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H. Schmid et al.
mice.10 Upon activation through glycolipids, iNKT cells regulate immune responses by the instant release of immunoregulatory cytokines or by direct cell killing.11-13
Several studies have shown the ability of iNKT cells to reduce the incidence of GvHD. In murine models, iNKT cells prevent acute and chronic GvHD, while promoting beneficial graft-versus-leukemia (GvL) effects.14-16 In humans, clinical studies have demonstrated that high iNKT-cell numbers are associated with a diminished occurrence of GvHD.17-19 Moreover, we recently showed that culture-expanded human iNKT cells are able to pre- vent T-cell activation and proliferation while exerting potent anti-leukemic activity.13,20
Nevertheless, the complex cellular and molecular mech- anisms of immune tolerance induction through iNKT cells remain poorly understood. In this study, we focused on how culture-expanded human iNKT cells modulate allore- active T-cell responses through DC in healthy volunteers and GvHD patients.
Methods
Research subjects
Human buffy coats from healthy volunteers were obtained from the Center of Clinical Transfusion Medicine Tuebingen. Samples from hematopoietic cell grafts and peripheral blood mononuclear cells (PBMC) from patients with GvHD were isolat- ed after written informed consent had been obtained. Human leukocyte antigen (HLA) typing was performed by the Center of Clinical Transfusion Medicine Tuebingen or the HLA laboratory of the Department of Medicine II of the University Hospital Tuebingen. The study was approved by our Institutional Review Board to be in accordance with ethical standards and with the Helsinki Declaration of 1975, as revised in 2013 (IRB approvals 483/2015BO2 and 137/2017BO2).
Flow cytometry
Antibodies and reagents used for flow cytometric analyses are described in the Online Supplementary Appendix.
Invariant natural killer T-cell expansion and enrichment
iNKT cells were expanded from third-party PBMC with some minor modifications as previously described (Online Supplementary Appendix).13 Culture-expanded iNKT cells were purified with anti- iNKT MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Alternatively, iNKT cells were stained with DAPI (4',6- diamidino-2-phenylindole, Merck, Darmstadt, Germany), anti- CD3, anti-CD4, anti-CD8 antibodies and PBS57-loaded CD1d tetramer allowing for enrichment of iNKT cells and their different subsets by fluorescence-activated cell sorting (FACS).
Generation of monocyte-derived dentritic cells and isolation of blood dendritic cells
Monocyte-derived dentritic cells (Mo-DC) were generated as described previously.13 Blood DC from healthy donors and patients were isolated using Blood Dendritic Cell Isolation Kit II (Miltenyi Biotech). Where indicated, HLA-DR+ blood DC were further sorted either as CD1c+ conventional DC (cDC) or CD303+ plasmacytoid DC (pDC).
CD3+ T-cell isolation
CD3+ T cells were isolated from human PBMC with anti-CD3 MicroBeads (Miltenyi Biotech). For proliferation analysis, T cells were marked with CFSE (carboxyfluorescein succinimidyl ester,
Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions and tested in a mixed lymphocyte reaction (MLR).
Mixed lymphocyte reaction
Major mismatched mo-DC or blood DC were plated together with allogeneic CD3+ T cells at a 1:1 ratio. Culture-expanded iNKT cells were added to the MLR at different doses, either direct- ly or separated from the MLR by 0.4 μm TC-Inserts (Sarstedt, Nuembrecht, Germany). Cells were analyzed by flow cytometry for activation markers (CD69 and CD25) and proliferation (CFSE). Alternatively, T cells were incubated with anti-CD3/CD28-coated beads (ThermoFisher Scientific, Waltham, MA, USA) in the pres- ence or absence of iNKT cells. For blocking assays, iNKT cells or DC were pre-treated with the respective antibodies or IgG control (Online Supplementary Appendix).
Apoptosis assays
Apoptosis was assessed with an annexin V-FITC/propidium iodide (PI) Staining Kit (BD Bioscience, Franklin Lakes, NJ, USA), by cell cycle analysis modified according to Nicoletti21 or by image stream analysis (Online Supplementary Appendix). The percentage of apoptotic cells was determined by flow cytometry.
Cytokine analysis
Cell culture supernatants from MLR were collected after 4 and 24 hours (h), respectively. In order to analyze cytokine production bead-based immunoassays were performed according to the man- ufacturer’s instructions. Cytokine release was measured by a LEGENDplex human CD8/NK-cell panel (BioLegend). Data were acquired using the Lyric flow cytometer with autosampler (BD Biosciences).
Statistical analysis
Student’s t-test and analysis of variance (ANOVA) were used for statistical analysis. P<0.05 was considered statistically significant. Data were analyzed with Prism 8 (GraphPad Software, La Jolla, CA, USA). All experiments were performed at least in technical duplicates and repeated independently at least three times using different iNKT-cell donors.
Results
Invariant natural killer T cells inhibit T-cell activation and proliferation in a cell contact-dependent manner
Human culture-expanded iNKT cells suppress alloreac- tive T-cell responses when T lymphocytes are stimulated by MHC-mismatched DC.13 As iNKT cells exhibit potent immunoregulatory properties through a rapid release of humoral mediators, we wondered whether this effect might be related to the inhibition of T-cell function.22 Therefore, we measured early (CD69 expression) and late activation (CD25 expression) as well as proliferation (CFSE dilution) of T cells co-incubated with DC in pres- ence or absence of iNKT cells. iNKT cells were either added directly to the MLR or separated through a TC- insert (transwell [TW]). Direct addition of iNKT cells at different ratios to the MLR reduced T-cell activation and proliferation in a dose-dependent manner. However, iNKT cells which were separated by a TW did not prevent T-cell activation and proliferation (Figure 1A to C; Online Supplementary Figure S1). We conclude from these experi- ments that iNKT cells mostly rely on direct cell contact to efficiently suppress alloreactive T-cell responses. In addi- tion, T-cell activation and proliferation initiated by artifi-
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haematologica | 2022; 107(2)