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Background: Addition of neoadjuvant immune checkpoint inhibition to standard-of-care interventions for locally advanced oral cancer could improve clinical outcome., Methods: In this study, 16 evaluable patients with stage III/IV oral cancer were treated with one dose of 480 mg nivolumab 3 weeks prior to surgery. Primary objectives were safety, feasibility, and suitability of programmed death receptor ligand-1 positron emission tomography (PD-L1 PET) as a biomarker for response. Imaging included 18 F-BMS-986192 (PD-L1) PET and 18 F-fluorodeoxyglucose (FDG) PET before and after nivolumab treatment. Secondary objectives included clinical and pathological response, and immune profiling of peripheral blood mononuclear cells (PBMCs) for response prediction. Baseline tumor biopsies and postnivolumab resection specimens were evaluated by histopathology., Results: Grade III or higher adverse events were not observed and treatment was not delayed in relation to nivolumab administration and other study procedures. Six patients (38%) had a pathological response, of whom three (19%) had a major (≥90%) pathological response (MPR). Tumor PD-L1 PET uptake (quantified using standard uptake value) was not statistically different in patients with or without MPR (median 5.3 vs 3.4). All major responders showed a significantly postnivolumab decreased signal on FDG PET. PBMC immune phenotyping showed higher levels of CD8 + T cell activation in MPR patients, evidenced by higher baseline expression levels of PD-1, TIGIT, IFNγ and lower levels of PD-L1., Conclusion: Together these data support that neoadjuvant treatment of advanced-stage oral cancers with nivolumab was safe and induced an MPR in a promising 19% of patients. Response was associated with decreased FDG PET uptake as well as activation status of peripheral T cell populations., Competing Interests: Competing interests: RvdV has received research funding from Genmab BV. TDdG is scientific advisor to Immunicum, GE Health, and Lava Therapeutics, holds stock from LAVA Therapeutics and received research funding from Idera Pharmaceuticals (now Aceragen). RHB received research grants from KWF Kankerbestrijding/Dutch Cancer Society, Cancer Center Amsterdam Foundation, ZonMW and NWO, Genmab BV and the Hanarth Foundation and is on the advisory board of Nanobiotix. He has a scientific collaboration with Orfenix BV and Qialix DoT. CRL received research grants from KWF Kankerbestrijding/Dutch Cancer Society, Cancer Center Amsterdam Foudation, Genmab BV, BMS and the Hanarth Foundation and is on the advisory board of Merck & Co. CWM-vdHvO received research grants from BMS, Boeringher Ingelheim, GSK, Pfizer and AstraZeneca and consulted for GE Health Care, Novartis and EliLilly. All other authors report no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Tertiary lymphoid structures (TLSs) are thought to stimulate antitumor immunity and positively impact prognosis and response to immune checkpoint blockade. In gastric cancers (GCs), however, TLSs are predominantly found in GC with poor prognosis and limited treatment response. We, therefore, hypothesize that immune cell composition and function of TLS depends on tumor location and the tumor immune environment., Methods: Spatial transcriptomics and immunohistochemistry were used to characterize the phenotype of CD45 + immune cells inside and outside of TLS using archival resection specimens from GC primary tumors and peritoneal metastases., Results: We identified significant intrapatient and interpatient diversity of the cellular composition and maturation status of TLS in GC. Tumor location (primary vs metastatic site) accounted for the majority of differences in TLS maturity, as TLS in peritoneal metastases were predominantly immature. This was associated with higher levels of tumor-infiltrating macrophages and Tregs and less plasma cells compared with tumors with mature TLS. Furthermore, mature TLSs were characterized by overexpression of antitumor immune pathways such as B cell-related pathways, MHC class II antigen presentation while immature TLS were associated with protumor pathways, including T cell exhaustion and enhancement of DNA repair pathways in the corresponding cancer., Conclusion: The observation that GC-derived peritoneal metastases often contain immature TLS which are associated with immune suppressive regulatory tumor-infiltrating leucocytes, is in keeping with the lack of response to immune checkpoint blockade and the poor prognostic features of peritoneal metastatic GC, which needs to be taken into account when optimizing immunomodulatory strategies for metastatic GC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Immune checkpoint inhibitors (ICIs) can elicit anticancer immune responses, but predictive biomarkers are needed. We measured programmed death ligand 1 (PD-L1) expression in organs and lymph nodes using 18 F-BMS-986192 positron emission tomography (PET)-imaging and looked for correlations with response and immune-related adverse events., Methods: Four 18 F-BMS-986192 PET studies in patients with melanoma, lung, pancreatic and oral cancer, receiving ICI treatment, were combined. Imaging data (organ standardized uptake value (SUV) mean , lymph node SUV max ) and clinical data (response to treatment and incidence of immune-related adverse events) were extracted., Results: Baseline PD-L1 uptake in the spleen was on average higher in non-responding patients than in responders (spleen SUV mean 16.1±4.4 vs 12.5±3.4, p=0.02). This effect was strongest in lung cancer, and not observed in oral cancer. In the oral cancer cohort, benign tumor-draining lymph nodes (TDLNs) had higher PD-L1 uptake (SUV max 3.3 IQR 2.5-3.9) compared with non-TDLNs (SUV max 1.8, IQR 1.4-2.8 p=0.04). Furthermore, in the same cohort non-responders showed an increase in PD-L1 uptake in benign TDLNs on-treatment with ICIs (+15%), while for responders the PD-L1 uptake decreased (-11%). PD-L1 uptake did not predict immune-related adverse events, though elevated thyroid uptake on-treatment correlated with pre-existing thyroid disease or toxicity., Conclusion: PD-L1 PET uptake in the spleen is a potential negative predictor of response to ICIs. On-treatment with ICIs, PD-L1 uptake in benign TDLNs increases in non-responders, while it decreases in responders, potentially indicating a mechanism for resistance to ICIs in patients with oral cancer., Competing Interests: Competing interests: RvdV: Research funding for Institute: Genmab B.V. TDdG: Research funding for Institute: Idera Pharmaceuticals (now Aceragen); Consultancy: GE Health, LAVA Therapeutics, Mendus (all to Institute); holds stocks from LAVA Therapeutics. GAPH: Research funding for Institute: Bristol-Myers Squibb, Seerave. Consultancy/advisory relationships with Amgen, Bristol-Myers Squibb, Roche, MSD, Pfizer, Novartis, Sanofi, Pierre Fabre. AJdL: Research funding for Institute: Bristol-Myers Squibb, MSD, Boehringer Ingelheim, AstraZeneca. Non-financial support from Merck Serono, non-financial support from Roche. CWM-vdHvO: Research funding for Institute: Bristol-Myers Squibb, Boehringer Ingelheim, GSK, Pfizer; AstraZeneca. Consultancy: GE Health Care, Novartis, Eli Lilly. IHCM, JEEP, AK, GJCZ, MCH, FEFT: No competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Intratumorally delivered immunotherapies have the potential to favorably alter the local tumor microenvironment and may stimulate systemic host immunity, offering an alternative or adjunct to other local and systemic treatments. Despite their potential, these therapies have had limited success in late-phase trials for advanced cancer resulting in few formal approvals. The Society for Immunotherapy of Cancer (SITC) convened a panel of experts to determine how to design clinical trials with the greatest chance of demonstrating the benefits of intratumoral immunotherapy for patients with cancers across all stages of pathogenesis., Methods: An Intratumoral Immunotherapy Clinical Trials Expert Panel composed of international key stakeholders from academia and industry was assembled. A multiple choice/free response survey was distributed to the panel, and the results of this survey were discussed during a half-day consensus meeting. Key discussion points are summarized in the following manuscript., Results: The panel determined unique clinical trial designs tailored to different stages of cancer development-from premalignant to unresectable/metastatic-that can maximize the chance of capturing the effect of intratumoral immunotherapies. Design elements discussed included study type, patient stratification and exclusion criteria, indications of randomization, study arm determination, endpoints, biological sample collection, and response assessment with biomarkers and imaging. Populations to prioritize for the study of intratumoral immunotherapy, including stage, type of cancer and line of treatment, were also discussed along with common barriers to the development of these local treatments., Conclusions: The SITC Intratumoral Immunotherapy Clinical Trials Expert Panel has identified key considerations for the design and implementation of studies that have the greatest potential to capture the effect of intratumorally delivered immunotherapies. With more effective and standardized trial designs, the potential of intratumoral immunotherapy can be realized and lead to regulatory approvals that will extend the benefit of these local treatments to the patients who need them the most., Competing Interests: Competing interests: JJL: Researcher: AbbVie, Astellas, Astrazeneca, Bristol-Myers Squibb, Corvus, Day 1, EMD Serono, Fstar, Genmab, Ikena, Immatics, Incyte, Kadmon, KAHR, Macrogenics, Merck, Moderna, Nektar, Next Cure, Numab, Palleon, Pfizer, Replimmune, Rubius, Servier, Scholar Rock, Synlogic, Takeda, Trishula, Tizona, XencorConsultant/Advisor/Speaker: Abbvie, Agenus, Alnylam, Atomwise, Bayer, Bristol-Myers Squibb, Castle, Checkmate, Codiak, Crown, Cugene, Curadev, Day One, Eisai, EMD Serono, Endeavor, Flame, G1 Therapeutics, Genentech, Gilead, Glenmark, HotSpot, Kadmon, KSQ, Janssen, Ikena, Inzen, Immatics, Immunocore, Incyte, Instil, IO Biotech, Macrogenics, Merck, Mersana, Nektar, Novartis, Partner, Pfizer, Pioneering Medicines, PsiOxus, Regeneron, Replimmune, Ribon, Roivant, Servier, STINGthera, Synlogic, Synthekine, 7 Hills, Affivant, Bright Peak, Exo, Fstar, Inzen, RefleXion, Xilio (stock) Actym, Alphamab Oncology, Arch Oncology, Duke Street Bio, Kanaph, Mavu, NeoTx, Onc.AI, OncoNano, physIQ, Pyxis, Saros, STipe, Tempest, Abbvie, Agenus, Amgen, Immutep, Evaxion. DD: Grants/Research Support (institutional): Arcus, Immunocore, Merck, Regeneron Pharmaceuticals, Tesaro/GSK. Consultant: ACM Bio, Ascendis, Castle, Clinical Care Options (CCO), Gerson Lehrman Group (GLG), Immunitas, Medical Learning Group (MLG), Replimmune, Trisalus, Xilio Therapeutics. CE Speakers’ Bureau: Castle Biosciences. Stockholder: None. Intellectual Property: US Patent 63/124,231, "Compositions and Methods for Treating Cancer", December 11, 2020 US Patent 63/208,719, "Compositions and Methods For Responsiveness to Immune Checkpoint Inhibitors (ICI), Increasing Effectiveness of ICI and Treating Cancer", June 9, 2021. AM: Owner: HiFiBio, Deka Bio, Hotspot Therapeutics, Shattuck Labs, Researcher: Astra Zeneca, BMS, Sanofi, Consultant/Advisor/Speaker: Gritstone, Innate Pharma, Neogene, Deka Bio, Hotspot therapeutics, J&J, Medicxi, Depth Charge, BiolineRx, Clover Pharma, Grey Wolf, Lytix, RedX, HiFiBio, ImCheck, Applied Materials, Takeda, Shattuck Labs, Marengo therapeutics, Pierre Fabre, Third Rock Ventures, SotioPublicly Traded Stocks: Centessa. RHA–Employee: Seven & Eight Biopharmaceuticals. Ownership Interest Less Than 5 Percent: Seven & Eight Biopharmaceuticals. NB: Consulting Fees: Novartis, Apricity, Rome Therapeutics, BreakBio, Carisma Therapeutics, CureVac, BioNTech, Gilead, Tempest Therapeutics, Boehringer Ingelheim, Contracted Research: Regeneron Pharmaceutics, Dragonfly Therapeutics, Harbour Biomed Sciences, Ownership Interest Less Than 5 Percent: BreakBio, Apricity. JDB: Researcher: Merck, Genentech, Astrazeneca, Kite/Gilead, BMS, Celldex, Oncovir, Seattle Genetics, ADC Therapeutics, Epizyme, Consultant/Advisor/Speaker: Merck, Genentech, Astrazeneca, Kite/Gilead, BMS, Seattle Genetics, ADC Therapeutics, Epizyme, Asgaard Therapeutics, Global BioAccess, SIRPant Immunotherapeutics. JC: Executive Role: UofL Health, Researcher: Amgen, Iovance, Fate, Replimune, Consultant/Advisor/Speaker: Replimune advisory board 2020-21, Royalties or Patent Beneficiary: US Patents: University of Louisville. RC: Employee: Replimune. TdB: Consultant/Advisor/Speaker: Terumo, Boston Scientific, Astra Zeneca, Nanobiotix, Quantum Surgical, Guerbet, Cook medical, Johnson & Johnson. TDdG: Patents: (1) The use of cytostatics for the accelerated differentiation of DC; WO2009019320-A2; WO2009019320-A3; AU2008285598-A1; EP2281030-A2; CA2724018-A1; US2011117051-A1. US8,470,789B2; DCprime BV. (2) Immunoglobulins binding human Vγ9VÎ’2 T cell receptors; P31885NL00. Consulting Fees: Mendus (formerly Immunicum, formerly DCPrime BV), Partner Therapeutics, GE Health, LAVA Therapeutics, Contracted Research: Idera Pharmaceuticals, Macrophage Parma, Ownership Interest Less Than 5 Percent: LAVA Therapeutics. MGF: Employee: Regeneron. GVG: Employee: Merck. Publicly Traded Stocks: Immunogen, Aveo, Beta Bionics. KJH: Researcher: AstraZeneca, Boehringer-Ingelheim, Replimune, Consultant/Advisor/Speaker: Arch Oncology, AstraZeneca, BMS, Boehringer-Ingelheim, Codiak, Eisai, Inzen, Merck-Serono, MSD, Oncolys, Pfizer, Replimune. HK: Employee: Ankyra Therapeutics. Consultant/Advisor/Speaker: Castle Biosciences, Marengo Therapeutics. CMK: Researcher: Amgen, Merck & Co., Kartos Pharmaceuticals, EMD Serono, Deciphera Pharmaceuticals, Eily lilly, Blueprint Medicines, Incyte Corporation, Clovis Oncology, BMS, Aadi Bioscience, Pfizer, Iterion Therapeutics, Springworks Therapeutics, Nektar Therapeutics, GSK, Adaptimmune, Medimmune, Bioatla, Oncternal Therapeutics, Daiichi- Sankyo Co. Traycon Pharm, Eisai. Ascentage Pharma, Hutchison Medipharma, Salarius Pharmaceuticals, Loxo Oncology, -BTG Specialty Pharmaceuticals, AStex Pharmaceuticals, Xencor, bayer healthcare, Athenex pharmaceuticals, servier, K- group Beta, Trilliium Pharmaceuticals, Ayala Pharmaceuticals, Ningbo Newbay Technology Development, Rain Therapeutics, Inhibrx, C4 Therapeutics, Foghorn Therapeutics, Theseus Pharmaceuticals, Cogent Therapeutics, Curadev PharmaRegeneron, Consultant/Advisor/Speaker: Chemocentryx, Kartos, Servier, Immunicom, Other: Spouse employed by Daichii Sankyo with stock options. ADK: Employee: Merck. KL: Employee: Marengo Therapeutics. SL: Executive Role: Big Against Cancer (Belgium), Breast Cancer Trials (Australia), International Breast Cancer Study Group (Switzerland) Researcher: Research funding to institution from Novartis, Bristol Meyers Squibb, Merck, Puma Biotechnology, Eli Lilly, Nektar Therapeutics, Astra Zeneca, Roche-Genentech and Seattle Genetics, Consultant/Advisor/Speaker: Consultant (not compensated) to Seattle Genetics, Novartis, Bristol Meyers Squibb, Merck, AstraZeneca, Eli Lilly, Pfizer, Gilead Therapeutics and Roche-Genentech. Consultant (paid to institution) to Aduro Biotech, Novartis, GlaxoSmithKline, Roche-Genentech, Astra Zeneca, Silverback Therapeutics, G1 Therapeutics, PUMA Biotechnologies, Pfizer, Gilead Therapeutics, Seattle Genetics, Daiichi Sankyo, Merck, Amunix, Tallac Therapeutics, Eli Lilly and Bristol Meyers Squibb, Role: Presenter (Speaker). GVL: Consultant/Advisor/Speaker: Consultant Advisor for Agenus, Amgen, Array Biopharma, AstraZeneca UK, Boehringer Ingelheim International, Bristol Myers Squibb, Evaxion Biotech A/S, Hexal AG (Sandoz Company), Highlight Therapeutics S.L., Innovent Biologics USA, Merck Sharpe & Dohme (Australia), Merck Sharpe & Dohme, Novartis Pharma AG, PHMR, Pierre Fabre, Provectus Australia, Qbiotics Group Limited, Regeneron Pharmaceuticals. IM: Consulting Fees: Bristol-Myers Squibb, F-STAR, Alligator, Pharma Mar, AstraZeneca, Numab Therapeutics, Roche, Amunix, Gossamer, Molecular Partners, Merck-Serono, Genmab, PharmaMar, Contracted Research: Roche, Bristol-Myers Squibb, Highlight Therapeutics, Alligator, Genmab, Astrazeneca. MRM: Researcher : Roche, Astrazeneca, Novartis, Immunocore, BMS, Pfizer, Merck/MSD, Regeneron, BiolineRx, Replimune, GRAIL, Alkermes, iOx, Vaccitech, Consultant/Advisor/Speaker: Infinitopes. BN: Researcher: As a principal investigator for clinical trials sponsored by UZ Brussel, my institution received support from Novartis, Pfizer, Bayer, BMS, MSD, Pierre-Fabre, Amgen, Consultant/Advisor/Speaker: Novartis, BMS, MSD, Pfizer, Pierre-Fabre, Amgen. DJP: Consulting Fees: ViiV Healthcare, Bayer, Hoffman La Roche, EISAI, H3B, MiNa Alpha Therapeutics, DaVolterra, Fees for Non CE Services: Hoffmann La Roche, EISAI, Contracted Research: Merck Sharpe and Dohme, Bristol Myers Squibb (to institution). RAS: Researcher: Boston ScientificConsultant/Advisor/Speaker: Cook Medical, TriSalus, Replimune, Medtronic. SBS: Owner: Aperture Medical Technology, Executive Role: Aperture Medical Technology, Researcher: GE Healthcare, Johnson & Johnson, Elesta, Consultant/Advisor/Speaker: GE Healthcare, XACT Robotics, Microbot, Candel Therapeutics, Varian, Merck & Co., Royalty and Patent Beneficiary: Aperture Medical Technology, Publicly Traded Stocks: Johnson & Johnson, Poseidx Therapeutics, Motus GI, Sientra, Avadel, Lantheus. PS: Employee: Johnson & Johnson, Publicly Traded Stocks: Johnson & Johnson. SITC staff members SMW, CG, AK, NL, EG and KJ have no disclosures., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: In patients with locally advanced unresectable non-small cell lung cancer (NSCLC), durvalumab, an anti-programmed cell death ligand-1 (PD-L1) antibody, has shown improved overall survival when used as consolidation therapy following concurrent chemoradiotherapy (CRT). However, it is unclear whether CRT itself upregulates PD-L1 expression. Therefore, this study aimed to explore the changes in the uptake of the anti PD-L1 antibody [ 89 Zr]Zr-durvalumab in tumors and healthy organs during CRT in patients with NSCLC., Methods: Patients with NSCLC scheduled to undergo CRT were scanned 7±1 days after administration of 37±1 MBq [ 89 Zr]Zr-durvalumab at baseline, 1-week on-treatment and 1 week after finishing 6 weeks of CRT. First, [ 89 Zr]Zr-durvalumab uptake was visually assessed in a low dose cohort with a mass dose of 2 mg durvalumab (0.13% of therapeutic dose) and subsequently, quantification was done in a high dose cohort with a mass dose of 22.5 mg durvalumab (1.5% of therapeutic dose). Tracer pharmacokinetics between injections were compared using venous blood samples drawn in the 22.5 mg cohort. Visual assessment included suspected lesion detectability. Positron emission tomography (PET) uptake in tumoral and healthy tissues was quantified using tumor to plasma ratio (TPR) and organ to plasma ratio, respectively., Results: In the 2 mg dose cohort, 88% of the 17 identified tumor lesions were positive at baseline, compared with 69% (9/13) for the 22.5 mg cohort. Although the absolute plasma concentrations between patients varied, the intrapatient variability was low. The ten quantitatively assessed lesions in the 22.5 mg cohort had a median TPR at baseline of 1.3 (IQR 0.7-1.5), on-treatment of 1.0 (IQR 0.7-1.4) and at the end of treatment of 0.7 (IQR 0.6-0.7). On-treatment, an increased uptake in bone marrow was seen in three out of five patients together with a decreased uptake in the spleen in four out of five patients., Conclusions: This study successfully imaged patients with NSCLC with [ 89 Zr]Zr-durvalumab PET before and during CRT. Our data did not show any increase in [ 89 Zr]Zr-durvalumab uptake in the tumor 1-week on-treatment and at the end of treatment. The changes observed in bone marrow and spleen may be due to an CRT-induced effect on immune cells., Trial Registration Number: EudraCT number: 2019-004284-51., Competing Interests: Competing interests: SMSH is on the advisory board and/or received institutional research support from AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, Janssen, GlaxoSmithKline, Merck Sharp & Dohme, Novartis, Roche and Takeda. DV received institutional research support from Bristol-Myers Squibb, Daiichi Sankyo, GlaxoSmithKline, Boehringer Ingelheim, Roche and Aplagon. SS is on the advisory board of AstraZeneca, receives institutional research support and a speakers fee from AstraZeneca. SS is on the advisory board of Bristol-Myers Squibb and receives institutional research support from Bristol-Myers Squibb. CWM-vdHvO is advisor for GEHealthcare and Eli Lilly and received institutional research support from GlaxoSmithKline, Boehringer Ingelheim, AstraZeneca, G1 Therapeutics and Bristol-Myers Squibb., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Natural killer (NK) cells are innate lymphocytes with a key role in the defense against tumors. Recently, allogeneic NK cell-based therapies have gained interest because of their ability to directly lyse tumor cells without inducing graft-versus-host disease. As NK cells are also able to influence the function of other immune cells (most notably dendritic cells (DC)), a better understanding of the effects of allogeneic NK cell products on the host immune system is required. In this study, we analyzed the effects of an allogeneic off-the-shelf NK cell product, on the tumor microenvironment (TME) of primary and metastatic colorectal cancer (pCRC and mCRC, respectively). Moreover, we explored if the combination of NK cells with R848, a toll-like receptors 7/8 ligand, could further enhance any pro-inflammatory effects., Methods: Ex vivo expanded umbilical cord blood stem cell derived NK cells were co-cultured with pCRC or mCRC single-cell suspensions in the presence or absence of R848 for 5 days, during and after which flow cytometry and cytokine release profiling were performed., Results: NK cells efficiently induced lysis of tumor cells in both pCRC and mCRC single-cell suspensions and thereby controlled growth rates during culture. They also induced differentiation of infiltrating monocytic cells to an activated DC phenotype. Importantly, this NK-mediated myeloid conversion was also apparent in cultures after tumor cell depletion and was further enhanced by combining NK cells with R848. Moreover, NK cells, and to a greater extent, the combination of NK cells and R848, triggered CD8 + and CD4 + T-cell activation as well as a reduction in activated regulatory T cell rates. Finally, the combination of NK cells and R848 induced a pro-inflammatory shift in the cytokine release profile resulting in higher levels of interferon (IFN)-γ, interleukin (IL)-2, IL-12p70, and IFN-α as well as a reduction in IL-6, in both pCRC and mCRC cultures., Conclusion: Allogeneic NK cells engaged in favorable myeloid crosstalk, displayed effective antitumor activity and, when combined with R848, induced a pro-inflammatory shift of the CRC TME. These findings prompt the investigation of NK cells and R848 as a combination therapy for solid tumors., Competing Interests: Competing interests: JS is chief scientific officer at Glycostem BV and HJvdV is chief scientific officer at Lava Therapeutics NV. TDdG is scientific advisor to Immunicum and Lava Therapeutics. AAvV is an employee of Glycostem BV., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach., Methods: To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d + tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography., Results: Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d + tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A'-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling., Conclusion: Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions., Competing Interests: Competing interests: DLH, PB and HJvdV are employees of LAVA Therapeutics, a company that develops bispecific gamma-delta T-cell engagers, and own LAVA Therapeutics shares and/or stock options. RL, TDdG and HJvdV are named inventors on international patent application WO 2016/122320 Al (‘Single domain antibodies targeting CD1d’) which partially relates to the work described in this paper. TDdG is a consultant for and a shareholder of LAVA Therapeutics. RL and MV were funded by a LAVA Therapeutics grant to Amsterdam UMC. JR has received funding from LAVA Therapeutics. DIG is a member of the scientific advisory board and a shareholder of Avalia Immunotherapies, a company working on NKT cell-based vaccines and is an inventor on patent applications WO 2021/127745 and WO 2021/127744 that describe effects of cross-linking CD1 molecules and WO2020/231274 that describes CD1d-dependent glycopeptide vaccines., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Therapeutic immune intervention is highly dependent on the T-cell priming and boosting capacity of tumor-draining lymph nodes (TDLN). In vulvar cancer, in-depth studies on the immune status of (pre)metastatic TDLN is lacking., Methods: We have phenotyped and enumerated various T-cell and myeloid subsets in tumor-free (LN-, n=27) and metastatic TDLN (LN+, n=11) using flow cytometry. Additionally, we studied chemokine and cytokine release profiles and assessed expression of indoleamine 2,3-dioxygenase (IDO) in relation to plasmacytoid dendritic cell (pDC) or myeloid subsets., Results: Metastatic involvement of TDLN was accompanied by an inflamed microenvironment with immune suppressive features, marked by hampered activation of migratory DC, increased cytokine/chemokine release, and closely correlated elevations of pDC and LN-resident conventional DC (LNR-cDC) activation state and frequencies, as well as of terminal CD8 + effector-memory T-cell (TemRA) differentiation, regulatory T-cell (Treg) rates, T-cell activation, and expression of cytotoxic T-lymphocyte protein-4 (CTLA-4) and programmed cell death protein-1 (PD-1) immune checkpoints. In addition, high indoleamine 2,3-dioxygenase (IDO) expression and increased frequencies of monocytic myeloid-derived suppressor cells (mMDSC) were observed. Correlation analyses with primary and metastatic tumor burden suggested respective roles for Tregs and suppression of inducible T cell costimulator (ICOS) + T helper cells in early metastatic niche formation and for CD14 + LNR-cDC and terminal T-cell differentiation in later stages of metastatic growth., Conclusions: Metastatic spread in vulvar TDLN is marked by an inflamed microenvironment with activated effector T cells, which are likely kept in check by an interplay of suppressive feedback mechanisms. Our data support (neoadjuvant) TDLN-targeted therapeutic interventions based on CTLA-4 and PD-1 blockade, to reinvigorate memory T cells and curb early metastatic spread and growth., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Usual vulvar intraepithelial neoplasia (uVIN) is a premalignancy caused by persistent infection with high-risk types of human papillomavirus (HPV), mainly type 16. Even though different treatment modalities are available (eg, surgical excision, laser evaporation or topical application of imiquimod), these treatments can be mutilating, patients often have recurrences and 2%-8% of patients develop vulvar carcinoma. Therefore, immunotherapeutic strategies targeting the pivotal oncogenic HPV proteins E6 and E7 are being explored to repress carcinogenesis., Method: In this phase I/II clinical trial, 14 patients with HPV16+ uVIN were treated with a genetically enhanced DNA vaccine targeting E6 and E7. Safety, clinical responses and immunogenicity were assessed. Patients received four intradermal HPV-16 E6/E7 DNA tattoo vaccinations, with a 2-week interval, alternating between both upper legs. Biopsies of the uVIN lesions were taken at screening and +3 months after last vaccination. Digital photography of the vulva was performed at every check-up until 12 months of follow-up for measurement of the lesions. HPV16-specific T-cell responses were measured in blood over time in ex vivo reactivity assays., Results: Vaccinations were well tolerated, although one grade 3 suspected unexpected serious adverse reaction was observed. Clinical responses were observed in 6/14 (43%) patients, with 2 complete responses and 4 partial responses (PR). 5/14 patients showed HPV-specific T-cell responses in blood, measured in ex vivo reactivity assays. Notably, all five patients with HPV-specific T-cell responses had a clinical response., Conclusions: Our results indicate that HPV-16 E6/E7 DNA tattoo vaccination is a biologically active and safe treatment strategy in patients with uVIN, and suggest that T-cell reactivity against the HPV oncogenes is associated with clinical benefit., Trial Registration Number: NTR4607., Competing Interests: Competing interests: JB is (part time) employee of Modra Pharmaceuticals and stockholder in Modra Pharmaceuticals. (not related to the manuscript). TS is advisor for Adaptive Biotechnologies, Allogene Therapeutics, Merus, Neogene Therapeutics, and Scenic Biotech; is a recipient of research support from Merck KgaA; is a stockholder in Allogene Therapeutics, Merus, Neogene Therapeutics and Scenic Biotech; and is venture partner at Third Rock Ventures, all not related to the current work. JH is advisor to Achilles Therapeutics, Bristol-Myers Squibb, BioNTech USA, Ipsen, Gadeta, Immunocore, MSD, Merck Serono, Molecular Partners, Neogene Therapeutics, Novartis, Pfizer, Roche/Genentech, Sanofi, Seattle Genetics, Third Rock Ventures, is stock holder in Neogene Therapeutics, and is a recipient of grant or research support from Bristol-Myers Squibb, MSD, Novartis and BioNTech USA. KDV reports research funding from Roche and is consultant for Third Rock Ventures, all outside the scope of this work. TDdG has served as advisor to TILT Biotherapeutics, LAVA Therapeutics, Macrophage Pharma and DCPrime, is a recipient of a grant from Idera Pharmaceuticals, and is co-founder and shareholder of LAVA Therapeutics. JHvdB is a recipient of grant or research support from BioNTech USA and Astra Zeneca, and is currently employed at CellPoint B.V., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: We previously reported CpG-B injection at the primary tumor excision site prior to re-excision and sentinel node biopsy to result in immune activation of the sentinel lymph node (SLN), increased melanoma-specific CD8 + T cell rates in peripheral blood, and prolonged recurrence-free survival. Here, we assessed recruitment and activation of antigen-presenting cell (APC) subsets in the SLN and at the injection site in relation to T cell infiltration., Methods: Re-excision skin specimens from patients with clinical stage I-II melanoma, collected 7 days after intradermal injection of either saline (n=10) or 8 mg CpG-B (CPG7909, n=12), were examined by immunohistochemistry, quantifying immune subsets in the epidermis, papillary, and reticular dermis. Counts were related to flow cytometric data from matched SLN samples. Additional in vitro cultures and transcriptional analyses on peripheral blood mononuclear cells (PBMCs) were performed to ascertain CpG-induced APC activation and chemokine profiles., Results: Significant increases in CD83 + , CD14 + , CD68 + , and CD123 + APC were observed in the reticular dermis of CpG-B-injected skin samples. Fluorescent double/triple staining revealed recruitment of both CD123 + BDCA2 + plasmacytoid dendritic cells (DCs) and BDCA3/CD141 + CLEC9A + type-1 conventional DC (cDC1), of which only the cDC1 showed considerable levels of CD83 expression. Simultaneous CpG-B-induced increases in T cell infiltration were strongly correlated with both cDC1 and CD14 counts. Moreover, cDC1 and CD14 + APC rates in the reticular dermis and matched SLN suspensions were positively correlated. Flow cytometric, transcriptional, and chemokine release analyses of PBMC, on in vitro or in vivo exposure to CpG-B, indicate a role for the activation and recruitment of both cDC1 and CD14 + monocyte-derived APCs in the release of CXCL10 and subsequent T cell infiltration., Conclusion: The CpG-B-induced concerted recruitment of cDC1 and CD14 + APC to the injection site and its draining lymph nodes may allow for both the (cross-)priming of T cells and their subsequent homing to effector sites., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Immunotherapy with checkpoint inhibitors has shown impressive results in patients with melanoma, but still many do not benefit from this line of treatment. A lack of tumor-infiltrating T cells is a common reason for therapy failure but also a loss of intratumoral dendritic cells (DCs) has been described., Methods: We used the transgenic tg(Grm1)EPv melanoma mouse strain that develops spontaneous, slow-growing tumors to perform immunological analysis during tumor progression. With flow cytometry, the frequencies of DCs and T cells at different tumor stages and the expression of the inhibitory molecules programmed cell death protein-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells were analyzed. This was complemented with RNA-sequencing (RNA-seq) and real-time quantitative PCR (RT-qPCR) analysis to investigate the immune status of the tumors. To boost DC numbers and function, we administered Fms-related tyrosine 3 ligand (Flt3L) plus an adjuvant mix of polyI:C and anti-CD40. To enhance T cell function, we tested several checkpoint blockade antibodies. Immunological alterations were characterized in tumor and tumor-draining lymph nodes (LNs) by flow cytometry, CyTOF, microarray and RT-qPCR to understand how immune cells can control tumor growth. The specific role of migratory skin DCs was investigated by coculture of sorted DC subsets with melanoma-specific CD8+ T cells., Results: Our study revealed that tumor progression is characterized by upregulation of checkpoint molecules and a gradual loss of the dermal conventional DC (cDC) 2 subset. Monotherapy with checkpoint blockade could not restore antitumor immunity, whereas boosting DC numbers and activation increased tumor immunogenicity. This was reflected by higher numbers of activated cDC1 and cDC2 as well as CD4+ and CD8+ T cells in treated tumors. At the same time, the DC boost approach reinforced migratory dermal DC subsets to prime gp100-specific CD8+ T cells in tumor-draining LNs that expressed PD-1/TIM-3 and produced interferon γ (IFNγ)/tumor necrosis factor α (TNFα). As a consequence, the combination of the DC boost with antibodies against PD-1 and TIM-3 released the brake from T cells, leading to improved function within the tumors and delayed tumor growth., Conclusions: Our results set forth the importance of skin DC in cancer immunotherapy, and demonstrates that restoring DC function is key to enhancing tumor immunogenicity and subsequently responsiveness to checkpoint blockade therapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Background: We previously showed selectively hampered activation of lymph node-resident (LNR) dendritic cell (DC) subsets in the breast cancer (BrC) sentinel lymph node (SLN) to precede a state of profound T cell anergy. Reactivating these DC subsets by intratumoral delivery of the Toll-like receptor-9 (TLR9) agonist CpG-B could potentially offer a promising immune therapeutic strategy to combat this immune suppression and prevent disease spread. Unfortunately, CpG-B can limit its own immune stimulatory activity through direct TLR9-mediated activation of signal transducer and activator of transcription 3 (STAT3), pinpointed as a key regulator of immune suppression in the tumor microenvironment. Here, we have investigated whether in vitro exposure to CpG-B, with or without simultaneous inhibition of STAT3 signaling, could overcome immune suppression in BrC SLN., Methods: Immune modulatory effects of CpG-B (CPG7909) with or without the JAK2/STAT3 inhibitor (STAT3i) AG490 were assessed in ex vivo cultured BrC SLN-derived single-cell suspensions (N=29). Multiparameter flow cytometric analyses were conducted for DC and T cell subset characterization and assessment of (intracellular) cytokine profiles. T cell reactivity against the BrC-associated antigen Mammaglobin-A was determined by means of interferon-γ ELISPOT assay., Results: Although CpG-B alone induced activation of all DC subsets, combined inhibition of the JAK2/STAT3 pathway resulted in superior DC maturation (ie, increased CD83 expression), with most profound activation and maturation of LNR DC subsets. Furthermore, combined CpG-B and JAK2/STAT3 inhibition promoted Th1 skewing by counterbalancing the CpG-induced Th2/regulatory T cell response and significantly enhanced Mammaglobin-A specific T cell reactivity., Conclusion: Ex vivo immune modulation of the SLN by CpG-B and simultaneous JAK2/STAT3 inhibition can effectively overcome BrC-induced immune suppression by preferential activation of LNR DC, ultimately restoring type 1-mediated antitumor immunity, thereby securing a BrC-specific T cell response. These findings provide a clear rationale for clinical exploration of SLN-immune potentiation through local CpG/STAT3i administration in patients with BrC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Immune regulated pathways influence both breast cancer (BrC) development and response to (neo)adjuvant chemotherapy. The sentinel lymph node (SLN), as the first metastatic site, is also the first site where BrC-induced suppression of immune effector subsets occurs. Since intricate knowledge of the phenotypic and functional status of these immune effector subsets is lacking, we set out to map the immune landscape of BrC SLN., Methods: Viable LN cells from BrC SLN (n = 58) were used for detailed flowcytometry-assisted mapping of the immune landscape of BrC SLN in a comparative analysis with healthy (i.e. prophylactic mastectomy-derived) axillary lymph nodes (HLN, n = 17). Findings were related to clinicopathological characteristics., Results: Our data show that BrC-induced immune suppression in tumor-involved SLN, as evidenced by increased Treg and MDSC rates as well as by a generalized state of T cell anergy, coincides with hampered activation of LN-resident (LNR) dendritic cell (DC) subsets rather than of migratory DC subsets. Importantly, suppression of these LN-resident DC subsets preceded profoundly disabled T cell effector functions in tumor-involved SLN. Furthermore, we provide evidence that the suppressed state of LNR-cDC is not only related to nodal involvement but is also related to high-risk breast cancer subtypes that lack expression of hormone receptors and may be a negative predictor of disease-free survival., Conclusion: These data thus provide new insights in the mechanisms underlying loco-regional immune suppression induced by BrC and how these relate to clinical outcome. They identify the LNR-cDC subset as a pivotal regulatory node in cellular immune suppressive pathways and therefore as a promising therapeutic target to combat immune suppression and secure the induction of effective antitumor immunity, e.g. in combination with neo-adjuvant chemotherapy. .

Background: Tumors avoid destruction by cytotoxic T cells (CTL) and natural killer (NK) cells by downregulation of classical human leukocyte antigens (HLA) and overexpression of non-classical HLA. This is the first study to investigate HLA expression in relation to histology (squamous cell carcinoma (SCC) vs. adenocarcinoma (AC)), clinicopathological parameters and survival in a large cervical cancer patient cohort., Methods: Classical (HLA-A and HLA-B/C)- and non-classical HLA molecules (HLA-E and HLA-G) were studied on primary tumors and paired lymph node (LN) metastases from cervical cancer patients ( n  = 136) by immunohistochemistry. The Chi 2 test was used for the comparison of clinicopathological characteristics between SCC and AC patients. The Related-Samples Wilcoxon Signed Rank test was used to compare HLA expression between the primary tumor and metastasis in LN. Patient survival rates were analyzed by Kaplan-Meier curves and Log Rank test. The Mann-Whitney U Test was used to compare the distribution of HLA class I expression between SCC and AC., Results: Decreased expression of HLA-A (SCC P  < 0.001), HLA-B/C (SCC P  < 0.01; AC P  < 0.01) and total classical HLA (SCC P  < 0.001; AC P  = 0.02) was apparent in metastatic tumor cells compared to the primary tumor. In primary SCC, there was a clear trend towards complete loss of HLA-A ( P  = 0.05). SCC metastases showed more complete loss of HLA-A, while AC metastases showed more complete loss of HLA-B/C ( P  = 0.04). In addition, tumor size and parametrium involvement were also related to aberrant HLA class I expression. No significant associations between HLA expression and disease-specific (DSS) or disease-free survival (DFS) were found in this advanced disease cohort. However, in the SCC group, samples showing loss of HLA-A or loss of total classical HLA but positive for HLA-G were linked to poor patient survival (DSS P  = 0.001 and P  = 0.01; DFS P  = 0.003 and P  = 0.01, for HLA-A and total classical HLA, respectively)., Conclusion: These results strengthen the idea of tumor immune escape variants leading to metastasis. Moreover, SCC tumors showing downregulation of HLA-A or total classical HLA in combination with HLA-G expression had poor prognosis. Our findings warrant further analysis of HLA expression as a biomarker for patient selection for CTL- and NK- cell based immunotherapeutic intervention.

Background: Cancer-related disturbances in myeloid lineage development, marked by high levels of myeloid-derived suppressor cells (MDSC) and impaired dendritic cell (DC) development, are associated with poor clinical outcome due to immune escape and therapy resistance. Redressing this balance may therefore be of clinical benefit. Here we investigated the effects of combined Prostate GVAX/ipilimumab immunotherapy on myeloid subsets in peripheral blood of castration-resistant prostate cancer (CRPC) patients as well as the putative predictive value of baseline and on-treatment myeloid parameters on clinical outcome., Methods: Patients with CRPC (n = 28) received thirteen intradermal administrations of Prostate GVAX, consisting of two allogeneic GM-CSF-transduced and irradiated prostate cancer cell lines (LN-CaP and PC3) and six infusions of escalating doses of anti-CTLA4/ipilimumab. Frequencies and activation status of peripheral blood DC (PBDC) and MDSC were determined before, during and after treatment by flowcytometric analysis and related to clinical benefit., Results: Significant treatment-induced activation of conventional and plasmacytoid DC subsets (cDC and pDC) was observed, which in the case of BDCA1/CD1c(+) cDC1 and MDC8(+)/6-sulfoLacNAc(+) inflammatory cDC3 was associated with significantly prolonged overall survival (OS), but also with the development of autoimmune-related adverse events. High pre-treatment levels of CD14(+)HLA-DR(-)monocytic MDSC (mMDSC) were associated with reduced OS. Unsupervised clustering of these myeloid biomarkers revealed particular survival advantage in a group of patients with high treatment-induced PBDC activation and low pretreatment frequencies of suppressive mMDSC in conjunction with our previously identified lymphoid biomarker of high pretreatment CD4(+)CTLA4(+) T cell frequencies., Conclusions: Our data demonstrate that DC and MDSC subsets are affected by prostate GVAX/ipilimumab therapy and that myeloid profiling may contribute to the identification of patients with possible clinical benefit of Prostate GVAX/ipilimumab treatment.