A lymphoproliferative disorder of T gamma cells with the phenotype of cytotoxic/suppressor T-cell

A 25-yr-old Japanese male showed unique T gamma cell proliferation different from cases reported previously. His clinical and hematological features were characterized by persistent high fever and the appearance of large lymphocytes with abundant cytoplasm and azurophilic granules in the peripheral blood (11% of the leukocyte differential count) and the ascitic fluid. These lymphocytes showed the ability to bind the Fc portion of IgG and they beared the antigen of cytotoxic/suppressor T-cell defined by monoclonal antibodies. T-cells from this patient suppressed the immunoglobulin production of normal B-cells by pokeweed mitogen, although a polyclonal hypergammaglobulinemia was observed in the serum. Chromosomal abnormality indicated the malignant nature of the proliferating T gamma cells in this patient. The clinical, hematological, and immunological findings characterized the disease of this patient as a distinct entity among the lymphoproliferative disorders of T-cell origin.     

In vitro expansion of gamma delta T cells with anti-myeloma cell activity by Phosphostim and IL-2 in patients with multiple myeloma

T-cell-mediated immunotherapy is a promising therapeutic option for multiple myeloma (MM). Gamma-delta T cells (gammadelta T cells) recognize phosphoantigens and display strong anti-tumour cytotoxicity. The synthetic agonist Phosphostim (bromohydrin pyrophosphate, BrHPP) has been shown to selectively activate Vgamma9Vdelta2 T cells. This study aimed to evaluate the expansion capacity and anti-myeloma cell cytotoxicity of circulating gammadelta T cells from MM patients at different time points throughout the disease, using Phosphostim and interleukin 2 (IL-2). Circulating gammadelta T cell counts in patients with newly diagnosed MM or in relapse did not differ from those in healthy donors. A 14-d culture of peripheral blood mononuclear cells with Phosphostim and IL-2 triggered a 100-fold expansion of gammadelta T cells in 78% of newly diagnosed patients. Gammadelta T cells harvested at the time of haematopoietic progenitor collection or in relapsing patients expanded less efficiently. Expanded gammadelta T cells killed 13/14 myeloma cell lines as well as primary myeloma cells, but not normal CD34 cells. Their killing efficiency was not affected by 2-d IL-2 starvation. This study demonstrated the ability of Phosphostim and IL-2 to expand gammadelta T cells from MM patients, and the efficient and stable killing of human myeloma cells by gd T cells.     

拉米夫定体外对慢性乙型肝炎患者树突状细胞功能的影响

目的:体外研究不同浓度拉米夫定(lamivu- dine,LAM)对慢性乙型肝炎(chronic hepatitis B,CHB)患者树突状细胞(dendritic cell,DC)功能影响．方法:分离慢乙肝患者外周血单核细胞,在含GM-CSF IL-4及不同浓度LAM(0,0．125, 0．25,0．5,1,2 mmol／L)培养条件下制备DC．观察DC形态学变化并用MTT法测定DC刺激同种异体淋巴细胞增殖能力,流式细胞仪(FCM) 测定其细胞表型分子CD1a,CD83,CD80及 HLA-DR的表达,ELISA法检测培养上清中 IL-12和IL-6含量．结果:在LAM 0．5 mmol／L组,DC表型分子 CD83,CD1a,HLA-DR的表达最高,CD80与 LAM未处理组相比无明显差异．与LAM未处理组相比,LAM 0．5 mmol／L处理组DC膜表面分子CD1a,CD83,HLA-DR表达增高(CD1a: 54．0±9．2 vs 33．6±10．1,P<0．05;CD83:20．3 ±6．1 vs 11．8±6．2,P<0．05;HLA-DR:74．5± 7．1 vs 52．9±7．7,P<0．05);其上清液中IL-12 分泌水平增高(810．0±91．5 ng／L vs 268．0± 34．3 ng／L,P<0．05),IL-6则显著降低(28．1± 2．6 ng／L vs 55．3±7．4 ng／L,P<0．05);刺激同种异体淋巴细胞增殖能力(SI)增强(1．9±0．6 vs 1．2±0．5,P<0．05)．结论:LAM体外可增强慢乙肝患者树突状细胞活性．     

恩替卡韦对慢性乙型肝炎患者树突状细胞功能的体外影响

目的:研究恩替卡韦(ETV)对慢性乙型肝炎(CHB)患者外周血树突状细胞(dendritic cell,DC)功能的影响.方法:体外常规分离CHB患者及健康人外周血单个核细胞,诱导扩增后常规培养.第4天将其与一定浓度的恩替卡韦共培养,第8天收获DC进行细胞表型、同种异体混合淋巴细胞反应等相关检测.结果:细胞培养8 d时DC形态分化健康对照组优于CHB ETV处理组,CHB ETV处理组优于CHB组;CHB组CD1a(35.73±3.12 vs 62.31±5.22,P<0.01),CD80(28.19±1.64 vs 45.38±3.10,P<0.01),CD83(22.24±2.14 vs 40.63±7.21,P<0.01)及HLA-DR(36.74±0.98 vs 56.05±3.89,P<0.01)表达明显低于健康对照组,而ETV处理组与CHB组相比CD83 (27.41±9.23 vs 22.24±2.14,P<0.05),CD80(32.67±7.82 vs 28.19±1.64,P<0.05)及HLA-DR(40.84±5.57 vs 36.74±0.98,P<0.01)显著高表达;淋巴细胞增殖能力测定ETV处理组DC刺激同种异体T淋巴细胞增殖能力较CHB组增强(1.53±0.09 vs 1.45±0.12,P<0.05).结论:恩替卡韦作为治疗CHB的新一代核苷类药物,除了直接抑制乙肝病毒DNA合成外,也能够增强CHB患者外周血DC的功能,通过调节机体的免疫系统发挥间接抗病毒作用.     

In vitro TNF blockade enhances ex vivo expansion of regulatory T cells in patients with immune thrombocytopenia

Tumour necrosis factor‐α (TNF) is an inflammatory cytokine that is elevated in a number of autoimmune diseases including immune thrombocytopenia (ITP), a bleeding disorder characterized by low platelet counts. In vitro TNF blockade increases expansion of the regulatory T cell (Treg) IKZF2 (also termed Helios) subset in T cell‐monocyte cocultures from healthy donors, but its role on proliferative responses of Tregs in ITP patients, who have altered immunoregulatory compartment, remains unclear. TNF in CD4+ T cells from patients with chronic ITP were elevated and negatively correlated with peripheral Treg frequencies, suggesting a possible inhibitory effect of TNF on ITP Tregs. In vitro antibody neutralization with anti‐TNF in T cell‐monocyte cocultures resulted in a robust expansion of pre‐existing ITP Tregs, higher than in healthy controls. Similar to the effects of anti‐TNF antibodies, TNF blockade with antibodies against TNFRSF1B (anti‐TNFRSF1B, previously termed anti‐TNFRII) almost doubled ITP Treg expansion whereas neutralization with anti‐TNFRSF1A (anti‐TNFRI) antibodies had no effect on proliferative responses of Tregs. In addition, TNFRSF1B levels on ITP Tregs were significantly elevated, which may explain the increased susceptibility of patient Tregs to the actions of TNF blockade. Altogether, these data raise the possibility that TNF blockers, through their ability to increase Treg proliferation, may be efficacious in ITP patients.     

乙型肝炎患者HBV X蛋白特异性CTL表位变异分析

目的 比较急性乙型肝炎、慢性乙型肝炎与慢性重型乙型肝炎HBV X 蛋白特异性CTL表位变异差异,探讨乙型肝炎重症化和慢性化的可能机理.方法 对393例乙型肝炎患者的血清样本进行HLA-A2分型;用巢式PCR扩增血清HBV前S/S基因与X基因并对PCR产物进行序列测定;根据HBV前S/S基因序列,用VirusBlast软件鉴定患者感染的HBV基因型;用Vector NTI软件对目前已知的6个HLA-A2限制性X蛋白特异性CTL表位序列分析,对患者各个表位变异的差异进行卡方检验.结果 190例(48.35%)患者HLA-A2阳性,其中急性乙型肝炎(AHB)67例,慢性乙型肝炎(CHB)52例,慢性重型乙型肝炎(CSHB)71例.CTL表位变异分析结果如下:对三组所有患者进行比较时,X92-100表位变异发生频数三组患者比较有非常显著性差异(P＜0.01);对三组中HBV C基因型患者进行比较时,X92-100和X115-123表位变异发生频数三组患者比较有显著性差异(P＜0.05);对三组中HBV B基因型患者进行比较时,各表位变异发生频数三组患者比较无显著差异.结论 某些HBV X蛋白特异性CTL表位在AHB、CHB和CSHB患者间变异有明显差异且受病毒基因型影响,CTL表位变异可能与乙型肝炎的重症化和慢性化机制相关.     

Impaired cytotoxic T lymphocyte inductivity by dendritic cells derived from patients with hepatitis C virus-positive hepatocellular carcinoma

Aim: Peptide-based therapeutic vaccines are being developed. The aim of this study was to determine the feasibility of immunotherapy to hepatitis C virus (HCV)-positive hepatocellular carcinoma (HCC) by assessing the inductivity of peptide-specific cytotoxic T lymphocyte (CTL) by dendritic cells. Methods: The inductivity of CTL was characterized in six patients with HCV-positive HCC, and compared to seven healthy volunteers and six patients with chronic HCV hepatitis (control). Results: Peptide-specific CTL was comparably induced in controls, but not induced in any patients with HCC. To characterize this, the cytokine profile and the expression of surface molecules interacting between dendritic and T cells were evaluated. Among the cytokines, production of interferon (IFN)-gamma was found to be impaired and closely related to the results of CTL assays, while the expression of surface molecules showed no significant changes. Conclusions: In HCV-positive HCC patients, CTL inductivity by dendritic cells is impaired. This may be related to the impaired production of IFN-gamma.     

Detection of monoclonal T populations in patients with KIR-restricted chronic lymphoproliferative disorder of NK cells

The etiology of chronic large granular lymphocyte proliferations is largely unknown. Although these disorders are characterized by the expansion of different cell types (T and natural killer) with specific genetic features and abnormalities, several lines of evidence suggest a common pathogenetic mechanism. According to this interpretation, we speculated that in patients with natural killer-type chronic lymphoproliferative disorder, together with natural killer cells, also T lymphocytes undergo a persistent antigenic pressure, possibly resulting in an ultimate clonal T-cell selection. To strengthen this hypothesis, we evaluated whether clonal T-cell populations were detectable in 48 patients with killer immunoglobulin-like receptor-restricted natural killer-type chronic lymphoproliferative disorder. At diagnosis, in half of the patients studied, we found a clearly defined clonal T-cell population, despite the fact that all cases presented with a well-characterized natural killer disorder. Follow-up analysis confirmed that the TCR gamma rearrangements were stable over the time period evaluated; furthermore, in 7 patients we demonstrated the appearance of a clonal T subset that progressively matures, leading to a switch between killer immunoglobulin-like receptor-restricted natural killer-type disorder to a monoclonal T-cell large granular lymphocytic leukemia. Our results support the hypothesis that a common mechanism is involved in the pathogenesis of these disorders.     

Expression and functional role of tumor necrosis factor receptors on leukemic cells from patients with type B chronic lymphoproliferative disorders

Two receptors for tumor necrosis factor (TNF) with different molecular weight (75-Kd and 55-Kd) and binding affinity have been recently discovered. To investigate the distribution and the functional role of these receptors on leukemic B cells from hairy cell leukemia (HCL) and B-cell chronic lymphocytic leukemia (B-CLL) patients, we evaluated: (1) the cytofluorimetric pattern of uncultured and cultured leukemic B cells incubated with utr-1 and htr-9 monoclonal antibodies (MoAbs), which specifically recognize the 75-Kd and 55-Kd TNF receptors (TNFR), respectively; (2) the effect of TNF-alpha and TNF-beta on leukemic B cells in an in vitro proliferation assay; (3) the role of anti-TNFR MoAbs on TNF-alpha and TNF-beta-driven B-cell growth; and (4) the proliferative effect of utr-1 and htr-9 MoAbs on in vitro cultured leukemic cells. Our study shows that the high affinity (75-Kd) but not the low affinity (55-Kd) TNFR molecules are expressed on freshly isolated leukemic B cells recovered from HCL and B-CLL patients. The expression of these receptors was neither upregulated nor downregulated by different stimuli, including TNF-alpha, TNF-beta, B-cell growth factor, and interleukin-2. TNF-alpha efficiently triggers the proliferation of HC and, to a lesser extent, the growth of B-CLL cells. TNF-beta was also able to transduce the proliferative signal in HCL, but not in B-CLL patients. TNF-alpha- and TNF-beta-driven B-cell proliferation was inhibited by the preincubation of leukemic B cells with utr-1 but not htr-9 MoAb. Moreover, anti-75-Kd, but not anti-55-Kd TNFR MoAb, was able to trigger the proliferation of leukemic B cells, and in particular of HC. These results show that leukemic B cells from patients with HCL and B-CLL are equipped with a fully functional high affinity TNFR.     

胸腺肽α1对慢性乙型肝炎患者树突状细胞功能的体外影响

树突状细胞(DC)是人体内抗原提呈能力最强的抗原提呈细胞(APC),在抗病毒的细胞免疫中起重要作用[1].慢性乙型肝炎(CHB)形成的重要原因之一是患者DC功能低下[2];胸腺肽α1是一种由28个氨基酸构成的胸腺多肽,被广泛用于调节机体免疫,促进T淋巴细胞功能可能是其重要机制之一.作为临床上治疗CHB的重要辅助用药,我们对胸腺肽α1调节CHB患者DC功能进行了研究.     

Stimulation in vitro of cells from patients with myeloproliferative disorders

Summary The response of peripheral leucocytes from patients with myeloproliferative disorders to the stimulus of phytohaemagglutinin, smallpox vaccine or normal leucocytes has been assesed.It was found that primitive myeloid cells did not respond to phytohaemagglutinin. Cells from patients with non leukaemic proliferative diseases appeared to have less reaction than normal cells. The response to phytohaemagglutinin was modified by previous therapy. Smallpox vaccine was found to produce variable results when introduced into cultures. The effect of normal leucocytes on cells from patients with proliferative disorders appeared to vary from patient to patient; cells from some patients did, however, react to normal leucocytes.

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Zusammenfassung Die Reaktion der peripheren Leukozyten von Patienten mit proliferierenden Störungen des Knochenmarkes auf den Reiz des Phytohämagglutins, Pockenimpfstoffes oder normaler Leukozyten wurde bestimmt. Es zeigte sich, daß primitive myeloide Zellen auf Phytohämagglutinin nicht reagieren. Zellen von Patienten mit nichtleukämischen proliferierenden Krankheiten scheinen weniger zu reagieren als normale Zellen. Die Reaktion auf Phytohämagglutinin wurde durch vorhergehende Behandlung modifiziert. Es zeigte sich, daß Pockenimpfstoff bei seinem Eintritt in Kulturen verschiedenartige Ergebnisse zeitigte. Die Wirkung von normalen Leukozyten auf Zellen von Patienten mit proliferierenden Störungen schienen von Patient zu Patient verschieden zu sein; dahingegen reagierten Zellen von manchen Patienten auf normale Leukozyten.

     

慢性乙型肝炎患者HBV核心区与聚合酶区特异性CTL表位变异分析

目的比较急性乙型肝炎、慢性乙型肝炎与慢性重型乙型肝炎患者HBV C蛋白和Pol蛋白特异性CTL表位变异的差异,以探讨乙型肝炎重症化和慢性化的可能机理。方法对516例乙型肝炎患者的血清进行HLA-A2和A11分型;用巢式PCR扩增血清HBV C基因与Pol基因并对PCR产物进行序列测定;根据HBV S基因序列,用VirusBlast软件鉴定患者感染的HBV基因型;用Vector NTI软件对目前已知的HLA-A2限制性的4个C蛋白和5个Pol蛋白特异性CTL表位与HLA-A11限制性的1个C蛋白表位进行序列分析。结果 247例（47.86%）患者HLA-A2阳性,其中AHB 67例,CHB 109例,CSHB 71例;220例（42.64%）患者HLA-A11阳性,其中AHB 67例,CHB 107例,CSHB 46例;CTL表位变异分析结果如下：①在3组HLA-A2阳性患者,表位变异发生率无显著性差异（P〉0.05）;②在三组HLA-A2阳性HBV B基因型患者,P455-463和P816-824表位变异有极显著性差异（P〈0.01）;③在三组HLA-A2阳性HBV C基因型患者,各表位变异无显著性差异;④在三组HLA-A11阳性患者,C88-96表位变异发生率有显著性差异（P〈0.05）,三组HBV C基因型患者,表位变异发生率有极显著性差异（P〈0.01）,而在HBV B基因型患者,各表位变异无显著性差异（P〉0.05）。结论某些HBV C蛋白和Pol蛋白特异性CTL表位在AHB、CHB和CSHB患者间变异有明显差异,并且受感染病毒基因型的影响。CTL表位变异可能与乙型肝炎的重症化和慢性化机制相关。     

Low-dose oral fludarabine plus cyclophosphamide in elderly patients with chronic lymphoproliferative disorders

A synergistic effect of fludarabine (FLU) and cyclophosphamide (CY) has been extensively demonstrated in the treatment of chronic lymphoproliferative disorders (CLD), although a high incidence of severe neutropenia and infectious complications, particularly in elderly patients, have been reported. Based on a previous clinical experience in elderly CLD patients treated with a combination of low-dose intravenous (i.v.) FLU and CY in whom we obtained good response rates and negligible toxicity, we tested efficacy and safety of the new oral formulation of FLU combined with CY at low doses. A total of 28 elderly patients with relapsed/refractory or untreated CLD were treated with oral FLU and CY (25 and 150 mg/m2 respectively, both for 4 days every 4 weeks). The treatment design consisted in four consecutive courses and the median value of courses per patient was 3. Overall, 25 out of 28 evaluable patients were responsive to the treatment (six CR and 19 PR; ORR 89%), while the remaining three patients did not show any appreciable response (two progressive and one stable disease). Hematological toxicity was low in the majority of patients (grade 2-3 neutropenia/anemia in 8/28 cases); however, two fatal infections occurred and one additional patient died because of disease progression. Extra-hematological toxicity was generally mild. This preliminary report suggests that oral combination of FLU and CY at low dose is effective as the i.v. formulation and standard doses, since it may induce rapid responses in about 90% of elderly patients with CLD, with an acceptable toxicity.     

In vitro cellular interactions among thyrocytes, T cells and monocytes from patients with Graves' disease

In order to investigate the cellular interactions among thyrocytes, T cells and monocytes in thyroid glands from patients with Graves' disease, we determined alterations of HLA-DR antigen expression on thyrocytes and T cells, and the production of interferon-gamma during coculture of thyrocytes, T cells and monocytes obtained from patients with Graves' disease. Thyroid glands were obtained at operation from 17 patients with Graves' disease. When thyrocytes and autologous peripheral blood T cells were cocultured for 7 days, the percentage of HLA-DR antigen expression on thyrocytes was significantly increased by T cells and that on T cells was also significantly increased by thyrocytes. Addition of autologous monocytes to the mixtures of thyrocytes and T cells significantly increased the expression of HLA-DR antigens on both thyrocytes and T cells compared with mixtures without monocytes. In culture supernatants, interferon-gamma was detected in 4 of 14 cocultures of thyrocytes and T cells, in 5 of 10 cocultures of thyrocytes, T cells and monocytes, but not in any cultures of thyrocytes alone, T cells alone and T cells and monocytes. Induction of HLA-DR antigen expression on thyrocytes and T cells was blocked by addition of anti-interferon-gamma monoclonal antibody to the mixture. These results suggest that HLA-DR antigen positive thyrocytes are able to induce the HLA-DR antigen expression of T cells in the presence of monocytes, and the activated T cells are capable of producing interferon-gamma which acts on thyrocytes to induce and maintain the expression of HLA-DR antigens.     

In vitro priming and expansion of cytomegalovirus-specific Th1 and Tc1 T cells from naive cord blood lymphocytes

Adoptive transfer of CMV-specific cytotoxic T cells (CTLs) expanded in vitro from memory donor T cells can reduce the incidence of CMV disease in allogeneic transplant recipients. However, this approach has been unavailable in the cord blood (CB) transplantation setting because CB T cells are antigen naive and biased toward Th2/Tc2 function. We developed a protocol to in vitro prime and expand CMV-specific CTLs from CB. T cells were primed with cytokines to trigger skewing toward Th1/Tc1 lineage before encountering monocyte and CD34+ progenitor-derived dendritic cells loaded with CMV antigen and its immune complex. CMV-pulsed cultures expanded significantly more over 4 to 6 weeks than CMV cultures despite identical cytokine milieu. T cells isolated from CMV+ cultures showed a preferential expansion of CD45RA-/RO+/CD27+ T cells compared to CMV- cultures. CMV-specific IFN-gamma- and TNF-alpha-producing CD4+ (Th1) and CD8+ (Tc1) T cells were enriched after 3 to 4 weeks and CMV-specific cytotoxicity developed 1 to 2 weeks later.     

In vitro killing of neuroblastoma cells by neutrophils derived from granulocyte colony-stimulating factor-treated cancer patients using an anti-disialoganglioside/anti-Fc gamma RI bispecific antibody

Neutrophils isolated from cancer patients treated with granulocyte colony-stimulating factor (G-CSF) express high levels of Fc gamma RI. They exhibited an efficient killing of GD2+ neuroblastoma cells in the presence of an antidisialoganglioside (GD2) mouse monoclonal antibody (MoAb; 7A4, IgG3 kappa). However, this cytotoxicity was totally blocked by human monomeric IgG. In contrast, a bispecific antibody (7A4 bis 22/MDX-260), prepared by chemically linking an F(ab') fragment of 7A4 with an F(ab') fragment of an anti-Fc gamma RI MoAb, 22, which binds outside the Fc binding domain, triggered antibody-dependent cell cytotoxicity, even when neutrophils were preincubated with human monomeric IgG. F(ab')2 22 MoAb abrogated the MDX-260 killing without affecting that of 7A4. The 3G8 MoAb, directed against the Fc gamma RIII binding site, did not inhibit the cytotoxicity induced by either antibody. Thus, these results indicate that G-CSF-activated neutrophils exert their cytotoxic effect against neuroblastoma cells through Fc gamma RI and not Fc gamma RIII, and that the saturation of the high affinity Fc gamma RI by monomeric IgG can be overcome by the use of bispecific antibodies binding epitopes outside the IgG Fc gamma RI binding site. A combined administration of such bispecific antibodies and G-CSF may be, therefore, an efficient therapeutic approach to trigger tumor lysis by cytotoxic neutrophils in vivo.     

Activation and adoptive transfer of Epstein–Barr virus-specific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease

The treatment of Epstein-Barr virus (EBV)-associated lymphoproliferative disease (PTLD) in EBV seronegative solid organ transplant recipients who acquire their EBV infection after engraftment poses a considerable challenge because of underlying immunosuppression that inhibits the virus-specific cytotoxic T cell (CTL) response in vivo. We have developed a protocol for activating autologous EBV-specific CTL lines from these patients and show their potential use for immunotherapy against PTLD in solid organ transplant patients. Peripheral blood mononuclear cells from a panel of solid organ transplant recipients with and without active PTLD were used to assess EBV-specific memory CTL responses. The activation protocol involved cocultivation of peripheral blood mononuclear cells with an autologous lymphoblastoid cell line under conditions that favored expansion of virus-specific CTL and hindered the proliferation of allospecific T cells. These CTL consistently showed (i) strong EBV-specificity, including reactivity through defined epitopes in spite of concurrent immunosuppressive therapy, and (ii) no alloreactivity toward donor alloantigens. More importantly, adoptive transfer of these autologous CTLs into a single patient with active PTLD was coincident with a very significant regression of the PTLD. These results demonstrate that a potent EBV-specific memory response can be expanded from solid organ recipients who have acquired their primary EBV infection under high levels of immunosuppressive therapy and that these T cells may have therapeutic potential against PTLD.