Proteasome alpha-type subunit C9 is a primary target of autoantibodies in sera of patients with myositis and systemic lupus erythematosus

Autoantibodies occur in low frequencies among patients with myositis characterizing only distinct subsets of this disease. Most of these known antibodies are directed to enzymatically active complexes. The 20S proteasome represents an essential cytoplasmatic protein complex for intracellular nonlysosomal protein degradation, and is involved in major histocompatibility complex class I restricted antigen processing. In this study we investigated whether the 20S proteasome complex is an antibody target in myositis and in other autoimmune diseases. 34 sera of poly/dermatomyositis patients were assayed for antiproteasomal antibodies using enzyme-linked immunosorbent assay, immunoblot, and two-dimensional non-equilibrium pH gradient electrophoresis (NEPHGE). Sera was from patients with systemic lupus erythematosus (SLE), mixed connective tissue disease, and rheumatoid arthritis; healthy volunteers served as controls. In 62% (21/34) of the cases sera from patients with myositis and in 58% (30/52) of the cases sera from patients with SLE reacted with the 20S proteasome. These frequencies exceeded those of sera from patients with mixed connective tissue disease, rheumatoid arthritis, and healthy controls. The alpha-type subunit C9 of the 20S proteasome was determined to be the predominant target of the autoimmune sera in myositis and SLE. Lacking other frequent autoantibodies in myositis, the antiproteasome antibodies are the most common humoral immune response so far detected in this disease entity

Preventing tumor escape by targeting a post-proteasomal trimming independent epitope

Adoptive T cell therapy (ATT) can achieve regression of large tumors in mice and humans; however, tumors frequently recur. High target peptide-major histocompatibility complex-I (pMHC) affinity and T cell receptor (TCR)-pMHC affinity are thought to be critical to preventing relapse. Here, we show that targeting two epitopes of the same antigen in the same cancer cells via monospecific T cells, which have similar pMHC and pMHC-TCR affinity, results in eradication of large, established tumors when targeting the apparently subdominant but not the dominant epitope. Only the escape but not the rejection epitope required postproteasomal trimming, which was regulated by IFN-gamma, allowing IFN-gamma-unresponsive cancer variants to evade. The data describe a novel immune escape mechanism and better define suitable target epitopes for ATT

High-affinity T-cell receptor specific for MyD88 L265P mutation for adoptive T-cell therapy of B-cell malignancies

BACKGROUND: Adoptive transfer of engineered T cells has shown remarkable success in B-cell malignancies. However, the most common strategy of targeting lineage-specific antigens can lead to undesirable side effects. Also, a substantial fraction of patients have refractory disease. Novel treatment approaches with more precise targeting may be an appealing alternative. Oncogenic somatic mutations represent ideal targets because of tumor specificity. Mutation-derived neoantigens can be recognized by T-cell receptors (TCRs) in the context of MHC-peptide presentation. METHODS: Here we have generated T-cell lines from healthy donors by autologous in vitro priming, targeting a missense mutation on the adaptor protein MyD88, changing leucine at position 265 to proline (MyD88 L265P), which is one of the most common driver mutations found in B-cell lymphomas. RESULTS: Generated T-cell lines were selectively reactive against the mutant HLA-B*07:02-restricted epitope but not against the corresponding wild-type peptide. Cloned TCRs from these cell lines led to mutation-specific and HLA-restricted reactivity with varying functional avidity. T cells engineered with a mutation-specific TCR (TCR-T cells) recognized and killed B-cell lymphoma cell lines characterized by intrinsic MyD88 L265P mutation. Furthermore, TCR-T cells showed promising therapeutic efficacy in xenograft mouse models. In addition, initial safety screening did not indicate any sign of off-target reactivity. CONCLUSION: Taken together, our data suggest that mutation-specific TCRs can be used to target the MyD88 L265P mutation, and hold promise for precision therapy in a significant subgroup of B-cell malignancies, possibly achieving the goal of absolute tumor specificity, a long sought-after dream of immunotherapy

In vitro proteasome processing of neo-splicetopes does not predict their presentation in vivo

Proteasome catalyzed peptide splicing (PCPS) of cancer-driving antigens could generate attractive neoepitopes to be targeted by TCR-based adoptive T cell therapy. Based on a spliced peptide prediction algorithm TCRs were generated against putative KRAS(G12V) and RAC2(P29L) derived neo-splicetopes with high HLA-A*02:01 binding affinity. TCRs generated in mice with a diverse human TCR repertoire specifically recognized the respective target peptides with high efficacy. However, we failed to detect any neo-splicetope specific T cell response when testing the in vivo neo-splicetope generation and obtained no experimental evidence that the putative KRAS(G12V)- and RAC2(P29L)-derived neo-splicetopes were naturally processed and presented. Furthermore, only the putative RAC2(P29L)-derived neo-splicetopes was generated by in vitro PCPS. The experiments pose severe questions on the notion that available algorithms or the in vitro PCPS reaction reliably simulate in vivo splicing and argue against the general applicability of an algorithm-driven 'reverse immunology' pipeline for the identification of cancer-specific neo-splicetopes

Development and tissue-specific distribution of mouse small heat shock protein hsp25

We have investigated the developmental and tissue-specific distribution of the mouse small hsp25 by immunohistology using an antibody that specifically identifies hsp25. Our analysis shows that the relative amount of hsp25 increases during embryogenesis. Through days 13-20 of embryogenesis, hsp25 accumulation is predominant in the various muscle tissues, including the heart, the bladder, and the back muscles. hsp25 is detectable also in neurons of the spinal cord and the purkinje cells. Furthermore analysis of the closely related alpha, B-crystallin shows that in several tissues, including the bladder, the notochordal sheath and the eye lens both proteins are coexpressed. Our studies demonstrate that mammalian hsp25 accumulation is developmentally regulated during mouse embryogenesis and support the view of an important functional role of small heat shock proteins in normal cell metabolism