MHC matching fails to prevent long-term rejection of iPSC-derived neurons in non-human primates

Abstract

Cell therapy products (CTP) derived from pluripotent stem cells (iPSCs) may constitute a renewable, specifically differentiated source of cells to potentially cure patients with neurodegenerative disorders. However, the immunogenicity of CTP remains a major issue for therapeutic approaches based on transplantation of non-autologous stem cell-derived neural grafts. Despite its considerable side-effects, long-term immunosuppression, appears indispensable to mitigate neuro-inflammation and prevent rejection of allogeneic CTP. Matching iPSC donors' and patients' HLA haplotypes has been proposed as a way to access CTP with enhanced immunological compatibility, ultimately reducing the need for immunosuppression. In the present work, we challenge this paradigm by grafting autologous, MHC-matched and mis-matched neuronal grafts in a primate model of Huntington's disease. Unlike previous reports in unlesioned hosts, we show that in the absence of immunosuppression MHC matching alone is insufficient to grant long-term survival of neuronal grafts in the lesioned brain.

Fig. 1

Generation and in vitro characterization of cell therapy products (CTPs) with specific MHC haplotypes. a Schematic representation of the experimental groups for intra-striatal cell transplantation in HD macaques (Mac) showing autologous (white, Mac 1–3), MHC-matched (gray, Mac 4–5) and MHC mismatched (black, Mac 6–7) recipients of CTPs derived from iPSC lines generated from PBMCs drawn from MHC homozygous (M1/M1; M3/M3) or heterozygous (M2/M5) cell donors (white). Survival time post-transplantation is indicated on each subject as 3 or 6 months (mo). b Immunohistochemistry results showing OCT4/DAPI double staining of each of the 3 iPSCs lines derived from PBMC NHP donors (Mac 1, 2, and 3) at passage (P) 14, 13, and 12 respectively. c Staining of CTPs derived from each of the iPSC batches at in vitro differentiation day (D.I.V.) 48 showing staining for Calbindin (CalB) and astrocytes (GFAP). Scale bar: 100 µm

Fig. 2

In vivo characterization of CTP batches derived from NHP iPSCs in quinolinic acid-lesioned nude rats (RNU). a Representative coronal slices showing the localization of the graft in the striatum and immunostaining with striatal markers at 10 weeks post-grafting (Calbindin, CalB; MAP2). b Immunohistochemistry results illustrating the composition of the graft (white dotted lines, white arrow) using striatal medium spiny neurons markers (CalB, DARPP32, FOXP1), striatal interneurons marker (Calretinin: CalRet), human and NHP cell nuclear marker (HNA) and the astrocyte marker GFAP. Scale bar: 200 µm

Fig. 3

Expression of immune-related surface antigens on CTPs. a Representative histograms showing the expression of MHC molecules by each batch of CTPs (Mafa-A Mafa-B counterparts of HLA-A and HLA-B, respectively and Mafa-DR counterpart of HLA-DR) and b costimulatory molecules (CD40, CD80 and CD86) on CTPs under normal culture conditions (Untreated- green histograms) or after exposure to IFNγ (100 ng ml⁻¹, blue histogram) for 48 h prior to analysis. The red histogram represents the isotype control

Fig. 4

Graft size, location in QA-lesioned NHPs analyzed in vivo with MRI. a Representative coronal images of the longitudinal MRI follow-up of CTP derivatives after intra-striatal transplantation in autologous (white), MHC-matched (gray) and MHC mis-matched (black) recipients showing areas of hyper- (red arrows) and hypo-intensity in the caudate (bilateral) and putamen (unilateral, left) corresponding to the transplanted CTPs at 1, 2, 3 and, where applicable, 6 months post-surgery. b Graft MRI images prior to euthanasia and on post mortem sections stained with CalRet in autologous (white), MHC-matched (gray) and MHC mismatched (black) recipients at end-point (3 months PG for Mac 1, 3, 6, 7; 6 months PG for Mac 2, 4, 5)

Fig. 5

Histological assessment of graft localization, survival and neuronal identity of the grafts in mismatched (3 months PG), autologous (6 months PG) and matched (6 months PG) NHP recipients. a, b Immunohistological staining of coronal sections of the brain at the level of graft with anti-FOXG1 (a) and Calretinin (b) antibodies. Red arrows indicate the location of the grafted cells. c Brain slices were stained with post-mitotic neuronal markers NeuN (cell nuclei), MAP2 (soma and neuritic extensions), HuC/D (peri-nuclear soma), SOX1 (immature neural cells), and PHH3 (proliferative cells) compared to Mafa-DR (MHC II). Red dotted lines represent graft contour. Scale bar: 100 µm

Fig. 6

Post mortem analysis of the immune markers in QA-lesioned NHPs after CTP transplantation. a Representative coronal sections of the NHP brain at the level of the graft (commissural) showing staining for microglia/macrophages (Iba1; CD68), MHC Class II positive cells (revealed by an anti HLA-DR antibody cross-reacting with Mafa-DR antigens), and cytotoxic T-cells (CD8, cartography,) in quinolinic acid lesioned (QA) untransplanted controls (green) and in autologous (AU, white), MHC-matched (MA, gray), and mismatched (MI, black) CTP recipients at 3 or 6 months post-grafting (PG). b–e Quantification of Iba1 (b), Mafa-DR (c), CD68 (d) and CD8 (e) immunoreactivity in AU, MA, MI CTP recipients at 3 or 6 months post-grafting (PG) and in untransplanted controls. Bar graphs represent mean value of the three regions of interest considered (left and right caudate, and left putamen) for each animal; gray dots represent individual values for each region (n = 3 biologically independent cell deposit per animal)

Fig. 7

Detection of hemolytic CTP-specific antibodies in the serum of transplanted macaques. The sera of AU, MA, MI recipients were collected before and at different time-points following transplantation. a The presence of anti-CTP IgG was detected by cell-based ELISA. b The capability of anti-graft antibodies to trigger the complement-dependent cytotoxicity was also evaluated by measuring the fluorescence signal of the Cell-Tox Green probe (for (a) and (b) data are expressed as mean ± s.e.m of n = 3 replicates, one-way ANOVA followed by Dunnett’s multiple comparisons post hoc test using day 0 as control; for b positive and negative controls: individual replicates shown) or c by measuring the percentage of the dead cells (propidium iodide-positive cells) by flow cytometry (n = 2 technical replicate of single biological samples, mean per time point)