Lymph nodes (LNs) are highly organized secondary lymphoid organs, and reflective of immune responses to infection, injuries, or the presence of cancer. Extensive molecular and morphological analyses of immune and stromal features in tumors and LNs of breast cancer patients have revealed novel patterns indicative of disease progression. Within LNs, there are dynamic structures called germinal centers (GCs), that act as the immunological hubs for B cell development and generation of affinity matured memory B and antibody-producing plasma cells. Acting as a bridge between systemic and local immunity, associations are observed between the frequency of GCs within cancer-free LNs, the levels of stromal tumor infiltrating lymphocytes, and cancer progression. Scattered throughout the tumor microenvironment (TME) or aggregated in clusters forming tertiary lymphoid structures (TLS), the occurrence of tumor infiltrating B cells (TIL-Bs) has been linked mostly to superior disease trajectories in solid cancers. Recent TIL-Bs profiling studies have revealed a plethora of different TIL-B populations, their functional roles, and whether they are derived from GC reactions in the LN, and/or locally from GC-like structures within the TME remains to be investigated. However, parallels between the immunogenic nature of LNs as a pre-metastatic niche, TIL-B populations within the TME, and the presence of TLS will help to decipher local and widespread TIL-Bs responses and their influence on cancer progression to the lymphatics. Therapies that enhance TIL-Bs responses in the LN GC and/or in GC-like structures in the TME are thus emerging management strategies for breast and other cancer patients.

During the anti-tumour response to breast cancer, the primary tumour, the peripheral blood, and the lymph nodes each play unique roles. Immunological features at each site reveal evidence of continuous immune cross-talk between them before, during and after treatment. As such, immune responses to breast cancer are found to be highly dynamic and truly systemic, integrating three distinct immune sites, complex cell-migration highways, as well as the temporal dimension of disease progression and treatment. In this review, we provide a connective summary of the dynamic immune environment triad of breast cancer. It is critical that future studies seek to establish dynamic immune profiles, constituting multiple sites, that capture the systemic immune response to breast cancer and define patient-selection parameters resulting in more significant overall responses and survival rates for breast cancer patients.

Introduction: Revlimid (Rev), binds to CRL4 CRBN E3 ligase leading to recruitment and proteasomal degradation of transcription factors Aiolos and Ikaros. This inhibits proliferation of malignant B cells and stimulates activity of T, NK and macrophage cells, thereby providing clinical activity of Rev as a single agent and in combination with CD19/CD20 antibodies in DLBCL and FL. Iberdomide (Iber), a new CELMoD with enhanced substrate degradation compared to Rev, is currently being studied in clinical trials for B-NHL and MM. Presented here is extensive in vitro and in vivo characterization of immune enhancement and antitumor effects of Iber with direct comparison to Rev. Results: In a panel of DLBCL cell lines, comprising ABC and GCB-DLBCL models, Iber degraded Aiolos/Ikaros with faster kinetics and to a greater depth than Rev, which led to enhanced antiproliferative and cytotoxic effects. Iber acted in a cell of origin independent manner, whereas Rev is preferentially active in ABC-DLBCL. To examine the molecular effects of Iber and Rev in immune cells, we performed RNAseq and proteomic based analyses on Iber and Rev treated T, NK and monocyte cell populations. These experiments revealed a complex series of immunomodulatory activities including promotion of pro-inflammatory cytokine production, activation marker expressions and migratory machinery with a trend of Iber exhibiting greater enhancements. We confirmed these findings by demonstrating that secretion of chemoattractants for T cells, NK cells and monocytes including CXCL9, 10 and 11 (10-90% increase) and CCL8 (30% increase, p<0.01) were higher in PBMCs treated with Iber compared to Rev. Additionally, functional chemotaxis assays demonstrated that Iber and Rev increased the trafficking capacity of T-cells compared to DMSO alone, with Iber demonstrating a greater increase than Rev (46% vs 21%, p<0.01). Furthermore, Iber increased the proliferative capacity of CD8+ T and NK cells compared to Rev (10 and 3.6-fold vs 4 and 2.8-fold, respectively). Functional co-culture assays with DLBCL cells showed that Iber induced NK cell mediated killing of DLBCL cells to a greater extent than Rev and each molecule enhanced ADCC with Rituximab compared to vehicle controls. Translational data from clinical trials of a related CELMoD, Avadomide, revealed significant trafficking of immune cells such a T cells, NK cells and monocytes to the tumor microenvironment (TME). To examine the effects of Rev and Iber in an in vivo DLBCL GEMM model, we developed a humanized CRBN (hCRBN) mouse capable of facilitating proteasomal degradation of target substrates upon treatment with a CELMoD. The hCRBN mouse was then crossed with the Eμ-Myc DLBCL mouse model resulting in Eμ-Myc/hCRBN progeny that then developed disease. Splenocytes were collected and transplanted to recipient hCRBN mice. The tumor cells were allowed to engraft for 5 days upon which 3 daily doses of vehicle, Rev and Iber were given prior to the mice being sacrificed. Non-transplanted hCRBN mice served as controls. Similar to human disease, DLBCL cells remodeled the myofibroblast-immune network within lymph node and the splenic tissues including activated podoplanin (PDPN)-expressing fibroblastic reticular cells (FRCs) and diminished CD8+ T cells and CD11c+ DCs within the lymphoid TMEs. Treatment with Iber resulted in significantly enhanced infiltration of DCs and notably, cytolytic granzyme B positive T cells into the TME compared to Rev or vehicle treated mice (Figure 1). Additional characterization of the immune (T cell, NK and monocyte)-stroma TME is on-going and will be presented. Conclusion: Our data demonstrate that Iber is more potent in substrate degradation and functionality in anti-proliferative activity against DLBCL cell line models and at triggering immunostimulatory activities in multiple lymphoid and myeloid populations. Additionally, we generated a humanized CRBN mouse model that revealed the ability of CELMoDs in inducing immune-rich TMEs supporting rational combination strategies with immune focused agents being explored in lymphoma such as SIRPα blockade, CAR T and CD3xCD20 bispecifics. Figure 1 Figure 1. Disclosures Nakayama: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Narla: Bristol Myers Squibb: Current Employment. Shakya: Bristol Myers Squibb: Current Employment. Gamez: Bristol Myers Squibb: Current Employment. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Gandhi: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.