Application of Flow Cytometry Combined Fluorescence in Situ Hybridization to Indentify the Lymphocyte Subtypies with Epstein-Barr Virus Infection / 中国实验血液学杂志

OBJECTIVE@#To establish the technique that take the advantages of flow cytometry combined fluorescence in situ hybridization (Flow-FISH) to identify the Epstein-Barr virus(EBV) infected lymphocyte subtypies in patients' peripheral blood sample.@*METHODS@#Peripheral Blood monocyte from 9 patients with EBV infection enrolled at Children's Hospital in Chongqing Medical University were isolated by Ficoll-paque centrifugal separation. The expressions of EBER1, EBER2 in cell were detected by qRT-PCR. The surface markers of cell were detected by Flow cytometry after staining with their antibodies. The cell was treated Fix-Permeabilization Buffer before hybridization with fluorescent labeled probe at 37 ℃ overnight. The cell status, surface markers and targeted mRNA are detected by flow cytometry and fluorescence microscope.@*RESULTS@#It was optimized that the Fix-Permeabilization Buffer and recipe with 0.2% Tween-20 were picked out as providing a good cell integrity and high resolution of surface markers. Hybridization with 20% formamide and 7% dextran sulfate at 37 ℃ overnight is the optimal hybridization condition as a good hybridization effect, a detectable cell integrity and a high resolution of cell markers under flow cytometry detection. Finally, upon the established Flow-FISH method, lymphocyte subpopulations of the EBV+ cells from cell lines and blood samples of patients were identified successfully.@*CONCLUSION@#A Flow-FISH technology is established, which can be applied in the identification of EBV infected cell subtypes. This research provides a foundmental for its application in clinical test in EBV+ related proliferative diseases.

Application of lymphocytes test in peripheral blood of patients with systemic sclerosis during the treatment / 北京大学学报(医学版)

OBJECTIVE@#To explore the significance of lymphocytes in systemic sclerosis (SSc), by detecting the levels of T lymphocytes, B lymphocytes and natural killer (NK) cells, and analyzing the correlation between the lymphocytes and clinical laboratory indexes.@*METHODS@#The numbers and proportion of T, CD4+T, CD8+T, B, and NK cells were detected by flow cytometry in peripheral blood of 32 SSc patients who had taken immunosuppressive drugs and 30 healthy controls (HC). The comparison of the lymphocyte subsets in SSc with them in the HC groups, and the correlation between the lymphocytes and other clinical and laboratory indicators were analyzed by the relevant statistical analysis.@*RESULTS@#Compared with the HC group, the numbers of T, CD4+T, CD8+T, and NK cells in peripheral blood of SSc group, who had taken immunosuppressive drugs, were significantly decreased (P < 0.05). More-over, the proportion of NK cells in peripheral blood of the SSc group was also significantly lower than that in the HC group (P=0.004). In addition, all the lymphocyte subsets were decreased in peripheral blood of more than 65% of the SSc patients who had taken immunosuppressive drugs. Compared with CD4+T normal group, the positivity of Raynaud's phenomenon, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) was significantly increased in CD4+T reduction group, respectively (P＝0.024, P < 0.001, P=0.018). ESR was higher in CD8+T reduction group than CD8+T normal group (P＝0.022). The prevalence of fingertip ulcer was significantly increased in B cell decrease group (P=0.019). Compared with NK cell normal group, the prevalence of fingertip ulcer was significantly increased in NK cell lower group (P=0.033), IgM was remarkablely decreased yet (P=0.049). The correlation analysis showed that ESR was negatively correlated with the counts of T lymphocytes (r＝－0.455, P＝0.009), CD4+T lymphocytes (r＝－0.416, P＝0.018), CD8+T lymphocytes (r＝－0.430, P=0.014), B cells (r＝－0.366, P＝0.039).@*CONCLUSION@#The number of CD4+T, CD8+T, B, and NK cells significantly decreased in peripheral blood of SSc patients who had used immunosuppressive drugs, some lymphocyte subsets might be related with Raynaud's phenomenon and fingertip ulcer, and reflected the disease activity by negatively correlated with ESR and CRP; the numbers of lymphocyte subsets in peripheral blood should be detected regularly in SSc patients who had taken immunosuppressive drugs.

Relationship between Changes of Lymphocyte Subsets and Early Hematologic Response in Immunosuppressive Therapy of Severe Aplastic Anemia Patients / 中国实验血液学杂志

OBJECTIVE@#To explore the relationship between the change of lymphocyte subsets before and after immunosuppressive therapy (IST) with disease severity of severe aplastic anemia (SAA) and hematologic response to IST.@*METHODS@#The clinical data of 94 patients with SAA/VSAA treated by r-ATG and CsA in our hospital from December 2009 to October 2011 was analyzed retrospectively. Among them, 26 patients who had sequential data of lymphocyte subsets and cytokines before and after treatment were enrolled. The relationship between lymphocyte subsets, cytokine level before IST and disease severity, as well as the relationship between changes if lymphocyte subsets, changes of cytokine and the HR after IST for 6 months was analyzed.@*RESULTS@#There were no statistical differences in the ratio and absolute count of lymphocyte, the ratio and absolute count of each lymphocyte subsets, including CD3@*CONCLUSION@#The hematopoietic recovery and early hematologic remission may be affected by the intensity of immune suppression reflected from the changes of lymphocyte subsets and the immune reconstruction reflected from the recovery of lymphocyte subsets. The immune reconstruction is most significant within 3 months after IST.

The Expression of CD40/CD40L in MM Patients and Its Significance to the Prognosis of MM Patients / 中国实验血液学杂志

OBJECTIVE@#To explore the expression of CD40/CD40L in multiple myeloma(MM) patients and its influence on prognosis.@*METHODS@#Thirty patients with MM treated in Cangzhou People's Hospital from May 2016 to June 2017 were selected and divided into MM group, then 30 healthy people with a physical examination in our hospital at the same time were selected as the normal group. The serum CD40/CD40L levels of the patients in the two groups was detected by flow cytometry, and its correlation with the lymphocyte population, pathological grade and prognostic significance of MM patients was anaysis.@*RESULTS@#The expression of CD40 in serum of the patients in MM group was significantly higher than those in normal group (P0.05). The levels of CD40 and CD40L in the patients before and after chemotherapy showed no difference(P>0.05). The levels of Ts and NK cells in the patients of MM group were lower than those in normal group (P0.05). The CD40 level was correlated with the serum total B lymphocyte level of the patients in MM group (r=0.877, P=0.005). There was a correlation with CD40L and Th cells in the serum of MM patients (r=-0.783, P=0.035). The expression of serum CD40 in the patients at phase III-IV was higher than those of the patients at phase I-II, the levels of serum CD40L in MM patients at different periods showed no significant difference(P>0.05). The survival rate of MM patients with high CD40 expression was lower than that of MM patients with low CD40 expression (χ@*CONCLUSION@#The increasing of CD40 level in MM patients is related to the pathological grade of the patients. Chemotherapy can reduce the level of CD40. The increasing of CD40 is an important factor for the poor prognosis of MM patients. CD40L level is not meaningful for MM treatment and prognosis.

Impact of post-thoracotomy analgesia with dexmedetomidine and morphine on immunocytes: a randomized clinical trial / Impacto da analgesia pós-toracotomia com dexmedetomidina e morfina em imunócitos: estudo randomizado

Abstract Objective This study aimed to investigate the impact of post-thoracotomy analgesia with dexmedetomidine and morphine on immunocytes. Methods A total of 118 patients with post-thoracotomy Patient-Controlled Intravenous Analgesia (PCIA) in our hospital from March 2016 to July 2018 were randomly selected and divided into the Composite (COM) Group (57 patients administered with dexmedetomidine [1.0 µg.kg-1 body weight] and morphine [0.48 mg.kg-1 body weight]) and the Morphine (MOR) group (61 patients administered with morphine [0.48 mg.kg-1]). The values of lymphocyte subsets (CD3+, CD4+, and CD8+) and Natural Killer cells in the peripheral blood of these two groups were detected by FACSCalibur flow cytometry at different time points (before anesthesia induction [T0], immediately after tracheal extubation [T1], 12 hours after surgery [T2], 24 hours after surgery [T3], 48 hours after surgery [T4], 72 hours after surgery [T5], and 7 days after surgery [T6]). The doses of morphine at T3 to T5 and the adverse reactions between the two groups were also recorded and compared. Results The CD3+ level and the CD4+/CD8+ ratio at T2 to T5 and the CD4+ level and NK cells at T3 to T5 were significantly higher in the COM Group than in the MOR Group (p< 0.05). The postoperative morphine dose and the incidence of postoperative itching, nausea, and vomiting were significantly lower in the COM Group than in the MOR Group (p< 0.05). Conclusions Dexmedetomidine combined with morphine for post-thoracotomy PCIA can improve the function of immunocytes, reduce morphine consumption, and reduce the adverse reactions during analgesia induction.

Resumo Objetivo Estudar o impacto em linfócitos causado pelo uso da dexmedetomidina associada à morfina para analgesia pós-toracotomia. Método Um total de 118 pacientes utilizando Analgesia Intravenosa Controlada pelo Paciente (AICP) pós-toracotomia em nosso hospital, de março de 2016 a julho de 2018, foram selecionados aleatoriamente e divididos em dois grupos: o Grupo Combinado [COM, 57 pacientes que receberam dexmedetomidina (1,0 µg.kg-1 de peso corpóreo) associada à morfina (0,48 mg.kg-1 de peso corpóreo)] e o Grupo Morfina [MOR, 61 pacientes, que receberam somente morfina (0,48 mg.kg-)]. Os valores dos subconjuntos de linfócitos (CD3+, CD4+ e CD8+) e das células NK no sangue periférico desses dois grupos foram medidos por citometria de fluxo FACSCalibur em diferentes momentos do estudo [antes da indução anestésica (T0), imediatamente após extubação traqueal (T1), 12 horas após a cirurgia (T2), 24 horas após a cirurgia (T3), 48 horas após a cirurgia (T4), 72 horas após a cirurgia (T5) e 7 dias após a cirurgia (T6)]. As doses de morfina do momento T3 ao T5 e as reações adversas entre os dois grupos também foram registradas e comparadas. Resultados O nível de CD3+ e a razão CD4+/CD8+ de T2 a T5, e o nível de CD4+ e as células NK de T3 a T5 do Grupo COM foram significantemente maiores (p< 0,05) quando comparados ao Grupo MOR. A dose de morfina no pós-operatório e a incidência de prurido, náusea e vômito no pós-operatório foram significantemente menores no grupo MOR (p< 0,05). Conclusões Dexmedetomidina combinada com morfina para AICP no período pós-toracotomia pode melhorar a função dos linfócitos, reduzir o consumo de morfina e diminuir reações adversas durante a analgesia.

Increased platelet distribution width and reduced IL-2 and IL-12 are associated with thrombocytopenia in Plasmodium vivax malaria

BACKGROUND Thrombocytopenia in malaria involves platelet destruction and consumption; however, the cellular response underlying this phenomenon has still not been elucidated. OBJECTIVE To find associations between platelet indices and unbalanced Th1/Th2/Th17 cytokines as a response to thrombocytopenia in Plasmodium vivax infected (Pv-MAL) patients. METHODS Platelet counts and quantification of Th1/Th2/Th17 cytokine levels were compared in 77 patients with uncomplicated P. vivax malaria and 37 healthy donors from the same area (endemic control group - ENCG). FINDINGS Thrombocytopenia was the main manifestation in 55 patients, but was not associated with parasitaemia. The Pv-MAL patients showed increases in the mean platelet volume (MPV), which may be consistent with larger or megaplatelets. Contrary to the findings regarding the endemic control group, MPV and platelet distribution width (PDW) did not show an inverse correlation, due the increase in the heterogeneity of platelet width. In addition, the Pv-MAL patients presented increased IL-1β and reduced IL-12p70 and IL-2 serum concentrations. Furthermore, the reduction of these cytokines was associated with PDW values. MAIN CONCLUSIONS Our data demonstrate that an increase in MPV and the association between reductions of IL-2 and IL-12 and PDW values may be an immune response to thrombocytopenia in uncomplicated P. vivax malaria.

Clinical Impact of Myeloid-Derived Suppressor Cells, Lymphocyte Subsets, and Neutrophil-to-Lymphocyte Ratio in Patients with Colorectal Cancer

Flow Cytometry for the Diagnosis of Primary Immunodeficiency Diseases: A Single Center Experience

PURPOSE: While there is an urgent need for diagnosis and therapeutic intervention in patients with primary immunodeficiency diseases (PIDs), current genetic tests have drawbacks. We retrospectively reviewed the usefulness of flow cytometry (FCM) as a quick tool for immunophenotyping and functional assays in patients suspected to have PIDs at a single tertiary care institute.METHODS: Between January 2001 and June 2018, patients suspected of having PIDs were subjected to FCM tests, including lymphocyte subset analysis, detection of surface- or intracellular-target proteins, and functional analysis of immune cells, at Samsung Medical Center, Seoul, Korea. The genetic diagnosis was performed using Sanger or diagnostic exome sequencing.RESULTS: Of 60 patients diagnosed with definite or probable PID according to the European Society of Immune Deficiencies criteria, 24 patients were provided with useful information about immunological dysfunction after initial FCM testing. In 10 patients, the PID diagnosis was based on abnormal findings in FCM testing without genetic tests. The FCM findings provided strong evidence for the diagnosis of severe combined immunodeficiency (n = 6), X-linked chronic granulomatous diseases (CGD) (n = 6), leukocyte adhesion deficiency type 1 (n = 3), X-linked agammaglobulinemia (n = 11), autoimmune lymphoproliferative syndrome-FASLG (n = 1), and familial hemophagocytic lymphohistiocytosis type 2 (n = 1), and probable evidence for autosomal recessive-CGD (n = 2), autosomal dominant-hyper-immunoglobulin E (IgE)-syndrome (n = 1), and STAT1 gain-of-function mutation (n = 1). In PIDs derived from PIK3CD (n = 2), LRBA (n = 2), and CTLA4 mutations (n = 3), the FCM test provided useful evidence of immune abnormalities and a tool for treatment monitoring.CONCLUSIONS: The initial application of FCM, particularly with known protein targets on immune cells, would facilitate the timely diagnosis of PIDs and thus would support clinical decisions and improve the clinical outcome.

Analysis of the correlation between lymphocyte subsets and severity of corona virus disease 19 / 北京大学学报(医学版)

OBJECTIVE@#To understand the differences in lymphocyte subsets in patients with different clinical classifications of corona virus disease 19 (COVID-19).@*METHODS@#Eighty-one patients with COVID-19 who were admitted to the isolation ward under the responsibility of three medical aid teams in the Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from February 8, 2020 to March 28, 2020, were selected to collect clinical data. According to the relevant diagnostic criteria, the disease status of the patients was classified into moderate cases (n=35), severe cases (n=39) and critical cases (n=7) when lymphocyte subset testing was performed. Their blood routine tests, lymphocyte subsets and other indicators were tested to compare whether there were differences in each indicator between the patients of different clinical classification groups.@*RESULTS@#The differences in the absolute count of total lymphocytes, T-lymphocytes, CD4+T-lymphocytes, CD8+T-lymphocytes and natural killer (NK) cells among the three groups of patients were all statistically significant (P < 0.05), and the critical cases were significantly lower than the moderate and severe cases in the above indicators, and the indicators showed a decreasing trend with the severity of the disease. In 22 patients, the six indicators of the absolute count of T-lymphocytes, B-lymphocytes, CD4+T-lymphocytes, CD8+T-lymphocytes and NK cells, CD4+/CD8+ ratio were all within the normal reference range in the first test, and 59 patients had abnormalities of the above indicators, with the absolute count of NK cells and CD8+ T lymphocytes decreasing most frequently (61%, 56%). The patients with the absolute count of NK cells and CD8+ T lymphocytes below the normal reference range were one group, and the remaining abnormal patients were the other group. There were more critical cases in the former group (moderate : severe : critical cases were 4 : 8 : 7 vs. 19 : 21 : 0, respectively, P=0.001), and all the deaths were in this group (6 cases vs. 0 case, P=0.001). The absolute B lymphocyte count was below the normal reference range in 15 patients, and the remaining 64 cases were within the normal range. The ratio of moderate, severe and critical cases in the reduced group was 4 : 7 : 4, and the ratio of critical cases was more in normal group which was 30 : 31 : 3, and the difference between the two groups was statistically significant (P=0.043).@*CONCLUSION@#The more critical the clinical subtype of patients with COVID-19, the lower the absolute count of each subset of lymphocytes.

New perspectives on traumatic bone infections / 中华创伤杂志(英文版)

In this paper, we review the results of previous studies and summarize the effects of various factors on the regulation of bone metabolism in traumatic bone infections. Infection-related bone destruction incorporates pathogens and iatrogenic factors in the process of bone resorption dominated by the skeletal and immune systems. The development of bone immunology has established a bridge of communication between the skeletal system and the immune system. Exploring the effects of pathogens, skeletal systems, immune systems, and antibacterials on bone repair in infectious conditions can help improve the treatment of these diseases.

Evaluation of lymphocyte apoptosis in patients with oral cancer

Abstract Objectives To evaluate apoptotic levels of peripheral blood mononuclear cells (PBMCs) and apoptotic regulatory proteins (Bax and Bcl-2) in lymphocyte subsets of oral cancer (OC) patients and healthy controls (HC). Methodology The percentage of apoptotic cells and lymphocyte counts were measured in the first cohort using PBMCs obtained from 23 OC patients and 6 HC. In the second cohort, (OC, 33; HC, 13), the mean fluorescence intensity (MFI) of Bax and Bcl-2 in CD19+ B, CD4+ T, CD8+ T, and CD16+56+ natural killer (NK) cells was determined via flow cytometry. Results The percentage of apoptotic cells was higher in the PBMCs of OC patients than in HC patients, particularly in patients with stage IV cancer (p<0.05). However, lymphocyte counts were significantly lower in stage IV patients (p<0.05). NK CD19+ B and CD16+56+ cell counts were significantly lower in OC patients compared with HC patients (p<0.001 and p<0.01, respectively), but CD4+ T cells were interestingly significantly higher in OC patients (p<0.001). While Bax MFI was slightly higher, Bcl-2 MFI was significantly lower for all four lymphocyte subsets in OC samples, particularly in stage IV patients, when compared with HC. Consequently, Bax/Bcl-2 ratios showed an upward trend from HC to OC patients, particularly those in stage IV. We found similar trends in Bax and Bcl-2 MFI for tumor stage, tumor size, and lymph node involvement. Conclusions The increased lymphocyte apoptosis in stage IV OC patients may be related to higher Bax levels and lower Bcl-2 levels. The Bax/Bcl-2 ratio in lymphocytes may be useful to determine the prognosis of OC patients, and could be considered a mean for supportive treatment in the future.

Distribution of peripheral blood lymphocyte subsets in 826 healthy children aged 0-6 years / 中国当代儿科杂志

OBJECTIVE@#To study the distribution of peripheral blood lymphocyte subsets in healthy children aged 0-6 years.@*METHODS@#A total of 826 healthy Han children aged 0-6 years were recruited. According to their age, the children were divided into four groups: newborn, infant, toddler and preschool. Their peripheral blood samples were collected to measure the percentages of lymphocyte subsets by flow cytometry.@*RESULTS@#There were significant differences in the percentages of CD3 T cells, CD3CD4 T cells and CD3CD19 B cells and the CD4/CD8 ratio between boys and girls (P<0.05). The girls had a lower percentage of CD3CD19 B cells, higher percentages of CD3 T cells and CD3CD4 T cells and a higher CD4/CD8 ratio than the boys. The newborn group had the highest percentages of CD3 T cells and CD3CD4 T cells and the highest CD4/CD8 ratio (P<0.05). The percentage of CD3CD4 T cells and the CD4/CD8 ratio gradually decreased with age and the preschool group had the lowest values (P<0.05). The newborn group had the lowest percentages of CD3CD19 B cells and CD3CD16CD56 NK cells (P<0.05). The percentage of CD3CD16CD56 NK cells gradually increased with age and the preschool group had the highest percentage (P<0.05). The percentage of CD3CD19 B cells reached the peak in the toddler period and then decreased with age (P<0.05). The preschool group had the highest percentage of CD3CD8 T cells (P<0.05). The variation trend of distribution of lymphocyte subsets in boys from different age groups was consistent with that in children from different age groups. For girls, the newborn group had the highest percentage of CD3CD4 T cells and CD4/CD8 ratio (P<0.05).@*CONCLUSIONS@#The distribution of peripheral blood lymphocyte subsets in healthy children is significantly different across ages and sexes. Therefore, the reference values should be established according to age and sex.

Value of absolute counts of lymphocyte subsets in the early prediction of refractory Mycoplasma pneumoniae pneumonia in children / 中国当代儿科杂志

OBJECTIVE@#To study the value of absolute counts of lymphocyte subsets in the early prediction of refractory Mycoplasma pneumoniae pneumonia (RMPP) in children.@*METHODS@#A retrospective analysis was performed for the clinical data of 244 children with Mycoplasma pneumoniae pneumonia (MPP). Among these children, 166 had MPP, and 58 had RMPP. The two groups were compared in terms of clinical features and laboratory markers such as lymphocyte subsets, lactate dehydrogenase, C-reactive protein, procalcitonin and immunoglobulin E (IgE). The receiver operating characteristic (ROC) curve was used to evaluate the specific indices for predicting RMMP.@*RESULTS@#There were significant differences between the two groups in the absolute counts of CD3, CD4, CD19, and CD56 lymphocytes and the serum levels of lactate dehydrogenase, C-reactive protein, and IgE (P<0.05). The ROC curve analysis showed that the absolute counts of CD3, CD4 and CD19 lymphocytes had an area under the ROC curve (AUC) of 0.866, 0.900 and 0.842 respectively in the differential diagnosis of RMPP and MPP, with a sensitivity of 86%, 90% and 82% respectively and a specificity of 75%, 70% and 80% respectively.@*CONCLUSIONS@#The absolute counts of CD3, CD4 and CD19 lymphocytes can be used to predict RMPP in children.

Levels of lymphocyte subsets, immunoglobulins, and complement C3 and C4 in children with hand-foot-mouth disease / 中国当代儿科杂志

OBJECTIVE@#To study the clinical value of lymphocyte subsets, immunoglobulins, and complement C3 and C4 in the evaluation of immune status in children with hand-foot-mouth disease (HFMD).@*METHODS@#A total of 282 children with HFMD were enrolled as the HFMD group, and 130 healthy children were enrolled as the healthy control group. The percentages of peripheral CD3, CD4, and CD8 T lymphocytes, CD19 B lymphocytes, and CD56 natural killer cells were measured. The CD4/CD8 ratio was calculated. The levels of immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G (IgG), and complement C3 and C4 were measured.@*RESULTS@#The multivariate analysis showed that compared with the healthy control group, the HFMD group had significantly lower percentages of CD3, CD4, and CD8 T lymphocytes and levels of complement C3 and C4 (P<0.05), as well as significantly higher percentage of CD56 natural killer cells and level of IgG (P<0.05). The individual effect analysis showed that the children aged 0-3 years in the HFMD group had a significantly higher CD4/CD8 ratio than the healthy control group (P<0.05); boys aged 0-3 and ≥3 years in the HFMD group had a significantly higher level of IgM than the healthy control group (P<0.05); boys aged ≥3 years and girls aged 0-3 years in the HFMD group had a significantly lower level of IgA than the healthy control group (P<0.05).@*CONCLUSIONS@#Cellular and humoral immunity disorders are observed in children with HFMD. The monitoring of lymphocyte subsets and immunoglobulin levels can provide a laboratory basis for immune status assessment in children with HFMD.

Correlation of Peripheral Blood T Lymphocyte Subsets with Prognosis of Elderly Patients with Newly Diagnosed Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression level of T lymphocyte subsets in elderly patients with newly diagnosed multiple myeloma (NDMM), and to evaluated the prognostic value of T lymphocytic abnormalities in elderly NDMM patients.@*METHODS@#Pretreated peripheral blood of 39 newly diagnosed elder patients with MM was tested by multi-parameter flow cytometry (MFC) to quantitatively detect T lymphocyte subsets, including CD4T cell, CD8T cell, and CD4/CD8 ratio. The prognostic values T-lymphocyte subset were evaluated in newly diagnosed elderly patients with MM.@*RESULTS@#The median follow-up time was 21.5 (range, 3.0-66.0) months. Absolute counts of CD4T cell and CD4/CD8 ratio positively correlated with prognosis. In the multivariate COX analysis, lower CD4/CD8 ratio and CD4T cell counts were identified to be independent adverse prognostic factors for OS.@*CONCLUSION@#Lower CD4/CD8 ratio and CD4T cell counts at initial diagnosis are independent unfavorable prognostic factors for elderly patients with MM, and T lymphocyte subsets are crucial indicators for MM patients' prognosis.

Single-cell Analysis of CAR-T Cell Activation Reveals A Mixed T1/T2 Response Independent of Differentiation / 基因组蛋白质组与生物信息学报·英文版

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 helper T (T) cells and CD8 cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T1 and T2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent T1 or T2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T1/T2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T1/T2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

Altered peripheral lymphocyte subsets in untreated systemic lupus erythematosus patients with infections

The leading cause of death in systemic lupus erythematosus (SLE) patients is infection. The objective of this study was to evaluate the distribution of lymphocyte subsets in untreated SLE patients with infections. This was a cross-sectional study. Data from January 2017 to May 2018 were collected. Flow cytometry was used to measure the peripheral lymphocyte subsets including CD3+T cells, CD4+T cells, CD8+T cells, CD19+B cells, CD3-CD16+CD56NK cells, and CD3+CD16+CD56NKT cells in 25 healthy controls and 52 treatment-naive SLE patients, among whom 13 were complicated with infections. Association between the lymphocyte subsets and infections was further analyzed. SLE patients with infections (n=13) showed a significantly higher incidence rate of fever (84.6 vs 28.2%) and serositis (84.6 vs 23.1%), increased level of erythrocyte sedimentation rate (60.5±30.1 vs 37.4±27.1 mm/h), serum C-reactive protein (CRP) (102.7±94.9 vs 9.4±14.9 mg/L), procalcitonin (PCT) (1.07±0.08 vs 0.16±0.13 μg/L), and lower blood hemoglobin (Hb) (93.0±20.5 vs 110.4±16.0 g/L) level compared with non-infection patients (n=39) (all P<0.05). In comparison with non-infectious SLE patients (387.9±261.6/μL), CD4+T cells count decreased significantly in infectious SLE patients (217.8±150.4/μL) (P<0.05), and it was negatively correlated with infection-related indicators including PCT (r=−0.573, P=0.041) and CRP (r=−0.596, P=0.032) levels. Our findings suggested that abnormalities of peripheral lymphocyte subsets were related to the immune disorder of lupus itself, regardless of immunosuppressive treatment. Monitoring lymphocyte subsets, especially CD4+T cells, may be helpful for identifying the presence of infection in SLE patients.

Circulating Low Absolute CD4⁺ T Cell Counts May Predict Poor Prognosis in Extranodal NK/T-Cell Lymphoma Patients Treating with Pegaspargase-Based Chemotherapy / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Extranodal natural killer/T-cell lymphoma, nasal type (ENKTL) is a rare subtype of non-Hodgkin lymphoma, and asparaginase-based regimens are the best first-line treatments. Data on the role of specific circulating lymphocyte subsets in the progression of ENKTL are limited. The aim of this study was to investigate the clinical correlation and distribution of circulating absolute CD4+ T-cell counts (ACD4Cs) in ENKTL. MATERIALS AND METHODS: We retrospectively searched medical records for 70 newly diagnosed ENKTL patients treated with pegaspargase-based regimens. Comparison of ACD4Cs as a continuous parameter in different groups was calculated. Univariate and multivariate analyses were used to assess prognostic factors for overall survival (OS) and progression-free survival (PFS). RESULTS: Stage III/IV, B symptoms, elevated lactate dehydrogenase, monocytopenia, high-intermediate and high risk International Prognostic Index (IPI) and Korean Prognostic Index (KPI), high risk Prognostic Index of Natural Killer Lymphoma (PINK), and lower lymphocytes were significantly associated with low ACD4C at diagnosis. With a median follow-up time of 32 months, patients who had an ACD4C < 0.30×109/L had a worse OS. Median OS was 11 months and median PFS was 5 months in the low ACD4C cohort. There were significant differences in both OS and PFS between the two cohorts. Moreover, multivariate Cox analysis identified ACD4Cs as an independent predictor for OS and PFS. CONCLUSION: Low ACD4Cs were associated with poorer survival and could act as a negative predictor for ENKTL patients treated with asparaginase-based regimens.

Effects of Different Stimulators on Proliferation of Peripheral Blood Lymphocyte Subsets / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of different stimultors (PHA, PMA and IL-2) and culture systems (PBMC and whole blood) on the proliferation of human peripheral blood lymphocyte subsets, so as to provide the experimental basis for selecting the appropriate system according to the experimental purposes.@*METHODS@#A total of 10 ml serum samples were collected from healthy volunteers (n=6). The 300 μl whole blood was directly used to detect lymphocyte subsets by flow cytometry. The 400 μl whole blood were inoculated respectively with 3 different stimulators at 37℃ and 5% CO2 for 60 h; Three different stimulators were also added to the PBMC which were isolated from 2 ml whole blood. Then the proliferation ability of lymphocyte subsets was analyzed by flow cytometry.@*RESULTS@#After the PBMC were stimulated with PHA, CD4CD8CD3 lymphocytes were the most subset; The proportion of CD3CD4 T lymphocytes and CD3CD19 B lymphocytes decreased after being stimulated by PMA (P<0.01, P<0.05); the lymphocyte subset ratio had no significant change after being stimulated by IL-2. After the whole blood system was stimulated with PHA, the CD4/CD8 T lymphoblasts were main subsets, the counts of B lymphocytes and NK cells were reduced; after being stimulated with PMA, the number of CD8CD3 T lymphoblast and CD4CD8T lymphocytes increased, the B/NK cells were not distinguished with the surface markers; after the whole blood system was stimulated with IL-2, the proportion of NK cells significantly increased (P<0.05).@*CONCLUSION@#Lymphocyte proliferation stimulated by PMA is the fastest, while the effect of IL-2 on the lymphocyte subset proportion stimulated by IL-2 is the minimal. After being stimulated by PHA the division cycles of lymphocyte are the most.