Two roads diverged: interferon alpha/beta- and interleukin 12-mediated pathways in promoting T cell interferon gamma responses during viral infection

Viral infections induce CD8 T cell expansion and interferon (IFN)-gamma production for defense, but the innate cytokines shaping these responses have not been identified. Although interleukin (IL)-12 has the potential to contribute, IL-12-dependent T cell IFN-gamma has not been detected during viral infections. Moreover, certain viruses fail to induce IL-12, and elicit high levels of IFN-alpha/beta to negatively regulate it. The endogenous factors promoting virus-induced T cell IFN-gamma production were defined in studies evaluating CD8 T cell responses during lymphocytic choriomeningitis virus infections of mice. Two divergent supporting pathways were characterized. Under normal conditions of infections, the CD8 T cell IFN-gamma response was dependent on endogenous IFN-alpha/beta effects, but was IL-12 independent. In contrast, in the absence of IFN-alpha/beta functions, an IL-12 response was revealed and substituted an alternative pathway to IFN-gamma. IFN-alpha/beta-mediated effects resulted in enhanced, but the alternative pathway also promoted, resistance to infection. These observations define uniquely important IFN-alpha/beta-controlled pathways shaping T cell responses during viral infections, and demonstrate plasticity of immune responses in accessing divergent innate mechanisms to achieve similar ultimate goals.     

Interferon beta 2/interleukin 6 modulates synthesis of alpha 1-antitrypsin in human mononuclear phagocytes and in human hepatoma cells.

The cytokine IFN beta 2/IL-6 has recently been shown to regulate the expression of genes encoding hepatic acute phase plasma proteins. INF beta 2/IL-6 has also been shown to be identical to MGI-2, a protein that induces differentiation of bone marrow precursor cells toward mature granulocytes and monocytes. Accordingly, we have examined the effect of IFN beta 2/IL-6 on expression of the IL-1- and tumor necrosis factor-unresponsive acute phase protein alpha 1-antitrypsin (alpha 1 AT) in human hepatoma-derived hepatocytes and in human mononuclear phagocytes. Purified human fibroblast and recombinant IFN beta 2/IL-6 each mediate a specific increase in steady-state levels of alpha 1 AT mRNA and a corresponding increase in net synthesis of alpha 1 AT in primary cultures of human peripheral blood monocytes as well as in HepG2 and Hep3B cells. Thus, the effect of IFN beta 2/IL-6 on alpha 1 AT gene expression in these cells is primarily due to an increase in accumulation of alpha 1 AT mRNA and can be distinguished from the direct, predominantly translational effect of bacterial lipopolysaccharide on expression of this gene in monocytes and macrophages. The results indicate that IFN beta 2/IL-6 regulates acute phase gene expression, specifically alpha 1 AT gene expression, in extrahepatic as well as hepatic cell types.     

Normal regulatory alpha/beta T cells effectively eliminate abnormally activated T cells lacking the interleukin 2 receptor beta in vivo

Although interleukin 2 (IL-2) has been thought to be the most important cytokine for T cell growth, animals lacking IL-2 or a component of its receptor molecules have more expanded T cells with activated memory phenotype, indicating an indispensable role for the IL-2/IL-2 receptor system in regulating the size and activity of the T cell population. In this study, we investigated the possible mechanism of abnormal expansion of activated T cells in IL-2 receptor beta chain (IL-2Rbeta)(-/-) mice using the systems of bone marrow transplantation and T cell transfer. Here, we show that IL-2Rbeta(2/-) T cells in mice reconstituted with a mixture of IL-2Rbeta(2/-) and IL-2Rbeta(1/+) bone marrow cells did not develop into an abnormally activated stage, and that already activated IL-2Rbeta(2/-) T cells were effectively eliminated by IL-2Rbeta(1/+) T cells when both cells were cotransferred to T cell-deficient host mice. This regulation and/or elimination was dependent on T cells bearing alpha/beta type T cell receptor, especially on CD8(+) T cells and independent of the Fas-Fas ligand (FasL) system. IL-2Rbeta(1/+) T cells that eliminated activated IL-2Rbeta(2/-) T cells expressed FasL, perforin, granzyme B, and tumor necrosis factor alpha/beta. These results indicate a novel function of IL-2Rbeta that is necessary for the induction of regulatory T cells acting to eliminate activated T cells.     

Involvement of the interleukin 2 pathway in the rearrangement and expression of both alpha/beta and gamma/delta T cell receptor genes in human T cell precursors

In this report, we have undertaken the phenotypic, functional and molecular characterization of a minor (less than 5%) subpopulation of adult thymocytes regarded as the earliest intrathymic T-cell precursors. Pro-T cells were immunoselected and shown to express different hematopoietic cell markers (CD45, CD38, CD7, CD5) and some activation-related molecules (4F2, Tr, HLA class II), but lack conventional T cell antigens (CD2-1-3-4-8-). TCR-gamma RNA messages are already expressed at this early ontogenic stage, while alpha and beta chain TCR genes remain in germline configuration. In vitro analyses of the growth requirements of pro-T cells demonstrated the involvement of the IL-2 pathway in promoting their proliferation and differentiation into CD3+ CD4+ or CD8+ mature thymocytes. Moreover, during the IL-2-mediated maturation process rearrangements and expression of both alpha and beta chain TCR genes occurred, and resulted in the acquisition of alpha/beta as well as gamma/delta (either disulphide-linked or non-disulphide-linked) heterodimeric TCR among the pro-T cell progeny.     

Dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon alpha/beta

Differentiation of dendritic cells (DCs) into particular subsets may act to shape innate and adaptive immune responses, but little is known about how this occurs during infections. Plasmacytoid dendritic cells (PDCs) are major producers of interferon (IFN)-alpha/beta in response to many viruses. Here, the functions of these and other splenic DC subsets are further analyzed after in vivo infection with murine cytomegalovirus (MCMV). Viral challenge induced PDC maturation, their production of high levels of innate cytokines, and their ability to activate natural killer (NK) cells. The conditions also licensed PDCs to efficiently activate CD8 T cells in vitro. Non-plasmacytoid DCs induced T lymphocyte activation in vitro. As MCMV preferentially infected CD8alpha+ DCs, however, restricted access to antigens may limit plasmacytoid and CD11b+ DC contribution to CD8 T cell activation. IFN-alpha/beta regulated multiple DC responses, limiting viral replication in all DC and IL-12 production especially in the CD11b+ subset but promoting PDC accumulation and CD8alpha+ DC maturation. Thus, during defense against a viral infection, PDCs appear specialized for initiation of innate, and as a result of their production of IFN-alpha/beta, regulate other DCs for induction of adaptive immunity. Therefore, they may orchestrate the DC subsets to shape endogenous immune responses to viruses.     

Transition from interleukin 1 beta (IL-1 beta) to IL-1 alpha production during maturation of inflammatory macrophages in vivo

In situ production of interleukin 1 alpha (IL-1 alpha) and IL-1 beta was investigated in Peyer's patches (PP) of mice undergoing an acute bacterial infection with Yersinia enterocolitica O8. Synthesis of IL-1 beta, as determined by immunohistochemistry, was found primarily in monocytes migrating into the inflamed PP. In comparison, synthesis of IL-1 alpha was temporarily delayed by at least 24 h and was only found in mature macrophages, which did not produce detectable levels of IL-1 beta. This indicates a transition from IL-1 beta to IL-1 alpha production during maturation of monocytes into inflammatory macrophages, and further emphasizes a dichotomy between IL-1 alpha and IL-1 beta.     

Mechanism of interleukin 12-mediated toxicities during experimental viral infections: role of tumor necrosis factor and glucocorticoids

Interleukin 12 (IL-12) doses in excess of 100 ng/d have been shown to induce profound immunotoxicities in mice infected with lymphocytic choriomeningitis virus (LCMV). These immunotoxicities are characterized by almost complete inhibition of virus-induced CD8+ T cell expansion and CTL activation, and up to 2 log increases in viral replication. They are accompanied by induction of serum tumor necrosis factor (TNF). The studies presented here were undertaken to characterize mechanisms for the IL-12-induced toxicities and to examine expression and function of TNF in this context. Several physiological changes were induced in IL-12-treated uninfected and dramatically elevated in IL-12-treated virus-infected mice. IL-12 induced (a) decreases in body weights, > 10% in uninfected and > 20% in LCMV-infected mice; (b) elevation of circulating glucocorticoid levels to > 10 micrograms/dl in uninfected and > 20 micrograms/dl in infected mice; and (c) decreases in thymic mass, > 30% in uninfected and up to 95% in infected mice. These changes are known to be associated with circulating TNF. Northern blot and in situ hybridization analyses demonstrated that IL-12 induced TNF-alpha expression and that LCMV infection synergized with IL-12 for induction of this factor. Antibodies neutralizing TNF reversed all of the IL-12- induced toxicities in LCMV-infected mice including the immunotoxicities against CD8+ T cells and anti-viral defenses. The TNF-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and CD8+ T cell subsets isolated from LCMV-infected mice were more sensitive to TNF- mediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. Experiments with the glucocorticoid type II receptor antagonist, RU486, demonstrated that endogenous glucocorticoids were secondary intermediaries in IL-12-induced thymic atrophy. Studies in IL-2-deficient mice showed that the synergism was dependent upon endogenous IL-2. The results delineate a unique mechanism of TNF-mediated toxicity. In addition, they have significant implications concerning potential detrimental consequences of in vivo TNF induction and of IL-12 administration for protective anti-viral responses.     

T helper type 2 cell differentiation occurs in the presence of interleukin 12 receptor beta2 chain expression and signaling

The differentiation of CD4(+) T cells into T helper type 1 (Th1) cells is driven by interleukin (IL)-12 through the IL-12 receptor beta2 (IL-12Rbeta2) chain, whereas differentiation into Th2 cells is driven by IL-4, which downregulates IL-12Rbeta2 chain. We reexamined such differentiation using IL-12Rbeta2 chain transgenic mice. We found that CD4(+) T cells from such mice were able to differentiate into Th2 cells when primed with IL-4 or IL-4 plus IL-12. In the latter case, the presence of IL-4 suppressed interferon (IFN)-gamma production 10-100-fold compared with cells cultured in IL-12 alone. Finally, in studies of the ability of IL-12 to convert Th2 cells bearing a competent IL-12R to the Th1 cells, we showed that: (a) T cells bearing the IL-12Rbeta2 chain transgene and primed under Th2 conditions could not be converted to Th1 cells by repeated restimulation under Th1 conditions; and (b) established Th2 clones transfected with the IL-12Rbeta2 chain construct continued to produce IL-4 when cultured with IL-12. These studies show that IL-4-driven Th2 differentiation can occur in the presence of persistent IL-12 signaling and that IL-4 inhibits IFN-gamma production under these circumstances. They also show that established Th2 cells cannot be converted to Th1 cells via IL-12 signaling.     

Regulation of the acute phase and immune responses in viral disease. Enhanced expression of the beta 2-interferon/hepatocyte-stimulating factor/interleukin 6 gene in virus-infected human fibroblasts

We have defined the expression of the mRNA for, and secretion of, IFN-beta 2/hepatocyte-stimulating factor/IL-6 (IFN-beta 2/IL-6) in human diploid fibroblasts (FS-4 strain) infected with different RNA- and DNA-containing viruses. RNA blot-hybridization analyses carried out 6-8 h after the beginning of infection showed that the RNA-containing Sendai virus (paramyxoviridae) enhanced IFN-beta 2/IL-6 mRNA levels 10-fold, followed, in decreasing order, by encephalomyocarditis (EMC, picornaviridae), vesicular stomatitis (VSV, rhabdoviridae), Newcastle disease virus (NDV, paramyxoviridae), and influenza A (Flu, myxoviridae) viruses. The DNA-containing pseudorabies virus (PR, herpesviridae) enhanced IFN-beta 2/IL-6 mRNA levels sixfold, while the effect of adenovirus type 5 (Ad5, adenoviridae) was considerably less and comparable with that of NDV or Flu. A rabbit antiserum raised against E. coli-derived human IFN-beta 2/IL-6 was used in immunoprecipitation experiments to monitor the secretion of 35S-methionine-pulse-labeled IFN-beta 2/IL-6 proteins by fibroblasts up to 7 h after the beginning of infection. Enhanced levels of secretion of IFN-beta 2/IL-6 (2-14-fold) were observed in every instance evaluated (Sendai, EMC, VSV, Flu, PR, Ad5 viruses). A biological consequence of enhanced secretion of IFN-beta 2/IL-6 was the ability of media from infected FS-4 cell cultures to enhance by 8-15-fold the synthesis and secretion of a typical acute phase plasma protein (alpha 1-antichymotrypsin) by human hepatoma Hep3B2 cells. These observations make it likely that IFN-beta 2/IL-6 mediates, in part, the host response to acute virus infections.     

Antigen-specific T helper cell function: differential cytokine expression in primary and memory responses

Distinguishing between the development of functional potential in antigen-specific T helper (Th) cells and the delivery of these specialized functions in vivo has been difficult to resolve. Here, we quantify the frequency of cytokine-producing cells within the primary and memory B10.BR Th cell response to pigeon cytochrome c (PCC). In vitro analysis of acquired functional potential indicated no Th1/Th2 cytokine polarity at the peak of the primary response with surprisingly little evidence for the selective preservation of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, IL-4, and interferon (IFN)-gamma potentials into the memory compartment. However, the expression of these functional potentials appears tightly regulated in vivo. The staggered appearance of primary response cytokines directly ex vivo contrasts markedly with their rapid coordinate expression in the memory response. Frequencies of IL-2-, TNF-alpha-, IFN-gamma-, and IL-10-expressing memory responders increased over their primary response counterparts, but were still markedly lower than revealed in vitro. IL-4-, IFN-gamma-, and IL-10-expressing Th cells remained at low but stable frequencies over the first 6 d of the memory response. Analysis of T cell receptor beta chain sequences of IL-4- and TNF-alpha-expressing PCC-specific Th cells provides evidence for early functional commitment among clonal progeny. These data indicate that the development of functional potential is a consequence of initial antigen experience, but delivery of specialized functions is differentially regulated in primary and memory immune responses.     

五种生物材料对大鼠巨噬细胞肿瘤坏死因子α和白细胞介素1 β表达的影响

背景：生物材料的免疫学研究主要包括免疫球蛋白、血清总补体及补体降解产物的血生化定量测定等方面，主要通过细胞学和组织学的检测手段，其评价指标比较单一，无特异性。 目的：以细胞因子（肿瘤坏死因子α和白细胞介素1β）为研究对象，从mRNA水平上对5种生物材料介导的炎症反应进行相关免疫学评价。 设计、时间及地点：对比观察实验，于2004—01／2005—03在上海生物材料研究测试中心完成。 材料：清洁级SD大鼠3只用于原代培养大鼠巨噬细胞。阳性材料：含8％有机锡的聚氟乙烯。阴性材料：聚苯乙烯。实验材料：美国NPG黄合金块、β-磷酸三钙、固化的磷酸钙骨水泥、聚丙交酯乙交酯及聚四氟乙烯。 方法：参照ISO 10993-12标准，用RPMI—1640按1g／5mL比例，37℃72h制备材料的浸提液。将大鼠腹腔巨噬细胞分别给予脂多糖刺激和无脂多糖刺激，脂多糖的终浓度为1．0mg／L，再用不同生物材料的浸提液予以刺激，作用2h后收集细胞，进行反转录-聚合酶链反应检测。 主要观察指标：大鼠巨噬细胞肿瘤坏死因子α和白细胞介素1β的表达强度。 结果：未经脂多糖诱导的大鼠腹腔巨噬细胞与阳性材料及聚四氟乙烯、NPG接触后肿瘤坏死因子α和白细胞介素1β的表达均高于阴性材料，差异均有非常显著性意义（P〈0．05）；聚丙交酯乙交酯中肿瘤坏死因子α的表达与阴性材料相比无明显升高（P〉0．05），白细胞介素1β的表达高于阴性材料，差异有显著性意义（P〈0．05）；β-磷酸三钙和固化的磷酸钙骨水泥中肿瘤坏死因子α和白细胞介素1β的表达与阴性材料比较差异无显著性意义（P〉0．05）。经脂多糖诱导的大鼠腹腔巨噬细胞与阳性材料及聚四氟乙烯、NPG、聚丙交酯乙交酯、β-磷酸三钙及固化的磷酸钙骨水泥接触后肿瘤坏死因子α和白细胞介素1β的表达均高于阴性材料，差异均有非常显著性意义（P〈0．01）。 结论：肿瘤坏死因子α、白细胞介素1β是在分子水平上衡量不同生物材料诱导免疫刺激反应的理想指标。聚四氟乙烯和NPG的生物相容性程度较差，β-磷酸三钙和固化的磷酸钙骨水泥的相容性较好，尤其是固化的磷酸钙骨水泥。     

小鼠皮肤损伤修复过程中白细胞介素1α和白细胞介素1β变化与损伤时间的关系

目的:研究表明,细胞因子在各种炎症反应、损伤修复及细胞突变过程中发挥着重要作用,并且有望成为推断损伤时间新的有效指标.实验拟验证皮肤损伤后损伤区组织内不同时间白细胞介素1α和白细胞介素1β基因表达的变化及其与损伤时间的关系.方法:实验于2006-03/2007-12在中国刑警学院法医系实验室完成.①实验材料:清洁级BALB/c纯系雄性小白鼠35只.体质量25～27g.②实验方法:建立动物皮肤损伤模型,在脊柱两侧1.0cm处,左右对称各做一处皮肤缺损损伤,损伤不伤及肌肉,每只鼠做3对损伤.损伤后分别在0,3,6,12 h,1,3,6 d(0 h作为对照)取5只动物麻醉断头处死.③实验评估:免疫组织化学染色后,显微镜下观察不同时间点白细胞的变化,实时定量聚合酶链反应检测白细胞介素1α和白细胞介素1β两种炎症因子的基因表达.结果:纳入BALB/c纯系雄性小白鼠35只,均进入结果分析.①伤后0～5 h,皮肤创缘呈弱阳性反应;伤后6～24 h,表皮阳性反应减弱,创伤局部以巨噬细胞为主的阳性细胞逐渐增多.伤后1 d可见嗜中性白细胞大量浸润及少量单核细胞;伤后2～4d,单核细胞数目逐渐增加,3 d达高峰.对照组皮肤表皮、毛囊、皮脂腺和汗腺均呈弱刚性反应.②皮肤损伤后在早期可诱导白细胞介素1α和白细胞介素1β的基因表达,6 h达第一次高峰,24 h下降,第3天再次出现高峰,第6天下降至正常水平.结论:损伤组织中白细胞介素1α和白细胞介素1β的基因表达随伤后不同时间呈现一定规律性变化,可为法医学案例早期损伤时间的推断提供参考.     

B lymphocytes regulate dendritic cell (DC) function in vivo: increased interleukin 12 production by DCs from B cell-deficient mice results in T helper cell type 1 deviation

Increasing evidence indicates that dendritic cells (DCs) are the antigen-presenting cells of the primary immune response. However, several reports suggest that B lymphocytes could be required for optimal T cell sensitization. We compared the immune responses of wild-type and B cell-deficient (muMT) mice, induced by antigen emulsified in adjuvant or pulsed on splenic dendritic cells. Our data show that lymph node cells from both control and muMT animals were primed, but each released distinct cytokine profiles. Lymph node T cells from control animals secreted interferon (IFN)-gamma, interleukin (IL)-2, and IL-4, whereas those from muMT mice produced IFN-gamma and IL-2 but no IL-4. To test whether B cells may influence the T helper cell type 1 (Th1)/Th2 balance by affecting the function of DCs, we immunized mice by transferring antigen-pulsed DCs from wild-type or mutant mice. Injection of control DCs induced the secretion of IL-4, IFN-gamma, and IL-2, whereas administration of DCs from muMT animals failed to sensitize cells to produce IL-4. Analysis of IL-12 production revealed that DCs from muMT mice produce higher levels of IL-12p70 than do DCs from wild-type animals. These data suggest that B lymphocytes regulate the capacity of DCs to promote IL-4 secretion, possibly by downregulating their secretion of IL-12, thereby favoring the induction of a nonpolarized immune response.     

Antibodies to interleukin 2. Effects on immune responses in vitro and in vivo

Antibodies to highly purified mouse interleukin 2 (IL-2) were raised in rabbits; a 1:500 dilution of antiserum completely blocked the in vitro mitogenic effect of 10(-9) M IL-2. The antisera functioned effectively to immunoprecipitate biosynthetically labeled IL-2 and the purified immunoglobulins were useful in the construction of affinity columns for the adsorption and one-step immunopurification of IL-2. The antibodies were apparently specific for IL-2 among the lymphokines, they did not block the biological effects of IL-1, IL-3, gamma-IFN, B cell stimulating factor(s), and cytotoxic T cell differentiation factor(s). When anti-IL-2 was added to the in vitro reactions, it blocked mixed leukocyte reactions (MLR) and associated lymphocyte proliferation, the in vitro generation of cytotoxic T cells, and antibody formation as assessed by erythrocyte-specific plaque-forming cells (PFC). When injected into mice, anti-IL-2 antibodies also reduced the formation of cytotoxic lymphocytes in response to allogeneic cells, suggesting that endogenous IL-2 participates in such reactions in vivo. Taken together, the results indicate that these IL-2 antibodies will be useful adjuncts in the analysis of immune response both in vivo and in vitro.     

腰椎间盘突出症患者血清白细胞介素1β、白细胞介素6、肿瘤坏死因子α表达与其疼痛的相关性

目的:分析腰椎间盘突出症患者血清细胞因子的表达及其与疼痛的关系,深化对其病变的认识。方法:于2005-02/12选择中国中医科学院望京医院及骨伤科研究所门诊和住院的腰椎间盘突出症患者43例,为试验组。腰椎间盘突出症根据临床症状结合CT和/或MRI影像学检查确诊。同期选择成年健康体检者30例,为正常组。应用放免法测定血清中白细胞介素1β、白细胞介素6、肿瘤坏死因子α的含量,采用目测类比评分法测定患者疼痛程度。结果:纳入患者43例和健康体检者30例,均进入结果分析。①试验组血清白细胞介素1β、白细胞介素6、肿瘤坏死因子α含量高于正常组,差异有显著性意义[分别为(0.40±0.17),(0.19±0.06)μg/L;(131.78±32.06),(108.85±41.48)μg/L;(1.84±0.49),(1.14±0.40)μg/L,P<0.01]。②疼痛和白细胞介素6依存性不显著(P>0.05),疼痛和白细胞介素1β、肿瘤坏死因子α呈数值依存性(P<0.01)。结论:腰椎间盘突出症患者血清白细胞介素1β、白细胞介素6、肿瘤坏死因子α异常升高,白细胞介素1β、肿瘤坏死因子α是导致疼痛的重要因素。     

Intercellular interactions and cytokine responsiveness of peritoneal alpha/beta and gamma/delta T cells from Listeria-infected mice: synergistic effects of interleukin 1 and 7 on gamma/delta T cells

Peritoneal gamma/delta T cells from Listeria-immune mice show an enhanced potential to expand when restimulated with antigens or mitogens in vitro (see companion paper [Skeen, M. J., and H. K. Ziegler. 1993. J. Exp. Med. 178:971]). When cocultured with peritoneal alpha/beta T cells, the gamma/delta T cell population expanded preferentially even when the in vitro stimulus was specific for the alpha/beta T cell population. Purified gamma/delta T cells did not respond to alpha/beta T cell-specific stimuli. If isolated T cell subsets were recombined in cell mixing experiments, the resulting proliferative response was greater than additive. Irradiated alpha/beta T cells could enhance the proliferation of responding gamma/delta T cells, but the effect was unidirectional; i.e., irradiated gamma/delta T cells did not stimulate responding gamma/delta T cells. This effect appeared to be cytokine mediated and did not require cell-cell contact. Both recombinant interleukin 2 (rIL-2) and rIL-7 could support the expansion of the gamma/delta T cells, while rIL-7 was only minimally stimulatory for the alpha/beta T cells. The magnitude of the response by gamma/delta T cells to rIL-7 exceeded the response to other in vitro stimuli, including immobilized anti-T cell receptor monoclonal antibody, and was 50-100-fold greater than the alpha/beta T cell response to IL-7. This unique sensitivity of gamma/delta T cells to IL- 7 was strongly enhanced by the presence of accessory cells. These cells could be replaced by rIL-1, establishing a synergy for IL-1 and IL-7 as factors that could uniquely stimulate this gamma/delta T cell population. Isolated peritoneal gamma/delta T cells from Listeria- immune mice react to heat-killed Listeria preparations in the presence of macrophages accessory cells in a non-H-2-restricted manner. Considered collectively, these results suggest a potential mechanism by which gamma/delta T cells can predominate in epithelial tissues and at sites of infection.     

Gene transfer to liver cancer cells of B7-1 plus interleukin 12 changes immunoeffector mechanisms and suppresses helper T cell type 1 cytokine production induced by interleukin 12 alone

To investigate the cooperative effect of B7-1 and IL-12 in the induction of antitumor activity, we have developed retroviral vectors encoding human B7-1, murine IL-12, or both B7-1 and IL-12 coordinately. Murine transformed liver cells (BNL) were engineered to stably express B7-1, IL-12, or both by infection with corresponding retroviruses. No tumor was observed in 20, 75, and 95% of mice receiving, respectively, B7-1-, IL-12-, and B7-1/IL-12-modified tumor cells after 250 days of inoculation. In contrast, injection of parental BNL or BNL/Neo cells resulted in lethal tumor progression in all mice. Protection against rechallenge with parental tumor cells was observed only in mice who had rejected BNL/IL-12, but not in animals that rejected BNL/B7-1 or BNL/B7-1-IL-12. Growth of parental tumor cells was significantly delayed by simultaneous injection in a distant site of irradiated tumor cells engineered to express IL-12 or both B7-1 and IL-12 but not B7-1 alone. BNL/B7-1 and BNL/B7-1-IL-12 showed similar efficacy in these experiments. Antitumor immunity induced by B7, with or without IL-12, was found to depend mainly on CD4+ T cells with a minor contribution of a non-T cell mechanism; whereas the effect of IL-12 was dependent on CD8+ T cells and on non-T cell effectors. Immunization of mice with IL-12-modified BNL cells induced secretion of a Thl pattern of cytokines while immunization with cells expressing both IL-12 and B7-1 resulted in inhibition of IFN-gamma production. Immunization with BNL/B7-1-IL-12 cells in the presence of anti-human B7-1 MAb resulted in restoration of IFN-gamma production to the levels found in animals injected with BNL/IL-12 cells. To summarize, in our model coexpression of B7-1 and IL-12 in tumor cells does not result in improved antitumoral activity as compared with expression of IL-12 alone. This may be related to the fact that B7-1 changes the mechanisms of antitumor immunity and inhibits IFN-gamma production induced by IL-12 in vivo.     

Gene transfer of Fc-fusion cytokine by in vivo electroporation: application to gene therapy for viral myocarditis

Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive and safe. Although combining direct DNA injection with in vivo electroporation increases the efficiency of gene transfer into muscle, applications of this method have remained limited because of the relatively low expression level. To overcome this problem, we developed a plasmid vector that expresses a secretory protein as a fusion protein with the noncytolytic immunoglobulin Fc portion and used it for electroporation-mediated viral interleukin 10 (vIL-10) expression in vivo. The fusion cytokine vIL-10/mutFc was successfully expressed and the peak serum concentration of vIL-10 was almost 100-fold (195 ng/ml) higher than with a non-fusion vIL-10 expression plasmid. The expressed fusion cytokine suppressed the phytohemagglutinin-induced IFN-gamma production by human peripheral blood mononuclear cells and decreased the mortality in a mouse viral myocarditis model as effectively as vIL-10 expression. These results demonstrate that the transfer of plasmid DNA expressing a noncytolytic Fc-fusion cytokine is useful to deliver enhanced levels of cytokine without altering general biological activities. This simple and efficient system should provide a new approach to gene therapy for human diseases and prove very useful for investigating the function of newly discovered secretory protein genes.