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Objective: Progressive supranuclear palsy (PSP) is a 4R‐tauopathy showing heterogeneous tau cytopathology commencing in the globus pallidus (GP) and the substantia nigra (SN), regions also associated with age‐related iron accumulation. Abnormal iron levels have been extensively associated with tau pathology in neurodegenerative brains, however, its role in PSP pathogenesis remains yet unknown. We perform the first cell type‐specific evaluation of PSP iron homeostasis and the closely related oxygen homeostasis, in relation to tau pathology in human postmortem PSP brains. Methods: In brain regions vulnerable to PSP pathology (GP, SN, and putamen), we visualized iron deposition in tau‐affected and unaffected neurons, astroglia, oligodendrocytes, and microglia, using a combination of iron staining with immunolabelling. To further explore molecular pathways underlying our observations, we examined the expression of key iron and oxygen homeostasis mRNA transcripts and proteins. Results: We found astrocytes as the major cell type accumulating iron in the early affected regions of PSP, highly associated with cellular tau pathology. The same regions are affected by dysregulated expression of alpha and beta hemoglobin and neuroglobin showing contrasting patterns. We discovered changes in iron and oxygen homeostasis‐related gene expression associated with aging of the brain, and identified dysregulated expression of rare neurodegeneration with brain iron accumulation (NBIA) genes associated with tau pathology to distinguish PSP from the healthy aging brain. Interpretation: We present novel aspects of PSP pathophysiology highlighting an overlap with NBIA pathways. Our findings reveal potential novel targets for therapy development and have implications beyond PSP for other iron‐associated neurodegenerative diseases. ANN NEUROL 2023;93:431–445 [ABSTRACT FROM AUTHOR]

The precise mechanisms by which the complement system contributes to the establishment of an immunosuppressive tumor microenvironment (TME) and promotes tumor progression remain unclear. In this study, we investigated the expression and function of complement C5a receptor 1 (C5aR1) in human and mouse cancer-associated dendritic cells (DCs). First, we observed an overexpression of C5aR1 in tumor-infiltrating DCs, compared to DCs from blood or spleen. C5aR1 expression was restricted to type 2 conventional DCs (cDC2) and monocyte-derived DCs (moDCs), which displayed a tolerogenic phenotype capable of inhibiting T-cell activation and promoting tumor growth. C5aR1 engagement in DCs drove their migration from tumors to tumor-draining lymph nodes, where C5a levels were higher. We used this knowledge to optimize an anticancer therapy aimed at enhancing DC activity. In three syngeneic tumor models, C5aR1 inhibition significantly enhanced the efficacy of poly I:C, a Toll-like receptor 3 (TLR3) agonist, in combination with PD-1/PD-L1 blockade. The contribution of C5aR1 inhibition to the antitumor activity of the combination treatment relied on type 1 conventional DCs (cDC1s) and antigen-specific CD8+ T cells, required lymphocyte egress from secondary lymphoid organs, and was associated with an increase in interferon gamma (IFNγ) signaling. In conclusion, our study highlights the importance of the C5a/C5aR1 axis in the biology of cancer-associated DCs and provides compelling evidence for the therapeutic potential of modulating the complement system to enhance DC-mediated immune responses against tumors.

Background: Immunodeficient mice engrafted with peripheral blood mononuclear cells (PBMCs) are models to study new cancer immunotherapy agents. However, this approach is associated with xenograft-versus-host disease (xGVHD), which starts early after PBMC transfer and limits the duration and interpretation of experiments. Here, we explore different approaches to overcome xGVHD and better support the development of cancer immunotherapies., Methods: Immunodeficient NOD-scid IL2Rg null (NSG) mice were intravenously transferred with human PBMCs and subcutaneously co-engrafted with HT29 human colon carcinoma cells. Diverse strategies to reduce xGVHD while preserving the antitumor activity of human immune cells were evaluated: (1) ex vivo immune graft modification by depleting CD4 + T cells pre-transfer using magnetic beads, (2) post-transplantation cyclophosphamide administration to eliminate proliferating xenoreactive T-cell clones and (3) using major histocompatibility complex (MHC) class I and II-deficient NSG mice: (K b D b ) null (IA) null (MHC-dKO NSG). Body weight and plasma murine alanine aminotransferase levels were measured as indicators of xGVHD and tumor size was measured every 2-3 days to monitor antitumor activity. The antitumor effects and pharmacodynamics of nivolumab plus ipilimumab and an anti-epithelial cell adhesion molecule (EpCAM)/CD3 T-cell engager (αEpCAM/CD3 bispecific antibody (BsAb)) were evaluated in the model., Results: CD4 + T-cell depletion attenuates xGVHD but also abrogates the antitumor activity. Cyclophosphamide limits the antitumor response and does not substantially prevent xGVHD. In contrast, xGVHD was significantly attenuated in MHC-dKO NSG recipients, while the antitumor effect of human PBMCs was preserved. Furthermore, the administration of nivolumab plus ipilimumab caused exacerbated xGVHD in conventional NSG mice, thereby precluding the observation of their antitumor effects. Severe xGVHD did not occur in MHC-dKO NSG mice thus enabling the study of complete and durable tumor rejections. Similarly, NSG mice treated with an αEpCAM/CD3 BsAb showed complete tumor regressions, but died due to xGVHD. In contrast, MHC-dKO NSG mice on treatment with the αEpCAM/CD3 BsAb achieved complete tumor responses without severe xGVHD. A significant proportion of mice rendered tumor-free showed tumor rejection on rechallenge with HT29 cells without further treatment. Finally, tumor-infiltrating CD8 + T-cell number increase, activation and CD137 upregulation were observed on αEpCAM/CD3 BsAb treatment., Conclusion: Humanized MHC-dKO immunodeficient mice allow and refine the preclinical testing of immunotherapy agents for which experimentation is precluded in conventional immunodeficient mice due to severe xGVHD., Competing Interests: Competing interests: IE-S, EFdP, GC, PM-M, MC, DC, IA-B, IRL, RS-V, CEDA, LÁ-G and PB declare no conflicts of interest. IM reports grants and personal fees from Genmab during the conduct of the study, as well as grants and personal fees from Bristol Myers Squibb, Roche, AstraZeneca, and Pharmamar and personal fees from F-Star, Numab, Pieris, Boehringer Ingelheim, Gossamer, Alligator, Hotspot, Biolinerx, Bioncotech, Dompe, Highlight Therapeutics, Bright Peaks and Boston Therapeutics outside the submitted work. MFS reports grants from Bristol Myers Squibb and Roche during the conduct of the study, as well as grants and personal fees from Roche and Bristol Myers Squibb, and personal fees from Numab outside the submitted work., (© 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: Activating and inhibitory receptors of natural killer (NK) cells such as NKp, NKG2, or CLEC are highly relevant to cold tumors including glioblastoma (GBM). Here, we aimed to characterize the expression of these receptors in GBM to gain insight into their potential role as modulators of the intratumoral microenvironment., Methods: We performed a transcriptomic analysis of several NK receptors with a focus on the activating receptor encoded by KLRC2, NKG2C, among bulk and single-cell RNA sequencing GBM data sets. We also evaluated the effects of KLRC2-overexpressing GL261 cells in mice treated with or without programmed cell death protein-1 (PD-1) monoclonal antibody (mAb). Finally, we analyzed samples from two clinical trials evaluating PD-1 mAb effects in patients with GBM to determine the potential of NKG2C to serve as a biomarker of response., Results: We observed significant expression of several inhibitory NK receptors on GBM-infiltrating NK and T cells, which contrasts with the strong expression of KLRC2 on tumor cells, mainly at the infiltrative margin. Neoplastic KLRC2 expression was associated with a reduction in the number of myeloid-derived suppressor cells and with a higher level of tumor-resident lymphocytes. A stronger antitumor activity after PD-1 mAb treatment was observed in NKG2C high -expressing tumors both in mouse models and patients with GBM whereas the expression of inhibitory NK receptors showed an inverse association., Conclusions: This study explored the role of neoplastic NKG2C/ KLRC2 expression in shaping the immune profile of GBM and suggests that it is a predictive biomarker for positive responses to immune checkpoint inhibitor treatment in patients with GBM. Future studies could further validate this finding in prospective trials., Competing Interests: Competing interests: MER-R reports personal fees from BMS and grants from Highlight-Therapeutics and Roche outside the submitted work. She also has received speaker’s bureau honoraria from BMS and ROCHE. The rest of the authors declare that they have 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.)

Introduction: The tissue immune microenvironment is associated with key aspects of tumor biology. The interaction between the immune system and cancer cells has predictive and prognostic potential across different tumor types. Spatially resolved tissue-based technologies allowed researchers to simultaneously quantify different immune populations in tumor samples. However, bare quantification fails to harness the spatial nature of tissue-based technologies. Tumor-immune interactions are associated with specific spatial patterns that can be measured. In recent years, several computational tools have been developed to increase our understanding of these spatial patterns., Topics Covered: In this review, we cover standard techniques as well as new advances in the field of spatial analysis of the immune microenvironment. We focused on marker quantification, spatial intratumor heterogeneity analysis, cell‒cell spatial interaction studies and neighborhood analyses., Competing Interests: Competing interests: No, there are 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.)

Purpose: The aim of our study was to elucidate the impact of bevacizumab added to neoadjuvant chemotherapy (NACT) on the tumor immune microenvironment and correlate the changes with the clinical outcome of the patients., Experimental Design: IHC and multiplex immunofluorescence for lymphoid and myeloid lineage markers were performed in matched tumor samples from 23 patients with ovarian cancer enrolled in GEICO 1205/NOVA clinical study before NACT and at the time of interval cytoreductive surgery., Results: Our results showed that the addition of bevacizumab to NACT plays a role mainly on lymphoid populations at the stromal compartment, detecting a significant decrease of CD4+ T cells, an increase of CD8+ T cells, and an upregulation in effector/regulatory cell ratio (CD8+/CD4+FOXP3+). None of the changes observed were detected in the intra-epithelial site in any arm (NACT or NACT-bevacizumab). No differences were found in myeloid lineage (macrophage-like). The percentage of Treg populations and effector/regulatory cell ratio in the stroma were the only two variables significantly associated with progression-free survival (PFS)., Conclusions: The addition of bevacizumab to NACT did not have an impact on PFS in the GEICO 1205 study. However, at the cellular level, changes in CD4+, CD8+ lymphocyte populations, and CD8+/CD4+FOXP3 ratio have been detected only at the stromal site. On the basis of our results, we hypothesize about the existence of mechanisms of resistance that could prevent the trafficking of T-effector cells into the epithelial component of the tumor as a potential explanation for the lack of efficacy of ICI in the first-line treatment of advanced epithelial ovarian cancer. See related commentary by Soberanis Pina and Oza, p. 12., (©2023 The Authors; Published by the American Association for Cancer Research.)

IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy., (©2022 American Association for Cancer Research.)

Background: Despite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear., Methods: To better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8 + and CD4 + EC - resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8 + and CD4 + TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8 + and CD4 + tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival., Results: We found that CD8 + TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1 - and PD-1 dim , respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8 + PD-1 dim CD39 + and PD-1 hi CD39 + T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4 + T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1 hi compared with PD-1 - or PD-1 dim T cells, and in fact, it was the CD4 + PD-1 hi subpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8 + PD-1 hi CD39+ and CD4 + PD-1 hi tumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1 hi , CD39, CXCL13 and CD103 by CD8 + TILs and PD-1 hi and CXCL13 by CD4 + Tconv TILs, correlated with prolonged survival of patients with EC., Conclusions: Our results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1 hi and CD39 or PD-1 hi can be used to select and expand CD8 + and CD4 + tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3 + , CD8 + and CD4 + lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity., Competing Interests: Competing interests: AG is a member of the scientific advisory board (SAB) of Achilles Therapeutics, SingulaBIO, RootPath, and BioNTech SE, and consults for PACT Pharma, and Instil BIo. AG is co-inventor of patents licensed and with royalties related to this work E-059-2013/0, E-085-2013/0, E-149-2015/0. IM reports grants from Roche, BMS, AstraZeneca and Genmab, as well as consultancy activities for Roche, BMS, AZ, Phamamar, F-sar, Merus, Amunix, Pieris, Numab, Third Rock and Highlight therapeutics. The authors declare that no other conflict of interest exists., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)