Immunosuppressive therapy (IST) is the standard treatment for acquired pure red cell aplasia (aPRCA), but predictors of treatment response and long-term prognosis remain unclear. The clinical significance of somatic mutations in aPRCA is not fully understood. We retrospectively analyzed 69 adult aPRCA patients who underwent targeted next-generation sequencing of 69 genes associated with clonal hematopoiesis and myeloid neoplasms. Somatic mutations, T-cell receptor (TCR) gene rearrangements, treatment response at 6 months, overall survival (OS), and progression-free survival (PFS) were evaluated. Somatic mutations were detected in 62.3% of patients, mainly in epigenetic regulators like DNMT3A and TET2. Although mutations did not predict 6-month response to IST, higher mutation burden was associated with worse treatment outcomes (p = 0.01). TP53 and DNMT3A mutations were linked to shorter OS and PFS. Survival worsened with increasing mutation numbers, while mutations in TET2, ASXL1, GATA2, and STAT3 had no significant effect. TCR gene rearrangements were common in large granular lymphocyte leukemia-associated aPRCA but did not affect treatment response or survival. Somatic mutations are common in aPRCA and reflect clonal hematopoiesis. Mutation burden is a key determinant of IST response, while TP53 and DNMT3A mutations signal poorer long-term outcomes. Mutational profiling may improve risk stratification and guide personalized management.

Background:Natural killer (NK) cells play important immunoregulatory roles in the immune pathogenesis of severe aplastic anemia (SAA). Our previous research showed that SAA caused a decrease in T cell immunoglobulin mucin-3 (TIM3) expression on NK cells. Here we investigated the expression of surface receptors, and the cytotoxicity of peripheral TIM3+ NK and TIM3- NK cells in patients with SAA.Methods:The expressions of surface receptors and cytoplasmic protein of TIM3+ NK and TIM3- NK cells from peripheral blood were detected by FCM. The functions of mDCs, and apoptosis rate of K562 cells after co-culture with TIM3+ NK and TIM3- NK cells were maesured by FCM. Westren-blot was used to detect the changes of TIM3+ NK and TIM3- NK signaling pathway proteins (AKT, P-AKT) and compare the functional activity of the two groups.Results:Activating receptors NKG2D and Granzyme B were higher, while inhibiting receptors NKG2A, CD158a and CD158b were lower on TIM3- NK cells compared with TIM3+ NK cells in patients with SAA. In SAA, the expression of CD80 and CD86 on mDCs (Myeloid dendritic cells) was significantly decreased after incubation with TIM3- NK cells. The apoptosis rate (AR) of K562 cells was significantly increased after being incubated with TIM3- NK cells in SAA. The level of signal pathway protein AKT of TIM3- NK cells in SAA was similar to that of TIM3+ NK cells, and the levels of P-AKT and P-AKT/AKT ratio of TIM3- NK cells were significantly higher than those of TIM3+ NK cells.Conclusions:Therefore, TIM3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. Low expression of TIM3 contributes to the enhancement of NK cell activity which in turn inhibits the immune activation state of SAA and improves the disease state. Our research may aid the development of new therapeutic strategies based on TIM3-NK cells infusion for the treatment of SAA.

Objective: To investigate the expression level of IRF1 in bone marrow NK cells of patients with severe aplastic anemia (SAA), and to explore its effect on NK cell proliferation and function and its molecular biological mechanism. Methods: The expression level of IRF1 and its correlation with clinical characteristic indexes were explored by analyzing bone marrow NK cells of SAA patients. The effects of IRF1 on NK cell proliferation and cycle were preliminarily analyzed by in vitro experiments. We searched for the downstream target proteins and related signaling pathways of IRF1 by high-throughput sequencing of transcriptome (RNA sequencing (RNA-seq)) combined with personalized analysis of PPI. Finally, the therapeutic effect of IRF1 inhibitor Rottlerin on SAA was further explored by constructing a mouse model of bone marrow failure. Results: By testing the bone marrow NK cells of SAA patients, it was found that IRF1 expression was significantly elevated in bone marrow NK cells of SAA patients, and was associated with the severity of the patient's condition.After sh-IRF1 lentivirus knockdown in YT-NK cells, the apoptosis rate of NK cells was higher than that of control. And five consecutive time points showed that the proliferative ability of YT-NK cells was significantly weakened after knockdown of the IRF1 gene. 72h after shIRF1 virus infection, flow assay revealed that the cell cycle of the knockdown group was slightly increased in the G1 phase and slightly decreased in the S phase. NKG2D, perforin was significantly lower in the shRNA-IRF1 group than in the NC group. The knockdown of IRF1 resulted in significant alterations in the NFkB signaling pathway. RNA-seq results of knockdown IRF1 showed that all differentially downregulated protein functions were enriched in RNA metabolism, multicellular biological processes, signaling molecules and interactions, NF-κB pathway, JAK/STAT pathway and IFN-α-mediated related pathways. Using string11.5 database for protein-protein interaction (PPI) analysis of genes enriched in the NF-κB signaling pathway with IRF1, we screened the top 10 core targets, which were IL-1β, CXCL8, CXCL1, CCL4, CXCL2, NFKBIA, VCAM1, MYD88, LY96, CD14. And then by protein-protein interaction study (CO-IP) technique, it was found that IRF1 might interact with IL-1β in the nucleus, and the rescue assay of overexpression of IL-1β after knocking down IRF1 showed that the proliferative activity of the cells was significantly weakened and apoptosis rate was significantly increased. In addition, the WB results showed that the NF-κB signaling pathway was phosphorylated and inhibited after IRF1 was knocked down, whereas overexpression of IL-1β could activate the NF-κB signaling pathway, and down-regulation of IRF1 and simultaneous up-regulation of IL-1β partially restored the inhibition of phosphorylation of the NF-κB signaling pathway. We applied Rotterin to treat mice with bone marrow failure and found that after injection of Rotterin, the blood count, bone marrow hematopoietic function, peripheral blood CD4/CD8 ratio, and IFN-γ expression level in CD8+T lymphocytes of mice were significantly restored compared to SAA mice. Conclusions: IRF1 may promote the phosphorylation level expression of NF - κ B pathway related proteins by interacting with IL-1 β, affecting the proliferation and function of bone marrow NK cells in SAA patients, and participating in the immunopathogenesis of SAA.

Pure red cell aplasia (PRCA) is a rare bone marrow disorder characterized by a severe reduction or absence of erythroid precursor cells, without affecting granulocytes and megakaryocytes. Immunosuppressive therapies, particularly cyclosporine, have demonstrated efficacy as a primary treatment. This study aims to develop a predictive model for assessing the efficacy of cyclosporine in acquired PRCA (aPRCA). This retrospective study encompasses newly treated aPRCA patients at the General Hospital of Tianjin Medical University. Diagnosis criteria include severe anemia, and absolute reticulocyte count below 10 × 109/L, with normal white blood cell and platelet counts, and a severe reduction in bone marrow erythroblasts. Cyclosporine therapy was administered, with dose adjustments based on blood concentration. Response to cyclosporine was evaluated according to established criteria. Statistical analysis involved logistic multi-factor regression, generating a predictive model. The study included 112 aPRCA patients with a median age of 63.5 years. Patients presented with severe anemia (median Hb, 56 g/L) and reduced reticulocyte levels. Eighty-six patients had no bone marrow nucleated erythroblasts. Primary PRCA accounted for 62 cases (55.4

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Natural killer (NK) cells play an essential immunoregulatory role in the pathogenesis of severe aplastic anaemia (SAA). NK cells exert a regulatory effect on adaptive immunity by secreting interferon (IFN) and stimulating cytotoxic T cell proliferation and activation. Our previous research showed that IFN regulatory factor 1 (IRF-1) was essential in developing NK cells. Moreover, an increased expression of IRF-1 was detected in NK cells, which was associated with NK cell dysfunction and the pathogenesis of SAA. Aims: In this study, the expression of IRF-1 was evaluated in the bone marrow NK cells of patients with SAA and murine model to further understand the pathogenesis of SAA. Methods: The expression of IRF-1 in NK cells from the bone marrow was detected by flow cytometry. The expression levels of IRF-1 in NK cells were detected using Rt-PCR and western blot.NK92MI cells were selected to induce IRF-1 overexpression in NK cells using IFN-α or to reduce IRF-1 expression using the IRF-1 inhibitor rottlerin. Fluorescence-activated cell sorting was used to detect the activation states of NKG2A and NKG2D in NK cells. CCK-8 was used to detect NK cell proliferation, while annexin V was used to detect NK cell apoptosis. The treated NK cells were co-cultured with CD8+T cells. Then, a cytometric bead array system was used to detect the levels of TNF-α, IFN-γ, IL-10, and IL-12 in vivo. The functions and apoptosis rates of the CD8+T cells cultured with the treated NK cells were measured. The murine model of AA was established,following IRF-1 inhibitor treatment, the probability of survival, general condition, blood routine, and bone marrow count of the mice in each group were compared. Results: The NK cells of the SAA initial treatment group had a higher expression of IRF-1 compared to the remission and control groups. IRF-1 expression in NK cells of SAA patients was negatively correlated with the peripheral blood platelet count as well as the proportion of neutrophils and reticulocytes,but positively correlated with the proportion of lymphocytes (P <0.05). The expression of NKG2A on the surface of NK92MI cells decreased. Meanwhile, the expression of NKG2D increased. Adding the IRF-1 blocker promoted NK-92MI cell proliferation and inhibited apoptosis. The levels of TNF-α, IFN-γ, IL-10, and IL-12 in the supernatant were significantly reduced after the addition of the IRF-1 blocker. The apoptosis rate of the CD8+T cells was increased. The level of signalling pathway protein NF-κB p65 in the NK cells in the IRF-1 inhibitor group was lower than that of the control group.The NK cells of the SAA mice had a higher IRF-1 expression than the TBI mice and normal controls. On the 17th day of model establishment, the weight, haemogram, and bone marrow cell count of the AA mice were lower than those of the NC group (P<0.05). The combination of the CsA and IRF-1 inhibitor infusion had a more significant therapeutic effect than CsA treatment alone. IRF-1 inhibitor infusion therapy possibly had a synergistic effect with CsA. Conclusion: The findings of this study confirmed that increased IRF-1 expression inhibited NK cells, thus contributing to the pathogenesis of SAA. Following IRF-1 inhibitor reinfusion, the probability of survival, general condition, blood cell count, and bone marrow cell count of the SAA mice improved. Additionally, the combined treatment with CsA increased the effectiveness of the treatment. The results of this study helped enhance the understanding of the pathogenesis of SAA, and new treatment targets for improving the efficacy of SAA treatment were identified.Keywords: Murine models, Aplastic anemia, Interferon regulatory factor, NK cell

Acquired pure red cell aplasia (aPRCA) is a rare hematological disorder characterized by normochromic, normocytic anemia, reticulocytopenia, and the absence of erythroblasts. The pathogenesis of aPRCA has remained elusive. This review delves into the intricate web of immune mechanisms underlying the development of this enigmatic condition. By exploring immune responses, cytotoxic effects, and antibody-mediated processes, we dissect the immune-driven assault on erythroid progenitors. The classification of aPRCA, including its primary and secondary forms, is elucidated, with a particular emphasis on etiological factors such as viruses, drugs, thymoma, and large granular lymphocytic leukemia. Furthermore, we discuss the implications of cytogenetic changes in erythroid progenitors and immune cells in the pathophysiology of aPRCA. This comprehensive overview aims to shed light on the complex interplay between immune dysregulation and erythroid failure in aPRCA, offering insights that will be crucial for better understanding and treating this disease.

BackgroundT-cell immunoglobulin and mucin-containing domain (TIM)-3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. In this study, we aimed to explore a novel treatment method of TIM-3(+) NK or TIM-3(-) NK cell infusion in combination with immunosuppressive therapy for bone marrow failure (BMF)/aplastic anemia (AA) mice. MethodsBMF/AA mouse model was constructed. The TIM-3 expression and functional molecules on TIM-3(+) and TIM-3(-) NK cells of the BMF group, total body irradiation (TBI) group, and normal control (NC) group mice were detected by flow cytometry. After treatment, the general condition, whole blood cell and bone marrow cell (BMC) count, and immune condition of mice from each group were compared. ResultsTIM-3 expression in the peripheral blood NK cells of BMF mice was significantly lower than that of the TBI and NC group mice. TIM-3(-) NK cells expressed more NKG2D receptors than TIM-3(+) NK cells. The levels of P-Akt and PI3K in TIM-3(-) NK cells were higher than those in TIM-3(+) NK cells. On the 17th day after BMF induction, the weight, peripheral whole blood cell count, and BMC count of BMF mice decreased significantly compared with that of the NC group mice. The therapeutic effect in the TIM-3(-) NK cell treatment group was better than that in the TIM-3(+) NK cell treatment and CsA treatment groups. Concurrently, the ratio of CD4(+)T and CD8(+)T cells of BMF mice was significantly lower than that of the NC group mice. The therapeutic effect in CsA + TIM-3(-) NK group was more significant than that of the CsA treatment and the CsA + TIM-3(+) NK groups. ConclusionsIn this study, we found that the general condition, peripheral whole blood cell and BMC count, and immune status of BMF mice improved significantly after CsA + TIM-3(-) NK cell treatment. These results may provide further insights into the immune pathogenesis of SAA and novel therapeutic ideas for improving SAA treatment.

Clinical and Translational MedicineVolume 12, Issue 12 e1092 LETTER TO THE EDITOROpen Access Single-cell transcriptomic analysis of PB and BM NK cells from severe aplastic anaemia patients Chunyan Liu, Chunyan Liu Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China Chunyan Liu and Yingying Chen contributed equally to the study.Search for more papers by this authorYingying Chen, Yingying Chen orcid.org/0000-0001-7575-0867 Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China Chunyan Liu and Yingying Chen contributed equally to the study.Search for more papers by this authorDan Lu, Dan Lu Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorBingnan Liu, Bingnan Liu Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorTian Zhang, Tian Zhang Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorLing Deng, Ling Deng Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorZixuan Liu, Zixuan Liu Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorCongwei Zhong, Congwei Zhong Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorRong Fu, Corresponding Author Rong Fu [email protected] Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China Correspondence Rong Fu, Department of Hematology, Tianjin Medical University General Hospital, 154 Anshan Street, Tianjin 300052, China. Email: [email protected]Search for more papers by this author Chunyan Liu, Chunyan Liu Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China Chunyan Liu and Yingying Chen contributed equally to the study.Search for more papers by this authorYingying Chen, Yingying Chen orcid.org/0000-0001-7575-0867 Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China Chunyan Liu and Yingying Chen contributed equally to the study.Search for more papers by this authorDan Lu, Dan Lu Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorBingnan Liu, Bingnan Liu Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorTian Zhang, Tian Zhang Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorLing Deng, Ling Deng Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorZixuan Liu, Zixuan Liu Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorCongwei Zhong, Congwei Zhong Department of Hematology, Tianjin Medical University General Hospital, Tianjin, ChinaSearch for more papers by this authorRong Fu, Corresponding Author Rong Fu [email protected] Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China Correspondence Rong Fu, Department of Hematology, Tianjin Medical University General Hospital, 154 Anshan Street, Tianjin 300052, China. Email: [email protected]Search for more papers by this author First published: 05 December 2022 https://doi.org/10.1002/ctm2.1092 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Dear Editor, In recently, we reported the important contribution of nature killer (NK) cells in the pathogenesis of severe aplastic anaemia (SAA). Our pilot research indicated that NK cell number and function were abnormal in SAA patients,1 and we also identified several proteins associated with NK cell dysfunction by proteomic analysis.2 To further explore the subgroups of NK cells that may be implicated in the pathogenesis of SAA and find the differences between bone marrow NK cells (BM-NK) and peripheral blood NK cells (PB-NK), we conducted single-cell RNA sequencing (scRNA-seq) on BM-NK and PB-NK of SAA patients and healthy volunteers (HC). In keeping with our preceding studies,1 the percentage of BM-NK was higher than that PB-NK in both HC and SAA, and the percentage of PB-NK and BM-NK were both markedly lower in the SAA than in the HC (Figure S1). A total of 9280 cells (6134-13100) from each sample were sequenced, averaging 1876 genes analyzed per cell. After Harmony integration and Scanpy filtering, 97424 cells were included for further analysis, and 20 clusters were obtained from the initial UMAP (Unifrom Manifold Approximation and Projection) and Louvain clustering (Figure S2). We removed the non-NK cell clusters (Table S1) and finally obtained eight groups of NK cells (Figure 1A). These eight groups of NK cells were detected in all samples and passed quality control (Figure S3). We didn't detect NCAM (CD56) in the dataset and used CD7/KLRD1/KLRF1/NGK7/GNLY as specific NK cell markers.3 As expected, all these eight clusters expressed high levels of NK cell-specific markers (Figure 1B). Referring to the previous literature of NK cell scRNA-seq,3-6 combined with the characteristic marker genes of each cluster, we named these eight NK clusters as transitional NK, adaptive NK, CD56bright NK, mature NK, cytokine-induced memory-like (CIML) NK1(normal CIML NK), CIML NK2(active CIML NK), active NK and terminal NK (Table 1, Figure 1C,D, Figure S4). The cell number and the proportion of each NK cell subsets are shown Figure 1E and Table S3. We used RNA rate analysis7 to describe the differentiation trajectory of NK cells (Figure 1F) and found that terminal NK and CIML NK had unique transcriptional characteristics. CIML NK2 maybe derive from CIML NK1, and SCENIC analysis8 also showed that these two clusters had similar regulons (Figure 1G). What's more, the results showed that CD56bright NK was in the early stage of NK cell differentiation, and transitional NK was a mixture of the intermediate process of NK differentiation, which is the transitional stage of CD56bright NK, adaptive NK and active NK to mature NK. FIGURE 1Open in figure viewerPowerPoint Overview of eight types of nature killer (NK) cells. (A) UMAP of 76156 cells from eight types of NK cells (left) and the corresponding lesion type of their donors (right). (B) Violin plot showing the expression level of six markers (NCAM1, CD7, KLRD1, KLRF1, NKG7 and GNLY) in eight types of NK cells. (C) Heatmap displaying the expression level of 63 target genes in eight types of NK cells. (D) Visualizing expression of genes associated with biological pathways. A dot plot showing the log-transformed expression values of marker genes (y-axis) from five subsets (labelled with different colours) across eight types of NK cells (x-axis). (E) Stacked bar plots showing the percentage of different NK cells in four groups, from left to right, AA-BM-NK, AA-PB-NK, HC-BM-NK and HC-PB-NK. (F) RNA velocity unveils the dynamics of differentiation of eight types of NK cells. Terminal NK and cytokine-induced memory-like (CIML) had unique transcriptional characteristics, among which CIML NK2 was transformed from CIML NK1. (G) TFs (transcription factors) were identified by SCENIC across two CIML NK cells. Left: Relative-specific scores (RSS) plot of TFs in two CIML NK cells. Right: Visualizing the TF with the highest RSS in two CIML NK cells on UMAP plot. A high RSS value indicates high correlation between TF and cells. TABLE 1. NK cell population and nomenclature Cluster Name Flow cytometric marker Characteristic marker genes Cluster 0 Transitional NK – TMIGD2, down-regulated marker genes of CD56bright NK, up-regulated genes of CD56dim NK Cluster 1 Adaptive NK CD56dimCD16+CD57- CXCR4, CD3D/3E/3G, KLRC2, CD52, IL32, CCL5, VIM, IGHA1, IGKC, LAG3 Cluster 2 CD56bright NK CD56brightCD16- IL7R,SELL, KLRC1, GZMK, LTB, COLT1, RGS1, XCL1, DUSP1, FOS, JUN, JUNB Cluster 3 Mature NK CD56dimCD16+CD57+ GNLY, NKG7, GZMB, PRF1, FGFBP2, CST7, KLRF1, KLRB1 Cluster 9 CIML NK1 CD56dimCD16+ GZMH, MYOM2, FGFBP2, IL2RB, FCGR3A, KIR2DL1, CD3G, S100 and keratin families Cluster 11 Active NK CD56dimCD16+CD57- CXCR4, CCL4, CCL3, CCL4L2, CCL3L1,CD69, XCL2, JUN, KLRB1, KLRD1, FOS, NR4A2, NFBKIA Cluster 14 Terminal NK CD56dimCD16+CD57+ ZEB2, HAVCR2, CX3CR1, high expression of microtubule- and cytoskeleton-related genes Cluster 16 CIML NK2 CD56dimCD16+ Same as CIML NK1, in addition, highly expressed S100B, SAA1, SAA2, FTH1, HSP90, PTGDS After identifying the subgroup of NK cells, we analyzed the BM-NK and PB-NK transcriptomic changes in HC, identified 417 differentially expressed genes (DEGs) (Figure 2A). Note that 88.97% (371/417) of these DEGs were identified in only one cell types, and most of these genes were the characteristic genes of each cluster. Except for terminal NK cells, the gene expression of NK subsets in BM (bone marrow) and PB (peripheral blood) was basically similar, indicating that the function of BM-NK and PB-NK were consistent, including immunomodulatory and cytotoxic functions (Figure 2B). Terminal NK has active DNA replication (Figure 2C). Interestingly, PB terminal NK cells had a stronger cytotoxic function and cell killing/ NK cell mediated cytotoxicity pathways were up-regulated, while BM terminal NK cells had stronger immune regulatory function, and immune-related pathways were up-regulated (Figure 2D,E). The results indicated that terminal NK cells with different localization have different functional tendencies. FIGURE 2Open in figure viewerPowerPoint Comparison of PB-nature killer (NK) and BM-NK cells in healthy volunteers (HC). (A) Volcano plot showing differential expression genes (DEGs) across NK cells in BM and PB samples collected from HCs. A total of 417 DEGs were identified in HC BM-NK versus HC PB-NK (Wilcoxon rank sum test, absolute log2FC ≥ .5, Bonferroni adjusted p value < .05), including 234 up-regulated genes in BM-NK and 183 up-regulated genes in PB-NK. (B) LogFC bar graph of DEGs identified in NK cells by comparing HC-BM cells with HC-PB cells. Genes with log2FC > .5 indicate that genes are up-regulated in HC-BM and vice versa in HC-PB. (C) Violin plots showing the cell cycle score for each type of NK cells. Left: G2M phase. Right: S phase. (D) Expression level of specific genes in terminal NK cells across BM and PB samples. Left: cytoskeleton and cell cycle-related genes. Middle: cytotoxicity genes. Right: immune-related gene. (E) Functional enrichment analysis of DEGs in terminal NK cells. From left to right, GO (Gene Ontology) enrichment analysis of the up-regulated genes in the PB terminal NK, GO enrichment analysis of the up-regulated genes in the BM terminal NK, KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis of the up-regulated genes in the PB terminal NK and KEGG enrichment analysis of the up-regulated genes in the BM terminal NK. The color of the bubble indicates the adjusted p-value of the GO terms or pathways and the size of the bubble signifies the number of genes associated with a term. Next, we identified 245 DEGs in BM-NK and 126 DEGs in PB-NK between SAA and HC (Figure 3A). Of these DEGs, 44.9% and 47.6% DEGs appeared in multiple subgroups, and 58.73% of DEGs in PB-NK cells were consistent with BM-NK (Figure 3B), suggesting that the same NK subgroup in different position (PB or BM) and different NK subgroups in the same position had partial similar functional changes under pathological conditions, especially in CD56bright NK, transitional NK, adaptive NK and mature NK (Figure S5). We found that, in SAA patients, both BM and PB-NK played a stronger immunomodulatory role, while the cytotoxic function was down-regulated, which may be disease-related functional depletion. What's more, in SAA BM-NK, we detected more up-regulation of immunomodulatory genes and more down-regulation of killing related genes and found several transcription factors and their ligands were up-regulated (Figure 3C,D). Therefore, we believe that BM-NK was more sensitive than PB-NK in the disease state. FIGURE 3Open in figure viewerPowerPoint Comparison of PB-nature killer (NK) and BM-NK cells between severe aplastic anaemia (SAA) and HC. (A) Volcano plot showing differentially expressed genes (DEGs) of BM-NK cells (left) and PB-NK cells (right) between SAA and HC. A total of 245 and 126 DEGs were identified in BM-NK and PB-NK between SAA and HC (Wilcoxon rank sum test, absolute log2FC ≥ .5, Bonferroni adjusted p value < .05), including 141 up-regulated genes and 104 down-regulated genes in BM-NK of SAA patients, 66 up-regulated genes and 60 down-regulated genes in PB-NK of SAA patients. (B) Summary of DEGs between SAA and HC. Left-Middle: Pie charts showing the distribution of differential expression genes (DEGs) isolated from BM (left) and PB (middle) samples across eight types of NK cells. Venn diagram showing the differential expressed genes (DEGs) detected by pair-wise comparison at three groups. (C) Box plots showing expression of the different function genes in SAA and HC. From left to right: immunoregulation-related genes, ribosomal component protein coding genes, cytotoxicity-related genes, transcription factors and their ligands genes. (D) Heatmap displaying the expression level of some DEGs in eight types of BM-NK cells (left) and PB-NK cells (right) based on significance and logFC values. Pair-wise comparisons of each type of NK cell between HC group and AA group in BM and PB samples were peformed to calculate the significance and logFC values of genes. (E) Prioritization of cell types responsive to SAA with Augur. Left: AUC value of NK cells collected from BM. Right: AUC value of NK cells collected from PB. A high AUC value indicates the high sensitivity of the cells responding to changes in the SAA. Both cytokine-induced memory-like (CIML) NK1 and CIML NK2 contributed significantly to disease phenotype in BM and PB (BM: AUC .815 and .810; PB: AUC: .725 and .772). (F) Heat map of CMIL NK cell-specific gene expression profiles in PB-NK and BM-NK of SAA patients/ HCs. (G) The heatmap showing the expression profiles of specific genes between the HC group and AA group in BM and PB samples. (H) The percentage of two CIML NK cells among four groups. (I) BarPlot showing the different percentages of two CIML NK cells collected from SAA patients. Left: BM CIML NK cells. Right: PB CIML NK cells Finally, through Augur9 analysis, we found that CIML NK1 and CIML NK2 were the most significantly cell clusters, which were contributed to disease phenotype in both BM and PB (BM: AUC (Area Under Curve) .815 and .810; PB: AUC: .725 and .772) (Figure 3E). Both in PB and BM, the percentage of CIML NK cells of SAA patients was higher than that in HC, but the ratio of active CIML NK/total CIML NK was decreased (Figure 3F). At the same time, the characteristic genes of BM and PB CIML NK cells in SAA were down-regulated and the pathways were enriched in cytokine stimulation and immune response (Figure 3G). Further analysis showed that among these three SAA patients, those with a higher proportion of CIML NK cells and a higher CIML NK1/CIML NK2 ratio had a better prognosis (Figure 3H, Table S4). So, we speculate that the increase proportion of CIML is a protective response in SAA, while the decrease in proportion and function of active CIML NK is a compensatory-related exhaustion. CIML NK cells may be reliable predictors of SAA treatment outcome. Exploration of the role of CIML NK in the occurrence and development of SAA will further clarify the immune pathogenesis of SAA and provide ideas for new therapeutic methods. In conclusion, scRNA-seq can more accurately reflect the function of NK subsets and enrich our understanding of NK cells. BM-NK and PB-NK cells have similar functions, and BM-NK cells are more sensitive in the disease state. Further investigation is warranted to examine whether CIML NK cells are associated with treatment response. Additional samples and further analysis are needed to validate the results of different NK subsets. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (grant numbers: 81770110, 81970115, 81870101, 81900125 and 81970116), the Scientific Research Program of Tianjin Education Commission (Natural Science) (grant number: 2019KJ199) and Tianjin Key Medical Discipline (Specialty) Construction Project (grant number: TJYXZDXK-028A). CONFLICT OF INTEREST The authors declare that they have no competing interests. Supporting Information Filename Description ctm21092-sup-0001-SuppMat.docx5.6 MB Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Liu C, Li Z, Sheng W, et al. Abnormalities of quantities and functions of natural killer cells in severe aplastic anemia. Immunol Invest. 2014; 43(5): 491- 503. 2Liu H, Zhang T, Chen Y, et al. Proteomics analysis reveals alterations of NK cells in patients with severe aplastic anemia. Int J Lab Hematol. 2020; 42(3): 308- 315. 3Yang C, Siebert JR, Burns R, et al. Heterogeneity of human bone marrow and blood natural killer cells defined by single-cell transcriptome. Nat Commun. 2019; 10(1): 3931. 4Crinier A, Dumas PY, Escalière B, et al. Single-cell profiling reveals the trajectories of natural killer cell differentiation in bone marrow and a stress signature induced by acute myeloid leukemia. Cell Mol Immunol. 2021; 18(5): 1290- 1304. 5Smith SL, Kennedy PR, Stacey KB, et al. Diversity of peripheral blood human NK cells identified by single-cell RNA sequencing. Blood Adv. 2020; 4(7): 1388- 1406. 6Crinier A, Milpied P, Escalière B, et al. High-dimensional single-cell analysis identifies organ-specific signatures and conserved NK cell subsets in humans and mice. Immunity. 2018; 49(5): 971- 986. 7La Manno G, Soldatov R, Zeisel A, et al. RNA velocity of single cells. Nature. 2018; 560(7719): 494- 498. 8Aibar S, González-Blas CB, Moerman T, et al. SCENIC: single-cell regulatory network inference and clustering. Nat Methods. 2017; 14(11): 1083- 1086. 9Skinnider MA, Squair JW, Kathe C, et al. Cell type prioritization in single-cell data. Nat Biotechnol. 2021; 39(1): 30- 34. Volume12, Issue12December 2022e1092 FiguresReferencesRelatedInformation

Introduction：To investigate the expression level of macrophage pyroptosis in patients with severe aplastic anemia (SAA) and its effect on downstream effector T cells, the study explored the mechanism of Toll like receptor 4 (TLR4) inducing macrophage pyroptosis and maintaining immune homeostasis of SAA through pyruvate kinase M2 (PKM2) at cellular and molecular levels, which provided a theoretical basis for the targeted therapy of macrophages in SAA. Three groups of bone marrow macrophages of new diagnosed SAA patients, remission patients and healthy controls were induced and cultured in vitro. qRT-PCR and Western Blot were used to detect pyroptosis-related indicators IL-1β, IL-18, NLRP3, Caspase-1 and GSDMD expression level; ELISA to detect the concentration of IL-1β and IL-18 in the culture supernatant.

OBJECTIVES:Our aim is to investigate the clinical characteristics of low- and intermediate-risk myelodysplastic syndrome (MDS) with pure red cell aplasia (PRCA).METHODS:We retrospectively reviewed the patients of low- and intermediate-risk MDS patients who had been diagnosed with PRCA in our hospital between January 2010 and December 2019.RESULTS:There were 6 low- and intermediate-risk MDS patients with PRCA in our study, 1 male and 5 females, with a median age of 63.5 (50-75) years. It accounted for 7.7% (6/78) of all diagnosed PRCA cases and 1.67% (6/359) of diagnosed MDS cases during the same period. All patients were treated with multiple drugs, including recombinant human erythropoietin, cyclosporine, glucocorticoids, androgen, sirolimus, intravenous immunoglobulin and decitabine. Two patients achieved complete remission, two patients achieved partial remission and became blood transfusion independent. Two patients had no response and one patient died.CONCLUSION:Low- and intermediate-risk MDS with PRCA was difficult to treat, but the prognosis was good.

Introduction Severe aplastic anemia (SAA) is a disease characterized by severe pancytopenia and hematopoietic failure of bone marrow. Natural killer (NK) cells are a class of large granular lymphocytes that perform killing and immunomodulatory functions. Our previous study demonstrated that NK cells played the "protective" role in SAA, which is weakened. However, the mechanism remains unclear. Methods Peripheral blood NK cells from SAA patients and normal controls were sorted and total proteins were extracted. Then, mass spectrometry was performed to screen differentially expressed proteins (DEPs). Results Significant differences in the expression levels of 93 proteins were observed in NK cells of SAA patients compared with normal controls. Among them, 48 were upregulated proteins, including histone H1.2, histone H1.3, heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), and interferon regulatory factor 1 (IRF-1), and 45 were downregulated proteins, including actin-related complex (ARP2/3), histone H3, histone H4, phosphoglycerate kinase 1 (PGK1), talin-1. Gene Ontology (GO) function indicated that the DEPs most involved were vesicle-mediated transport, innate immune response, and DNA binding. KEGG analysis showed 3 upregulated and 12 downregulated pathways, in which cell endocytosis and FC-gamma receptor-mediated phagocytosis were most closely related to NK cell functions. Conclusion Our study is the first analysis of proteomic profile in NK cells in SAA and found many DEPs involving in dysfunction of NK cells, which provides potential targets for deeper research of inadequate immunomodulation.

Severe aplastic anemia (SAA) is a rare disease characterized by severe pancytopenia and bone marrow failure. Most patients with AA respond to immunosuppressive therapy (IST), usually as antithymocyte globulin (ATG) and cyclosporine (CsA), but some relapse on CsA withdrawal or require long-term administration of CsA to maintain blood counts. Recent research has found that rapamycin (Rapa) was an effective therapy in mouse models of immune-mediated bone marrow failure. However, it has not achieved a satisfactory effect in clinical application. At present, many studies have confirmed that eltrombopag (ELT) combined with IST can improve the curative effect of AA patients. Then, whether Rapa combined Elt in the treatment of AA will acquire better efficacy than a single drug application remains unclear. In this study, an immune attack-mediated AA mouse model was constructed by total body irradiation (TBI) and allo-lymphocyte infusion. In our study, we tested the efficacy of Rapa combined with Elt as a new treatment in mouse models of immune-mediated bone marrow failure. It showed that treatment with Rapa in combination Elt in the AA mouse model ameliorated pancytopenia and extended animal survival in a manner comparable to the standard dose of CsA and Rapa alone. However, there was no significant improvement effect on the number and function of NK cells and their subsets, mDCs, and CD4+/CD8+ ratio in AA mice after the therapy of Rapa combined with Elt compared with Rapa alone. Furthermore, the secretion of IL-10 of Tregs in AA mice increased significantly after the therapy of Rapa combined with Elt, but there was no significant difference in the number of Treg cells. We did not observe the difference in the curative effect of the Rapa group and CsA group, but for IL-10/Tregs ratio, the Rapa group was superior to the CsA group. And the IFN-r secretion of CD8+T cells in AA mice decreased significantly after the combination therapy of Rapa and Elt than Rapa alone. Compared with the AA group, the level of plasma IFN-γ, IL-2, and TNF-α decreased significantly (P < 0.05), but IL-10, IL-4, IL-5, and IL-1β increased significantly in the Rapa group (P < 0.05). As for IL-10, IL-12p70, IL-2, IL-6, KC/GRO, and TNF-α, the therapy of Rapa combined with Elt showed a more significant effect than Rapa alone in AA mice. To some extent, this study had shown a relatively better synergistic effect in murine models of immune-mediated bone marrow failure after the combination therapy of Rapa and Elt, which was a promising clinical utility in SAA treatment.

Abstract MDSCs, which are defined as a kind of negatively regulatory cells, could suppress T cell immune response in many tumor-bearing animal models and cancer patients. We supposed that MDSCs also contributed to the impaired antitumor immunity in MDS. Here we demonstrated that STAT3-ARG1 pathway could be a critical signal transduction pathway that regulated MDSCs-mediated immunosuppression. Increased MDSCs was revealed in MDS patients when compared to healthy controls. Especially, MDSCs performed higher activated STAT3 and CCR2 expression in high-risk MDS patients. The CCL2 and IL-6 levels in MDS patients were also higher than in healthy controls, which could drive recruitment and activation of MDSCs. Meanwhile, lower expression levels of CD3ζ chain, perforin and granzyme B were demonstrated in MDS patients, which were associated with downregulated activation of CD8+ T lymphocytes. The results were supported by the decreased perforin, granzyme B and IFN-γ levels in the mixed-culture system of MDSCs and CD8+ T lymphocytes in vitro. Notably, targeting STAT3 pathway by selective inhibitor could decrease ARG1 expression in MDSCs and partially reverse the lower expression levels of effector molecules on CD8+ T lymphocytes. Therefore, this study revealed the potential STAT3-ARG1 mechanism behind MDSCs and provided a promising STAT3 targeting strategy in MDS.

OBJECTIVE:We studied bone marrow plasma (BMP) cytokines in severe aplastic anemia (SAA) patients and healthy volunteers to investigate differences in the cytokine profiles between them and propose a cytokine signature of SAA. METHODS:A Bio-Plex suspension array system was used to measure 27 analytes in BMP samples from 47 SAA patients and 30 healthy donors. RESULTS:Compared to healthy people, SAA patients had higher levels of tumor necrosis factor α (TNF-α (TNF-γ (IFN-γ (IFN-β (MIP-1β (MIP-1α (TNF-α (TNF-β (MIP-1β (MIP-1β (MIP-1γ (IFN-α (TNF. CONCLUSIONS:The current study demonstrated distinct cytokine profiles among untreated SAA patients, recovering SAA (RSAA) patients, and healthy people. The cytokines of RSAA patients showed similar characteristics to those of untreated SAA patients and healthy people, respectively, which may reflect that the immune status of RSAA patients is in different stages of recovery after IST; thus, it may provide an important tool in diagnosing and evaluating or predicting curative effects in clinics.

In this study, we intend to detect the expression of TIM3 on peripheral blood NK cells in SAA patients to reveal the further immune pathogenesis of SAA. Furthermore, we tried to further elucidate the changes of functions of TIM3+ NK and TIM3-NK cells in SAA by measuring the functional molecules and cytotoxic activity of TIM3+ NK and TIM3-NK cells. Finally, we observed the therapeutic effects of TIM3 blocker, TIM3+ NK infusion and TIM3-NK infusion on SAA mice model. 1.The TIM3 expression on NK cells in SAA untreated patients was significantly lower than that in SAA remission patients (P<0.05) and normal controls (P<0.01). 2. TIM3-NK cells expressed higher NKG2D and Granzyme B than TIM3+ NK cells in untreated SAA patients. The expression of NKG2A, CD158a and CD158b on TIM3-NK cells were lower than TIM3+ NK cells. 3. The expression of CD80 and CD86 were significantly decreased after being incubated with TIM3-NK and TIM3+NK cells in SAA, especially mDC+ TIM3-NK group, significantly lower than mDC+TIM3+NK group(P<0.01). 4. The apoptosis rate (AR) of K562 cells were significantly increased after being incubated with TIM3-NK and TIM3+ NK cells in SAA, especially K562+TIM3-NK group, significantly higher than K562+TIM3+NK groups(P<0.01). 5. There was no significant difference in the level of AKT of receptor post-signal pathway protein between TIM3-NK and TIM3+ NK cells in patients with SAA, but the level of P-AKT in TIM3-NK cells is higher than TIM3+ NK cells. 6. AA mice model was established. The TIM3 expression on peripheral blood NK cells in SAA mice was significantly lower than that in TBI mice (P<0.05) and normal controls (P<0.05). TIM3-NK cells expressed higher NKG2D than TIM3+ NK cells (P<0.05). The level of P-AKT and PI3K in TIM3-NK cells is higher than TIM3+NK cells. 7. On the 17th day of model establishment, the weight, hemogram and bone marrow cells count of AA mice were significantly lower than that of NC group (p<0.05).The weight, hemogram and bone marrow cells count of CsA treatment group, TIM3+ NK cell infusion treatment group, TIM3-NK cell infusion treatment group, CsA combined with TIM3-NK cell infusion treatment group, CsA combined with TIM3 blocker treatment group has some improvement, TIM3 blocker alone treatment of AA mice slightly increased, the effect is not significant(P>0.05), and combined with CsA has no significant synergistic effect. The therapeutic effect of TIM3-NK cell infusion group was better than that of TIM3+ NK cell infusion group. The therapeutic effect of CsA combined with TIM3-NK cell infusion group was more significant than that of CsA alone group. TIM3-NK cell infusion therapy may have some synergistic effect with CSA. Conclusions 1. In this study, we found that untreated patients with SAA had lower TIM3 expression on NK cells compared with normal controls, andwere correlated with the severity of pancytopenia of SAA. 2. We further confirmed that the expression of activation molecules on TIM3-NK cells was increased and the killing function was enhanced compared with TIM3+NK cells. In addition, TIM3-NK cells have enhanced inhibition of mDCs and K562 cells and play an immunomodulatory role in SAA. Therefore, TIM3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. Low expression of TIM3 contributes to the enhancement of NK cell function, which in turn inhibits the immune activation state of SAA and improves the disease level. 3. The expression of TIM3 on NK cells of AA mice decreased, and the activity of TIM3-NK cells was stronger than that of TIM3+ NK cells, which was consistent with the decrease of TIM3 on NK cells of SAA patients and the strong activity of TIM3-NK cells. After TIM3-NK cell reinfusion, the general condition, blood cell count and bone marrow cell count of SAA mice were improved, and the combined treatment of CsA was more effective. It may further clarify the immune pathogenesis of SAA and provide a new treatment target to improve the efficacy of SAA treatment. Disclosures No relevant conflicts of interest to declare.

OBJECTIVES:We performed a retrospective analysis to investigate the clinical characteristics and therapeutic strategies of Chinese paroxysmal nocturnal hemoglobinuria (PNH) patients, and assessed the efficacy and safety of glucocorticoid in PNH patients. METHODS:The clinical data of 92 PNH cases in our hospital were analyzed, including clinical manifestation, laboratory examination, treatment efficacy, and survival. RESULTS:The main clinical manifestations of these patients included hemoglobinuria, anemia, fatigue, dyspnea, headache, abdominal pain, and erectile dysfunction. Glucocorticoid is still the first-line treatment for PNH patients to control hemolytic attack, and the short-term remission rate (12 months) is 79.01% (64/81). Meanwhile, the overall survival (OS) of 10 years after diagnosis was estimated as 70.77% (46/65). Moreover, Cox proportional risk model for multivariate analysis showed that the increase in LDH multiple, thrombosis complications, and complicated with bone marrow failure were the independent adverse prognostic factors affecting the survival of PNH patients. CONCLUSION:Paroxysmal nocturnal hemoglobinuria patients in mainland China have various clinical features, while lower incidences of thrombosis and renal damage. Thrombosis and bone marrow failure are two complications with worse prognosis.

The roles of natural killer (NK) cells in shaping the immune system had raised wide interests. Here we intended to explore the regulatory functions of NK cells on CD8+ T cells in severe aplastic anemia (SAA) using human participants and lymphocyte infusion-induced bone marrow failure (BMF) mouse model. In SAA patients, NK cells had over-expressions of NKG2D and NKp46, under-expression of NKG2A and enhanced cytotoxicity. NK cells limited autologous CD8+ T cell immunity in an effector/target ratio manner. The suppression was dependent on the existence of NKG2D. We also observed upregulated MICA expression on activated CD8+ T cells, which were susceptible to NK cell mediated lysis in SAA. Animal model concurred with the data from patients. Infusion of NK cells suppressed the proliferation of CD8+ T cells and decreased IFN-γ production. In conclusion, NK cells served NKG2D-dependent immunoregulatory roles by attenuating autologous CD8+ T cell response in SAA.

Microvesicles (MVs) were involved in the pathogenesis of many diseases, such as cardiovascular diseases and diabetes. Oxidative stress played a key role in the development and progression of diabetic nephropathy (DN). Our aim of the present study was to investigate whether high glucose (HG) could provoke MVs generation from podocytes and its potential mechanism. Mouse podocyte clone 5 (MPC-5) was stimulated by HG. The intracellular reactive oxygen species (ROS) of podocytes were measured by fluorescence microscopy with the probe of CM-H2DCFDA and MitoSOX™. Antioxidants N-Acetyl-l-cysteine (NAC) and α lipoic acid (α-LA) were used to treat podocytes after HG stimulation. The rate of podocyte apoptosis was evaluated with Annexin V-FITC by flow cytometry. NOX4 expression was examined and siRNA were performed to explore the mechanism of MVs generation. The quantities of MVs from MPC-5 cells was significantly increased (P<0.05) by 4.6-times after 30 mM glucose stimulation, accompanied with double increased apoptosis. Cellular ROS generation was increased by HG at the peak of 48 h stimulation. HG-induced MVs were significantly decreased by 52.9% after pretreatment by antioxidant NAC. Nevertheless, mitochondrial ROS in podocytes reached a peak at 4 h stimulation, but specific antioxidant α-LA had no effect on the production of MVs (P>0.05). Levels of NOX4 mRNA and protein expression were significantly up-regulated by HG (P<0.05). Podocyte-derived MVs by HG were eliminated by NOX4 siRNA. HG can provoke MVs generation from glomerular podocytes through ROS/NOX4 pathway, not from mitochondrial pathway.

Severe aplastic anemia (SAA) is characterized by pancytopenia and failure of hematopoietic function and is caused by excessive functioning of cytotoxic T lymphocytes (CTLs). EBNA-1, a nucleoprotein of the Epstein Barr virus (EBV), can influence the proliferation and function of lymphocytes. We therefore tested the number of EBV copies in the CD8+ T cells of 27 patients with SAA and 10 healthy control subjects and observed the influences of EBNA-1 upon the CD8+ T cells of patients with SAA. The results showed that more EBV copies were found in the CD8+ T cells of patients with untreated SAA than in patients with SAA in remission or in the healthy control subjects. Their copy number was positively correlated with the expression of granzyme B and perforin, the secretion level of interferon- γ in CD8+ T cells, and the viability of CD8+ T cells, whereas no correlation was seen between the copy number and the interleukin 4 secretion level or the apoptosis rate. Meanwhile, the expression of granzyme B and perforin was reduced after EBNA-1 gene knockdown, whereas the interferon- γ secretion level and cell viability declined. Therefore, we infer that EBV infection may be a factor in the activation of CTLs and in damaging the bone marrow hematopoietic function of patients with SAA.

Severe aplastic anemia (SAA) is a primary disorder of severe bone marrow failure characterizing with extreme pancytopenia and a profound diminution of bone marrow progenitor cells, which is associated with T cell hyper-function. Abnormal telomere shortening of bone marrow mononuclear cell has been reported in AA, which may lead to genomic instability, and result in cell senescence or apoptosis. Notably, certain studies identfieid that lymphocytes of shortening telomere length have undergone apoptosis escape in autoimmune diseases. In order to investigate the association between telomere lengths and function of T lymphocytes in SAA, the relative telomere lengths (RTLs) of different subtypes of T lymphocytes were investigated by flow-fluorescent in situ hybridization in 30 patients with SAA and 25 healthy controls. Then the levels of expression of cluster of differentiation 28 (CD28), CD158 and CD70 were measured, which represent the function of T lymphocytes. The apoptosis rate and the cell cycle progression of CD8(+)T lymphocytes, and the level of secretion interferon- and tumor necrosis factor- were also measured. Finally, the correlation between telomere length and these functional events of CD8(+)T lymphocytes was analyzed in patients with SAA. The results showed that RTLs of CD8(+)T lymphocytes in SAA were significantly shorter compared with those in controls. Furthermore, in patients with SAA, CD8(+)T lymphocytes are associated with T cell hyper-function, which is related to the RTL. Thus, the shorter RTLs of CD8(+)T lymphocytes in SAA may be associated with hyper-function of these cells, which contribute to the pathogenesis of SAA.