Killer-immunoglobulin-like receptors (KIRs) are critical natural killer (NK) cell regulators. The expression of KIRs is a dynamic process influenced by many factors. Their ligands-HLA(Human Leukocyte Antigen) class I molecules-are expressed on all nucleated cells that keep NK cells under control. In hematopoietic stem cell transplantation (HSCT), NK cells play an essential role in relapse protection. In the presented pilot study, we characterized the dynamic expression of inhibitory KIRS (iKIRs), which protect cells against untoward lysis, in donors and patients during the first three months after HSCT using flow cytometry. The expression of all iKIRs was highly variable and sometimes correlated with patients' clinical presentation and therapy regiment. Cyclophosphamide (Cy) in the graft-versus-host disease (GvHD) prevention protocol downregulated KIR2DL1 to just 25% of the original donor value, and the FEAM (Fludarabine + Etoposid + Ara-C + Melphalan) conditioning protocol reduced KIR2DL3. In lymphoid neoplasms, there was a slightly increased KIR2DL3 expression compared to myeloid malignancies. Additionally, we showed that the ex vivo activation of NK cells did not alter the level of iKIRs. Our study shows the influence of pre- and post-transplantation protocols on iKIR expression on the surface of NK cells and the importance of monitoring their cell surface.

Department of Haematology and Oncology University Hospital Pilsen 304 60 Pilsen Czech Republic

Department of Histology and Embryology Faculty of Medicine in Pilsen Charles University 301 66 Pilsen Czech Republic

Department of Immunology and Inflammation Faculty of Medicine Imperial College London London W12 0NN UK

Laboratory of Tumor Biology and Immunotherapy Biomedical Center Faculty of Medicine in Pilsen Charles University 323 00 Pilsen Czech Republic

NTIS Faculty of Applied Science University of West Bohemia 301 00 Pilsen Czech Republic

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Karre K. Immunology. A perfect mismatch. Science. 2002;295:2029–2031. doi: 10.1126/science.1070538. PubMed DOI

Caligiuri M.A., Velardi A., Scheinberg D.A., Borrello I.M. Immunotherapeutic approaches for hematologic malignancies. Hematol. Am. Soc. Hematol. Educ. Program. 2004;1:337–353. doi: 10.1182/asheducation-2004.1.337. PubMed DOI

Stern L., McGuire H., Avdic S., Rizzetto S., Fazekas de St Groth B., Luciani F., Slobedman B., Blyth E. Mass Cytometry for the Assessment of Immune Reconstitution After Hematopoietic Stem Cell Transplantation. Front. Immunol. 2018;9:1672. doi: 10.3389/fimmu.2018.01672. PubMed DOI PMC

Della Chiesa M., Muccio L., Moretta A. CMV induces rapid NK cell maturation in HSCT recipients. Immunol. Lett. 2013;155:11–13. doi: 10.1016/j.imlet.2013.09.020. PubMed DOI

Vitale C., Chiossone L., Cantoni C., Morreale G., Cottalasso F., Moretti S., Pistorio A., Haupt R., Lanino E., Dini G., et al. The corticosteroid-induced inhibitory effect on NK cell function reflects down-regulation and/or dysfunction of triggering receptors involved in natural cytotoxicity. Eur. J. Immunol. 2004;34:3028–3038. doi: 10.1002/eji.200425418. PubMed DOI

Paul S., Lal G. The Molecular Mechanism of Natural Killer Cells Function and Its Importance in Cancer Immunotherapy. Front. Immunol. 2017;8:1124. doi: 10.3389/fimmu.2017.01124. PubMed DOI PMC

Horton N.C., Mathew P.A. NKp44 and Natural Cytotoxicity Receptors as Damage-Associated Molecular Pattern Recognition Receptors. Front. Immunol. 2015;6:31. doi: 10.3389/fimmu.2015.00031. PubMed DOI PMC

Moretta A., Locatelli F., Moretta L. Human NK cells: From HLA class I-specific killer Ig-like receptors to the therapy of acute leukemias. Immunol. Rev. 2008;224:58–69. doi: 10.1111/j.1600-065X.2008.00651.x. PubMed DOI

Middleton D., Gonzelez F. The extensive polymorphism of KIR genes. Immunology. 2010;129:8–19. doi: 10.1111/j.1365-2567.2009.03208.x. PubMed DOI PMC

Shilling H.G., Guethlein L.A., Cheng N.W., Gardiner C.M., Rodriguez R., Tyan D., Parham P. Allelic polymorphism synergizes with variable gene content to individualize human KIR genotype. J. Immunol. 2002;168:2307–2315. doi: 10.4049/jimmunol.168.5.2307. PubMed DOI

Impola U., Turpeinen H., Alakulppi N., Linjama T., Volin L., Niittyvuopio R., Partanen J., Koskela S. Donor Haplotype B of NK KIR Receptor Reduces the Relapse Risk in HLA-Identical Sibling Hematopoietic Stem Cell Transplantation of AML Patients. Front. Immunol. 2014;5:405. doi: 10.3389/fimmu.2014.00405. PubMed DOI PMC

Beziat V., Traherne J.A., Liu L.L., Jayaraman J., Enqvist M., Larsson S., Trowsdale J., Malmberg K.J. Influence of KIR gene copy number on natural killer cell education. Blood. 2013;121:4703–4707. doi: 10.1182/blood-2012-10-461442. PubMed DOI PMC

Koutsakos M., McWilliam H.E.G., Aktepe T.E., Fritzlar S., Illing P.T., Mifsud N.A., Purcell A.W., Rockman S., Reading P.C., Vivian J.P., et al. Downregulation of MHC Class I Expression by Influenza A and B Viruses. Front. Immunol. 2019;10:1158. doi: 10.3389/fimmu.2019.01158. PubMed DOI PMC

Gaafar A., Sheereen A., Almohareb F., Eldali A., Chaudhri N., Mohamed S.Y., Hanbali A., Shaheen M., Alfraih F., El Fakih R., et al. Prognostic role of KIR genes and HLA-C after hematopoietic stem cell transplantation in a patient cohort with acute myeloid leukemia from a consanguineous community. Bone Marrow Transplant. 2018;53:1170–1179. doi: 10.1038/s41409-018-0123-7. PubMed DOI

Boudreau J.E., Hsu K.C. Natural killer cell education in human health and disease. Curr. Opin. Immunol. 2018;50:102–111. doi: 10.1016/j.coi.2017.11.003. PubMed DOI PMC

Ewen E.M., Pahl J.H.W., Miller M., Watzl C., Cerwenka A. KIR downregulation by IL-12/15/18 unleashes human NK cells from KIR/HLA-I inhibition and enhances killing of tumor cells. Eur. J. Immunol. 2018;48:355–365. doi: 10.1002/eji.201747128. PubMed DOI

Ullah M.A., Hill G.R., Tey S.K. Functional Reconstitution of Natural Killer Cells in Allogeneic Hematopoietic Stem Cell Transplantation. Front. Immunol. 2016;7:144. doi: 10.3389/fimmu.2016.00144. PubMed DOI PMC

Daher M., Rezvani K. Next generation natural killer cells for cancer immunotherapy: The promise of genetic engineering. Curr. Opin. Immunol. 2018;51:146–153. doi: 10.1016/j.coi.2018.03.013. PubMed DOI PMC

Maniangou B., Legrand N., Alizadeh M., Guyet U., Willem C., David G., Charpentier E., Walencik A., Retiere C., Gagne K. Killer Immunoglobulin-Like Receptor Allele Determination Using Next-Generation Sequencing Technology. Front. Immunol. 2017;8:547. doi: 10.3389/fimmu.2017.00547. PubMed DOI PMC

Wagner I., Schefzyk D., Pruschke J., Schofl G., Schone B., Gruber N., Lang K., Hofmann J., Gnahm C., Heyn B., et al. Allele-Level KIR Genotyping of More Than a Million Samples: Workflow, Algorithm, and Observations. Front. Immunol. 2018;9:2843. doi: 10.3389/fimmu.2018.02843. PubMed DOI PMC

R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing, Vienna, Austria. [(accessed on 12 March 2020)];2018 Available online: https://www.R-project.org/

Wickham H. ggplot2: Elegant Graphics for Data Analysis. Springer; New York, NY, USA: 2016.

Pende D., Falco M., Vitale M., Cantoni C., Vitale C., Munari E., Bertaina A., Moretta F., del Zotto G., Pietra G., et al. Killer Ig-Like Receptors (KIRs): Their Role in NK Cell Modulation and Developments Leading to Their Clinical Exploitation. Front. Immunol. 2019;10:1179. doi: 10.3389/fimmu.2019.01179. PubMed DOI PMC

Falco M., Moretta L., Moretta A., Bottino C. KIR and KIR ligand polymorphism: A new area for clinical applications? Tissue Antigens. 2013;82:363–373. doi: 10.1111/tan.12262. PubMed DOI

Solloch U.V., Schefzyk D., Schäfer G., Massalski C., Kohler M., Pruschke J., Heidl A., Schetelig J., Schmidt A.H., Lange V., et al. Estimation of German KIR Allele Group Haplotype Frequencies. Front. Immunol. 2020;11:1–429. doi: 10.3389/fimmu.2020.00429. PubMed DOI PMC

Vierra-Green C., Roe D., Hou L., Hurley C.K., Rajalingam R., Reed E., Lebedeva T., Yu N., Stewart M., Noreen H., et al. Allele-level haplotype frequencies and pairwise linkage disequilibrium for 14 KIR loci in 506 European-American individuals. PLoS ONE. 2012;7:e47491. doi: 10.1371/journal.pone.0047491. PubMed DOI PMC

Li Y., Wang T., Hu X., Zhang H., Chen L., Bao X., He J. Study of KIR gene expression at the mRNA level in specific donor-derived NK cells after allogeneic HSCT. Immunogenetics. 2020;72:135–141. doi: 10.1007/s00251-019-01153-6. PubMed DOI

Nguyen S., Achour A., Souchet L., Vigouroux S., Chevallier P., Furst S., Sirvent A., Bay J.O., Socié G., Ceballos P., et al. Clinical impact of NK-cell reconstitution after reduced intensity conditioned unrelated cord blood transplantation in patients with acute myeloid leukemia: Analysis of a prospective phase II multicenter trial on behalf of the Societe Francaise de Greffe de Moelle Osseuse et Therapie Cellulaire and Eurocord. Bone Marrow Transplant. 2017;52:1428–1435. PubMed

Giebel S., Dziaczkowska J., Czerw T., Wojnar J., Krawczyk-Kulis M., Nowak I., Holowiecka A., Segatti A., Kyrcz-Krzemien S., Kusnierczyk P., et al. Sequential recovery of NK cell receptor repertoire after allogeneic hematopoietic SCT. Bone Marrow Transplant. 2010;45:1022–1030. doi: 10.1038/bmt.2009.384. PubMed DOI

Nutalai R., Gaudieri S., Jumnainsong A., Leelayuwat C. Regulation of KIR3DL3 Expression via Mirna. Genes. 2019;10:603. doi: 10.3390/genes10080603. PubMed DOI PMC

Charoudeh H.N., Terszowski G., Czaja K., Gonzalez A., Schmitter K., Stern M. Modulation of the natural killer cell KIR repertoire by cytomegalovirus infection. Eur. J. Immunol. 2013;43:480–487. doi: 10.1002/eji.201242389. PubMed DOI

Moesta A.K., Parham P. Diverse functionality among human NK cell receptors for the C1 epitope of HLA-C: KIR2DS2, KIR2DL2, and KIR2DL3. Front. Immunol. 2012;3:336. doi: 10.3389/fimmu.2012.00336. PubMed DOI PMC

Jones D.C., Peacock S., Hughes D., Traherne J.A., Allen R.L., Barnardo M.C., Friend P., Taylor C.J., Fuggle S., Trowsdale J., et al. Killer immunoglobulin-like receptor gene repertoire influences viral load of primary human cytomegalovirus infection in renal transplant patients. Genes Immun. 2014;15:562–568. doi: 10.1038/gene.2014.53. PubMed DOI

Behrendt C.E., Nakamura R., Forman S.J., Zaia J.A. Donor killer immunoglobulin-like receptor genes and reactivation of cytomegalovirus after HLA-matched hematopoietic stem-cell transplantation: HLA-C allotype is an essential cofactor. Front. Immunol. 2013;4:36. doi: 10.3389/fimmu.2013.00036. PubMed DOI PMC

Cisneros E., Moraru M., Gomez-Lozano N., Lopez-Botet M., Vilches C. KIR2DL5: An Orphan Inhibitory Receptor Displaying Complex Patterns of Polymorphism and Expression. Front. Immunol. 2012;3:289. doi: 10.3389/fimmu.2012.00289. PubMed DOI PMC

El-Serafi I., Abedi-Valugerdi M., Potacova Z., Afsharian P., Mattsson J., Moshfegh A., Hassan M. Cyclophosphamide alters the gene expression profile in patients treated with high doses prior to stem cell transplantation. PLoS ONE. 2014;9:e86619. doi: 10.1371/journal.pone.0086619. PubMed DOI PMC

Neuchel C., Furst D., Niederwieser D., Bunjes D., Tsamadou C., Wulf G., Pfreundschuh M., Wagner E., Stuhler G., Einsele H., et al. Impact of Donor Activating KIR Genes on HSCT Outcome in C1-Ligand Negative Myeloid Disease Patients Transplanted with Unrelated Donors-A Retrospective Study. PLoS ONE. 2017;12:e0169512. doi: 10.1371/journal.pone.0169512. PubMed DOI PMC

Verheyden S., Demanet C. Susceptibility to myeloid and lymphoid leukemia is mediated by distinct inhibitory KIR-HLA ligand interactions. Leukemia. 2006;20:1437–1438. doi: 10.1038/sj.leu.2404279. PubMed DOI

Vey N., Karlin L., Sadot-Lebouvier S., Broussais F., Berton-Rigaud D., Rey J., Charbonnier A., Marie D., Andre P., Paturel C., et al. A phase 1 study of lirilumab (antibody against killer immunoglobulin-like receptor antibody KIR2D.; IPH2102) in patients with solid tumors and hematologic malignancies. Oncotarget. 2018;9:17675–17688. doi: 10.18632/oncotarget.24832. PubMed DOI PMC

Holubova M., Leba M., Gmucova H., Caputo V.S., Jindra P., Lysak D. Improving the Clinical Application of Natural Killer Cells by Modulating Signals Signal from Target Cells. Int. J. Mol. Sci. 2019;20:3472. doi: 10.3390/ijms20143472. PubMed DOI PMC