CD40 agonism enhances immune checkpoint blockade and generates immunologic memory via CD4+ T cells in ERα+ mammary tumors

Abstract

There has been marked improvement in the clinical outcome of triple-negative breast cancer (TNBC) with the use of immune checkpoint blockade (ICB) although serious immune-related adverse effects are not uncommon. Unlike TNBC, ERα + breast tumors are largely unresponsive to ICB. Here we demonstrate defective priming by cross-presenting conventional dendritic cells (cDCs) and a blunted response to ICB in ERα + mouse mammary tumors compared to TNBC. Systemic administration of an agonistic CD40 antibody (aCD40) induced T cell proliferation and activation in tumor-draining lymph nodes and attracted effector T cells to the tumor bed from the periphery. This effect was largely due to activation, maturation and migration of type 1 conventional dendritic cells (cDC1s). aCD40 alone slowed tumor growth in ERα + tumors but its combination with ICB cured tumor-bearing mice, accomplishing a "vaccine effect" and the immune-mediated rejection of tumor rechallenge. The anti-tumor effect of aCD40 effect was cDC1 and CD8 + T cell-dependent, whereas the rejection of secondary tumor rechallenge in cured mice required CD4 + T cells. Importantly, intra-tumoral administration of aCD40 combined with systemic or intra-tumoral ICB - to mimic neoadjuvant therapeutic approaches-induced complete regressions of both treated and distant tumors. These findings indicate that aCD40 achieves DC activation required for the response to immunotherapy in ERα + tumors and further supports intra-tumoral administration of both aCD40 and ICB as an effective treatment that might limit systemic exposure and lower risk of immune-related toxicity.

Figure 1

aCD40 increases ERα+ mammary tumor sensitivity to ICB. A-C. Proportions of indicated cell types in the TME of Brpkp110 and E0771 tumors by flow cytometry on day 13 post implantation (n=7–8). D. Proportions of Ki67+ proliferating EpCAM+ tumor cells in vivo measured by flow cytometry on day 13 post implantation (n=7–8). E. Tumor volume changes compared to pretreatment on day 29 post implantation (left) and growth curves (right) of E0771 tumor cells. Indicated treatments initiated on day 8 post implantation (n=12–15, data representative of 2 experiments with similar results). F. Tumor volume changes compared to pretreatment on day 26 post implantation (left) and growth curves (right) of Brpkp110 tumor cells. Indicated treatments initiated on day 7 post implantation (n=18–20, data representative of 3 experiments with similar results). Data: (A-D) median, (E-F, left) each column represents individual tumor, (E-F, right) mean ± SEM. For all panels, p<0.05 was considered statistically significant, and * p<0.05, ** p<0.01, ***p<0.001 and ****p<0.0001.

Figure 2

aCD40 increases tumor-infiltrating cytotoxic T-cells in orthotopic ERα+ Brpkp110 tumors. A. Tumor volume changes compared to pretreatment on day 25 post implantation (left) and growth curves (right) of Brpkp110 tumor cells. aCD40 or vehicle treatments initiated on day 6 post implantation (n=16–18, data representative of 3 experiments with similar results). B. Flow cytometry analysis of implanted control and aCD40 treated Brpkp110 tumors on day 7 post treatment (n=10, data representative of 2 experiments with similar results). C. Cleaved caspase 3 measured by IHC in control and aCD40 treated Brpkp110 tumors on day 5 posttreatment (n=6–7, data representative of 2 experiments with similar results). D. DC subtype proportions measured by flow cytometry in implanted control and aCD40 treated Brpkp110 tumors on day 7 post-treatment (n=10, data representative of 2 experiments with similar results). E. Proportions of CD103+ cDC1s measured in Brpkp110 tumors (left) and TDLN (right) on days 2, 5, and 7 post aCD40 administration, compared to untreated (control) tumors (n=6–7). F. Proportions of CD3+, CD8+, and CD4+ T cells by flow cytometry in control and aCD40 treated Brpkp110 tumors on day 13 post treatment (n=10, data representative of 2 experiments with similar results). G. FoxP3+ CD4+ regulatory T cells on day 7 post-treatment (n=7, data representative of 2 experiments with similar results). H. Proportions of Granzyme B+ T cells in subpopulations by flow cytometry in control and aCD40 treated Brpkp110 tumors on day 7 post implantation (n=10, data representative of 2 experiments with similar results). I. Images of immunofluorescent staining for CD8 (red) and nuclei (blue) and CD8 staining quantification in untreated (control) and aCD40 treated Brpkp110 tumors on day 7 post treatment (n=4–6). J. Quantification of CD8 immunofluorescent staining in outer, middle, and inner thirds of control and aCD40 treated tumors on day 7 post treatment (n=5–6). K. After 7 days of treatment with aCD40, Brpkp110 tumors were minced and cultured ex vivo. Supernatant was collected and pooled for each treatment group after 48 hours and cytokines were measured. (n=2, with 3 tumors pooled per group). Data: (A, left) each column represents individual tumor and (A, right) mean±SEM, (B-G, H right, K) median, (I, J) mean±SEM. * p<0.05, ** p<0.01, *** p< 0.001, and ****p<0.0001.

Figure 3

cDC1s and T cells are critical for the anti-tumor activity of aCD40. A. DC maturation and activation markers measured by flow cytometry in cDC1 and cDC2 cell populations of control and aCD40 treated Brpkp110 TDLN on day 2 post-treatment (n=8–10, data representative of 2 experiments with similar results). B. T cell activation and proliferation markers measured by flow cytometry in CD4+ and CD8+ T cell populations of control and aCD40 treated Brpkp110 TDLN on day 7 post-treatment (n=10, data representative of 2 experiments with similar results). C. Growth curves of control and aCD40 treated Brpkp110 tumors implanted into WT hosts with or without T cell depletions (n=17–20). D. Growth curves of control and aCD40 treated Brpkp110 tumors implanted into WT and BATF3 KO hosts (n=16–20). Data: (A-B) median, (C-D) mean±SEM. * p<0.05, ** p<0.01, *** p< 0.001, and ****p<0.0001.

Figure 4

aCD40 and ICB results in a vaccine effect and rejection of a secondary tumor rechallenge. A. Brpkp110 tumor growth curves (left) and volume changes compared to pretreatment (right) on day 27 post implantation in control and aCD40+ICB treated hosts with and without CD8+ T cell depletions. Indicated treatments initiated on day 7 post implantation (n=12–15). B. Brpkp110 tumor growth curves (left) and volume changes compared to pretreatment (right) on day 25 post implantation in control and aCD40+ICB treated hosts with and without CD4+ T cell depletions. Indicated treatments initiated on day 8 post implantation (n=14–18). C. Brpkp110 tumor growth curves (left) and volume changes compared to pretreatment (right) on day 27 post implantation in control and aCD40+ICB treated hosts with and without CD4+ and CD8+ T cell depletions. Indicated treatments initiated on day 7 post implantation (n=12–18). D. Tumor growth curves (left) and volume changes compared to pretreatment (right) on day 26 post Brpkp110 tumor implantation into WT and BATF3 KO hosts. Indicated treatments initiated on day 7 post implantation (n=14–18). E. Proportions of circulating effector memory (CD44+CD62L−), central memory (CD44+CD62L+), and naïve (CD44−CD62L−) CD4+ (left) and CD8+ (right) in blood, 3 months post treatment induced tumor clearance (n=5–6, data representative of 2 experiments with similar results). F. Secondary Brpkp110 tumor rechallenge of naïve and previously Brpkp110 tumor bearing mice cured after aCD40 + ICB, at least 2 months post primary tumor clearance (n=12–14, data representative of 3 experiments with similar results). G. Control and rechallenge tumor growth in T cell sufficient (n=6–12) and T cell depleted hosts (n=12–14, data representative of 2 experiments with similar results). H. Brpkp110 tumor growth curves in intra-tumoral (IT) vehicle (control) and IT aCD40 treated hosts. aCD40 administered tumors denoted as aCD40 IT and contralateral untreated tumors denoted as CD40 IT Distant (n=9–12, data representative of 2 experiments with similar results). I. Brpkp110 tumor growth curves in intra-tumoral (IT) vehicle (control) and IT or intraperitoneal (IP) aCD40 or ICB received hosts (n=4–9, data representative of 2 experiments with similar results). * p<0.05, ** p<0.01, *** p< 0.001, and ****p<0.0001.