The platelet transcriptome after stem cell transplantation

Abstract

Dear Editor,

Platelets play a role in haemostasis, vascular biology and immunity.¹ Activated platelets may worsen or modulate immune reactions. Stem cell transplantation is an established treatment for leukaemia, lymphoma and some other conditions. It is unclear if the platelet transcriptome displays markers of immune activation in the recovery period after transplantation. The aim of our study was to identify changes in the platelet transcriptome (being a corollary of events happening in megakaryocytes in the bone marrow) after transplantation. The study of the platelet transcriptome was found to be useful for discoveries in human health and disease.² In sepsis, altered transcriptome of human and mouse platelets was found. The sepsis-induced upregulation of the ITGA2B gene on platelets predicts mortality.³ To our knowledge the platelet transcriptome has never been studied systematically after hematopoietic stem cell transplantation (HSCT). Autologous transplantation permits to deliver high-dose chemotherapy. Allogeneic transplantation in addition generates an immune response most pronounced in myeloid leukaemias. Both types of transplantation have a potential for cure. An earlier study investigated platelet markers after allogeneic transplantation using flow cytometry and found platelet receptor and granule defects.⁴ In a recent Japanese study early thrombocytopenia was a risk factor for a poor outcome after allogeneic HSCT.⁵ We hypothesized that an altered transcriptome could correlate with complications and possibly predict the outcome after autologous and allogeneic HSCT.

Platelets were isolated and purified according to standard methods.⁶ This includes negative bead selection for GPA and LCA. A total of 8 patients were studied before and after HSCT. Four patients underwent an autologous HSCT, 4 patients underwent an allogeneic HSCT. Details about these patients can be found in Table 1. RNA was sequenced using an Illumina NovaSeq 6000 and transcriptomes were analysed with R, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA). Following the isolation of platelets and RNA, sequencing generated raw gene datasets that were compared using an R algorithm. All isolations were performed in duplicate. In the first comparison, we examined gene expression, before versus after two types of HSCT, autologous (Figure 1A) and allogeneic (Figure 1C) by aggregating all data based on type of transplant. A threshold of log2 (FC) > 1 and FDR < 0.01 (blue dots) or FDR < 0.05 (red dots) was applied. Further the top differentially expressed genes were identified and ranked according to the log2FC (Figure 1B,D). From this analysis, autologous HSCT causes fewer changes in gene expression than allogenic stem cell transplantation (SCT). In the autologous cohort, we identified 7 downregulated genes and 8 upregulated genes in the platelet transcriptomes. In the allogeneic transplanted patients, the platelet transcriptomes showed only 6 downregulated genes but there were 58 upregulated genes (Figure 1). In the autologous HSCT group, TCL1A, CFP and CD4 were upregulated genes, while OLFM4, LOC100420587 and TPTEP1 were significantly downregulated (Figure 1B). In the allogeneic HSCT group, TCN1, SLP1 and MPO were highly upregulated and MYL4, PDZK1IP1 and FAM110B were significantly downregulated (Figure 1D). Figure 1E illustrates the expression changes of eight selected genes TCL1A, CD4, OLFM4, MYL4, TCN1, LTF, CAMP and VCAN in platelets before and after autologous and allogeneic transplants. Notably, TCL1A, CD4, TCN1, LTF, CAMP and VCAN show significant upregulation post-transplant in autologous and allogeneic conditions. Conversely, OLFM4 and MYL4 display significant downregulation.

To further understand the variation of platelet genes expressed in autologous and allogeneic HSCT groups, we conducted functional annotation using GO, KEGG and GSEA. These analyses were performed on the datasets utilizing Shiny GO version 0.80, GSEA 4.3.2 version and an R package. The gene sets used in these analyses were derived from the upregulated genes in each group, comparing expression levels before and after transplantation. This approach allowed us to capture the dynamic changes in gene expression associated with the transplantation process. By focusing on genes upregulated after transplant, we aimed to reveal key pathways involved in immune response, cellular repair and hematopoietic recovery.

GO analysis was conducted to annotate the biological roles of upregulated genes, including biological process, cellular component and molecular function. In Figures 2 and 3, the respective genes are shown for the autologous and allogeneic groups. The KEGG database integrates genomic, chemical and systemic functional information. Our results indicated that upregulated genes in the autologous group were primarily enriched in pathways such as primary immunodeficiency, antigen processing and presentation, as well as hematopoietic cell lineage. Downregulated genes in the autologous group were mainly enriched in pathways such as inflammatory bowel disease, ECM-receptor interaction, and Legionellosis (Figure S1A). In the allogeneic group, downregulated genes were mainly enriched in pathways such as cardiac muscle contraction, vascular smooth muscle contraction and adrenergic signalling in cardiomyocytes (Figure S1B).

GSEA was next performed on the ranked gene lists to extract biological insights at the pathway level. GSEA results for IL6_JAK_STAT3_SIGNALING pathways of the ranked gene lists in autologous and allogeneic HSCT groups are shown in Figure 4. For the autologous cohort, the IL6–JAK–STAT3 signalling pathway showed negative enrichment (NES = –1.56, FDR = 0.00), indicating significant downregulation of the pathway. Core enriched genes included IFNGR1, IL10RB, and IL7. For the allogeneic cohort, the pathway also displayed negative enrichment (NES = –1.72, FDR = 0.05), similarly suggesting downregulation. Core enriched genes included CD38, IL12RB1, and ACVR1B. Together, these findings demonstrate a consistently reduced IL6–JAK–STAT3 signalling signature in both transplant groups.

In conclusion, multiple platelet-associated genes were altered during HSCT, with a broader transcriptional shift observed in the allogeneic cohort compared with the autologous cohort. Importantly, contrary to our initial expectation, the GSEA analysis indicates that the IL6–JAK–STAT3 signalling pathway is downregulated rather than activated in both groups. The JAK–STAT axis is known to regulate haematopoiesis and immune reconstitution⁷; therefore, suppression of this pathway in platelets may reflect altered cytokine responsiveness or compensatory downmodulation of deleterious immune reactions following HSCT.⁸ Interestingly, several genes that changed significantly during HSCT, such as TCL1A, OLFM4, TCN1 and MYL4, are not classical platelet genes. This suggests that platelets may acquire transcriptional programs derived from hematopoietic or immune compartments during engraftment. For example, the increased expression of TCL1A,⁹ a known promoter of HSC expansion, may point toward broader interactions between platelets and stem cell biology. Our study is limited by the relatively small cohort size, variable time points and disease heterogeneity; nonetheless, it provides new insight into how platelet-associated transcriptional signatures may reflect immune and engraftment dynamics during HSCT.

LL and RM performed research; all authors wrote and appproved the manuscript.

The authors declare no conflicts of interest.

This study was approved by the Institutional Review Board of the University of Kentucky (IRB # 61338). Informed consent was obtained from all study participants.

Research materials and supporting information can be made available by contacting the corresponding author.