Abstract SY16-02: Generation of B cell immunity in tertiary lymphoid structures supports response to immunotherapy
Authors: Fridman Wolf Herman , Meylan Maxime , Vano Yann , Pupier Guilhem , Bougouin Antoine , Calvez Anne , Petitprez Florent , Sun Cheng Ming , Mathieu Margot , Vanhersecke Lucile , Loarer François Le , Bessede Alban , Oudard Stephane , Italiano Antoine , Sautès-Fridman Catherine 
Published: April 2023

Abstract
The presence of tertiary lymphoid structures (TLSs) and a high intratumoral density of B lymphocytes have been associated with improved survival in several tumor types. TLSs are heterogenous organized lymphoid aggregates, their mature form containing a germinal center with follicular B cells juxtaposing CD4+ follicular helper T cells and intricated with CD21+CD23+ follicular dendritic cells. We investigated the impact of TLS on responses to immunotherapy.
By performing a large-scale retrospective analysis of three independent cohorts of patients with different cancer types (including soft-tissue sarcomas, lung, bladder, colorectal, head and neck, breast cancer and others, n &gt; 500) treated with anti-PD-1 or anti-PD-L1 antibodies, we showed that the presence of mature TLSs was associated with higher objective response rates (ORR), progression-free survival (PFS) and overall survival (OS), independently of PD-L1 expression status and CD8+ T cell density.
To decipher the role of B cells in supporting responses to immunotherapy by immune check-point blockers and patients’ survival, we focused on two cancers: clear cell Renal Cell Carcinoma (ccRCC) and Soft Tissue Sarcoma (STS):The randomized phase II BIONIKK trial (NCT02960906) has recently shown that molecular grouping of tumors enabled enhanced response rate to Nivolumab (N), Nivolumab + Ipilimumab (NI) and VEGFR-Tyrosine Kinase Inhibitor (TKI) in patients with frontline metastatic RCC (mRCC). 199 pts were randomized and treated with N (40 pts), NI (101pts) and TKI (58pts). After a median follow-up of 46.5 months, 86 (43%) patients died: 27/58 (46.5%), 39/101 (39%) and 20/40 (50%) in the N, NI, and TKI arm, respectively. Median OS was 35 months with N, not reached with NI and 45 months with TKI. 160/199 mRCC patients had available FFPE samples (N: 42, NI: 84, TKI: 34). In N-treated patients, number of TLS&gt;2 was associated with higher ORR (73% versus 15%, p=0.01), longer PFS (p=0.015) and less upfront progressions (no progression at 6 weeks, p=0.02). In patients treated with NI, TLS&gt;2 correlated with higher ORR (71% versus 40%, p=0.02), fewer events of early progressions (no progression at 6 months) (p=0.04) and a high TLS transcriptomic signature correlated with higher ORR (79%) versus 25% in TKI-treated patients. Analyzing Immunoglobulin (Ig) gene expression and repertoire, we showed that TLS+ tumors depicted higher numbers of IgM, IgG1 and IgA unique clonotypes and a higher number of over-represented clones found more than 30 times in the same tumor. We also performed spatial transcriptomics and examined the nature of B cell responses within TLS structures. B cells were enriched inside TLS, and therein we could identify all B cell maturation stages from naïve B cells to plasma cells (PCs) formation. B cell repertoire analysis revealed clonal diversification, selection, expansion in TLS and the presence of fully mature clonotypes at distance. In TLS+ tumors, IgG producing PCs disseminated into the tumor beds along fibroblastic tracks. TLS+ tumors exhibited high frequencies of IgG-producing PCs and high numbers of IgG-stained and apoptotic malignant cells, suggestive of anti-tumor effector activity. Proximity analyses revealed the presence of activated macrophages located close to IgG-coated cleaved caspase 3 positive tumor cells, reflecting activation of antibody-dependent cell mediated cytotoxicity. Patients with high numbers of IgG-stained tumor cells (above 60%) exhibited higher ORR (62% versus 18%, p=0.03) and PFS (17 versus 6 months, p=0.03) when treated with NI.PEMBROSARC is a multi-cohort phase II study of pembrolizumab combined with low-dose cyclophosphamide in patients with advanced STS. The 6-months non-progression rate (NPR) and the ORR previously reported in cohorts including all comers were 4.9% and 2.4%, respectively. In the cohort enrolling patients selected based on the presence of TLS, the 6-month NPR was 40% and the ORR was 30%. Exploratory analyses revealed that infiltration by mature dendritic cells, activated macrophages and IgG -producing PCs was associated with improved clinical outcome. the Presence of IgG antibodies on tumor cells was documented in some cases.
Altogether, these data establish that maturation, selection and amplification of B cells in intratumoral TLSs results in PC generation which may produce anti-tumor antibodies. Macrophage-dependent killing of tumor cells results in the release of immune complexes of IgG with tumor-associated antigens; the latter are endocytosed by dendritic cells that present tumor-specific and self-antigens to T cells in a very efficient way, amplifying “in situ” antitumor immune responses and lowering the threshold for T lymphocytes reinvigoration by anti-checkpoint inhibitors. It represents an amplification loop that may reinforce therapeutic responses to checkpoint inhibitors, particularly in tumors with low mutational burden. Mechanisms underlying B cell maturation, plasma cell generation and antibody production and their regulation in TLS will be discussed.
Citation Format: Wolf Herman Fridman, Maxime Meylan, Yann Vano, Guilhem Pupier, Antoine Bougouin, Anne Calvez, Florent Petitprez, Cheng Ming Sun, Margot Mathieu, Lucile Vanhersecke, François Le Loarer, Alban Bessede, Stephane Oudard, Antoine Italiano, Catherine Sautès-Fridman. Generation of B cell immunity in tertiary lymphoid structures supports response to immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr SY16-02.