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Project titleT-cell response to immunodominant minor histocompatibility antigens after allogeneic hematopoietic stem cell transplantation

AffiliationFederal State Budget Institution National Research center for Hematology Ministry of Health Russion Federation,

Keywordsgenomic polymorphisms, alloreactivity, allogeneic hematopoietic stem cell transplantation, minor histocompatibility antigens, next generation sequencing, graft versus tumor effect, GVT, graft versus host disease, GVHD, major histocompatibility complex, MHC-multimers, T cell receptor repertiores

Annotation
Currently allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents the only one potentially curative option for a number of hematopoietic malignancies. The efficiency of allo-HSCT therapy relies on antigen-specific alloreactive immune response of the donor cells towards patient’s hematopoietic system, including residual cancer cells that have survived pre-transplant chemotherapy. At the same time such alloreactive immune response, apart of hematopoietic tissues, can damage healthy tissues of recipient in the course of graft versus host disease (GvHD) that represents one of the main complications of allo-HSCT. The targets of T lymphocyte mediated immune response, including alloreactive one, are peptides displayed on the surface of the cells in the context of major histocompatibility complex (MHC, in humans the genes encoding these proteins are designated as HLA, human leukocyte antigens). Allo-HSCT from a donor bearing HLA-alleles different from recipient’s HLA-alleles results in severe forms of GvHD. Accordingly, the majority of all transplantations nowadays is performed either from HLA-identical sibling, either from HLA-matching donor selected with the use of bone marrow donor databases. In the case of HLA-matched transplantation the remaining molecular targets of alloreactive response are recipient’s unique peptides presented in HLA molecules that are recognized as foreign by donor immune cells. These peptides are called minor histocompatibility antigens (MiHA). Their occurrence is explained by the genome polymorphism in humans, mainly by the existence of nonsynonymous single nucleotide polymorphisms (nsSNP): translation and proteasomal degradation of polymorphic proteins lead to the generation of unique sets of peptides presented in the MHC complexes on the tissues of every individual. In the context of allo-HSCT unique recipient peptides are seen as foreign by the donor T cells. At the moment we know around 50 immunogenic nonsynonymous single nucleotide polymorphisms, i.e. polymorphisms coding minor antigens of histocompatibility. Though some of those MiHAs have been already used as targets for experimental immunotherapy of malignant hematological diseases, the molecular mechanism of nsSNP immunogenicity is still poorly understood. When we take into account only known MiHAs, every pair of donor and recipient will mismatch in average by one or two known anti-MiHA. However, the latest mass-spectrometric analysis of immunopeptidome data points to the fact that each donor and recipient differs for a few dozens of such immunogenic peptides. Besides, each donor-recipient pair shall bear significant number of unique or rare nsSNP, and their contribution to alloreactive response have never been studied. To be able to measure immunogenicity of these nsSNP and to estimate their contribution to alloreactive response we propose a detailed study of alloreactive immune response in sufficient number of donor-recipient pairs. Moreover the very important open question is the number of MiHAs that could induce simultaneous productive responses in vivo, i.e. to which extent the alloreactive immune response is immunodominant. It is also unknown which factors determine immunodominance and whether we can predict immunogenic antigens in silico or in vitro. In the course of the current project we plan to study alloreactive responses within several donor-recipient pairs that undergo allo-HSCT, including analysis of specific responses towards known MiHA and towards dozens of in silico predicted immunogenic minor histocompatibility antigens (including those encoded by rare and individual nsSNP). The majority of allo-HSCT studies up to date were mainly focused on the search and analysis of new and frequently occurring MiHAs that could be potentially used in immunotherapy. Current work aims to quantitatively describe fundamentals of alloreactive immune response. The understanding of factors that influence alloreactive immune response could improve predictions of immunodominant antigens for a specific donor-recipient pair, and thus can increase the efficiency of allo-HSCT.

Expected results
In the course of the present project we will study the number of alloreactive cells, clonality of alloreactive response and its variability in several donor-patient pairs undergoing HLA-matched allo-HSCT, both by in vitro and in vivo methods. This will allow, on the one hand, to obtain an upper estimate for the number of antigens targeted by alloreactive response (the number of T cell clones activated by allogeneic cells), on the other hand, to assess the number of significantly expanded clones (the response to the immunodominant MiHAs), and finally we will study the variability of this parameters between the donor-patient pairs. These previously unexplored, but important parameters can be associated with the severity GVHD and GVL effects. One of the main features of the proposed project is the comparison of results of in vitro and in vivo alloreactivity assays. We will obtain an intersection of full alloreactive repertoire in vivo/in vitro, and intersections of antigen-specific repertoires for already known minor histocompatibility antigens. It is expected that the sequences of the most expanded clones will be found both in vivo and in vitro. These data will allow to respond to an extremely interesting question: is it possible to predict the clones that will become alloreactive in vivo in advance, before the transplantation, based only on laboratory tests with donor and patient blood samples. If alloreactivity can be predicted, in the future, such tests could form the basis of the protocol for the depletion of the cells specific to the most immunogenic targets from the graft. Another important result will be the evaluation of the contribution to the alloreactive response made by rare and or unique for the donor-recipient pair immunogenic nsSNPs. Earlier, studying the publicly available genomic data, we predicted that rare MiHAs should cover the majority of the targets of alloreactive response. In this project we aim to for the first time to confirm these results experimentally by testing immunogenicity of in silico predicted individual MiHAs. Previously, researchers focused exclusively on the search for MiHAs common in the population, which is explained by the practical value associated with the wide clinical applicability of these antigens. However, the study of individual MiHAs is not only an important fundamental problem, but also might have practical applications: individual MiHAs can be used as additional targets of immunotherapy to improve its efficacy or in the case where mismatches at common MiHAs are absent in a particular pair. The information about antigen-specific TCR repertoires accumulated during the work will allow us to find out, if there is a correlation between the immunogenicity of the epitopes (the ability to induce T cell proliferation in vivo) and the number of the specific naïve antigen-specific cells in the initial repertoire of the donor. This hypothesis was previously confirmed in murine models of alloreactivity, and its confirmation for the human will have both fundamental value and practical application. An additional result of this work could be the discovery of new clinically relevant minor histocompatibility antigens.

Annotation of the results obtained in 2019
We predicted 19 new histocompatibility minor antigens. With the use of synthetic peptides, it was confirmed that all predicted antigens are capable of MHC binding. An analysis of the endogenous processing of proteasome peptides and their ability to elicit an immune response was carried out using co-cultivation of naive donor CD8 + lymphocytes with dendritic cells expressing the studied antigens. For this, tandem minigens were constructed that encode the predicted antigens surrounded by amino acids from the original proteins. For the three predicted minor antigens, it was confirmed that they are processed by the proteasome and are capable of eliciting an immune response, thus they represent the new described minor antigens. For another four minor antigens, an immune response developed with the addition of synthetic antigenic peptides. At the same time, most of the studied peptides were non-immunogenic. Which, most likely, is associated with an extremely low frequency of antigen-specific lymphocytes. Antigen-specific cells were sorted by flow cytometry, after which the sequences encoding the T-cell receptor were analyzed using NGS. All receptors obtained had low probability of recombination, which is consistent with their low in vitro immunogenicity. A receptor for one of the antigens was cloned into a lentiviral vector. It was confirmed that the transduced lymphocytes specifically bind the antigen.

Publications

1. Dmitrii S. Romaniuk, Anna M. Postovskaya, Alexandra A. Khmelevskaya, Dmitry B. Malko and Grigory A. Efimov Rapid Multiplex Genotyping of 20 HLA-A*02:01 Restricted Minor Histocompatibility Antigens Frontiers in Immunology, - (year - 2019) https://doi.org/10.3389/fimmu.2019.01226

2. Romaniuk D.S., Khmelevskaya A.A., Postovskaya A.M., Malko D.B., Kuzminova E.P., Khamaganova E.G., Efimov G.A. КЛИНИЧЕСКИ ЗНАЧИМЫЕ МИНОРНЫЕ АНТИГЕНЫ ГИСТОСОВМЕСТИМОСТИ ДЛЯ РОССИЙСКИХ ПАЦИЕНТОВ, ПОЛУЧАЮЩИХ ТРАНСПЛАНТАЦИЮ СТВОЛОВЫХ КЛЕТОК КРОВИ Медицинская иммунология, Т. 21, No 5, стр. 847-860 (year - 2019) https://doi.org/10.15789/1563-0625-2019-5-847-860

3. Zoya Konova, Nadia Bykova, Murad Vagida, Larisa Kuzmina, Mikhail Drokov, Grigory Efimov, Elena Parovichnikova, Valery Savchenko T-cell subpopulation, specific for patients after allogeneic hematopoietic stem cell transplantation Bone Marrow Transplantation, 54 S, 491-492 (year - 2019) https://doi.org/10.1038/s41409-019-0559-4

4. - СпособидентификациииммуногенныхнесоответствийДНКвпарахдонор-пациентприпланировании трансплантации гемопоэтических стволовых клеток -, 2 675 597 (year - )

5. - Российские медики выяснили, как улучшить пересадку костного мозга РИА НОВОСТИ, - (year - )

6. - Создан способ найти отличия между клетками донора костного мозга и пациента Газета.Ru, - (year - )

7. - Найден способ быстрого обнаружения различий в ДНК донора и реципиента при пересадке костного мозга RT (Russia Today), - (year - )

8. - Новый метод анализа генетической совместимости повысит эффективность пересадки при лейкозе ТАСС, - (year - )

9. - В НМИЦ гематологии нашли быстрый способ определения различий в генетике донора и пациента МЕДВЕСТНИК, - (year - )

10. - Российские иммунологи разработали новый метод выявления генетических различий Indicator, - (year - )

Annotation of the results obtained in 2017
In unrelated HLA-matched hematopoietic stem cell transplantation there are thousands of mismatches of non- synonymous SNPs between donor and patient, some of which result in polymorphic peptides presented in MHC context, known as Minor Histocompatibility Antigens (MiHAs). As these peptides are foreign to the donor immune cells, they may drive allogeneic immune response, causing both graft-versus-host disease and beneficial graft-versus-tumor effect. But due to the phenomenon of immunodominance some of MiHAs are much more immunogenic than the others. We have selected seven fully HLA-matched donor-patient pairs undergoing allogeneic hematopoietic stem cell transplantation and analyzed in vitro response to previously described MiHAs. To this end, we first developed a method of rapid multiplex MiHA genotyping of immunogenic MiHA mismatches between donors and patient based on real-time PCR with fluorescent probes. The donor-patient pairs were genotyped for the panel of MiHAs presented in common HLA-A alleles, and potentially immunogenic mismatches were found for each pair. Then dendritic cells were derived from donor peripheral blood monocytes, loaded with synthesized antigenic peptides and co-cultured with autologous naive T cells in extreme limiting dilution assay, which allowed us to calculate naive T cell frequencies. Furthermore, we have separated antigen-specific CD8+ T lymphocytes by the use of magnetic sorting of activated T cells cloned and sequenced variable fragments of alpha and beta chains of their T cell receptor (TCR). Dominant clones were also tested for antigen-specific effector functions. We have demonstrated that frequencies of naive T cells specific for immunodominant antigens (HA-2, HA-1, LB-ADIR) was substantially higher than for subdominant antigens (ACC-1Y) and variability between antigens is drastically higher than between donors. The variation of frequencies of naive T cells specific to various MiHAs could be explained either by the peptide properties or by the influence of the negative selection on initial T cell repertoire. ACC-1Y has the alternative allelic variant (ACC-1C) presented in the thymus, which leads to the elimination of all cross-reactive clones, whereas alternative allelic variant of HA-2, HA-1 and LB-ADIR are not presented to the immune system (HA-2 is cleaved by proteasome other two have low affinity to HLA-A*02 allele). The obtained data indicate that the phenomenon of immunodominance of MiHAs may be dictated by the frequencies of MIHA-specific T cells in turn influenced by negative selection in the thymus. The analysis of TCR sequences of MiHA-specific cell clones demonstrated that TCR alpha chain most likely is paramount to the recognition of HA-2 antigen. Antigen-specific clones were substantially enriched TRAV21 and TRAJ42. Previously published HA-2 specific TCR sequences also were skewed towards the usage of these V and J genes in alpha-chain. We have demonstrated that MiHA-specific T cells express IFN-gamma upon stimulation with cells, expressing mismatched MiHA and could mediate cytotoxicity of target cells, while autologous cells (presenting peptide with a single amino acid substitution in their MHC) are protected from the cytotoxicity. This indicates that in MiHA-specific T cell cultures only the cells that did not encounter cognate antigen in the thymus are expanded i.e. no breach of tolerance is occurring. Thus these cells may be used for T cell therapy post MiHA-mismatched transplantations. Besides, during the first year of the project, the valuable collection of cell samples or donor-patient pars was accumulated, which would be used during the second and third years.

Publications

1. A. S. Vdovin, N. A. Bykova, G. A. E mov Т-ЛИМФОЦИТЫ С МОДИФИЦИРОВАННОЙ СПЕЦИФИЧНОСТЬЮ В ТЕРАПИИ ЗЛОКАЧЕСТВЕННЫХ ЗАБОЛЕВАНИЙ МОЛЕКУЛЯРНАЯ БИОЛОГИЯ, том 51, No 6, с. 1–17 (year - 2017) https://doi.org/10.7868/S002689841706012X

2. Anna Kuchmiy, Savely Sheetikov, Dmitrii Romaniuk, Anna Postovskaya, Sergey Filkin, Nadezhda Bykova, Grigory Efimov IMMUNOGENICITY OF MINOR HISTOCOMPATIBILITY ANTIGENS MAY BE DEPENDENT ON THE FREQUENCIES OF NAIVE PRECURSOR CELLS IN THE DONOR REPERTOIRE Human Immunology, 78 (2017) 51–254 (year - 2017) https://doi.org/10.1016/j.humimm.2017.06.088

3. Anna Kuchmiy, Savely Sheetikov, Dmitrii Romaniuk, Anna Postovskaya, Sergey Filkin, Nadia Bykova, Grigory Efimov and Valeriy G. Savchenko Highly Variable Frequencies of Donor Antigen-Specific Naive Cells May Dictate the Immunogenicity of Minor Histocompatibility Antigens after Hematopoietic Stem Cell Transplantation Blood, - (year - 2017)

4. Nadezhda Bykova, Dmitry Malko, Grigory Efimov DISCREPANCY BETWEEN IN SILICO PREDICTIONS AND EXPERIMENTAL ESTIMATION OF DOMINANT/CODOMINANT RATIO OF POTENTIAL MINOR HISTOCOMPATIBILITY ANTIGENS Human Immunology, 78 (2017) 1–50 (year - 2017) https://doi.org/10.1016/j.humimm.2017.06.036

Annotation of the results obtained in 2018
In the course of the second year of the project, we estimated the frequencies of naive precursors that recognize minor histocompatibility antigens (MiHA), for which we used two independent methods: NGS sequencing of T-lymphocyte receptor repertoires from the antigen-specific expansions, as well as the limiting dilution assay followed by the statistical calculation of the frequency of antigen-specific clones. It was shown that the frequency of precursors for different MiHAs differs by several orders of magnitude. This means that when predicting new MiHAs, it is necessary to take into account not only the binding constant of the peptide to the MHC molecule and peptide processing by the proteasome, but also the potential frequency of naive precursors in the repertoire. We obtained 149 sequences encoding T-cell receptors specific for 5 minor histocompatibility antigens and the degree of their homology was analyzed. We showed that TCR alpha chains specific for the minor HA-2 antigen have a high level of amino acid homology, which allows predicting the specificity of the newly sequenced clones. At the same time, no such homology was found for the other minor antigens studied. Using antigen-specific expansion and subsequent sequencing of TCR repertoires, the response to the minor antigens HA-1 and HA-2 in vivo was detected in patients after allogeneic transplantation. It was shown that the number of antigen-specific cells circulating in the blood does not allow detection of antigen-specific clones directly ex vivo. The response to both of these antigens is in vivo was polyclonal, which is consistent with the high frequency of precursors found in the donors. We demonstrated that after the transplantation, patients had a significantly increased population of cytotoxic lymphocytes, expressing both co-stimulatory markers and inhibitory markers. The proportion of cells with this phenotype significantly increases over time since the transplantation. The full exome sequencing of 3 donor-recipient pairs was performed and the polymorphisms that most likely could encode previously unknown minor antigens were predicted. The most promising polymorphisms were selected for analysis on the 3rd year. We developed a method for obtaining antigen-specific expansions on minor antigens using autologous dendritic cells transfected with RNA encoding tandem minigens. Previously similar method was described only for validation of the immunogenicity of neoantigens.

Publications

1. Cytotoxic lymphocyte modification with the receptor specific to the minor histocompatibility antigen ACC-Y МОДИФИКАЦИЯ ЦИТОТОКСИЧЕСКИХ ЛИМФОЦИТОВ РЕЦЕПТОРОМ, СПЕЦИФИЧНЫМ К МИНОРНОМУ АНТИГЕНУ ГИСТОСОВМЕСТИМОСТИ ACC1-Y Молекулярная биология, - (year - 2019)

2. In silico analysis of the T-cell receptor sequences, specific to the minor histocompatibility antigen HA In silico анализ последовательностей Т-клеточных рецепторов, специфичных к минорному антигену гистосовместимости HA-2 Российский иммунологический журнал, - (year - 2019)

3. Nadia A. Bykova*, Dmitry B. Malko, Grigory A. Efimov In Silico analysis of the Minor histocompatibility antigen landscape Based on the 1000 genomes Project Frontiers in Immunology, August 2018 | Volume 9 | Article 1819 (year - 2018) https://doi.org/10.3389/fimmu.2018.01819

4. Nadia Bykova, Dmitrii Romaniuk, Grigory Efimov, Zoya Konova Discovery of a new minor histocompatibility antigen presented in HLA-B*13:02 allele Bone Marrow Transplantation, - (year - 2018) https://doi.org/10.1038/s41409-018-0354-7

5. - Способ идентификации иммуногенных несоответствий ДНК в парах донор-пациент при планировании трансплантации гемопоэтических стволовых клеток -, - (year - )