VISTA drives macrophages towards a pro-tumoral phenotype that promotes cancer cell phagocytosis yet down-regulates T cell responses.

BACKGROUND: VISTA is a well-known immune checkpoint in T cell biology, but its role in innate immunity is less established. Here, we investigated the role of VISTA on anticancer macrophage immunity, with a focus on phagocytosis, macrophage polarization and concomitant T cell activation. METHODS: Macrophages, differentiated from VISTA overexpressed THP-1 cells and cord blood CD34+ cell-derived monocytes, were used in phagocytosis assay using B lymphoma target cells opsonized with Rituximab. PBMC-derived macrophages were used to assess the correlation between phagocytosis and VISTA expression. qRT-PCR, flow cytometry, and enzyme-linked immunosorbent assay were performed to analyze the impact of VISTA on other checkpoints and M1/M2-like macrophage biology. Additionally, flow cytometry was used to assess the frequency of CD14+ monocytes expressing VISTA in PBMCs from 65 lymphoma patients and 37 healthy donors. RESULTS: Ectopic expression of VISTA in the monocytic model cell line THP-1 or in primary monocytes triggered differentiation towards the macrophage lineage, with a marked increase in M2-like macrophage-related gene expression and decrease in M1-like macrophage-related gene expression. VISTA expression in THP-1 and monocyte-derived macrophages strongly downregulated expression of SIRPα, a prominent 'don't eat me' signal, and augmented phagocytic activity of macrophages against cancer cells. Intriguingly, expression of VISTA's extracellular domain alone sufficed to trigger phagocytosis in ∼ 50% of cell lines, with those cell lines also directly binding to recombinant human VISTA, indicating ligand-dependent and -independent mechanisms. Endogenous VISTA expression was predominantly higher in M2-like macrophages compared to M0- or M1-like macrophages, with a positive correlation observed between VISTA expression in M2c macrophages and their phagocytic activity. VISTA-expressing macrophages demonstrated a unique cytokine profile, characterized by reduced IL-1ß and elevated IL-10 secretion. Furthermore, VISTA interacted with MHC-I and downregulated its surface expression, leading to diminished T cell activation. Notably, VISTA surface expression was identified in monocytes from all lymphoma patients but was less prevalent in healthy donors. CONCLUSIONS: Collectively, VISTA expression associates with and drives M2-like activation of macrophages with a high phagocytic capacity yet a decrease in antigen presentation capability to T cells. Therefore, VISTA is a negative immune checkpoint regulator in macrophage-mediated immune suppression.

Tumor infiltrating CD8/CD103/TIM-3-expressing lymphocytes in epithelial ovarian cancer co-express CXCL13 and associate with improved survival.

Reactivation of tumor infiltrating T lymphocytes (TILs) with immune checkpoint inhibitors or co-stimulators has proven to be an effective anti-cancer strategy for a broad range of malignancies. However, epithelial ovarian cancer (EOC) remains largely refractory to current T cell-targeting immunotherapeutics. Therefore, identification of novel immune checkpoint targets and biomarkers with prognostic value for EOC is warranted. Combining multicolor immunofluorescent staining's with single cell RNA-sequencing analysis, we here identified a TIM-3/CXCL13-positive tissue-resident memory (CD8/CD103-positive) T cell (Trm) population in EOC. Analysis of a cohort of ~175 patients with high-grade serous EOC revealed TIM-3-positive Trm were significantly associated with improved patient survival. As CXCL13-positive CD8-positive T cells have been strongly linked to patient response to anti-PD1 immune checkpoint blockade, combinatorial TIM-3 and PD-1 blockade therapy may be of interest for the (re)activation of anti-cancer immunity in EOC.

CD24 Is a Potential Immunotherapeutic Target for Mantle Cell Lymphoma.

CD24 and its ligand Siglec-10 were described as an innate immune checkpoint in carcinoma. Here, we investigated this axis in B-cell lymphoma by assessing CD24 expression and evaluating pro-phagocytic effects of CD24 antibody treatment in comparison to hallmark immune checkpoint CD47. In mantle cell lymphoma (MCL) and follicular lymphoma patients, high mRNA expression of CD24 correlated with poor overall survival, whereas CD47 expression did not. Conversely, CD24 expression did not correlate with survival in diffuse large B-cell lymphoma (DLBCL), whereas CD47 did. CD24 was also highly expressed on MCL cell lines, where treatment with CD24 antibody clones SN3 or ML5 potently induced phagocytosis, with SN3 yielding >90% removal of MCL cells and triggering phagocytosis of primary patient-derived MCL cells by autologous macrophages. Treatment with CD24 mAb was superior to CD47 mAb in MCL and was comparable in magnitude to the effect observed in carcinoma lines. Reversely, CD24 mAb treatment was less effective than CD47 mAb treatment in DLBCL. Finally, phagocytic activity of clone SN3 appeared at least partly independent of antibody-dependent cellular phagocytosis (ADCP), suggesting CD24/Siglec-10 checkpoint activity, whereas clone ML5 solely induced ADCP. In conclusion, CD24 is an immunotherapeutic target of potential clinical relevance for MCL, but not DLBCL.

CD20 positive CD8 T cells are a unique and transcriptionally-distinct subset of T cells with distinct transmigration properties.

The presence of T cells that are dimly positive for the B cell marker CD20 is well-established in autoimmunity and correlates with disease severity in various diseases. Further, we previously identified that the level of CD20-positive T cells was three-fourfold elevated in ascites fluid of ovarian carcinoma patients, together suggesting a role in both autoimmunity and cancer. In this respect, treatment of autoimmune patients with the CD20-targeting antibody Rituximab has also been shown to target and deplete CD20-positive T cells, previously identified as IFN-gamma producing, low proliferative, CD8 cytotoxic T cells with an effector memory (EM) differentiation state. However, the exact phenotype and relevance of CD20-positive T cells remains unclear. Here, we set out to identify the transcriptomic profile of CD20-positive T cells using RNA sequencing. Further, to gain insight into potential functional properties of CD20 expression in T cells, CD20 was ectopically expressed on healthy human T cells and phenotypic, functional, migratory and adhesive properties were determined in vitro and in vivo. Together, these assays revealed a reduced transmigration and an enhanced adhesive profile combined with an enhanced activation status for CD20-positive T cells.