Interferon Signaling is Diminished with Age and is Associated with Immune Checkpoint Blockade Efficacy in Triple-Negative Breast Cancer

ABSTRACT
Immune checkpoint blockade (ICB) therapy, which targets T-cell inhibitory receptors, has revolutionized cancer treatment. Among the breast cancer subtypes, evaluation of ICB has been of greatest interest in triple negative breast cancer (TNBC) due to its immunogenicity, as evidenced by the presence of tumor infiltrating lymphocytes and elevated PD-L1 expression relative to other subtypes. TNBC incidence is equally distributed across the age spectrum, affecting 10-15% of women in all age groups. Here we report that increased immune dysfunction with age limits ICB efficacy in aged TNBC-bearing mice. The tumor microenvironment in both aged mice and TNBC patients shows decreased interferon (IFN) signaling and antigen presentation, suggesting failed innate immune activation with age. Triggering innate immune priming with a STING agonist restored response to ICB in aged mice. Our data implicate age-related immune dysfunction as a mechanism of ICB resistance in mice and suggest potential prognostic utility of assessing IFN-related genes in TNBC patients receiving ICB therapy. These data demonstrate for the first time that age determines the T cell inflamed phenotype in TNBC and impacts response to ICB in mice. Evaluating IFN-related genes from tumor genomic data may aid identification of patients for whom combination therapy including an IFN pathway activator with ICB may be required.

INTRODUCTION
Recent clinical successes using immune checkpoint blockade (ICB) have led to FDA- approval of antibody-based therapeutics that target T-cell inhibitory receptors, including cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD-1), and its 1igand PD-L1. These therapies have revolutionized cancer treatment, particularly for melanoma and non-small cell lung cancer (NSCLC) (1-5). Focus has recently shifted to applying ICB to other tumor types including breast cancer. Breast cancers that do not express hormone receptors (ER, PR) and lack amplification of Her2 are classified as triple-negative breast cancer (TNBC), which lacks targeted therapy and is the most aggressive subtype of the disease (6). Evaluation of ICB has been of greatest interest in TNBC due to higher numbers of tumor infiltrating lymphocytes and elevated PD-L1 expression – which correlate with response to ICB – relative to other breast cancer subtypes (7-10). ICB monotherapy has proven effective for ~10% of TNBC patients (11,12). The IMpassion130 trial recently reported that patients with metastatic TNBC whose tumors express PD-L1 significantly benefited from anti-PD-L1 (atezolizumab) when administered with the chemotherapy, nab-paclitaxel (13), leading to recent FDA approval of atezolizumab as first-line therapy for metastatic TNBC. ICB represents an improvement over standard therapeutic strategies for TNBC; however, the reasons why ICB therapy has had selected efficacy for TNBC patients (14) are unknown.

Age is associated with increasing immune dysfunction that includes significant changes to both innate and adaptive immunity (15-17). Most notably, hematopoiesis is skewed toward myeloid production while lymphopoiesis retracts with age. Some of the age-related changes specific to peripheral T-cell populations include fewer naïve T cells, increased numbers of terminally differentiated memory T cells, reduced expression of the co-stimulatory molecule, CD28, and increased exhaustion, evidenced by PD-1 expression (18). Antigen-presenting cells including dendritic cells (DCs) and macrophages also have decreased activity, while immunosuppressive populations, such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) are increased with age in cancer patients (19). While most aspects of innate and adaptive immunity are different between young and elderly individuals, the clinical implications of these age-related changes are unknown. Age-related immune dysfunction could therefore present particular challenges to efficacy of immunotherapy in breast cancer, as over 50 percent of patients are older than 60 years of age at diagnosis (20). However, most clinical studies either exclude or under-enroll older patients (21) and do not include tissue-based analyses that can help predict for efficacy. Given the limited number of older patients enrolled on clinical trials evaluating ICB in TNBC, it is not known whether age impacts response to therapy. We therefore initiated a study in murine models of TNBC to understand whether age-related immune dysfunction influences ICB efficacy in TNBC.

RESULTS
In order to identify murine models correlating with age-associated immune changes seen in humans, we first assessed age-associated differences in peripheral immune cells from young (8-10 weeks) and aged (>12 months) Balb/C and FVB mice. Lymphocytes comprised the majority of circulating white blood cells (WBCs) in both strains of mice, irrespective of age (Fig. 1A, B). However, total lymphocyte counts, including both CD4+ and CD8+ T cells, were significantly reduced in both strains of aged mice compared to young mice (Fig. 1A-D). Thecirculating myeloid cell compartment was significantly expanded in both strains with age (Fig. 1A, B). Within the myeloid compartment, neutrophil counts were elevated in both strains while monocyte counts were altered in a strain-specific manner (Fig 1A, B).Given that aged mice reflected the myeloid bias and decreased lymphocyte production observed in humans with age (22-24), we proceeded to test whether age also affects tumor progression and development of the tumor microenvironment (TME). To do so, we used 4T1 and Met1 transplantable orthotopic models of TNBC in Balb/C and FVB mice, respectively.In the Balb/C mice, 4T1 mammary carcinoma onset and incidence (100%) were similar between young and aged cohorts (Fig. 1E). Tumor growth kinetics were modestly increased in the aged cohort during the early stages of tumor development, but ultimately were similar in both young and aged cohorts by late stage disease (Fig. 1E, Suppl. Fig. 1A).

In the FVB mice, Met1 tumor onset and incidence (100%) were the same in both cohorts; however, tumor growth rates were significantly reduced in the aged mice (Fig. 1F, Suppl. Fig. 1B). Testing various other mammary carcinoma cells revealed similar strain-specific differences; 4T07 and 67NR tumors grew similarly in young and aged Balb/C mice and McNeuA tumors grew more slowly in aged than in young FVB mice (Suppl. Fig. 1C, D), suggesting that age-related host physiology may affect tumor growth.We next assessed tumor-related changes to the peripheral immune system with age. Relative to cancer-free animals, circulating lymphocytes were reduced in young tumor-bearing mice and this reduction was even more enhanced in aged tumor-bearing mice (Fig. 1G, H; Suppl. Fig. 1E). Young and aged tumor-bearing Balb/C and FVB mice exhibited neutrophilia, which was more pronounced in the aged cohort than in the young cohort (Fig. 1I, J; Suppl. Fig. F). In young Balb/C mice, we observed a reduction in circulating monocytes with tumorigenesis;however, in aged Balb/C mice, which already had lower monocyte counts, these levels were unchanged with tumorigenesis (Fig. 1K; Suppl Fig 1G). In the FVB mice, neither age nor tumorigenesis resulted in altered monocyte levels (Fig. 1L).We next examined the TME by immunohistochemistry. There were no significant age- dependent differences in the percentage of Ki67+ proliferating cells or myofibroblasts (α-SMA) in 4T1 or Met1 tumors (Suppl. Fig. 1H,I).

Macrophage (F4/80+) infiltration was significantly reduced in 4T1 tumors from aged Balb/C mice relative to young, while unchanged in Met1 tumors from FVB mice (Suppl. Fig. 1H,I). We observed an increase in the overall percentage of intratumoral neutrophils (MPO+) in 4T1 tumors compared to Met1 tumors (Suppl. Fig 1H, I), but no difference when comparing young and aged mice in the respective models. All mice also exhibited tumor-dependent neutrophilia in the blood (Suppl. Fig. 1H, I). Intratumoral infiltration of CD3+ and CD8+ T-cells was also unchanged with age in both strains of mice (Fig. 1M-P). Numbers of regulatory T cells (Tregs) were significantly reduced in 4T1 tumors from aged mice and increased in the aged Met1 tumor-bearing mice (Fig. 1M-P); nevertheless, these changes were not sufficient to significantly alter the intratumoral CD8:Treg ratio in either strain (Fig. 1M-P).The age- and tumor-dependent changes to peripheral immunity and the TME that we observed raised the question of whether age influences response to immunotherapy, particularly checkpoint blockade. We first verified that the 4T1 and Met1 cells express PD-L1 and significantly increased cell-surface PD-L1 and MHC I expression in response to interferongamma (IFNγ) (Suppl. Fig. 2A-D), indicating their potential to be recognized by immune cells (CD8+ T cells).We generated orthotopic 4T1 and Met1 TNBC in cohorts of young (8-10 weeks) and aged (>12 months) mice.

Once tumors reached ~5mm3 in volume, mice were randomized into treatment cohorts and administered four doses of anti-PD-L1, anti-CTLA-4, or isotype antibody control (Suppl. Fig. 2E-G).In four independent experiments in the Balb/C mice, only the young cohort exhibited significantly decreased 4T1 tumor growth after either anti-PD-L1 or anti-CTLA-4 treatment compared to isotype controls; neither treatment was effective in the aged mice (Fig. 2 A, B; Suppl. Fig. 2H). In separate experiments to measure survival, anti-PD-L1 and anti-CTLA-4 each improved overall survival of young mice relative to the young isotype controls; however, neither treatment affected survival of the aged mice (Fig. 2C; Suppl. Fig. 2I).We had earlier observed that Met1 tumor growth was attenuated in aged mice relative to young mice (Fig. 1F); therefore, we did not directly compare young and aged FVB cohorts to one another and instead, only made comparisons within each age cohort. In four independent experiments, anti-PD-L1 and anti-CTLA4 treatment significantly slowed the rate of Met1 tumor growth in young mice, although no complete regressions were observed (Fig. 2D, E; Suppl. Fig. 2J). In the aged mice from these experiments, anti-PD-L1 treatment resulted in a slight reduction in tumor growth, although this was not statistically significant, and the aged mice did not respond to anti CTLA-4 (Fig. 2F, G; Suppl. Fig. 2J).

In separate experiments to measure survival, anti-PD-L1 and anti-CTLA-4 treatment significantly improved overall survival in young mice (Fig. 2H; Suppl. Fig. 2K). Aged FVB mice gained significant overall survival benefit from anti-PD-L1 but not anti-CTLA-4 therapy (Fig. 2I; Suppl. Fig. 2K).To determine if adaptive immunity is necessary for response to ICB, we first orthotopically injected 4T1 or Met1 TNBC cells into young and aged nude mice, which lack mature T cells, and administered ICB according to our protocol (Suppl. Fig. 2E). Aged nude mice bearing 4T1 tumors did not respond to anti-PD-L1 treatment (Fig. 2J; Suppl. Fig. 2L). The young nude mice showed a statistically significant reduction in tumor volume with anti-PD-L1 treatment only at the experimental end point; however, this reduction was modest relative to immunocompetent mice (Fig. 2J; Suppl. Fig. 2L). Likewise, both young and aged cohorts of Met1 tumor-bearing nude mice failed to respond to anti-PD-L1 or anti-CTLA4 treatment (Suppl. Fig. 2M-O). By neutralizing CD8+ T cells in young Balb/C mice (Suppl. Fig. 2P, Q), we validated that CD8+ T cells were necessary for response to anti-PD-L1 (Fig. 2K; Suppl. Fig. 2R).Given that the adaptive immune response is necessary for effective ICB therapy, we characterized peripheral and tumor-infiltrating lymphocytes in 4T1 tumor-bearing mice. We focused efforts on the 4T1 model due to the fact that tumor growth was similar between young and aged untreated Balb/C mice, and 4T1 tumors demonstrated more T cell activity in general in the TME compared to Met1 tumors in FVB mice, which grew more slowly and showed very minimal T cell and myeloid cell infiltration in the TME (Fig. 1; Suppl. Fig 1).

Accordingly, we collected tumors from young and aged Balb/C mice 19 days after tumor cell injection, a time point at which significant differences in response to anti-PD-L1 and anti-CTLA-4 were manifest. We first analyzed age- and treatment-associated changes in T-cell phenotypes in peripheral blood cells by flow cytometry (Suppl. Fig. 3A). We focused on analysis of naïve T- cells (no prior antigen exposure), memory phenotypes (prior exposure to antigen), and cell-surface checkpoint protein expression (indicating active immune responses as well as loss of both proliferative potential and effector function). Age alone was associated with ~3-fold increased numbers of circulating effector memory (EM) CD8+ cells (CD45+/CD3+/CD8+/CD44+/CD62L-) and ~2-fold increases in CD8+/PD-1+ T-cells relative to young isotype-treated cohorts (Fig. 3A, Suppl. Fig. 3B). Anti-CTLA-4 treatment resulted in ~2- fold increased numbers of circulating naïve CD8+ T cells (CD45+/CD3+/CD8+/CD44-/CD62L+) and ~2-fold increases in PD-1+/CD8+ cells in the young cohort, but not in the aged cohort, (Fig. 3A, Suppl. Fig. 3B).We similarly analyzed spleen cells. Isotype-treated aged mice had ~4-fold higher numbers of splenic CD8+/PD-1+/TIM3+ T-cells, ~7-fold higher numbers of CD8+ EM cells, and ~4-fold higher numbers of PD-1+ EM cells than young mice (Fig. 3B, Suppl. Fig. 3C). In response to ICB, the numbers of PD1+ naïve T-cells (CD45+/CD3+/CD8+/CD44-/CD62L+/PD- 1+) were significantly reduced only in spleens of young mice (Fig. 3B, Suppl. Fig. 3C).

In response to anti-PD-L1, the numbers of CD3+/PD-1+/TIM3+ cells were enhanced ~2.5-fold in the aged mice but not in the young mice (Fig. 3B, Suppl. Fig. 3C).Collectively, our analysis of blood and spleen lymphocytes indicated that age is associated with enhanced CD8+ T-cell EM and exhaustion phenotypes, as previously reported in tumor-free aging models of immune dysfunction (25). Importantly, peripheral T-cell phenotypes with ICB treatment were reflective of active immune response in young but not aged mice.Given these results, we tested the proliferation capacity of CD8+ T cells from spleen and lymph nodes of young and aged mice. CD8+ T cell proliferation in response to CD3/CD28 stimulation ex vivo was not significantly different between cohorts (Fig. 3C). However, the number of checkpoint proteins (PD-1, CTLA4, LAG3 and/or TIM3) expressed per cell wassignificantly increased on CD8+ T cells from aged mice, but not young mice, after stimulation (Fig. 3D, E). Among the CD8+ populations from aged mice, PD1+/TIM3+, PD-1+/CTLA4+, and PD-1+/CTLA4+/TIM3+ cells underwent the greatest expansion in response to stimulation (Fig. 3F). The PD-1+/CTLA4+ CD8 cells from young mice expanded most significantly following stimulation (Fig. 3F). Collectively, these results demonstrated that CD8+ T cells from aged mice are capable of proliferation but also concurrently increase expression of checkpoint proteins more so than T cells from young mice in response to stimulation.We next characterized TIL populations (CD3+, FOXP3+ (Tregs), and CD8+ T cells) in 4T1 tumor sections by immunohistochemistry. Total numbers of CD3+/FOXP3+ Tregs were lower in aged mice and unchanged with treatment (Fig. 3G-I).

Moreover, CD8+ T cells in these tumor sections were not elevated with ICB treatment in the aged mice (Fig. 3J-K). These results translated to a significantly increased CD8+:Treg ratio in the aged mice (Fig. 3L), but when considered collectively, our results suggested this ratio was more a function of lower Treg numbers rather than a robust CD8+ T cell response. In contrast, both CD8+ T cells and Tregs increased with ICB therapy, particularly with anti-CTLA-4 treatment, in young mice (Fig. 3G- L).We therefore scored intratumoral (T), stromally restricted (S), and peripherally restricted(P) localization of CD8+ T cells in tumor sections from all cohorts and stratified sections into 5 groups: “cold” (no CD8+ cells), “restricted” (P or S only), “minor infiltration” (S or P > T), “medium” infiltration (T≈S), and “hot” (T > S or P). Tumors from both young and aged isotype- treated mice displayed mixed phenotypes by this scoring method and were not significantly different from one another (Fig. 3M). After ICB treatment, tumors from young mice were ~3- fold enriched for the “medium” phenotype, while “cold” and “minor” phenotypes disappeared(Fig. 3M). In sharp contrast, tumors from aged 4T1 tumor-bearing mice treated with anti-PD-L1 or anti-CTLA-4 failed to expand the “medium” phenotype and in fact, emergence of “cold” phenotypes lacking TILs became obvious (Fig. 3M).Further characterization of TILs revealed that PD1+/TIM3+ CD8+ T cells increased ~3- 5-fold with ICB treatment in young mice (Fig. 3N, Suppl. Fig. 3D).

While ICB treatment did not enhance the PD1+/TIM3+ CD8+ T cell population in aged mice, these cells were already ~7-fold elevated in tumors from aged isotype-treated mice relative to young isotype-treated mice (Fig. 3N, Suppl. Fig. 3D). PD-1+ TILs in 4T1 tumors from aged mice also demonstrated a memory- like phenotype particularly after ICB therapy (Fig. 3N, Suppl. Fig. 3D). Overall these data demonstrated that ICB therapy does very little to change the immune contexture of the TME in aged mice.To gain further insights into the tumor immune microenvironment at the molecular level that could explain lack of response to ICB therapy in aged mice, we performed whole tissue RNAseq on tumors from young and aged mice treated with isotype, anti-PD-L1, or anti-CTLA-4 (GSE130472). Multiple genes were significantly differentially expressed between young and aged cohorts in response to treatment and hierarchical clustering of these genes revealed strong associations based on age and treatment, indicating consistency of effect (Suppl. Fig. 4A, B; Suppl File 1). Gene Set Enrichment Analysis (GSEA) revealed that some of the enriched gene sets shared in common between young and aged mice included UV response and EMT with anti- PD-L1 (Suppl. Fig. 4C, D) and TNF signaling and EMT with anti-CTLA4 (Suppl. Fig. 4E, F).The most significantly enriched signatures in young mice treated with anti-PD-L1 or anti- CTLA4 compared to the isotype control cohorts included gene sets involved in IFN response and inflammatory response (Fig. 4A, B).

In contrast, these same signatures were not enriched in the tumors from aged mice with either ICB treatment (Fig. 4C, D). Moreover, when we stratified the young ICB treatment cohorts into responders and non-responders, IFN response, IFNα response, inflammatory response, and allograft rejection were significantly enriched in the responders (Fig. 4E, F, Suppl. Fig. 4G, H).We calculated 24 immune cell-related gene expression signatures via the nCounter Mouse PanCancer Immune Profiling Panel from Nanostring® and compared relative activity of each signature across all samples (see Methods). Tumors from young mice treated with either anti-PD-L1 or anti-CTLA4 were largely enriched for both innate and adaptive immune cell signatures compared to the young isotype treated mice (Fig. 4G). However, these immune signatures were not enriched in the aged mice treated with anti-PD-L1 or anti-CTLA4 compared to the respective isotype treated cohort (Fig. 4G).Intracellular flow cytometric analysis of CD8+ TILs were consistent with these findings. Specifically, tumors from aged mice had a ~9-fold reduction in CD8+/IFN+ cells than those from young mice, and numbers of these cells were not increased with ICB treatment as they were in young mice (Fig. 4H).

Moreover, CD8+ T cells in tumors from aged mice stored significantly more of the cytotoxic granule component, granzyme B (GrzB), in response to either ICB treatment, which was not evident in the young ICB treated mice (Fig. 4I).Given that IFN production and release of GrzB are critical for anti-tumor immune responses, our data indicated that ICB was less effective at triggering anti-tumor responses in aged mice. Moreover, our results established an important correlation between expression ofinflammatory response and IFN signaling pathways in the TME and the ability of TNBC tumors to respond to ICB therapy.We next asked whether age-dependent differences in critical ICB response pathways would be evident even prior to treatment. Principal component analysis (PCA) of the RNAseq data from murine tumors confirmed that tumors from young and aged isotype treated mice formed distinct clusters that stratified with age (Fig. 5A). Strikingly, GSEA revealed that the tumors from young isotype-treated mice were enriched for IFN, IFN, and inflammatory response genes relative to those from aged isotype-treated mice (Fig. 5B). We also applied a previously reported IFN pathway signature that defined response to anti-CTLA4 in melanoma patients (26). Again, we found that tumors from young isotype-treated mice were enriched for this IFN response pathway compared to tumors from aged mice (Fig. 5B).Processing and presentation of tumor antigens is also critical for initiating anti-tumor immune responses. Therefore, we also examined genes associated with antigen processing and presentation and found significant differences in expression of a number of these genes between young and aged isotype-treated mice (Fig. 5C).

Together, these results established that the critical mediators of response to ICB therapy could be prospectively identified in tumor gene expression data even prior to treatment. We therefore interrogated the METABRIC database for TNBC patients and stratified them into two groups based on age at initial diagnosis: ≤40 and ≥65 years of age. Two distinct clusters of themost differentially expressed genes were observed in patients ≤40 whereas these genes did not cluster in the patients ≥65 (Suppl. Fig. 5).In patients ≤40, IFN response, IFN response, inflammatory response, allograft rejection, IL6-JAK-STAT3 signaling, and IL2-STAT5 signaling gene sets were the most highly enriched relative to those ≥65 (Fig. 5D), similar to our pre-clinical observations. To further understand which immune cell subtypes may be associated with these changes, we applied CIBERSORT to characterize 22 inferred immune subsets for each patient (27). In patients ≥65 relative to those ≤40, M1 macrophage and plasma cell signatures were significantly reduced while activated NK cells and M0 macrophage signatures were significantly enriched (Fig. 5E).Taken together, these results established that the immune contexture of the TME in TNBC is significantly different between young and older patients, suggesting that age-related immune changes may lead to reduced efficacy of ICB in older individuals.Our results raised the question of whether stimulation of IFN signaling, which was enriched in young mice but lacking in aged mice, is sufficient to trigger response to ICB in aged mice.

In particular, Type I interferons (IFN and IFN) play an important role in T-cell priming and our results suggested that failed innate immune priming results in the lack of response to ICB in aged mice. Activation of the stimulator of interferon genes (STING) pathway promotes the transcription of Type I IFNs and has previously been reported to promote anti-tumor immunity in other preclinical models (28). To enhance IFN signaling, we used intratumoral injection of the mouse-specific STING agonist, 5,6-Dimethylxanthenone-4-acetic acid (DMXAA).We first tested DMXAA monotherapy and combination with either anti-PD-L1 or anti- CTLA4 in aged Balb/C mice with 4T1 TNBC, according to our protocol (Suppl. Fig. 6A-D). As observed repeatedly, neither anti-CTLA-4 nor anti-PD-L1 monotherapy was efficacious in aged mice (Fig. 6A-F; Suppl. Fig. 6E-J). DMXAA monotherapy significantly slowed tumor growth only during the course of treatment (Fig. 6A, B, D, E; Suppl. Fig. 6E-H) but did not significantly improve overall survival (Fig. 6C, F). Importantly, both DMXAA + anti-PD-L1 and DMXAA + anti-CTLA4 combination therapies significantly reduced 4T1 tumor growth, resulting in significantly improved overall survival of aged mice (Fig. 6A-F; Suppl. Fig. 6E-H).Interestingly, in 4 independent experiments in young mice, DMXAA monotherapy did not alter tumor growth relative to the isotype control cohort (Fig. 6G, Suppl. Fig. 6I, K). Anti- CTLA-4 and the combination DMXAA + anti-CTLA-4 significantly reduced tumor growth to the same degree in the young mice, suggesting there is no additive effect of DMXAA in young mice (Fig. 6G, Suppl. Fig. 6J, L).To ensure that DMXAA treatment was affecting Type I IFN expression, we performed qPCR on whole tumors one day following intratumoral injection of DMXAA (Suppl. Fig. 6M,N). Importantly, expression of both IFNα and IFNβ were significantly enhanced after DMXAA injection in both young and aged mice, with no age-associated differences in relative expression observed (Fig. 6H, I). Together, these results established that response to ICB in aged mice with TNBC is rescued by stimulating Type I IFNs within the tumor microenvironment.

DISCUSSION
The present work revealed a critical role for physiological aging in reducing ICB efficacy in murine models of TNBC. Tumors from young mice with TNBC and TNBC patients <40 years display enrichment of genes associated with antigen presentation, inflammation, and IFN response pathways, suggesting efficient innate priming of CD8+ T-cells (Fig. 7, top left). In young mice, these pathways are further enriched with ICB therapy and together with increased infiltration of CD8+ T-cells, results in a robust anti-tumor response (Fig. 7, top right). Tumors from aged mice and TNBC patients >65 years lack enrichment for genes associated with antigen presentation, inflammation, or IFN response pathways, suggesting that T-cell priming by antigen-presenting cells is not as efficient as in young mice (Fig. 7, bottom left). Aging, also results in an immunologically cold tumor microenvironment, defined by peripherally restricted TILs that have increased checkpoint protein expression (Fig. 7, middle). The age-dependent tumor microenvironment fails to generate an anti-tumor response to ICB (Fig 7, bottom right). Stimulating Type I interferon signaling with a STING agonist (DMXAA) promotes response to ICB in the aged mice (Fig. 7, bottom right).

To date, only a handful of studies have investigated age-related immune dysfunction as a potential mechanism for resistance to ICB. In advanced NSCLC, where anti-PD-1 antibodies are now standard-of-care for patients who progress on or after platinum-based chemotherapy, patients > 75 years old demonstrated less benefit after nivolumab therapy compared to younger patients, however overall survival (OS) and toxicity were not different (29-32). One meta- analysis of data from over 5000 patients treated with ICB found no difference in OS between young and older patients in multiple cancer types (33). Another meta-analysis with greater representation of patients over 65 years of age also showed comparable efficacy of anti-PD-1 or anti-PD-L1 therapy in young and older patients (34). In addition, comparative benefit and toxicity analyses have only shown modestly higher rates of treatment discontinuation and immune-related adverse events in melanoma patients older than 80 years of age when treated with ICB (35). Conversely, another recent study showed increased rates of response to ICB in older melanoma patients (36). It is worth noting that most of the studies of age and response to ICB, be it patient data or mouse models, have been done in NSCLC or melanoma. These cancer types generally have a higher mutational load and increased immunogenicity compared to breast cancer (37), which, among other things, makes them more susceptible to ICB, which is now part of standard-of-care therapy in these cancers. In fact, it is now well accepted that tissue-specific differences in the immune microenvironment critically affect cancer progression and therapeutic responses (38). Likewise, mouse strain and life span, tumor models, complexity of the immune microenvironment, and specific ICB therapy are important considerations when drawing conclusions from pre-clinical studies.

For example, decreased numbers of tumor-infiltrating T- regulatory cells (Tregs) were associated with improved response to anti-PD-1 therapy in ten- month old C57Bl/6 mice in a recent melanoma study (36). However, decreased Tregs in the 4T1 tumors from aged (>12 months) compared to young Balb/C mice did not lead to improved responses to anti-PD-L1 or anti-CTLA4 in our study. In fact, decreased Tregs with age appeared to be a function of a “cold” immune environment in our models, which is supported by our observation of reduced IFN signatures with age. Importantly, the young mice bearing either 4T1 or Met1 tumors responded to ICB unlike young mice in the melanoma study (36). Conversely, the 4T1 tumor model results in neutrophilia in Balb/C mice, which can also limit the effects of ICB in general, although we observed that intratumoral neutrophils did not change in young compared to aged mice, while response to ICB did. Therefore while neutrophilia may contribute to the overall limited immunogenicity of the 4T1 model, it does not explain the age-specific response to ICB we observed, or that intratumoral stimulation of Type I IFNs could recover responses to ICB in aged mice. Clearly however, the caveats to pre-clinical models highlight the need for more detailed clinical data from ICB trials in different cancers. The application of ICB to TNBC is still under active investigation, with atezolizumab in combination with abraxane recently receiving FDA-approval for first-line treatment of metastatic TNBC (13). ICB monotherapy in breast cancer shows response rates of only 5-10% in unselected cohorts (12,39,40).

In selected cohorts of patients with PD-L1+ metastatic TNBC in the Phase II KEYNOTE 086 trial, pembrolizumab as a first-line therapy showed an objective response rate (ORR) of 21.4% (41). While not significant, the authors note the ORR was numerically higher in patients who were >65 or postmenopausal. This difference was not as apparent when a cut-off of >50 was used. Age stratification in the Impassion130 trial showed that both young (18-40 years) and older (>65 years) women performed better with combination therapy (18-40: 3.7 vs 3.6 months; ≥65: 9.1 vs 6.2 months) (13). However, as with nearly all breast cancer clinical trials, young and older women were underrepresented (12.6% and 24% respectively) in this trial. TNBC also presents unique challenges to ICB therapy due to age-associated differences in disease progression as well as the composition of the tumor microenvironment (42), as we, and others, have reported (43). Correlative studies from TNBC trials of ICB that include longitudinal molecular and histopathological assessment have not yet been reported. Therefore, the baseline immune phenotype of tumors from the reported studies is unknown, making interpretation of their results difficult in the context of our data. Interpretation of these types of analyses are further complicated by the fact that we do not actually know where the appropriate cut-off for “old” is, therefore grouping all patients above or below a certain age may mask any actual age-specific effects. In fact, a recent study demonstrated considerable differences in the rate of immune aging between healthy individuals (24)(24)(24)(24) supporting the notion that immune age does not necessarily reflect chronological age, and that in fact immunological age may be a better predictor of response to immunotherapy.

The age-stratified responses to ICB that we observed in our TNBC mouse models appear to be due to defective innate immune priming with age, as tumors from aged mice (without therapy) showed overall decreased antigen processing and presentation as well as decreased IFN and IFN signaling, resulting in an overall “cold” TME that was not responsive to ICB monotherapy. Only the addition of the STING agonist, DMXAA, which drives Type I IFN signaling, showed efficacy in combination with ICB in aged mice. Our parallel findings in tumors from older TNBC patients in the METABRIC cohort, suggest that older TNBC patients exhibit a “cold” TME characterized by poor T cell infiltrate and a lack of IFN signaling. To the best of our knowledge, these data demonstrate for the first time that age is a defining factor in determining the T cell inflamed phenotype in TNBC and could be an important determinant of ICB therapy response. Intratumoral expression of IFN pathway genes is closely associated with response to ICB in the clinic. Intratumoral loss of IFN signaling is associated with primary resistance to anti- CTLA4 therapy (26), while a T cell-inflamed TME characterized by active IFN signaling and antigen presentation, is a common feature associated with tumors that are responsive to anti-PD- 1 (44). In line with these studies, our data suggest that analysis of IFN-related genes taken from tumor genomic data may provide prognostic utility for patients selected to receive ICB monotherapy or potentially ICB therapy in combination with an IFN pathway activator, such as a STING agonist, to generate a T cell response in “cold” tumors.

Clinical trials utilizing intratumoral injection of synthetic CDN STING agonists alone or in combination with anti-PD-1 are now underway. One of these trials (NCT03010176) using the STING agonist MK-1454 also includes TNBC patients and early reports suggest the combination with pembrolizumab is more effective than MK-1454 alone, which lacked efficacy. Likewise, our study indicated that the mouse STING agonist, DMXAA, as monotherapy did not impact tumor growth in young mice, but significantly improved response to ICB in aged mice. Given these results, one might speculate that efficacy of STING agonists stratifies with age or that patients whose tumors lack enrichment for IFN signatures would benefit from these therapies. Nevertheless, the implications of our findings for TNBC patients whose tumors reflect the biology we observed has yet to be determined. A critical aspect of effective T cell responses is antigen presentation, primarily via professional antigen presenting cells, including macrophages and dendritic cells (DCs). Macrophages have been shown to exhibit decreased antigen presentation and MHC Class II expression with age (45). Notably, we observed decreased expression of antigen processing and presentation genes in tumors from aged mice. In line with these observations, we provide gene expression-based evidence of reduced tumor-infiltrating M1 macrophages in older TNBC patients in our analysis of the METABRIC cohort, and in TNBC tumors from aged mice compared to young. The ability of DCs to prime T cells (among other functions) has also been reported to decrease with age (46). This aspect of tumor immunology remains to be further investigated in our models.

While ICB therapy presents an emerging treatment option for older TNBC patients, and older cancer patients in general in terms of efficacy and safety compared to chemotherapy, how the aging immune system responds in cancer and more specifically, after ICB treatment, remains poorly understood. Immune-related adverse events and ICB-related toxicities may also worsen with age, but the lack of elderly patients enrolled in clinical trials hinders progress in this area. With an aging global population predicted to rise from 10% in 2000 to over 22% by 2050 (47), this is an increasingly pressing concern. Building on the preclinical results and data presented here, it may be possible to stratify TNBC patients into those requiring combination therapy with an immune stimulatory agent such as a STING agonist using tumor genomic data for IFN pathway genes, and therefore make ICB therapy applicable to TNBC patients of all ages. Female Balb/C CyJ, FVB/NJ, C57Bl/6 and NCR-NU (nude) mice were purchased from Jackson Labs. Balb/C mice were aged in house or provided by the National Institute of Aging (NIA) at 12 months of age. FVB/NJ and NCR-NU mice were aged in house. For all experiments, young mice were defined as 8-12 weeks of age; aged mice >12 months of age.

All experiments were conducted in accordance with regulations of the Children’s Hospital Institutional Animal Care and Use Committee (protocol # 12-11-2308R) and the Brigham and Women’s Hospital (protocol # 2017N000056) 4T1 tumor cells were provided by F.Miller and maintained as previously described(48). The Met1 TNBC cell line was a gift from J.Joyce with permission from A.Borowsky and maintained as previously described(49). The McNeuA cell line was provided by Michael Campbell and maintained as previously described(50). The 4T07 and 67NR cell lines were provided by Robert Weinberg and maintained as previously described(51). Met1 cells were generated from a spontaneously arising tumor in an FVB/N-Tg (MMTV-PyVmT) mouse(49) and 4T1 cells are a variant line from a single spontaneously arising mammary tumor from a BALB/cfC3H mouse(52). All cells were routinely tested for mycoplasma and confirmed negative when used in experiments. For IFN stimulation to assess PD-L1 and MHC I expression, Vadimezan tumor cells were incubated for 24 hours with 1ng/ml recombinant mouse IFN (BioLegend, 575304) and expression of cell surface receptors analyzed by flow cytometry.