Celiac disease driven by an antigluten T cell response is strongly associated with the histocompatibility antigen HLA-DQ2.5 but is barely associated with HLA-DQ2.2. Yet these molecules have very similar peptide-binding motifs and both present gluten T cell epitopes. We found that DQ2.5(+) antigen-presenting cells (APCs) had greater stability of bound peptides and protracted gluten presentation relative to that of DQ2.2(+) cells. The improved ability of DQ2.5 to retain its peptide cargo can be ascribed to a polymorphism of DQalpha22 whereby DQ2.5 (tyrosine) can establish a hydrogen bond to the peptide main chain but DQ2.2 (phenylalanine) cannot. Our findings suggest that the kinetic stability of complexes of peptide and major histocompatibility complex (MHC) is of importance for the association of HLA with disease.
Rab9 is a small GTPase that localizes to the trans-Golgi Network (TGN) and late endosomes. Its main function has long been connected to the recycling of mannose-6-phosphate receptors (MPRs). However, recent studies link Rab9 also to autophagy and lysosome biogenesis. In this paper, using confocal imaging, we characterize for the first time the live dynamics of the Rab9 constitutively active mutant, Rab9Q66L. We find that it localizes predominantly to late endosomes and that its expression in HeLa cells disperses TGN46 and cation-independent (CI-MPR) away from the Golgi yet, has no effect on the retrograde transport of CI-MPR. We also show that CI-MPR and Rab9 enter the endosomal pathway together at the transition stage between early, Rab5-positive, and late, Rab7a-positive, endosomes. CI-MPR localizes transiently to separate domains on these endosomes, where vesicles carrying CI-MPR attach and detach within seconds. Taken together, our results demonstrate that Rab9 mediates the delivery of CI-MPR to the endosomal pathway, entering the maturing endosome at the early-to-late transition.
BackgroundTargeted thorium-227 conjugates (TTCs) are an emerging class of targeted alpha therapies (TATs). Their unique mode of action (MoA) is the induction of difficult-to-repair clustered DNA double-strand breaks. However, thus far, their effects on the immune system are largely unknown. Here, we investigated the immunostimulatory effects of the mesothelin-targeted thorium-227 conjugate (MSLN-TTC)in vitroandin vivoin monotherapy and in combination with an inhibitor of the immune checkpoint programmed death receptor ligand 1 (PD-L1) in immunocompetent mice.MethodsThe murine cell line MC38 was transfected with the human gene encoding for MSLN (hMSLN) to enable binding of the non-cross-reactive MSLN-TTC. The immunostimulatory effects of MSLN-TTC were studiedin vitroon human cancer cell lines and MC38-hMSLN cells. The efficacy and MoA of MSLN-TTC were studiedin vivoas monotherapy or in combination with anti-PD-L1 in MC38-hMSLN tumor-bearing immunocompetent C57BL/6 mice. Experiments were supported by RNA sequencing, flow cytometry, immunohistochemistry, mesoscale, and TaqMan PCR analyses to study the underlying immunostimulatory effects.In vivodepletion of CD8+ T cells and studies with Rag2/Il2Rg double knockout C57BL/6 mice were conducted to investigate the importance of immune cells to the efficacy of MSLN-TTC.ResultsMSLN-TTC treatment induced upregulation of DNA sensing pathway transcripts (IL-6,CCL20,CXCL10, and stimulator of interferon genes (STING)-related genes)in vitroas determined by RNASeq analysis. The results, including phospho-STING activation, were confirmed on the protein level. Danger-associated molecular pattern molecules were upregulated in parallel, leading to dendritic cell (DC) activationin vitro. MSLN-TTC showed strong antitumor activity (T:C 0.38, p<0.05) as a single agent in human MSLN-expressing MC38 tumor-bearing immunocompetent mice. Combining MSLN-TTC with anti-PD-L1 further enhanced the efficacy (T:C 0.08, p<0.001) as evidenced by the increased number of tumor-free surviving animals. MSLN-TTC monotherapy caused migration of CD103+ cDC1 DCs and infiltration of CD8+ T cells into tumors, which was enhanced on combination with anti-PD-L1. Intriguingly, CD8+ T-cell depletion decreased antitumor efficacy.ConclusionsThesein vitroandin vivodata on MSLN-TTC demonstrate that the MoA of TTCs involves activation of the immune system. The findings are of relevance for other targeted radiotherapies and may guide clinical combination strategies.
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