Scientists from the National Research Center for Hematology (Moscow) have shared their findings of a new way to rapidly identify genetic differences between the donor and recipient in the journal Frontiers in Immunology. This method may improve the results of hematopoietic stem cell transplantation. The studies were supported by a grant from the Russian Science Foundation.
Hematopoietic stem cell transplantation is often used to treat blood cancer. Donated immune blood cells (white blood cells) then target the patient's malignant cells and destroy them, causing the “graft-versus-leukemia” effect. Unfortunately, these white cells can also target healthy host cells. This is known as “graft-versus-host disease”. The induction of the immune response is associated with the recognition of antigens: small fragments of the cell’s internal proteins, which are carried to its surface by major histocompatibility complex (MHC) molecules, which are highly variable among individuals, so are the presented antigens. This is why most of the attention in hematopoietic stem cell transplantation is given to this complex. However, donor-recipient compatibility is affected not only by the major complex but also by minor histocompatibility antigens, they are inner cell peptides, but due to DNA polymorphisms, they can differ in a pair of fully MHC matched people. There are more than 60 known minor antigens. Their effects on the immune response and possible uses in cancer immunotherapy are being studied at the laboratory of Grigory Efimov, a researcher at the National Research Centre for Hematology under the Ministry of Health of the Russian Federation.
The effect produced by the transplant depends on the degree of dissimilarity between minor histocompatibility antigens of the donor and recipient. Existing methods for detecting such differences have many disadvantages, most notably the time it takes to conduct the tests. This situation creates a demand for rapid and reliable genotyping for the minor histocompatibility antigens.
The method developed by the Russian scientists allows differences in the donor and recipient genes to be detected within one hour, obviating the need for more labor-intensive and expensive techniques. This method is a modification of the polymerase chain reaction. During this reaction, the number of copies of the DNA fragment of interest grows by a large factor. The new method combines two types of reaction: the allele-specific and the real-time PCR. Primers, specially synthesized short DNA sequences complementary to the fragment under investigation, are required to start the process. The PCR succeeds only if the allele-specific primer matches a particular variation of the gene. A real-time PCR makes it possible to detect the DNA sequences of interest, in addition to multiplexing reactions.
The Moscow-based immunologists selected suitable primers for 20 minor antigens presented via one of the most common MHC alleles amongst Caucasians. They are mixed with the DNA submitted for analysis, and then real-time PCR is carried out. The investigators monitor the course of the reaction and see which primers result in successful runs, thereby determining which minor antigens are present in a particular patient.
“The method we developed makes it possible to rapidly genotype donor-recipient pairs and detect genetic differences that may induce an immune response after transplantation. On the one hand, the test allows us to predict the intensity of the immune response and the possible outcome of the transplantation and make appropriate adjustments to immunosuppressive therapy that is required to avoid graft-versus-host disease. On the other hand, it will make it possible to choose optimum immunotherapy targets for each patient, targeting minor histocompatibility antigens. This approach is being developed at our laboratory,” said the scientists.
The proposed approach can also detect differences in other genes, which may encourage its application in a variety of laboratory studies.