弓形虫多表位DNA疫苗的构建及其免疫保护作用

目的:构建弓形虫多表位DNA疫苗并研究其免疫保护效果.方法:将编码含弓形虫多个T、B细胞表位的6段弓形虫多肽基因,以5个甘氨酸编码基因相间隔相连接,克隆入真核表达质粒pcDNA3.1( )中,构建成多表位弓形虫DNA疫苗.免疫BALB/c小鼠,测定其诱导的特异性抗体水平及T淋巴细胞增殖状况,同时进行弓形虫攻击感染保护实验.结果:成功构建了包含多个弓形虫表位的真核表达质粒pcDNA3.1/T-ME,以其作为DNA疫苗免疫小鼠,可诱导机体产生弓形虫特异性的体液及细胞免疫应答,产生有效的抗弓形虫保护性免疫应答.结论:构建的弓形虫多表位DNA疫苗能诱导机体产生有效的保护性免疫应答,在控制弓形虫感染上具有可行性.     

减毒鼠伤寒沙门氏菌运送CD8+T细胞表位的细胞免疫应答

目的： 探索减毒沙门氏菌运送CD8＋ T 细胞表位诱导机体产生特异性细胞免疫应答的规律性.方法： 通过构建融合表达OVA 257～264aa和LCMV NP 118～132aa CD8＋ T 细胞表位的原核表达质粒ptG2F, 以电穿孔法转化减毒鼠伤寒沙门氏菌SL7207, 筛选重组菌SL7207（ptG2F）.采用静脉注射免疫C57BL/6和BALB/c小鼠, 间隔2 wk, 分别于第2次和第3次免疫后, 取免疫小鼠脾细胞, 用ELISPOT法检测特异性IFN-γ分泌细胞和IL- 4分泌细胞.结果： 携带CD8＋ T细胞表位的重组菌SL7207（ptG2F）能诱导产生细胞免疫应答.在提呈OVA CD8＋ T细胞表位时, 2次免疫后, 诱导产生的细胞免疫应答趋向于Th1; 而在3次免疫后, 呈现Th1/Th2的平衡转换.在提呈LCMV NP CD8＋ T细胞表位过程中, Th2免疫应答水平高于Th1, 且有增强趋势.结论： 减毒沙门氏菌可以有效运送CD8＋ T细胞表位并诱导产生特异细胞免疫应答, 这为减毒细菌作为运送载体的研究提供了参考依据.     

肿瘤环境影响抗感染CD8~+T细胞应答启动的初步研究

目的探讨肿瘤环境下,机体对非肿瘤来源的外来微生物抗原特异性CD8+T细胞应答的变化及其可能机制,为肿瘤患者免疫调节治疗提供理论依据。方法在建立小鼠荷瘤模型的基础上,尾静脉感染李斯特菌(OVA-Listeria Monocytogenes,OVA-LM),观察其生存曲线的变化;在荷瘤后感染小鼠模型中,通过流式胞内细胞因子染色和Pentamer染色技术检测小鼠抗OVA257-264表位特异性CD8+T细胞免疫应答的变化;通过标记CFSE的OT-Ⅰ细胞过继转移小鼠,观察体内抗原特异性CD8+T细胞增殖和抗原呈递情况;FACS分析荷瘤情况下小鼠脾脏CD11b+Gr-1+细胞及Treg细胞比例和数量变化。结果荷瘤小鼠感染OVA-LM菌后生存期明显缩短;荷瘤小鼠脾脏抗OVA257-264表位特异性CD8+T细胞免疫应答显著下调;抗原特异性CD8+T细胞增殖速率没有显著变化,但是细胞总数明显下降;荷瘤小鼠脾脏调节性T细胞比例未见明显变化,但CD11b+Gr-1+细胞显著上调。结论荷瘤小鼠抗感染CD8+T细胞免疫反应下降,其脾脏CD11b+Gr-1+细胞显著上调,提示肿瘤环境下脾脏CD11b+Gr-1+细胞升高可能是影响抗感染CD8+T细胞应答的重要因素。     

HIV gag DNA疫苗诱导小鼠特异性细胞免疫应答的研究

目的 研究小鼠注射HIV gag DNA疫苗后的抗原特异性细胞免疫应答.方法 C57BL/6小鼠以初免/加强的策略经肌肉注射HIV gag DNA疫苗,一周后获取其脾与肺的单个细胞,体外经Gag抗原多肽刺激后,采用ELISA法检测细胞培养上清中IFN-γ的水平,ELISPOT法检测IFN-γ分泌细胞的频率,流式细胞仪分析特异性T细胞的亚群.结果 经Gag多肽刺激后,加强免疫组IFN-γ产生的总体水平和分泌细胞频率均高于对照组及初次免疫组.HIV gag DNA疫苗可同时诱导产生Gag-特异性CD4 和CD8 T细胞.结论 HIV gagDNA疫苗免疫小鼠后可诱导抗原特异性效应性T细胞应答.     

乙肝病毒核心抗原DNA疫苗诱导小鼠抗原特异性CD8+T细胞动态变化和动态分布

目的：研究乙肝病毒核心抗原(HBcAg)DNA疫苗诱导的细胞免疫应答。方法：用表达乙肝病毒核心抗原N端144个氨基酸的重组质粒DNA(简称pHBc144)100ug免疫C57BL／6小鼠，用细胞内细胞因子染色技术检测小鼠体内抗原特异性CD8^ T细胞的动态变化。结果：pHBc144免疫后抗原特异性CD8^ T细胞逐渐增多，在初次免疫的第14天出现第一个免疫应答高峰，此后抗原特异性CD8^ T细胞数量逐渐下降进入免疫记忆期并在1年内维持在稳定水平。加强免疫后在第10天抗原特异性CD8^ T细胞数量达到高峰，约是初次免疫应答高峰的2倍，此后抗原特异性CD8^ T细胞数量逐渐下降，但下降的速度比初次免疫应答减缓。结论：pHBc144DNA疫苗可诱导有效的细胞免疫应答。     

人免疫缺陷病毒Ⅱ型核心蛋白DNA疫苗的实验免疫研究

目的　检测HIV 2核心蛋白DNA疫苗诱导Balb c小鼠免疫应答的能力。方法　将表达HIV 2核心蛋白DNA疫苗质粒pVAXIgag肌注Balb c小鼠 ,分析CD4 、CD8 T淋巴细胞的数量、脾特异性CTL反应、血清中HIV 2的特异性抗体水平。结果　重组质粒pVAXI gag免疫组与空载体pVAXI及PBS对照组相比较差异显著 ,血清抗体滴度及淋巴细胞杀伤效应为P <0 .0 1,脾T细胞亚群的数量为P <0 .0 5。结论　HIV 2核心蛋白DNA疫苗能诱导Balb c小鼠产生特异性细胞免疫应答和体液免疫应答     

肿瘤5-氟尿嘧啶治疗后可提高CD8~+T细胞免疫应答

目的探讨荷瘤小鼠5-氟尿嘧啶治疗后,机体细胞免疫应答的改变及初步机制,为化疗结合免疫治疗提供理论依据。方法皮下注射Lewis肺癌细胞,不同时相点观察肿瘤大小的变化,流式细胞仪检测脾脏髓源抑制细胞(myeloid derived suppressor cell,MDSC)及CD8+T细胞的比例;尾静脉感染李斯特菌,胞内细胞因子染色检测抗原特异性CD8+T细胞免疫应答;标记CFSE的OT-I细胞过继转移实验,观察体内抗原特异性CD8+T细胞增殖。结果 5-氟尿嘧啶处理的荷瘤小鼠,肿瘤生长显著受到抑制;MDSC细胞的比例显著减少;机体总CD8+T细胞的比例没有明显的改变,感染李斯特菌感染后,5-氟尿嘧啶处理组,抗原特异性CD8+T细胞显著增多;抗原特异性CD8+T细胞体内增殖速率明显增强。结论荷瘤小鼠5-氟尿嘧啶治疗后,可以显著抑制MDSC,提高机体CD8+T细胞免疫应答。     

TNF-α缺失减少抗李斯特菌CD8~+T细胞应答并增加效应记忆细胞形成

目的探讨TNF-α在李斯特菌(Listeria monocytogenes,LM)感染模型中对CD8~+T细胞应答的影响。方法采用尾静脉注射重组OVA的李斯特菌株感染TNF-α基因敲除(TNF-αKO)小鼠和野生型(WT)对照小鼠建立系统性感染模型。运用流式细胞术检测不同时相点TNF-αKO小鼠和WT小鼠中内源性和外源性特异性CD8+T细胞应答反应情况。采用体内细胞杀伤实验和检测细菌滴度的方法评估TNF-αKO小鼠和WT小鼠感染后记忆期CD8~+T细胞的效应功能。接着分析记忆期和增殖期CD8~+T细胞的KLRG-1和CD127等分子的表达变化。结果与WT对照小鼠相比,TNF-αKO小鼠中CD8~+T细胞应答减少,记忆细胞增多;TNF-αKO小鼠中记忆CD8~+T细胞的效应功能正常;TNF-αKO小鼠记忆CD8~+T细胞中含有更多比例的KLRG-1+CD127-CD62L-效应记忆细胞,而第7天TNF-αKO小鼠特异性CD8~+T细胞的KLRG-1和CD127的表达与对照组无明显差别。结论TNF-αKO小鼠初次应答降低,形成更多记忆细胞,且效应记忆细胞增多。其机制与早期效应CD8~+T细胞分化无关,可能是TNF-αKO小鼠更多的KLRG-1+CD127-细胞在应答收缩期存活所致。     

重组弓形虫棒状体蛋白5诱导小鼠的免疫应答及其抗弓形虫感染的保护作用

为探讨重组弓形虫棒状体蛋白5诱导的免疫应答及其免疫保护效应,本研究克隆表达了弓形虫棒状体蛋白5,并分析其诱导的细胞免疫、体液免疫应答和抗弓形虫感染的保护作用。采用PCR方法从刚地弓形虫cDNA中扩增出ROP5基因片段,将该基因片段克隆至pET-28a原核表达载体表达,用重组ROP5蛋白免疫BALB／c小鼠,ELISA检测免疫后小鼠抗体和细胞因子的变化。以强毒型RH株弓形虫攻击感染免疫小鼠,统计小鼠不同时间存活率,评价重组蛋白产生的免疫保护性。结果表明ROP5重组蛋白疫苗免疫BALB／c小鼠诱导机体产生高水平的IgG、IgG1、IgG2a抗体和IFN—y、IL-2及IL-10细胞因子。与PBS及佐剂对照组相比,重组蛋白免疫组小鼠的存活时间明显延长。本实验证实了ROP5重组蛋白免疫BALB／c小鼠能够诱导其产生高水平的体液免疫和细胞免疫应答,以及一定的抗弓形虫感染保护作用。     

SARS-CoV S DNA疫苗诱导特异性T细胞及相关细胞因子的特性研究

目的 小鼠经皮下SARS-CoV S DNA疫苗免疫后，研究其特异性T细胞及相关细胞因子的特性。方法SARS-CoV S DNA疫苗免疫BALB／c小鼠后，获取淋巴细胞悬液。经S抗原多肽刺激后，采用ELISA检测细胞培养上清液中IFN-γ／的水平，利用流式细胞仪在单个细胞水平上检测IFN-γ和IL-2的表达及其关系。结果 当S混合多肽刺激后，DNA疫苗免疫小鼠的淋巴细胞产生大量的IFN-γ，与对照鼠相比差异有统计学意义（P〈0．01）。细胞亚群分析的结果表明，IFN-γ^＋和IL-2^＋的CD4^＋T细胞百分率明显高于CD8^＋T细胞。单独产生IL-2的细胞占大多数，其次为IFN-γ和IL-2双阳性细胞，只产生IFN-γ的细胞很少。结论 SARS-CoV S DNA疫苗免疫小鼠后可以诱导抗原特异性CD4^＋和CD8^＋T细胞的产生。     

Chronic pneumonia despite adaptive immune response to Mycobacterium bovis BCG in MyD88-deficient mice

To assess the role of Toll-like receptor (TLR) signalling in host response to mycobacterial infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with the vaccine strain Mycobacterium bovis (BCG), and the immune response and bacterial burden were investigated. Macrophages and dendritic cells from MyD88-deficient mice stimulated in vitro with BCG mycobacterial antigens produced very low levels of proinflammatory cytokines, while the expression of costimulatory molecules such as CD40 and CD86 was preserved. Upon systemic infection with BCG (2 x 10(6) CFU i.v.) MyD88-deficient mice developed confluent chronic pneumonia with two log higher CFU than wild-type mice. Interestingly, the infection was controlled in liver and spleen and there was efficient systemic T-cell priming with high IFNgamma production by CD4+ splenic T cells in MyD88-deficient mice. Lung infiltrating cells showed IFNgamma production by pulmonary CD4+ T cells upon specific restimulation, and a reduced capacity to produce nitric oxide and IL-10. In summary, despite the dramatic reduction of the innate immune response, MyD88-deficient mice were able to mount an efficient T-cell response to mycobacterial antigens, which was however insufficient to control infection in the lung, resulting in chronic pneumonia in MyD88-deficient mice.     

Opportunistic infections and retrovirus-induced immunodeficiency: studies of acute and chronic infections with Toxoplasma gondii in mice infected with LP-BM5 murine leukemia viruses.

Mice infected with LP-BM5 murine leukemia viruses develop a syndrome, termed mouse AIDS (MAIDS), characterized by increasingly severe immunodeficiency and progressive lymphoproliferation. Virus-infected mice were examined for the ability to resist acute infection and to control chronic infection with the protozoan Toxoplasma gondii, a major opportunistic pathogen of individuals infected with human immunodeficiency virus. Mice infected with the retroviruses for 2 or 4 weeks responded normally to challenge with the parasite, but mice inoculated with the protozoan 8 or 12 weeks after viral infection died with acute disease due to T. gondii. Increased sensitivity to acute infection was associated with a reduced ability to produce gamma interferon (IFN-gamma) and with established changes in CD4+ T-cell function. Mice latently infected with T. gondii and then inoculated with the retrovirus mixture were found to reactivate the parasite infection, with 30 to 40% of dually infected animals dying between 5 and 16 weeks after viral infection. Reactivation was associated with reduced proliferation and impaired production of IFN-gamma in response to stimulation with soluble T. gondii antigens or to concanavalin A. Continuing resistance to lethal reactivation in the remaining mice was shown to require CD8+ T cells and expression of IFN-gamma. In addition, it was found that chronic infection with T. gondii altered the course of MAIDS by inhibiting the progression of splenomegaly and immunodeficiency and reducing the expression of both the helper and etiologic defective viruses. These results support previous studies which indicate that infection with T. gondii is controlled by synergistic interactions between CD4+ and CD8+ T cells, the functions of which are progressively impaired during the course of MAIDS.     

CD8+-T-Cell Immunity against Toxoplasma gondii Can Be Induced but Not Maintained in Mice Lacking Conventional CD4+ T Cells

T-cell immunity is critical for survival of hosts infected with Toxoplasma gondii. Among the cells in the T-cell population, CD8(+) T cells are considered the major effector cells against this parasite. It is believed that CD4(+) T cells may be crucial for induction of the CD8(+)-T-cell response against T. gondii. In the present study, CD4(-/-) mice were used to evaluate the role of conventional CD4(+) T cells in the immune response against T. gondii infection. CD4(-/-) mice infected with T. gondii exhibited lower gamma interferon (IFN-gamma) messages in the majority of their tissues. As a result, mortality due to a hyperinflammatory response was prevented in these animals. Interestingly, T. gondii infection induced a normal antigen-specific CD8(+)-T-cell immune response in CD4(-/-) mice. No difference in generation of precursor cytotoxic T lymphocytes (pCTL) or in IFN-gamma production by the CD8(+)-T-cell populations from the knockout and wild-type animals was observed. However, the mutant mice were not able to sustain CD8(+)-T-cell immunity. At 180 days after infection, the CD8(+)-T-cell response in the knockout mice was depressed, as determined by pCTL and IFN-gamma assays. Loss of CD8(+)-T-cell immunity at this time was confirmed by adoptive transfer experiments. Purified CD8(+) T cells from CD4(-/-) donors that had been immunized 180 days earlier failed to protect the recipient mice against a lethal infection. Our study demonstrated that although CD8(+)-T-cell immunity can be induced in the absence of conventional CD4(+) T cells, it cannot be maintained without such cells.     

Depletion of L3T4+ (CD4+) T lymphocytes prevents development of resistance to Toxoplasma gondii in mice.

The role of L3T4+ (CD4+) T lymphocytes in the resistance of mice of different haplotypes to Toxoplasma gondii was examined, using the monoclonal antibody GK 1.5. Outbred Swiss-Webster or inbred CBA/Ca (H-2k), BALB/c (H-2d), and C57BL/6 (H-2b) mice injected with monoclonal antibody GK 1.5 24 h before and 24 h and 7, 15, and 21 days after oral inoculation with 10 live cysts of the low-virulence strain ME49 of T. gondii exhibited an almost complete abrogation of their antibody and cell-mediated immune responses to the parasite. Delayed-type hypersensitivity response and lymphocyte stimulation assays showed significantly reduced values compared with those of control mice inoculated with ME49 cysts but not treated with the monoclonal antibody. The number of T. gondii cysts in the brains of GK 1.5-treated mice was significantly higher than in controls. Challenge of the GK 1.5-treated and chronically infected mice with the virulent RH strain of T. gondii resulted in 100% mortality, whereas 100% of chronically infected controls survived the infection. These results suggest that L3T4+ (CD4+) T lymphocytes actively participate in the development of resistance to T. gondii and in the mechanisms controlling the formation of cysts of the parasite in the brains of mice.     

The major histocompatibility complex haplotype affects T-cell recognition of mycobacterial antigens but not resistance to Mycobacterium tuberculosis in C3H mice

Both innate and adaptive immunity play an important role in host resistance to Mycobacterium tuberculosis infection. Although several studies have suggested that the major histocompatibility complex (MHC) haplotype affects susceptibility to infection, it remains unclear whether the modulation of T-cell immunity by the MHC locus determines the host's susceptibility to tuberculosis. To determine whether allelic differences in the MHC locus affect the T-cell immune response after M. tuberculosis infection, we infected inbred and H-2 congenic mouse strains by the respiratory route. The H-2 locus has a profound effect on the antigen-specific CD4+-T-cell response after M. tuberculosis infection. CD4+ T cells from infected mice of the H-2(b) haplotype produced more gamma interferon (IFN-gamma) after in vitro stimulation with mycobacterial antigens than mice of the H-2(k) haplotype. A higher level of IFN-gamma was also detected in bronchoalveolar lavage fluid from infected mice of the H-2(b) haplotype. Furthermore, C3.SW-H2(b)/SnJ mice generate and recruit activated T cells to the lung after infection. Despite a robust immune response, C3.SW-H2(b)/SnJ mice succumbed to infection early and were similarly susceptible to infection as other C3H (H-2(k)) substrains. These results suggest that although the MHC haplotype has a profound impact on the T-cell recognition of M. tuberculosis antigens, the susceptibility of C3H mice to infection is MHC independent.     

Prophylactic and therapeutic vaccination with dendritic cells against hepatitis C virus infection

Antigen uptake and presentation capacities enable DC to prime and activate T cells. Recently, several studies demonstrated a diminished DC function in hepatitis C virus (HCV) infected patients showing impaired abilities to stimulate allogenic T cells and to produce IFN-gamma in HCV infected patients. Moreover, DC of patients who have resolved HCV infection behave like DC from healthy donors responding to maturation stimuli, decrease antigen uptake, up-regulate expression of appropriate surface marker, and are potent stimulators of allogenic T cells. A number of studies have demonstrated in tumour models and models of infectious diseases strong induction of immune responses after DC vaccination. Because DC are essential for T-cell activation and since viral clearance in HCV infected patients is associated with a vigorous T-cell response, we propose a new type of HCV vaccine based on ex vivo stimulated and matured DC loaded with HCV specific antigens. This vaccine circumvents the impaired DC maturation and the down regulated DC function of HCV infected patients in vivo by giving the necessary maturation stimuli and the HCV antigens in a different setting and location ex vivo. Strong humoral and cellular immune responses were detected after HCV core DC vaccination. Furthermore, DC vaccination shows partial protection in a therapeutic and prophylactic model of HCV infection. In conclusion, mice immunized with HCV core pulsed DC generated a specific antiviral response in a mouse HCV challenge model. Our results indicate that HCV core pulsed DC may serve as a new modality for immunotherapy of HCV especially in chronically infected patients.     

The CD40/CD40 ligand interaction is required for resistance to toxoplasmic encephalitis

Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-gamma), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-gamma when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-gamma or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L(-/-) mice infected with T. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-gamma but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L(-/-) mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-gamma, to activate macrophages in vitro to control replication of T. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.     

Mucosal and systemic cellular immune responses induced by Toxoplasma gondii antigens in cyst orally infected mice.

This study was performed to determine the T-cellular immune responses following Toxoplasma gondii oral infection and to assess further toxoplasma antigens on their ability to stimulate in vitro mucosal and systemic T-cell immunity. Parasite-specific cellular immune responses in Peyer's patches (PP), in mesenteric lymph nodes (MLN) and in spleen (SPL) were investigated using a lymphoblastic transformation test following oral infection of mice with strain 76K cysts of T. gondii. An early toxoplasma sonicate-induced mucosal T-cell proliferation occurred in MLN and PP with a peak responsiveness on day 6 post-infection (PI) and rapidly reached background levels on day 7 PI in PP and on day 8 PI in mesenteric lymph nodes. A later splenic cellular blastogenesis was observed from day 28 PI and persisted throughout the experiment (day 91). At the time of T-cell proliferation, FACS analyses revealed a decrease in the relative percentages of CD4+ and CD8+ T cells with a predominance of CD8+ lymphocytes which leads to an inversion of the CD4/CD8 ratios. We found that CBA/J is a high responder mouse strain in the induction of mesenteric and splenic T-lymphocyte blastogenesis compared to the intermediate responder BALB/c and low responder C57BL/6. Toxoplasma gondii antigens SAG1 (30,000 MW) and GRA4 (40,000-41,000 MW), which are known to induce locally IgA antibodies, are shown to stimulate primed mucosal T lymphocytes from CBA/J and BALB/c mice whereas no proliferation was demonstrated with C57BL/6 T cells. 229-242 peptide, derived from the deduced amino acid sequence of GRA4, only induces detectable proliferation of primed-CBA/J T lymphocytes. Following oral experimental infection, the in vitro mesenteric response to a toxoplasma sonicate is dominated by a Th2-type cytokine pattern whereas a predominant Th1 cytokine response is observed in the spleen. Finally, in vitro stimulation of mesenteric T cells with the three defined toxoplasma antigens resulted in secretion of interleukin-5 (IL-5) and IL-6 (except for SAG1) and interferon-gamma (IFN-gamma) whereas no detectable IL-2 or IL-4 was observed.     

Organ- and disease-stage-specific regulation of Toxoplasma gondii-specific CD8-T-cell responses by CD4 T cells

Toxoplasma gondii induces a persistent central nervous system infection, which may be lethally reactivated in AIDS patients with low CD4 T-cell numbers. To analyze the role of CD4 T cells for the regulation of parasite-specific CD8 T cells, mice were infected with transgenic T. gondii expressing the CD8 T-cell antigen beta-galactosidase (beta-Gal). Depletion of CD4 T cells prior to infection did not affect frequencies of beta-Gal(876-884)-specific (consisting of residues 876 to 884 of beta-Gal) CD8 T cells but resulted in a pronounced reduction of intracerebral beta-Gal-specific gamma interferon (IFN-gamma)-producing and cytolytic CD8 T cells. After cessation of anti-CD4 treatment a normal T. gondii-specific CD4 T-cell response developed, but IFN-gamma production of intracerebral beta-Gal-specific CD8 T cells remained impaired. The important supportive role of CD4 T cells for the optimal functional activity of intracerebral CD8 T cells was also observed in mice that had been depleted of CD4 T cells during chronic toxoplasmosis. Reinfection of chronically infected mice that had been depleted of CD4 T cells during either the acute or chronic stage of infection resulted in an enhanced proliferation of beta-Gal-specific IFN-gamma-producing splenic CD8 T cells. However, reinfection of chronically infected mice that had been depleted of CD4 T cells in the acute stage of infection did not reverse the impaired IFN-gamma production of intracerebral CD8 T cells. Collectively, these findings illustrate that CD4 T cells are not required for the induction and maintenance of parasite-specific CD8 T cells but, depending on the stage of infection, the infected organ and parasite challenge infection regulate the functional activity of intracerebral CD8 T cells.     

A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection

Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-gamma). The contribution of CD4(+) and CD8(+) T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4(+) and CD8(+) T-cell subsets produced IFN-gamma upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8(+) T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8(+) T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4(+) T cells led to an increase in brain cyst burden during the chronic phase of infection.