Volume 85, Issue 4, Pages 888-897 (April 2014) Rapamycin induces ILT3highILT4high dendritic cells promoting a new immunoregulatory pathway  Giovanni Stallone, Paola Pontrelli, Barbara Infante, Margherita Gigante, Giuseppe S. Netti, Elena Ranieri, Giuseppe Grandaliano, Loreto Gesualdo  Kidney International  Volume 85, Issue 4, Pages 888-897 (April 2014) DOI: 10.1038/ki.2013.337 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Flow cytometric analysis of circulating dendritic cell (DC) subsets (a–e) and Ig-like transcript 3 (ILT3) and ILT4 expression in circulating DCs (f–i). (a) Four-color flow cytometry was used to distinguish BDCA1+ and BDCA2+ DC subsets within freshly isolated peripheral blood mononuclear cells (PBMCs) using specific antibodies (data from one patient/group, representative of the whole group). Percentage (b and c) and absolute numbers (d and e) of circulating BDCA2+ (b and d) and BDCA1+ cells (c and e) in control subjects and in transplant recipients at the time of conversion (T0) and after 24 months (T24) in the two groups of patients. °P<0.0001 versus group A and B at T0 and T24 (Kruskal–Wallis and Mann–Whitney test); *P<0.001 group B T24 versus T0 (Wilcoxon test); †P<0.0001 group B versus group A at 24 months after conversion (Kruskal–Wallis and Mann–Whitney test); ‡P=0.001 group B T24 versus T0 (Wilcoxon test); §P=0.0001 group B versus group A at 24 months after conversion (Kruskal–Wallis and Mann–Whitney test). Percentage (f and g) and absolute numbers (h and i) of circulating DCs expressing ILT3 (f and h) and ILT4 (g and i) in control subjects and in transplant recipients at the time of conversion (T0) and after 24 months (T24) in the two groups of patients enrolled. *P<0.0001 group B T24 versus T0 (Wilcoxon test); †P=0.0001 group B versus group A patients at T24 (Kruskal–Wallis and Mann–Whitney test); ‡P=0.001 group B T24 versus T0; §P=0.0002 group B versus group A patients at T24 (Kruskal–Wallis and Mann–Whitney test). CHS, control healthy subjects. Kidney International 2014 85, 888-897DOI: (10.1038/ki.2013.337) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Flow cytometric analysis of Ig-like transcript 3 (ILT3)/ILT4 (a,b) and CD40 (c,d) expression on circulating plasmacytoid DCs. (a) ILT3 and ILT4 were evaluated on gated BDCA2+ cells from isolated peripheral blood mononuclear cells (PBMCs) of patients in groups A and B both at T0 and at T24. (a and b) Percentage (a) and absolute numbers (b) of circulating BDCA2+ DCs expressing ILT3/ILT4 at the time of conversion (T0) and after 24 months (T24) in the two groups of patients. (c and d) Percentage (c) and absolute numbers (d) of BDCA2+ DCs expressing CD40 at the time of conversion (T0) and after 24 months (T24) in the two patient groups. (a and b) *P=0.003 group B T24 versus T0 (Wilcoxon test); P=0.0001 group B versus group A patients at T24 (Mann–Whitney test). (c) *P=0.002 group B T24 versus T0 (Wilcoxon test); †P=0.003 group B versus group A patients at T24 (Mann–Whitney test). Kidney International 2014 85, 888-897DOI: (10.1038/ki.2013.337) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Flow cytometric analysis of circulating CD4+CD25+Foxp3+CTLA4+ Tregs (a–d) and CD8+CD28− T cells (e and f). (a) Peripheral blood mononuclear cells (PBMCs) isolated from Group A patients at T24 were gated on CD4/CD25 T cells. Foxp3 and CTLA4 were analyzed within CD4+/CD25high+-gated population of T cells by intracellular staining, as described in Patients and Methods. (b) PBMCs isolated from group B patients at T24 were gated on CD4/CD25 T cells. Foxp3 and CTLA4 were analyzed within CD4+/CD25high+−gated population of T cells by intracellular staining, as described in Patients and Methods. Percentage (c) and absolute (d) numbers of CD4+CD25+Foxp3+CTLA4+ Tregs in PBMCs isolated from the two groups of patients enrolled at both times considered (T0 and T24). *P=0.0001 group B T24 versus T0 (Wilcoxon test); †P=0.0001 group B versus group A patients at T24 (Mann–Whitney test). Percentage (e) and absolute numbers (f) of CD8+CD28− T cells in PBMCs isolated from the two groups of patients enrolled at both times considered (T0 and T24), as described in Patients and Methods. *P=0.001 group B T24 versus T0 (Wilcoxon test); †P=0.002 group B versus group A patients at T24 (Mann–Whitney test). (g) CD8 and CD28 were evaluated on T cells gated from isolated PBMCs. Quadrants were based on cell staining with isotype control monoclonal antibodies (negative control) (data from one patient/group, representative of the whole group). Kidney International 2014 85, 888-897DOI: (10.1038/ki.2013.337) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Ig-like transcript 3 (ILT3) (a and c) and ILT4 (b and d) protein expression in graft biopsies of group B patients at T0 (a and b) and T24 (c and d). Indirect immunofluorescence and confocal microscopy was performed as described in Patients and Methods. ILT3 and ILT4 expression was barely present at the tubulointerstitial level at T0. At T24, we observed a marked upregulation of both signals, mainly localized within the tubulointerstitial infiltrate and at the endothelial cell level. Quantification of both ILT3- and ILT4-specific immunofluorescence was performed as described in Patients and Methods (e). Values are expressed as mean±standard deviation (mean± s.d.). *P=0.001 T24 versus T0 (Wilcoxon test); †P=0.002 T24 versus T0 (Wilcoxon test). Scale bar= × 60. Kidney International 2014 85, 888-897DOI: (10.1038/ki.2013.337) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 GATA-3 (a, c, f, and h) and Tbet (b, d, g, and i) protein expression at the tubulointerstitial level in graft biopsies of group A and B patients at T0 (a, b, f, and g) and T24 (c, d, h, and i). For each treatment group, all the images are from a single patient and are representative of the whole group of patients. Quantification of both GATA3- and Tbet-specific staining was performed as described in Patients and Methods. Values are expressed as means±s.d. (anti-GATA3, original magnification, × 40; anti-Tbet, original magnification, × 40). In group A graft biopsies, we observed no variations of GATA-3 expression compared with basal levels (a and c) and an increase of Tbet expression after 2 years (b and d). GATA-3 and Tbet protein expression was quantified as described in the Patients and Methods section (e). *P=0.002 T24 versus T0 (Wilcoxon test). In group B graft biopsies, we did not observe any variation of Tbet expression compared with basal levels (g and i), whereas there was an increase of GATA-3 expression after 2 years (f and h). GATA-3 and Tbet protein expression was quantified as described in the Patients and Methods section (j). †P=0.0001 T24 versus T0 (Wilcoxon test). Hpf, high-power field. Kidney International 2014 85, 888-897DOI: (10.1038/ki.2013.337) Copyright © 2014 International Society of Nephrology Terms and Conditions