Construction of NKG2D CAR-NK92 cells and its killing effect on multiple myeloma cells / 细胞与分子免疫学杂志

Objective This study aims to construct and identify the chimeric antigen receptor NK92 (CAR-NK92) cells targeting NKG2D ligand (NKG2DL) (secreting IL-15Ra-IL-15) and verify the killing activity of NKG2D CAR-NK92 cells against multiple myeloma cells. Methods The extracellular segment of NKG2D was employed to connect 4-1BB and CD3Z, as well as IL-15Ra-IL-15 sequence to obtain a CAR expression framework. The lentivirus was packaged and transduced into NK92 cells to obtain NKG2D CAR-NK92 cells. The proliferation of NKG2D CAR-NK92 cells was detected by CCK-8 assay, IL-15Ra secretion was detected by ELISA and killing efficiency was detected by lactate dehydrogenase (LDH) assay. The molecular markers of NKp30, NKp44, NKp46, the ratio of apoptotic cell population, CD107a, and the secretion level of granzyme B and perforin were detected using flow cytometry. In addition, the cytotoxic mechanism of NKG2D CAR-NK92 cells on the tumor was verified by measuring the degranulation ability. Moreover, after NKG2D antibody inhibited effector cells and histamine inhibited tumor cells, LDH assay was utilized to detect the effect on cell-killing efficiency. Finally, the multiple myeloma tumor xenograft model was constructed to verify its anti-tumor activity in vivo. Results Lentiviral transduction significantly increased NKG2D expression in NK92 cells. Compared with NK92 cells, the proliferation ability of NKG2D CAR-NK92 cells was weaker. The early apoptotic cell population of NKG2D CAR-NK92 cells was less, and NKG2D CAR-NK92 cells had stronger cytotoxicity to multiple myeloma cells. Additionally, IL-15Ra secretion could be detected in its culture supernatant. NKp44 protein expression in NKG2D CAR-NK92 cells was clearly increased, demonstrating an enhanced activation level. Inhibition test revealed that the cytotoxicity of CAR-NK92 cells to MHC-I chain-related protein A (MICA) and MICB-positive tumor cells was more dependent on the interaction between NKG2D CAR and NKG2DL. After stimulating NKG2D CAR-NK92 cells with tumor cells, granzyme B and perforin expression increased, and NK cells obviously upregulated CD107α. Furthermore, multiple myeloma tumor xenograft model revealed that the tumors of mice treated with NKG2D CAR-NK92 cells were significantly reduced, and the cell therapy did not sensibly affect the weight of the mice. Conclusion A type of CAR-NK92 cell targeting NKG2DL (secreting IL-15Ra-IL-15) is successfully constructed, indicating the effective killing of multiple myeloid cells.

A Case of Peripheral T-cell Lymphoma, Unspecified Presenting as Multiple Ulcerative Tumors / 대한피부과학회지

Peripheral T cell lymphoma, unspecified (PTCL-U) comprises a heterogenous group of mature T-cell lymphomas that do not match with any defined T-cell entities in the current classification system. A 68-year-old man presented with extensive erythematous to brownish ulcerative tumors with yellowish discharge on the neck, trunk, and both upper extremities that had persisted for the past 7 months. Histological findings showed medium- to large-sized pleomorphic lymphocytes with cellular atypia infiltrating the deep dermis and subcutis. Immunohistochemical analysis of specimens from this patient revealed positive staining for CD2, CD45, and granzyme B and mildly positive staining for CD3, CD4, CD30, and CD79a. Based on these clinico-pathological findings, the patient was finally diagnosed with PTCL-U. We report herein a rare case of PTCL-U presenting as multiple ulcerative tumors.

Primary Peripheral Gamma Delta T-Cell Lymphoma of the Central Nervous System: Report of a Case Involving the Intramedullary Spinal Cord and Presenting with Myelopathy

Primary central nervous system lymphoma of T-cell origin (T-PCNSL) is rare, and its clinicopathological features remain unclear. Peripheral T-cell lymphoma of γδ T-cell origin is an aggressive lymphoma mainly involving extranodal sites. Here, we report a case of γδ T-PCNSL involving the intramedullary spinal cord and presenting with paraplegia. A 75-year-old Korean woman visited the hospital complaining of back pain and lower extremity weakness. Magnetic resonance imaging revealed multifocal enhancing intramedullary nodular lesions in the thoracic and lumbar spinal cord. An enhancing nodular lesion was observed in the periventricular white matter of the lateral ventricle in the brain. There were no other abnormalities in systemic organs or skin. Laminectomy and tumor removal were performed. The tumor consisted of monomorphic, medium-to-large atypical lymphocytes with pale-to-eosinophilic cytoplasm. Immunohistochemically, the tumor cells were CD3(+), TCRβF1(-), TCRγ(+), CD30(-), CD4(-), CD8(-), CD56(+), TIA1(+), granzyme B(+), and CD103(+). Epstein-Barr virus in situ was negative. This case represents a unique T-PCNSL of γδ T-cell origin involving the spinal cord.

Resposta imune e mecanismos de evasão na carcinogênese de lábio: um estudo imunoistoquímico / Immune response and evasion mechanisms in lip carcinogenesis: an immunohistochemical study

A vigilância imunológica, principalmente mediada por linfócitos T CD8+ , reconhece e destrói células malignas ou alteradas. Contudo, através de estratégias imunossupressoras, como as vias de sinalização do ligante de morte celular programada-1 (PD-L1) e do antígeno leucocitário humano-G (HLA-G), estas células mutadas conseguem escapar da resposta imune antitumoral. Este estudo investigou a imunoexpressão de PD-L1, HLA-G, CD8 e granzima B (GrB) no microambiente de carcinomas de células escamosas (CCEs) de lábio (n = 40), de queilites actínicas (QAs; n = 55) e de mucosa labial saudável (MLS; n = 10). As amostras foram submetidas à técnica da imunoistoquímica e as análises das imunomarcações seguiram métodos semi-quantitativos (PD-L1 e HLA-G) e quantitativos (CD8 e GrB). A expressão das proteínas foi comparada entre os três grupos de amostras, bem como com parâmetros clinicopatológicos das lesões e sobrevida global dos pacientes com CCE de lábio. A correlação entre as proteínas e o tipo do microambiente tumoral de acordo com a presença de PD-L1 e CD8 também foram avaliados. Os testes estatísticos incluíram o exato de Fisher, Mann-Whitney, Kruskal-Wallis, correlação de Spearman e log-rank para comparação das curvas de sobrevida global construídas pelo método Kaplan-Meier. O nível de significância foi estabelecido em 5%. Os números de células CD8+ e GrB+ aumentaram progressivamente de MLS para CCEs de lábio, com QAs exibindo números intermediários (p < 0,01). A menor expressão dessas proteínas foi associada à metástase para linfonodos e tumores pobremente diferenciados (p < 0,05). A expressão de PD-L1 e HLA-G em células neoplásicas/ceratinócitos e estroma/tecido conjuntivo foi significativamente maior em CCEs de lábio e QAs, em comparação com MLSs (p < 0,05). PDL1 não foi significativamente associado aos aspectos clinicopatológicos das lesões. A maioria dos CCEs de lábio mostrou coexistência de células PD-L1+ e CD8+ (72,5%) no microambiente tumoral. A expressão de PD-L1 foi diretamente correlacionada à infiltração linfocítica CD8+ e GrB+ em CCEs de lábio (p < 0,05). A expressão das proteínas não foi associada com a sobrevida global dos pacientes com CCEs de lábio (p > 0,05). Nossos achados sugerem que as moléculas imunossupressoras PD-L1 e HLA-G estão consistentemente expressas desde QAs e se mantém até fases avançadas dos CCE de lábio. A correlação entre a expressão de PD-L1 e a expressão de CD8 e GrB nos carcinomas sugere que PD-L1 pode surgir como um mecanismo de escape frente a uma resposta antitumoral ativa (AU).

Immune surveillance, mainly mediated by CD8 + T lymphocytes, recognize and destroy malignant or altered cells. However, through immunosuppressive strategies, such as the signaling pathways of the programmed cell death ligand-1 (PD-L1) and human leukocyte antigen-G (HLA-G), these mutated cells often escape the antitumor immune response. The aim of this study was to investigate and compare the immunoexpression of PD-L1, HLA-G, CD8 and granzyme B (GrB) in the microenvironment of lip squamous cell carcinomas (LSCCs; n = 40), actinic cheilitis (ACs; n = 55), and healthy lip mucosa (HLM; n = 10). The samples were submitted to immunohistochemistry and the analysis followed a semi-quantitative (PD-L1 and HLA-G) and quantitative methods (CD8 and GrB). Protein expression was compared between the three groups of samples, as well as with the lesion's clinicopathologic parameters and overall survival of patients with LSCC. Correlation between proteins and the type of tumor microenvironment according to a presence of PD-L1 and CD8 were also evaluated. Statistical tests included Fisher's exact, Mann-Whitney, Kruskal-Wallis, Spearman's correlation, as well as the log-rank for comparison of the overall survival built through Kaplan-Meier method. Significance was set at p < 0.05. The CD8+ and GrB+ cell numbers progressively increased from HLMs to LSCCs, with ACs exhibiting intermediate numbers (p < 0.01). Lower expression of these proteins was associated with lymph node metastasis and poor tumor differentiation (p < 0.05). PD-L1 and HLA-G expression in neoplastic cells/keratinocytes and stroma/connective tissue was significantly higher in LSCCs and ACs, compared to HLMs (p < 0.05). PD-L1 was not significantly associated with clinicopathological aspects of the lesions. Most LSCCs showed coexistence of PD-L1+ and CD8+ cells (72.5%) in the tumor microenvironment. PDL1 was directly correlated to CD8+ and GrB+ lymphocytic infiltration in LSCCs (p < 0.05). Proteins expression was not associated with overall survival of LSCCs patients (p > 0.05). Our findings suggest that immunosuppressive molecules PD-L1 and HLA-G are consistently expressed from ACs and are maintained until advanced stages of LSCCs. The correlation between PD-L1 expression and the expression of CD8 and GrB in carcinomas suggests that that PD-L1 may appear as an escape mechanism against an active antitumor response (AU).

Potential Utility of FDG PET-CT as a Non-invasive Tool for Monitoring Local Immune Responses

PURPOSE: The tumor microenvironment is known to be associated with the metabolic activity of cancer cells and local immune reactions. We hypothesized that glucose metabolism measured by 2-deoxy-2-(¹⁸F)fluoro-D-glucose (¹⁸F-FDG) positron emission tomography (PET)-computed tomography (CT) (¹⁸F-FDG PET-CT) would be associated with local immune responses evaluated according to the presence of tumor infiltrating lymphocytes (TILs). MATERIALS AND METHODS: We retrospectively reviewed 56 patients who underwent ¹⁸F-FDG PET-CT prior to gastrectomy. In resected tumor specimens, TIL subsets, including cluster of differentiation (CD) 3, CD4, CD8, Forkhead box P3 (Foxp3), and granzyme B, were subjected to immunohistochemical analysis. The prognostic nutritional index (PNI) was calculated as: (10×serum albumin value)+(0.005×peripheral lymphocyte counts). Additionally, the maximum standard uptake value (SUVmax) was calculated to evaluate the metabolic activity of cancer cells. RESULTS: The SUVmax was positively correlated with larger tumor size (R=0.293; P=0.029) and negatively correlated with PNI (R=−0.407; P=0.002). A higher SUVmax showed a marginal association with higher CD3 (+) T lymphocyte counts (R=0.227; P=0.092) and a significant association with higher Foxp3 (+) T lymphocyte counts (R=0.431; P=0.009). No other clinicopathological characteristics were associated with SUVmax or TILs. Survival analysis, however, indicated that neither SUVmax nor Foxp3 held prognostic significance. CONCLUSIONS: FDG uptake on PET-CT could be associated with TILs, especially regulatory T cells, in gastric cancer. This finding may suggest that PET-CT could be of use as a non-invasive tool for monitoring the tumor microenvironment in patients with gastric cancer.

Diffuse large B-cell lymphoma with aberrant expression of CD56: a clinicopathologic and immunohistochemical study / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinicopathologic features and significance of aberrant CD56 expression in diffuse large B-cell lymphoma (DLBCL).</p><p><b>METHODS</b>The clinical and pathologic profiles of 10 cases of DLBCL with aberrant expression of CD56 were investigated. Immunohistochemical staining, in-situ hybridization for Epstein-Barr virus encoded RNA and gene rearrangement for IgH and Igκ were carried out.</p><p><b>RESULTS</b>There were 6 male and 4 female patients. The medium age of patients was 46 years. All of them presented with extranodal lymphoma involvement, with gastrointestinal tract being the commonest site (5/10). Histologic examination showed that most of the atypical lymphoid cells were centroblast-like and demonstrated a diffuse growth pattern. Apoptosis and necrosis were identified in some cases. Immunohistochemical study showed that the tumor cells were positive for CD20 or CD79α and aberrantly expressed CD56. Five cases had the GCB phenotype while the remaining cases had the non-GCB phenotype, according to Hans classification. Bcl-6 was positive in most cases (9/10). All cases showed a high proliferation index by Ki-67. The tumor cells were negative for CD3ε, CD138 and granzyme B. In-situ hybridization for Epstein-Barr virus encoded RNA was performed in 7 cases and none of them showed positive signals. IgH gene rearranged bands were detected in 4 cases (4/6) and Igκ was detected in 3 cases (3/6). Follow-up data were available in 8 patients. Two patients died of disease progression within 5 to 13 months after diagnosis and the other 6 patients were alive 8 to 60 months after therapy.</p><p><b>CONCLUSIONS</b>DLBCL with aberrant expression of CD56 is rare. Most of them present with extranodal involvement, show high frequency of bcl-6 expression and high proliferation index. The patients often have good response to chemotherapy.</p>

Clinicopathologic characteristics and immunophenotypes of histiocytic necrotizing lymphadenitis: an analysis of 84 cases / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinical manifestation, pathologic features and immunophenotype of histiocytic necrotizing lymphadenitis (HNL).</p><p><b>METHODS</b>The clinicopathologic data of 84 patients with HNL from 2005 to 2014 were retrospectively studied. Immunohistochemical staining using EliVision method for CD20, PAX5, CD3, CD45RO, CD4, CD8, CD56, CD68, CD123, granzyme-B, TIA1 and MPO was carried out. In-situ hybridization for Epstein-Barr virus RNA was performed on archival lymph node biopsy tissue.</p><p><b>RESULTS</b>Immunohistochemical study showed that the lesional cells were predominantly histiocytes (CD68+), plasmacytoid dendritic cells (CD123+) and T lymphocytes (CD3+ and CD45RO+). Clusters of CD68-positive cells with strong and diffuse MPO expression were identified. T lymphocytes with CD4 and CD8 positivity were noted. CD56+ natural killer cells and CD20+/PAX5 B cells were rare. Apoptosis-related markers, including TIA1 and granzyme B were expressed by T lymphocytes and histiocytes in lymph nodes of HNL. In-situ hybridization for Epstein-Barr virus RNA was positive in only 10.0% of the cases.</p><p><b>CONCLUSIONS</b>HNL shows no specific clinical and laboratory findings. Recognition of the characteristic histopathologic changes in lymph node biopsy of HNL is the key to correct diagnosis. Immunohistochemical study using a panel of markers, including CD3, CD4, CD8, MPO, CD123, granzyme-B and TIA1, is helpful in the differential diagnosis of HNL.</p>

Effects of Iron Overload on the Apoptosis and Function of Splenic CD8+ T Cells in Mice / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of iron overload on apoptosis and function of splenic CD8+ T cells in mice.</p><p><b>METHODS</b>Forty C57BL/6 mice were randomly divided into control groups, Iron overload (IO), IO+NAC and IO+DFX groups. The iron overload model was established by intraperitoneal injection of iron dextran, and saline was injected as the control. The levels of intracellular reactive oxygen species (ROS) and labile iron pool (LIP) were analyzed by measuring the mean fluorescence intensity (MFI) of 2-7 dichlorofluorescein (DCF) or calcein. The ratio of CD8+ T cells and the levels of IFN-γ, TNF-α, Granzyme-B, and perforin in CD8+ T cells were detected by flow cytometry. The CD8+ T cell apoptosis was determined by flow cytometry with Annexin V/PI double staining. Real-time PCR was used to detect the expression of IFN-γ, TNF-α, Granzyme-B, perforin, BCL-2, and bax at mRNA level in CD8+ T cells.</p><p><b>RESULTS</b>Iron overload was found by spleen iron staining and flow cytometry. The level of intracellular ROS in iron overload (IO) groups was higher than that of the control groups (P<0.01). The percentage of CD8+ T cells in spleen from mice with IO was lower than that in control groups (P<0.05). The expression of IFN-γ and Granzyme-B in CD8+ T cells in IO group were lower than that in control group, the expression of IFN-γ and Granzyme-B at mRNA level in CD8+ T cells was lower than that of control group (P<0.05). CD8+ T cell apoptosis in iron overload group was significantly higher than that in control groups (P<0.01); the expression of BCL-2 at mRNA level was lower than that in control group, but the expression of BAX at mRNA level was higher than that in control group (P<0.05). These effects could be reversed after treating iron-overloaded mice with DFX or NAC.</p><p><b>CONCLUSION</b>Iron overload can inhibit the ratio of CD8+ T cells of splenic cells in mice, decrease the expression of IFN-γ, Granzyme-B, increase the apoptosis of CD3+ CD8+/CD8-. These effects may be regulated through increasing the intracellular ROS level, and can be partially reversed after treating iron-overloaded mice with DFX or NAC.</p>

Increased circulating macrophage migration inhibitory factor levels are associated with coronary artery disease

BACKGROUND: To evaluate the macrophage migration inhibitory factor and E-selectin levels in patients with acute coronary syndrome. MATERIALS/METHODS: We examined the plasma migration inhibitory factor and E-selectin levels in 87 patients who presented with chest pain at our hospital. The patients were classified into two groups according to their cardiac status. Sixty-five patients had acute myocardial infarction, and 22 patients had non-cardiac chest pain (non-coronary disease). We designated the latter group of patients as the control group. The patients who presented with acute myocardial infarction were further divided into two subgroups: ST-elevated myocardial infarction (n = 30) and non-ST elevated myocardial infarction (n = 35). RESULTS: We found higher plasma migration inhibitory factor levels in both acute myocardial infarction subgroups than in the control group. However, the E-selectin levels were similar between the acute myocardial infarction and control patients. In addition, we did not find a significant difference in the plasma migration inhibitory factor levels between the ST elevated myocardial infarction and NST-elevated myocardial infarction subgroups. DISCUSSION: The circulating concentrations of migration inhibitory factor were significantly increased in acute myocardial infarction patients, whereas the soluble E-selectin levels were similar between acute myocardial infarction patients and control subjects. Our results suggest that migration inhibitory factor may play a role in the atherosclerotic process. .

Expression of perforin and granzyme-B in peripheral blood lymphocyte in patients with prostate cancer and the clinical significance / 中南大学学报(医学版)

OBJECTIVE@#To explore the expression of perforin and granzyme-B in peripheral blood lymphocyte (PBL) in patients with prostate cancer (PCa) and the clinical significance.@*METHODS@#The expressions of perforin and granzyme-B in PBL were detected by fluorescence quantitative reverse transcription polymerase chain reaction. The results of perforin and granzyme-B expression were compared among patients with PCa (n=60), patients with BPH (benign prostatic hyperplasia, n=40) and healthy controls (n=20).@*RESULTS@#Th e expressions of perforin and granzyme-B in patients with PCa were significantly lower than that in patients with BPH or that in the healthy controls (P<0.05), respectively. Furthermore, in PCa patients with low pathological grade, the expressions of perforin and granzyme-B in PBL was statistically higher than that in patients with high pathological grade (P<0.05). The expressions of perforin and granzyme-B in PCa patients at high clinical stage was statistically lower than that in PCa patients at low clinical stage (P<0.05).@*CONCLUSION@#The results of this study suggest that development and progression of PCa might be associated with poor immune status of patients.

Comparative analysis of serum proteomic profiles between patients with silicosis and chronic bronchitis / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To analyze the differences in serum proteomic profiles between patients with silicosis and chronic bronchitis and to investigate the pathogenesis, clinical diagnosis, and treatment of these two disease.</p><p><b>METHODS</b>Serum samples from patients with stage I silicosis and chronic bronchitis were collected. Two-dimensional gel electrophoresis was performed and protein plots with expression differences higher than 2-fold were identified and further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.</p><p><b>RESULTS</b>Compared with the silicosis group, the chronic bronchitis group had 11 up-regulated proteins and 23 down-regulated proteins. The chronic bronchitis group had high expression of proteins such as interferon beta precursor, apolipoprotein precursor, and transforming growth factor beta1 precursor. The silicosis group had high expression of proteins such as interleukin-6, granzyme A, cathepsin G, and glycoprotein precursor.</p><p><b>CONCLUSION</b>The differentially expressed proteins are mainly involved in the activity of serine enzymes, cytotoxicity, inflammation response, and apolipoprotein transfer and play different roles in silicosis and chronic bronchitis.</p>

Changes in serum protease and cytokine in patients with silicosis, tuberculosis, and lung cancer / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the changes in serum protease and cytokine in patients with silicosis, tuberculosis, and lung cancer.</p><p><b>METHODS</b>Serum samples of patients with silicosis, tuberculosis, and lung cancer were collected. The variation trends of the expression of granzyme A, cathepsin G, apolipoprotein A, and interferon-β (IFN-β) were analyzed using enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>The concentration of apolipoprotein A of the silicosis group was 200 µg/ml, significantly higher than those of the tuberculosis and lung cancer groups (P < 0.05), and the lung cancer group had a significantly higher concentration of apolipoprotein A compared with the tuberculosis group (P < 0.05). The silicosis group had significantly higher expression of cathepsin G compared with the tuberculosis and lung cancer groups (P < 0.05), and the tuberculosis group and lung cancer group showed no significant difference in the concentration of cathepsin G (P > 0.05). The tuberculosis group had a significantly higher concentration of granzyme A than the silicosis and lung cancer groups (P < 0.05), and the silicosis group and lung cancer group had similar protein concentration trends (P > 0.05). The tuberculosis group and lung cancer group had significantly higher concentration of IFN-β compared with the silicosis group (P < 0.05), and the tuberculosis group and lung cancer group showed no significant difference in IFN-β concentration (P > 0.05).</p><p><b>CONCLUSION</b>This study may offer diagnostic markers for the clinical diagnosis of silicosis, tuberculosis, and lung cancer, and could provide a basis for the research, as well as potential molecular targets for the diagnosis and treatment of these diseases.</p>

Attenuation of Peripheral Regulatory T-Cell Suppression of Skin-Homing CD8+T Cells in Atopic Dermatitis

PURPOSE: Cutaneous lymphocyte-associated antigen (CLA)-expressing CD8+T cells have been known to play an important role in the pathogenesis of atopic dermatitis (AD). However, the mechanisms underlying the loss of self-tolerance remain unclear. Regulatory T cells (Tregs) play a key role in the development of homeostasis in the immune system. We, therefore, hypothesized that a reduced ability of Tregs to inhibit autologous CD8+CLA+T cells might be underlying mechanism in AD. MATERIALS AND METHODS: CD8+CLA+T cells and Tregs were obtained from the peripheral blood of AD patients and control volunteers. The frequencies of CD8+CLA+T cells were evaluated. The proliferative responses of CD8+CLA+T cells were assessed by flow cytometry, and the levels of transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) in culture supernatants were detected by enzyme-linked immunosorbent assay. RESULTS: Our results revealed higher frequency and increased expression of perforin and granzyme-B in peripheral CD8+CLA+T cells in AD, and lower inhibitory ability of Tregs on proliferation of CD8+CLA+T cells in AD. Meanwhile, the levels of TGF-beta1 produced by Tregs were significantly lower in AD, and anti-TGF-beta1 abolished such suppression. CONCLUSION: The attenuated inhibitory ability of Tregs on hyper-activated autologous CD8+CLA+T cells, mediated by TGF-beta1, plays an important role in the pathogenesis of AD.

Association between Chemotherapy-Response Assays and Subsets of Tumor-Infiltrating Lymphocytes in Gastric Cancer: A Pilot Study

PURPOSE: The purpose of this pilot study was to evaluate the association between adenosine triphosphate-based chemotherapy response assays (ATP-CRAs) and subsets of tumor infiltrating lymphocytes (TILs) in gastric cancer. MATERIALS AND METHODS: In total, 15 gastric cancer tissue samples were obtained from gastrectomies performed between February 2007 and January 2011. Chemotherapy response assays were performed on tumor cells from these samples using 11 chemotherapeutic agents, including etoposide, doxorubicin, epirubicin, mitomycin, 5-fluorouracil (5-FU), oxaliplatin, irinotecan, docetaxel, paclitaxel, methotrexate, and cisplatin. TILs in the tissue samples were evaluated using antibodies specific for CD3, CD4, CD8, Foxp3, and Granzyme B. RESULTS: The highest cancer cell death rates were induced by etoposide (44.8%), 5-FU (43.1%), and mitomycin (39.9%). Samples from 10 patients who were treated with 5-FU were divided into 5-FU-sensitive and -insensitive groups according to median cell death rate. No difference was observed in survival between the two groups (P=0.216). Only two patients were treated with a chemotherapeutic agent determined by an ATP-CRA and there was no significant difference in overall survival compared with that of patients treated with their physician's choice of chemotherapeutic agent (P=0.105). However, a high number of CD3 TILs was a favorable prognostic factor (P=0.008). Pearson's correlation analyses showed no association between cancer cell death rates in response to chemotherapeutic agents and subsets of TILs. CONCLUSIONS: Cancer cell death rates in response to specific chemotherapeutic agents were not significantly associated with the distribution of TIL subsets.

Expansion and function of MHC restricted killer T cells derived from umbilical cord blood / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was to expand the cytotoxic T lymphocytes (CTL) through inducing the differentiation of umbilical blood monomuclear cells (UBMNC) by using various combination of cytokines, and to investigate the functions of expanded CTL.</p><p><b>METHODS</b>The MNC were isolated by ficoll density gradient centrifugation. Then, the PHA-P, IFN-γ combined with IL-2, IL-15 and other cytokines were used for induction and expansion of the cord blood-derived CTL. The biological function of CTL was examined by phenotype analysis, cytotoxic tests and real-time fluorescence quantitative PCR.</p><p><b>RESULTS</b>After expansion for 15 days, the cell number increased by 1522% ± 137%. The content of CD3(-)CD8(-) cells in uncultured cord blood MNC was 95%, and the CD3(+)CD8(+) CTL cells reached 82.77% in cultured cord blood MNC after expansion for 15 days. The expanded CTL cell showed the cytotoxic activity against K562 and HeLa cell line. The killing rate of MNC was 61.88 ± 1.08%. After expansion, the killing rate could reach to 90% with the average value of 90.33 ± 2.02%. The expanded CTL cells highly expressed some key cytokines, such as granzyme A, granzyme B, GM-CSF, granulysin, IFN-γ, TGF-β, TNF-α and perforin. Compared with the control group, the expression of IFN-γ and TGF-β significantly increased (P < 0.05), and the other factors dramatically increased (P < 0.01).</p><p><b>CONCLUSION</b>The cord blood-derived CTL can be expanded by different combinations of cytokines. These protocols may provide alternative choices for CTL cell expansion in tumor adoptive immunotherapy.</p>

Intravascular Cytotoxic T-Cell Lymphoma in a Young Immunocompetent Woman

Intravascular lymphoma (IVL) is a rare disorder characterized by the presence of large neoplastic lymphoid cells restricted to the lumens of small vessels with a predilection for the skin and the central nervous system. While the vast majority of cases involving IVL are of B-cell lineage, the disease rarely affects the T-cell, the histiocytes, and the natural killer cells. We report a case of intravascular T-cell lymphoma (IVTL) associated with Epstein-Barr virus (EBV). A 23-year-old healthy woman presented with tender indurated erythematous patches with overlying telangiectasia on her right breast, abdomen, both the upper and the lower extremities and the back for 3 months. The pathology revealed an infiltration of dermal and subcutaneous vessels by large and atypical lymphoid cells with immunohistochemical features of the T-cell lineage with a cytotoxic phenotype (CD3+, CD8+, granzyme B+, TIA-1+, CD4-, CD5-, CD20-, CD56-). Interestingly, the DNA extracted from the skin biopsies demonstrated evidence of a monoclonal immunoglobulin heavy chain gene rearrangement, but no T-cell receptor gene rearrangement was found. In situ hybridization study for EBV-encoded RNA was positive. She was diagnosed with an EBV-associated IVTL. The patient's skin lesions were refractory to the combination of chemotherapy and autologous stem cell transplant, and she expired. The findings in the present case may highlight the unique clinicopathologic aspects of EBV-associated cytotoxic IVTL that occurred in a young, immunocompetent woman.

Development of NK cell expansion methods using feeder cells from human myelogenous leukemia cell line

BACKGROUND: Natural killer (NK) cells constantly survey surrounding tissues and remove newly generated cancer cells, independent of cancer antigen recognition. Although there have been a number of attempts to apply NK cells for cancer therapy, clinical application has been somewhat limited because of the difficulty in preparing a sufficient number of NK cells. Therefore, ex vivo NK cell expansion is one of the important steps for developing NK cell therapeutics. METHODS: CD3+ depleted lymphocytes were cocultured with IL-2 and with feeder cells (peripheral blood mononuclear cells [PBMCs], K562, and Jurkat) for 15 days. Expanded NK cells were tested for cytotoxicity against cancer cell lines. RESULTS: We compared feeder activities of three different cells-PBMC, K562, and Jurkat. K562 expanded NK cells by almost 20 fold and also showed powerful cytotoxic activity against cancer cells. K562-NK cells remarkably expressed the NK cell activation receptors, NKG2D, and DNAM-1. K562-NK cells exhibited more than two-fold production of cytotoxic granules compared with Jurkat-NK cells, producing more perforin and granzyme B than naive NK cells. CONCLUSION: Our findings suggest that K562 are more efficient feeder cells than Jurkat or PBMCs. K562 feeder cells expanded NK cells by almost 20 fold and showed powerful cytotoxic activity against cancer cells. We herein propose an intriguing approach for a design of NK cell expansion.

CD8+ T cells in situ in different clinical forms of human cutaneous leishmaniasis

Introduction Leishmania braziliensis infection induces a large spectrum of lesions that clinically manifest as nodules or papules that progress to ulcers. Although it is already known that T helper cells predominate in the lesions, cytotoxic T cells have also been reported to be present, and their role in leishmaniasis immunopathogenesis is not well known. This study investigated the amounts of CD8+ and granzyme B+ cells in different clinical forms of human cutaneous leishmaniasis (CL). Methods Forty tissue fragments from early (E-CL) and late CL (L-CL) lesions and from disseminated leishmaniasis (DL) - papules and ulcers - were characterized. The inflamed area per fragment was calculated, and the CD8 and granzyme B expression levels in the infiltrates were quantified by counting positive cells in 15 fields. The localization of CD8 and granzyme B was graded subjectively. Results Inflammation was higher in L-CL and DL ulcers. CD8 expression was increased in late ulcerated lesions compared to recent lesions. The increase in CD8+ cells also correlated with the duration of the lesion. Papules had a higher frequency of granzyme B+ cells than E-CL lesions, although the frequency was similar to those for late and DL ulcers. CD8+ cells were mostly found in the papillary dermis. Conclusions CD8+ T and granzyme B+ cells are present in the inflammatory infiltrates of CL and DL and may participate in the immunopathogenesis of Leishmania infection. .

Clinicopathologic features of intestinal natural killer/T-cell lymphoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinicopathologic features, diagnosis and differential diagnosis of intestinal natural killer (NK)/T-cell lymphoma.</p><p><b>METHODS</b>The clinical features, histopathology, immunohistochemical findings and follow-up data of 14 cases of intestinal NK/T-cell lymphoma were retrospectively reviewed.</p><p><b>RESULTS</b>The male-to-female ratio was 9:5. The medium age of patients was 45 years. The sites of involvement included small intestine (6 cases), colon (6 cases) or both (2 cases). The main clinical manifestations were an abdominal mass, other gastrointestinal symptoms such as abdominal pain, as well as systemic symptoms such as fever and cachexia. Intestinal perforation complicated by acute peritonitis might occur in advanced disease. Histologically, the intestinal wall showed full-thickness infiltration by medium-sized atypical lymphoid cells with pleomorphic nuclei, prominent inflammatory background, angiocentric/angiodestructive growth pattern and coagulative necrosis. Immunohistochemical study showed that the tumor cells were positive for CD3ε, CD43, CD56, granzyme B and perforin. They were negative for CD20, CD79α and MPO. In-situ hybridization for Epstein-Barr virus encoded RNA (EBER) showed negative signals. A high proliferative index was demonstrated by Ki-67 immunostaining. Follow-up data of 8 cases were available, with duration of follow up ranging from 0.5 to 36 months. Five patients died within 20 months.</p><p><b>CONCLUSIONS</b>Extranodal NK/T-cell lymphoma, nasal-type primarily involving intestine is rare and tends to carry an aggressive clinical course. The relatively non-specific clinical manifestations of intestinal NK/T-cell lymphoma may result in misdiagnosis in some cases. A comprehensive evaluation of clinical manifestations, pathologic features and immunohistochemical findings is essential for definitive diagnosis.</p>

IL-23 alone or with IL-2 induces the killing effect of hPBMNC on K562 cells / 中国实验血液学杂志

This study was aimed to explore the killing effect of PBMNC induced by IL-23 alone or combined with IL-2 on K562 cells and its mechanism. The PBMNC were induced in vitro by IL-23 (50 ng/ml) alone or IL-23 combined with IL-2 (100 U/ml) for 72 h, and then were co-cultured with leukemia cell line K562. The CCK-8 method was used to detect the effect of PBMNC induced at different times on K562 cells, the ELISA was performed for detecting IFN-γ level in culture supernatant, and the perforin and granzymes B were detected by RQ-PCR. The results showed that the killing effect of PBMNC induced by IL-23 alone or IL-23 combined with IL-2 on K562 cells was observed, and obviously enhanced with prolonging of time, moreover, there was statistical difference among different time points (P < 0.05). The IFN-γ level in supernatant of PBMNC cultured with cytokines significantly increased, and the IFN-γ levels in group of IL-23 combined with IL-2 were higher than that in other groups (P < 0.05). The mRNA expressions level of perforin and granzymes B of the expanded PBMNC in groups cultured with cytokines were higher than that in control group (P < 0.05), and the mRNA expressions of perforin and granzymes B in group of IL-23 combined with IL-2 were significantly higher than that in others (P < 0.05). It is concluded that IL-23 can promote the killing effect of PBMNC on K562 cells. The combination of IL-2 with IL-23 displays synergic effect and a time-dependent manner. IL-23 also enhances the expression of IFN-γ, perforin and granzyme B in PBMNC. Its combination with IL-2 displays synergistic effect, suggesting that the anti-leukemic activity of IL-23 may be realized through inducing PBMNC to express IFN-γ, perforin and granzyme B.