Page 90 - 2022_02-Haematologica-web

H. Schmid et al.
cial antigen-presenting cells (aAPC, e.g., Dynabeads) was only affected when higher numbers of iNKT cells were added to the culture (Figure 1D and E; Online Supplementary Figure S2). In particular, proliferation speed was decreased with a predominance of early daughter generations (Online Supplementary Figure S2). Our findings suggest that the interaction of iNKT cells with DC largely contributes to the control of alloreactive T cells although a minor direct impact of iNKT cells on T cells could be observed.
Invariant natural killer T cells induce apoptosis of allogeneic dendritic cells in a dose-dependent manner
We performed flow cytometry to determine the pheno- type of DC challenged with iNKT cells. Notably, DC numbers were highly reduced (Figure 2A) and we suspect- ed induction of apoptosis through iNKT cells. Annexin V assays showed that culture-expanded iNKT cells rapidly induced apoptosis of allogeneic DC, while co-culture of DC with conventional allogeneic CD3+ T cells did not, suggesting that apoptosis induction is not only dose- dependent but also specific to iNKT cells (Figure 2B and C). The Nicoletti assay revealed that DC start to defrag- ment their DNA after co-culture with iNKT cells, which represents a further hallmark of apoptosis (Figure 2D). Interestingly, an increase of spontaneous DNA defrag- mentation could be observed in DC without iNKT cells after 18 h which could be explained by the lack of specific stimuli. Image stream analysis also confirmed morpholog- ic changes in DC after co-culture with iNKT cells. Whereas DC cultured alone presented a healthy and round morphology, DC co-cultured with iNKT cells were small, squashed and with a blobbing membrane. Further, upregulation of Annexin V and loss of nuclear integrity in DC co-incubated with iNKT cells could be observed in image stream assays, confirming our previous assumption (Figure 2E). Next, we investigated whether the induction of apoptosis is specific to certain iNKT-cell subpopula- tions. Therefore, culture-expanded iNKT cells were sorted into double negative, CD4+CD8– and CD4–CD8+ subsets and co-cultured separately with allogeneic DC: all iNKT- cell subsets were able to induce apoptosis of DC with comparable efficiency (Figure 2F).
Induction of dendritic cell apoptosis is cell contact-dependent and mediated by cytotoxic effector molecules
In order to further elucidate the cellular and molecular mechanisms responsible for iNKT-cell-induced DC apop- tosis, we first analyzed image stream data visualizing dou- blets consisting of DC and iNKT cells. Image stream analysis revealed a direct binding of iNKT cells (PBS57- loaded CD1d tetramer+) to the surface of allogeneic DC (HLA-DR+), which subsequently revealed positive surface staining for the apoptosis marker annexin V (Figure 3A). In order to test whether this direct cellular interaction is required, iNKT cells and DC were separated by a tran- swell insert demonstrating that iNKT cells were unable to induce DC apoptosis anymore (Figure 3B). We further per- formed blocking experiments of common key molecules to identify critical pathways responsible for apoptosis induction through iNKT cells. It was observed that block- ing FasL, TRAIL or NKG2D did not significantly reduce apoptosis of DC exposed to iNKT cells. However, block- ing the CD1d and invariant T-cell receptor interaction
reduced DC apoptosis significantly indicating that T-cell receptor engagement contributes to efficient lysis. Further, blocking apoptosis via caspase inhibitor zVAD-fmk (N- benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylke- tone) or inhibition of the perforin pathway via CMA (con- canamycin A) also diminished iNKT-cell-mediated cell death of DC. Moreover, the inhibition of iNKT-cell degranulation by monensin and brefeldin A was shown to impede apoptosis induction most efficiently (Figure 3C; Online Supplementary Figure S3). By adding supernatant of DC-triggered degranulated iNKT cells to viable DC, we could show that iNKT cells released cytotoxic factors dur- ing degranulation which further induced apoptosis in DC (Figure 3D). In order to identify these factors, we per- formed bead-based multiplex assays and thereby revealed the release of interferon-g (IFN-g), granzyme B, perforin and granulysin (Figure 3E).
Invariant natural killer T cells induce preferential apoptosis of blood conventional dendritic cells in healthy donors and graft-versus-host disease patients
Our previous observations are based on ex vivo cultured mo-DC. In order to support our findings, we additionally performed MLR and apoptosis assays using blood DC iso- lated from PBMC of healthy donors and GvHD patients following allogeneic HCT. Blood DC are mainly com- posed of cDC and pDC with the latter expressing lower levels of CD1d (Online Supplementary Figure S4A). Also, human blood DC of healthy volunteers induce activation and proliferation of MHC-mismatched T cells that can be diminished through the addition of iNKT cells (Figure 4A). Given that iNKT cells interact with DC through CD1d and CD1d engagement contributes to efficient lysis of tar- get cells, we aimed to determine how human blood pDC and cDC are susceptible to iNKT-cell apoptosis induction. For this purpose, we isolated HLA-DR+ pDC (CD303+) and cDC (CD1c+) by FACS, co-cultured them separately with iNKT cells for 4 h and stained with annexin V and PI. We observed preferential apoptosis induction of cDC, while pDC were less affected by the addition of iNKT cells (Figure 4B). Further, we wondered whether preferen- tial apoptosis of cDC by iNKT cells would also affect T- cell alloreactivity using fresh human blood DC as stimula- tors. We observed that only allogeneic cDC in contrast to pDC could induce significant T-cell activation and prolif- eration (Online Supplementary Figure S4B). Consequently, co-culture of these distinct blood DC subsets with allo- geneic T cells and iNKT cells revealed that iNKT cells were also able to suppress activation and proliferation of alloreactive T cells induced by cDC (Figure 4C).
Finally, we tested whether our findings also apply to patients with acute GvHD having received grafts from HLA-matched donors. Therefore, we isolated blood DC from PBMC obtained from patients with clinical manifes- tations of acute GvHD grade ≥2 prior to induction of sys- temic treatment with steroids. At the time point of blood collection patients had complete donor chimerism in their peripheral blood. cDC from GvHD patients also showed higher expression levels of CD1d (Online Supplementary Figure S4C) and were more susceptible to iNKT-cell- induced apoptosis than pDC (Figure 4D), similarly as demonstrated in our previous experiments with cells from healthy donors. Next, T cells derived from donors prior to transplantation were co-cultured with blood DC from GvHD patients. Importantly, adding iNKT cells from
432
haematologica | 2022; 107(2)