Indirect Presentation of Y-Chromosome Antigen Dby Is Regulated By Hsc70 and Mediated through CD63 Positive Exosomes
Authors: Kretschmann Sascha , Bruns Heiko , Schlötzer-Schrehardt Ursula , Mackensen Andreas , Griffioen Marieke , Kremer Anita 
Published: October 2019

Abstract
Introduction:
Recent studies have demonstrated that CD4 T cells can efficiently reject MHC class II negative tumors in vivo. This requires presentation of tumor-associated antigens on surrounding antigen presenting cells (APC). However, whether intercellular antigen transfer is a consequence of cell-death-mediated antigen release or a regulated mechanism remains poorly characterized. Recently, heat shock cognate protein 70 (hsc70) has been shown to deliver distinct cytosolic proteins to the vesicles of late endosomes. This microautophagy-like pathway happened at the endosomal limiting membrane and was selective through binding of hsc70 to cytosolic target proteins via KFERQ-like motifs. Hence, we hypothesized that intercellular antigen transfer is an active process which might be regulated by hsc70 in a microautophagy-related manner.
Methods:
We generated tumor cell lines retrovirally transduced with human DBY, its X-chromosome homologue DBX, a CD4 T cell epitope of DBY (25-mer) and full length DBY mutated in either one or both putative hsc70 binding sites. Expression of transgenes was verified by flow cytometry, western blot analysis, qPCR and immunofluorescence. The capacity of our antigens to be processed and presented was confirmed by T cell recognition after transduction into HLA class II positive EBV-LCL. To study indirect antigen presentation, we co-cultured antigen transduced HLA class II negative HeLa cells with antigen negative, HLA class II positive EBV-LCL and tested T cell recognition. Protein interaction of hsc70 and DBY mutants was assessed using an in situ proximity ligation assay. Crude exosomes were isolated from serum-free culture supernatants by differential ultracentrifugation and characterized by flow cytometry, western blot analysis and antigen presentation assays.
Results:
All transgenes were highly and comparably expressed and were able to activate the DBY-specific CD4 T cell clone upon direct presentation. In contrast, while we observed strong T cell activation after intercellular transfer of full length DBY and one of the DBY mutants, we showed that activation of the T cell clone was significantly reduced for the other mutation, and completely abolished for the 25-mer encoding the DBY T cell epitope. We addressed the question whether intercellular antigen transfer is reliant on cell-cell contact and could demonstrate T cell activation after application of culture supernatants from transgene positive HeLa cells to APC, indicating extracellular release of the antigen. We hypothesized that intercellular transfer might be mediated by secreted vesicles. To further validate this, we purified exosomes by differential ultracentrifugation and characterized the pellets by flow cytometry on the basis of three exosomal marker proteins. By doing so, we qualitatively showed purification of CD9, CD63 and CD81 positive exosomes. We were able to recover full length DBY in the exosomal pellet by western blot analysis. Furthermore, exosomal pellets were applied to EBV-LCL to analyze T cell recognition. Comparably to our indirect presentation assays, full length DBY was recognized, one DBY mutant showed clearly reduced T cell activation whereas the 25-mer epitope again did not activate the T cell clone at all. Further studies performed with the proximity ligation assay revealed that protein-protein interaction of hsc70 and the sensitive DBY mutant was remarkably reduced and even absent in case of the 25-mer epitope, suggesting that indeed interaction with hsc70 plays a role in the release via secreted microvesicles.
Conclusions:
Our data indicate that indirect recognition of tumor-associated antigens on surrounding APC can be the result of an active process of antigen transfer and not unselective antigen release after tumor cell-death. We showed that binding of hsc70 to putative KFERQ-like motifs on DBY correlates with intercellular antigen transfer indicating that binding to hsc70 is a possible mechanism for the transmission of cytosolic HLA class II-restricted antigens to surrounding APC. Furthermore, we provided evidence that CD63 positive exosomes mediate the transfer of our full-length antigen suggesting recruitment of cytosolic proteins to intraluminal vesicles of late endosomes. This regulation might be highly relevant in the context of HLA class II negative tumor eradication.

Disclosures
No relevant conflicts of interest to declare.