原发性肝癌和乙型肝炎肝硬化患者外周血淋巴细胞凋亡率、T细胞亚群和NK细胞的水平变化和临床意义

目的通过检测乙型肝炎肝硬化和合并乙型肝炎病毒感染的原发性肝细胞癌患者的外周血的粒细胞、单核细胞、NK细胞、T细胞及其亚群和淋巴细胞及其凋亡率,探讨两者在细胞免疫方面的差异.方法用Ficoll Hypaque离心法分离外周血单个核细胞(PBMNC),流式细胞仪检测外周血T细胞及其亚群、NK细胞和淋巴细胞、单核细胞和粒细胞,AnnexinV/FITCKit检测凋亡细胞.结果乙型肝炎肝硬化患者的外周血单核细胞、粒细胞、淋巴细胞、CD3-CD16 CD56 NK细胞、CD3 T细胞、CD3 CD4 T细胞、CD3 CD4 T细胞和CD4 CD8 T细胞比值,均与正常对照组无显著性差异(P＞0.05);但淋巴细胞凋亡率明显低于对照组(P＜0.01).原发性肝癌外周血CD3-CD16 CD56 NK细胞、单核细胞和CD4 /CD8 T细胞比值与肝硬化组和正常对照组比较,均无显著性差异(P＞0.05),而粒细胞明显升高(P＜0.05);CD3 T细胞、CD3 CD4 T细胞和CD3 CD8 T细胞均较另两组明显减少(P＜0.05),淋巴细胞及其凋亡率均明显低于另两组(P＜0.01).结论乙型肝炎肝硬化患者的外周血细胞免疫只发生不显著的变化,但淋巴细胞的凋亡率明显降低.原发性肝癌外周血的细胞免疫和淋巴细胞凋亡率均明显低下.     

Role of monocytes in the expansion of human activated natural killer cells.

We have studied the mechanisms underlying expansion of recombinant interleukin-2 (rIL-2)-stimulated natural killer (NK) cells in vitro. A population of NK cells expressing the CD56+/CD3-phenotype (98.9% +/- 0.42%) was obtained from normal human peripheral blood mononuclear cells (PBMNC) by fluorescence-activated cell sorting (FACS). Culture of NK cells in media containing rIL-2 (1,000 U/mL) for 18 days resulted in a population of activated NK cells (ANK) with significantly enhanced cytotoxicity, but only 2.6 +/- 0.56-fold expansion of cell number compared with the starting NK population. Culture of starting NK populations and autologous PBMNC in a Transwell system (Costar, Cambridge, MA), providing separation of the two cell populations by a 0.4-microns pore membrane, resulted in a dose-dependent increase in fold expansion of ANK (expansion = 19.9 +/- 4.0-fold; P < .001; n = 22) significantly greater than that observed when NK were cultured alone. Further experiments using the Transwell system showed that the stimulatory effect of autologous PBMNC on ANK progenitor proliferation resides in the CD14+ monocyte fraction (maximal expansion = 14.5 +/- 1.5-fold; n = 17) and not in the CD5+ T-lymphocyte or CD19+ B-lymphocyte fractions. Direct coculture of purified NK and autologous monocytes in the same compartment, thus permitting cell-cell contact, resulted in significantly greater expansion of the ANK population (30.6 +/- 4.7-fold expansion, P < .001; n = 10) than that observed when NK and monocytes were separated by the Transwell membrane. Finally, depletion of PBMNC of cells bearing CD5 and CD8 by panning on antibody-coated plastic flasks resulted in a starting cell population enriched for NK progenitors and for monocytes. Cultures of this resultant population for 18 days in the presence of rIL-2 yielded an ANK population similar to that obtained when CD56+/CD3- cells obtained by FACS were cocultured with autologous monocytes. These results suggest that proliferation of ANK requires autologous monocytes and is in part mediated by humoral factors, but is enhanced when NK and monocytes are in direct cell-cell contact. Depletion of cells bearing CD5 and CD8 from PBMNC is a single efficient method for obtaining a starting population capable of producing large numbers of ANK in culture that may lead to new therapeutic uses for the ANK population.     

Peripheral lymphocytes and intracellular cytokines in C282Y homozygous hemochromatosis patients

BACKGROUND/AIMS: Several abnormalities in the immune status of hereditary hemochromatosis patients have been reported. We evaluated the peripheral blood lymphocytes phenotype and cytokine profile of CD8(+) and CD4(+) T cells in C282Y homozygous hereditary hemochromatosis patients compared to control subjects. METHODS: Peripheral blood lymphocytes from 17 asymptomatic patients and 14 control subjects were analyzed. We determined the distribution of lymphocyte subsets and investigated at single-cell level by flow-cytometry the potential of cytokines production. The frequency of cytokine (interferon gamma, tumor necrosis factor alpha, interleukin 2 (IL-2), IL-4, IL-5, IL-10 and IL-13) producing cells was assessed in total T-lymphocytes, CD3(+)CD8(+) and CD3(+)CD4(+) subsets. RESULTS: The patients studied showed a significant decrease of total lymphocyte count, T CD4(+)CD3(+), CD28(+), CD8(+)CD28(+) lymphocytes and natural killer (NK) CD56(+)CD16(+)CD3(-) cells. The reduction of CD28(+) and CD8(+)CD28(+) lymphocyte count was inversely related to transferrin saturation index. An increase in the ability of T-cells to produce all the cytokines studied and a major increase in IL-4 and IL-10 production in the CD3(+)CD8(+) subset was found. Our results demonstrate that activated Th1 and Th2 lymphocytes coexist in the peripheral blood of hereditary hemochromatosis patients and that T-cytotoxic (Tc) 2 subset is more expanded than in control population. CONCLUSIONS: The association of a decreased number of T CD8(+) cytotoxic lymphocytes and NK cells, and the development of Tc2 cells in asymptomatic C282Y homozygous patients represents an imbalance in their immune function that might contribute to the high incidence of hepatocarcinoma.     

Granulocyte-macrophage colony-stimulating factor and interleukin-3 mRNAs are produced by a small fraction of blood mononuclear cells

Northern blot analysis has identified granulocyte macrophage colony stimulating factor (GM-CSF) mRNA in monocytes and both GM-CSF and interleukin-3 (IL-3) mRNA in lymphocytes. However, these results have not addressed whether all cells or a subset of the population is capable of hematopoietic growth factor (HGF) production. To resolve this question, we applied in situ hybridization of radiolabeled antisense RNA probes to centrifuged preparations of total blood mononuclear cells (BMCs) and fractionated lymphocyte subpopulations. Without stimulation, no circulating cells expressed detectable levels of GM-CSF or IL-3 mRNA. On stimulation of BMCs with phorbol myristate acetate (PMA) and phytohemagglutinin or PMA and the calcium ionophore ionomycin, approximately 5% expressed GM-CSF mRNA and approximately 1% IL-3 mRNA. Control sense probes produced no labeled cells. To determine the subsets of lymphocytes capable of GM-CSF and IL-3 expression, BMCs were fractionated by FACS into CD8+ and CD4+ lymphocyte subsets and CD16+ (NK) cells. The unfractionated cells and cell fractions were then stimulated with PMA and ionomycin. Results demonstrated that 3% to 5% of the CD16+, CD8+, and CD4+ lymphocytes produced GM-CSF mRNA. However, the number of IL-3 mRNA-positive cells in the FACS-sorted subsets was greatly reduced (0.02% to 0.05%) as compared with the unseparated cells (1%). Treatment of BMCs with high-dose interleukin-2 (IL-2) for 1 week followed by PMA plus ionomycin resulted in a lymphocyte population in which 50% and 3% of cells expressed GM-CSF and IL-3 mRNA, respectively. Thus, GM-CSF and IL-3 mRNA expression in T cells and NK cells is restricted to a small fraction of cells that can be greatly expanded by IL-2 stimulation. These results suggest a possible physiologic mechanism for increasing HGF production by circulating lymphocytes.     

Natural killer cell activity in Sj?gren's syndrome and systemic lupus erythematosus: stimulation with interferons and interleukin-2 and correlation with immune complexes.

Natural killer (NK) cell activity and its stimulation by interferons (IFNs) and interleukin-2 (IL-2) are diminished in Sjögren''s syndrome and systemic lupus erythematosus (SLE). Serum samples of these patients often contain circulating immune complexes, which may influence NK cell activity. Sixteen patients with Sjögren''s syndrome (14/16 immune complex positive), 14 with SLE (9/14 immune complex positive), and 11 controls (immune complex negative) were studied. Mononuclear cells collected from a Percoll gradient were preincubated with recombinant IFN-alpha (rIFN-alpha) (100 U/ml), rIFN-gamma (1000 U/ml), rIL-2 (100 U/ml), or without cytokine. Natural killer cell activity was determined by incubating the mononuclear cells with carboxyfluorescein labelled K562 cells, and the percentage decrease of fluorescence was measured on an FACS Analyzer. In patients with Sjögren''s syndrome and SLE NK cell activity and the numbers of cells expressing the NK cell associated antigens CD16 and Leu7 were diminished compared with the controls. Interleukin-2 stimulated NK cell activity significantly in comparison with the non-stimulated value in all studied groups, whereas IFN-gamma only stimulated NK cell activity in both patient groups and IFN-alpha only in patients with Sjögren''s syndrome. There was no correlation between NK cell activity, with or without stimulation, and the immune complex concentrations. It is concluded that NK cell activity is decreased in Sjögren''s syndrome and SLE and that it may be partially restored by IL-2 and IFN-gamma in both diseases, and by IFN-alpha in Sjögren''s syndrome. The decrease of NK cell activity did not correlate with immune complex concentrations; on the other hand, decreased numbers of NK cells (CD16+ or Leu7+) and of cytokine concentrations might be important in the impaired NK cell activity in both diseases.     

腹腔镜和开腹直肠癌根治术对机体免疫功能的影响

目的探讨腹腔镜直肠癌根治术(Miles)围手术期免疫功能的变化。方法40例行Miles的患者分为2组,腹腔镜组(L组)和传统开腹组(O组),每组20例。分别于术前1d,术后第1天、第2天、第3天抽取患者外周静脉血,检测辅助T淋巴细胞(TH)2个亚群Th1、Th2细胞含量、Th1/Th2及白细胞介素-2(IL-2)、IL-6含量。术前1d和术后第3天检测全血CD3 CD56 T细胞和CD3-CD56 自然杀伤(NK)细胞的百分比。IL-2、IL-6的检测用酶联免疫吸附法(ELISA),CD3 CD56 T细胞和CD3-CD56 NK细胞用流式细胞仪进行检测。结果术后第1天,L组Th1细胞含量高于O组(P<0.05);术后第1天、第2天、第3天,L组Th2细胞含量低于O组,Th1/Th2高于O组(P<0.05);术后第2天,L组IL-2含量高于O组,术后第1天L组IL-6含量低于O组(P<0.05)。2组手术对CD3 CD56 T细胞、CD3-CD56 NK细胞的影响差异有显著性意义(P<0.05)。结论Miles对患者术后免疫功能的抑制有一定的保护作用,对肿瘤患者的预后有益。     

Immune reconstitution following allogeneic peripheral blood stem cell transplants

Growth factor-mobilized peripheral blood stem cells (PBSCs) engraft rapidly in myeloablated recipients compared to conventional BM, but this procedure also mobilizes mature lymphocytes and monocytes which can impact immune reconstitution and GVHD. Hence, we serially evaluated immune reconstitution and cytokine expression in PBSCT recipients in the first year. Engraftment of neutrophils and monocytes stabilized early but NK cells, B cells and CD4+ T cell numbers were significantly (P < 0.05) low with persistently reversed CD4:CD8 ratios. NK function remained low throughout the first year. The quantitative decrease in CD4+ T cells resulted in significantly decreased proliferation in response to mitogens and alloHLA antigens. Yet, a qualitative analysis of T cell function measured by Ca++ influx after T cell activation with antiCD3 as well as T-dependent polyclonal Ig secretion by mitogen-stimulated B cells was preserved even early post transplant. TNF alpha mRNA was detected in almost all recipients in the first year. IL-10 mRNA was detected in 77%, IL-2 in 22% and IFN gamma in 44% of recipients in the first 6 months. Only 30% expressed IL-10 in the second 6 months post transplant while expression of IL-2 and IFN gamma was detected in 38% and 46% respectively. Thirty-seven percent of PBSCT recipients developed grades II-IV acute GVHD but 72% went on to develop chronic extensive GVHD at a median of 120 days. Sixty-two percent developed CMV viremia and 5.4% developed overt CMV disease in the first year post PBSCT. Lymphocyte engraftment is quantitatively delayed but CD4 functions are preserved while NK numbers and function are compromised post PBSCT. IL-10 expression decreases after the first 6 months post transplant while TNF alpha is continually expressed. The balance between quantitative lymphocyte reconstitution and qualitative lymphocyte functions as well as changes in lymphokine patterns may influence infection and GVHD and thus the clinical outcome post PBSCT.     

Effect of low-dose IL-2 immunotherapy on frequency and phenotype of regulatory T cells and NK cells in HIV/HCV-coinfected patients

We evaluated the effect of low-dose IL-2 therapy (daily 1.2 MIU/m(2), subcutaneously) on the number and phenotype of regulatory T cells (T(regs)) and natural killer (NK) cells in HIV/HCV-coinfected patients taking antiretroviral therapy. The frequency and phenotype of circulating T(regs) (defined as CD3(+) CD4(+) CD25(high) or CD3(+) CD4(+) FOXP3(+)) and NK cells (CD3(-) CD16(+)/CD56(+)) were evaluated at baseline and after 12 weeks of treatment. The expression of CD25, CTLA-4, and granzymes A and B by CD4(+) FOXP3(+) cells, as well as the expression of KIR receptors (NKB1, CD158a, and NKAT2) on NK cells, was evaluated. Low doses of IL-2 resulted in the augmented frequency and absolute number of T(regs) in coinfected individuals. FOXP3 levels per cell as well as augmented CD25 and CTLA-4 expression by T(regs) suggested that IL-2 may lead to both expansion and activation of T(regs), although changes in the proportion of CD4(+) FOXP3(+) cells were not associated with changes in HCV viral load and CD4(+) cells between baseline and week 12. NK cell frequency also increased after IL-2 therapy. Interestingly, the pattern of expression of KIR receptors was changed by IL-2 treatment, since the frequency of NK cells expressing NKB1 augmented whereas the frequency of NK expressing CD158a and NKAT2 decreased.     

Analysis of immunologic mechanisms of high natural killer cell activity in tuberculous pleural effusions

We found natural killer (NK) activity to be high in tuberculous pleural effusions from which Mycobacterium tuberculosis was cultured. In this study, we investigated and compared the mechanisms of this NK activity in tuberculous pleural effusions and peripheral blood. Cytotoxicity was augmented in tuberculous pleural effusion mononuclear cells (PEMNC) and peripheral blood mononuclear cells (PBMNC) by culture with purified protein derivative (PPD). Prior to culture with PPD, there were many more interleukin-2 receptors (IL-2R, p55) on Leu 11+ (CD16+) cells in tuberculous PEMNC than in tuberculous PBMNC. After 5 days of culture with PPD, the numbers of IL-2R increased in both PEMNC and PBMNC. PPD-induced cytotoxicity was inhibited by the addition of anti-IL-2R monoclonal antibody (anti-IL-2R mAb). Interferon-gamma (IFN-gamma) production was abundant in the supernatants of cultured tuberculous PEMNC and PBMNC, but very little interleukin-2 (IL-2) and IFN-alpha and -beta were produced. Considerably more IFN-gamma was produced in tuberculous PEMNC than in tuberculous PBMNC, and PPD-induced cytotoxicity was inhibited by anti-IFN-gamma monoclonal antibody (anti-IFN-gamma mAb). Cell surface analysis after PPD-induced cytotoxicity was performed by complement-mediated cytolysis. Leu 11+ cells exhibited essential cytotoxicity in the induction and effector phases, but neither OKT4+ (CD4+) nor OKT8+ (CD8+) cells displayed such cytotoxicity. These data suggest that Leu 11+ cells in tuberculous PEMNC are activated by M. tuberculosis through IFN-gamma and that IL-2R plays an important role in the activation of NK cells.     

Intracellular cytokine production and cytokine receptor interaction of cord mononuclear cells: relevance to cord blood transplantation

A 'cytokine storm' consisting of IL-1, IL-2, IL-12, IFNgamma and TNFalpha is considered important in the development of graft-versus-host disease (GvHD). These cytokines activate effector cells or damage host tissues. Cord blood transplantation has been associated with a low incidence of GvHD. We hypothesized that the low incidence of GvHD relates to the cord mononuclear cells being poor producers of pro-inflammatory cytokines. The cytokine profile (IL-1alpha/beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFNgamma and TNFalpha) of cord blood cells induced by immune stimuli was determined in heparinized whole blood. Compared to adult, cord blood CD3+ and NK cells produced less IFNgamma, less cord blood CD3+ cells and monocytes produced TNFalpha and less monocytes produced IL-1alpha/beta. Although more cord T cells produced IL-2 compared to adult T cells at 4 h, adult T cells produced more at 24 h. Cord blood had similar proportions of monocytes to adult producing IL-6, IL-10 and IL-12. Both adult and cord mononuclear cells constitutively expressed receptors for IFNgamma and TNFalpha but not IL-12. In contrast to the adult cells, cord CD3+ and NK cells did not express IL-12 receptor but did up-regulate IL-10 receptor after mitogenic stimulation. The findings of this study indicate that the cord blood cytokine-receptor network is biased towards anti-inflammatory activity compared to adult and helps to explain the decreased incidence of GVHD in cord blood transplantation.     

HCV感染患者免疫细胞部分细胞因子分泌情况的初步研究

目的研究与正常人比较丙型肝炎病毒（HCV）感染患者免疫细胞分泌γ干扰素（IFN-γ）、白细胞介素10（IL-10）、肿瘤坏死因子α（TNF-α）及白细胞介素4（IL-4）的细胞频数情况,了解HCV感染对其影响。方法分离外周血单核细胞（PBMCs）,应用IL-10、IFN-γ、TNF-α及IL-4流式抗体进行细胞内因子染色,应用FACSCalibur流式细胞仪及FACSCalibur软件进行检测分析。结果HCV感染患者分泌IL-10、IFN-γ、IL-4的细胞频数在CD4＋CD8-T细胞、CD4-CD8＋T细胞、NK细胞和NKT细胞均发生明显下降;分泌TNF-α的细胞频数在CD4-CD8＋T淋巴细胞及NK细胞出现下降;HCV感染患者NK细胞、NKT细胞分泌IL-10和IFN-γ的细胞频数较CD4＋CD8-T淋巴细胞、CD4-CD8＋T淋巴细胞下降得更加明显。结论HCV感染患者的细胞免疫功能受到明显的损害,细胞因子分泌能力明显减低;固有免疫细胞功能受损可能是HCV感染慢性化的重要原因;细胞因子分泌的调节是抗HCV感染免疫治疗过程中需要调节的方向。     

Impaired activity of natural killer cells in patients with acute brucellosis.

Researchers have claimed that natural killer (NK) cells are involved in the mechanisms of defense of the host against infections. We have investigated the activity of NK cells in peripheral blood mononuclear cells (PBMNC) from 12 patients for whom acute brucellar infection has been diagnosed and from 14 healthy controls. The sera of eight of the patients were also analyzed 3 months after initiation of a 45-day course of antibiotic treatment, at which time they had no evidence of relapse. PBMNC from patients with acute brucellar infection showed a significantly depressed NK cell activity (P < .01) when compared with those from healthy controls; this depressed activity was not related to a deficient number of NK cells since the numbers of CD56+ and CD16+ cells present in PBMNC were similar in patients and controls. Incubation of PBMNC from patients with acute brucellar infection with recombinant interleukin-2, but not with interferon-gamma, can correct this impaired cytotoxic activity. In treated patients, there was a significant enhancement (P < .05) and normalization of the previously defective NK cell activity. It is concluded that acute brucellar infection is associated with a deficient cytotoxic activity of NK cells that can be overcome by in vitro incubation with interleukin-2 and that reverts to normal after antibiotic treatment.     

Comparative evaluation of immune response after laparoscopical and open total mesorectal excisions with anal sphincter preservation in patients with rectal cancer

AIM: The study of immune response of open versus laparoscopical total mesorectal excision with anal sphincter preservation in patients with rectal cancer has not been reported yet. The dissected retroperitoneal area that contacts directly with carbon dioxide is extensive in laparoscopic total mesorectal excision with anal sphincter preservation surgery. Tt is important to clarify whether the immune response of laparoscopic total mesorectal excision with anal sphincter preservation (LTME with ASP) in patients with rectal cancer is suppressed more severely than that of open surgery (OTME with ASP). This study was designed to compare the immune functions after laparoscopic and open total mesorectal excision with anal sphincter preservation for rectal cancer.METHODS: This study involved 45 patients undergoing laparoscopic (n=20) and open (n=25) total mesorectal excisions with anal sphincter preservation for rectal cancer.Serum interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor α (TNFα) were assayed preoperatively and on days 1 and 5 postoperatively. CD3+ and CD56+ T lymphocyte count, CD3- and CD56+ natural killer cell (NK)count and immunoglobulin (IgG/IgM/IgA) were assayed preoperatively and on day 5 postoperatively. The numbers of CD3+ and CD56+ T lymphocytes and CD3- and CD56+ NK cells were counted using flow cytometry. An enzyme-linked immunosorbent assay (ELISA) was used for IL-2, TL-6 and TNFα determination. And IgG, IgM, and IgA were assayed using immunonephelometry.RESULTS: The demographic data of the two groups had no difference. The preoperative levels of CD3+ and CD56+ T lymphocyte count, CD3- and CD56+ NK count, serum IgG,IgM, IgA, IL-2, IL-6 and TNFα also had no significant difference in the two groups (P＞0.05). The CD3+ and CD56+ T lymphocyte counts had no obvious changes after surgery in laparoscopic (d=-0.79±3.83 %) and open (d=0.42±2.09 %)groups. The CD3- and CD56+ NK counts were decreased postoperatively in both laparoscopic (d=-7.23±11.33 %) and open (d=-9.21±13.93 %) groups. The differences of the determined values of serum IgG, IgM and IgA on the fifth day after operation subtracted those before operation were -2.56±2.14 g/L, -252.35±392.94 mg/L, -506.15±912.24 mg/L in laparoscopic group, and -1.81±2.10 g/L, -282.72±356.75mg/L, -252.20±396.28 mg/L in open group, respectively. The levels of IL-2 were decreased after operation in both groups.However, the levels of IL-6 were decreased after laparoscopic surgery (d1=-23.14±263.97 ng/L and d5=-40.08±272.03 ng/L),and increased after open surgery (d1=27.38±129.14 ng/L and d5=21.67±234.31 ng/L). The TNFα levels were not elevated after surgery in both groups. There were no significant differences in the numbers of CD3+ and CD56+ T lymphocytes and CD3- and CD56+ NK cells, the levels of IgG, IgM, IgA,IL-2, IL-6 and TNFα between the two groups (P＞0.05).CONCLUSION: There are no differences in immune responses between the patients having laparoscopic total mesorectal excision with anal sphincter preservation and those undergone open surgery for rectal cancer.     

Functional impairment of natural killer cells in active ulcerative colitis: reversion of the defective natural killer activity by interleukin 2.

We have studied the functional characteristics and clinical importance of the natural killer (NK) cytotoxicity of peripheral blood mononuclear cells (PBMNC) from patients with ulcerative colitis. Normal NK activity was observed in PBMNC from patients with inactive disease, but a pronounced decrease was found in those with active disease. Clinical change from active to inactive disease was associated with enhancement of the depressed NK activity. The impairment of NK cytotoxicity found in patients with active disese could not be ascribed to a deficient number of NK cells as the amounts of HNK-1+, CD16+ (Leu 11), and CD11b (OKM1) cells in PBMNC were within normal ranges. This defective cytotoxic PBMNC activity was normalised by short term (18 hour) incubation with recombinant interleukin 2 (rIL-2). Moreover, long term (5 day) incubation of these effector cells with rIL-2 induced strong cytotoxic activity against NK resistant and NK sensitive target cells in patients with active and inactive disease. We also found that both precursors and effectors of cytotoxic activity promoted by short term and long term incubation with rIL-2 of PBMNC from the patients showed the phenotype of NK cells (CD16+, CD3-). Taken together, these results show that active ulcerative colitis is associated with a defective function of NK cells that is found to be normal in the inactive stage of the disease. The possible pathogenic and therapeutic implications of these findings are discussed.     

从Th1/Th2、Treg/Th17之间的平衡研究轻、中度慢性乙型肝炎患者CD4~+T淋巴细胞的免疫失衡状态

目的从Th17及其相关细胞因子与Th1、Th2、Treg的相互关系,探讨轻、中度CHB患者CD4~+T淋巴细胞的整体免疫失衡状态。方法选择轻、中度CHB患者,与健康志愿者进行比较,观察其外周血各免疫细胞(Th17、Th1、Th2、Treg、Th1/Th2、Treg/Th17)、相关细胞因子(IL-17、IL-6、TGF-β、IFN-γ、IL-4)、主要效应分子(穿孔素、颗粒酶B)和核转录因子(孤独核受体RORγt、Foxp3等)的变化及其相互关系。结果与健康志愿者相比,CHB患者CD8~+T淋巴细胞比例更高,CD4~+/CD8~+比例显著下降,差异有统计学意义(P0.01);与健康志愿者相比,CHB患者Th1细胞比例更高,Th1/Th2显著升高,差异有统计学意义(P0.01);与健康志愿者相比,CHB患者Th17细胞比例更高,Treg/Th17比例显著下降,差异有统计学意义(P0.01);与健康志愿者相比,CHB患者穿孔素和颗粒酶B的表达更高,差异有统计学意义(P0.01);与健康志愿者相比,CHB患者FOXP3、IL17mRNA和RORγt的表达差异无统计学意义(P0.05);与健康志愿者相比,CHB患者TGF-B1、IL-6、IL-17显著增高,差异有统计学意义(P0.01);IFN-γ和IL-4的表达差异无统计学意义(P0.05)。结论轻、中度CHB患者在免疫细胞及其相关细胞因子、效应分子等各环节,尤其是Treg/Th17比例,均处于严重的免疫失衡状态。     

CD16- CD56+ natural killer cells after bone marrow transplantation.

Natural killer (NK) cells are phenotypically defined as lymphocytes expressing the antigens CD56 and mostly CD16 (Fc gamma RIII), but lacking CD3. A small CD3- CD16- CD56+ NK cell subset has been described in normal individuals representing less than 2% of peripheral blood lymphocytes. We analyzed here 70 patients for their reconstitution of the immune system during follow-up after autologous or allogeneic bone marrow transplantation. In 35% of these patients, two different NK cell subsets, namely CD56+dim and CD56+bright cells, were observed. The mean duration of these two subsets after transplant was 4 months. Sixty-five percent of the patients exhibited an increased number of NK cells, but only the typical CD16+ CD56+dim population. The CD56+bright subpopulation represented a particular CD3- CD16- NK subset, with posttransplant frequencies up to 70% of all NK cells and 40% of peripheral blood lymphocytes, respectively. In contrast to normal CD56+dim NK cells, CD56+bright cells coexpressed the activation antigens p75 beta-chain of interleukin-2 receptor (IL-2R), CD2R, and CD26, but were negative for CD16. NK and antibody-dependent cellular cytotoxicity activity of CD56+bright cells was low compared with CD56+dim NK cells. But using IL-2 and interferon gamma, their cytotoxicity could be enhanced even more than in CD56+dim lymphocytes. These different subsets may reflect distinct activation or differentiation steps of NK cells during reconstitution of the immune system. Their differential response to IL-2 may be of functional importance for posttransplant cytokine therapy.     

Local and systemic effects of an allogeneic tumor cell vaccine combining transgenic human lymphotactin with interleukin-2 in patients with advanced or refractory neuroblastoma

In murine models, transgenic chemokine-cytokine tumor vaccines overcome many of the limitations of single-agent immunotherapy by producing the sequence of T-cell attraction followed by proliferation. The safety and immunologic effects of this approach in humans were tested in 21 patients with relapsed or refractory neuroblastoma. They received up to 8 subcutaneous injections of a vaccine combining lymphotactin (Lptn)- and interleukin-2 (IL-2)-secreting allogeneic neuroblastoma cells in a dose-escalating scheme. Severe adverse reactions were limited to reversible panniculitis in 5 patients and bone pain in 1 patient. Injection-site biopsies revealed increased cellularity caused by infiltration of CD4+ and CD8+ lymphocytes, eosinophils, and Langerhans cells. Systemically, the vaccine produced a 2-fold (P =.035) expansion of CD4+ T cells, a 3.5-fold (P =.039) expansion of natural killer (NK) cells, a 2.1-fold (P =.014) expansion of eosinophils, and a 1.6-fold (P =.049) increase in serum IL-5. When restimulated in vitro by the immunizing cell line, T cells collected after vaccination showed a 2.3-fold increase (P =.02) of T-helper (TH2)-type CD3+IL-4+ cells. Supernatant collected from restimulated cells showed increased amounts of IL-4 (11.4-fold; P =.021) and IL-5 (8.7-fold; P =.002). Six patients had significant increases in NK cytolytic activity. Fifteen patients made immunoglobulin G (IgG) antibodies that bound to the immunizing cell line. Measurable tumor responses included complete remission in 2 patients and partial response in 1 patient. Hence, allogeneic tumor cell vaccines combining transgenic Lptn with IL-2 appear to have little toxicity in humans and can induce an antitumor immune response.