Correction: Single-cell RNAseq and longitudinal proteomic analysis of a novel semi-spontaneous urothelial cancer model reveals tumor cell heterogeneity and pretumoral urine protein alterations.

[This corrects the article DOI: 10.1371/journal.pone.0253178.].

MALT1 inhibition suppresses antigen-specific T cell responses.

The aim of this study was to assess the potential use of a selective small molecule MALT1 inhibitor in solid tumor treatment as an immunotherapy targeting regulatory T-cells (Tregs). In vitro, MALT1 inhibition suppressed the proteolytic cleavage of the MALT1-substrate HOIL1 and blocked IL-2 secretion in Jurkat cells. It selectively suppressed the proliferation of PBMC-derived Tregs, with no effect on conventional CD4+T-cells. In vivo, however, no evident anti-tumor effect was achieved by MALT1 inhibition monotherapy or in combination with anti-CTLA4 in the MB49 cancer model. Despite decreased Treg-frequencies in lymph nodes of tumor-bearing animals, intratumoral Treg depletion was not observed. We also showed that MALT1-inhibition caused a reduction of antigen-specific CD8+T-cells in an adoptive T-cell transfer model. Thus, selective targeting of Tregs would be required to improve the immunotherapeutic effect of MALT1-inhibition. Also, various dosing schedules and combination therapy strategies should be carefully designed and evaluated further.

Long-term effects of immunotherapy with a brain penetrating Aß antibody in a mouse model of Alzheimer's disease.

BACKGROUND: Brain-directed immunotherapy is a promising strategy to target amyloid-ß (Aß) deposits in Alzheimer's disease (AD). In the present study, we compared the therapeutic efficacy of the Aß protofibril targeting antibody RmAb158 with its bispecific variant RmAb158-scFv8D3, which enters the brain by transferrin receptor-mediated transcytosis. METHODS: AppNL-G-F knock-in mice received RmAb158, RmAb158-scFv8D3, or PBS in three treatment regimens. First, to assess the acute therapeutic effect, a single antibody dose was given to 5 months old AppNL-G-F mice, with evaluation after 3 days. Second, to assess the antibodies' ability to halt the progression of Aß pathology, 3 months old AppNL-G-F mice received three doses during a week, with evaluation after 2 months. Reduction of RmAb158-scFv8D3 immunogenicity was explored by introducing mutations in the antibody or by depletion of CD4+ T cells. Third, to study the effects of chronic treatment, 7-month-old AppNL-G-F mice were CD4+ T cell depleted and treated with weekly antibody injections for 8 weeks, including a final diagnostic dose of [125I]RmAb158-scFv8D3, to determine its brain uptake ex vivo. Soluble Aß aggregates and total Aß42 were quantified with ELISA and immunostaining. RESULTS: Neither RmAb158-scFv8D3 nor RmAb158 reduced soluble Aß protofibrils or insoluble Aß1-42 after a single injection treatment. After three successive injections, Aß1-42 was reduced in mice treated with RmAb158, with a similar trend in RmAb158-scFv8D3-treated mice. Bispecific antibody immunogenicity was somewhat reduced by directed mutations, but CD4+ T cell depletion was used for long-term therapy. CD4+ T cell-depleted mice, chronically treated with RmAb158-scFv8D3, showed a dose-dependent increase in blood concentration of the diagnostic [125I]RmAb158-scFv8D3, while concentration was low in plasma and brain. Chronic treatment did not affect soluble Aß aggregates, but a reduction in total Aß42 was seen in the cortex of mice treated with both antibodies. CONCLUSIONS: Both RmAb158 and its bispecific variant RmAb158-scFv8D3 achieved positive effects of long-term treatment. Despite its ability to efficiently enter the brain, the benefit of using the bispecific antibody in chronic treatment was limited by its reduced plasma exposure, which may be a result of interactions with TfR or the immune system. Future research will focus in new antibody formats to further improve Aß immunotherapy.

Long-term SARS-CoV-2-specific and cross-reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology.

BACKGROUND: Cellular immune memory responses post coronavirus disease 2019 (COVID-19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large-scale long-term follow-up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific peptide pool that contains no overlapping peptides with endemic human coronaviruses. METHODS: Peptide stimulated memory T cell responses were assessed with dual interferon-gamma (IFNγ) and interleukin (IL)-2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array. RESULTS: Our work demonstrates that long-term SARS-CoV-2-specific memory T cell responses feature dual IFNγ and IL-2 responses, whereas cross-reactive memory T cell responses primarily generate IFNγ in response to SARS-CoV-2 peptide stimulation. T cell responses correlated to long-term humoral immune responses. Disease severity as well as specific COVID-19 symptoms correlated with the magnitude of the SARS-CoV-2-specific memory T cell response four to five months post seroconversion. CONCLUSION: Using a large cohort and a SARS-CoV-2-specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS-CoV-2-specific memory T cell responses.

Single-cell RNAseq and longitudinal proteomic analysis of a novel semi-spontaneous urothelial cancer model reveals tumor cell heterogeneity and pretumoral urine protein alterations.

Bladder cancer, one of the most prevalent malignancies worldwide, remains hard to classify due to a staggering molecular complexity. Despite a plethora of diagnostic tools and therapies, it is hard to outline the key steps leading up to the transition from high-risk non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC). Carcinogen-induced murine models can recapitulate urothelial carcinogenesis and natural anti-tumor immunity. Herein, we have developed and profiled a novel model of progressive NMIBC based on 10 weeks of OH-BBN exposure in hepatocyte growth factor/cyclin dependent kinase 4 (R24C) (Hgf-Cdk4R24C) mice. The profiling of the model was performed by histology grading, single cell transcriptomic and proteomic analysis, while the derivation of a tumorigenic cell line was validated and used to assess in vivo anti-tumor effects in response to immunotherapy. Established NMIBC was present in females at 10 weeks post OH-BBN exposure while neoplasia was not as advanced in male mice, however all mice progressed to MIBC. Single cell RNA sequencing analysis revealed an intratumoral heterogeneity also described in the human disease trajectory. Moreover, although immune activation biomarkers were elevated in urine during carcinogen exposure, anti-programmed cell death protein 1 (anti-PD1) monotherapy did not prevent tumor progression. Furthermore, anti-PD1 immunotherapy did not control the growth of subcutaneous tumors formed by the newly derived urothelial cancer cell line. However, treatment with CpG-oligodeoxynucleotides (ODN) significantly decreased tumor volume, but only in females. In conclusion, the molecular map of this novel preclinical model of bladder cancer provides an opportunity to further investigate pharmacological therapies ahead with regards to both targeted drugs and immunotherapies to improve the strategies of how we should tackle the heterogeneous tumor microenvironment in urothelial bladder cancer to improve responses rates in the clinic.

Wide-Ranging Effects on the Brain Proteome in a Transgenic Mouse Model of Alzheimer's Disease Following Treatment with a Brain-Targeting Somatostatin Peptide.

Alzheimer's disease is the most common neurodegenerative disorder characterized by the pathological aggregation of amyloid-ß (Aß) peptide. A potential therapeutic intervention in Alzheimer's disease is to enhance Aß degradation by increasing the activity of Aß-degrading enzymes, including neprilysin. The somatostatin (SST) peptide has been identified as an activator of neprilysin. Recently, we demonstrated the ability of a brain-penetrating SST peptide (SST-scFv8D3) to increase neprilysin activity and membrane-bound Aß42 degradation in the hippocampus of mice overexpressing the Aß-precursor protein with the Swedish mutation (APPswe). Using LC-MS, we further evaluated the anti-Alzheimer's disease effects of SST-scFv8D3. Following a triple intravenous injection of SST-scFv8D3, the LC-MS analysis of the brain proteome revealed that the majority of downregulated proteins consisted of mitochondrial proteins regulating fatty acid oxidation, which are otherwise upregulated in APPswe mice compared to wild-type mice. Moreover, treatment with SST-scFv8D3 significantly increased hippocampal levels of synaptic proteins regulating cell membrane trafficking and neuronal development. Finally, hippocampal concentrations of growth-regulated α (KC/GRO) chemokine and degradation of neuropeptide-Y were elevated after SST-scFv8D3 treatment. In summary, our results demonstrate a multifaceted effect profile in regulating mitochondrial function and neurogenesis following treatment with SST-scFv8D3, further suggesting the development of Alzheimer's disease therapies based on SST peptides.