Surface membrane antigen alteration on blood basophils in patients with Hymenoptera venom allergy under immunotherapy

BACKGROUND: Venom immunotherapy (VIT) provides widespread protection against systemic anaphylactic reactions after a sting of the respective insect. This effect is attributed to a shift from T(H)2 to T(H)1. However, because basophils also produce and release cytokines, such as IL-4 and IL-13, they may be part of the cytokine network. The cytokines may regulate basophilic granulocytes, as suggested by the presence of cytokine receptors IL-2Ralpha, GM-CSFRalpha, IL-1RII, IL-3R, IL-4R, IL-5R, and IL-6R on basophils from nonallergic donors. OBJECTIVE: The purpose of this study was to demonstrate that human basophils from subjects allergic to wasp venom undergoing VIT are regulated by cytokines, as shown by the alteration of the expression of cytokine receptors (and other markers). METHODS: The expression of the surface interleukin receptors and activation antigens on basophils from 19 nonallergic subjects and 48 patients with wasp venom allergy was investigated before, immediately after, and 1 week after VIT (20 patients only). RESULTS: Basophilic granulocytes in allergic subjects, compared with those in healthy persons, showed elevated expression of CD32 (FcgammaRII), CD122 (IL-2Rbeta), CD124 (IL-4Ralpha), CD130 (IL-6 and 11Rbeta), CD154 (CD40L), and HLA-DR. Activation of basophils clearly increased during VIT indicated by increased expression of CD32, CD33, CD35 (CR1), CD63, CD116 (GM-CSFRalpha), CD122, CD124, CD130, and CD154. HLA-DR expression also tended to increase. The expression of IL-5R (CD125) decreased. A significant decrease of the basophilic surface antigens CD11c, CD32, CD35, CD63, CD116, CD122, CD124, CD130, and CD132 (interleukin receptor gamma) was detected 1 week after the end of rush VIT. CONCLUSION: The rise in CD63 during VIT indicates a partial basophil degranulation with release of stored protein mediators, including IL-4. IL-4 may cause a transient upregulation of different surface antigens in an autocrine manner. Thereafter, cytokines released by T cells, which as a result of VIT have changed from a T(H)2 type to a more T(H)1 type, downregulate the activation of the basophilic granulocytes.     

Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom

Background: Specific immunotherapy with honeybee venom (BV) is highly effective, but allergic side effects can occur during treatment. Immunotherapy with peptides containing major T-cell epitopes of the relevant allergen or allergens provides an alternative strategy without these problems. Objective: The study investigates the immunologic mechanisms and clinical effects of immunotherapy with T-cell epitope peptides of the major BV allergen, the phospholipase A2 (PLA). Methods: Five patients with IgE-mediated systemic allergic reactions to bee stings were treated with a mixture of three T-cell epitope peptides of PLA. Ten patients allergic to BV receiving whole BV immunotherapy served as control subjects. Increasing doses of the peptide mixture, up to a maintenance dose of 100 μg, were administered subcutaneously within 2 months. The patients were then challenged with PLA and 1 week later with a bee sting. The cellular and humoral immune response was measured in vitro. Results: No allergic side effects were caused by the peptide immunotherapy, and all patients tolerated the challenge with PLA without systemic allergic symptoms. Two patients developed mild systemic allergic reactions after the bee sting challenge. After peptide immunotherapy, specific proliferative responses to PLA and the peptides in peripheral blood mononuclear cells were decreased in successfully treated patients. The production of T_H2 and T _H1 cytokines was inhibited, and B cells were not affected in their capacity to produce specific IgE and IgG4 antibodies. Their levels increased after allergen challenge in favor of IgG4. Conclusions: Immunotherapy of BV allergy with short T-cell peptides of PLA induces epitope-specific anergy in peripheral T cells and changes the specific isotype ratio in a fashion similar to that of conventional immunotherapy in successfully treated patients. (J Allergy Clin Immunol 1998;101:747-54.)     

Risk Assessment of Hymenoptera Re-Sting Frequency: Implications for Decision-Making in Venom Immunotherapy

Background: Venom immunotherapy is highly efficacious in preventing anaphylactic sting reactions. However, there is an ongoing discussion regarding patient selection and whether and how to apply a cost-benefit analysis of venom immunotherapy. In order to help decision-making, we investigated the re-sting frequency of hymenoptera-venom-allergic patients to single out those at high risk. Methods: In this retrospective study, re-sting data of 96 bee-venom-allergic patients and 95 vespid-venom-allergic patients living mainly in a rural area of Switzerland were analyzed. Hymenoptera venom allergy status was rated according to the classification system of H.L. Mueller [J Asthma Res 1966;3:331-333]. Different risk-groups were defined according to sting exposure and their median sting-free interval was calculated. Results: The risk factors for a wasp or bee re-sting were outdoor occupation, beekeeping and habitation close to a bee-house. Half of all vespid-venom-allergic outdoor workers were re-stung within 3.75 years compared to 7.5 years for indoor workers. Similarly, 50% of the bee-venom-allergic beekeepers or subjects with a bee-house in the vicinity suffered a bee re-sting within 5.25 years compared to 10.75 years for individuals who were not beekeepers. Conclusions: The high degree of exposure of vespid-venom-allergic outdoor workers and bee-venom-allergic beekeepers and subjects living close to bee-houses underlines the high benefit of venom immunotherapy for these patients even if they suffered a non-life-threatening grade II reaction. Yet, bee-venom-allergic individuals with no proximity to bee-houses and with an indoor occupation face a very low exposure risk, which justifies epinephrine rescue treatment for these patients especially if they have suffered from grade II sting reactions.     

Systemic T-cell unresponsiveness during rush bee-venom immunotherapy

By rush bee-venom immunotherapy, subjects reacting allergically to the venom can be effectively anergized, although the mechanism of action is not known. Here we analyzed the systemic effects of rush desensitization on the T cells of allergic patients. In most patients, we found reduced frequencies of T cells recalled to express CD69 and the cytokines interleukin (IL)-4 and interferon-gamma (IFN-γ) after stimulation of peripheral blood mononuclear cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin, as compared with normal donors. These frequencies are progressively reduced during immunotherapy. The frequency of cells expressing IL-2 does not change. A few patients show a different response to immunotherapy: frequencies of cells expressing CD69, IL-4, or IFN-γ do not change, and remain similar to those of normal donors. However, the frequency of cells able to express IL-2 is increased. The analysis of cytokine expression in CD45RO* vs CD45RO' T-cell populations revealed differences between normal and allergic donors. In allergic patients, higher frequencies of IL-4- and IFN-γ-expressing cells among the CD45RO subpopulation were found than in normal donors. This situation is not modified by immunotherapy. The results reveal a certain degree of heterogeneity in the response of allergic patients to bee-venom rush immunotherapy; however, all are clearly differentiated from normal controls as judged by cytokine expression of CD45RO T cells. In most allergic patients, a considerable percentage of TTi cells become unresponsive to mitogenic stimulation, and may be responsible for the desensitization itself     

小儿慢性丙型肝炎外周血T细胞亚群和TH1/TH2型细胞因子的研究

目的通过研究小儿慢性丙型肝炎外周血T细胞亚群及TH1/TH2型细胞因子的表达,进一步探讨小儿慢性丙型肝炎的免疫发病机制。方法(1)流式细胞仪(FACS)检测16例慢性丙型肝炎患儿及10例正常对照外周血T细胞亚群。(2)将慢性丙型肝炎患儿和正常对照外周血单个核细胞(PBMC)体外培养72h后,用ELISA法检测培养上清中TH1型细胞因子(IFN-γ、IL-2、IL-12和TNF-γ)和TH2型细胞因子(IL-4、IL-10)的浓度。结果(1)CD4 细胞无明显变化。CD8 细胞与正常对照比较明显升高(P<0.05)。CD3 细胞升高,CD4 /CD8 比值下降,但与正常对照比较无统计学意义(P>0.05)。(2)PBMC培养上清中IFN-γ、IL-10和TNF-α的水平明显升高(P<0.01),而没有检测到IL-2、IL-4、IL-12的基础分泌。结论慢性丙型肝炎患儿体内T淋巴细胞存在数量和功能的异常,CD8 细胞数升高,CD4 细胞功能异常,表现在以TH2型细胞因子的分泌为主。这可能与丙肝病毒(HCV)感染的慢性化有关。     

Activated B cells mediate efficient expansion of rare antigen-specific T cells

Potent professional antigen-presenting cells (APC) are essential tools to activate and expand antigen-specific T cells in vitro for use in adoptive immunotherapy. CD40-activated B cells can be easily generated and propagated from human donors and have been successfully used to generate antigen-specific T-cell cultures. Here we show that CD40-activated B cells strongly and specifically expand rare populations of antigen-specific CD8 T cells, with frequencies of less than 1 in 20,000 CD8 T cells in peripheral blood. We focused on T cells recognizing an epitope from the human papillomavirus 16 (HPV-16) E7 protein. In 6 of 6 healthy donors, epitope-specific CD8+ T cells were found to be "rare" by this criterion, as shown by staining with human leukocyte antigen (HLA)/peptide multimers. Using peptide-loaded CD40-activated B cells, epitope-specific T cells could be selectively expanded in all donors up to 10(6) fold, and the resulting T-cell cultures contained up to 88% specific T cells. These results strongly encourage the use of CD40-stimulated B cells as APCs in immunotherapy.     

Histamine release from peripheral blood leukocytes with purified bee venom allergens: effect of hyperimmune beekeeper plasma

The response of 15 strongly bee-venom-allergic patients to highly purified venom allergens was compared using skin prick test titration, peripheral blood leukocyte (PBL) histamine release and radioallergosorbent test with three highly purified bee venom allergens: phospholipase (PLA2), hyaluronidase (HYAL) and acid phosphatase (ACID P). Sensitivity to the three allergens ranked in the same order for all three tests and in each case PLA2 was found to the most potent allergen. In the presence of hyperimmune beekeeper plasma, maximum histamine release was reduced significantly for all three allergens (p less than 0.001). Furthermore, hyperimmune beekeeper plasma increased the amount of allergen required for a comparable release of histamine (mean shift in dilution curve PLA2 917-fold; HYAL, 492-fold; ACID P, 61-fold). The release of histamine from whole blood was also compared with PBL + 10% normal human serum (NHS). For all three allergens maximum release was much lower from whole blood compared with washed cells + 10% NHS (p less than 0.001). These data confirm PLA2 as the major bee venom allergen by all three tests. Hyperimmune beekeeper plasma reduces maximum histamine release and increases its threshold. Histamine release in response to ACID P appears harder to block with hyperimmune beekeeper plasma than that provoked by PLA2 or HYAL (p less than 0.01). Whole blood releases less histamine and requires more allergen than washed cells, indicating that sensitivity of PBL in vivo is unlikely to be as great as washed PBL in vitro.     

CD40‐activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential

Cellular adjuvants such as dendritic cells (DC) are in the focus of tumour immunotherapy. In DC‐vaccine trials, induction of tumour antigen‐specific immunity is observed frequently and well‐documented clinical responses have been reported. However, the overall response rate is less than 3%, therefore alternative strategies are being investigated. CD40‐activated B cells (CD40‐B) have been characterized previously as an interesting alternative because they present antigen efficiently and can be expanded by several logs from small amounts of peripheral blood. To determine the central technical challenges of cell‐based vaccines we performed a single‐patient analysis of 502 patients from DC‐based tumour vaccine trials and identified at least three factors contributing to their limited efficiency: (1) lack of cell numbers; (2) lack of documented purity thus high contamination of bystander cells; and (3) lack of quality control and thus heterogeneous or unknown expression of important surface molecules such as major histocompatibility complex (MHC) and chemokine receptors. Based on these findings we re‐evaluated the CD40‐B approach in cancer patients. Here, we show that proliferation of B cells from cancer patients is equivalent to that observed in healthy donors. Purity is always > 90% after 2 weeks and remains stable for several weeks. They have comparable antigen‐presenting capability determined phenotypically and by allogeneic mixed lymphocyte reaction. Expression of CCR7 and CD62L was detected in all samples and B cells migrated towards the relevant homing chemokines. Taken together, CD40‐B cells from cancer patients can be expanded in virtually unlimited numbers at high purity and full function concerning antigen‐presentation and migratory properties.     

Expansion of cytotoxic CD3+ CD56+ cells from peripheral blood progenitor cells of patients undergoing autologous hematopoietic cell transplantation.

Immunotherapy may potentially improve the outcome of autologous hematopoietic cell transplantation (HCT). Poor effector cell proliferation and marginal antitumor activity limit attempts to use immunotherapy. We have characterized the ex vivo expansion, up to 1000-fold, of CD3+ CD56+ lymphocytes from the peripheral blood lymphocytes (PBL) of healthy donors. Expanded cells termed cytokine-induced killer (CIK) cells induce non-major histocompatibility complex-restricted lysis of tumor cells and demonstrate cytolytic activity superior to lymphokine-activated killer cells without the requirement of interleukin (IL)-2 treatment in vivo. To determine whether cytolytic cells could be expanded from patient material, we evaluated samples of peripheral blood progenitor cells (PBPCs) from 25 patients undergoing autologous HCT. The PBPCs were expanded by priming with interferon-gamma followed by anti-CD3 monoclonal antibody and IL-2 the next day. Fluorescence-activated cell sorting analysis was performed on days 0, 15, 21, and 28 of cell culture. The median T-cell content rose from 15.3% (range, 1.1% to 89.7%) on day 0 to 97.2% (range, 83.6% to 99.5%) by day 15. By day 21, T cells expanded 21.8-fold (range, 1.7- to 420.0-fold) and CD3+ CD56+ cells expanded 44.8-fold (range, 5.1- to 747.0-fold). CIK cells were used as effector cells against B-cell lymphoma targets (OCI-Ly8) with a median of 24% (range, 3% to 67%) and 42% (range, 6% to 96%) specific lysis of target cells on days 21 and 28, respectively. CIK cells derived from PBL of 2 additional patients with acute myelogenous leukemia demonstrated 39% and 78% specific lysis of OCI-Ly8 and 26% and 58% specific lysis of autologous leukemic blasts at an effector:target ratio of 40:1. CIK cells may be expanded from granulocyte colony-stimulating factor-mobilized PBPCs of patients undergoing autologous HCT. CIK cells may provide a potent tool for use in posttransplantation adoptive immunotherapy.     

Analysis of function-associated receptor molecules on peripheral blood and synovial fluid granulocytes from patients with rheumatoid and reactive arthritis

In this study we report the expression pattern of 13 different function-associated surface molecules on synovial fluid and peripheral blood granulocytes from rheumatoid and reactive arthritis patients. We found increased expression of the complement receptors 1 (CD35) and 3 (CD11b) and of the activation-associated antigens CD67, CD24, and M5 on synovial fluid granulocytes from rheumatoid and/or reactive arthritis patients compared to autologous peripheral blood granulocytes. In addition, synovial fluid granulocytes expressed IgG Fc receptor 1 (CD64) and complement receptor 4 (CD11c), neither of which can be found on peripheral blood granulocytes. Peripheral blood granulocytes from rheumatoid and reactive arthritis patients expressed higher levels of leucocyte function-associated antigen 1 (CD11a) and of the membrane proteins CD31, CD24, M5, and M6 compared to peripheral blood granulocytes from healthy controls and patients with degenerative joint disease. No significant differences in the expression of any of the molecules studied could be observed between cells from rheumatoid and cells from reactive arthritis patients, suggesting a similar activation process for granulocytes in these two diseases.     

Induction of T ''regulatory'' cells by standardized house dust mite immunotherapy: an increase in CD4+CD25+ interleukin-10+ T cells expressing peripheral tissue trafficking markers

BACKGROUND: Clinically effective subcutaneous allergen-specific immunotherapy (SIT) is associated with altered circulating T cell cytokine production and altered local cytokine responses with increased IL-10 following allergen challenge in target organs. OBJECTIVE: This study aimed to elucidate mechanisms for these T cell changes, by examining surface expression of markers for peripheral tissue trafficking on circulating cytokine-positive T cells following standardized house dust mite- (HDM-) SIT. METHODS: A randomized conventional HDM immunotherapy study was performed on a panel of 12 HDM-allergic subjects. Nine subjects received treatment with conventional HDM immunotherapy using a standardized extract and three subjects were treated by standard pharmacotherapy alone. Symptom and medication scores and allergen-induced cutaneous late-phase responses were assessed before and 9 months after institution of therapy. Before and at 3 and 9 months of SIT, peripheral blood mononuclear cells were cultured for 14 days with HDM extract and CD4+ and CD8+ T cell expression of CD62L, CD49d and CCR5 and production of IL-10, IFN-gamma and IL-4 were analysed by flow cytometry. Allergen-specific T cell proliferation was assessed by 3H-thymidine incorporation. RESULTS: At 9 months, all SIT-treated patients showed reduced symptom scores and late-phase cutaneous responses to HDM compared with baseline levels. The proportions of CD4+ T cells which were IL-10+ were increased (P < 0.01), and the proportions of CD4+ and CD8+ T cells which were IL-4+ decreased (P < 0.05) compared with baseline. CD4+ and CD8+ T cell IFN-gamma production, expression of surface markers for peripheral tissue trafficking and allergen-specific proliferation remained unchanged during SIT treatment. However, increased proportions of CD4+CD62L(-), CD4+CD49d(hi), CD4+CCR5+ T cells expressing IL-10 were detected at 9 months of SIT compared with baseline (P < 0.05). IL-10 staining co-localized with CD4+CD25+ T cells. CONCLUSION: Clinically effective subcutaneous immunotherapy with a standardized HDM Dermatophagoides pteronyssinus preparation results in decreased numbers of IL-4+ T cells and expansion of CD4+IL-10+ T cells expressing a peripheral tissue trafficking phenotype. The co-localization of IL-10+ staining to CD4+CD25+ T cells is consistent with the induction of a T regulatory cell population by SIT.     

Low Number of Peripheral Blood B Lymphocytes in Patients with Pulmonary Tuberculosis

The cellular immune response plays a critical role in the containment of persistent Mycobacterium tuberculosis infection; however, the immunological mechanisms that lead to its control are not completely identified. The goal of this study was to evaluate B (CD19+) and T (CD3+) peripheral blood lymphocyte profiles and T-cell subsets (CD4+ and CD8+) in patients with pulmonary tuberculosis (TB). Percentages (p = 0.02) and absolute numbers (p = 0.005) of B cells were significantly lower in patients with pulmonary TB than in healthy donors. In contrast, percentages (p = 0.12) and absolute numbers (p = 0.14) of T cells were similar in TB patients and healthy donors. No significant differences in percentages of CD4+ (p = 0.19) or CD8+ (p = 0.85) T cells between patients and healthy donors were observed. In summary, patients with pulmonary tuberculosis had a lower number of peripheral blood B lymphocytes than healthy controls.     

Delay and not deficiency in cap formation of peripheral blood B cells in patients with multiple myeloma

A major problem in the study of peripheral blood (PB) B cells from patients with multiple myeloma (MM) is the distinction between the cells really able to synthesize membrane (m) immunoglobulins (Ig) and those able only to absorb serum Ig passively, since the lymphocytes of such patients are bathed in very high concentrations of monoclonal Ig. In order to reappraise PB B cells (including putative pre-B cells) in MM, we have used three different criteria: (a) the capacity of PB B cells to cap mIg when triggered by an anti-Ig; (b) the presence of B-cell differentiation antigens (CD19, CD20, CD21, and CD37) as specific B-cell markers; and (c) the expression of cytoplasmic heavy chain as a marker of pre-B cells. We have found that, in active myeloma (N=13), the percentages and absolute numbers of PB B cells able to cap mIg (4.25%; 45.43 cells/mm³) were significantly lower than those in healthy donors (8.4%; 151.2 cells/mm³) and those in stable MM (7.67%; 134.39 cells/mm³). In addition, the capping formation in patients with stable or active MM was significantly delayed compared to that in healthy donors. For all the normal individuals and patients investigated, there has been found an excellent correlation between the percentages and absolute numbers of PB B cells able to cap their mIg and those of PB mononuclear cells bearing the four B cell-specific differentiation antigens: CD19, CD20, CD21, and CD37. Finally, virtually no pre-B cells bearing cytoplasmic chains have been identified in the peripheral blood from healthy donors and patients with MM.     

自体造血干细胞移植治疗恶性淋巴瘤后大剂量白细胞介素2过继免疫对长期生存的影响

背景：过继免疫治疗是目前肿瘤免疫治疗的热点,白细胞介素2是一种具有多种生物学活性的细胞因子,在机体的抗肿瘤免疫中起到重要作用。 目的：评价比较淋巴瘤自体造血干细胞移植治疗后应用与不应用大剂量白介素2行免疫治疗的临床疗效。 方法：回顾分析30例恶性淋巴瘤患者(治疗组)自体造血干细胞移植后行大剂量白细胞介素2 治疗,与随机挑选30例患者(对照组)自体造血干细胞移植后未行白细胞介素2治疗进行对比,检测两组患者外周血T淋巴细胞亚群,观察两组免疫功能的变化,并对所有患者进行随访观察。 结果与结论：自体造血干细胞移植后白细胞介素2治疗组外周血T淋巴细胞亚群CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺水平明显提升。随访结束时统计复发率：治疗组13.3%,对照组26.7%;中位生存期：治疗组14~98 (42±2)个月,对照组8~78 (28±2)个月。提示恶性淋巴瘤自体造血干细胞移植后行大剂量白细胞介素2治疗能提高患者的免疫功能,减少移植后复发率,并有望延长生存期。     

原发性肝癌患者CTCs与CD4~+/CD8~+、NLR、TTV及肿瘤分期的相关性研究

目的:探讨原发性肝癌患者外周血循环肿瘤细胞(CTCs)与T细胞亚群CD4~+/CD8~+比值、中性粒细胞与淋巴细胞比值(NLR)、肿瘤总体积(TTV)及肿瘤分期的关系。方法:收集80例原发性肝癌患者外周血,采用益善生物技术公司使用的"免疫去除结合纳米过滤法"Can PatrolTM行CTCs检测。同时使用流式细胞术检测外周血中CD4+T细胞和CD8~+T细胞数量;收集患者外周血中性粒细胞数、淋巴细胞数、肿瘤最大直径及肿瘤分期等临床病理参数。计算出外周血CD4~+/CD8~+比值、NLR及TTV。根据CD4~+/CD8~+平均值及TTV、NLR中位数将CD4+/CD8+比值、TTV和NLR分为高、低2组;根据肝癌第7版TMN分期标准将患者分为I+II期和III+IV期2组。分别比较CTCs在2组中的差别。结果:高CD4~+/CD8~+比值组外周血CTCs数及间质型CTCs数明显低于低CD4~+/CD8~+比值组(P0.05)。高TTV组外周血的CTCs数、间质型CTCs数及混合型CTCs数明显高于低TTV组(P0.05);肿瘤分期I、II期组外周血的CTCs数、间质型CTCs数及混合型CTCs数明显低于III、IV期组(P0.05)。高NLR组与低NLR组外周血的CTCs数、间质型CTCs数、混合型CTCs数和上皮型CTCs数间的差异无统计学显著性。结论:原发性肝癌患者外周血中存在CTCs;外周血CTCs与T细胞免疫、TTV及肿瘤分期有显著相关:T细胞免疫越差、TTV越大及肿瘤分期越晚,外周血的CTCs越多。