OBJECTIVE:To investigate the changes in function of CD8+ T cell subsets, and explore its significance in pathogenesis of secondary hemophagocytic lymphohistiocytosis (sHLH).METHODS:Flow cytometry was used to detect the expressions of PD-1, TIM-3, and LAG-3, which were the markers of exhausted CD8+ T cell, as well as the secretion levels of interferon γ (IFN-γ) and expression of ΔCD107a after the stimulation; the numbers of effector-memory CD8+ T cells, regulatory T cells and double negative T cells were also detected.RESULTS:The expressions of inhibitory receptors (PD-1, TIM3 and LAG-3) on CD8+ T cells of sHLH patients were 40.73±22.64, 15.97±14.45 and 0.73 (0-37.41), respectively, which were significantly higher than those of the control group (P<0.001). In contrast, the expression of ΔCD107a (4.49±2.71 vs 6.07±2.14, P=0.035) and secretion level of IFN-γ (37.30±24.46 vs 55.17±22.23, P=0.034) were significantly lower. The number of effector-memory CD8+ T cells in sHLH patients was also lower as compared with that in control group (14.35±10.37 vs 22.92±11.12, P=0.016). But there was no significant difference in the number of regulatory T cell and double negative T cell. In addition, the expressions of PD-1, TIM3 and LAG-3 in active stage of sHLH were 38.09±21.87, 14.35±13.70 and 0.82 (0-13.22), respectively, which were significant higher than those in remission stage [24.27±17.23 (P=0.03), 8.64±5.60 (P=0.014) and 0.13 (0-3.69)].CONCLUSION:The exhausted CD8+ T lymphocytes may play a critical role in the development of sHLH.

Background Primary hemophagocytic lymphohistiocytosis (pHLH) is a genetic disorder that is classically diagnosed by genetic testing. Secondary HLH (sHLH) is usually caused by infections, malignancies, or autoimmune disorders, but may display some mutations or polymorphisms. Rapid immunological assays examining natural killer (NK) cell activity, degranulation function (CD107a), and protein expression related to genetic deficiencies have been recommended for early pHLH identification. Methods A retrospective analysis of 311 HLH patients from a Chinese population was performed to evaluate the potential correlations between genetic testing and rapid immunological assays; genotyping characteristics, age of onset, and etiology were examined. Results Among the 128 (128/311) patients who were positive in the genetic screening, the most frequently detected mutant gene was UNC13D (29%), followed by LYST (21%), PRF1 (17%), and STXBP2 (10%). Among pHLH patients ( n = 39), the majority (67%) had PRF1 and UNC13D defects. FHL-2 was predominant (12/27, 44%) in patients aged under 18, while FHL-3 was the most common (6/12, 50%) in adults. Differences in genetic variant types and etiological components were noted in HLH patients based on the age of onset. NK cell activity and CD107a were observed to show a consistent trend ( P trend < 0.001) when grouping patients according to the severity of the genetic variant type. Moreover, NK cell activity was generally consistent within a certain range of ΔCD107a values ( P trend < 0.001). The PPV for bi-allelic degranulation gene mutations in patients with CD107a < 5% was 38.9% (7/18), while the PPV in patients with CD107a ≤10% was 16.7% (13/78). The PPV for pHLH was 41.4% (29/70) with NK cell activity ≤13%. To further evaluate the diagnostic efficacy of NK cell activity assay in pHLH, a receiver operating characteristic (ROC) curve was generated and showed an area under the curve (AUC) of 0.872, and the optimal cutoff value was determined to be 13.425% with a sensitivity of 84.21% and specificity of 80.67% when the corresponding Youden index was maximized. Flow cytometry screening for deficient proteins, including perforin, SAP, and XIAP, showed a relatively high sensitivity (83.33–93.33%). The positive predictive values (PPVs) of perforin and XIAP were relatively low (20.83–26.92%), but the negative predictive values (NPVs) for all three were excellent (all > 98%). Conclusions Various immunological indicators have different clinical prediction and application values for the diagnosis of pHLH. The degree of reduction of immunological indicators also needs attention, and choosing appropriate cutoff value may be of important significance in guiding clinical judgment for pHLH.

OBJECTIVE:To establish a secondary hemophagocytic lymphohistiocytosis(HLH) mouse model, and to investigate the effect of ruxolitinib on the disease manifestation of model mice.METHODS:Wild type C57BL/6 mice were randomly divided into 4 groups: two groups of mice were intraperitoneally injected with CpG oligodeoxynucleotide 1826 (CpG-ODN1826) every other day to induce HLH, and other two groups were control groups. One group of the CpG-ODN1826 groups and one of the control groups were given ruxolitinib, and other two groups were given the same amount of PBS. Blood samples, serum ferritin and hepatic/spleen weights of experimental mice were detected and serum cytokine levels were measured by ELISA.RESULTS:Compared with the control groups, the levels of white blood cells, hemoglobin and platelets in the CpG-ODN1826 groups were significantly lower (P＜0.05); and liver/body weight, spleen/body weight, serum ferritin, sCD25, IL-10, IL-1β, IFN-Ƴ, IL-12p70, GM-CSF, TNF-α and IL-18 levels significantly increased (P＜0.05). There was no significant difference in the levels of IL-2, IL-4, IL-5, IL-6, IL-22, IL-13, IL-27 and IL-23 between the two groups (P＞0.05). The spleen in CpG group had disordered internal structure, expanding red pulp and hyperplastic nucleated cells. The liver had severe perivascular inflammations. The spleen/weight of the ruxolitinib-treated mice in the CpG-ODN1826 group was significantly smaller than that of the unapplied ruxolitinib (P＜0.05).CONCLUSION:The CpG-ODN1826 can induce secondary HLH symptoms in wild type C57BL/6 mice. Ruxolitinib can alleviate the symptoms of splenomegaly in HLH model mice.

XLP-2 is known as a rare primary immunodeficiency disease, which is characterized by the susceptibility to EBV infection and potential development into the pHLH. The existing studies believe that the dysfunction of XIAP represents one of the most significant pathogenic mechanisms of XLP-2, and allo-HSCT is regarded as a crucial treatment for the long-term survival in XLP-2 patients. In our present study, a 2-year-old male patient was diagnosed with XLP-2. After receiving chemotherapy by using HLH-2004 without allo-HSCT, he reached a complete remission, and his EBV load was brought under control. Our family survey revealed a novel frameshift mutation in the XIAP gene in this patient, as well as in his cousin and grandfather. Until now, the patient has been followed up for 22 months with no recurrence reported yet. Based on these findings, it is believed that for child pHLH patients with XLP-2, the treatment by controlling symptoms alone without allo-HSCT and with regular monitoring of EBV load could be conducive to a long-term survival.

Hemophagocytic lymphohistiocytosis (HLH) includes primary HLH (pHLH) and secondary HLH (sHLH). Mutations that cause abnormal functions in natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) are frequently identified in pHLH. However, why NK cells and CTLs exhibit abnormal functions in sHLH remains unclear. Here, we demonstrated that NK cells in sHLH exhibited a high expression of inhibitory receptor NKG2A and a low expression of activating receptor NKG2D. Besides, the expression of HLA-E on lymphocyte, the adaptor of NKG2A on NK cells, was elevated in sHLH. Moreover, CTLs in sHLH patients expressed a higher level of functional exhaustion markers PD-1, TIM-3 and LAG-3 as well as a lower secretion of IFN-γ and CD107a upon stimulation. In addition, the expression of MHC-I on lymphocytes was decreased. Taken together, our study indicates a potentially pathological mechanism of sHLH and may open up new avenues for the development of immunotherapies against sHLH.

The differentiation of primary hemophagocytic lymphohistiocytosis (pHLH) and macrophage activation syndrome (MAS) poses a challenge to hematologists. The aim of this study was (1) to compare the levels of soluble ST2 (sST2), sCD163, IFN-γ, IL-10, IL-18, TNF-α and Serum soluble interleukin-2 receptor (sCD25) in patients with pHLH and MAS and (2) to investigate whether they can help differentiate the two diseases. A total of 52 participants were recruited in this study, including 12 pHLH patients, 20 MAS patients, and 20 healthy subjects. We measured the levels of sST2, sCD163 and sCD25 in serum by ELISA. The serum levels of IFN-γ, IL-10, IL-18, and TNF-α were detected using a Luminex 200 instrument. The serum levels of sST2 and sCD163 in MAS patients were markedly higher than that in pHLH patients (363.13 ± 307.24 ng/ml vs 80.75 ± 87.04 ng/ml, P = 0.004; 3532.72 ± 2479.68 ng/ml vs 1731.96 ± 1262.07 ng/ml, P = 0.046). There was no significant difference in the expression of IFN-γ (306.89 ± 281.60 pg/ml vs 562.43 ± 399.86 pg/ml), IL-10 (20.40 ± 30.49 pg/ml vs 8.3 ± 13.14 pg/ml), IL-18 (463.33 ± 597.04 pg/ml vs 1247.82 ± 1318.58 pg/ml), TNF-α (61.48 ± 84.69 pg/ml vs 106.10 ± 77.21 pg/ml), and sCD25 (21062.1 ± 18515.26 pg/ml vs 11074.78 ± 11149.96 pg/ml) between pHLH and MAS. Patients with pHLH and MAS show some differences in cytokine profiles. The elevated levels of IFN-γ, IL-10, TNF-α, IL-18, and sCD25 can contribute to the diagnosis of HLH, but may not discriminate pHLH from MAS. Levels of sST2 and sCD163 may serve as markers to distinguish pHLH from MAS.

Background: This study investigated the clinical characteristics of primary hemophagocytic lymphohistiocytosis (HLH) in adults, including immunological markers, pedigree findings, and conditions of allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Methods: The study included clinical data of 18 adult patients with primary HLH treated in our center from June 2010 to January 2017. Results: Of these 18 cases, pathogenic variants were found in the following genes: PRF1 (n = 11), UNC13D (n = 5), SH2D1A (n = 2), RAB27a (n = 1), and LYST (n = 2). One patient had pathogenic variants in both PRF1 and UNC13D genes, one patient had pathogenic variants in both LYST and UNC13D genes and another patient had pathogenic variants in both PRF1 and SH2D1A genes. Additionally, 3 of the 18 cases involved homozygous pathogenic variants, while 2 cases involved hemizygous pathogenic variants. The remaining 13 cases involved compound heterozygous pathogenic variants. The natural killer (NK) cell activity test was conducted in all 18 cases where 14(77.8%) patients showed reduction in NK cell activity. Furthermore, this article presents 3 representative results of the pedigree findings from 12 patients who underwent family surveys. The 8 patients who underwent Allo-HSCT had a median survival of 27.2 months, as compared with the median survival of 7 months for the 10 patients who did not undergo Allo-HSCT, a significant difference between the two groups of patients (p = 0.006). Conclusion: PRF1 was one of the most commonly mutated gene in adult patients with primary HLH. Family surveys and immunological markers were important for the HLH diagnosis and the selection of an appropriate donor. Allo-HSCT was an effective therapy for adult primary HLH.

EB病毒(Epstein-Barr virus,EBV)属于4型疱疹病毒,除与传染性单核细胞增多症(infectious mononucleosis,IM)、EBV相关淋巴组织增殖性疾病(Epstein-Barr virus-related lymphoproliferative disorders,EBV-LPD)、Burkitt淋巴瘤和鼻咽癌等多种疾病密切相关外,还与慢性活动性EBV感染(chronic active EBV infection syndrome,CAEBV)、EBV相关噬血细胞性淋巴组织细胞增多症(Epstein-Barr virus-related haemophagocytic lymphohistiocytosis,EBV-HLH)和EBV相关T淋巴细胞淋巴瘤(Epstein-Barr virus genome-positive T-cell lymphoma)、NK细胞白血病/淋巴瘤、霍奇金淋巴瘤等疾病有关[1].此外,有研究表明,EBV DNA<1×105拷贝/mL的患者和EBV DNA>1×105拷贝/mL的患者相比,高拷贝数的患者生存时间显著缩短.有研究显示,EBV DNA水平的下降与患者预后密切相关[2].而儿童EBV感染具有自限性,部分EBV DNA阳性的儿童在不治疗的情况下可以转阴,定量检测EBV DNA对儿童患者也不可缺少.因此,EBV检测对于EBV感染相关疾病的诊断、监测和不良预后的控制具有重要意义.

OBJECTIVES:Encouraging progress has been made in application of splenectomy in the treatment of relapsed hemophagocytic lymphohistiocytosis (HLH) of unknown cause. The aim was to determine the roles of lymphocyte subpopulations and inflammatory cytokines in splenectomy. METHODS:We retrospectively analyzed changes in lymphocyte subpopulations and levels of inflammatory cytokines at different time-points before and after splenectomy in the patients with relapsed HLH of unknown cause, as well as the correlations between these changes and the disease prognosis. RESULTS:During the period from June 2006 to June 2016, we enrolled 107 patients with relapsed HLH of unknown cause, of whom 29 were treated with splenectomy. Among the 29 patients, 7 cases were non-Hodgkin lymphomas based on spleen pathology, 1 case withdrew and the remaining 21 non-lymphoma cases were available for analysis. Results showed a significant increase in both percentage of CD16+CD56+ NK cells (P = 0.003) and NK cell activity (P = 0.028) at 24 wk after splenectomy compared to their baseline pre-surgery levels. We also examined seven patients for the changes in cytokine levels before and after splenectomy and found that IL-21 and IL-1α decreased at 4 wk after splenectomy (P < 0.05). Seven non-lymphoma patients determined as no response to treatment (NR) prior to splenectomy had significantly longer survival (P = 0.001) compared to the 24 patients with relapsed HLH of unknown cause who were also determined as NR but not treated by splenectomy. DISCUSSION:Splenectomy can improve clinical symptoms and survival of patients with relapsed HLH of unknown cause. The mechanism is likely related to the changes in percent NK cells and cytokines (IL-21 and IL-1α) after surgery.

Low or absent natural killer (NK) cell activity is included as one of the HLH-2004 diagnostic criteria. To improve the diagnosis of HLH, we aimed to establish a rapid and reliable NK cell activity assay that avoids the use of radioactivity. The K562 cell line, as standard NK target cells, was engineered to stably express enhanced green fluorescent protein (EGFP), which can be quantified by flow cytometry. The EGFP-flow cytometry method for measuring NK cell activity was improved by double staining of early and late apoptotic target cells. Whole-blood samples from healthy volunteers were assessed with this method, which demonstrated that optimal conditions were effector–target ratio of 10:1 and incubation time of 4 h. This method was further evaluated for samples from 113 HLH patients and 64 healthy volunteers. Mean NK cell activity in either primary or secondary HLH patients was significantly lower (P < 0.001) than in healthy individuals (20.23 ± 4.12%). Furthermore, primary HLH patients (10.76 ± 2.54%) exhibited even lower (P < 0.001) NK cell activity compared with secondary HLH patients (15.01 ± 3.62%). We have optimized and implemented this method in clinically relevant samples.

OBJECTIVE:To investigate the significance of pedigree genetic screening and rapid immunological parameters in the diagnosis of primary hemophagocytic lymphohistiocytosis (HLH).METHODS:Four cases of primary HLH patients with PRF1, UNC13D and SH2D1A gene mutations were conducted pedigree investigation, including family genetic screening and detections of immunological parameters (NK cell activity, CD107a degranulation and expression of HLH related defective protein), to evaluate the significance of these different indicators in the diagnosis of primary HLH and explore their correlations.RESULTS:The DNA mutations of the four families included missense mutation c.T172C (p.S58P) and non- frameshift deletions c.1083_1094del (p.361_365del), missense mutation c.C1349T (p.T450M) and frameshift mutation c.1090_1091delCT (p.T364fsX93) in PRF1 gene, missense mutation c.G2588A (p.G863D) in UNC13D gene and hemizygous mutation c.32T>G (p.I11S) in SH2D1A gene. The patients and their family members presented decreased NK cell activities. Individuals who carried mutations of PRF1 gene and SH2D1A gene showed low expression of perforin (PRF1) and signaling lymphocytic activation molecule associated protein (SAP). And the patient with UNC13D gene mutation and his family member with identical mutation showed significant reducing cytotoxic degranulation function (expression of CD107a).CONCLUSION:Pedigree genetic screening and rapid detection of immunological parameters might play an important role in the diagnosis of primary HLH, and both of them had good consistency. As an efficient detection means, the rapid immunological detection indicators would provide reliable basis for the early diagnosis of the primary HLH.

目的:探讨原发性噬血细胞综合征(HLH)合并中枢神经系统病变诊断要点以及异基因造血干细胞移植(Allo-HSCT)治疗情况.方法:对1例原发性HLH合并中枢神经系统病变患者的临床特点进行分析,完善基因测序、免疫学指标检测和家系调查,进行Allo-HSCT.结果:11岁男性病例,表现为反复发热、全血细胞减少,脾大、骨髓中可见噬血现象,自然杀伤(NK)细胞活性下降(10.39％) 基因检测和家系调查显示患者携带分别来自父系和母系的PRF1基因的复杂杂合改变,两位胞姐各自携带不同突变位点;全家成员穿孔素蛋白表达量均有不同程度下降.病程中出现癫痫,头颅核磁共振提示多发病变.确诊原发性HLH合并中枢神经系统病变.给予HLH-2004方案治疗后,接受胞姐HLA 5/10相合Allo-HSCT.目前移植后14个月,一般情况良好.结论:对于合并中枢神经系统病变的原发性HLH,尽早进行Allo-HSCT是获得长期生存及治愈的唯一方法

目的 探究自体造血干细胞移植(AHSCT)治疗复发难治性朗格汉斯组织细胞增多症(LCH)的疗效。 方法 报道1例AHSCT治疗复发难治性LCH患者的治疗经过，并复习相关文献。 结果 患者经过多次化疗后疗效欠佳，进行AHSCT治疗后完全缓解。 结论 AHSCT治疗可作为复发难治性LCH的有效治疗方案。

It is a challenge in diagnosis of hemophagocytic lymphohistocytosis (HLH).Rapid diagnosis is the key to make decisions in clinical management of HLH.The timely and reliable diagnosis of HLH can be assisted by laboratory tests including nature killer (NK) cell activity, sCD25, hemophagocytosis, ferritin, surface CD107a, and genetic testing.Here is introduced lab assays and evaluate the clinical applications in HLH.The aim is to raise awareness of the values of laboratory evaluations in diagnosing HLH.