Immune checkpoint blockade in triple negative breast cancer influenced by B cells through myeloid-de

Triple negative breast cancer holds a dismal clinical outcome and as such, patients routinely undergo aggressive, highly toxic treatment regimens. Clinical trials for TNBC employing immune checkpoint blockade in combination with chemotherapy show modest prognostic benefit, but the percentage of patients that respond to treatment is low, and patients often succumb to relapsed disease. Here, we show that a combination immunotherapy platform utilizing low dose chemotherapy (FEC) combined with oncolytic virotherapy (oHSV-1) increases tumor-infiltrating lymphocytes, in otherwise immune-bare tumors, allowing 60% of mice to achieve durable tumor regression when treated with immune checkpoint blockade. Whole-tumor RNA sequencing of mice treated with FEC + oHSV-1 shows an upregulation of B cell receptor signaling pathways and depletion of B cells prior to the start of treatment in mice results in complete loss of therapeutic efficacy and expansion of myeloid-derived suppressor cells. Additionally, RNA sequencing data shows that FEC + oHSV-1 suppresses genes associated with myeloid-derived suppressor cells, a key population of cells that drive immune escape and mediate therapeutic resistance. These findings highlight the importance of tumor-infiltrating B cells as drivers of antitumor immunity and their potential role in the regulation of myeloid-derived suppressor cells.

Triple-negative breast cancer (TNBC) is an aggressive disease with dismal clinical outcome due to limited therapeutic options1,2. Immune checkpoint blockade (CP), which limits inhibitory pathways on CD8+ and CD4+ tumor-infiltrating lymphocytes (TILs), has demonstrated unparalleled clinical success in a wide variety of cancer types. Antibodies blocking the PD1: PDL1 inhibitory axis aim to target PD1 on T cells and PDL1 on tumor cells. However, only ~9% of patients with advanced TNBC express PDL1 on tumor cells3. Recently approved therapies for TNBC are limited (PARPi, atezolizumab, and sacituzumab-govitecan) and only benefit 10–20% of patients treated3. For this reason, a deeper understanding of the immune landscape in TNBC tumors is required to develop novel, effective therapies and delineate prognostic biomarkers of disease.

Many clinical trials are currently underway using CP as a standalone, adjuvant or neoadjuvant therapy for various tumor types4. However, the percentage of patients that respond to CP as a monotherapy is low and is generally limited to cancers with a favorable mutational landscape and high rates of cell turnover, such as melanoma and non-small cell lung cancer. In contrast, most breast lesions establish a highly suppressive tumor microenvironment (TME) with high levels of myeloid-derived suppressor cells (MDSCs), which has been shown to directly correlate with disease stage and overall tumor burden5,6. Additionally, clinical studies have shown the extent of immune cell infiltration and activation in breast tumors to be a key indicator of long-term survival and a predictor of therapeutic efficacy7,8,9,10. The use of therapies that can decrease MDSCs, increase TILs, and induce potent immunogenic cell death (ICD) may allow immunogenically cold breast tumors to better respond to CP.

A phase III clinical trial has shown that chemotherapy works to enhance the antitumor activity of CP in metastatic TNBC patients3. However, this combination of chemotherapy and immunotherapy only confers a modest overall survival benefit3. These results further highlight the need for additional strategies to treat TNBC and to mediate intrinsic resistance mechanisms. To this end, a more thorough analysis of the immune environment in TNBC patients beyond CD8+ TILs is necessary.

Oncolytic viruses (OVs) have risen as an interesting therapeutic option with widespread clinical application and safe toxicity profiles due to their ability to preferentially target cancer cells over normal healthy cells. We have developed herpesvirus-based OVs that target multiple tumor types11,12,13,14 and identified a strong correlation between OV-mediated ICD and antitumor immunity14,15,16,17,18,19. Moreover, oncolytic HSV-1s, such as oHSV-1 dICP014, synergize with low-dose chemotherapy to overcome immune tolerance14,16,17,18. Our early studies used mitoxantrone, a well-characterized immunogenic agent18,20,21,22. However, mitoxantrone is not normally used for breast cancer therapy, decreasing the translational value of our findings. Using a chemotherapy cocktail that is routinely used for TNBC patients will allow for ease of translation and biological analysis of current clinical applications.

Additional to the need for improved therapeutic approaches for TNBC patients is the simultaneous need to better understand the foundational biology that allows some patients to respond to treatment while their phenotypically identical counterparts do not. Here, we focused on the development of clinically relevant therapies that can be used to induce the inflamed microenvironment needed for tumors to respond to CP. We have used our therapeutic platform as a mechanism to study and better understand the complex biological processes that mediate therapeutic responses, as well as the interactions of key immune cell populations within the TME. This approach facilitates translation of findings to the clinic to improve immunotherapy responses in solid tumors.

FEC + oHSV-1 improves survival outcome in TNBC tumor-bearing mice

While chemotherapies are often given in dose-dense cytotoxic regimens, many studies have described the immunostimulatory properties of various chemotherapeutics through the use of low-dose intervention23,24,25,26. Based on current clinical practice, two of the most commonly used chemotherapy cocktails for breast cancer patients are FEC (5-fluorouracil, epirubicin, and cyclophosphamide) and AC (doxorubicin and cyclophosphamide)27. In an effort to reverse translate these regimens from the clinic into murine models, we performed optimization studies to find dosing levels and schedules that showed no acute cytotoxicity to murine hosts, as determined through close monitoring of body weights and scoring (Supplementary Fig. 1). In an effort to mimic clinical dosing, the individual ratios of the drugs to one another were the same as commonly prescribed in the clinic. The therapeutic potential of each regimen was evaluated in combination with oHSV-1 (Fig. 1a) using the E0771 TNBC syngeneic murine model. While no monotherapy showed efficacy, the addition of oHSV-1 to the clinical FEC regimen (FEC + oHSV-1) delayed tumor growth in some mice (Fig. 1b) and significantly extended survival, with 10% of mice resulting in durable tumor regression (Fig. 1c). Since epirubicin and doxorubicin are chemical analogues of one another, the main difference between these two regimens is in the addition of 5-fluorouracil to the FEC regimen. Indeed, a single dose of 5-fluorouracil alone has been shown to promote an antitumor immune response28.

a C57/Bl6 mice bearing E0771 tumors were treated with saline, chemotherapy (FEC or AC), oncolytic virus (oHSV-1 dICP0), or chemotherapy + oncolytic virus. *Created using BioRender.com. b Tumor volumes were measured every 2–3 days from the start of treatment until mice reached endpoint. Each line represents an individual mouse within the group. c Kaplan–Meier survival curves of each group. *Mantel–Cox test was used for statistical analyses.

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FEC + oHSV-1 sensitizes tumors to checkpoint blockade therapy

Although TNBC tumors were previously thought to be immunologically cold, recent clinical studies have shown that they do indeed express various immunogenic markers, such as PD-L129. However, the expression of these markers is low and it is not diffuse throughout the tumor, but rather clumped in focal areas limited to a small proportion of cancer cells30. Further, clinical trials have reported both the efficiency and necessity of combined therapeutic modalities (e.g., immunotherapy and chemotherapy), as TNBC patients often have low, short-lived responses to CP on its own31. Based on our preliminary studies, we hypothesized that FEC + oHSV-1 is capable of sensitizing tumors to CP by turning an immune-cold tumor into an immunogenic one. Survival studies were performed with the addition of dual CP targeting the non-redundant pathways of cytotoxic T-lymphocyte antigen 4 and programmed death ligand-1 (with anti-CTLA4 and anti-PD-L1 antibodies, respectively). While neither CP alone, nor in combination with FEC or oHSV-1, showed therapeutic efficacy, the combination of FEC + oHSV-1 + CP resulted in greatly improved responses with 60% of mice achieving durable tumor regression (Fig. 2a–c). Furthermore, mice that showed durable regression of tumors subsequently rejected the parental (E0771) tumor cells when re-challenged, suggesting that responding mice generated immune memory against the tumor (Fig. 2d). Interestingly, when this triple combination therapy was tried with singular administration of CP (anti-CTLA4 mAb or anti-PD-L1 mAb) no therapeutic efficacy was seen (Supplementary Fig. 2), suggesting that targeting both pathways simultaneously is beneficial in overcoming resistance mechanisms in this aggressive tumor model.

a C57/Bl6 mice bearing E0771 tumors were treated with saline, CP (anti-CTLA4 and anti-PD-L1), FEC + CP, oHSV-1 + CP, FEC + oHSV-1, or FEC + oHSV-1 + CP. *Created using BioRender.com. b Tumor volumes were measured every 2–3 days from the start of treatment until mice reached endpoint. Each line represents an individual mouse within the group. c Kaplan–Meier survival curves of each group. dMice who achieved durable tumor regression with FEC + oHSV-1 + CP therapy were subsequently re-challenged with E0771 cells on day 63 days. Naïve mice of similar age were used as a control for tumor implantation and growth. *Mantel–Cox test was used for statistical analyses.

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To identify pathways essential for therapeutic efficacy, cytokine analysis was performed on tumor lysates obtained from mice treated with either saline or FEC + oHSV-1 + CP (Fig. 3; full dataset shown in Supplementary Fig. 3). Mice treated with FEC + oHSV-1 + CP had significantly changed expression levels of cytokines related to myeloid cell differentiation and chemotaxis, macrophage activation, and inflammatory pathways.

C57/Bl6 mice bearing E0771 tumors were treated with saline or FEC + oHSV-1 + CP. Mice were sacrificed on day 10 treatment and tumors flash frozen. Tumor lysates were made and sent for cytokines expression analysis. All values are reported as a concentration (pg/mL). Two-tailed paired ttest was used for statistical analyses. Error bars are representative of the standard deviation.

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Immunogenic therapy induces TILs into otherwise immune-bare tumors

To investigate the immune landscape of untreated and treated E0771 tumors, histologic assessment was performed. Tumors were harvested on days 7 and 10 from mice treated with saline, FEC, oHSV-1, FEC + oHSV-1, or FEC + oHSV-1 + CP (Day 7, Fig. 4a; Day 10, Supplementary Fig. 4). Analysis of whole tumor sections harvested on day 7 and stained with hematoxylin and eosin (H&E) (Supplementary Fig. 5A) shows that saline and FEC-treated mice have large, densely packed tumors with many cells actively undergoing cellular division. Conversely, mice treated with oHSV-1, FEC + oHSV-1, and FEC + oHSV-1 + CP present with shrinking cellular structures and large areas of necrosis, likely due to direct viral oncolysis from intratumoral administration of the virus. When these same groups are again analyzed on day 10 (Supplementary Fig. 5B) it is clear that saline and FEC-treated mice have continued to progress towards tumor endpoint with highly vascularized tumors undergoing extensive angiogenesis, displayed by the appearance of many new microvessels and infiltrating vasculature. oHSV-1-treated mice still have areas of necrosis in the center, but the remaining tumor tissue has begun to progress towards tumor endpoint, with large tumors that are densely packed with viable cells. Interestingly, FEC + oHSV-1-treated tumors now present two distinct populations within mice, with 80% of tumors shifting towards an untreated phenotype with increased proliferation and density, similar in appearance to saline and FEC-treated tumors. The remaining 20% of tumors show increased areas of necrosis and decreased overall tumor size. As expected from survival outcomes, tumors harvested from mice that were treated with FEC + oHSV-1 + CP present with increased necrosis and very small tumors (Supplementary Fig. 5C).

C57/Bl6 mice bearing E0771 tumors were treated with saline, FEC, oHSV-1, FEC + oHSV-1 or FEC + oHSV-1 + CP and tumors were harvested on day 7 (n = 5 per group). Tumors were sectioned and stained with H&E for pathological analysis. Sections were then further stained with antibodies for CD3, CD4, CD8α, and FOXP3. a Representative images for tumors harvested on day 7. Each image shows a whole section of an individual tumor. b Whole tumor sections were scanned and quantified using HALO quantification software. Each symbol represents an individual mouse within that group. Two-tailed paired t test was used for statistical analyses. Error bars are representative of the standard deviation.

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Tumors were further stained with antibodies for CD3, CD4, and CD8α to assess the level of immune cell infiltration across the treatment groups (Fig. 4a). As expected, saline and FEC-treated mice presented with immune-bare tumors with almost no immune cells present in the tumor bulk or surrounding connective tissue. Tumors treated with oHSV-1, FEC + oHSV-1, or FEC + oHSV-1 + CP, however, show increased levels of T-cell infiltration throughout the remaining viable tumor tissue. Quantification of these stains (Fig. 4b) reveals that tumors treated with oHSV-1, FEC + oHSV-1, or FEC + oHSV-1 + CP have statistically significant recruitment of immune cells into the tumor, as compared to saline-treated tumors. Clinical studies have shown the ratio of CD8+ T cells to FOXP3+ regulatory T cells to be a significant indicator of prognostic outcome in TNBC patients32,33. For this reason, all tumor slices stained for CD8α were co-stained for FOXP3 expression and the ratio between the two was assessed within individual mice (Fig. 4b). As shown, mice treated with oHSV-1, FEC + oHSV-1, or FEC + oHSV-1 + CP had statistically significant increases in their ratio of CD8α+/FOXP3+ infiltrates at day 7, but only with the combination therapies (FEC + oHSV-1 and FEC + oHSV-1 + CP) is this increase sustained out to day 10. This observation suggests that while oncolytic virotherapy is able to prime the tumor microenvironment, additional therapeutic intervention may be required for durable response to immunotherapy treatments.

FEC + oHSV-1 induces a B-cell signature in whole tumor RNA sequencing

Immunohistochemistry (IHC) clearly demonstrates that treatment with oHSV-1 creates an initial influx of T cells into the TME. However, IHC is unable to determine the functionality and activation state of these T cells and more specifically, whether they are capable of contributing to a robust antitumor immune response. The therapeutic efficacy of oHSV-1 as a monotherapy (as shown in Fig. 1c) shows that it is insufficient to limit tumor progression. To begin to understand how the addition of FEC mechanistically alters therapeutic responses to oHSV-1 treatment, we performed RNA sequencing analysis. Tumors were treated with saline, FEC, oHSV-1, or FEC + oHSV-1 and harvested on day 5. Principal component analysis (PCA) shows that while saline and FEC-treated tumors cluster closely together, both oHSV-1 and FEC + oHSV-1 show distinct clustering patterns (Fig. 5a). Interestingly, within the FEC + oHSV-1 group we see two separate clusters of mice, one that has a similar expression profile to saline and FEC-treated mice (thought to be non-responders to treatment) and one that clusters distinctly (thought to be responders to treatment), consistent with previous data that FEC + oHSV-1 therapy results in a dichotomous response.

a 3-D cluster plot showing the RNA expression correlations between mice treated with saline (gray; n = 5), FEC (pink; n = 10), oHSV-1 (teal; n = 5) and FEC + oHSV-1 (purple; n = 10). b Bar plot illustrating the results of pathway enrichment analysis performed on samples from mice treated with FEC + oHSV-1, compared to those treated with saline alone. c Heat map showing the normalized expression values of genes across all samples. d Heat map showing the normalized expression values of genes associated with B-cell receptor pathways. e Kaplan–Meier survival plot for TNBC patient RFS (n = 220) microarray data based on mean mRNA expression level of the top 29 available genes in Table S1. fKaplan–Meier survival plot for all breast cancer patients RFS (n = 2032) based on mean mRNA expression levels of the top 29 available genes in Table S1. *BCR B-Cell Receptor.

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Pathway enrichment analysis identified many immune pathways and processes that were upregulated with FEC + oHSV-1 therapy (as compared to saline, Fig. 5b). Assessment of the differentially expressed gene pool (Fig. 5c) reveals that mice treated with FEC show similar genomic profiles to those treated with saline alone. However, treatment with either oHSV-1 or FEC + oHSV-1 shows a markedly different gene signature, with both oHSV-1 and FEC + oHSV-1 treatments upregulating many genes associated with the B-cell lineage (Fig. 5D, Supplementary Data 1), including Ifitm1, Il1b, CD24a, CXCL12, FGF7, and Nrg1. A signature comprising the top 29 upregulated genes from this analysis was surveyed across a large dataset of publicly available breast cancer patient data for prognostic relevance34. Breast cancer patients with mean expression of this signature in the highest tertile had significantly improved relapse-free survival (RFS) in TNBC [HR = 0.47 (0.26–0.86); logrank p value = 0.015] (Fig. 5e), while mean expression above median across all patients was also correlated with improved RFS [HR = 0.58 (0.50–0.68); logrank p value < 0.01] (Fig. 5f).

Depletion of B cells results in loss of therapeutic efficacy

While B cells play many different roles in the body, their primary function is in antibody production. To follow-up on the results of RNA sequencing analysis and to determine whether or not B cells play a key part in the efficacy of our combination therapies, we performed B-cell depletion studies. Subcutaneous E0771 tumors were grown in C57/Bl6 mice and circulating B cells were depleted using an anti-CD20 antibody (Fig. 6a).35 Analysis of the overall survival shows that the depletion of B cells diminished therapeutic outcome in tumor-bearing mice (Fig. 6b, c, Supplementary Fig. 6). Depletion was confirmed via flow cytometry (Fig. 6d) and an isotype antibody was used as a control. Bulk antibody production levels were also assessed using an indirect ELISA assay. Serum was taken from naïve mice, tumor-bearing mice, and from therapeutically treated mice on day 15 and protein lysates from E0771 cells were used to coat the plates. Results of the ELISA assay indicates that FEC alone has no effect on antibody production levels. However, treatment with oHSV-1, CP, FEC + oHSV-1, and FEC + oHSV-1 + CP all show a statistically significant increase in antibody production levels, which is lower in the absence of circulating B cells (Fig. 6e). Since oHSV-1 or CP alone confer no therapeutic benefit, these data suggest that antibody production, while affected by our therapies, is not the most important function of B cells in relation to our therapeutic efficacy.

a C57/Bl6 mice bearing E0771 tumors were treated with either an anti-CD20 antibody or isotype antibody, followed by treatment with saline, FEC, oHSV-1, CP, FEC + oHSV-1, or FEC + oHSV-1 + CP therapy. *Created using BioRender.com. b Tumor volumes were measured every 2–3 days from the start of treatment until mice reached endpoint. Each line represents individual mice within the group. c Kaplan–Meier survival curves of combination therapy treatments, with both the depletion and isotype antibodies. d Flow cytometry confirmed that a singular dose of an anti-CD20 antibody is sufficient to deplete B cells within 24 h of administration. e Bar plot showing IgG production levels from naïve mice, tumor-bearing mice, and therapeutically treated mice. *Mantel–Cox test was used for statistical analyses in c, two-tailed paired t test was used for statistical analyses in d, and two-tailed unpaired t test was used for statistical analyses in e. Error bars are representative of the standard deviation.

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To further assess the role B cells play in our treatment, mice were injected with either an anti-CD20 antibody or an isotype antibody, treated with saline or FEC + oHSV-1 and tumors harvested on day 7. Quantification of IHC images shows that B-cell depletion results in decreased levels of tumor-infiltrating T cells and increased levels of Ly6G+ myeloid cells (Supplementary Fig. 7A). Immunofluorescence (IF) staining was performed on whole tumor sections and stained for CD3, CD8, PNAd, Pax5, and CD11b. Analysis of multiplex images further corroborates the IHC quantification data and shows that mice treated with FEC + oHSV-1, in the presence of the isotype antibody, had formation of immature tertiary lymphoid structures (as evidenced by the presence of PNAd+ high endothelial venules) (Supplementary Fig. 7B). Conversely, mice treated with FEC + oHSV-1, in the presence of the anti-CD20 antibody, have no PNAd present and no organized lymphoid aggregates (Supplementary Fig. 7C).

B cells are required for control of MDSCs and resultant therapeutic efficacy

Depletion of circulating B cells results in a complete loss of therapeutic efficacy and disruption to immune cell organization in mice treated with FEC + oHSV-1 therapy. What remains unknown is how the addition of FEC mechanistically alters therapeutic outcome to oHSV-1 therapy and what effector cell functions are being regulated by tumor-infiltrating B cells (TIL-Bs). We performed a deeper assessment into the differentially expressed gene pool from RNA sequencing data, paying particular attention to the differences between oHSV-1 and FEC + oHSV-1 therapy. In this comparison we see many genes that are implicated in immunosuppressive MDSCs. Interestingly, some of these genes (S100A8, CXCL2, CXCL1, Ly6G, Slpi, Fpr2) are upregulated in oHSV-1 vs. saline but downregulated in FEC + oHSV-1 vs. oHSV-1 (Table 1, Fig. 7a), suggesting that FEC + oHSV-1 therapy is able to not only upregulate B-cell receptor signaling pathways, but also regulate the maturation and migration of MDSCs. It is well established in the literature that some chemotherapies (including 5-fluorouracil in particular) directly deplete MDSCs in both animal models as well as in patients36,37,38. However, in such studies chemotherapy is given in dose-dense cytotoxic regimens. In our hands, chemotherapy is being used as a low dose, immune-stimulatory intervention and shows no therapeutic efficacy alone, suggesting that it is unable to suppress MDSCs without the addition of oHSV-1.

To further investigate the relationship between these distinct cell types, we performed immune analysis studies. E0771 tumors were grown in C57/Bl6 mice and treated with saline, FEC, oHSV-1, CP, FEC + oHSV-1, or FEC + oHSV-1 + CP in both the presence (isotype mAb) and absence (anti-CD20 mAb) of circulating B cells. Blood was drawn on days 6, 10, and 15 and peripheral blood mononuclear cells (PBMCs) were analyzed via flow cytometry. While this analysis confirmed the depletion of circulating B cells in mice treated with our anti-CD20 mAB (Fig. 7b), it also revealed a striking and consistent difference in the population of MDSCs (Ly6GhiLy6Cintcells) between mice treated with isotype and anti-CD20 antibodies (Fig. 7c). Analysis of the frequency of these populations shows that circulating B cells in isotype-treated mice decrease over time in all treatment groups, except for those mice treated with FEC + oHSV-1 + CP (Fig. 7d). On the contrary, mice treated with the triple combination therapy see a re-population of circulating B cells by day 15. The frequency of MDSCs in the blood (Fig. 7e) confirms that there is rapid expansion of the granulocytic MDSC population, with the frequency more than doubling in B-cell-depleted mice treated with triple therapy. Levels of other immune cell populations in the blood (CD4s, CD8s, monocytes, and DCs) are consistent with expected findings, with no clear disturbance in their frequencies in the absence of B cells (Supplementary Fig. 8).

Further highlighting the clinical impact of these findings, we assessed the prognostic significance of the MDSC gene signature downregulated by FEC + oHSV-1 therapy (Table 1) in a combined cohort of publicly available clinical microarray data34. Mean expression of this signature above median was associated with worse overall survival (OS) in TNBC restricted patient data [HR = 2.08 (1.05–4.10); logrank p value = 0.031] (Fig. 7f), and generally across all breast cancer patients combined [HR = 1.73 (1.32–2.27); logrank p value < 0.01] (Fig. 7g). This suggests that attenuation of the MDSC signature by FEC + oHSV-1 could have positive prognostic implications.

To rule out the clonal effect and assess whether or not the correlation between B cells and MDSCs holds true across more than one TNBC tumor model, we also conducted immune analysis using the PY230 murine breast cancer line (Supplementary Fig. 9). Here too, we see that when mice are treated with FEC + oHSV-1 + CP and depleted of circulating B cells, there is a prominent expansion of MDSCs in the blood. These data suggest that the correlation between B cells and MDSCs is not due to tumor clonality, but rather carries over between different models.

B cells are required to alleviate tumor immunosuppressive mechanisms

To assess whether changes seen in the peripheral blood were consistent and representative of what was happening in the tumor, immune analysis was performed on TILs. E0771 tumors were grown in C57/Bl6 mice and treated with saline, FEC, oHSV-1, CP, FEC + oHSV-1, or FEC + oHSV-1 + CP in both the presence (isotype mAb) and absence (anti-CD20 mAb) of circulating B cells. Mice were sacrificed on day 10 and TILs were processed and stained for analysis via flow cytometry. Consistent with previous findings, treatment with FEC + oHSV-1 or FEC + oHSV-1 + CP increases the level of TIL-Bs (Fig. 8a), particularly those of a memory phenotype (CD19+CD27+). Interestingly, whether or not mice were depleted of circulating B cells did not significantly affect the level of TIL-Bs. However, in line with findings from the peripheral blood, depletion of circulating B cells did result in a rapid expansion of MDSCs (Fig. 8b, c). Irrespective of whether mice were treated with the depletion antibody or not, the triple combination therapy was also shown to significantly reduce immunosuppressive populations in the tumor, such as the CD244.2+ immunoregulatory receptor, PDL1+ immune checkpoint, and F4/80+tumor-associated macrophages. The relationship between B cells and myeloid cells was further shown in both splenocytes (Fig. 8e, f) and tumor-draining lymph nodes (TDLNs) (Fig. 8g–i). The levels of other immune cell populations in the tumor, spleen, and TDLN (CD4s, CD8s, and DCs) are consistent with expected findings, with no clear disturbance in their frequencies in the absence of B cells (Supplementary Fig. 10).

FEC + oHSV-1 responders present with a B-cell gene signature and control of MDSCs

Consistent with clinical outcomes, mice treated with FEC + oHSV-1 have dichotomous responses to therapy, shown by the survival outcomes (Fig. 1), IHC quantification (Fig. 4), and gene expression profiles (Fig. 5). To further identify genomic features influencing outcome to therapy, we have sub-grouped these mice into what we believe to be responders and non-responders to treatment, as based on their likeness to the gene expression profiles of saline-treated mice (Fig. 9a). Assessment of the differentially expressed gene list shows that responders have upregulation of many genes associated with B-cell receptor signaling pathways (Fig. 9b), as consistent with previous findings. Additional to this and perhaps even more notable, the responders also downregulate Siglec15, a critical immune suppressor that is commonly upregulated on human cancer cells and tumor-infiltrating myeloid cells39.

We utilized a publicly available cohort of combined clinical microarray data containing response rates to various therapies40. While there are no large datasets available for breast cancer patients treated with oncolytic virotherapy, evaluation of CD19 mRNA expression in patients treated with FEC therapy indicated CD19 trended towards being a predictor of complete pathological response in a Her2(-) ER(-) restricted patient group [AUC = 0.657, ROC pvalue = 0.02, Mann–Whitney p value = 0.054], and was a predictor of pathological response across combined breast cancer patients treated with FEC [AUC = 0.729, ROC p value < 0.01, Mann–Whitney p value < 0.01].

Immune checkpoint blockade has surged to the forefront of cancer therapy with astonishing clinical success rates and low toxicity profiles. However, this highly efficacious therapy only works in a fraction of patients and we have yet to fully elucidate the underlying biology that allows some patients to respond to therapy, while others do not. Helmink and colleagues have eloquently shown that B cells play an important role in promoting efficacious responses to immune checkpoint blockade in melanoma and renal cell carcinoma patients41. In line with these findings, our data suggest that promoting a B-cell signature within the tumor allows for successful treatment with combination immunotherapy platforms. Using the clinical chemotherapy cocktail FEC, in combination with oHSV-1, we were able to elicit the upregulation of B-cell receptor signaling pathways that allowed our otherwise non-responsive TNBC tumors to respond to CP. In particular, mice treated with the triple combination therapy had a re-population of circulating B cells over time and an increase in memory B cells within the tumor, suggesting that the sustained presence of B cells may be required to achieve durable responses and improved prognostic outcomes.

In vivo depletion studies in which an anti-CD20 antibody was used to deplete mice of circulating B cells have shown that groups of mice that would otherwise achieve complete responses to treatment instead had a complete loss of therapeutic efficacy. Deeper investigation into this phenomenon revealed a strong correlation between the presence of B cells and control of immunosuppressive MDSCs. These data suggest that B cells are required to suppress the rapid expansion of immunosuppressive myeloid cell populations in the TME. Indeed, RNA sequencing data identified that FEC + oHSV-1 is able to not only upregulate genes associated with the B-cell lineage, but also to downregulate genes that are known to be key players in tumor immunosuppression. When further looking at the population of mice with the most distinct RNA profile (as thought to be responders to treatment) we find that amongst the downregulated MDSC genes is Siglec15, a known immune suppressor broadly expressed on human cancer cells and tumor infiltrating myeloid cells39. As Chen and colleagues have shown, Siglec15 overexpression has been documented in numerous human cancers and its expression is mutually exclusive of PD-L1, suggesting that it may be a potential therapeutic target for patients who are refractive to anti-PD-1/PD-L1 checkpoint blockade therapy42. Interestingly, our therapy downregulates this key immune modulator, suggesting a therapeutic platform that can be used to target this pathway of tumor immune escape. Along with downregulation of Siglec15, immune analysis studies showed that our triple combination therapy significantly reduces levels of CD244.2, an immunoregulatory receptor found on a variety of immune cells, including exhausted CD8+ T cells and MDSCs43.

Current immunotherapies do not target or consider B cells, despite their predominance in the TME and key role in the adaptive immune response. In TNBC, evidence suggests that TIL-Bs generate a robust humoral response to amplify antitumor immunity44 and mediate immunotherapy outcomes45. TIL-Bs have been correlated with enhanced overall survival46, but findings in the literature suggest conflicting roles with TIL-Bs having both pro- and anti-tumorigenic functions. TIL-Bs have been identified as mediators of malignancy in several cancer types, with TIL-B depletion yielding positive outcomes47. Conversely, coordinated antibody and T-cell responses have been documented in cancer patients and TIL-Bs have been correlated with improved outcome41,45,48,49,50. Further, clinical studies have reported that TIL-Bs are influenced by the TME and immunotherapy, a discovery consistent with our findings. Indeed, our B-cell gene signature correlates with improved RFS in TNBC patients as well as all breast cancer subtypes combined, highlighting the clinical impact of these findings.

MDSCs are a heterogenous population of immature myeloid cells that accumulate in tumor beds and lymphoid organs (such as the spleen) of tumor-bearing hosts. MDSCs have long been attributed to tumor immunosuppression, most commonly due to their ability to suppress T-cell-mediated immune responses. However, studies have also shown that granulocytic MDSCs can suppress antitumor B-cell responses, predominantly through the secretion of nitric oxide (NO), arginase (Arg), and IL151,52,53. While there are no documented studies to our knowledge looking at the reverse regulation of this phenomenon, our data suggests that while MDSCs can suppress TIL-Bs through the secretion of NO, Arg, and IL1 perhaps the opposite is also true. We have shown that our therapeutic platform upregulates B-cell receptor signaling pathways, suppressing key genes such as NOS2, Arg2, and IL1R1 (Table 1), which we believe to be essential for the regulation and control of MDSCs. Indeed, the MDSC gene signature that we identified from our RNA sequencing data showed a strong correlation with clinical datasets, with low expression of those genes correlating with improved OS in TNBC patients as well as all breast cancer subtypes combined.

We have used a multi-pronged therapeutic approach to treat triple-negative breast tumors (Fig. 10). In this approach, we have combined low-dose chemotherapies that work to enhance tumor immunogenicity and antigen presentation aiding in BCR-recognition of TAAs. This, coupled with our oncolytic virotherapy initiates a cascade of DC and B-cell recruitment and ultimately clonal expansion of B cells. B-cell and T-cell priming results in the release of antitumor chemokines driving effector cell function, while simultaneously suppressing cytokines responsible for the accumulation of MDSCs in the TME. This immune-activating cascade of events leads to an immune landscape conducive to successful treatment with CP.

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Our studies are limited by the nature of murine hosts and their inability to fully recapitulate human biology. It is common to have patients with phenotypically similar cancers present with varying response levels to treatment. This phenomenon was also seen in our studies, in which mice with identical genetic makeup had dichotomous responses to the same treatment. This inter-mouse heterogeneity may be due to the individual metabolic activity and immune profile of each individual mouse. Our data were generated from subcutaneous tumors and should be further analyzed in metastatic and spontaneously arising tumor models to assess these core immunological interactions in a more naturally occurring setting. We aim to further assess these fascinating findings as we continue to phenotype our B-cell populations and assess their interactions with various myeloid cell populations in cancer-bearing hosts. We believe that clinical studies should focus not only on the levels of T cells and their correlated effects to prognostic outcomes, but also look at B cells as a key biomarker to predict responses to immunotherapy treatments.