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The clinical success of cancer immune checkpoint blockade (ICB) has refocused attention on tumor-infiltrating lymphocytes (TILs) across cancer types. The outcome of immune checkpoint inhibitor therapy in cancer patients has been linked to the quality and magnitude of T cell, NK cell, and more recently, B cell responses within the tumor microenvironment. State-of-the-art single-cell analysis of TIL gene expression profiles and clonality has revealed a remarkable degree of cellular heterogeneity and distinct patterns of immune activation and exhaustion. Many of these states are conserved across tumor types, in line with the broad responses observed clinically. Despite this homology, not all cancer types with similar TIL landscapes respond similarly to immunotherapy, highlighting the complexity of the underlying tumor-immune interactions. This observation is further confounded by the strong prognostic benefit of TILs observed for tumor types that have so far respond poorly to immunotherapy. Thus, while a holistic view of lymphocyte infiltration and dysfunction on a single-cell level is emerging, the search for response and prognostic biomarkers is just beginning. Within this review, we discuss recent advances in the understanding of TIL biology, their prognostic benefit, and their predictive value for therapy.The schematic representation of the tumor immune environment shows the composition and function of a tertiary lymphoid structures (TLS), who are usually found peritumorally in the stroma and/or in the invasive margin. The chemokine CXCL13, produced by CD8+ T cells, induces chemotaxis by binding to the receptor CXCR5, mainly expressed by B cells and TFH cells, and regulates the organization of B cells inside the follicles of lymphoid tissues. The TLS consists out of a T cell-rich zone containing mature dendritic cells (DCs), in close proximity to GC containing follicle like-B cells, intermingled with follicular dendritic cells (FDCs) and surrounded by plasma cells and helper-innate lymphoid cell group 3 (ILC3) at the edge of the TLS. In the optimally organized TLS immune structure, DCs, FDCs, T cells and B cells interact and activate each other, promoting a local sustained immune response including the induction of T cell effector function, antibody generation, and clonal expansion. The stroma surrounding the tumor epithelium and the invasive margin further harbors cellular immune components including NK cells, macrophages, ILC1s and ILC2s, and a nonimmune cellular component, including fibroblasts. Within the tumor epithelium ILCs, NK cells, B cells, and different T cell subsets are present, including TEX cells,-tumor-specific CD103+CD39+ TRM CTLs and CD103+CD39- bystander TRM cells. Upon ICB, both T and B cell signaling increases. TCF1-expressing TPE cells expand and differentiate into TRM cells migrating to the tumor, where they can exert their cytolytic potential. The ICB response also increases B cell receptor diversity by means of SMH and CSR and induces their clonal expansion and differentiation into advanced antibody-producing plasma cells. TLS: tertiary lymphoid structure, TFH cells: follicular helper T cells, DCs: dendritic cells, GC: germinal center, FDCs: follicular dendritic cells, ILC3: helper-innate lymphoid cell group 3, NK cells: natural killer cells, ILC1: helper-innate lymphoid cell group 1, ILC2: helper-innate lymphoid cell group 2, TEX: terminally exhausted T cells, CTLs: cytotoxic lymphocytes, TRM: tissue resident memory, ICB: immune checkpoint blockade, TCF1: transcription factor 1, TPE: progenitor STEM-like exhausted cells, SMH: somatic hypermutation, RCS: recombinant class switch

B-cells play a key role in cancer suppression, particularly when aggregated in tertiary lymphoid structures (TLS). Here, we investigate the role of B-cells and TLS in endometrial cancer (EC). Single cell RNA-sequencing of B-cells shows presence of naïve B-cells, cycling/germinal center B-cells and antibody-secreting cells. Differential gene expression analysis shows association of TLS with L1CAM overexpression. Immunohistochemistry and co-immunofluorescence show L1CAM expression in mature TLS, independent of L1CAM expression in the tumor. Using L1CAM as a marker, 378 of the 411 molecularly classified ECs from the PORTEC-3 biobank are evaluated, TLS are found in 19%. L1CAM expressing TLS are most common in mismatch-repair deficient (29/127, 23%) and polymerase-epsilon mutant EC (24/47, 51%). Multivariable Cox regression analysis shows strong favorable prognostic impact of TLS, independent of clinicopathological and molecular factors. Our data suggests a pivotal role of TLS in outcome of EC patients, and establishes L1CAM as a simple biomarker. Tertiary lymphoid structures (TLS) are associated with a reduced risk of cancer recurrence and improved response to immune checkpoint blockade in several tumor types. Here the authors identify L1CAM as a marker for mature TLS and show that the presence of TLS is associated with favorable prognosis in patients with endometrial cancer from the PORTEC-3 trial.

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.

CD47 is a prominent new target in cancer immunotherapy, with antagonistic antibodies currently being evaluated in clinical trials. For effective evaluation of this strategy it is crucial to identify which patients are suited for CD47-targeted therapy. In this respect, expression of the pro-phagocytic signal SLAMF7 on both macrophages and cancer cells was recently reported to be a requisite for CD47 antibody-mediated phagocytosis. Here, we demonstrate that in fact SLAMF7 expression on cancer cells is not required and does not impact on CD47 antibody therapy. Moreover, SLAMF7 also does not impact on phagocytosis induction by CD20 antibody rituximab nor associates with overall survival of Diffuse Large B-Cell Lymphoma patients. In contrast, expression of CD47 negatively impacts on overall and progression free survival. In conclusion, cancer cell expression of SLAMF7 is not required for phagocytosis and, in contrast to CD47 expression, should not be used as selection criterion for CD47-targeted therapy. CD47 is a promising new target in cancer immunotherapy and recently the pro-phagocytic signal SLAMF7 has been shown to have a crucial role in phagocytosis induced by CD47-blocking antibody in hematological tumors. In this study, the authors demonstrate that SLAMF7 expressed by cancer cells is not required for phagocytosis suggesting that, in contrast to CD47 expression, SLAMF7 should not be used as selection criterion for CD47-targeted therapy.

Bulk transcriptomic analyses of high-grade serous ovarian cancer (HGSOC) so far have not uncovered potential drug targets, possibly because subtle, disease-relevant transcriptional patterns are overshadowed by dominant, non-relevant ones. Our aim was to uncover disease-outcome-related patterns in HGSOC transcriptomes that may reveal novel drug targets. Using consensus-independent component analysis, we dissected 678 HGSOC transcriptomes of systemic therapy naïve patients—sourced from public repositories—into statistically independent transcriptional components (TCs). To enhance c-ICA’s robustness, we added 447 transcriptomes from non-serous histotypes, low-grade serous, and non-cancerous ovarian tissues. Cox regression and survival tree analysis were performed to determine the association between TC activity and overall survival (OS). Finally, we determined the activity of the OS-associated TCs in 11 publicly available spatially resolved ovarian cancer transcriptomes. We identified 374 TCs, capturing prominent and subtle transcriptional patterns linked to specific biological processes. Six TCs, age, and tumor stage stratified patients with HGSOC receiving platinum-based chemotherapy into ten distinct OS groups. Three TCs were linked to copy-number alterations affecting expression levels of genes involved in replication, apoptosis, proliferation, immune activity, and replication stress. Notably, the TC identifying patients with the shortest OS captured a novel transcriptional pattern linked to synaptic signaling, which was active in tumor regions within all spatially resolved transcriptomes. The association between a synaptic signaling-related TC and OS supports the emerging role of neurons and their axons as cancer hallmark-inducing constituents of the tumor microenvironment. These constituents might offer a novel drug target for patients with HGSOC.

BackgroundTreatment of high grade serous ovarian cancer (HGSOC) after debulking and chemotherapy remains challenging. This phase 1 trial (NCT04739527) evaluates the use of a cell-based cancer vaccine, vididencel, to prevent disease recurrence after primary treatment. Vididencel expresses Tumor Associated Antigens (TAA), such as WT1 and PRAME, and induced immune responses to these antigens correlated with clinical responses in a study in AML patients. As these antigens are also frequently upregulated in HGSOC, vididencel is evaluated in this patient population.MethodsPatients with HGSOC after primary debulking and chemotherapy, were given vididencel four times biweekly (week 0, 2, 4 and 6) at 25 million cells/vaccination, followed by 2 boosters at week 14 and 18 at 10 million cells/vaccination. Peripheral blood mononuclear cells (PBMC) were obtained at week 0, 4, 10, 14, 18 and 22.IFNγ ELISpot was performed on isolated, frozen PBMC after restimulation with WT1, PRAME, MAGEA3/4 and NY-ESO1. Vaccine induced T-cell response (VIR) were calculated as ≥2-fold increase of the mock-corrected baseline response. Flow cytometry was being performed using a 40-marker panel to evaluate the immune profiles of innate and adaptive immune system, activation and exhaustion markers and memory profiles.ResultsAs of June 23 2023, in total 13 patients have been enrolled, of which 9 had completed the treatment phase (up to week 22). All these patients were still alive and three patients showed disease recurrence before week 22.Immune response assessment to WT1 and PRAME, showed VIR in 6/8 patients analysed. Additionally, NY-ESO1 and MAGEA3/A4 responses, although not expressed by vididencel, were induced in 4/8 patients.Analysis of the immune profile by flow cytometry indicated an increase in B-cells after vaccination, with a phenotypes resembling memory B-cells (CD19+CD38++CD24-IgM-IgD-CD20dim/- CD27++) or plasmablasts (CD19+ CD38+CD24+IgM+IgD-CD20+CD27-).ConclusionsIFN secreting T-cells to TAA expressed by vididencel were increased in the majority of patients. Importantly, VIR to antigens not expressed in these cells, namely NY-ESO1 and MAGE A3/A4, were also increased. These results could indicate antigen spreading by tumor cell lysis, as also previously shown in AML. Except for induced T-cell responses, B-cell responses were observed by increased B-cell memory and plasmablast frequencies. These humoral and cellular responses show activation of both the innate and adaptive immune system. Vididencel thus showed the possibility of induction of a broad immune response to target antigens known to be expressed in ovarian cancer.Trial RegistrationNCT04739527Ethics ApprovalThis study was approved by the central committee on research involving human subjects (CCMO) Ethics Board; approval number NL74250.000.20

BackgroundCD103 is an integrin specifically expressed on the surface of cancer-reactive T cells. The number of CD103+ T cells significantly increases during successful immunotherapy and might therefore be an attractive biomarker for noninvasive PET imaging of immunotherapy response. Since the long half-life of antibodies preclude repeat imaging of CD103+ T cell dynamics early in therapy, we therefore here explored PET imaging with CD103 Fab fragments radiolabeled with a longer (89Zr) and shorter-lived radionuclide (68Ga).MethodsAntihuman CD103 Fab fragment Fab01A was radiolabeled with 89Zr or 68Ga, generating [89Zr]Zr-hCD103.Fab01A and [68Ga]Ga-hCD103.Fab01A, respectively. In vivo evaluation of these tracers was performed in male nude mice (BALB/cOlaHsd-Foxn1nu) with established CD103-expressing CHO (CHO.CD103) or CHO-wildtype (CHO.K1) xenografts, followed by serial PET imaging and ex vivo bio-distribution.Results[89Zr]Zr-hCD103.Fab01A showed high tracer uptake in CD103+ xenografts as early as 3 h post-injection. However, the background signal remained high in the 3- and 6-h scans. The background was relatively low at 24 h after injection with sufficient tumor uptake. [68Ga]Ga-hCD103.Fab01Ashowed acceptable uptake and signal-to-noise ratio in CD103+ xenografts after 3 h, which decreased at subsequent time points.Conclusion[89Zr]Zr-hCD103.Fab01A demonstrated a relatively low background and high xenograft uptake in scans as early as 6 h post-injection and could be explored for repeat imaging during immunotherapy in clinical trials. 18F or 64Cu could be explored as alternative to 68Ga in optimizing half-life and radiation burden of the tracer.

Background Hepatic ischemia-reperfusion injury (I/Ri) is a serious complication occurring during liver surgery that may lead to liver failure. Hepatic I/Ri induces formation of reactive oxygen species, hepatocyte apoptosis, and release of pro-inflammatory cytokines, which together causes liver damage and organ dysfunction. A potential strategy to alleviate hepatic I/Ri is to exploit the potent anti-inflammatory and cytoprotective effects of carbon monoxide (CO) by application of so-called CO-releasing molecules (CORMs). Here, we assessed whether CO released from CORM-2 protects against hepatic I/Ri in a rat model. Methods Forty male Wistar rats were randomly assigned into four groups (n = 10). Sham group underwent a sham operation and received saline. I/R group underwent hepatic I/R procedure by partial clamping of portal structures to the left and median lobes with a microvascular clip for 60 minutes, yielding ~70% hepatic ischemia and subsequently received saline. CORM-2 group underwent the same procedure and received 8 mg/kg of CORM-2 at time of reperfusion. iCORM-2 group underwent the same procedure and received iCORM-2 (8 mg/kg), which does not release CO. Therapeutic effects of CORM-2 on hepatic I/Ri was assessed by measuring serum damage markers AST and ALT, liver histology score, TUNEL-scoring of apoptotic cells, NFkB-activity in nuclear liver extracts, serum levels of pro-inflammatory cytokines TNF-α and IL-6, and hepatic neutrophil infiltration. Results A single systemic infusion with CORM-2 protected the liver from I/Ri as evidenced by a reduction in serum AST/ALT levels and an improved liver histology score. Treatment with CORM-2 also up-regulated expression of the anti-apoptotic protein Bcl-2, down-regulated caspase-3 activation, and significantly reduced the levels of apoptosis after I/Ri. Furthermore, treatment with CORM-2 significantly inhibited the activity of the pro-inflammatory transcription factor NF-κB as measured in nuclear extracts of liver homogenates. Moreover, CORM-2 treatment resulted in reduced serum levels of pro-inflammatory cytokines TNF-α and IL-6 and down-regulation of the adhesion molecule ICAM-1 in the endothelial cells of liver. In line with these findings, CORM-2 treatment reduced the accumulation of neutrophils in the liver upon I/Ri. Similar treatment with an inactive variant of CORM-2 (iCORM-2) did not have any beneficial effect on the extent of liver I/Ri. Conclusions CORM-2 treatment at the time of reperfusion had several distinct beneficial effects on severity of hepatic I/Ri that may be of therapeutic value for the prevention of tissue damage as a result of I/Ri during hepatic surgery.

Genomic instability is a driver and accelerator of tumorigenesis and influences disease outcomes across cancer types. Although genomic instability has been associated with immune evasion and worsened disease prognosis, emerging evidence shows that genomic instability instigates pro-inflammatory signaling and enhances the immunogenicity of tumor cells, making them more susceptible to immune recognition. While this paradoxical role of genomic instability in cancer is complex and likely context-dependent, understanding it is essential for improving the success rates of cancer immunotherapy. In this review, we provide an overview of the underlying mechanisms that link genomic instability to pro-inflammatory signaling and increased immune surveillance in the context of cancer, as well as discuss how genomically unstable tumors evade the immune system. A better understanding of the molecular crosstalk between genomic instability, inflammatory signaling, and immune surveillance could guide the exploitation of immunotherapeutic vulnerabilities in cancer.