Study revealed that two genes commonly overexpressed in iPSCs trigger a hefty T cell-mediated attack on implanted cells.

Researchers are reporting that cell therapies based on induced pluripotent stem cells (iPSCs) could trigger immune rejection reactions even when the iPSCs have been derived from a patient’s own cells. It has been assumed that autologous iPSCs would be safe from a recipient’s immune system, but the new work in mice, by a team at the University of California, San Francisco demonstrated that overexpression of some genes by iPSCs triggers a hefty CD4+ and CD8+ T-cell attack on the cells following their implantation in the same strain of animal. Conversely, embryonic stem cells didn’t lead to the same rejection issues.

Biology professor Yang Xu, Ph.D., and colleagues claim their findings mean “the assumption that cells derived from iPSCs are totally immune tolerant has to be re-evaluated before considering human trials.” The team’s work is published in Nature in a paper titled “Immunogenicity of induced pluripotent stem cells.”

Dr. Xu and colleagues reprogrammed B6 mouse embryonic fibroblasts (MEFs) into iPSCs using either a retroviral approach or an episomal technique that doesn’t involve genomic integration.  They then implanted the resulting cells back into mice of the same inbred strain from which the MEFs had been taken and evaluated the ability of the cells to develop into teratomas.   

What they found was that while embryonic stem cells readily formed teratomas in recipient animals, the retroviral-generated iPSCs (ViPSCs) were rejected by the immune system, and most of the teratomas formed by the episomal iPSCs (EiPSCs) were immunogenic and showed evidence of T-cell infiltration and immune response-led tissue damage and tumor regression.  

Genetic analysis of the teratomas revealed that the iPSCs overexpressed a number of genes. Expression analysis of six regressing teratomas formed by two different EiPSCs indicated that nine of the 23 tested genes (Lce1f, Spt1, Cyp3a11, Zg16, Lce3a, Chi3L4, Olr1, Retn, and Hormad1) were commonly overexpressed. Interestingly, Hormad1 has been identified as a tumor antigen and Spt1 as a tissue-specific antigen, the authors note.

They then ectopically expressed seven of these genes in ESCs and their teratomas to evaluate whether this would impact on the immune response of recipient animals. Unlike unadulterated ESCs, which escaped immune recognition as evidenced by their ability to readily form teratomas, over 80% of Zg16-expressing ESC implants and 50% of Hormad1 or Cyp3a11 ESC implants failed to form visible teratomas. Extensive T-cell infiltration and widespread necrosis were detected in the teratomas that did form from the Zg16- and Hormad1- expressing ESCs.

The immune attack on ViPSCs and the Zg16- and Hormad1-expressing cells was abolished when the cells were instead implanted in mice deficient in CD8 and CD4 cells. The team says this confirms the critical role of CD4+ helper T cells and CD8+ cytotoxic T cells in immune rejection. Similarly, there was no regression of teratomas formed by EiPSCs implanted in CD4 and CD8 cell-deficient animals.  “These findings also indicate that the innate immunity does not have an important role in the immune rejection of the cells derived from iPSCs,” the authors note.

Separate studies subsequently confirmed that Hormad1 was also overexpressed in most teratomas formed by four independently generated, integration-free iPSCs reprogrammed with adenoviral vectors, recombinant proteins, or plasmid vectors, while Zg16 was overexpressed in most teratomas formed by iPSCs reprogrammed with recombinant proteins.

Dr. Xu’s group admits that their findings haven’t identified the specific peptides responsible for activating T cells but say the studies do demonstrate that abnormal expression of Hormad1 and Zg16 contributes directly to the immunogenicity of cells derived from iPSCs.

“Therefore, the abnormal expression of such immunogenic proteins could represent a common mechanism to induce T cell-mediated immune responses to cells derived from iPSCs,” they conclude. “The T-dependent immune response is likely due to the abnormal expression of antigens not expressed during normal development or differentiation of ESCs, leading to the break of peripheral tolerance. The expression of these minor antigens could be due to the subtle yet apparent epigenetic difference between iPSCs and ESCs.”

The researchers stress that current reprogramming technology will need to be optimized to minimize the epigenetic differences between iPSCs and ESCs with respect to the clinical development of iPSC-derived therapies. They suggest their mouse-based immune response assay could provide “a robust screening platform for improving the reprogramming technology.”

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