxic T cells (CTLs), but the microenvironment of established tumours promotes immune tolerance through poorly understood mechanisms. Recently developed therapeutics that overcome tolerogenic mechanisms activate tumour-directed CTLs and are effective in some human cancers. Immune mechanisms also affect treatment outcome, and certain chemotherapeutic drugs stimulate cancer-specific immune responses by inducing immunogenic cell death and other effector mechanisms. Our previous studies revealed that B cells recruited by the chemokine CXCL13 into prostate cancer tumours promote the progression of castrate-resistant prostate cancer by producing lymphotoxin, which activates an IκB kinase α (IKKα)-BMI1 module in prostate cancer stem cells. Because castrate-resistant prostate cancer is refractory to most therapies, we examined B cell involvement in the acquisition of chemotherapy resistance. Here we focus on oxaliplatin, an immunogenic chemotherapeutic agent that is effective in aggressive prostate cancer. We show that mouse B cells modulate the response to low-dose oxaliplatin, which promotes tumour-directed CTL activation by inducing immunogenic cell death. Three different mouse prostate cancer models were refractory to oxaliplatin unless genetically or pharmacologically depleted of B cells. The crucial immunosuppressive B cells are plasmocytes that express IgA, interleukin (IL)-10 and programmed death ligand 1 (PD-L1), the appearance of which depends on TGFβ receptor signalling. Elimination of these cells, which also infiltrate human-therapy-resistant prostate cancer, allows CTL-dependent eradication of oxaliplatin-treated tumours.

Extended Data Figure 1. Treatment schemes and characterization of tumors and mouse survival before and after treatment

a, Early and late treatment schemes for TRAMP mice. b, TRAMP mice (n= 3-6/group) were subjected to early oxaliplatin treatment as described in (a) and prostate weights were determined at 14 weeks, one week after completion of 4 treatment cycles. Dashed red line indicates prostate weight of tumor-free controls (n=33 in total). c-d, Histopathology of TRAMP tumors. c, Representative images of H&E stained prostate sections from TRAMP mice are shown. Magnification bars: 100 μm. PIN, prostatic intraepithelial neoplasia; WD, well differentiated adenocarcinoma; PD, poorly differentiated adenocarcinoma. d, Histopathological assessment of early and late treated TRAMP tumors in WT and Jh^-/- mice without or with oxaliplatin treatment. The percentages of the different histotypes shown in (c) are depicted (n=3-7/group). Fisher chi-square analysis was used to calculate statistical significance. e, Early and late treatment schemes for mice bearing s.c. MC tumors. f, MC cells were s.c. transplanted into WT and Jh^-/- mice (n= 3-7/group) that were subjected to early oxaliplatin treatment when tumor volume was 100 mm³. 48 hrs after completion of 3 treatment cycles, mice were sacrificed and tumor volumes (mm³) were measured (n=19 in total). Prostate weight in (f) is shown in a Log 2 scale. g, MC tumors from indicated mice were stained for CD45 (green) and cleaved caspase 3 (CC3; red) (n=4-6/group). h,i, MC tumors (n=3-5/group) grown in WT, Jh^-/- and Cd8a^-/- mice were stained for CD45 (green) and p-γH2AX (red), and the p-γH2AX⁺ foci in CD45^- cells were enumerated (i). Magnification bars: 100 μm. All results are means ± s.e.m. j, Representative images of s.c. MC tumors (n=5-6/group) from WT and Jh^-/- mice, with or without oxaliplatin treatment stained for αSMA (green) and CD31 (red). k, l, Frequency of αSMA (k) and CD31 (l) positive cells within tumors from (j). Shown are median values ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance indicated by *P, 0.05; **P, 0.01; ***P, 0.001. m, TRAMP mice (WT, Cd8a^-/- or Jh^-/-; n=6-14/group) were treated weekly with low-dose oxaliplatin. Moribund mice were sacrificed, and survival was compared by Kaplan-Meyer analysis and significance was determined (WT: n.s.; Cd8a^-/-: n.s.; Jh^-/-; p<0.002;**). n, Survival curves for the different TRAMP groups before and after oxaliplatin treatment. Significant differences in survival times are indicated on the right. No statistically significant differences in survival were found between WT and Jh^-/- or Jh^-/- and Cd8a^-/- mice without treatment. Significant differences in survival times were observed between all three oxaliplatin-treated groups (WT, Cd8a^-/- or Jh^-/-; indicated on the right).

Extended Data Figure 2. B cells attenuate oxaliplatin-triggered CTL activation

a, Flow cytometry of CD8⁺ T lymphocytes in prostates of 20 weeks old TRAMP mice after 4 cycles of oxaliplatin treatment (n=5-7/group) normalized to prostate weights. b, Late s.c. MC tumors from WT and Jh^-/- mice were stained for CD8 and analyzed by immunofluorescent microscopy. In the upper left areas (white square), single CD8 staining (green) without DAPI counterstain is shown. Tumoral CD8⁺ cells were counted in 3-4 HMF (200×)/tumor (n=4-5 tumors/group). Magnification bars: 100 μm. c,d, Late s.c. MC tumors were analyzed by flow cytometry for CD4⁺ lymphocytes in spleens (c) and tumors (d) after 3 oxaliplatin treatment cycles (n=4-7/group). The results show percentages of CD4⁺ cells in the CD45⁺ population. e, Flow cytometric analysis of TNF and IFNγ expression by CD8⁺ cells in MC tumors from WT and Jh^-/- mice treated as above (n=6-8) and re-stimulated in vitro with tumor cell lysate. f, Flow cytometry of STAT1 phosphorylation in CD8⁺ cells from MC tumors of treated and untreated WT, and Jh^-/- mice (for isotype controls, see E.D. Fig. 10u). The results are summarized in the right panel (n=3 mice/group). g, Expression of GrzBand Ki-67in CD8⁺ T effector cells (CD8⁺CD44⁺) from spleens of MC inoculated mice after oxaliplatin treatment. h,i, Flow cytometry of PD-1 and Tim-3 expression by CD8⁺ effector cells (CD8⁺CD44⁺ cells) in spleen (h) and MC tumors (i) as indicated with or without oxaliplatin treatment. Shown are percentages of the corresponding CD8⁺ T cells in the CD8⁺CD44⁺ population (n=3-5/group). j, The experimental scheme for B cell immunodepletion in tumor-bearing mice. MC tumors were raised in WT or Cd8a^-/- mice, 16 days after s.c. tumor cell inoculation. B cells were depleted by twice weekly administration of antibodies directed against CD19, CD20, CD22 and B220. Four days after first antibody treatment, mice were treated with oxaliplatin (n=4-7 mice/group, total: 44). After 3 weekly chemotherapy cycles, mice were sacrificed. k, Flow cytometry analysis of tumor-infiltrating CD45⁺CD8⁺ T cells stained for IFNγ (left) or IFNγ and TNF (right) after in vitro restimulation with PMA/ionomycin (n=4-6 mice/group). l-n, Flow cytometry analysis of CD19⁺ (l,m) and IgA⁺ (n) cells in spleens and tumors isolated from the WT mice described above, confirming depletion of CD19⁺ B cells and oxaliplatin-induced IgA⁺ cells in spleen and tumors. o, Serum IgA concentrations in the mice described in i (n=3-5/group). p, Flow cytometry analyses of CD19⁺ B cells in tumors isolated from Cd8a^-/- mice subjected to B cell depletion or not, confirmed the efficient depletion of tumoral CD19⁺ B cells. All results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance indicated by *P, 0.05; **P, 0.01; ***P, 0.001.

Extended Data Figure 3. Immunogenic chemotherapy induces tumor infiltration by immunosuppressive CD19⁺CD20^lowB220^lowIgA⁺ B cells

a,b, MC tumors (n=4-9/group) raised in WT mice without or with oxaliplatin treatment were stained for B220 (a), and tumor-infiltrating B220⁺ cells per HMF were enumerated (b). In panel a, single B220 staining (above) and combined staining B220/DAPI (below) are shown. Magnification bars: 100 μm. c,d, The flow cytometry plots and gating strategy for analysis of splenic B cell populations using CD19, IgA, B220, CD138 and CD20 antibodies. Results from WT mice bearing MC tumors are shown in panel (c) and from oxaliplatin-treated mice in panel (d) (n=8 mice/group). Oxaliplatin treatment modestly increased the amount of splenic IgA⁺ cells. Splenic IgA⁺ cells expressed CD138 as expected and showed lower levels of B220 and CD20, in either control or oxaliplatin-treated mice. e, The gating strategies for analysis of tumoral B cells using CD19, IgA, B220 and CD138 antibodies. Results from MC tumors in two representative oxaliplatin-treated WT mice are shown (n=8 mice/group), demonstrating the presence of IgA⁺ cells in oxaliplatin-treated tumors with a typical CD138⁺B220^low phenotype. f-i, Flow cytometry plots and gating strategies for analysis of tumoral B cell populations using CD19, B220, CD138, IgA and PD-L1 antibodies. Results from WT mice bearing MC tumors without (f) or with oxaliplatin treatment (g) (n=6 mice/group) and Iga^-/- mice bearing MC tumors without (h) or with oxaliplatin treatment (i) (n=6 mice/group) are shown. Oxaliplatin treatment increased the amount of tumoral IgA⁺CD138⁺B220^lowPD-L1⁺cells in WT mice. j, Flow cytometric analysis of PD-L1 and IL-10 expression in IgA⁺B220^- and B220⁺IgA^- B cells from oxaliplatin-treated TRAMP tumors (n=4). k, Flow cytometric analysis of IgA and CD138 expression by TRAMP tumor-infiltrating B cells. Shown are percentages of IgA⁺ cells amongst all tumor-infiltrating CD19⁺CD138⁺ cells. l, WT mice bearing MC tumors were treated with oxaliplatin as above. Two days after the first or last oxaliplatin cycle, mice were sacrificed, tumors were isolated and analyzed by flow cytometry as indicated (n=6/group). After dead cell exclusion, tumor-infiltrating B cells were stained with CD19, CD20, B220, IgA and IgM antibodies. Shown are the results for control (left panels), one cycle (middle panels), and 3 cycles (right panels) of oxaliplatin treatment, demonstrating the presence of tumoral IgA⁺ cells with a CD19⁺CD20^lowB220^low IgA⁺ cell phenotype within 48 hrs after oxaliplatin treatment.

Extended Data Figure 4. Immunogenic chemotherapy induces tumoral and systemic IgA production through class switch recombination

a, Ex vivo analysis of IgA released by tumor single cell suspension isolated from oxaliplatin-treated TRAMP tumors. Single cell suspension from non-treated tumors and culture medium without cells were used as controls. b,c, Serum IgA (b) and IgG (c) in treated and untreated TRAMP mice and age-matched naïve FVB controls (n=7-14/group). d, Serum IgA amounts in control or oxaliplatin-treated mice bearing MC tumors (n=5-7/group) were determined and compared to age-matched naïve FVB controls (n=7). e. Serum IgG amounts in control or oxaliplatin-treated mice bearing MC tumors (n=5-7 mice/group) were determined and compared to age-matched naïve FVB controls. a-e, Results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance. f,g, Immunofluorescence analysis of activation-induced cytidine deaminase (AID, green) and IgA (red) expression in spleen (f, used as a positive control) and MC tumors from oxaliplatin-treated WT mice (g). Magnification bars: 10 μm except in panel f (right) where it is 100 μm. Arrows point to IgA⁺AID⁺ cells. Shown are representative results of spleens and tumors isolated from 4 mice/group. h, Q-RT-PCR analysis of Tgfb1 mRNA in MC tumors raised in WT or Jh^-/- mice without or with oxaliplatin treatment (n=3-7 mice/group). Results are means ± s.e.m. i, Flow cytometry of SMAD2/3 phosphorylation in MC tumor-infiltrating B cells from WT mice before and after oxaliplatin treatment (n=4/group). Shown are the mean fluorescence intensities (MFI) and percentages (see Fig. 3e). j, Flow cytometry of SMAD2/3 phosphorylation and PD-L1 in MC tumor-infiltrating B cells from WT mice before and after oxaliplatin treatment (n=4/group). Shown are the percentages of PD-L1⁺p-SMAD2/3⁺ cells within CD45⁺CD19⁺ cells. k, Q-RT-PCR analysis of Il21 mRNA in MC tumors raised in WT or Jh^-/- mice without or with oxaliplatin treatment (n=4-7 mice/group). Chemotherapy-induced Il21 mRNA mainly in WT mice. l,m, Flow cytometry of tumor-infiltrating B cells stained for phospho-STAT3 and IL-10 (n=5-6/group) before and after oxaliplatin treatment. n, Flow cytometry analysis of β-actin mRNA, IL-10 protein and Il10 mRNA in MC tumor-infiltrating IgA⁺ cells using PrimeFlow™ RNA technology (pooled data of 4 mice/group, after oxaliplatin treatment). Left panel: β-actin mRNA gated on CD45⁺ cells; middle panel: Il10 mRNA and IL-10 protein expression after 1 hr stimulation with PMA/ion/LPS gated on IgA⁺ cells, right panel: Il10 mRNA and IL-10 protein expression after 5 hrs stimulation with PMA/ion/LPS, gated on IgA⁺ cells. o,p, Flow cytometric analysis of tumor-infiltrating B cells in TRAMP mice (n=4-5/group) stained for CD19, B220, IL-10 and LTαβ (o). The percentage of tumor-infiltrating LTαβ⁺ cells amongst all tumor-infiltrating B cells was determined (p). q, Flow cytometric analyses of CD5 expression by B cells from spleen and MC-tumor of WT mice after oxaliplatin treatment (n=4-5/group). Shown are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance indicated by *P, 0.05; **P, 0.01; ***P, 0.001.

Extended Data Figure 5. Immunogenic chemotherapy or B cell deficiency has marginal effects on Tregs, NK and myeloid cells

a,b, Q-RT-PCR analyses of Nos2 (a) and Arg1 (b) mRNAcontent of MC tumors (n=4-7 mice/group). Chemotherapy induced Nos2 and Arg1 expression in WT and Jh^-/- mice and no significant and consistent differences were found between both groups. c,d, Q-RT-PCR analyses of Il12p40 (c), Il12p35 (d) mRNAin MC tumors grown in WT and Jh^-/- mice (n=4-6 mice/group). e-i, Flow cytometry analyses of tumor-infiltrating or splenic lymphocytes and monocytes: tumoral Nk1.1⁺ cells (e), tumoral CD11b⁺CD11c⁺ MHCII⁺ cells (f), tumoral CD11b⁺GR-1⁺ cells (g), CD4⁺FoxP3⁺ cells (splenic, h; tumoral, i). Cells in panels e-i are from tumor-bearing mice subjected to oxaliplatin treatment and/or B cell depletion as indicated (B cell depletion + oxaliplatin; n=4-6 mice/group). Results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance.

Extended Data Figure 6. Analyses of B and T cells in human prostate cancer specimens

a-h, Tissue microarrays of tumor and non-tumor tissue from 110 PC patients were stained for CD8 and CD20 (5-6 spots/patient = 3-4 tumor tissue + 2 non-tumor tissue). a,b, Representative examples of CD8 (a) and CD20 (b) IHC of PC tissue microarrays (left). Magnification bar: 200 μm. Right, computer assisted image analysis with ad hoc developed image software. Tumor tissue is represented in yellow and CD8⁺ and CD20⁺ cells are represented in red. The percentages of immune reactive area (IRA) occupied by CD8⁺ or CD20⁺ cells are shown. Magnification bars: 200 μ. c,d, comparison of CD8 and CD20 IRAs in matched non-tumor and tumor tissues from each early stage PC (E-PC) patient (n=87). e-h, Patients were divided into three subgroups: E-PC (n=86); therapy-resistant-PC (TR-PC; n=9), and metastatic-PC (M-PC; n=15). e, CD8⁺ cell infiltration into tumor tissues of the different groups. f, CD20⁺ cell infiltration into tumor tissues of the different groups. g, The CD8⁺/CD20⁺ ratio for the different groups. Each dot represents one patient. Line indicates the median value. Mann-Whitney test was used to calculate statistical significance between the two groups. Kruskal-Wallis test was used to calculate statistical significance between the three groups. h, IHC analysis of low risk (n=5) and high risk (n=5) human PC specimens using IgA (red) and αSMA (black). Nuclei were counterstained with hematoxylin. Magnification bar: 100 μm i, IF analysis of human PC showing IL-10 (red) –expressing IgA⁺ (green) CD138⁺ (turquoise) plasma cells (n=6). Representative images are shown. White arrow indicates IL-10-expressing IgA⁺ cells. Magnification bars: 50 μm. j, Human normal prostate (n=3-5), and human PC (n=5), were stained for IgA and CD8. Typical images are shown. Red and green arrows indicate IgA⁺ and CD8⁺ cells, respectively. Magnification bar: 100 μm. k, Human normal prostate (n=3), and human PC (n=5), were stained for IgA (red arrow) and CD20 (green arrow). Magnification bar: 100 μm. l, Flow cytometric analysis of human prostate tumor-infiltrating CD19⁺ B cells and IgA⁺ cells. The percentages of IL-10-expressing B cells in CD19⁺IgA⁺ (2 different samples) and CD19⁺IgA^- B cells are shown. m, Summary of results obtained from human blood samples taken from healthy donors (n=3) and PC patients (n=5) and prostate tissue specimens (benign, malignant; n=4) analyzed by flow cytometry for IL-10 expression in CD19⁺IgA^- and CD19⁺IgA⁺ B cells. n,o, Tissue microarrays from 110 PC patients (described above) were stained for IgA and CD138. Patients were divided into three subgroups: E-PC (n=86); TR-PC (n=9), and M-PC (n=15). (n) Representative images of IgA (immunoperoxidase) and CD138 (alkaline phosphatase) double staining of tumor tissues from each group. CD138⁺ and IgA⁺ double positive cells in the PC stroma are indicated by the white arrows (hematoxylin counterstain). Magnification bar: 100 μm. o, Frequencies of IgA⁺ and CD138⁺ double positive cells in the tumor stroma of the different PC patient groups. p, PC patient specimens were divided into two groups: IgA^-/low (n=64) and IgA^+/hi (n=46). Shown is the CD8⁺/CD20⁺ ratio for each group. Each dot represents one patient. Line indicates the median value. q, IgA mRNA expression (IGHA1) is significantly elevated in human PC tissue relative to healthy or benign prostate tissue in 5 out of 15 studies evaluated via Oncomine. Results from one significant study are presented. Chi square test and Fischer exact test were used to calculate statistical significance shown by *P, 0.05; **P, 0.01; ***P, 0.001.

Extended Data Figure 7. Effects of TGFβR2, IgA, PD-L1 and IL-10 ablations on tumor-infiltrating lymphocytes

MC tumors were raised in WT, Tgfbr2^ΔB or Iga^-/- mice (n= 5-11/group). Mice were subjected to 3 cycles of late oxaliplatin treatment after which splenic (Spl) and tumoral (Tu) B cells were analyzed. After dead cell exclusion, splenic (a,b) and tumoral (c,d) B cells were stained with CD19, B220, IgA, IgG2a and IgG1 antibodies and analyzed by flow cytometry. e, Serum IgG concentrations in control or oxaliplatin-treated WT, Tgfbr2^ΔB or Iga^-/- mice bearing MC tumors (n=5-9/group). f, Flow cytometry of tumor-infiltrating CD19⁺ B cells from WT, Tgfbr2^ΔB or Iga^-/- MC tumor-bearing mice (n=4-7/group) analyzed for PD-L1 expression, revealing lower PD-L1 surface expression on Tgfbr2^Δ and Iga^-/- B cells after oxaliplatin treatment. g, Flow cytometry of tumor-infiltrating B220^hi B cells (left) and IgA⁺B220^low B cells (right) from WT, Tgfbr2^ΔB or Iga^-/- MC tumor-bearing mice (n=4-7/group) analyzed for IL-10 expression, revealing no difference in IL-10 expression by B220^hiIgA^- B cells in the corresponding groups, and lower IL-10 expression by Tgfbr2^Δ B cells after oxaliplatin treatment compared to WT mice. Results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance. h, The experimental scheme. WT mice bearing MC tumors were divided into four treatment groups (n=7-8/group): 1) isotype control (IgG2a); 2) oxaliplatin (weekly); 3) anti-PD-L1 (twice weekly); 4) oxaliplatin plus anti-PD-L1 (weekly and twice weekly, respectively). After 3 treatment cycles, mice were sacrificed and analyzed. i, Tumor growth curves of tumor-bearing mice and gross appearance of untreated and treated mice. Significance was determined by Mann-Whitney and t tests. j, Flow cytometric analysis for GrzB expression by tumor-infiltrating CD8⁺ T effector cells (CD8⁺CD44⁺) from MC tumor-bearing mice treated as described above. Results are shown either as percentages of GrzB⁺ cells amongst CD8⁺ T cells (black), or percentages of GrzB⁺CD8⁺CD44⁺ T cells amongst tumoral CD45⁺ cells (red). k, Flow cytometry of PD-L1 expression on tumor-infiltrating IgA⁺ CD19⁺ B cells in the different treatment groups. l,m, Serum IgA (l), and IgG (m) concentrations in the different treatment groups described in panel h. n, The experimental scheme for the experiment whose results are shown in Fig. 4g,h. B cells were isolated from WT, Pdl1/2^-/- and Il10^-/- mice and 5 × 10⁶ cells (purity 98%) were i.p. transferred into MC tumor-bearing Jh^-/- mice (16 days after MC cell inoculation). After 2 days (day 18), the mice were given 3 oxaliplatin treatment cycles and analyzed. o, Flow cytometric analysis of splenocytes after staining with CD45 and CD19 antibodies, confirming presence of B cells in the ABCT groups. Shown are percentages and absolute B cell numbers in spleen. p, Tumor infiltrating CD8⁺ cells from MC tumor-bearing Jh^-/- mice transplanted with B cells and treated as above were re-stimulated for 4 hrs with PMA/ionomycin before flow cytometry (n=4-6 mice/group). Results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance.

Extended Data Figure 8. Low dose cisplatin treatment is devoid of immunogenic activity and low dose oxaliplatin does not affect gut barrier function

a, Flow cytometry of MC cells stained with Annexin V and propidium iodide 24 hrs after treatment with either oxaliplatin or cisplatin (both at 20 μM). b, Flow cytometry analysis of MC cells treated as above and stained with antibody to the autophagy marker LC3A. c-e, MC tumors were raised in WT and Jh^-/- mice until 400 mm³ in size, after which the mice were treated with either cisplatin or oxaliplatin at 6 mg/kg (n=4-5/group). After 3 weekly chemotherapy cycles, mice were sacrificed. c, Tumor weights; left panel: WT mice; right panel: Jh^-/- mice. d, e, Flow cytometry of tumor-infiltrating CD8 (d) and CD4 (e) cells. Left panel: WT mice, right panel: Jh^-/- mice. f, Gut permeability was measured in WT mice before and after low (LD) and high (HD) dose oxaliplatin treatment using orally administered fluorescein isothiocyanate (FITC)–dextran. Shown are FITC-dextran concentrations in serum (μg/ml) (n=5 mice/group). g, Serum IgA concentrations in naïve WT (FVB) and Tgfbr2^ΔB mice before and after oxaliplatin treatment. h, IgA staining of colon sections of untreated or LD oxaliplatin-treated WT mice. Magnification bars: 100 μm. i-k, Flow cytometry of CD8⁺ (i), CD4⁺ (j) and Nk1.1⁺ (k) cells in spleens of naïve WT and Tgfbr2^Δ mice without or with oxaliplatin treatment. All results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance shown as *P, 0.05; **P, 0.01; ***P, 0.001.

Extended Data Figure 9. Immunogenic chemotherapy supports adoptive T cell transfer only in the absence of B cells

a, The experimental scheme. Immunogenic TRAMP-C2 cells were s.c. inoculated into WT or Tcrβ^-/- mice. After 30 days, the mice were divided into 4 groups (n=4-5/group): 1) control, 2) oxaliplatin (weekly), 3) ATCT, 4) ATCT plus oxaliplatin (weekly). The first oxaliplatin cycle was given at day 31. Two days after the second cycle, CD8⁺ T cells from CD45.1×CD45.2 WT mice (3 × 10⁶ cells) were transferred into tumor-bearing mice and this was followed by two more oxaliplatin cycles after which mice were sacrificed for analysis on day 59. b, Tumor volumes (mm³) c,d, Flow cytometric analysis of spleen (c) and tumor (d) cells after staining with CD45.1, CD45.2, CD8 and TCRαβ antibodies, confirming expansion of adoptively transferred T cells. e, tumor growth curves. f, The experimental scheme. Immunogenic TRAMP-C2 cells were s.c. inoculated into WT or Rag1^-/- × OT-1 mice (no B cells), that harbor CD8⁺ T cells specific for chicken ovalbumin which is not expressed by TRAMP-C2 cells. After 30 days, tumor-bearing Rag1^-/- × OT-1 mice were divided into 4 groups (n=3-4 mice per group): 1) control, 2) oxaliplatin treatment, 3) ATCT, 4) oxaliplatin treatment plus ATCT. The first oxaliplatin cycle was given at day 31. Two days after the second oxaliplatin cycle, CD8⁺ T cells (3 × 10⁶) from CD45.1×CD45.2 mice were adoptively transferred into tumor-bearing mice, which were sacrificed on day 59 and analyzed. g, Flow cytometric analysisof tumor-infiltrating cells stained with CD45.1, CD45.2, CD8 and TCRαβ antibodies, confirming infiltration of adoptively transferred T cells. h, Flow cytometric analysis of GrzB expression in adoptively transferred, tumor-infiltrating, CD8⁺ effector cells (CD45.1⁺CD8⁺CD44⁺) from tumor-bearing mice treated as above. i, Tumor volumes (mm³). j, tumor growth curves. k, The experimental scheme for Fig. 5a-f. Sixteen weeks old TRAMP;Rag1^-/- mice (no B and T cells) were treated with oxaliplatin (weekly). One day after the 1^st treatment cycle, CFSE-labeled splenocytes from either WT (B and T cells, SP-WT) or Jh^-/- (T but no B cells, SP-Jh^-/-) mice were transferred into the tumor-bearing mice (5 × 10⁶ T cells per mouse; 4-5 mice per group). l, m, After 6 days, one mouse from each group was sacrificed, and the proliferation of CD8⁺ (l) and CD4⁺ (m) T cells in bone marrow (BM), spleen and prostates was analyzed by CFSE staining and flow cytometry. n-r, After 3 more oxaliplatin cycles (4 weeks in total), the mice were sacrificed and analyzed. n, Frequency of adoptively transferred CD19⁺ cells amongst CD45⁺ cells in spleens and prostates 30 days after ACT. o, Flow cytometric analyses of CD19⁺ B lymphocytes for TIM-1 expression in spleens (left) and prostates (right) of abovemice. p-r, Flow cytometric analyses of T lymphocytes. Percentages of CD8⁺ and CD4⁺ T cells in LN (p); spleens (q); prostates (r) of above TRAMP;Rag1^-/- mice. Red:splenocytes from WT mice (T and B cell transfer), blue: splenocytes from Jh^-/- mice (T cell transfer). Results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance.

Extended Data Figure 10. Immunogenic chemotherapy supports adoptive T cell transfer only in the absence of B cells and analysis of lymphocytes and monocytes in tumor-free mice

a, The experimental scheme for Fig. 5g. MC tumor-bearing Rag1^-/- mice (no B and T cells) were treated with oxaliplatin (weekly). One day after 1^st oxaliplatin treatment, 5 × 10⁶ T cells (negative selection) from WT mice immunized with MC cell lysate were adoptively transferred into tumor-bearing mice (4-5 mice/group), alone or in combination with 5 × 10⁶ B cells from WT or Tgfbr2^ΔB mice (purity 98%). After 2 more oxaliplatin cycles (3 weeks total), the mice were sacrificed and analyzed. b, Serum IgG analysis of above mice. c, Flow cytometric analysis of splenocytes after staining with CD45 and CD19 antibodies. All results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance. Statistical significance is given by *P, 0.05; **P, 0.01; ***P, 0.001. d-p, WT, Jh^-/-, Iga^-/- and Tgfbr2^ΔB mice in the FVB background and WT, Pdl1/2^-/-, Il10^-/- and Iga^-/- in the C57BL/6 background were analyzed for the distribution of immune markers. d, Spleen weights of WT, Jh^-/- and Tgfbr2^ΔB mice in the FVB background. e, Flow cytometry of splenocytes for the following markers: CD3 (left), CD8 (middle), CD4 (right), gated on the splenic CD45⁺ population. f, Absolute cell numbers of splenic CD3⁺ (left), CD8⁺ (middle), and CD4⁺ (right) cells are shown (percentage × cell count of whole spleen). g,h Flow cytometry for TNF and IFNγ in CD8⁺ cells from tumor-free WT, Jh^-/-, Tgfbr2^ΔB and Iga^-/- mice (n=6-8) that were re-stimulated in vitro with PMA/ionomycin and the representative flow cytometry panels (e). i,j, Flow cytometry of splenocytes from WT and Tgfbr2^ΔB for: CD19⁺IgM⁺ cells (i) and IgA (j) gated on the splenic CD45⁺ population. k-n, Flow cytometry of splenocytes from WT, Pdl1/2^-/- and Il10^-/- mice for: CD45⁺CD19⁺IgM⁺ cells (k), CD45⁺IgA⁺ cells (l), PD-L1 expression by CD19⁺IgM⁺ cells (m), and IL-10 expression by CD19⁺ cells (n). o,p, Serum IgA and IgG concentrations were analyzed in WT, Pdl1/2^-/- and Il10^-/- mice (n=4-5 mice/group). All results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance shown as *P, 0.05; **P, 0.01; ***P, 0.001. The different gating strategies and staining controls are shown. q, Gating strategies for tumor-infiltrating lymphocytes: lymphocyte gate, dead cell exclusion, doublets exclusion, and gating on the CD45⁺ population. r, Flow cytometric analysis of IL-10 and IgA expression, gated on the CD45⁺ population: 1) isotype control (no staining), 2) non-stimulated splenocytes: showing IgA staining, but not IL-10. 3) stimulated splenocytes from Il10^-/- mice showing IgA staining, but not IL-10. 4) stimulated splenocytes from WT mice showing IgA and IL-10 staining. s, Flow cytometric analysis of IL-10 and CD19 expression, gated on the CD19⁺B220⁺ population. left: stimulated cells from Il10^-/- mice, showing B cell staining, but not IL-10; right: stimulated cells from WT mice showing B cell staining and IL-10 staining. t, Flow cytometric analysis of IL-10 and IgA expression, gated on the IgA⁺ population: left: stimulated cells from Il10^-/- mice, showing IgA cell staining, but not IL-10; right: stimulated cells from WT mice showing IgA and IL-10 staining. These results confirm IL-10 production by IgA⁺ cells. u, Flow cytometric analysis of p-STAT1 staining with corresponding isotype control.

Figure 1. B cells inhibit oxaliplatin-induced tumor regression

a, TRAMP (FVB) mice (TR-WT, TR-Jh^-/-, and TR-Cd8a^-/-; n=7-15/group) received weekly oxaliplatin (6 mg/kg), starting at week 16. After 4 weeks, prostate weights measured. Dashed red line = prostate weight of naïve controls. b, Tumor growth in mice transplanted with MC cells and treated with oxaliplatin as in Extended Data Fig. 1f (late treatment) or 5% dextrose (n=7-11/group). c, Weights of MC tumors after oxaliplatin or vehicle treatment (n=5-7/group). d, Mice of indicated genotypes bearing MC tumors (n=7-11/group) were treated as above. After 3 cycles, tumor volumes (mm³) were determined. e, Numbers of cleaved caspase 3 (CC3) CD45^- cells per high magnification field (HMF; 200×) in tumors from Extended Data Fig. 1g. f, MC tumors were inoculated into WT (left) or Cd8a^-/- (right) mice. After 16 days, B cells were depleted with antibodies against CD19, CD20, CD22 and B220. Four days after first twice-weekly antibody treatment, mice received weekly oxaliplatin (n=4-7/group, total: 42), and sacrificed 3 weeks later. Tumor volumes were analyzed by Kruskal-Wallis test: P=0.007**. Results are means ± s.e.m. Mann-Whitney and t tests were used to determine significance indicated as *P, 0.05; **P, 0.01; ***P, 0.001.

Figure 2. B cells inhibit oxaliplatin-induced T cell activation

a, CD8⁺ cells in TRAMP prostates (WT, Jh^-/-; n=4-6/group) from mice treated as in 1a, enumerated by flow cytometry and normalized to CD45⁺ cells. b, Mice (n=6-8/group) bearing MC tumors were analyzed as above for CD8⁺ cells in spleens and tumors after 3 chemotherapy cycles. c, d, Q-RT-PCR analysis of Perforin and Ifnγ mRNA in MC tumors collected as in (b) (n=4-7). e, IFNγ expression by CD8⁺ cells from tumors (n=6-8) from (b) after in vitro re-stimulation with tumor cell lysate. f-h, Expression of GrzB and Ki-67 (f), PD-1and Tim-3 (g) and BTLA (h) in CD8⁺ T effector cells (CD8⁺CD44⁺; f,g) or total CD8⁺ cells (h) from tumors of MC inoculated mice (b). Results are percentages of positive cells in tumoral CD8⁺ cells or mean fluorescence intensities (MFI) and are means ± s.e.m of 3 independent experiments (n=6-8 mice/group). Mann-Whitney and t tests were used to determine significance shown as above.

Figure 3. Oxaliplatin induces tumor infiltration with IgA⁺PD-L1⁺IL-10-producing plasmocytes

a, B220⁺CD19⁺ B lymphocytes in 20 weeks old TRAMP prostates after 4 oxaliplatin cycles (n=5-7/group) normalized to prostate weights. b,c, B220, CD19, CD138 and IgA expression in tumoral B cells from (a). Values are % of tumoral CD45⁺ (b) or CD19⁺ (c) cells. d, MC tumors (n=4-5/group) stained for αSMA (green) and IgA (red). Arrows: IgA⁺ cells whose number per HMF is displayed on the bottom. e, p-SMAD2/3 in tumor-infiltrating B cells (n=3-4/group). f, Il10 mRNAin MC tumors (n=5-6/group). g, Tumor-infiltrating IL-10⁺CD19⁺B cells in MC-WT mice, as percentages of CD45⁺ cells. h, Percentages of IL-10-producing cells in tumoral (MC-WT) CD19⁺IgA⁺ and CD19⁺IgA^- cells i, IL-10 expression by tumoral (MC-WT) IgA⁺ and IgA^- B cells (n=4-6/group). j, PD-L1 and FAS-L expression in B cells from TRAMP tumors. k, Pdl1 mRNA in MC tumors (n=5-6/group). l, Low (n=5) and high (n=5) risk human PC specimens stained with IgA (red) and αSMA (green) antibodies. Arrows: IgA⁺ cells. All results are means ± s.e.m of at least three independent experiments. Magnification bars: 100 μm. Mann-Whitney and t tests were used to calculate statistical significance shown as above.

Figure 4. TGFβR signaling and IgA CSR are required for immunosuppressive plasmocyte development

a, WT, Tgfbr2^ΔB or Iga^-/- mice bearing late MC tumors were given 3 weekly oxaliplatin cycles (n=5-11/group, total: 48), and tumor volumes at treatment end were analyzed (Kruskal-Wallis test: P=0.0004***). b, c, Tumoral CD19⁺ (b) and IgA⁺ (c) cells, depicted as percentages of tumoral CD45⁺ cells (b) or total vital cells (c) (n=4-7/group). d, Serum IgA in MC-WT and MC-Tgfbr2^ΔB mice (n=5-8/group). Tumor-free WT and Tgfbr2^ΔB mice served as controls. e, Frequency of tumoral CD8⁺ cells in mice from (a). f, CD8⁺ cells (5 × 10⁶/well) from (a) were re-stimulated with either MC lysate (left) or PMA/ionomycin (right) and analyzed for indicated markers. Percentages of marker positive cells within tumoral CD8⁺ cells are shown (n=4-7 mice/group). g, B cells (5 × 10⁶; 98% pure) from WT, Pdl1/2^-/- and Il10^-/- mice were transferred into MC tumor-bearing Jh^-/- mice (16 days after inoculation) that received oxaliplatin 2 days later. Tumor volumes were determined on day 30 (n=4-6 mice/group). Results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance.

Figure 5. Adoptively transferred B cells inhibit T cell-dependent tumor eradication

a,b TRAMP;Rag1^-/- mice (16 weeks old) received weekly oxaliplatin. One day after 1^st treatment, CFSE-labeled splenocytes from WT or Jh^-/- mice were adoptively transferred (ACT) into tumor-bearing mice (4-5/group). After 3 more oxaliplatin cyclesthe prostates were photographed (a) and tumor weight measured (b). c, Serum IgA in both ACT groups and FVB-WT mice. d,e Serum anti-SV40-Tag IgA and IgG concentrations in indicated strains with or without ACT and/or oxaliplatin treatment. f, Frequency of CD8⁺ cells amongst CD45⁺ cells in TRAMP;Rag1^-/- prostates after ACT and oxaliplatin treatment. g, MC tumor-bearing Rag1^-/- mice were oxaliplatin treated. One day later, mice (4-5/group) received activated T cells from WT mice immunized with MC cell extract without or with B cells from WT or Tgfbr2^ΔB mice. After 2 more treatments, mice were sacrificed and tumor volumes determined. h, IFNγ in tumoral CD8⁺ cells of above mice. Cells were re-stimulated with PMA/ionomycin before determining percentages of IFNγ-expressing cells in total CD8⁺ cells (n=5-8/group). j, Serum IgA in above mice. Results are means ± s.e.m. Mann-Whitney and t tests were used to determine significance. n.d. not detectable.