Background There is an increasing interest in local tumor ablative treatment modalities that induce immunogenic cell death and the generation of antitumor immune responses. Methods We report six recurrent glioblastoma patients who were treated with intracavitary thermotherapy after coating the resection cavity wall with superparamagnetic iron oxide nanoparticles (“NanoPaste” technique). Patients underwent six 1-h hyperthermia sessions in an alternating magnetic field and, if possible, received concurrent fractionated radiotherapy at a dose of 39.6 Gy. Results There were no major side effects during active treatment. However, after 2–5 months, patients developed increasing clinical symptoms. CT scans showed tumor flare reactions with prominent edema around nanoparticle deposits. Patients were treated with dexamethasone and, if necessary, underwent re-surgery to remove nanoparticles. Histopathology revealed sustained necrosis directly adjacent to aggregated nanoparticles without evidence for tumor activity. Immunohistochemistry showed upregulation of Caspase-3 and heat shock protein 70, prominent infiltration of macrophages with ingested nanoparticles and CD3⁺ T-cells. Flow cytometric analysis of freshly prepared tumor cell suspensions revealed increased intracellular ratios of IFN-γ to IL-4 in CD4⁺ and CD8⁺ memory T cells, and activation of tumor-associated myeloid cells and microglia with upregulation of HLA-DR and PD-L1. Two patients had long-lasting treatment responses > 23 months without receiving any further therapy. Conclusion Intracavitary thermotherapy combined with radiotherapy can induce a prominent inflammatory reaction around the resection cavity which might trigger potent antitumor immune responses possibly leading to long-term stabilization of recurrent GBM patients. These results warrant further investigations in a prospective phase-I trial.

Myeloid-derived suppressor cells (MDSCs) comprise a heterogeneous population of myeloid cells that are significantly expanded in cancer patients and are associated with tumor progression. Multicolor flow cytometry was used to study the frequency, phenotype, and function of MDSCs in peripheral blood and freshly resected tumors of 52 participants with primary glioblastoma (GBM). The frequency of CD14^highCD15^pos monocytic and CD14^lowCD15^pos granulocytic MDSCs was significantly higher in peripheral blood of GBM participants compared with healthy donors. The majority of granulocytic MDSCs consisted of CD14^lowCD15^high neutrophilic MDSCs with high T-cell suppressive capacities. At the tumor side, we found an increase in CD14^highCD15^pos monocytic MDSCs and high frequencies of CD14^lowCD15^pos granulocytic MDSCs that displayed an activated phenotype with downregulation of CD16 and upregulation of HLA-DR molecules, which did not inhibit T-cell proliferative responses in vitro. However, a strong association between granulocytic MDSCs and CD4⁺ effector memory T-cells (T_EM) within the tumors was detected. Tumor-derived CD4⁺ T_EM expressed high levels of PD-1 when compared with their blood-derived counterparts and were functionally exhausted. The respective ligand, PD-L1, was significantly upregulated on tumor-derived MDSCs, and T-cell co-culture experiments confirmed that glioma-infiltrating MDSCs can induce PD-1 expression on CD4⁺ T_EM. Our findings provide a detailed characterization of different MDSC subsets in GBM patients and indicate that both granulocytic MDSCs in peripheral blood and at the tumor site play a major role in GBM-induced T-cell suppression.

Lactate formation in highly proliferative tumors such as malignant gliomas is associated with poor survival and contributes to the suppression of local immunity. Here, we report that diclofenac used at nontoxic concentrations significantly decreased lactate production in murine glioma cells and inhibited the expression of lactate dehydrogenase‐A in vitro. Lactate reduction was accompanied by a dose‐dependent inhibition of cell growth and a cell cycle arrest at the G2/M checkpoint. In the presence of diclofenac, murine bone marrow‐derived dendritic cells (DCs) showed enhanced IL‐12, but decreased IL‐10 secretion on Toll‐like receptor stimulation with R848 that correlated with reduced lactate levels in the glioma cell coculture and a blockade of signal transducers and activators of transcription 3 phosphorylation. In vivo, diclofenac treatment diminished intratumoral lactate levels and resulted in a significant delay of glioma growth. Ex vivo analyses revealed that tumor‐infiltrating DCs regained their capacity to produce IL‐12 on R848 stimulation. Moreover, diclofenac reduced the number of tumor‐infiltrating regulatory T cells and impaired the upregulation of the Treg activation marker CD25. Nevertheless, a single intratumoral injection of R848 combined with diclofenac failed to induce an additional survival advantage in glioma‐bearing mice. Further analyses illustrated that the presence of diclofenac during T‐cell activation compromised INF‐γ production and T‐cell proliferation, indicating that immunotherapeutic approaches have to be carefully timed when combined with diclofenac. In summary, diclofenac appears as an attractive agent for targeting lactate production and counteracting local immune suppression in malignant gliomas.

Substitution of lysine 27 to methionine in histone H3 (H3K27M) defines an aggressive subtype of diffuse glioma. Previous studies have shown that a H3K27M-specific long peptide vaccine (H3K27M-vac) induces mutation-specific immune responses that control H3K27M⁺ tumors in major histocompatibility complex-humanized mice. Here we describe a first-in-human treatment with H3K27M-vac of eight adult patients with progressive H3K27M⁺ diffuse midline glioma on a compassionate use basis. Five patients received H3K27M-vac combined with anti-PD-1 treatment based on physician’s discretion. Repeat vaccinations with H3K27M-vac were safe and induced CD4⁺ T cell-dominated, mutation-specific immune responses in five of eight patients across multiple human leukocyte antigen types. Median progression-free survival after vaccination was 6.2 months and median overall survival was 12.8 months. One patient with a strong mutation-specific T cell response after H3K27M-vac showed pseudoprogression followed by sustained complete remission for >31 months. Our data demonstrate safety and immunogenicity of H3K27M-vac in patients with progressive H3K27M⁺ diffuse midline glioma.

Background: Primary central nervous system lymphoma (PCNSL) is a rare lymphoma of the central nervous system, usually of diffuse large B cell phenotype. Stereotactic biopsy followed by histopathology is the diagnostic standard. However, limited material is available from CNS biopsies, thus impeding an in-depth characterization of PCNSL. Methods: We performed flow cytometry, single-cell RNA sequencing, and B cell receptor sequencing of PCNSL cells released from biopsy material, blood, and cerebrospinal fluid (CSF), and spatial transcriptomics of biopsy samples. Results: PCNSL-released cells were predominantly activated CD19⁺CD20⁺CD38⁺CD27⁺ B cells. In single-cell RNA sequencing, PCNSL cells were transcriptionally heterogeneous, forming multiple malignant B cell clusters. Hyperexpanded B cell clones were shared between biopsy- and CSF- but not blood-derived cells. T cells in the tumor microenvironment upregulated immune checkpoint molecules, thereby recognizing immune evasion signals from PCNSL cells. Spatial transcriptomics revealed heterogeneous spatial organization of malignant B cell clusters, mirroring their transcriptional heterogeneity across patients, and pronounced expression of T cell exhaustion markers, co-localizing with a highly malignant B cell cluster. Conclusions: Malignant B cells in PCNSL show transcriptional and spatial intratumor heterogeneity. T cell exhaustion is frequent in the PCNSL microenvironment, co-localizes with malignant cells, and highlights the potential of personalized treatments.

Objective: HIV‐associated neurocognitive disorders (HAND) remain a challenge despite combination antiretroviral therapy (cART). Immune cell activation has been implicated to play a major role in the development of HAND.Methods: In this study, we used multicolor flow cytometry on peripheral blood (PB) and cerebrospinal fluid (CSF) samples to determine the expression of HLA‐DR and programmed death‐1 (PD‐1) on CD4+ and CD8+ T cells in patients with chronic HIV infection. Expression levels were correlated with HI virus load in PB and CSF, classification of HAND and severity of magnetic resonance imaging (MRI) signal abnormalities.Results: In a cohort of 86 HIV patients we found that the grade of neurocognitive impairment and the severity of MRI signal abnormalities correlated with decreasing CD4/CD8‐ratios and increased frequencies of HLA‐DR expressing CD4+ and CD8+ T cells reaching the highest values in the CSF samples. Importantly, HLA‐DR upregulation was still detectable in virologically suppressed HIV patients. Further, T‐cell subpopulation analysis of 40 HIV patients showed a significant shift from naïve to effector memory (EM) T cells that was negatively correlated with the grade of neurocognitive impairment in the PB samples. Moreover, PD‐1 was significantly increased on CD4+ memory T cells with highest levels on EM T cells in HIV patients with mild or severe neurocognitive alterations.Interpretation: The CD4/CD8 ratio, the proportion of EM to naïve T cells and the immune activation profile of CD4+ and CD8+ T cells in PB and CSF might be useful parameters to monitor the efficacy of cART and to identify HIV patients at risk of further neurocognitive deterioration.