HLA-G expression levels influence the tolerogenic activity of human DC-10

Abstract

ABSTRACTand CD8 low suppressor T cells, 25 interleukin-10 (IL-10)-producing T regulatory type 1 (Tr1) cells, DC-10 are a subset of human tolerogenic DC that are present in vivo [27][28][29] and can be differentiated in vitro by culturing monocytes in the presence of IL-10. DC-10 secrete IL-10, are CD11c + , express CD14, CD16, HLA-G and ILT4 and, although not activated, display a mature phenotype, being CD86+ and HLA-DR + . The secretion of IL-10 and the expression of membrane-bound HLA-G and ILT4 are critical factors involved in DC-10-mediated induction of Tr1 cells. 27 In the present study, we investigated the role of HLA-G in DC-10-mediated Tr1 cell induction and whether polymorphisms present at the 3'UTR of the gene influence the expression of membrane-bound HLA-G on DC-10. MethodsThe methods are described in full in the Online Supplementary Appendix. Peripheral blood was obtained after informed consent in accordance with the Declaration of Helsinki under protocols approved by the ethical committee of the San Raffaele Telethon Institute for Gene Therapy. Dendritic cell differentiation CD14+ monocytes were isolated from peripheral blood mononuclear cells by positive selection using CD14 MicroBeads (Miltenyi Biotech, Germany) according to the manufacturer's instructions. Cells were cultured in RPMI 1640 (Lonza, Italy) supplemented with 10% fetal bovine serum (FBS) (Lonza, Italy) or with 5% human serum (HS) (EuroClone, Italy), 100 U/mL penicillin/streptomycin (Lonza, Italy), 2 mM L-glutamine (Lonza, Italy), (DC medium) at 37°C in the presence of 10 ng/mL recombinant human (rh)IL-4 (R&D Systems, Minneapolis MN, USA) and 100 ng/mL rhGM-CSF (Genzyme, Seattle, WA, USA) with 10 ng/mL of rhIL-10 (BD, Bioscience, CA, USA) for 7 days to differentiate DC-10. Cells cultured with rhIL-4 and rhGM-CSF on day 5 were matured with 1 mg/mL of lipopolysaccharide (Sigma, CA, USA) for 2 more days to generate mature dendritic cells (mDC). At day 7, DC were collected, phenotypically analyzed, and used to stimulate T cells. Statistical analysisSample mean results were compared using the non-parametric Mann-Whitney U test for continuous variables. HLA-G 3'UTR allele and genotype frequencies were obtained by direct counting. Allele and genotype frequencies between populations were compared using the χ 2 test. The correlation between membranebound HLA-G and ILT4 expression was determined using the Spearman correlation test. FBS and DC-10 HS were compared using a paired t-test. All results are presented as mean values ± standard error of mean (SEM). Differences were regarded as statistically significant at *P<0.05, **P<0.01, and ***P<0.001. The results were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc. La Jolla, CA, USA). Results In vitro differentiated DC-10 express variable levels of membrane-bound HLA-GIndependently of the donor, DC-10 differentiated in vitro as described in the Methods section were Figure 1A,B). High variability in the expression of membrane-bound HLA-G (ranging from 3.5% to 97.7%) and of ILT4 (rangin...

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“…Furthermore, Tr1 cells are reported to be induced at mucosal sites in response to Ag stimulation in the presence of IL-10. We observed that IL-10-deficient DCs promoted IL-10-secreting CD49b + Treg cell expansion in several lymphoid organs of wild-type animals, suggesting that, in contrast to Tr1 cells (64) + Treg cells and thus might also play an important role in the suppressive function of CD49b + Treg cells. Furthermore, CD103 expression could also be implicated in their suppressive function as its expression was shown to be responsible for the retention of Treg cells in inflamed tissue by interaction with its ligand E-cadherin (65,66).…”

The Biology of T Regulatory Type 1 Cells and Their Therapeutic Application in Immune-Mediated Diseases

“…DC-10 secrete IL-10 in the absence of IL-12 and express the tolerogenic molecules ILT-4 and HLA-G . Resting and activated (Amodio and Gregori, 2012) DC-10 efficiently promote the differentiation of Tr1 cells from naive CD4 + T cells in vitro, which requires IL-10 production and expression of ILT-4 and HLA-G (Amodio et al, 2015;Gregori et al, 2010).…”

“…generated in vitro can be purified from bulk cultures using CD49b and LAG-3 antibodies (Amodio et al, 2015;Gagliani et al, 2013b;Mfarrej et al, 2017;Petrelli et al, 2015). The frequency of CD4 + CD45RA -CD49b + LAG-3 + cells correlates with the expression of IL-10 in human (Amodio et al, 2015;Mfarrej et al, 2017;Petrelli et al, 2015;Sutavani et al, 2013;Tousa et al, 2017;Yao et al, 2015;Tousa et al, 2017), and with induction of immunological tolerance in different preclinical and clinical settings Gagliani et al, 2013b;Kim et al, 2018;Koch et al, 2015;Zhu et al, 2018). Using single-cell mass cytometry, we have shown that CD49b and LAG-3 were sufficient to identify a population of CD4 + CD45RAcells, which is distinct from Th1, Th2, Th17, Tfh, and Treg cells.…”

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“…Despite the presence of IL-10 during differentiation, DC-10 are mature myeloid cells that express CD86 and HLA-DR. DC-10 also express the tolerogenic molecules immunoglobulin-like transcript (ILT)2, ILT3, ILT4, and HLA-G [24]. The high expression levels of HLA-G and the high IL-10/IL-12 production ratio render DC-10 potent inducers of allo-specific Tr1 cells [24,83]. The clinical use of DC-10 relies on this latter feature, and clinical-grade protocols for the induction of allo-specific Tr1 cells, suitable for cell therapy, using DC-10 have been developed [84,85].…”