Week 9: Pseudo-Homozygous HLA Cells

Hello! This week I’ll revisit HLA matching and discuss strategies of engineering HLA-matched cells for “Off-the-Shelf” CAR T-cells.

Why is HLA matching necessary? Human Leukocyte Antigens (HLA) are responsible for helping the immune system distinguish itself from foreign bodies. When cells with foreign HLA genes are found in the immune system, they are attacked by NK cells or T cells and induce Graft v. Host disease.

Homozygous HLA Cells

Cell banks that house different cell lines that are homozygous for common alleles may be a great way to create “Off-the-Shelf” CAR T-cells. But which alleles must be matched to avoid immune rejection? Many scientists predict that HLA-A, HLA-B, HLA-C, and HLA-DR are the most important for rejection. However, there are no clinical trials that evaluate the feasibility or outcome of HLA matching for “Off-the-Shelf” CAR T-cell therapy.

For some genes, such as HLA-A and HLA-B, there are hundreds of alleles that could be expressed in a given person. However, most of the HLA alleles are in linkage disequilibrium where certain alleles commonly appear together in individuals due to evolution. As a result, it’s easier to bank common cell lines. For example, one study found that Caucasians commonly have the HLA-A*02, 24, 01 alleles and the HLA-B*35, 44, 51 alleles. Other races also have common alleles. (Bardi et al., 2012) Figure 1 (below) illustrates homozygous HLA matching along the HLA-A, HLA-B, and HLA-DRB1 genes.

Pseudo-Homozygous HLA Cells

Finding HLA homozygous cell lines that fit diverse populations is difficult. As a result, it is necessary to explore strategies to artificially create homozygous cell lines. One group of scientists has proposed engineering HLA class I pseudo-homozygous iPSCs from HLA heterozygous donors through allele-specific genetic engineering. They knocked out a single allele of HLA-A, HLA-B, and HLA-C via gene editing and using CRISPR-Cas9 to transfect (introduce nucleic acids to eukaryotic cells through non-viral methods) the HLA-A*01, HLA-B*07, and HLA-C*07 alleles. These newly engineered HLA-ABC cells were then placed in a solution of peripheral blood mononuclear cells (PBMC), blood cells with a round nucleus which include T cells, B cells, and NK cells. The researchers performed Chromium-51 release assays on the sample to access cytotoxicity, and it was determined that HLA-ABC cells could evade T cell toxicities. (Xu et al., 2019)

Another group of researchers utilized a different method to create pseudo-homozygous Mesenchymal stem cells (multipotent stem cells found in the bone marrow that contain only HLA class I). Instead of knocking out and replacing HLA alleles, they only knocked out one set of HLA alleles for HLA-A, -B, and -C using CRISPR-Cas9. The study found that the mesenchymal stem cells were still able to differentiate in vitro and therefore resisted rejection. (Kwon et al., 2020) While pseudo-homozygous HLA strategies have shown to evade T cell toxicity, these methods have not been tested in the clinic and may not correlate with iPSC allogeneic CAR T-cells where additional HLA matching may be necessary.

Conclusion

This week, I accessed the extent of HLA matching that is required to engineer “Off-the-Shelf” CAR T-cells. Ultimately, it seems reasonable to start with matches at HLA-A, -B, -C, and -DR allele locations. Pseudo-homozygous methods may be a way to expand HLA banks and expand banking to more diverse populations. Thanks for reading! Tune in next week as I continue my quest to “Off-the-Shelf” CAR T-cells!

Sidharth