Divergent cancer etiologies drive distinct B cell signatures and tertiary lymphoid structures

Abstract

26Current immunotherapy paradigms aim to reinvigorate CD8+ T cells, but the contribution 27 of humoral immunity to antitumor immunity remains understudied1,2. Head and neck 28 squamous cell carcinoma (HNSCC) is caused by either human papillomavirus (HPV+) or 29 environmental carcinogens (i.e. tobacco and alcohol; HPV-)3,4. Here, we demonstrate that 30 HPV+ HNSCC patients have transcriptional signatures of germinal center (GC) tumor 31 infiltrating B cells (TIL-Bs) and spatial organization of immune cells consistent with GC-32 like tertiary lymphoid structures (TLS), both of which correlate with favorable outcomes in 33 HNSCC patients. Further, our single-cell RNAseq data also indicate that GC TIL-Bs are 34 characterized by distinct waves of gene expression consistent with dark zone, light zone 35 and a transitional state of GC B cells. High-dimensional spectral flow cytometry permitted 36 in depth characterization of activated, memory and GC TIL-Bs. Further, single cell RNAseq 37 analysis and subsequent protein validation identified a role for semaphorin 4a (Sema4a) 38 in the differentiation of GC TIL-Bs and indicated that expression of Sema4a was enhanced 39 on GC TIL-Bs and within GC-like TLS in the TME. Thus, in contrast to some reports on the 40 detrimental role of TIL-Bs in human tumors, our findings suggest that TIL-Bs play an 41 instrumental role in antitumor immunity5,6. Novel therapeutics to enhance TIL-B responses 42 in HNSCC should be prioritized as a compliment to current T-cell mediated 43 immunotherapies. 65 organization that form outside of secondary lymphoid organs (SLOs) in response to chronic 66 inflammation or infection20,21. TLS are characterized by organization patterns similar to SLOs with 67 defined T cell zones, B cell rich follicles and mature dendritic cells (DCs)22,23. TLS have been 68 shown to also correlate with increased patient survival in many human tumors24,25. Recent studies 69 have demonstrated that the presence of B cells and TLS in melanoma, renal cell carcinoma, 70 sarcoma, and HNSCC are associated with better responses to immune checkpoint blockade 71 Ruffin et al. Cancer etiologies drive B cells and TLS 4 (ICB)10,11,26,27. However, TLS are quite heterogeneous structures28, and the composition of TIL-Bs 72 within these structures has not been fully elucidated. Characterization of TLS in the TME, 73 including their composition, spatial organization, maturity, and phenotypes of immune cells 74 involved would provide critical insight into the roles these structures play in antitumor immunity. 75 Additionally, understanding the factors that drive formation of TLS within the TME would permit 76 the identification of therapeutic avenues to foster an influx of TIL-Bs into the proper spatial 77 organization. 78 One feature associated with mature TLS is the formation and presence of germinal centers 79 (GCs)29. GCs are typically found in SLOs and are responsible for producing affinity matured and 80 class switched B cells that effectively recognize their cognate antigen, leading to m...

Neoantigen driven B cell and CD4+T follicular helper cell collaboration promotes robust anti-tumor CD8+T cell responses

“…We found tumor-infiltrating TFH cells and GC B cells in our analyses of human LUAD and our autochthonous KP model. The former is in line with the observations that NSCLC and other cancer types contain tumor-associated TFH-like cells with an enrichment of a TFH signature (i.e., PDCD1, ICOS, CD200, MAF and IL21) (Bindea et al, 2013;Cillo et al, 2020;Gu-Trantien et al, 2013;Gu-Trantien et al, 2017;Guo et al, 2018;Li et al, 2019;Satpathy et al, 2019;Thommen et al, 2018). In human studies including autoimmune diseases, TFH-like cells have been shown to express CXCL13, the ligand of CXCR5, which was associated with CXCR5 + B cell infiltration and tertiary lymphoid structure (TLS) formation (Bocharnikov et al, 2019;Li et al, 2015;Manzo et al, 2008;Morita et al, 2011;Rao et al, 2017).…”

“…To examine whether GC B cells and TFH cells associate with clinical outcomes of LUAD patients, we established gene expression signatures for GC B cells and TFH cells based on previous human studies in lymphoid tissues and cancers (Cabrita et al, 2020;Cillo et al, 2020;Milpied et al, 2018;Vella et al, 2019), and refined them based on the data in Figures 1D -1E to reflect the gene expression of GC B cells and TFH cells in LUAD. Analysis of 478 LUAD cases from TCGA showed significant correlations between prolonged overall survival in LUAD patients and GC B cell-or TFH-signatures ( Figure 1F and 1G, respectively).…”

“…Tumor-associated TLSs were observed in many types of cancers, including up to 70% of NSCLC patients, and their presence correlated with favorable clinical outcomes (Dieu-Nosjean et al, 2008;Germain et al, 2014;Goc et al, 2014;Sautes-Fridman et al, 2016;Sautes-Fridman et al, 2019). Moreover, TLSs with mature GCs were further associated with more clinical benefits for lung cancer and HPV + HNSCC patients (Ruffin et al, 2020;Silina et al, 2018). Thus, the follicular structures of TLSs could support interactions between IL-21producing TFH cells and CXCR5 + CD8 + T cells in tumors.…”

“…A growing body of evidence has highlighted the potential importance of CD4 + T helper (Th) cells and B cells in cancer and immunotherapy (Alspach et al, 2019;Borst et al, 2018;Ferris et al, 2020;Helmink et al, 2020;Tran et al, 2014;Wieland et al, 2020). Much of the mechanistic research on CD4 + T cells has focused on Th1, Th2 and Th17 cells (Galon et al, 2013;Goc et al, 2014;Guo et al, 2018;Liu et al, 2020;Martin-Orozco et al, 2009;Oh et al, 2020), and relatively little is known about the functional role of TFH cells in cancer. In non-cancer contexts, TFH cells provide necessary help for B cell maturation and function.…”

“…In a wide range of human cancers, the presence of B cells and TFH cells is correlated with prolonged survival and favorable therapeutic responses, including in NSCLC (Germain et al, 2014), breast cancer (Garaud et al, 2019;Gu-Trantien et al, 2013;Gu-Trantien et al, 2017;Hollern et al, 2019;Lu et al, 2020), melanoma (Cabrita et al, 2020Griss et al, 2019;Helmink et al, 2020), sarcoma (Petitprez et al, 2020), head and neck cancer (Cillo et al, 2020), colorectal cancer (Bindea et al, 2013), gastric cancer (Hennequin et al, 2016) and ovarian cancer (Kroeger et al, 2016;Truxova et al, 2018). This correlation is especially strong when TFH cells and B cells are located in tumor-associated tertiary lymphoid structures (TLSs), as these structures could facilitate TFH cell-B cell interactions with subsequent effector functions in tumors (Ruffin et al, 2020;Sautes-Fridman et al, 2016;Sautes-Fridman et al, 2019;Sharonov et al, 2020). Yet, the functional importance of tumor-specific TFH cells remains uncertain, as does the importance of their interactions with B cells.…”