GZMB and PRF1 mRNA expression and donor genotypes and their associations with post-transplant complications in allogeneic hematopoietic stem cell transplant recipients

GZMB and PRF1 mRNA expression and donor genotypes and their associations with post-transplant complications in allogeneic hematopoietic stem cell transplant recipients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article GZMB and PRF1 mRNA expression and donor genotypes and their associations with post-transplant complications in allogeneic hematopoietic stem cell transplant recipients Ioulia K Gavriilidi, Piotr Łacina, Jagoda Siemaszko, Agnieszka Szeremet, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6556505/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Graft-versus-host disease (GvHD) and cytomegalovirus (CMV) infection are serious complications after allogeneic hematopoietic stem cell transplantation (HSCT) and NK cells are involved in their pathogenesis. As granzyme B (GzmB) and perforin are markers of cytotoxicity, we examined polymorphism and expression of GzmB and perforin genes in the context of HSCT outcome. A number of 247 patients and 117 donors were genotyped for GZMB rs8192917 and PRF1 rs885822 variants, and GZMB and PRF1 mRNA expression as well as serum levels were assessed 30 and 90 days post-transplantation. Chronic GvHD (cGvHD) and CMV infection were associated with donor GZMB rs8192917 TT genotype (p < 0.0001) while donor PRF1 rs885822 GG was predominant in patients with acute GvHD (p < 0.0001). GZMB expression decreased post-transplantation in recipients with cGvHD (p = 0.0237) while patients with CMV exhibited higher expression of GZMB (p = 0.0027) and PRF1 (p = 0.0035) on day 90 in comparison to recipients without complications. Strong correlations were observed between the expression of GZMB and PRF1 (p < 0.0001, R = 0.767) and between IFNG (interferon-γ) and GZMB (p = 0.0007, R = 0.69) and PRF1 (p = 0.0013, R = 0.67). Serum GzmB concentration decreased significantly over time in patients (p < 0.0001). These results suggest that GZMB and PRF1 genotypes and expression may be potential markers of post-transplant complications and have roles in cGvHD and CMV pathogenesis. granzyme B perforin NK cells allogeneic HSCT GvHD cytomegalovirus Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Allogeneic hematopoietic stem cell transplant (HSCT) is a procedure involving the transplantation of healthy donor stem cells to a recipient whose bone marrow function is disrupted or depleted. The process can restore hematopoiesis and is used for the treatment of various malignant and non-malignant hematological disorders. Importantly, HSCT has become a standard treatment for hematological malignancies where hematopoietic stem cells of a genetically similar donor are transferred into patient’s bloodstream in order to develop an immunological response against malignant cells in graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) effect [ 1 , 2 ]. The rate of survival ten years after the procedure ranges from 76–86% and, due to advances in medicine, it increases over time. However, a variety of risk factors including post-transplantation complications such as graft-versus-host disease (GvHD) and cytomegalovirus infection (CMV), increase patient morbidity and can lead to death[ 3 ]. A range of 20–50% of long-term surviving patients develops chronic GvHD (cGvHD) while 35–50% develop acute GvHD (aGvHD) where fatality rates can be as high as 50%[ 4 – 5 ]. Before HSCT is performed, the patient undergoes conditioning treatment (chemotherapy, radiotherapy or both) which suppresses their immune system. After the procedure, host immunity begins to reconstitute, with Natural Killer (NK) cells being the first lymphoid cells to dynamically recover, typically around 30 days post-transplantation, followed by T cells which begin to recover around 60 to 90 days post-transplantation [ 6 – 7 ].Defects in immune reconstitution are closely related to the development of post-HSCT complications and NK cells are believed to influence transplant outcomes [ 8 – 10 ]. NK cells are part of innate immunity with adaptive properties, high cytotoxic potential and immunoregulatory capacity. Following HSCT, they are the first line of defence against viruses and are capable of killing residual malignant cells both of which happen by direct lysis or proinflammatory cytokine secretion. They also possess both protective and detrimental roles in GvHD, as they can promote lysis of alloreactive T lymphocytes but also contribute to tissue damage through cytokine secretion. Furthermore, NK cells are known to produce serine proteases such as granzyme B (GzmB) and glycoproteins such as perforin which, in combination, induce apoptosis in targeted cells under inflammatory conditions [ 10 – 12 ]. While, T cells also produce GzmB and perforin, it has been proposed that GzmB presence enhances GvHD mediated by CD8 + but not CD4+ [ 13 ]. It must be noted however, that the production of GzmB and perforin in mainly attributed to NK cells [ 14 ]. Moreover, the activation of the granzyme/perforin lytic pathway was reported in early NK cells after umbilical cord blood transplantation [ 15 ]. Characteristic of NK cells function is also the production of cytokines such interferon-γ (IFN-γ) and tumor necrosis factors α (TNF-α) which mediate immune response [ 16 ]. Although GzmB and perforin are both markers of cytotoxicity, they have not yet been examined in more detail in the context of allogeneic HSCT outcome. Despite the fact that HSCT is constantly optimized and implemented in clinical settings, the reduction of the risk of relapse remains essential. Therefore, understanding the behaviour of NK cells and T cells post-transplant, their cytotoxic and secretory roles and identifying potential risk factors is of essence. Here, we aim to examine the role of GzmB and perforin in relation to the development of post-transplant complications in adult patients undergoing HSCT. 2 Methods 2.1 Study Group For this study, a number of 274 adult allogeneic hematopoietic stem cell transplant patients, as well as 117 donors, were incorporated. The patients were diagnosed with various hematological disorders, including cancer, which qualified them for HSCT. Among the patients, 72,42% exhibited post-HSCT complications including aGvHD, cGvHD, CMV infection and relapse. A control group of 233 healthy individuals was included. The patient samples were collected from five Polish transplantation centres and the study was approved by the Wroclaw Medical University Ethics Committee (identification code KB-561/2019). Detailed patients’ characteristics can be found on Table 1 , shown below. Table 1 Patients’ Characteristics. N = 274 Age (years, median, range) 48, 18–73 Sex (M/F) 160 (58,39%) / 114 (41,60%) Type of Donor MSD 110 (40,14%) MUD 85 (31,02%) Haploidentical 57 (20,80%) MMSD 2 (0,72%) MMUD 14 (5,10%) Diagnosis AML 110 (40,14%) ALL 36 (13,13%) MDS 27 (9,85%) NHL 26 (9,48%) MPN 29 (10,58%) HL 14 (5,10%) PCM 9 (3,28%) Other 24 (8,75%) Conditioning (RIC/MAC/NMA) 124 (45,25%) / 141 (51,45%) / 2 (0,72%) Post-transplant complications aGvHD (I-IV) 106 (38,68%) aGvHD (II-IV) 48 (17,51%) cGvHD 57 (20,80%) CMV 96 (35,6%) Relapse 42 (15,32%) Death 46 (16,78%) No complications 48 (17,51%) 3 Results 3.1 Allele and genotype distribution Patients and donors were genotyped for GZMB rs8192917 and PRF1 rs885822 SNPs. The studied groups follow the Hardy-Weinberg equilibrium. For rs8192917 polymorphism, the distributions of both alleles and genotypes were similar in patients and donors. The same did not apply to rs885822 SNP where allele frequencies were close but genotype frequencies differed for AA and AG variants. This difference, however, was not statistically significant (p=0.2659) (Table 2). Majority of patient-donor pairs were matched for GZMB polymorphism. This compatibility was observed in 80.43% of pairs tested. In 68.56% of pairs, patients and donors were matched for the PRF1 genotypes. However, genotype incompatibility did not affect HSCT outcomes. Table 2. Distribution of GZMB and PRF1 alleles and genotypes in HSCT recipients and donors Recipients N = 241 Donors N = 103 GZMB rs8192917 Genotypes 14 (5.8%) 8 (7.7%) 84 (34.85%) 38 (36.89%) 143 (59.33%) 57(55.33%) Alleles 98 (29.87%) 46 (32.62%) 227 (69.2%) 95 (67.37%) PRF1 rs885822 Recipients N = 268 Donors N = 111 Genotypes 96 (35.42%) 48 (43.24%) 123 (46.49%) 42 (37.86%) 49 (18.08%) 21 (18.91%) Alleles 222 (55.91%) 90 (58.82%) 175 (44.08%) 63 (41.17%) 3.2 GZMB and PRF1 genotypes and post-HSCT complications Further statistical analysis revealed that the donor GZMB rs8192917 genotype was associated with post-transplant complications in HSCT patients. Specifically, it was observed that donor TT homozygosity was more common in patients who developed cGvHD compared to patients with no cGvHD (85.41% vs 14.58% , p<0.0001) (Fig. 1a). Similarly, donor TT genotype was more common in patients with CMV infection compared to patients with without infection (69.49% vs 30.50%, p<0.0001) (Fig. 1b). An association was also observed between donors’ genotype status in PRF1 rs885822 and aGvHD stage. The AA/AG genotypes dominated among donors of patients without or with mild aGvHD (grades 0-I) while the GG genotype was predominant in the group with more severe (grades II-IV) disease (p<0.0001) (Fig. 1c). 3.4 mRNA expression of GZMB and PRF1 genes – association with cGvHD and CMV infection It was observed that GZMB expression strongly correlated with expression of PRF1 (p<0.0001, R=0.767) (Fig. 2a). Furthermore, the expression analysis revealed that the relative expression of GZMB was lower on the 90 th day after transplantation in comparison to the 30 th day, in patients with cGvHD (p=0.0237) (Fig. 2b). A similar observation was noted for patients with no complications, although non-significantly (p=0.069) (Fig. 2c). The CMV group exhibited higher expression of GZMB (p=0.0027) and PRF1 (p=0.0035) on the 90 th post-transplant in comparison to individuals without any post-transplant complications (Fig. 2d and 2e) . It was also observed that in patients with CMV infection the expression of both GZMB (p=0.0007, R=0.69) and PRF1 (p=0.0013, R=0.67) correlated positively with the expression of IFNG (Fig. 3a and 3b). No statistically significant relationship was observed between the expression of genes and genotype status of rs819219 and rs885822. 3.5 Granzyme B and Perforin serum levels Significant difference was observed in serum Granzyme B (GzmB) concentrations between HSCT recipients and healthy individuals, with notable decrease over time (median +30 days = 51,98 pg/ml, +90 days = 31,04 pg/ml and controls =12,57 pg/ml, p<0.0001) (Fig. 4). Furthermore, a significant decrease of GzmB between the 30 th and 90 th day after HSCT was observed in all subgroups when tested separately (aGvHD, cGvHD, CMV and no complications) (p<0.0001) (Table 3). The patients exhibiting cGvHD and those without any complications had the largest difference in concentrations between 30 th and 90 th day. Regarding serum perforin levels, no significant differences between patients and controls or across groups were observed. Median serum concentrations in patients at day +30, day +90 post-HSCT and in controls equalled: +30 days = 2419.71 pg/ml, +90 days = 2272.4 pg/ml and controls = 2234.61 pg/ml. Table 3 . Serum GzmB concentrations in patients post-HSCT. 30 days after transplantation 90 days after transplantation Median (pg/ml) SD Std error Median (pg/ml) SD Std error P value aGvHD 48.97 36.12 9.65 28.08 60.18 9.65 0.0226 cGvHD 48.30 38.19 11.02 19.97 12.97 3.74 0.0210 CMV 60.35 35.21 9.40 41.07 18.80 5.42 0.0034 No complications 66.29 64.16 16.04 29.32 27.32 6.62 0.0009 All groups 51.98 52.20 6.85 31.04 34.80 4.73 <0.0001 Abbreviations. HSCT: hematopoietic stem cell transplant; aGvHD: acute graft-versus-host disease; cGvHD: chronic graft-versus-host-disease; CMV: cytomegalovirus. 4 Discussion Hematopoietic stem cells transplantation (HSCT) is a standard procedure for the treatment of hematological disorders, including malignancies. Previous studies have demonstrated the importance of NK cells and their receptors in the development of post-transplantation complications in patients undergoing HSCT[22-25]. It has been also proposed that GzmB positive T regulatory cells can be a prognostic marker for aGvHD and relapse[26,27]. Although some studies have also investigated the roles of GzmB and perforin in acute organ transplant rejection [28,29] and in aGvHD in mice [13] thus far, the potential implication of GzmB and perforin in post-transplant complications has not been further explored. Here, we present the results of our investigation regarding this matter. NK cells are the main producers of GzmB and perforin, followed by T cells[14,30]. GzmB is a serine protease with strong apoptotic activity, capable of inducing targeted cell death. Perforin on the other hand is a pore-forming glycoprotein which facilitates the entrance of GzmB into targeted cells. While perforin co-operates with GzmB to activate pro-apoptotic pathways, GzmB has additional roles such as cleavage of substrates in the extracellular matrix[31-32]. Both proteins, have been associated with the pathogenesis of various inflammatory and autoimmune conditions such as: chronic obstructive pulmonary disease, chronic idiopathic thrombocytopenic purpura, atopic dermatitis, rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis and Sjögren syndrome [33]. After HSCT, NK cells reconstitute rapidly within the first few weeks. In allogeneic transplant, their main source are donor’s hematopoietic stem cells or residual host cells in situations where mixed chimerism occurs[10,12]. In healthy individuals, normal levels of GzmB range from 0 to 30 pg/ml with the lowest baseline being 1-5 pg/ml[33-34]. Increased GzmB detected in patients’ blood may signify inflammatory conditions (with the participation of NK cells) which occur during CMV and Epstein-Barr virus infection, GvHD, GvL and graft rejection. It is generally recognized that, primarily, the dominating subset of NK cells post HSCT is CD56 bright , which is an immature form with lower cytotoxic activity[35]. However, NK cells after transplantation are very capable of targeting residual cancer cells in GvL, and expand in the case of CMV. This can potentially explain the increase in production of GzmB and perforin[9,36,37]. The observed decrease 90 days after transplantation may be indicative of immune rebalancing. At this time adaptive immunity begins to develop with the reconstitution of T cells and B cells[38]. Additionally, at this time, complications that were detected at an early stage after transplantation are already treated with medication that decreases inflammation, as well as GzmB and perforin production. The expression of GZMB and PRF1 was correlated with the expression of IFNG in patients with CMV infection. IFNG codes for interferon-γ, a pro-inflammatory cytokine crucial in immune response to viral infections. Furthermore, interferon-γ is considered in terms of monitoring and assessment of CMV infection[39-42]. Therefore, the correlation with IFNG can be indicative of viral reactivation and increased inflammatory conditions associated with it, which promotes the expression of GZMB and PRF1 . In the present study, the serum levels of perforin were not significantly increased in patients undergoing allogeneic HSCT when compared to healthy controls. This can be possibly explained by the fact that perforin in comparison to GzmB is an unstable molecule, degraded immediately after pore formation. In addition, GzmB is more readily released into circulation and extracellular space as it also has extracellular functions[43]. Perforin, on the other hand, is less likely to be released into serum as it has a localized action at immune synapse and is membrane bound. GzmB is also independently secreted without the need of perforin which is not true the other way around[31-33]. Finally, although we would expect higher levels of perforin in the serum of HSCT patients, it must be considered that these patients undergo conditioning treatment before the procedure and receive glucocorticoid medication. Such treatments decrease calcium levels, and calcium is a major regulator of perforin[44,45]. Perhaps, the implementation of a different method, such as detection of intracellular or membrane bound perforin through flow cytometry could generate contrasting results. The two SNPs that were chosen for analysis, GZMB rs819217 and PRF1 rs885822, are missense variants (C>T, A>G) that lead to changes in amino acid sequence, arginine to glutamine and histidine to glutamine, respectively. Both of them have been previously reported to be associated with autoimmune diseases[46-49]. In the present study both polymorphisms were also found to be associated with clinical outcomes in HSCT. For rs819217, donor’s T T homozygosity seems to have an unfavorable effect on the development of cGvHD and CMV infection in transplant recipients while the C C genotype seems to play a protective role. For rs885822, the wild type GG homozygosity was predominant in donors of patients with aGvHD grades II-IV. Our results suggest an association of GZMB rs819217 polymorphism with cGvHD and PRF1 rs885822 with aGvHD. While GzmB and perforin often work synergistically their roles in GvHD might differ. A recent study by Thompson et al explored the effect of PRF1 and GZMB KO on aGvHD in mice and demonstrated that PRF1 KO in donor cells reduced aGvHD while DKO of GZMB and PRF1 did not have that effect [50]. In another study it has been indicated that the levels of GzmB are higher in post-HSCT patients who never developed aGvHD [51]. We did not observe any associations between gene expression and occurrence of the GZMB / PRF1 genotypes, although this could be due to the relatively small sample size. Granzyme B, alongside perforin, is involved in the cytotoxic process by inducing apoptosis in recipient tissues, possibly contributing to the pathogenesis of post-transplant complications. With further investigation, the mRNA expression and serum protein levels of granzyme B can serve as biomarkers of GvHD and CMV severity, or as markers for ongoing inflammation. The same applies to genetic polymorphisms in rs819217 and rs885822 SNPs which can be potential markers of the development of complications after the HSCT. Declarations The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Authors’ contribution IKG performed the assessment of granzyme B and perforin serum levels, genotyping studies and data analyses, drafted, edited and finalized the manuscript; PŁ performed mRNA expression analysis, contributed to data analyses, edited and finalised the manuscript; JS performed mRNA expression, DNA isolation, edited and finalised the manuscript; AS, MM, AC, MSK, WF, IS, BNA, PS, MB, AT, GWB, SG and TW provided patients’ clinical samples and clinical data; KBK conceived, designed and supervised the study, analysed the data, drafted, edited and finalized the manuscript and secured funding. All authors approved the final version of the manuscript. Funding Supported by National Science Centre (Poland) project No. 2018/31/B/NZ2/03065. The authors do not declare any conflict of interest. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Wroclaw Medical University Ethics Committee (identification code KB-561/2019). Consent to participate Informed consent was obtained from all individual participants included in the study. Consent for publication Informed consent was obtained from all individual participants/family members. Data availability statement The data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request. Data are located in controlled access data storage at Hirszfeld Institute of Immunology and Experimental Therapy. References Giralt S, Bishop MR. Principles and Overview of Allogeneic Hematopoietic Stem Cell Transplantation. In: Bishop MR, editor. Hematopoietic Stem Cell Transplantation [Internet]. Boston, MA: Springer US; 2009 [cited 2024 Nov 18]. pp. 1–21. (Cancer Treatment and Research; vol. 144). Available from: http://link.springer.com/ 10.1007/978-0-387-78580-6_1 Khaddour K, Hana CK, Mewawalla P. Hematopoietic Stem Cell Transplantation. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 [cited 2024 Nov 18]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK536951/ Grønvold BL, Ali MM, Myklebust TÅ, Lenartova A, Remberger M, Abrahamsen IW, et al. Allogeneic stem cell transplant recipients surviving at least 2 years without relapse: outcome and risk factors. EJHaem. 2024;5(1):117–24. Langer R, Lelas A, Rittenschober M, Piekarska A, Sadowska-Klasa A, Sabol I, et al. Retrospective analysis of the incidence and outcome of late acute and chronic graft-versus-host disease—an analysis from transplant centers across Europe. Front Transpl. 2024;3:1332181. for the Infectious Diseases Working, Party EBMT, Styczyński J, Tridello G, Koster L, Iacobelli S, Van Biezen A, et al. Death after hematopoietic stem cell transplantation: changes over calendar year time, infections and associated factors. Bone Marrow Transpl. 2020;55(1):126–36. Dekker L, de Koning C, Lindemans C, Nierkens S. Reconstitution of T Cell Subsets Following Allogeneic Hematopoietic Cell Transplantation. Cancers. 2020;12(7):1974. Hattori N, Saito B, Sasaki Y, Shimada S, Murai S, Abe M, et al. Status of Natural Killer Cell Recovery in Day 21 Bone Marrow after Allogeneic Hematopoietic Stem Cell Transplantation Predicts Clinical Outcome. Biol Blood Marrow Transpl. 2018;24(9):1841–7. Kim HO, Oh HJ, Lee JW, Jang PS, Chung NG, Cho B, et al. Immune reconstitution after allogeneic hematopoietic stem cell transplantation in children: a single institution study of 59 patients. Korean J Pediatr. 2013;56(1):26. Locatelli F, Pende D, Falco M, Della Chiesa M, Moretta A, Moretta L. NK Cells Mediate a Crucial Graft-versus-Leukemia Effect in Haploidentical-HSCT to Cure High-Risk Acute Leukemia. Trends Immunol. 2018;39(7):577–90. Hadjis AD, McCurdy SR. The role and novel use of natural killer cells in graft-versus-leukemia reactions after allogeneic transplantation. Front Immunol. 2024;15:1358668. Tibbs E, Cao X. Emerging Canonical and Non-Canonical Roles of Granzyme B in Health and Disease. Cancers. 2022;14(6):1436. Gail LM, Schell KJ, Łacina P, Strobl J, Bolton SJ, Steinbakk Ulriksen E, et al. Complex interactions of cellular players in chronic Graft-versus-Host Disease. Front Immunol. 2023;14:1199422. Du W, Leigh ND, Bian G, O’Neill RE, Mei L, Qiu J et al. Granzyme B-Mediated Activation-Induced Death of CD4 + T Cells Inhibits Murine Acute Graft-versus-Host Disease. J Immunol Baltim Md. 1950. 2015;195(9):4514–23. Lin L, Couturier J, Yu X, Medina MA, Kozinetz CA, Lewis DE. Granzyme B secretion by human memory CD4 T cells is less strictly regulated compared to memory CD8 T cells. BMC Immunol. 2014;15(1):36. Brahmi Z, Hommel-Berrey G, Smith F, Thomson B. NK cells recover early and mediate cytotoxicity via perforin/granzyme and Fas/FasL pathways in umbilical cord blood recipients. Hum Immunol. 2001;62(8):782–90. Almishri W, Santodomingo-Garzon T, Le T, Stack D, Mody CH, Swain MG. TNFα Augments Cytokine-Induced NK Cell IFNγ Production through TNFR2. J Innate Immun. 2016;8(6):617–29. Faul F, Erdfelder E, Buchner A, Lang AG. Statistical power analyses using G*Power 3.1: Tests for correlation and regression analyses. Behav Res Methods. 2009;41(4):1149–60. Xu Z, Taylor JA. SNPinfo: integrating GWAS and candidate gene information into functional SNP selection for genetic association studies. Nucleic Acids Res. 2009;37(suppl2):W600–5. Glare EM, Divjak M, Bailey MJ, Walters EH. beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels. Thorax. 2002;57(9):765–70. Sikand K, Singh J, Ebron JS, Shukla GC. Housekeeping gene selection advisory: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are targets of miR-644a. PLoS ONE. 2012;7(10):e47510. Song R, He S, Wu Y, Chen W, Song J, Zhu Y, et al. Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of septic patients. Heliyon. 2023;9(4):e15269. Nihtilä J, Penna L, Salmenniemi U, Itälä-Remes M, Crossland RE, Gallardo D, et al. Effect of NK cell receptor genetic variation on allogeneic stem cell transplantation outcome and in vitro NK cell cytotoxicity. Sci Rep. 2024;14(1):26988. Siemaszko J, Ussowicz M, Rybka B, Ryczan-Krawczyk R, Kałwak K, Bogunia-Kubik K. The impact of NKG2A and NKG2D receptors and HLA-E and MICA ligands polymorphisms on post-transplant complications after paediatric allogeneic HSCT: a single-centre experience. Front Genet. 2023;14:1186123. Siemaszko J, Łacina P, Szymczak D, Szeremet A, Majcherek M, Czyż A, et al. Soluble MICA concentrations and genetic variability of MICA and its NKG2D receptor as factors affecting Graft-versus-Host Disease development after allogeneic haematopoietic stem cell transplantation. Hum Immunol. 2024;85(6):111147. Xie DH, Wang J, Sun K, Shi ZY, Wang YZ, Chang Y, et al. The Functional and Prognostic Impact of TIGIT Expression on Bone Marrow NK Cells in Core Binding Factor-Acute Myeloid Leukemia Patients at Diagnosis. Biomedicines. 2024;12(10):2207. Drokov MY, Davydova JO, Kuzmina LA, Galtseva IV, Kapranov NM, Vasilyeva VA, et al. Level of Granzyme B-positive T-regulatory cells is a strong predictor biomarker of acute Graft-versus-host disease after day + 30 after allo-HSCT. Leuk Res. 2017;54:25–9. Drokov M, Davydova Y, Popova N, Kapranov N, Starikova O, Mikhaltsova E, et al. High expression of granzyme B in conventional CD4 + T cells is associated with increased relapses after allogeneic stem cells transplantation in patients with hematological malignancies. Transpl Immunol. 2021;65:101295. Kummer JA, Wever PC, Kamp AM, Ten Berge IJM, Hack CE, Weening JJ. Expression of granzyme A and B proteins by cytotoxic lymphocytes involved in acute renal allograft rejection. Kidney Int. 1995;47(1):70–7. Mac QD, Mathews DV, Kahla JA, Stoffers CM, Delmas OM, Holt BA, et al. Non-invasive early detection of acute transplant rejection via nanosensors of granzyme B activity. Nat Biomed Eng. 2019;3(4):281–91. Campos TM, Novais FO, Saldanha M, Costa R, Lordelo M, Celestino D, et al. Granzyme B Produced by Natural Killer Cells Enhances Inflammatory Response and Contributes to the Immunopathology of Cutaneous Leishmaniasis. J Infect Dis. 2020;221(6):973–82. Velotti F, Barchetta I, Cimini FA, Cavallo MG. Granzyme B in Inflammatory Diseases: Apoptosis, Inflammation, Extracellular Matrix Remodeling, Epithelial-to-Mesenchymal Transition and Fibrosis. Front Immunol. 2020;11:587581. Thompson R, Cao X. Reassessing granzyme B: unveiling perforin-independent versatility in immune responses and therapeutic potentials. Front Immunol. 2024;15:1392535. Boivin WA, Cooper DM, Hiebert PR, Granville DJ. Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma. Lab Invest. 2009;89(11):1195–220. Spaeny-Dekking EHA, Hanna WL, Wolbink AM, Wever PC, Kummer AJ, Swaak AJG, et al. Extracellular Granzymes A and B in Humans: Detection of Native Species During CTL Responses In Vitro and In Vivo. J Immunol. 1998;160(7):3610–6. Mushtaq MU, Shahzad M, Shah AY, Chaudhary SG, Zafar MU, Anwar I, et al. Impact of natural killer cells on outcomes after allogeneic hematopoietic stem cell transplantation: A systematic review and meta-analysis. Front Immunol. 2022;13:1005031. Pende D, Marcenaro S, Falco M, Martini S, Bernardo ME, Montagna D, et al. Anti-leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for pediatric patients: evaluation of the functional role of activating KIR and redefinition of inhibitory KIR specificity. Blood. 2009;113(13):3119–29. Minculescu L, Marquart HV, Friis LS, Petersen SL, Schiødt I, Ryder LP, et al. Early Natural Killer Cell Reconstitution Predicts Overall Survival in T Cell–Replete Allogeneic Hematopoietic Stem Cell Transplantation. Biol Blood Marrow Transpl. 2016;22(12):2187–93. Bosch M, Khan FM, Storek J. Immune reconstitution after hematopoietic cell transplantation. Curr Opin Hematol. 2012;19(4):324–35. Kim SH, Lee HS, Lee HJ, Kim SM, Shin S, Park SH, et al. Clinical applications of interferon-γ releasing assays for cytomegalovirus to differentiate cytomegalovirus disease from bystander activation: a pilot proof-of-concept study. Korean J Intern Med. 2017;32(5):900–9. Yong MK, Cameron PU, Slavin M, Morrissey CO, Bergin K, Spencer A, et al. Identifying Cytomegalovirus Complications Using the Quantiferon-CMV Assay After Allogeneic Hematopoietic Stem Cell Transplantation. J Infect Dis. 2017;215(11):1684–94. De Gracia-Guindo MdC, Ruiz-Fuentes MdC, Galindo-Sacristán P, Osorio-Moratalla JM, Ruiz-Fuentes N, Rodriguez Granger J et al. Cytomegalovirus Infection Monitoring Based on Interferon Gamma Release Assay in Kidney Transplantation. Transplant Proc. 2018;50(2):578–80. Kim SH. Interferon-γ Release Assay for Cytomegalovirus (IGRA-CMV) for Risk Stratification of Posttransplant CMV Infection: Is It Time to Apply IGRA-CMV in Routine Clinical Practice? Clin Infect Dis. 2020;71(9):2386–8. Buzza MS, Zamurs L, Sun J, Bird CH, Smith AI, Trapani JA, et al. Extracellular Matrix Remodeling by Human Granzyme B via Cleavage of Vitronectin, Fibronectin, and Laminin. J Biol Chem. 2005;280(25):23549–58. Law RHP, Lukoyanova N, Voskoboinik I, Caradoc-Davies TT, Baran K, Dunstone MA, et al. The structural basis for membrane binding and pore formation by lymphocyte perforin. Nature. 2010;468(7322):447–51. Miglietta F, Iamartino L, Palmini G, Giusti F, Marini F, Iantomasi T, et al. Endocrine sequelae of hematopoietic stem cell transplantation: Effects on mineral homeostasis and bone metabolism. Front Endocrinol. 2023;13:1085315. Corrales-Tellez E, Vu D, Shah T, Hutchinson I, Min DI. Association between granzyme B and perforin I polymorphisms and allograft outcomes in Hispanic kidney transplant recipients. Clin Transplant [Internet]. 2013 May [cited 2024 Nov 25];27(3). Available from: https://onlinelibrary.wiley.com/doi/ 10.1111/ctr.12114 Dai D, Wen F, Liu S, Zhou S. Brain damage resembling acute necrotizing encephalopathy as a specific manifestation of haemophagocytic lymphohistiocytosis - induced by hypersensitivity. Ital J Pediatr. 2016;42(1):79. Jaworowska A, Pastorczak A, Trelinska J, Wypyszczak K, Borowiec M, Fendler W, et al. Perforin gene variation influences survival in childhood acute lymphoblastic leukemia. Leuk Res. 2018;65:29–33. Jeong KH, Kim SK, Seo JK, Shin MK, Lee MH. Association of GZMB polymorphisms and susceptibility to non-segmental vitiligo in a Korean population. Sci Rep. 2021;11(1):397. Thompson R, Puppala E, Mustapha H. Assessing Perforin-independent Granzyme B function in Acute Graft-Versus-Host Disease: Impact on Allogeneic Hematopoietic Cell Transplantation. J Immunol. 2024;212(1Supplement):12705459–12705459. Drokov MYu, Parovichnikova EN, Davydova J, Kuzmina LA, Galtseva IV, Kapranov NM, et al. Granzyme B Expression in T-Regulatory Cells Is a Strong Predictor of Acute Graft-Versus-Host Disease after Day + 30 in Patients with Classic Immunosuppression after Allo-HSCT. Blood. 2016;128(22):2238–2238. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6556505","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":450768403,"identity":"122a7eff-3d41-4e99-9cf3-9e5564d7166b","order_by":0,"name":"Ioulia K Gavriilidi","email":"","orcid":"","institution":"Ludwik Hirszfeld Institute of Immunology and Experimental Therapy","correspondingAuthor":false,"prefix":"","firstName":"Ioulia","middleName":"K","lastName":"Gavriilidi","suffix":""},{"id":450768404,"identity":"46420712-a4d0-4a1d-a11e-336ccdec6407","order_by":1,"name":"Piotr Łacina","email":"","orcid":"","institution":"Ludwik Hirszfeld Institute of Immunology and Experimental Therapy","correspondingAuthor":false,"prefix":"","firstName":"Piotr","middleName":"","lastName":"Łacina","suffix":""},{"id":450768405,"identity":"63ecb470-90f2-4ccb-b494-13d2dba5ac43","order_by":2,"name":"Jagoda Siemaszko","email":"","orcid":"","institution":"Ludwik Hirszfeld Institute of Immunology and Experimental Therapy","correspondingAuthor":false,"prefix":"","firstName":"Jagoda","middleName":"","lastName":"Siemaszko","suffix":""},{"id":450768406,"identity":"782e0b7a-d2ab-4e2f-a90e-a60f09be6979","order_by":3,"name":"Agnieszka Szeremet","email":"","orcid":"","institution":"Wroclaw Medical University","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Szeremet","suffix":""},{"id":450768407,"identity":"5cfce2b4-12d2-4041-a080-e0329bd1faf3","order_by":4,"name":"Maciej Majcherek","email":"","orcid":"","institution":"Wroclaw Medical University","correspondingAuthor":false,"prefix":"","firstName":"Maciej","middleName":"","lastName":"Majcherek","suffix":""},{"id":450768408,"identity":"5b3a1db3-4be1-4243-8515-79ab78a52d95","order_by":5,"name":"Anna Czyż","email":"","orcid":"","institution":"Wroclaw Medical University","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Czyż","suffix":""},{"id":450768409,"identity":"830b3842-10a1-4cf8-a5ec-2b5a44b8bb8b","order_by":6,"name":"Małgorzata Sobczyk-Kruszelnicka","email":"","orcid":"","institution":"Maria Sklodowska- Curie Memorial Cancer Center and Institute of Oncology","correspondingAuthor":false,"prefix":"","firstName":"Małgorzata","middleName":"","lastName":"Sobczyk-Kruszelnicka","suffix":""},{"id":450768410,"identity":"6477731f-6bfc-4cff-ae4a-542bea1acc06","order_by":7,"name":"Wojciech Fidyk","email":"","orcid":"","institution":"Maria Sklodowska- Curie Memorial Cancer Center and Institute of Oncology","correspondingAuthor":false,"prefix":"","firstName":"Wojciech","middleName":"","lastName":"Fidyk","suffix":""},{"id":450768411,"identity":"34dac1ea-9f90-4515-bd04-df878d49da37","order_by":8,"name":"Iwona Solarska","email":"","orcid":"","institution":"Institute of Hematology and Blood Transfusion Medicine","correspondingAuthor":false,"prefix":"","firstName":"Iwona","middleName":"","lastName":"Solarska","suffix":""},{"id":450768412,"identity":"373e2743-294e-4c5f-b900-b140c86eec10","order_by":9,"name":"Barbara Nasiłowska-Adamska","email":"","orcid":"","institution":"Institute of Hematology and Blood Transfusion Medicine","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Nasiłowska-Adamska","suffix":""},{"id":450768413,"identity":"87f43914-86c0-4c10-a4b8-787cfefb0b9e","order_by":10,"name":"Patrycja Skowrońska","email":"","orcid":"","institution":"University Medical Center in Gdansk","correspondingAuthor":false,"prefix":"","firstName":"Patrycja","middleName":"","lastName":"Skowrońska","suffix":""},{"id":450768414,"identity":"1511ed41-3746-4c9a-88f0-76f2410808cd","order_by":11,"name":"Maria Bieniaszewska","email":"","orcid":"","institution":"Medical University of Gdansk","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Bieniaszewska","suffix":""},{"id":450768417,"identity":"d0c9ffb9-c81e-4a52-b0f3-5b4519eef15e","order_by":12,"name":"Agnieszka Tomaszewska","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Tomaszewska","suffix":""},{"id":450768420,"identity":"37250220-2ff8-40f0-ab0b-5328fe03840a","order_by":13,"name":"Grzegorz W. Basak","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Grzegorz","middleName":"W.","lastName":"Basak","suffix":""},{"id":450768424,"identity":"7069b050-50af-4a19-96dd-0debe28b6410","order_by":14,"name":"Sebastian Giebel","email":"","orcid":"","institution":"Maria Sklodowska- Curie Memorial Cancer Center and Institute of Oncology","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Giebel","suffix":""},{"id":450768427,"identity":"5f0c12ef-03ab-4544-a201-1201967f8c26","order_by":15,"name":"Tomasz Wróbel","email":"","orcid":"","institution":"Institute of Hematology and Blood Transfusion Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tomasz","middleName":"","lastName":"Wróbel","suffix":""},{"id":450768428,"identity":"b7f1b50c-61dd-496e-a31b-148356b90c28","order_by":16,"name":"Katarzyna Bogunia-Kubik","email":"data:image/png;base64,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","orcid":"","institution":"Ludwik Hirszfeld Institute of Immunology and Experimental Therapy","correspondingAuthor":true,"prefix":"","firstName":"Katarzyna","middleName":"","lastName":"Bogunia-Kubik","suffix":""}],"badges":[],"createdAt":"2025-04-29 12:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6556505/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6556505/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82299582,"identity":"773dd152-fc28-4057-b4fc-2a5c1be8e93f","added_by":"auto","created_at":"2025-05-08 20:36:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43628,"visible":true,"origin":"","legend":"\u003cp\u003eRelationships between donor’s genotypes and post-transplantation complications in HSCT recipients. \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e) Recipients whose donors possess the \u003cem\u003eTT\u003c/em\u003e genotype in rs819217 exhibit cGvHD and CMV more frequently. \u003cstrong\u003ec\u003c/strong\u003e) In case of rs885822, recipients grafted from \u003cem\u003eGG\u003c/em\u003e homozygous donors develop aGvHD grade II-IV more frequently than those with donors carrying \u003cem\u003eAA/AG\u003c/em\u003e genotypes\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6556505/v1/f294a9df80cce44c059bdb5d.png"},{"id":82299583,"identity":"f8e2dfa1-2cdf-46ed-8c6d-3d7c8b17b89a","added_by":"auto","created_at":"2025-05-08 20:36:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":32578,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression of \u003cem\u003eGZMB \u003c/em\u003eand\u003cem\u003e PRF1 \u003c/em\u003eand associations with HSCT complications. \u003cstrong\u003ea)\u003c/strong\u003e Correlation between \u003cem\u003eGZMB\u003c/em\u003e and \u003cem\u003ePRF1\u003c/em\u003e expression 30 days after transplantation. \u003cstrong\u003eb, c)\u003c/strong\u003e \u003cem\u003eGZMB \u003c/em\u003eexpression is lower at 90 days after transplantation than after 30 days in patients with cGvHD and without post-transplant complications. \u003cstrong\u003ed, e)\u003c/strong\u003e The expression of both \u003cem\u003eGZMB \u003c/em\u003eand \u003cem\u003ePRF1 \u003c/em\u003eis higher in patients with CMV compared to patients without complications, 90 days after transplantation\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6556505/v1/48d69e4027da5a3841c26e9a.png"},{"id":82299586,"identity":"9f997956-8b8e-4f57-b2bd-938d6ae7bec1","added_by":"auto","created_at":"2025-05-08 20:36:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16106,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between the expression of \u003cem\u003eGZMB\u003c/em\u003e/\u003cem\u003ePRF1\u003c/em\u003eand \u003cem\u003eIFNG, \u003c/em\u003ein post-HSCT patients with CMV infection 90 days after transplantation. \u003cem\u003eIFNG\u003c/em\u003e expression correlated strongly with the expression of \u003cem\u003eGZMB\u003c/em\u003e \u003cstrong\u003e(a)\u003c/strong\u003e and \u003cem\u003ePRF1\u003c/em\u003e \u003cstrong\u003e(b)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6556505/v1/84ffd34393fcc196fda977f6.png"},{"id":82299585,"identity":"ec1c67d9-c401-493c-a13f-48db36937bb7","added_by":"auto","created_at":"2025-05-08 20:36:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":95171,"visible":true,"origin":"","legend":"\u003cp\u003eThe levels of serum granzyme B (pg/ml) are higher in patients compared to healthy controls, and decrease over time after HSCT procedure (median day +30 = 51,98 pg/ml and day +90 = 31,04 pg/ml vs controls =12,57 pg/ml, p\u0026lt;0,0001)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6556505/v1/b17316c0f7094cbe56aa1f87.png"},{"id":88235748,"identity":"8112b432-7fc0-462d-a76b-78226ac32067","added_by":"auto","created_at":"2025-08-04 10:17:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1117447,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6556505/v1/129ef063-95e4-47fe-935b-608ac47341e5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"GZMB and PRF1 mRNA expression and donor genotypes and their associations with post-transplant complications in allogeneic hematopoietic stem cell transplant recipients","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAllogeneic hematopoietic stem cell transplant (HSCT) is a procedure involving the transplantation of healthy donor stem cells to a recipient whose bone marrow function is disrupted or depleted. The process can restore hematopoiesis and is used for the treatment of various malignant and non-malignant hematological disorders. Importantly, HSCT has become a standard treatment for hematological malignancies where hematopoietic stem cells of a genetically similar donor are transferred into patient\u0026rsquo;s bloodstream in order to develop an immunological response against malignant cells in graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) effect [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe rate of survival ten years after the procedure ranges from 76\u0026ndash;86% and, due to advances in medicine, it increases over time. However, a variety of risk factors including post-transplantation complications such as graft-versus-host disease (GvHD) and cytomegalovirus infection (CMV), increase patient morbidity and can lead to death[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A range of 20\u0026ndash;50% of long-term surviving patients develops chronic GvHD (cGvHD) while 35\u0026ndash;50% develop acute GvHD (aGvHD) where fatality rates can be as high as 50%[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBefore HSCT is performed, the patient undergoes conditioning treatment (chemotherapy, radiotherapy or both) which suppresses their immune system. After the procedure, host immunity begins to reconstitute, with Natural Killer (NK) cells being the first lymphoid cells to dynamically recover, typically around 30 days post-transplantation, followed by T cells which begin to recover around 60 to 90 days post-transplantation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].Defects in immune reconstitution are closely related to the development of post-HSCT complications and NK cells are believed to influence transplant outcomes [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNK cells are part of innate immunity with adaptive properties, high cytotoxic potential and immunoregulatory capacity. Following HSCT, they are the first line of defence against viruses and are capable of killing residual malignant cells both of which happen by direct lysis or proinflammatory cytokine secretion. They also possess both protective and detrimental roles in GvHD, as they can promote lysis of alloreactive T lymphocytes but also contribute to tissue damage through cytokine secretion. Furthermore, NK cells are known to produce serine proteases such as granzyme B (GzmB) and glycoproteins such as perforin which, in combination, induce apoptosis in targeted cells under inflammatory conditions [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While, T cells also produce GzmB and perforin, it has been proposed that GzmB presence enhances GvHD mediated by CD8\u0026thinsp;+\u0026thinsp;but not CD4+ [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It must be noted however, that the production of GzmB and perforin in mainly attributed to NK cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, the activation of the granzyme/perforin lytic pathway was reported in early NK cells after umbilical cord blood transplantation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Characteristic of NK cells function is also the production of cytokines such interferon-γ (IFN-γ) and tumor necrosis factors α (TNF-α) which mediate immune response [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Although GzmB and perforin are both markers of cytotoxicity, they have not yet been examined in more detail in the context of allogeneic HSCT outcome.\u003c/p\u003e \u003cp\u003eDespite the fact that HSCT is constantly optimized and implemented in clinical settings, the reduction of the risk of relapse remains essential. Therefore, understanding the behaviour of NK cells and T cells post-transplant, their cytotoxic and secretory roles and identifying potential risk factors is of essence. Here, we aim to examine the role of GzmB and perforin in relation to the development of post-transplant complications in adult patients undergoing HSCT.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study Group\u003c/h2\u003e \u003cp\u003eFor this study, a number of 274 adult allogeneic hematopoietic stem cell transplant patients, as well as 117 donors, were incorporated. The patients were diagnosed with various hematological disorders, including cancer, which qualified them for HSCT. Among the patients, 72,42% exhibited post-HSCT complications including aGvHD, cGvHD, CMV infection and relapse. A control group of 233 healthy individuals was included. The patient samples were collected from five Polish transplantation centres and the study was approved by the Wroclaw Medical University Ethics Committee (identification code KB-561/2019). Detailed patients\u0026rsquo; characteristics can be found on Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, shown below.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatients\u0026rsquo; Characteristics.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;274\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years, median, range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48, 18\u0026ndash;73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex (M/F)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e160 (58,39%) / 114 (41,60%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of Donor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110 (40,14%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMUD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85 (31,02%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHaploidentical\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57 (20,80%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (0,72%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMUD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (5,10%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiagnosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAML\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110 (40,14%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36 (13,13%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27 (9,85%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26 (9,48%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMPN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29 (10,58%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (5,10%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (3,28%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24 (8,75%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConditioning (RIC/MAC/NMA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e124 (45,25%) / 141 (51,45%) / 2 (0,72%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePost-transplant complications\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eaGvHD (I-IV)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e106 (38,68%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eaGvHD (II-IV)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48 (17,51%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecGvHD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57 (20,80%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCMV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96 (35,6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRelapse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42 (15,32%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDeath\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46 (16,78%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo complications\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48 (17,51%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Allele and genotype distribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients and donors were genotyped for \u003cem\u003eGZMB\u003c/em\u003e rs8192917 and \u003cem\u003ePRF1\u003c/em\u003e rs885822 SNPs. The studied groups follow the Hardy-Weinberg equilibrium. For rs8192917 polymorphism, the distributions of both alleles and genotypes were similar in patients and donors. The same did not apply to rs885822 SNP where allele frequencies were close but genotype frequencies differed for \u003cem\u003eAA\u003c/em\u003e and \u003cem\u003eAG\u003c/em\u003e variants. This difference, however, was not statistically significant (p=0.2659) (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMajority of patient-donor pairs were matched for \u003cem\u003eGZMB\u0026nbsp;\u003c/em\u003epolymorphism. This compatibility was observed in 80.43% of pairs tested. In 68.56% of pairs, patients and donors were matched for the \u003cem\u003ePRF1\u003c/em\u003e genotypes. However, genotype incompatibility did not affect HSCT outcomes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Distribution of \u003cem\u003eGZMB\u003c/em\u003e and \u003cem\u003ePRF1\u003c/em\u003e alleles and genotypes in HSCT recipients and donors\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecipients N = 241\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDonors N = 103\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGZMB\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;rs8192917\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eGenotypes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e14 (5.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e8 (7.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e84 (34.85%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e38 (36.89%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e143 (59.33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e57(55.33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eAlleles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e98 (29.87%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e46 (32.62%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e227 (69.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e95 (67.37%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePRF1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;rs885822\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecipients N = \u0026nbsp;268\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDonors N = 111\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eGenotypes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e96 (35.42%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e48 (43.24%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e123 (46.49%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e42 (37.86%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e49 (18.08%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e21 (18.91%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eAlleles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e222 (55.91%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e90 (58.82%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 33.3333%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 36.9444%;\"\u003e\n \u003cp\u003e175 (44.08%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.7222%;\"\u003e\n \u003cp\u003e63 (41.17%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 \u003cem\u003eGZMB\u003c/em\u003e and \u003cem\u003ePRF1\u003c/em\u003e genotypes and post-HSCT complications\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther statistical analysis revealed that the donor \u003cem\u003eGZMB\u003c/em\u003e rs8192917 genotype was associated with post-transplant complications in HSCT patients. Specifically, it was observed that donor \u003cem\u003eTT\u0026nbsp;\u003c/em\u003ehomozygosity was more common in patients who developed cGvHD compared to patients with no cGvHD (85.41% vs 14.58%\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003ep\u0026lt;0.0001) (Fig. 1a). Similarly, donor \u003cem\u003eTT\u003c/em\u003e genotype was more common in patients with CMV infection compared to patients with without infection (69.49% vs 30.50%, p\u0026lt;0.0001) (Fig. 1b). An association was also observed between donors\u0026rsquo; genotype status in \u003cem\u003ePRF1\u003c/em\u003e rs885822 and aGvHD stage. The \u003cem\u003eAA/AG\u003c/em\u003e genotypes dominated among donors of patients without or with mild aGvHD (grades 0-I) while the \u003cem\u003eGG\u003c/em\u003e genotype was predominant in the group with more severe (grades II-IV) disease (p\u0026lt;0.0001) (Fig. 1c). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 mRNA expression of \u003cem\u003eGZMB\u003c/em\u003e and \u003cem\u003ePRF1\u003c/em\u003e genes \u0026ndash; association with cGvHD and CMV infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was observed that \u003cem\u003eGZMB\u003c/em\u003e expression strongly correlated with expression of \u003cem\u003ePRF1\u003c/em\u003e (p\u0026lt;0.0001, R=0.767) (Fig. 2a). Furthermore, the expression analysis revealed that the relative expression of \u003cem\u003eGZMB\u003c/em\u003e was lower on the 90\u003csup\u003eth\u003c/sup\u003e day after transplantation in comparison to the 30\u003csup\u003eth\u003c/sup\u003e day, in patients with cGvHD (p=0.0237) (Fig. 2b). A similar observation was noted for patients \u0026nbsp;with no complications, although non-significantly (p=0.069) (Fig. 2c). The CMV group exhibited higher expression of \u003cem\u003eGZMB\u003c/em\u003e (p=0.0027) and \u003cem\u003ePRF1\u003c/em\u003e (p=0.0035) on the 90\u003csup\u003eth\u003c/sup\u003e post-transplant in comparison to individuals without any post-transplant complications (Fig. 2d and 2e)\u003cs\u003e.\u003c/s\u003e It was also observed that in patients with CMV infection the expression of both \u003cem\u003eGZMB\u003c/em\u003e (p=0.0007, R=0.69) and \u003cem\u003ePRF1\u0026nbsp;\u003c/em\u003e(p=0.0013, R=0.67) correlated positively with the expression of \u003cem\u003eIFNG\u0026nbsp;\u003c/em\u003e(Fig. 3a and 3b). No statistically significant relationship was observed between the expression of genes and genotype status of rs819219 and rs885822.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Granzyme B and Perforin serum levels\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificant difference was observed in serum Granzyme B (GzmB) concentrations between HSCT recipients and healthy individuals, with notable decrease over time (median +30 days = 51,98 pg/ml, \u0026nbsp;+90 days = 31,04 pg/ml and \u0026nbsp;controls =12,57 pg/ml, \u0026nbsp;p\u0026lt;0.0001) (Fig. 4). Furthermore, a significant decrease of GzmB between the 30\u003csup\u003eth\u003c/sup\u003e\u0026nbsp; and 90\u003csup\u003eth\u003c/sup\u003e\u0026nbsp; day after HSCT was observed in all subgroups when tested separately (aGvHD, cGvHD, CMV and no complications) (p\u0026lt;0.0001) (Table 3). The patients exhibiting cGvHD and those without any complications had the largest difference in concentrations between 30\u003csup\u003eth\u003c/sup\u003e and 90\u003csup\u003eth\u0026nbsp;\u003c/sup\u003eday.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding serum perforin levels, no significant differences between patients and controls or across groups were observed. Median serum concentrations in patients at day +30, day +90 post-HSCT and in controls equalled: +30 days = 2419.71 pg/ml, +90 days = 2272.4 pg/ml and controls = 2234.61 pg/ml.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e. Serum GzmB concentrations in patients post-HSCT.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e30 days after transplantation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e90 days after transplantation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMedian (pg/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eStd error\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMedian (pg/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eStd error\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eaGvHD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e48.97\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e36.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e28.08\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e60.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0226\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ecGvHD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e48.30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e38.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e19.97\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0210\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCMV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e60.35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e35.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e41.07\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0034\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo complications\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e66.29\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e64.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e29.32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e27.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.0009\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAll groups\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e51.98\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e52.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e31.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations. HSCT: hematopoietic stem cell transplant; aGvHD: acute graft-versus-host disease; cGvHD: chronic graft-versus-host-disease; CMV: cytomegalovirus.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eHematopoietic stem cells transplantation (HSCT) is a standard procedure for the treatment of hematological disorders, including malignancies. Previous studies have demonstrated the importance of NK cells and their receptors in the development of post-transplantation complications in patients undergoing HSCT[22-25]. It has been also proposed that GzmB positive T regulatory cells can be a prognostic marker for aGvHD and relapse[26,27]. Although some studies have also investigated the roles of GzmB and perforin in acute organ transplant rejection [28,29] and in aGvHD in mice [13] thus far, the potential implication of GzmB and perforin in post-transplant complications has not been further explored. Here, we \u0026nbsp;present the results of our investigation regarding this matter.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNK cells are the main producers of GzmB and perforin, followed by T cells[14,30]. GzmB is a serine protease with strong apoptotic activity, capable of inducing targeted cell death. Perforin on the other hand is a pore-forming glycoprotein which facilitates the entrance of GzmB into targeted cells. While perforin co-operates with GzmB to activate pro-apoptotic pathways, GzmB has additional roles such as cleavage of substrates in the extracellular matrix[31-32]. Both proteins, have been associated with the pathogenesis of various inflammatory and autoimmune conditions such as: chronic obstructive pulmonary disease, chronic idiopathic thrombocytopenic purpura, atopic dermatitis, rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis and Sjögren syndrome \u0026nbsp;[33].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter HSCT, NK cells reconstitute rapidly within the first few weeks. In allogeneic transplant, their main source are donor’s hematopoietic stem cells or residual host cells in situations where mixed chimerism occurs[10,12]. In healthy individuals, normal levels of GzmB range from 0 to 30 pg/ml with the lowest baseline being 1-5 pg/ml[33-34]. Increased GzmB detected in patients’ blood may signify inflammatory conditions (with the participation of NK cells) which occur during CMV and Epstein-Barr virus infection, GvHD, GvL and graft rejection. It is generally recognized that, primarily, the dominating subset of NK cells post HSCT is CD56\u003csup\u003ebright\u003c/sup\u003e, which is an immature form with lower cytotoxic activity[35]. However, NK cells after transplantation are very capable of targeting residual cancer cells in GvL, and expand in the case of CMV. This can potentially explain the increase in production of GzmB and perforin[9,36,37]. The observed decrease 90 days after transplantation may be indicative of immune rebalancing. At this time adaptive immunity begins to develop with the reconstitution of T cells and B cells[38]. Additionally, at this time, complications that were detected at an early stage after transplantation are already treated with medication that decreases inflammation, as well as GzmB and perforin production.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe expression of \u003cem\u003eGZMB\u003c/em\u003e and \u003cem\u003ePRF1\u003c/em\u003e was correlated with the expression of \u003cem\u003eIFNG\u003c/em\u003e in patients with CMV infection. \u003cem\u003eIFNG\u003c/em\u003e codes for interferon-γ, a pro-inflammatory cytokine crucial in immune response to viral infections. Furthermore, interferon-γ is considered in terms of monitoring and assessment of CMV infection[39-42]. Therefore, the correlation with \u003cem\u003eIFNG\u003c/em\u003e can be indicative of viral reactivation and increased inflammatory conditions associated with it, which promotes the expression of \u003cem\u003eGZMB\u003c/em\u003e and \u003cem\u003ePRF1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn the present study, the serum levels of perforin were not significantly increased in patients undergoing allogeneic HSCT when compared to healthy controls. This can be possibly explained by the fact that perforin in comparison to GzmB is an unstable molecule, degraded immediately after pore formation. In addition, GzmB is more readily released into circulation and extracellular space as it also has extracellular functions[43]. Perforin, on the other hand, is less likely to be released into serum as it has a localized action at immune synapse and is membrane bound. GzmB is also independently secreted without the need of perforin which is not true the other way around[31-33]. Finally, although we would expect higher levels of perforin in the serum of HSCT patients, it must be considered that these patients undergo conditioning treatment before the procedure and receive glucocorticoid medication. Such treatments decrease calcium levels, and calcium is a major regulator of perforin[44,45]. Perhaps, the implementation of a different method, such as detection of intracellular or membrane bound perforin through flow cytometry could generate contrasting results.\u003c/p\u003e\n\u003cp\u003eThe two SNPs that were chosen for analysis, \u003cem\u003eGZMB\u003c/em\u003e rs819217 and \u003cem\u003ePRF1\u003c/em\u003e rs885822, are missense variants (C\u0026gt;T, A\u0026gt;G) that lead to changes in amino acid sequence, arginine to glutamine and histidine to glutamine, respectively. Both of them have been previously reported to be associated with autoimmune diseases[46-49]. In the present study both polymorphisms were also found to be associated with clinical outcomes in HSCT. For rs819217, donor’s \u003cem\u003eT\u003c/em\u003e\u003cem\u003eT\u0026nbsp;\u003c/em\u003ehomozygosity seems to have an unfavorable effect on the development of cGvHD and CMV infection in transplant recipients while the \u003cem\u003eC\u003c/em\u003e\u003cem\u003eC\u003c/em\u003e genotype seems to play a protective role. For rs885822, the wild type \u003cem\u003eGG\u0026nbsp;\u003c/em\u003ehomozygosity was predominant in donors of patients with aGvHD grades II-IV. Our results suggest an association of \u003cem\u003eGZMB\u0026nbsp;\u003c/em\u003ers819217 polymorphism with cGvHD and \u003cem\u003ePRF1\u003c/em\u003e rs885822 with aGvHD. While GzmB and perforin often work synergistically their roles in GvHD might differ. A recent study by Thompson et al explored the effect of \u003cem\u003ePRF1\u003c/em\u003e and \u003cem\u003eGZMB\u003c/em\u003e KO on aGvHD in mice and demonstrated that PRF1 KO in donor cells reduced aGvHD while DKO of GZMB and PRF1 did not have that effect [50]. In another study it has been indicated that the levels of GzmB are higher in post-HSCT patients who never developed aGvHD [51]. We did not observe any associations between gene expression and occurrence of the \u003cem\u003eGZMB\u003c/em\u003e/\u003cem\u003ePRF1\u003c/em\u003e genotypes, although this could be due to the relatively small sample size.\u003c/p\u003e\n\u003cp\u003eGranzyme B, alongside perforin, is involved in the cytotoxic process by inducing apoptosis in recipient tissues, possibly contributing to the pathogenesis of post-transplant complications. With further investigation, the mRNA expression and serum protein levels of granzyme B can serve as biomarkers of GvHD and CMV severity, or as markers for ongoing inflammation. The same applies to genetic polymorphisms in rs819217 and rs885822 SNPs which can be potential markers of the development of complications after the HSCT.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIKG performed the assessment of granzyme B and perforin serum levels, genotyping studies and data analyses, drafted, edited and finalized the manuscript; PŁ performed mRNA expression analysis, contributed to data analyses, edited and finalised the manuscript; JS performed mRNA expression, DNA isolation, edited and finalised the manuscript; AS, MM, AC, MSK, WF, IS, BNA, PS, MB, AT, GWB, SG and TW provided patients’ clinical samples and clinical data; KBK conceived, designed and supervised the study, analysed the data, drafted, edited and finalized the manuscript and secured funding. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupported by National Science Centre (Poland) project No. 2018/31/B/NZ2/03065. The authors do not declare any conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of\u0026nbsp;Wroclaw Medical University Ethics Committee (identification code KB-561/2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants/family members.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request. Data are located in controlled access data storage at Hirszfeld Institute of Immunology and Experimental Therapy.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGiralt S, Bishop MR. Principles and Overview of Allogeneic Hematopoietic Stem Cell Transplantation. In: Bishop MR, editor. Hematopoietic Stem Cell Transplantation [Internet]. Boston, MA: Springer US; 2009 [cited 2024 Nov 18]. pp. 1\u0026ndash;21. (Cancer Treatment and Research; vol. 144). Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://link.springer.com/\u003c/span\u003e\u003cspan address=\"http://link.springer.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-0-387-78580-6_1\u003c/span\u003e\u003cspan address=\"10.1007/978-0-387-78580-6_1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhaddour K, Hana CK, Mewawalla P. Hematopoietic Stem Cell Transplantation. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 [cited 2024 Nov 18]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/books/NBK536951/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/books/NBK536951/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGr\u0026oslash;nvold BL, Ali MM, Myklebust T\u0026Aring;, Lenartova A, Remberger M, Abrahamsen IW, et al. Allogeneic stem cell transplant recipients surviving at least 2 years without relapse: outcome and risk factors. EJHaem. 2024;5(1):117\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanger R, Lelas A, Rittenschober M, Piekarska A, Sadowska-Klasa A, Sabol I, et al. Retrospective analysis of the incidence and outcome of late acute and chronic graft-versus-host disease\u0026mdash;an analysis from transplant centers across Europe. Front Transpl. 2024;3:1332181.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003efor the Infectious Diseases Working, Party EBMT, Styczyński J, Tridello G, Koster L, Iacobelli S, Van Biezen A, et al. Death after hematopoietic stem cell transplantation: changes over calendar year time, infections and associated factors. Bone Marrow Transpl. 2020;55(1):126\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDekker L, de Koning C, Lindemans C, Nierkens S. Reconstitution of T Cell Subsets Following Allogeneic Hematopoietic Cell Transplantation. Cancers. 2020;12(7):1974.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHattori N, Saito B, Sasaki Y, Shimada S, Murai S, Abe M, et al. Status of Natural Killer Cell Recovery in Day 21 Bone Marrow after Allogeneic Hematopoietic Stem Cell Transplantation Predicts Clinical Outcome. Biol Blood Marrow Transpl. 2018;24(9):1841\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim HO, Oh HJ, Lee JW, Jang PS, Chung NG, Cho B, et al. Immune reconstitution after allogeneic hematopoietic stem cell transplantation in children: a single institution study of 59 patients. Korean J Pediatr. 2013;56(1):26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLocatelli F, Pende D, Falco M, Della Chiesa M, Moretta A, Moretta L. NK Cells Mediate a Crucial Graft-versus-Leukemia Effect in Haploidentical-HSCT to Cure High-Risk Acute Leukemia. Trends Immunol. 2018;39(7):577\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHadjis AD, McCurdy SR. The role and novel use of natural killer cells in graft-versus-leukemia reactions after allogeneic transplantation. Front Immunol. 2024;15:1358668.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTibbs E, Cao X. Emerging Canonical and Non-Canonical Roles of Granzyme B in Health and Disease. Cancers. 2022;14(6):1436.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGail LM, Schell KJ, Łacina P, Strobl J, Bolton SJ, Steinbakk Ulriksen E, et al. Complex interactions of cellular players in chronic Graft-versus-Host Disease. Front Immunol. 2023;14:1199422.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu W, Leigh ND, Bian G, O\u0026rsquo;Neill RE, Mei L, Qiu J et al. Granzyme B-Mediated Activation-Induced Death of CD4\u0026thinsp;+\u0026thinsp;T Cells Inhibits Murine Acute Graft-versus-Host Disease. J Immunol Baltim Md. 1950. 2015;195(9):4514\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin L, Couturier J, Yu X, Medina MA, Kozinetz CA, Lewis DE. Granzyme B secretion by human memory CD4 T cells is less strictly regulated compared to memory CD8 T cells. BMC Immunol. 2014;15(1):36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrahmi Z, Hommel-Berrey G, Smith F, Thomson B. NK cells recover early and mediate cytotoxicity via perforin/granzyme and Fas/FasL pathways in umbilical cord blood recipients. Hum Immunol. 2001;62(8):782\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlmishri W, Santodomingo-Garzon T, Le T, Stack D, Mody CH, Swain MG. TNFα Augments Cytokine-Induced NK Cell IFNγ Production through TNFR2. J Innate Immun. 2016;8(6):617\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaul F, Erdfelder E, Buchner A, Lang AG. Statistical power analyses using G*Power 3.1: Tests for correlation and regression analyses. Behav Res Methods. 2009;41(4):1149\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Z, Taylor JA. SNPinfo: integrating GWAS and candidate gene information into functional SNP selection for genetic association studies. Nucleic Acids Res. 2009;37(suppl2):W600\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlare EM, Divjak M, Bailey MJ, Walters EH. beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels. Thorax. 2002;57(9):765\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSikand K, Singh J, Ebron JS, Shukla GC. Housekeeping gene selection advisory: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are targets of miR-644a. PLoS ONE. 2012;7(10):e47510.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong R, He S, Wu Y, Chen W, Song J, Zhu Y, et al. Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of septic patients. Heliyon. 2023;9(4):e15269.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNihtil\u0026auml; J, Penna L, Salmenniemi U, It\u0026auml;l\u0026auml;-Remes M, Crossland RE, Gallardo D, et al. Effect of NK cell receptor genetic variation on allogeneic stem cell transplantation outcome and in vitro NK cell cytotoxicity. Sci Rep. 2024;14(1):26988.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiemaszko J, Ussowicz M, Rybka B, Ryczan-Krawczyk R, Kałwak K, Bogunia-Kubik K. The impact of NKG2A and NKG2D receptors and HLA-E and MICA ligands polymorphisms on post-transplant complications after paediatric allogeneic HSCT: a single-centre experience. Front Genet. 2023;14:1186123.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiemaszko J, Łacina P, Szymczak D, Szeremet A, Majcherek M, Czyż A, et al. Soluble MICA concentrations and genetic variability of MICA and its NKG2D receptor as factors affecting Graft-versus-Host Disease development after allogeneic haematopoietic stem cell transplantation. Hum Immunol. 2024;85(6):111147.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie DH, Wang J, Sun K, Shi ZY, Wang YZ, Chang Y, et al. The Functional and Prognostic Impact of TIGIT Expression on Bone Marrow NK Cells in Core Binding Factor-Acute Myeloid Leukemia Patients at Diagnosis. Biomedicines. 2024;12(10):2207.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrokov MY, Davydova JO, Kuzmina LA, Galtseva IV, Kapranov NM, Vasilyeva VA, et al. Level of Granzyme B-positive T-regulatory cells is a strong predictor biomarker of acute Graft-versus-host disease after day\u0026thinsp;+\u0026thinsp;30 after allo-HSCT. Leuk Res. 2017;54:25\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrokov M, Davydova Y, Popova N, Kapranov N, Starikova O, Mikhaltsova E, et al. High expression of granzyme B in conventional CD4\u0026thinsp;+\u0026thinsp;T cells is associated with increased relapses after allogeneic stem cells transplantation in patients with hematological malignancies. Transpl Immunol. 2021;65:101295.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKummer JA, Wever PC, Kamp AM, Ten Berge IJM, Hack CE, Weening JJ. Expression of granzyme A and B proteins by cytotoxic lymphocytes involved in acute renal allograft rejection. Kidney Int. 1995;47(1):70\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMac QD, Mathews DV, Kahla JA, Stoffers CM, Delmas OM, Holt BA, et al. Non-invasive early detection of acute transplant rejection via nanosensors of granzyme B activity. Nat Biomed Eng. 2019;3(4):281\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampos TM, Novais FO, Saldanha M, Costa R, Lordelo M, Celestino D, et al. Granzyme B Produced by Natural Killer Cells Enhances Inflammatory Response and Contributes to the Immunopathology of Cutaneous Leishmaniasis. J Infect Dis. 2020;221(6):973\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVelotti F, Barchetta I, Cimini FA, Cavallo MG. Granzyme B in Inflammatory Diseases: Apoptosis, Inflammation, Extracellular Matrix Remodeling, Epithelial-to-Mesenchymal Transition and Fibrosis. Front Immunol. 2020;11:587581.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson R, Cao X. Reassessing granzyme B: unveiling perforin-independent versatility in immune responses and therapeutic potentials. Front Immunol. 2024;15:1392535.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoivin WA, Cooper DM, Hiebert PR, Granville DJ. Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma. Lab Invest. 2009;89(11):1195\u0026ndash;220.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpaeny-Dekking EHA, Hanna WL, Wolbink AM, Wever PC, Kummer AJ, Swaak AJG, et al. Extracellular Granzymes A and B in Humans: Detection of Native Species During CTL Responses In Vitro and In Vivo. J Immunol. 1998;160(7):3610\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMushtaq MU, Shahzad M, Shah AY, Chaudhary SG, Zafar MU, Anwar I, et al. Impact of natural killer cells on outcomes after allogeneic hematopoietic stem cell transplantation: A systematic review and meta-analysis. Front Immunol. 2022;13:1005031.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePende D, Marcenaro S, Falco M, Martini S, Bernardo ME, Montagna D, et al. Anti-leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for pediatric patients: evaluation of the functional role of activating KIR and redefinition of inhibitory KIR specificity. Blood. 2009;113(13):3119\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinculescu L, Marquart HV, Friis LS, Petersen SL, Schi\u0026oslash;dt I, Ryder LP, et al. Early Natural Killer Cell Reconstitution Predicts Overall Survival in T Cell\u0026ndash;Replete Allogeneic Hematopoietic Stem Cell Transplantation. Biol Blood Marrow Transpl. 2016;22(12):2187\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBosch M, Khan FM, Storek J. Immune reconstitution after hematopoietic cell transplantation. Curr Opin Hematol. 2012;19(4):324\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SH, Lee HS, Lee HJ, Kim SM, Shin S, Park SH, et al. Clinical applications of interferon-γ releasing assays for cytomegalovirus to differentiate cytomegalovirus disease from bystander activation: a pilot proof-of-concept study. Korean J Intern Med. 2017;32(5):900\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYong MK, Cameron PU, Slavin M, Morrissey CO, Bergin K, Spencer A, et al. Identifying Cytomegalovirus Complications Using the Quantiferon-CMV Assay After Allogeneic Hematopoietic Stem Cell Transplantation. J Infect Dis. 2017;215(11):1684\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Gracia-Guindo MdC, Ruiz-Fuentes MdC, Galindo-Sacrist\u0026aacute;n P, Osorio-Moratalla JM, Ruiz-Fuentes N, Rodriguez Granger J et al. Cytomegalovirus Infection Monitoring Based on Interferon Gamma Release Assay in Kidney Transplantation. Transplant Proc. 2018;50(2):578\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SH. Interferon-γ Release Assay for Cytomegalovirus (IGRA-CMV) for Risk Stratification of Posttransplant CMV Infection: Is It Time to Apply IGRA-CMV in Routine Clinical Practice? Clin Infect Dis. 2020;71(9):2386\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuzza MS, Zamurs L, Sun J, Bird CH, Smith AI, Trapani JA, et al. Extracellular Matrix Remodeling by Human Granzyme B via Cleavage of Vitronectin, Fibronectin, and Laminin. J Biol Chem. 2005;280(25):23549\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaw RHP, Lukoyanova N, Voskoboinik I, Caradoc-Davies TT, Baran K, Dunstone MA, et al. The structural basis for membrane binding and pore formation by lymphocyte perforin. Nature. 2010;468(7322):447\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiglietta F, Iamartino L, Palmini G, Giusti F, Marini F, Iantomasi T, et al. Endocrine sequelae of hematopoietic stem cell transplantation: Effects on mineral homeostasis and bone metabolism. Front Endocrinol. 2023;13:1085315.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorrales-Tellez E, Vu D, Shah T, Hutchinson I, Min DI. Association between granzyme B and perforin I polymorphisms and allograft outcomes in Hispanic kidney transplant recipients. Clin Transplant [Internet]. 2013 May [cited 2024 Nov 25];27(3). Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://onlinelibrary.wiley.com/doi/\u003c/span\u003e\u003cspan address=\"https://onlinelibrary.wiley.com/doi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/ctr.12114\u003c/span\u003e\u003cspan address=\"10.1111/ctr.12114\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai D, Wen F, Liu S, Zhou S. Brain damage resembling acute necrotizing encephalopathy as a specific manifestation of haemophagocytic lymphohistiocytosis - induced by hypersensitivity. Ital J Pediatr. 2016;42(1):79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaworowska A, Pastorczak A, Trelinska J, Wypyszczak K, Borowiec M, Fendler W, et al. Perforin gene variation influences survival in childhood acute lymphoblastic leukemia. Leuk Res. 2018;65:29\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeong KH, Kim SK, Seo JK, Shin MK, Lee MH. Association of GZMB polymorphisms and susceptibility to non-segmental vitiligo in a Korean population. Sci Rep. 2021;11(1):397.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson R, Puppala E, Mustapha H. Assessing Perforin-independent Granzyme B function in Acute Graft-Versus-Host Disease: Impact on Allogeneic Hematopoietic Cell Transplantation. J Immunol. 2024;212(1Supplement):12705459\u0026ndash;12705459.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrokov MYu, Parovichnikova EN, Davydova J, Kuzmina LA, Galtseva IV, Kapranov NM, et al. Granzyme B Expression in T-Regulatory Cells Is a Strong Predictor of Acute Graft-Versus-Host Disease after Day\u0026thinsp;+\u0026thinsp;30 in Patients with Classic Immunosuppression after Allo-HSCT. Blood. 2016;128(22):2238\u0026ndash;2238.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"granzyme B, perforin, NK cells, allogeneic HSCT, GvHD, cytomegalovirus","lastPublishedDoi":"10.21203/rs.3.rs-6556505/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6556505/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGraft-versus-host disease (GvHD) and cytomegalovirus (CMV) infection are serious complications after allogeneic hematopoietic stem cell transplantation (HSCT) and NK cells are involved in their pathogenesis. As granzyme B (GzmB) and perforin are markers of cytotoxicity, we examined polymorphism and expression of GzmB and perforin genes in the context of HSCT outcome. A number of 247 patients and 117 donors were genotyped for \u003cem\u003eGZMB\u003c/em\u003e rs8192917 and \u003cem\u003ePRF1\u003c/em\u003e rs885822 variants, and \u003cem\u003eGZMB\u003c/em\u003e and \u003cem\u003ePRF1\u003c/em\u003e mRNA expression as well as serum levels were assessed 30 and 90 days post-transplantation. Chronic GvHD (cGvHD) and CMV infection were associated with donor \u003cem\u003eGZMB\u003c/em\u003e rs8192917 \u003cem\u003eTT\u003c/em\u003e genotype (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) while donor \u003cem\u003ePRF1\u003c/em\u003e rs885822 \u003cem\u003eGG\u003c/em\u003e was predominant in patients with acute GvHD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). \u003cem\u003eGZMB\u003c/em\u003e expression decreased post-transplantation in recipients with cGvHD (p\u0026thinsp;=\u0026thinsp;0.0237) while patients with CMV exhibited higher expression of \u003cem\u003eGZMB\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0027) and \u003cem\u003ePRF1\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0035) on day 90 in comparison to recipients without complications. Strong correlations were observed between the expression of \u003cem\u003eGZMB\u003c/em\u003e and \u003cem\u003ePRF1\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u0026thinsp;=\u0026thinsp;0.767) and between \u003cem\u003eIFNG\u003c/em\u003e (interferon-γ) and \u003cem\u003eGZMB\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0007, R\u0026thinsp;=\u0026thinsp;0.69) and \u003cem\u003ePRF1\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0013, R\u0026thinsp;=\u0026thinsp;0.67). Serum GzmB concentration decreased significantly over time in patients (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These results suggest that \u003cem\u003eGZMB\u003c/em\u003e and \u003cem\u003ePRF1\u003c/em\u003e genotypes and expression may be potential markers of post-transplant complications and have roles in cGvHD and CMV pathogenesis.\u003c/p\u003e","manuscriptTitle":"GZMB and PRF1 mRNA expression and donor genotypes and their associations with post-transplant complications in allogeneic hematopoietic stem cell transplant recipients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-08 20:36:46","doi":"10.21203/rs.3.rs-6556505/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"13c5582d-2407-443c-89d3-0e267dea6746","owner":[],"postedDate":"May 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T10:09:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-08 20:36:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6556505","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6556505","identity":"rs-6556505","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}