抑制性杀伤细胞受体在NK细胞和T细胞上的表达及其与急性移植物抗宿主病的关系

 目的 研究异基因造血干细胞移植（allo-HSCT）患者移植前后外周血NK及T细胞上4种抑制性杀伤细胞受体（CD158a、CD158b、NKB1和CD94／NKG2A）的表达及其与急性移植物抗宿主病（aGVHD）的关系。方法 采用流式细胞术检测NK及T细胞上抑制性杀伤细胞受体的表达。结果 NK细胞上CD158a和CD158b的表达于移植后3～4个月、NKB1和CD94／NKG2A的表达于移植后2个月恢复移植前水平。移植前后CD158a和NKB1在CD+3 T细胞上持续低水平表达;移植前CD158b和CD94／NKG2A在CD+3 T细胞上的表达水平较高,且主要表达于CD+8 T细胞上,移植后其在CD+8 T细胞上的表达增加。CD+8 T细胞上CD158b的表达在发生Ⅰ度aGVHD时显著增高,与无aGVHD组及Ⅱ~Ⅳ度aGVHD组相比,差异均有统计学意义（P均＜0.05）;在发生Ⅱ~Ⅳ度aGVHD时增高不明显,与无aGVHD组相比,差异无统计学意义（P＞0.05）。结论 CD158b在CD+8 T细胞上高表达可能有助于降低T细胞同种反应性,减轻aGVHD的程度。     

Ex vivo expansion of CD8+CD56+ and CD8+CD56- natural killer T cells specific for MUC1 mucin

Prostate cancers express MUC1, but nearly all metastatic cells lack HLA class I molecules. Thus, a lymphocyte population that can sense its antigenic environment, while also able to react to stimuli of natural killer (NK) cells, may be a more versatile effector cell population for antitumor immune responses. Herein, we report that tumor-specific MUC1 peptide, interleukin 2, and interleukin 12 act synergistically to stimulate the ex vivo expansion of CD8(+)CD56(-) T cells and CD8(+)CD56(+) natural killer T (NKT) cells from the peripheral blood mononuclear cells of prostate cancer patients, as well as healthy male and female donors. Both the CD56(+) NKT cells and CD56(-) T cells lysed allogeneic mucin-bearing target cells, as well as NK target cells, but not lymphokine-activated killer target cells. However, the CD56(+) NKT cells displayed a 2-fold greater cytolytic activity than the CD56(-) T cells. The mucin-specific cytolytic activity and NK cytolytic activities for both lymphocyte populations were independent of HLA class I and CD1 molecules. The CD56(-) T cells up-regulated CD56 with continued antigenic stimulation in the presence of interleukin 12, suggesting that CD8(+)CD56(-) T cells are NKT cells. However, CD56(+) NKT cells expand poorly to continued stimulation. All mucin-stimulated NKT cells exhibited the activated/memory CD45RO phenotype. The NKT cell lines express the alpha/beta T-cell receptor (TCR). The TCR repertoire was limited and varied with cell line, but was not the V alpha 24V beta 11 TCR typically associated with NKT cells. Whereas CD161 is generally considered a marker of NKT cells, the mucin-stimulated NKT cells did not express this marker. Thus, we have described two phenotypically distinct NKT types that do not display a biased TCR repertoire, but do display specificity for a tumor-specific peptide antigen (CTL-like activity), as well as HLA class I-deficient target cells (NK-like activity).     

Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentration-effect relationship

The FCGR3A gene dimorphism generates two allotypes: FcgammaRIIIa-158V and FcgammaRIIIa-158F. The genotype homozygous for FcgammaRIIIa-158V (VV) is associated with higher clinical response to rituximab, a chimeric anti-CD20 IgG1 used in the treatment of B lymphoproliferative malignancies. Our objective was to determine whether this genetic association relates to rituximab-dependent cytotoxicity mediated by FcgammaRIIIa/CD16a+ cells. The number of CD16+ circulating monocytes, T cells, and natural killer (NK) cells in 54 donors was first shown to be unrelated to FCGR3A polymorphism. We then demonstrated that FcgammaRIIIa-158V displays higher affinity for rituximab than FcgammaRIIIa-158F by comparing rituximab concentrations inhibiting the binding of 3G8 mAb (anti-CD16) with VV NK cells and NK cells homozygous for FcgammaRIIIa-158F (FF). VV and FF NK cells killed Daudi cells similarly after FcgammaRIIIa engagement by saturating concentrations of rituximab or 3G8. However, the rituximab concentration resulting in 50% lysis (EC(50)) observed with NK cells from VV donors was 4.2 times lower than that observed with NK cells from FF donors (on average 0.00096 and 0.00402 microg/ml, respectively, P = 0.0043). Finally, the functional difference between VV and FF NK cells was restricted to rituximab concentrations weakly sensitizing CD20. This study supports the conclusion that FCGR3A genotype is associated with response to rituximab because it affects the relationship between rituximab concentration and NK cell-mediated lysis of CD20+ cells. Rituximab administration could therefore be adjusted according to FCGR3A genotype.     

Optimized protocols for generation of cord blood-derived cytokine-induced killer/natural killer cells

The efficacy of various combinations of stem cell factor (SCF), FLT3 ligand, interleukin (IL)-2, IL-7 and IL-15 to induce and expand cord blood-derived cytokine-induced killer (CIK) cells was investigated. There were three treatment groups: group A: SCF combined with IL-2, IL-7 and IL-15; group B: SCF, FLT3 ligand combined with IL-2, IL-7 and IL-15, and group C: IL-2, IL-7 and IL-15, the control group. Proliferation rates of CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) natural killer (NK) cells were highest in group B; expansion of CIK cells increased 796.1 ± 278.5-fold, and that of NK cells increased 36.6 ± 3.5-fold. All expanded cord blood-derived CIK/NK cells showed cytotoxic activity against the K562 cell line. Interestingly, the cytotoxicity of group A was highest and significantly higher than that of other groups. These protocols might provide an alternative choice for CIK/NK cell expansion.     

Increased frequency of CD4+ cells expressing CD161 in cancer patients.

PURPOSE: Although the function of natural killer receptors on T cells infiltrating tumors and their potential effect on antitumor immunity has been investigated, little is known about T cells expressing NKR-P1A (CD161) in cancer patients. In the present study, we examined T cells expressing CD161 in the peripheral blood, the tumor tissue and in malignant effusions of patients with several types of malignancies. EXPERIMENTAL DESIGN: Expression of CD161 in CD4(+) or CD8(+) (lacking CD56) T cells isolated from peripheral blood (n = 61), tumor specimens (n = 8), and malignant effusions (n = 37) of cancer patients was examined using four-color flow cytometry. Proliferative capacity and cytokine production of purified CD4(+)CD161(+)CD56(-) cells were studied after weak or strong stimulation, with or without costimulation, in the presence or absence of interleukin 2. The possible regulatory function of activated CD4(+)CD161(+)CD56(-) cells on T-cell alloresponses was also investigated. RESULTS: CD4(+) cells expressing CD161 were increased in cancer patients, compared with healthy individuals. This increase in the peripheral blood of cancer patients positively correlated with disease stage and was augmented at the tumor site. Phenotypic analysis revealed that CD4(+)CD161(+) cells are memory T cells, with low expression of activation markers. CD4(+)CD161(+) cells play an immunoregulatory role through cytokine production, because upon receiving costimulatory signals via CD28, they exert suppressive activity on autologous peripheral blood mononuclear cell alloresponses. CONCLUSIONS: CD4(+)CD161(+)CD56(-) cells represent a distinct memory T-cell population significantly increased in cancer patients. Depending on the type of signals provided by the tumor microenvironment, CD4(+)CD161(+) cells may regulate the immune response.     

Local administration of PF-3512676 CpG-B instigates tumor-specific CD8+ T-cell reactivity in melanoma patients

PURPOSE: Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. Local administration of PF-3512676 (formerly known as CpG 7909) has shown immunostimulatory effects of both dendritic cell and T-cell subsets in the melanoma SLN. Here, we set out to ascertain whether these PF-3512676-induced immunostimulatory effects translate into higher frequencies of melanoma-specific CD8(+) T cells. EXPERIMENTAL DESIGN: Twenty-four stage I to III melanoma patients were randomized to preoperative local administration of either PF-3512676 or saline. CD8(+) T cells from SLN and peripheral blood were tested for reactivity by IFN-gamma ELISPOT assay against several HLA-A1/A2/A3-restricted epitopes derived from various melanoma-associated antigens (MAA) in 21 of 24 enrolled patients. Frequencies of natural killer (NK) cells and frequencies and maturation state of dendritic cell subsets in the SLN were determined by flow cytometry. RESULTS: Melanoma-specific CD8(+) T-cell response rates against >1 MAA epitope in the SLN were 0 of 11 for the saline group versus 5 of 10 for the PF-3512676-administered group (P = 0.012). Of these 5 responding patients, 4 also had a measurable response to >1 MAA epitope in the blood. Increased frequencies in the SLN of both MAA-specific CD8(+) T cells and NK cells correlated to CpG-induced plasmacytoid dendritic cell maturation. CONCLUSIONS: These data show an increase in melanoma-specific CD8(+) T-cell frequencies as well as an increased effector NK cell rate after a single dose of PF-3512676 and thus support the utility of local PF-3512676 administration as adjuvant treatment in early-stage melanoma to try and halt metastatic spread.     

Interleukin-12-responding asialoGM1+CD8+ central memory-type T cells as precursor cells for interferon-gamma-producing killer T cells

While investigating CD8(+) memory T cells in unimmunized C57BL/6 mice, we found that there were unique memory-type CD8(+) T cells expressing asialoGM1 (ASGM1), CD62L and CCR7 cell surface molecules, which occupied approximately 10% of CD8(+) T cells and 35% of CD44(+) memory CD8(+) T cells. Culture of freshly isolated ASGM1(+)CD8(+) T cells with interleukin (IL)-12 plus IL-2 caused the proliferation and generation of killer T cells. Moreover, ASGM1(+)CD8(+) T cells, but not ASGM1(-)CD8(+) T cells, produced high levels of interferon (IFN)-gamma in response to IL-12 plus IL-2. Although ASGM1(+)CD8(+) T cells showed no significant responses to IL-12 alone or IL-2 alone, pulse incubation of ASGM1(+)CD8(+) T cells with IL-12 at an earlier time (0-12 h), and subsequently with IL-2 at a later time (12-24 h), caused the same levels of proliferation, killer cell generation and IFN-gamma production as when they were incubated simultaneously with IL-12 plus IL-2 for 24 h. Thus, ASGM1(+)CD8(+) T cells appeared to respond to IL-12 directly to acquire IL-2 responsiveness and differentiate into IFN-gamma-producing killer T cells. Indeed, freshly isolated ASGM1(+)CD8(+) T cells, but not ASGM1(-)CD8(+) T cells, expressed higher levels of IL-12R beta2 mRNA. The fact that IL-12 administration in vivo caused the generation of ASGM1(+)CD8(+) killer T cells in an IFN-gamma-dependent manner further indicated a physiological significance of ASGM1(+)CD8(+) central memory-type T cells in IL-12-induced immunoregulation for the therapy of tumors and infectious diseases.     

Role of NKG2D in cytokine-induced killer cells against multiple myeloma cells

The cytokine-induced killer cells (CIK) have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanisms that CIK cell recognizing MM cells remain unknown. Recent studies indicated that the interaction between NKG2D receptor and NKG2D ligands plays an important role in inducing cytotoxicity against various target cells by natural killer cells (NK). We suspect whether NKG2D receptor and NKG2D ligands interaction is also responsible for the killing of MM cells by CIK as the same way did NK cells. We expanded CIK cells from healthy controls with interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and interleukin-2 (IL-2), and checked expression of NK cell receptors on CIK cells by flow cytometry. About 86% bulk CIK cells expressed NKG2D receptor but not other NK receptors, such as CD158a, CD158b and NCRs. We analyzed NKG2D ligands expression in MM patients by flow cytometry, primary plasma cells from 8 out of 13 (62%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. Interestingly, when stimulated with MM cell line U266 that expressed some levels of MICA/B, only NKG2D expressing CIK cells released IFN-γ. CIK cells showed cytotoxicity against NKG2D ligands expressing U266 and primary MM cells, and the cytotoxicity was partially blocked by treating CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligand interaction may be one of the mechanisms by which CIK cells kill MM cells.     

Increase of regulatory T cells in the peripheral blood of cancer patients.

PURPOSE: T cells constitutively expressing both CD4 and CD25are essential for maintenance of self-tolerance and therefore have been referred to as regulatory T cells (Treg). Experimental tumor models in mice revealed that Tregs are potent inhibitors of an antitumor immune response. The current study was designed to determine whether cancer patients exhibit an expanded Treg pool. EXPERIMENTAL DESIGN: The frequency of Tregs in the peripheral blood of 42 patients suffering from epithelial malignancies and from 34 healthy controls was determined by flow cytometry. The immunoregulatory properties of CD4(+)CD25(+) and CD4(+)CD25(-) T cells were characterized by proliferation and suppression assays. Cocultures with natural killer (NK) cells were performed to determine the impact of Tregs on NK-mediated cytotoxicity. RESULTS: Patients with epithelial malignancies show an increase of CD4(+)CD25(+) T cells in the peripheral blood with characteristics of Tregs, i.e., they are CD45RA(-), CTLA-4(+), and transforming growth factor beta(+). Notably, CD4(+) T cells from cancer patients are characterized by an impaired proliferative capacity, which is restored to the extend of CD25-depleted CD4(+) T cells from control persons by prior removal of CD25(+) T cells. In contrast to CD4(+)CD25(-) T cells, isolated CD4(+)CD25(+) T cells from cancer patients were anergic towards T cell receptor stimulation. In addition, CD4(+)CD25(+) T cells suppressed the proliferation of CD4(+)CD25(-) T cells. When cultured together with CD56(+) NK-cells, CD4(+)CD25(+) T cells from cancer patients effectively inhibited NK-cell-mediated cytotoxicity. CONCLUSIONS: Thus, we provide evidence of an increased pool of CD4(+)CD25(+) regulatory T cells in the peripheral blood of cancer patients with potent immunosuppressive features. These findings should be considered for the design of immunomodulatory therapies such as dendritic cell vaccination.     

In vivo antitumor activity of interleukin 21 mediated by natural killer cells

Immunotherapy with high-dose interleukin (IL) 2 has been shown to successfully treat tumors in animal models and cause dramatic tumor regressions in some patients with metastatic melanoma, renal cell carcinoma, and non-Hodgkin's lymphoma. However, toxicity associated with IL-2 administration has compromised its widespread use in the clinic. IL-21 is a more recently discovered cytokine produced by activated CD4(+) T cells that shares significant sequence homology to IL-2, IL-4, and IL-15. Because IL-21 and IL-2 and their receptors share significant sequence similarities and both cytokines can stimulate T and natural killer (NK) cells, we sought to study whether IL-21, like IL-2, exhibits antitumor effects in vivo. In this study, we treated established s.c. tumor in mice by systemically administering plasmid DNA encoding murine IL-21 using a hydrodynamics-based gene delivery technique. Administration of IL-21 plasmid DNA resulted in high levels of circulating IL-21 in vivo. Treatment of tumor-bearing mice with IL-21 plasmid DNA significantly inhibited the growth of B16 melanoma and MCA205 fibrosarcoma in a dose-dependent manner without significant toxicity and increased the survival rate, compared with mice treated with control plasmid DNA. In vivo depletion of either CD4(+) or CD8(+) T cells did not affect IL-21-mediated antitumor activity. However, depletion of NK cells completely abolished IL-21-induced tumor inhibition. Consistent with this, the antitumor activity of IL-21 seemed to be mediated through enhanced cytolytic activity of NK cells. Our study suggests that IL-21 has significant antitumor activity and may have therapeutic potentials as an antitumor agent in the clinic.     

The increase of CD57+ T cells in the peripheral blood and their impaired immune functions in patients with advanced gastric cancer

In addition to natural killer (NK) cells, T cells expressing natural killer cell markers, CD56 or CD57 (NK type T cells), have been considered to play an important role in antitumor immunity. We examined the proportion of NK cell and NK type T cell subsets in the peripheral blood from patients with gastric cancer. The IFN-gamma production capacity and population of cytoplasmic perforin positive cells in peripheral blood mononuclear cells (PBMC) were evaluated. Peripheral blood samples were obtained from 56 patients with gastric cancer and 21 healthy volunteers. The proportion of CD56- CD57+ T cells (CD57+ T cells) was significantly higher in advanced gastric cancer patients than those in healthy volunteers and patients with early stage gastric cancer, whereas no correlation was observed between the proportion of CD56+ T cells or NK cells and tumor progression. Furthermore, a significant decrease of CD8+ CD57+ T cells was found in patients with advanced gastric cancer. The proportion of CD57+ T cells did not correlate with interferon-gamma (IFN-gamma) production from PBMC in gastric cancer patients, although a significant correlation was found between them in healthy volunteers. The proportion of perforin positive CD57+ T cells, especially CD8+ CD57+ T cells, in patients with gastric cancer was markedly lower than that in healthy volunteers. Collectively, although the proportion of CD57+ T cells in PBMC was found to increase with tumor progression, their function in antitumor immunity is impaired in patients with gastric cancer.     

Pilot trial of interleukin-2 with granulocyte colony-stimulating factor for the mobilization of progenitor cells in advanced breast cancer patients undergoing high-dose chemotherapy: expansion of immune effectors within the stem-cell graft and post-stem-cell infusion.

PURPOSE: To evaluate whether administration of interleukin-2 (IL-2) with granulocyte colony-stimulating factor (G-CSF) improves mobilization of immune effector cells into the stem-cell graft of patients undergoing high-dose chemotherapy and autografting. PATIENTS AND METHODS: We performed a trial of stem-cell mobilization with IL-2 and G-CSF in advanced breast cancer patients receiving high-dose chemotherapy with cyclophosphamide, thiotepa, and carboplatin and stem cells followed by IL-2. The trial defined immune, hematologic, and clinical effects of IL-2 in this setting. RESULTS: Of 32 patients enrolled, nine received G-CSF alone for mobilization. Twenty-one of 23 patients mobilized with IL-2 plus G-CSF had stem cells collected with more mononuclear cells than those receiving G-CSF (19.3 v 10.4 x 10(8)/kg; P =.006), but fewer CD34(+) progenitor cells (6.9 v 22.0 x 10(6)/kg; P =.049). The IL-2 plus G-CSF-mobilized patients had greater numbers of activated T (CD3(+)/CD25(+)) cells (P =.009), natural killer (NK; CD56(+)) cells (P =.007), and activated NK (CD56 bright(+)) cells (P: =.039) than those patients mobilized with G-CSF. NK (P =.042) and lymphokine-activated killer (LAK) (P =.016) activity was increased in those mobilized with IL-2 + G-CSF, whereas G-CSF-mobilized patients had a decline in cytolytic activity. In the third week posttransplantation, immune reconstitution was superior in those mobilized with IL-2 plus G-CSF based on greater numbers of activated T cells (P =.003), activated NK cells (P =.04), and greater LAK activity (P =.003). The 16 of 21 IL-2 + G-CSF-mobilized patients with adequate numbers of stem cells (> 1.5 x 10(6) CD34(+) cells/kg) collected engrafted rapidly posttransplantation. CONCLUSION: The results demonstrate that G-CSF + IL-2 can enhance the number and function of antitumor effector cells in a mobilized autograft without impairing the hematologic engraftment, provided that CD34 cell counts are more than 1.5 x 10(6) cells/kg. Mobilization of CD34(+) stem cells does seem to be adversely affected. In those mobilized with IL-2 and G-CSF, post-stem-cell immune reconstitution of antitumor immune effector cells was enhanced.     

Killer cell Ig-like receptors ligand-mismatched, alloreactive natural killer cells lyse primary solid tumors

BACKGROUND: Donor alloreactive natural killer (NK) cells have a potent antileukemic effect in haploidentical stem cell transplantation. Whether alloreactive NK cells are able to specifically kill fresh tumor cells from primary solid tumors was analyzed. METHODS: NK cells were purified from healthy donors for the expression of inhibitory killer cell immunoglobulin (Ig)-like receptors (KIRs), ex vivo expanded, and used as effector cells. Their cytotoxic effect on tumor cells freshly obtained from surgical specimens was assessed by means of a single-cell cytotoxic assay (SCCA). RESULTS: Tumor cells from 1 ovarian, 1 gastric, 3 colon, and 4 renal cell cancers were analyzed and found susceptible to alloreactive NK cell killing (>20% lysis at an effector cell to target cell [E:T] ratio of 10:1 for tumor cells not expressing at least 1 human lymphocyte antigen [HLA] class I KIR-ligand group). Remarkably, NK cells that recognized specific HLA-C group mismatches were able to kill HLA-C KIR ligand-mismatched tumor cells, whereas no lysis of target cells occurred with KIR ligand-matched tumor targets. CONCLUSIONS: Alloreactive NK-cell mediated antitumor effects might provide useful insights for designing new cell therapy approaches against solid tumors.     

Natural killer cell lymphoma in the duodenum

We present a case of duodenal non-Hodgkin lymphoma in a 71-year-old woman. Immunohistochemistry characterized the lymphoma cells as CD2(+); surface CD3(-) but cytoplasmic CD3(+); CD7(+); and CD56(+) without a rearrangement of the T-cell receptor gene. Cells had a high N/C ratio and irregular nuclear outlines and lacked azurophilic granules and these features indicated that the lymphoma cells arose from natural killer (NK) cells. She was treated with intensive chemotherapy including pirarubicin, cyclophosphamide, vincristine, and prednisolone, but died three weeks after diagnosis. CD56(+) lymphomas originate from NK or cytotoxic T cells and are designated "extranodal NK/T-cell lymphoma, nasal type" in the WHO classification. Nasal NK cell lymphoma is most common in East Asians and CD56(+) lymphomas usually occur in the nasal area. Extranasal forms such as gastrointestinal lymphomas are very rare and usually carry a poor prognosis. Extranodal NK/T-cell lymphoma, nasal type, is characterized by a broad morphologic spectrum and have variable prognosis. These lymphomas constitute an heterogeneous group, and their subclassification has not yet been established.     

Impaired perforin-dependent NK cell cytotoxicity and proliferative activity of peripheral blood T cells is associated with metastatic melanoma.

AIMS AND BACKGROUND: Patients with metastatic melanoma often have defects in the percentage and function of peripheral blood NK cells, which are involved in the non-specific innate antitumor immune response, and T cells, which participate in the specific acquired antitumor immune response. The aim of this study was to investigate in more detail not only the percentage but also the activation status and function of NK and T cells in patients with metastatic melanoma prior to therapy. METHODS: The percentage of peripheral blood CD56+ NK cells, CD3+ T cells and their CD4+ and CD8+ subsets, as well as the expression of the activation antigens CD69, CD38 and HLA-DR were analyzed by flow cytometry. The functional capacity of NK cells was evaluated by the 51-chromium release cytotoxicity assay, while the proliferative activity of T cells was estimated by the lymphocyte transformation test to mitogen phytohemagglutinin. RESULTS: The results obtained in this study have revealed a new aspect of NK and T cell dysfunction that is not, as commonly reported, associated with a decrease in their percentage. Moreover, a significant number of the investigated patients had a higher percentage of NK cells that did not lead to improved NK cell cytotoxicity as a result of the detected defect in the NK cell perforin-mediated cytotoxic mechanism of tumor cell lysis. The impaired proliferative response of T cells was associated with a decreased expression of the activation antigen HLA-DR. CONCLUSION: The novel finding in this study of melanoma patients with metastatic disease is the impaired perforin-dependent NK cell cytotoxic mechanism, which was recently shown to be primarily responsible for preventing metastasis. Another interesting finding was the generally hyporeactive status of T cells, possibly resulting from persistent antigenic stimulation. The observed dysfunction of NK and T cells in patients with metastatic melanoma prior to therapy point to the need to supplement chemotherapy with appropriate immunotherapeutic agents in order to overcome the immunosuppression associated with advanced malignancy.     

选择性扩增人自然杀伤细胞的实验研究

背景与目的:自然杀伤(naturalkiller,NK)细胞具有很强的杀瘤活性,但体外扩增NK细胞的数量难以达到治疗肿瘤的目的。本实验探讨由人外周血单个核细胞(peripheralbloodmononuclearcells,PBMCs)和附壁Wilms肿瘤细胞株HFWT混合培养能否有效地诱导NK细胞。方法:采用PBMCs和HFWT细胞混合培养诱导扩增NK细胞,51Cr释放法和结晶紫染色法测定NK细胞的杀瘤活性;流式细胞计数仪检测培养细胞中CD3 、CD4 、CD8 、CD16 和CD56 细胞的比例。结果:HFWT对人NK细胞特别敏感,它比不表达MHC-Ⅰ类分子的K562细胞和其他附壁生长的肿瘤细胞株更有效地刺激PBMCs,从而选择性扩增NK细胞。健康人PBMCs在HFWT细胞中培养10～21天后,CD56 CD16 细胞占细胞总数50%以上。当效靶比为2∶1时,NK细胞杀伤80%的HFWT细胞,NK细胞的扩增需要HFWT细胞反复刺激PBMC。结论:从HFWT细胞株中可选择性扩增人NK细胞,将有助于开展临床肿瘤过继免疫疗法。     

A clinical study of cytokine-induced killer cells for the treatment of refractory lymphoma

Cytokine-induced killer (CIK) cell therapy, an adoptive T-cell immunotherapy, has been reported to be a safe and effective mode of treatment for patients with metastatic diseases, lymphoma and acute leukaemia. To investigate the clinical efficacy of cytokine-induced killer cells for the treatment of refractory lymphoma, the present clinical study was conducted. A total of 8 male patients with a mean age of 41 years (range 22-65) who were pathologically diagnosed with malignant lymphoma (Hodgkin's disease, 2 and non-Hodgkin's lymphoma, 6) were enrolled. CIK cells were expanded by priming with IFN-γ, monoclonal antibody (mAb) to CD3 and IL-1α, followed by the addition of IL-2 the following day using peripheral blood mononuclear cells (PBMCs) of the 8 male patients. The CIK cells were then transfused back to the patients as treatment. On day 13, the CIK cell count reached 7-18×10(19) (mean, 12.7×10(9)), a 44- to 140-fold increase (mean, 98-fold). The average percentage of cells expressing CD3(+), CD4(+), CD8(+) and CD3(+)CD56(+) were also increased from 50.9±3.5, 29.9±1.7, 41.3±3.2, 1.6±0.2% to 90.2±1.6, 40.6±5.5, 52.8±4.9 and 33.1±4.0%, respectively. Patients showed measurable radiographic tumor reduction, increased T-cell subset levels, and relief of symptoms after treatment. No severe toxicity or side effects were reported. CIK cells developed by this culture method have a high in vitro proliferation rate and tumor-killing capacity. In conclusion, CIK cell treatment of patients with malignant lymphoma achieves effective clinical responses, causing few side effects.     

CD8(+) T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1

Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T-cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8(+) T-cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8(+) T-cell expansion and function in melanoma. BTLA-expressing PD-1(+)Tim-3(-) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in patients with melanoma. These cells were partially dysfunctional, producing less IFN-γ than BTLA(-) T cells but more IFN-γ, TNF, and interleukin-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T-cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8(+) T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.     

Natural killer cells play a critical role in the immune response following immunization with melanoma-antigen-engineered dendritic cells

Tumor antigen gene-modified dendritic cells (DC) generates robust antigen-specific protective antitumor responses. Though the role of CD4 positive and CD8 positive cells in the immunological response to gene-modified DC has been well-characterized, the role of NK cells in this response has been somewhat less clear. Owing to the significant contribution of innate immunity in other model systems, we postulated that NK cells would hold a critical position in the generation of an immune response following immunization with tumor antigen-engineered DC. Immunization with MART-1 melanoma antigen-engineered DC in C57BL/6 mice resulted in the generation of antigen-specific cytotoxic T lymphocytes and in vivo protective responses to the murine B16 melanoma. These responses were dependent on the presence of functional NK cells, although NK cells alone were not sufficient in generating protective responses. Adoptive transfer of NK cells into an NK-deficient but T-cell-competent environment restored the protective response to gene-modified DC immunization. In conclusion, protective immunity after tumor antigen gene-modified DC immunization requires collaboration between CD4+ and CD8+ T cells and NK cells.