Anti-CD20 antibody promotes cancer escape via enrichment of tumor-evoked regulatory B cells expressing low levels of CD20 and CD137L

Abstract

The possible therapeutic benefits of B-cell depletion in combating tumoral immune escape have been debated. In support of this concept, metastasis of highly aggressive 4T1 breast cancer cells in mice can be abrogated by inactivation of tumor-evoked regulatory B cells (tBreg). Here, we report the unexpected finding that B-cell depletion by CD20 antibody will greatly enhance cancer progression and metastasis. Both murine and human tBregs express low levels of CD20 and, as such, anti-CD20 mostly enriches for these cells. In the 4T1 model of murine breast cancer, this effect of enriching for tBregs suggests that B-cell depletion by anti-CD20 may not be beneficial at all in some cancers. In contrast, we show that in vivo-targeted stimulation of B cells with CXCL13-coupled CpG oligonucleotides (CpG-ODN) can block cancer metastasis by inhibiting CD20(Low) tBregs. Mechanistic investigations suggested that CpG-ODN upregulates low surface levels of 4-1BBL on tBregs to elicit granzyme B-expressing cytolytic CD8(+) T cells, offering some explanative power for the effect. These findings underscore the immunotherapeutic importance of tBreg inactivation as a strategy to enhance cancer therapy by targeting both the regulatory and activating arms of the immune system in vivo.

FIGURE 1. Depletion of CD20⁺ B cells promotes metastasis

4T1.2 cells (5×10⁴) were subcutaneously injected in the fourth mammary gland of Jh KO (A,B) and BALB/c (A–E) mice to assess tumor progression (A,D) and number of metastatic foci (at day 32 post challenge, B,C,E). To evaluate the role of tBregs (A,B) or anti-CD20 Ab –mediated B cell depletion (C–E), 4T1.2 tumor-bearing Jh KO mice were adoptively transferred with tBregs (A,B) or BALB/c mice were intraperitoneally injected with 250 µg/mouse anti-CD20 Ab or control isotype-matched antibody (IgG, mock) at 5, 10 and 15 days post tumor challenge (D–E) or at -10, -6 and -2 before tumor challenge (C). Y-axis shows tumor size (A,D), or number of metastatic foci (B,C,E) ± SEM of four-eight mice per group experiments reproduced at least three times. From here on, * P<0.05 of indicated differences between groups are considered significant.

FIGURE 2. Depletion of CD20⁺ B cells after tumor challenge enriches for tBregs

Anti-CD20 Ab efficiently depletes CD19⁺ cells in various organs (A) of 4T1.2 tumor-bearing BALB/c mice used in Figure 1D,E, but enriches for tBregs (CD81^hiCD25⁺ within CD19⁺ gated cells, B). CD19⁺ cells isolated from lymph nodes of tumor-bearing mice suppressed proliferation of syngeneic CD4⁺ and CD8⁺ T cells (C). In particular, B cells from anti-CD20 Ab –treated mice were much more suppressive for both CD4⁺ and CD8⁺ T cells (C). Proliferation was assessed using CFSE dilution assay of T cells stimulated with anti-CD3 Ab and incubated with equal numbers of B cells for 5 days. CD19⁺ cells from the spleen of tumor-bearing mice -treated with anti-CD20 Ab expressed low levels of surface CD20 as comaperd with naïve mice (Naïve, D) and non-treated tumor-bearing mice (Tumor-bearing, D). A representative FACS staining dot plots of CD20⁺ cells within CD19⁺ gated cells (D). Y-axis shows % of CD19⁺ cells (A), tBregs (B), or proliferated CD4⁺ and CD8⁺ T cells (C) ± SEM of four-eight mice per group experiments reproduced at least three times.

FIGURE 3. Human tBregs also express low levels of CD20

Ex vivo generated human tBregs (from healthy donor B cells -treated with CM of MDA-MB-231 cells (17)) not only suppressed proliferation of human CD4⁺ and CD8⁺ T cells stimulated with anti-CD3 Ab (B-MDA, A), but also reduced cells expressing high levels of CD20 within CD81^hiCD25⁺ CD19⁺ cells (B). The suppressive activity (for CD4⁺ and CD8⁺ T cells) of human tBregs was retained within CD20^Low B cells (C), as shown by FACS sorting for CD20^Low B cells (enrichment >95%). Y-axis shows (%) Ki67⁺ CD4⁺ and CD8⁺ T cells ± SEM (C) of triplicate experiments. (D–F) Rituximab enriches for CD20^Low tBregs in patients with B-CLL, as shown by the increased presence of B cells expressing CD20^Low (broken line, D). In particular, compared with B cells before the treatment (continuous line, middle panel, D), the majority of B cells of patient 1 were CD20^Low after rituximab treatment (broken line, D). These CD20^Low cells efficiently suppressed proliferation of allogeneic CD4⁺ and CD8⁺ T cells (E) and induced FoxP3⁺ Treg conversion when mixed with CD25⁻ CD4⁺ T cells (% of FoxP3⁺ within CD4⁺, F). All data shown here were from triplicate experiments reproduced at least three times.

FIGURE 4. CXCR5-targeted delivery of CpG-ODN blocks tBregs and activates B cells eliciting cytolytic CD8⁺ T cells

(A,B) Ex vivo generation of murine tBregs was done in the absence or presence of various TLR ligands (5 µg/ml of ssRNA40; 10 µg/ml of Pam3CSK4 or FSL-1; 5 µg/ml of LPS; 1 µg/ml ODN1826 PS (CpG) and control CpG K) or 10 µg/ml anti-IgM. After 48h, cells were stained for CD20 expression (A) and evaluated for the ability to suppress proliferation of CD3⁺ T cells stimulated with anti-CD3 Ab (B). FITC- labeled CpG is better uptaken by tBregs when coupled with BLC-arp (continuous line, C), as compared with free CpG (broken line, C). Shaded area is for untreated control cells (C). BLC-arp/CpG (3 µg/ml ODN1826 PS) blocks activity of murine tBregs in vitro, at the same extent as free CpG, as shown by the inability to inhibit proliferation of CFSE-labeled CD3⁺ T cells (D). Controls were B cells treated with BAFF (B-Mock) and untreated tBregs.

FIGURE 5. In vivo-targeted delivery of CpG-ODN blocks lung metastasis

(A,E). BALB/c mice with established 4T1.2 cancer (as in Fig.1) were i.v. injected with BLC-Arp (30 µg) coupled with 5 µg ODN1826 PS (CpG) control CpG (CpG K) on days 3, 7, 12, 16 and 21 post tumor challenge to test for metastasis suppression (A). CD20 expression within CD19⁺ B cells in blood of BLC-arp/CpG-treated tumor-bearing mice (broken line, B) compared with the untreated tumor-bearing mice (mock, A, and dotted line, B). BLC-arp/CpG retarded lung metastasis (black bar, A) and increased CD20 expression on B cells (broken line, B). Expression of CD20 in B cells of naïve mice is in continuous line (B). Importantly, draining lymph node B cells from BLC-arp/CpG –treated tumor-bearing mice did not suppress proliferation of CD4⁺ and CD8⁺ T cells (C), instead becoming stimulatory for CD8⁺ T cells (p<0.05, naïve B cells vs. BLC-arp/CpG –treated tBregs, C). The inability of 4T1.2 cancer to induce cytolytic CD8⁺ T cells (compare tumor vs naïve, D) is reversed in 4T1.2 cancer-bearing mice treated with BLC-arp/CpG (D). Y-axis shows % of specific lysis (Cr⁵¹ release) ± SEM of 4T1.2 cells incubated with purified CD8⁺ T cells at indicated E:T ratio. To test the stimulatory activity of BLC-arp/CpG and to show that the effect is due to direct B cell stimulation 4T1.2 tumor –bearing mice were first depleted of CD20⁺ B cells by treating with anti-CD20 Ab on days 4 and 6 post tumor challenge (E). Then, mice were separated into two random groups and adoptively transferred with syngeneic B cells pretreated ex-vivo for 3h with 10 µg/ml CpG (B cells CpG) or PBS (B cells PBS). The cells were thoroughly washed with PBS to remove free CpG before i.v. injections of 5×10⁶ B cells at days 10 and 15. Y-axis shows number of lung metastatic foci ± SEM in the lungs of 4–6 mice per group at day 32. All data shown here were reproduced at least three times.

FIGURE 6. CpG renders tBregs stimulatory by up regulating 4-1BBL on tBregs

Compared with LPS-stimulated B cells (B-LPS), ex vivo-generated murine tBregs reduce surface expression of 4-1BBL (A). Shown is % ± SEM of 4-1BBL⁺ within CD25⁺ CD19⁺ B cells (B) of a triplicate experiment. (B) Compared with naïve mice, 4T1.2 cancer-bearing BALB/c mice have less B cells expressing 4-1BBL. Shown is % ± SEM (4 mice/group) of 4-1BBL⁺ within CD25⁺ CD19⁺ B cells in blood, spleen, draining lymph nodes (LN), lungs and liver. The low levels of 4-1BBL on ex vivo generated human (B-MDA, C) and murine tBregs from mice with 4T1.2 cancer (D) were drastically reversed by in vitro (C) and in vivo (D) BLC-arp/CpG treatment, respectively. The suppressive activity of tBregs was blocked in the presence of agonistic anti-4-1BB (CD137) Ab that presumably supplemented the missing 4-1BBL signaling to T cells (E). All data shown here were from triplicate experiments reproduced at least three times.