De novo mutations in mitochondrial DNA of iPSCs produce immunogenic neoepitopes in mice and humans

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Abstract

The utility of autologous induced pluripotent stem cell (iPSC) therapies for tissue regeneration depends on reliable production of immunologically silent functional iPSC derivatives. However, rejection of autologous iPSC-derived cells has been reported, although the mechanism underlying rejection is largely unknown. We hypothesized that de novo mutations in mitochondrial DNA (mtDNA), which has far less reliable repair mechanisms than chromosomal DNA, might produce neoantigens capable of eliciting immune recognition and rejection. Here we present evidence in mice and humans that nonsynonymous mtDNA mutations can arise and become enriched during reprogramming to the iPSC stage, long-term culture and differentiation into target cells. These mtDNA mutations encode neoantigens that provoke an immune response that is highly specific and dependent on the host major histocompatibility complex genotype. Our results reveal that autologous iPSCs and their derivatives are not inherently immunologically inert for autologous transplantation and suggest that iPSC-derived products should be screened for mtDNA mutations.

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Fig. 1: Immune response to allogeneic mtDNA-derived proteins in mice.
Fig. 2: Immune response to allogeneic mtDNA-derived proteins in human transplant recipients.
Fig. 3: Antigenicity of low- and high-passage iPSCs and their iEC derivatives in mice.
Fig. 4: Occurrence and antigenicity of mtDNA-derived neoantigens in human iPSCs and iECs.

Data availability

All epitope sequences and 20-residue oligomer sequences are presented in the paper. All data supporting the findings of this study are available in the paper and its supplementary information files. Sequencing data are available from the Sequence Read Archive under accession code PRJNA544330.

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Acknowledgements

We thank C. Pahrmann for cell culture work, assistance with experiments and immunoblots; G. Tediashvili for assistance with the in vivo experiments; and M.S. Mahadevan for computer assistance. Special thanks to R. Jaenisch (MIT, Department of Biology, MIT, Cambridge, MA) for providing the NT-ESCs and to J. Wu (Stanford Cardiovascular Institute, Stanford, CA) and P.-L. So (Stem Cell Core, Gladstone Institutes, San Francsico, CA) for providing the mouse and human iPSCs. Special thanks to D. Sun and X. Wu for their help in developing the targeted mtDNA sequencing and droplet digital PCR assays. We thank R. Nelson and K. Copeland for their support with in vivo imaging and M. Lewis for his support with the Xcelligence experiments. S.S. received research grants from the Deutsche Forschungsgemeinschaft (DFG; grants SCHR992/3-1, SCHR992/4-1), the Fondation Leducq (CDA 2013-2015) and the DZHK (German Center for Cardiovascular Research; grant FKZ 81Z2710105). D.W. was supported by the Max-Kade-Foundation (DFG). M.H.S. received an NIH shared instrumentation grant (1S10OD018040-01). B.P. and Z.K.-Y. were supported by the NIH (grant 1R21AI134127-01). S.A.-E., A. M., Y. Y. and H. V. were supported by National Heart Lung and Blood Institute (NHLBI) Intramural Research Program.

Author information

T.D. and S.S. designed the experiments, supervised the project and wrote the manuscript. X.H. performed the immunobiology experiments, molecular biology, imaging studies, cell culture work and analyzed the data. S.A.E., A.M., Y.Y. and H.V. performed the mtDNA sequencing and analysis. M.H.S. performed cytometry by time of flight experiments. A.G. did imaging studies and cell culture work. M.A. performed bioinformatics analyses. B.P. and Z.K.-Y. performed epitope predictions and designed 20-residue oligomers. R.R. performed HLA typing. D.W. performed the in vivo and immunofluorescence imaging studies (confocal microscopy). M.K., B.N. and R.K. performed the human kidney and liver transplant study. H.R. and I.L.W. gave technical support and conceptual advice. All authors contributed to editing the manuscript.

Correspondence to Sonja Schrepfer.

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Integrated supplementary information

Supplementary Figure 1 System-wide analysis of the immune response by mass cytometry.

a-b, B/c splenocytes from animals receiving syngeneic fibroblasts overexpressing syngeneic B/c or allogeneic B6 Co3 (a) or Cytb proteins (b) were harvested and analyzed by mass cytometry (representative pictures of 6 animals per group). Statistical scaffold analysis was performed on the resulting data to identify immune cell populations of differing frequencies. Immune cell subsets that are significantly increased or decreased in animals receiving B6-overexpressing cells are shown in red and blue, respectively. Landmark cell populations manually identified in the data are shown in black and facilitate interpretation of the map.

Supplementary Figure 2 Animals challenged with allogeneic proteins have expanded effector / memory T cell populations.

a-d, Animals were immunized with B/c or B6 Co3 proteins (a, c) or Cytb (b, d). Mass cytometry data were analyzed for the frequency of effector / memory CD4 (a, b) and CD8 (c, d) T cell subsets in the spleen (mean ± s.d., 6 animals per group, two-tailed Student’s t-test). Frequencies of total antigen experienced (CD44+), short-lived effector (CD45+CD27), effector memory (CD45+CD27+CD62L), and central memory (CD45+CD27+CD62L+) CD4 or CD8 T cells are shown.

Supplementary Figure 3 There is no cross-reactivity between fetal nuclear gene-derived antigens and the Co3 and Cytb 20mers.

B/c mice were immunized with B/c ESCs, which were suspended in saline and injected into the thigh muscle. After 5 days, ELISpot assays were performed with recipient splenocytes to test reactivity against 20mers carrying the B6-specific (blue) or B/c-specific (red) amino acid of the SNP at position 6 or 15 (mean ± s.d., quadruplicates of 4 animals per protein, two-tailed Student’s t-test).

Supplementary Figure 4 mtDNA sequencing of human donor-recipient pairs.

a, Total DNA was extracted from donor tissue and recipient blood. mtDNA was enriched and amplified and mtDNA libraries were created. After sequencing, raw reads underwent quality control and map reads were aligned to human rCRS. After removal of duplicates and pileup of variants, either heteroplasmic or homoplasmic donor-recipient SNP variants were identified. b, Of 15 patient pairs, the number of homoplasmic nonsynonymous mtDNA SNPs is shown. c, Nonsynonymous homoplasmic SNPs were found at different frequencies in 12 mitochondrial proteins. d, The number of nonsynonymous mtDNA SNPs in donor-recipient pairs by ethnic background is shown.

Supplementary Figure 5 In vitro reactivation of PBMCs of immunosuppressed organ recipients.

Blood was drawn from three organ recipients 3 weeks after transplantation. All patients were on a regimen including tacrolimus, mycophenolate mofetil, and steroids with therapeutic tacrolimus trough levels (mean ± s.d., n = 3 patients). PBMCs were isolated using Ficoll gradient centrifugation and one fraction was immediately tested for their immune responsiveness (0 h). Another fraction of PBMCs was washed and cultured in the presence of anti-CD3 and anti-CD28 for 24 h before being tested (24 h). PBMCs were unspecifically stimulated with PMA and ionomycin and their IL2 expression was quantified by PCR (mean ± s.d., n = 3 patients, two-tailed Student’s t-test) and their IFNγ release by ELISpot assays (mean ± s.d., quadruplicates of n = 3 patients, two-tailed Student’s t-test).

Supplementary Figure 6 Additional patients for the human transplant study.

Five more patients are displayed from the human transplant study outlined in Figure 2 (mean ± s.d., quadruplicates per 20mer and patient, two-tailed Student’s t-test). Blue shades correspond to donor, red shades to recipient 20mers. ** P < 0.01.

Supplementary Figure 7 Confirmatory patient study on the specificity of the immune response.

Six patients with 4-5 nonsynonymous mtDNA SNPs to their donor were chosen for this study. Blood was drawn 6 months after liver or kidney transplantation and PBMCs underwent in vitro reactivation. In Elispot assays, PBMCs were challenged with autologous (red) or two sets of allogeneic 20mers reflecting mtDNA SNPs of their donor (blue) or another unconnected donor (green; mean ± s.d., quadruplicates per 20mer and patient, two-tailed Student’s t-test). Among the depicted SNPs, yellow asterisks mark SNPs that are identical between the patient’s donor and the unconnected donor. ** P < 0.01.

Supplementary Figure 8 In silico antigenicity predictions of human and mouse SNPs.

a, Blood was drawn from two volunteers for 4-digit HLA typing. Using in silico prediction models, the top 5 peptides with the highest predicted immunogenicity and bottom 5 SNPs with the lowest predicted immunogenicity from the annotated human SNP library were identified for each volunteer. b, 20mers were generated to cover either the mutant or autologous SNP and used in extended naïve in vitro immunization Elispot assays with volunteer PBMCs (Volunteer 1: mean ± s.d., triplicates per 20mer, two-tailed Student’s t-test; Volunteer 2: mean ± s.d., quadruplicates per 20mer, two-tailed Student’s t-test). Blue shades correspond to autologous, yellow shades to mutant 20mers. c, In silico predictions for B6 MHC binding was performed for the 3 neoantigenic SNP candidates identified in P37 iPSCs. d, B6 mice were immunized with autologous P7 or P37 iPSCs and after 5 days, splenocytes were incubated in Elispot assays with 20mers from the 2 neoantigen candidate SNPs or the B6 reference SNPs. e, Elispot assays from mice immunized with P7 iPSCs (mean ± s.d., quadruplicates of 6 animals per 20mer, two-tailed Student’s t-test). f, Elispot assays from mice immunized with P37 iPSCs (mean ± s.d., quadruplicates of 6 animals per 20mer, two-tailed Student’s t-test). g, FVB mice were immunized with autologous P13 or P38 iPSCs and after 5 days, splenocytes were incubated in Elispot assays with 20mers from the neoantigen candidate SNP or the FVB reference SNP. h, Elispot assays from mice immunized with P13 iPSCs (mean ± s.d., quadruplicates of 6 animals per 20mer, two-tailed Student’s t-test). i, Elispot assays from mice immunized with P38 iPSCs (mean ± s.d., quadruplicates of 6 animals per 20mer, two-tailed Student’s t-test). Blue shades correspond to the B6 reference 20mer, green to the FVB reference 20mer, and yellow shades to the neoantigenic 20mer. ** P < 0.01.

Supplementary Figure 9 Survival of low- and high-passage FVB iPSCs.

a, FVB mice received different cell amounts of autologous low-passage (P13) or high-passage (P38) iPSCs grafts injected into the thigh muscle and were followed for immune response and teratoma development. b, After 5 days, splenocytes were recovered for Elispot assays (mean ± s.d., quadruplicates of 6 animals per group, two-tailed Student’s t-test). c, Teratoma growth is depicted for every animal and the percentage of teratoma formation for each group is shown in a separate bar graph (n = 6 per cell amount and iPSC group).

Supplementary Figure 10 Proliferative capacity of B6 iPSCs P7 and P37 in vivo and in vitro.

a, B6 iPSC grafts from P7 and P37 were injected into the thigh muscle of immunodeficient SCID-beige mice. b, Teratoma growth of iPSCs P7 and P37 was followed and the percentage of teratoma formation for each group is shown in a separate bar graph (Individual animals are shown, n = 4 per group). c, In vitro growth of P7 and P37 iPSC cultures was captured with time-laps life cell imaging and the confluency at 0 min and 24 h is shown (representative pictures of two independent experiments). d, The calculated cell proliferation after 24 h is shown (mean ± s.d., 5 replicates per group, two-tailed Student’s t-test).

Supplementary Figure 11 The immunogenic nature of B6 iPSC P37 fate after transplantation.

a, iPSC grafts from P7 and P37 were injected below the kidney capsule of syngeneic B6 recipients. b-d, Infiltration of P7 and P37 iPSC grafts with CD3+ lymphocytes (b), CD335+ natural killer cells (c), and F4/80+ macrophages (d) by immunofluorescence staining is shown. Scale bar 25 μm. e, P37 iPSC grafts at two low cell amounts, which were completely or mostly rejected in immunocompetent B6 mice in Figure 3, were injected into the thigh muscle of B6 recipients immunosuppressed with tacrolimus. f, Elispot assays with splenocytes recovered after 5 days are shown (mean ± s.d., quadruplicates of 5 animals per group). g, Teratoma growth is depicted for every animal and the percentage of teratoma formation for each group is shown in a separate bar graph (n = 5 animals per group). h, P37 iPSC grafts at the same two low cell amounts were then transplanted into immunocompromized CD4 knockout recipients on B6 background. i, Elispot assays with splenocytes recovered after 5 days are shown (mean ± s.d., quadruplicates of 5 animals per group). j, Teratoma growth is depicted for every animal and the percentage of teratoma formation for each group is shown in a separate bar graph (n = 5 animals per group).

Supplementary Figure 12 Presentation of mtDNA neoantigens via MHC.

a, iPSCs (P7) underwent CRISPR-Cas9 inactivation of the B2m and Ciita genes to generate double MHC knockout (dKO) iPSCs and iEC (dKO) were differentiated. b, The knockouts of B2m and Ciita in iEC (dKO) were confirmed by PCR (representative gel of two independent experiments). c, Surface expression of MHC class I and II was assessed by flow cytometry (mean ± s.d., 4 independent experiments per group, two-tailed Student’s t-test). d, B/6 mice were immunized with either iEC (P7) or iEC (dKO) overexpressing B/c (red) or B6 (blue) Co3 and Cytb. The splenocyte response after 5 days against the same overexpressed proteins in either iEC (P7) or iEC (dKO) was assessed in Elispot assays to determine the mechanistic role of MHC. e, Elispot assays of B/6 mice immunized with iEC (P7) or iEC (dKO) overexpressing syngeneic B/6 proteins are shown (mean ± s.d., quadruplicates of 3 animals per group, two-tailed Student’s t-test). f, Elispot assays of B/6 mice immunized with iEC (P7) or iEC (dKO) overexpressing allogeneic B/c proteins are shown. Unstimulated responder splenocytes served a control (mean ± s.d., quadruplicates of 3 animals per group, two-tailed Student’s t-test).

Supplementary Figure 13 Longitudinal mtDNA sequencing of human PBMC SNPs in vivo.

PBMCs were repeatedly isolated from 6 volunteers over a minimum of 6 months and mtDNA sequencing was performed. SNPs in the non-coding D-loop are marked in red. SNPs with < 1% heteroplasmy in the first specimen that increased to > 1% in subsequent specimens were considered de novo mutations and are marked in green. SNPs that have been present in the first PBMC specimen and changed their heteroplasmy >1% over time are marked in black. None of the volunteers developed candidate SNPs for neoantigens in vivo.

Supplementary information

Supplementary Information

Supplementary Figs. 1–13 and Supplementary Notes 1–5

Reporting Summary

Supplementary Tables

Supplementary Tables 1–18

Supplementary Video 1: In vitro proliferation of B6 iPSCs P7 and P37

The proliferation of B6 P7 and P37 iPSCs in vitro was recorded over 24 h and then quantified.

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