scholarly journals Indirect Presentation of Y-Chromosome Antigen Dby Is Regulated By Hsc70 and Mediated through CD63 Positive Exosomes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3712-3712
Author(s):  
Sascha Kretschmann ◽  
Heiko Bruns ◽  
Ursula Schlötzer-Schrehardt ◽  
Andreas Mackensen ◽  
Marieke Griffioen ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Clone ◽  
Class Ii ◽  
Hla Class Ii ◽  
Full Length ◽  
T Cell Recognition ◽  
T Cell Clone ◽  
Antigen Transfer

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.

scholarly journals IL4I1 Accelerates the Expansion of Effector CD8+ T Cells at the Expense of Memory Precursors by Increasing the Threshold of T-Cell Activation

2020 ◽  
Vol 11 ◽  
Author(s):  
Marie-Line Puiffe ◽  
Aurélie Dupont ◽  
Nouhoum Sako ◽  
Jérôme Gatineau ◽  
José L. Cohen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cell Clone ◽  
Cd8 T Cell ◽  
Lymphocytic Choriomeningitis ◽  
T Cell Clone

IL4I1 is an immunoregulatory enzyme that inhibits CD8 T-cell proliferation in vitro and in the tumoral context. Here, we dissected the effect of IL4I1 on CD8 T-cell priming by studying the differentiation of a transgenic CD8 T-cell clone and the endogenous repertoire in a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. Unexpectedly, we show that IL4I1 accelerates the expansion of functional effector CD8 T cells during the first several days after infection and increases the average affinity of the elicited repertoire, supporting more efficient LCMV clearance in WT mice than IL4I1-deficient mice. Conversely, IL4I1 restrains the differentiation of CD8 T-cells into long-lived memory precursors and favors the memory response to the most immunodominant peptides. IL4I1 expression does not affect the phenotype or antigen-presenting functions of dendritic cells (DCs), but directly reduces the stability of T-DC immune synapses in vitro, thus dampening T-cell activation. Overall, our results support a model in which IL4I1 increases the threshold of T-cell activation, indirectly promoting the priming of high-affinity clones while limiting memory T-cell differentiation.

scholarly journals Immunologically relevant peptide antigen exists on the presenting cell in a manner accessible to macromolecules in solution.

1986 ◽  
Vol 164 (5) ◽  
pp. 1440-1454 ◽  
Author(s):  
K B Cease ◽  
G Buckenmeyer ◽  
I Berkower ◽  
J York-Jolley ◽  
J A Berzofsky
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
Antigen Presentation ◽  
T Cell Activation ◽  
Antigen Processing ◽  
Cell Activation ◽  
Cell Clone ◽  
Sperm Whale ◽  
Amino Terminal ◽  
T Cell Clone

Although studies of the association of antigen with APC have been complicated by antigen-processing requirements, recent studies have suggested that immunologically relevant antigen should be present on the APC surface. Nevertheless, blocking of antigen presentation with antibody to the antigen has not been demonstrable in most systems. To study this problem we developed a system using avidin to block presentation of amino-terminal biotinylated synthetic peptide 132-146 of sperm whale myoglobin (B132) to a murine T cell clone specific for this site in association with I-Ed. greater than 95% specific inhibition was observed with doses of B132 equipotent to unmodified peptide. Specific blocking could be observed: (a) after pulsing APC with antigen, washing, and incubating for a chase period of 8-16 h before addition of avidin and T cells to assure adequate time for intracellular trafficking and maximal display of antigen on the cell surface, or (b) when monensin is present during the antigen pulse to inhibit such traffic. Therefore, the inhibition appeared to be occurring at the cell surface unless dissociation and reassociation were constantly occurring. To distinguish these, B10.GD APC (I-Ed-negative) were pulsed with antigen and cocultured with B10.D2 APC (I-Ed-positive). No detectable antigen presentation resulted. Thus, minimal dissociation and reassociation between antigen and APC occurs and, consequently, blocking by extracellular solution-phase binding of avidin to antigen is unlikely. Taken together, these data suggest that the blocking is occurring at the cell surface. Thus, under physiologic conditions, immunologically relevant antigen necessary for T cell activation appears to be present on the APC surface and is freely accessible to macromolecules the size of avidin. These findings hold specific implications for models of antigen presentation for T cell recognition.

scholarly journals The Fab fragment of a directly activating monoclonal antibody that precipitates a disulfide-linked heterodimer from a helper T cell clone blocks activation by either allogeneic Ia or antigen and self-Ia.

1984 ◽  
Vol 159 (5) ◽  
pp. 1397-1412 ◽  
Author(s):  
J Kaye ◽  
C A Janeway
Keyword(s):  
Monoclonal Antibody ◽  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Cell Clone ◽  
Antigen Receptors ◽  
Helper T Cell ◽  
Fab Fragment ◽  
T Cell Clone

We characterize a monoclonal antibody directed against the antigen/Ia receptor of a cloned helper T cell line that induced T cell clone proliferation and T cell clone-dependent B cell proliferation at antibody concentrations as low as 10(-11) M. A Fab fragment of this antibody was not stimulatory, implicating cross-linking of antigen receptors as the primary signal for T cell activation. The Fab fragment inhibited activation of this clone by both allogeneic Ia and antigen plus self-Ia, but not by the nonspecific stimulators concanavalin A and rabbit anti-mouse brain serum. This strongly supports the hypothesis that a single molecule mediates both self-Ia plus antigen and non-self-Ia recognition. This molecule is presumably the disulfide-linked heterodimer comprised of 42,000 mol wt acidic and basic subunits precipitated by this monoclonal antibody. The cell surface and internal precursor forms of this protein are also identified. In addition, the response to allogeneic Ia stimulation was more readily inhibited by the Fab fragment than was the response to antigen plus self-Ia, suggesting that alloreactivity reflects a low affinity interaction with a ligand represented at high frequency on the stimulatory cell.

scholarly journals Interleukin 2–mediated Uncoupling of T Cell Receptor α/β from CD3 Signaling

1998 ◽  
Vol 188 (9) ◽  
pp. 1575-1586 ◽  
Author(s):  
Loralee Haughn ◽  
Bernadine Leung ◽  
Lawrence Boise ◽  
André Veillette ◽  
Craig Thompson ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
T Cell ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Cell Clone ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
T Cell Clone

T cell activation and clonal expansion is the result of the coordinated functions of the receptors for antigen and interleukin (IL)-2. The protein tyrosine kinase p56lck is critical for the generation of signals emanating from the T cell antigen receptor (TCR) and has also been demonstrated to play a role in IL-2 receptor signaling. We demonstrate that an IL-2–dependent, antigen-specific CD4+ T cell clone is not responsive to anti-TCR induced growth when propagated in IL-2, but remains responsive to both antigen and CD3ε-specific monoclonal antibody. Survival of this IL-2–dependent clone in the absence of IL-2 was supported by overexpression of exogenous Bcl-xL. Culture of this clonal variant in the absence of IL-2 rendered it susceptible to anti-TCR–induced signaling, and correlated with the presence of kinase-active Lck associated with the plasma membrane. The same phenotype is observed in primary, resting CD4+ T cells. Furthermore, the presence of kinase active Lck associated with the plasma membrane correlates with the presence of ZAP 70–pp21ζ complexes in both primary T cells and T cell clones in circumstances of responsive anti-TCR signaling. The results presented demonstrate that IL-2 signal transduction results in the functional uncoupling of the TCR complex through altering the subcellular distribution of kinase-active Lck.

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