What’s Your Type: HLA Typing for Translational Medicine

Human leukocyte antigens (HLA) are found on the cell surface of almost all cells within an individual and are how your immune system can tell which cells belong to ‘self’ and which are foreign invaders. The various genes that make up these antigens are highly polymorphic, meaning that everyone’s ‘code’ is unique and your body would attack cells that came from another person or organism. This is what makes organ and tissue transplants so challenging, because finding a close enough match, even amongst siblings and other close family members, may not be possible.  Tissue registries can help find a suitably matched unrelated donor, but are often unsuccessful, especially for certain ethnicities that are underrepresented in such databases. 

What is HLA typing?

The HLA system represents the cell membrane glycoproteins that makeup the major histocompatibility complex (MHC) .(1) These predominantly include the class I and class II molecules where class I molecules are always presented on all nucleated cells, and class II can be conditionally expressed on all cell types, but are typically expressed only on antigen-presenting cells, like macrophages, B cells and dendritic cells. The three MHC class I molecules are composed of HLA A, B, C genes that together orchestrate immune regulation, activating adaptive immune responses and allowing for activation of the immune response to foreign invaders. In addition, there are several other non-classical genes that make up the MHC complex, which also contribute to the HLA phenotype.

HLA typing has applications across basic and translational research as well as in the clinical care setting. Deciding which method of HLA typing to employ depends on the specific needs of the researcher or clinician. 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 HLA combinations possible, HLA matching can be very complex and difficult to coordinate for a successful transplant match. For research purposes, researchers may be interested in typing for a specific HLA variant that has already been associated with a particular disease, or they may want to sequence all HLA gene variants to be able to identify novel disease-associated variants.

How to test for HLA variants

For typing several MHC class I and II related loci, PCR-rSSO (reverse sequencing specific oligonucleotide) represents a commonly used method.(2) These are lower throughput and are often batch tested on a single PCR plate for efficiency and reduced costs. Commercial rSSO kits (i.e. Luminex® platform) with DNA probes that bind to specific variant-related sequences can help automate the process; however some typing ambiguities may still exist and additional primers or testing may be needed to confirm the results. Standard qPCR or PCR-SSP (sequence-specific amplification) can rapidly provide low-to-medium resolution typing, which may be suitable in some deceased transplant donor scenarios or for certain research needs that need quick results. Both PCR-SSP and qPCR use sequence-specific primers to determine the presence or absence of a particular HLA allele or allele groups. 

Sequence based typing (SBT) can also be performed through Sanger sequencing or next-generation sequencing (NGS).(3) Although still dependent on PCR, these methods take an unbiased approach to produce a high-resolution DNA sequence for each allele of interest. With NGS, multi-gene testing for all classical HLA genes can be performed within a single reaction with only 50ng of genomic DNA, while Sanger sequencing would yield the exact sequence at each specific locus individually, requiring separate reactions for each HLA gene of interest. 

Overall, SBT methods, particularly NGS, may often have a longer turnaround time, given the cost and preference to batch samples together in a single plate. Ambiguities in the typing results are much less likely due to the clonal nature from PCR amplification and the high-resolution nucleotide results produced. As NGS becomes more affordable, it is likely to replace other methods as it has very low error rates and high sensitivity, eliminating the need to repeat test samples that may come back inconclusive for certain variants. 

Considerations for HLA matching

Different types of transplants can tolerate different levels of HLA mismatching based on a variety of factors. HLA matching is most important for hematopoietic stem cell transplants to restore impaired bone marrow transplants, with the greatest clinical impact on success rates if the donor and recipient are well-matched for many HLA loci.(4) These donated stem cells engraft in the recipient’s bone marrow cavity, seeding and producing a new, healthy immune system. As such, the donor’s circulating leukocytes could identify the recipient as foreign and systemically attack many organs. This phenomenon, known as graft-vs-host disease, can range in severity from mild to life-threatening. With more difficult to obtain organs, such as heart or lung transplants, a higher degree of mismatch in HLA type is acceptable  as there are other considerations such as clinical urgency and cytomegalovirus (CMV) compatibility that take a higher precedence. On the other hand, corneal transplants can be performed without any HLA matching as the eye is considered an immunologically privileged site where immune rejection of a transplant is rare. 

Not only is it important to consider the HLA type of the donor and recipient, but it is also critical to test for circulating HLA antibodies the recipient.(4) If antibodies against specific HLA proteins exist in the recipient, they would be primed to attack and reject the transplant if the donor contained those specific HLA proteins. This is why it is also important to minimize HLA mismatches for each transplant, as it reduces the chance that the recipient will develop reactive antibodies that could preclude the individual from receiving future transplants. 

Applications in Research

Certain HLA alleles can affect the susceptibility and severity of viral infections, while others are associated with distinct genetic predispositions to diseases like cancer and autoimmune conditions.(5) Understanding which haplotypes may be at higher risk of more severe disease or a poorer prognosis can be particularly useful in translational applications when developing therapeutics or studying certain therapeutic modalities. Identifying HLA haplotypes associated with certain diseases can be used clinically to identify risk factors or therapeutic approaches through personalized medicine approaches. Understanding the immunopeptidome could also help improve immunotherapies and help in next generation vaccine development against cancer, autoimmune or infectious diseases. 

Overall, HLA typing can be a valuable source of data across many basic and translational research studies. Sanguine has experience with HLA typing for various research applications and 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: http://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: http://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.