Novel, Non-Gene-Destructive Knock-In Reporter Mice Refute the Concept of Monoallelic Gata3 Expression.

Accurately tuned expression levels of the transcription factor GATA-3 are crucial at several stages of T cell and innate lymphoid cell development and differentiation. Moreover, several lines of evidence suggest that Gata3 expression might provide a reliable molecular marker for the identification of elusive progenitor cell subsets at the earliest stages of T lineage commitment. To be able to faithfully monitor Gata3 expression noninvasively at the single-cell level, we have generated a novel strain of knock-in reporter mice, termed GATIR, by inserting an expression cassette encoding a bright fluorescent marker into the 3'-untranslated region of the endogenous Gata3 locus. Importantly, in contrast to three previously published strains of Gata3 reporter mice, GATIR mice preserve physiological Gata3 expression on the targeted allele. In this study, we show that GATIR mice faithfully reflect endogenous Gata3 expression without disturbing the development of GATA-3-dependent lymphoid cell populations. We further show that GATIR mice provide an ideal tool for noninvasive monitoring of Th2 polarization and straightforward identification of innate lymphoid cell 2 progenitor populations. Finally, as our reporter is non-gene-destructive, GATIR mice can be bred to homozygosity, not feasible with previously published strains of Gata3 reporter mice harboring disrupted alleles. The availability of hetero- and homozygous Gata3 reporter mice with an exceptionally bright fluorescent marker, allowed us to visualize allelic Gata3 expression in individual cells simply by flow cytometry. The unambiguous results obtained provide compelling evidence against previously postulated monoallelic Gata3 expression in early T lineage and hematopoietic stem cell subsets.

Multi-omics analysis of overexpressed tumor-associated proteins: gene expression, immunopeptide presentation, and antibody response in oropharyngeal squamous cell carcinoma, with a focus on cancer-testis antigens.

Introduction: The human leukocyte antigen complex (HLA) is essential for inducing specific immune responses to cancer by presenting tumor-associated peptides (TAP) to T cells. Overexpressed tumor associated antigens, mainly cancer-testis antigens (CTA), are outlined as essential targets for immunotherapy in oropharyngeal squamous cell carcinoma (OPSCC). This study assessed the degree to which presentation, gene expression, and antibody response (AR) of TAP, mainly CTA, are correlated in OPSCC patients to evaluate their potential as immunotherapy targets. Materials and methods: Snap-frozen tumor (NLigand/RNA=40), healthy mucosa (NRNA=6), and healthy tonsils (NLigand=5) samples were obtained. RNA-Seq was performed using Illumina HiSeq 2500/NovaSeq 6000 and whole exome sequencing (WES) utilizing NextSeq500. HLA ligands were isolated from tumor tissue using immunoaffinity purification, UHPLC, and analyzed by tandem MS. Antibodies were measured in serum (NAb=27) utilizing the KREX™ CT262 protein array. Data analysis focused on 312 proteins (KREX™ CT262 panel + overexpressed self-proteins). Results: 183 and 94 of HLA class I and II TAP were identified by comparative profiling with healthy tonsils. Genes from 26 TAP were overexpressed in tumors compared to healthy mucosa (LFC>1; FDR<0.05). Low concordance (r=0.25; p<0.0001) was found between upregulated mRNA and class I TAP. The specific mode of correlation of TAP was found to be dependent on clinical parameters. A lack of correlation was observed both between mRNA and class II TAP, as well as between class II tumor-unique TAP (TAP-U) presentation and antibody response (AR) levels. Discussion: This study demonstrates that focusing exclusively on gene transcript levels fails to capture the full extent of TAP presentation in OPSCC. Furthermore, our findings reveal that although CTA are presented at relatively low levels, a few CTA TAP-U show potential as targets for immunotherapy.

Suppression of prostate cancer and amelioration of the immunosuppressive tumor microenvironment through selective immunoproteasome inhibition.

New treatment options to battle hormone-refractory prostate carcinoma (PC) are a pressing medical need. Chronic inflammation has been implicated in PC etiology. The pro-inflammatory cytokines IL-6, IL-23 and IL-17 are key mediators to promote growth of PC. Here, we evaluate the potential of immunoproteasome inhibition for anti-inflammatory and direct anti-tumorigenic therapy of PC. The anti-tumor effect of immunoproteasome inhibitor ONX 0914 was tested in mouse and human PC cells and the in vivo therapeutic efficacy of immunoproteasome inhibition was analyzed in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice in preventive and therapeutic settings and in castration-resistant (CR)PC after castration. Inhibition of the immunoproteasome subunit LMP7 induced apoptotic cell death in PC cell lines. In TRAMP mice, ONX 0914-treatment resulted in significant inhibition of PC growth with a decreased frequency of malignant prostatic lesions and inhibition of metastasis formation. The number of immunosuppressive myeloid cells in PC was greatly reduced in response to ONX 0914. Thus, immunoproteasome inhibition shows remarkable efficacy against PC progression in vivo and impedes tumor recurrence in CRPC-TRAMP mice by blocking the immunosuppressive inflammatory response in the tumor microenvironment. In conclusion, we show that the immunoproteasome is a promising drug target for the treatment of PC.

The ubiquitin-like modifier FAT10 is degraded by the 20S proteasome in vitro but not in cellulo.

Ubiquitin-independent protein degradation via the 20S proteasome without the 19S regulatory particle has gained increasing attention over the last years. The degradation of the ubiquitin-like modifier FAT10 by the 20S proteasome was investigated in this study. We found that FAT10 was rapidly degraded by purified 20S proteasomes in vitro, which was attributed to the weak folding of FAT10 and the N-terminally disordered tail. To confirm our results in cellulo, we established an inducible RNA interference system in which the AAA-ATPase Rpt2 of the 19S regulatory particle is knocked down to impair the function of the 26S proteasome. Using this system, degradation of FAT10 in cellulo was strongly dependent on functional 26S proteasome. Our data indicate that in vitro degradation studies with purified proteins do not necessarily reflect biological degradation mechanisms occurring in cells and, therefore, cautious data interpretation is required when 20S proteasome function is studied in vitro.

Immunoproteasome Inhibition Reduces the T Helper 2 Response in Mouse Models of Allergic Airway Inflammation.

Background: Allergic asthma is a chronic disease and medical treatment often fails to fully control the disease in the long term, leading to a great need for new therapeutic approaches. Immunoproteasome inhibition impairs T helper cell function and is effective in many (auto-) inflammatory settings but its effect on allergic airway inflammation is unknown. Methods: Immunoproteasome expression was analyzed in in vitro polarized T helper cell subsets. To study Th2 cells in vivo acute allergic airway inflammation was induced in GATIR (GATA-3-vYFP reporter) mice using ovalbumin and house dust mite extract. Mice were treated with the immunoproteasome inhibitor ONX 0914 or vehicle during the challenge phase and the induction of airway inflammation was analyzed. Results: In vitro polarized T helper cell subsets (Th1, Th2, Th17, and Treg) express high levels of immunoproteasome subunits. GATIR mice proved to be a useful tool for identification of Th2 cells. Immunoproteasome inhibition reduced the Th2 response in both airway inflammation models. Furthermore, T cell activation and antigen-specific cytokine secretion was impaired and a reduced infiltration of eosinophils and professional antigen-presenting cells into the lung and the bronchoalveolar space was observed in the ovalbumin model. Conclusion: These results show the importance of the immunoproteasome in Th2 cells and airway inflammation. Our data provides first insight into the potential of using immunoproteasome inhibition to target the aberrant Th2 response, e.g. in allergic airway inflammation.