What’s Your Type: HLA Typing for Translational Medicine

Human leukocyte antigens (HLA) sit on the cell surface of almost all cells in your body. They’re how your immune system tells which cells belong to “self” and which are foreign invaders. The genes that make up these antigens are highly polymorphic — everyone’s “code” is unique, and your body will attack cells that come from another person or organism. This is what makes organ and tissue transplants so challenging: finding a close enough match, even among siblings and other close family members, may not be possible. Tissue registries can help find a suitably matched unrelated donor, but they’re often unsuccessful, especially for certain ethnicities that are underrepresented in these databases.

What is HLA typing?

The HLA system represents the cell membrane glycoproteins that make up the major histocompatibility complex (MHC).(1) These mainly include class I and class II molecules. Class I molecules are always present on all nucleated cells. Class II molecules can be conditionally expressed on any cell type, but they’re typically expressed only on antigen-presenting cells, like macrophages, B cells, and dendritic cells.

The three MHC class I molecules are made up of HLA A, B, and C genes, which together orchestrate immune regulation and activate adaptive immune responses against foreign invaders. Several other non-classical genes also make up the MHC complex and contribute to the HLA phenotype.

HLA typing has applications across basic and translational research, as well as in clinical care. Which method a researcher or clinician chooses depends on their specific needs. HLA typing — with a particular focus on class I molecules — is necessary for both the donor and recipient of a stem cell or solid organ transplant, to reduce the risk of host rejection. With thousands of possible HLA combinations, matching can be very complex and hard to coordinate for a successful transplant. For research, scientists may want to type for a specific HLA variant already linked to a particular disease, or sequence all HLA gene variants to identify novel disease-associated ones.

How to test for HLA variants

For typing several MHC class I and II related loci, PCR-rSSO (reverse sequencing specific oligonucleotide) is a commonly used method.(2) It has lower throughput and is often batch-tested on a single PCR plate for efficiency and reduced cost. Commercial rSSO kits (like the Luminex® platform) use DNA probes that bind to specific variant-related sequences, which helps automate the process. Some typing ambiguities can still occur, though, and may need additional primers or testing to confirm results.

Standard qPCR or PCR-SSP (sequence-specific amplification) can quickly provide low-to-medium resolution typing, which works well for certain deceased transplant donor scenarios or research needs that require fast results. Both PCR-SSP and qPCR use sequence-specific primers to detect whether a particular HLA allele or allele group is present.

Sequence based typing (SBT) can also be done through Sanger sequencing or next-generation sequencing (NGS).(3) These methods still depend on PCR, but they take an unbiased approach that produces a high-resolution DNA sequence for each allele of interest. With NGS, researchers can test multiple genes for all classical HLA genes in a single reaction using only 50ng of genomic DNA. Sanger sequencing, by contrast, yields the exact sequence at each specific locus individually, requiring a separate reaction for each HLA gene of interest.

Overall, SBT methods — particularly NGS — often take longer to turn around, given the cost and preference for batching samples on a single plate. But ambiguities in the results are much less likely, thanks to the clonal nature of PCR amplification and the high-resolution nucleotide results these methods produce. As NGS becomes more affordable, it’s likely to replace other methods, since it has very low error rates and high sensitivity — eliminating the need to retest samples that come back inconclusive for certain variants.

Considerations for HLA matching

Different types of transplants can tolerate different levels of HLA mismatching, depending on several factors. HLA matching matters most for hematopoietic stem cell transplants used to restore impaired bone marrow, where success rates improve the most when donor and recipient are well-matched across many HLA loci.(4) These donated stem cells engraft in the recipient’s bone marrow cavity, seeding and producing a new, healthy immune system. Because of this, the donor’s circulating leukocytes could identify the recipient’s body as foreign and attack many organs systemically — a condition known as graft-vs-host disease, which ranges from mild to life-threatening.

For organs that are harder to obtain, like hearts or lungs, a higher degree of HLA mismatch is acceptable, since other factors — like clinical urgency and cytomegalovirus (CMV) compatibility — take higher priority. Corneal transplants, on the other hand, can be performed without any HLA matching at all, since the eye is considered an immunologically privileged site where transplant rejection rarely happens.

It’s not just the HLA type of donor and recipient that matters — it’s also critical to test the recipient for circulating HLA antibodies.(4) If the recipient already has antibodies against specific HLA proteins, those antibodies would be primed to attack and reject the transplant if the donor’s tissue contained those same HLA proteins. This is another reason to minimize HLA mismatches for every transplant: it reduces the chance that the recipient develops reactive antibodies that could rule them out for future transplants.

Applications in Research

Certain HLA alleles can affect how susceptible someone is to viral infections, and how severe those infections become, while others are linked to distinct genetic predispositions to diseases like cancer and autoimmune conditions.(5) Understanding which haplotypes carry a higher risk of severe disease or a poorer prognosis can be especially useful in translational research, when developing therapeutics or studying certain treatment approaches. Identifying HLA haplotypes linked to specific diseases can help clinicians spot risk factors or guide therapeutic approaches through personalized medicine. Understanding the immunopeptidome could also help improve immunotherapies and support next-generation vaccine development against cancer, autoimmune conditions, or infectious disease.

Overall, HLA typing is a valuable source of data across many basic and translational research studies. Sanguine has experience with HLA typing for a wide range of research applications, and we can meet your specific research needs with rapid turnaround times.

References

1. Howell WM, Carter V, Clark B. The HLA system: immunobiology, HLA typing, antibody screening and crossmatching techniques. J Clin Pathol. 2010 May;63(5):387–90.
2. Dunckley H. HLA Typing by SSO and SSP Methods. In: Christiansen FT, Tait BD, editors. Immunogenetics [Internet]. Totowa, NJ: Humana Press; 2012 [cited 2022 Jul 13]. p. 9–25. (Methods in Molecular BiologyTM; vol. 882). Available from: https://link.springer.com/10.1007/978-1-61779-842-9_2
3. Gabriel C, Fürst D, Faé I, Wenda S, Zollikofer C, Mytilineos J, et al. HLA typing by next-generation sequencing – getting closer to reality: HLA typing by NGS. Tissue Antigens. 2014 Feb;83(2):65–75.
4. Sheldon S, Poulton K. HLA Typing and Its Influence on Organ Transplantation. In: Transplantation Immunology [Internet]. New Jersey: Humana Press; 2006 [cited 2022 Jul 13]. p. 157–74. Available from: https://link.springer.com/10.1385/1-59745-049-9:157
5. Dendrou CA, Petersen J, Rossjohn J, Fugger L. HLA variation and disease. Nat Rev Immunol. 2018 May;18(5):325–39.