Short-Chain Fatty Acids Regulate Cytokines and Th17/Treg Cells in Human Peripheral Blood Mononuclear Cells in vitro

Short-chain fatty acids (SCFAs) have been recognized as mediators of immune responses, including pathways of cytokine production. In this study, we investigated the immune-regulatory effects of SCFAs on human peripheral blood mononuclear cells (PBMCs) from buffy coat of healthy donors. PBMCs were exposed to varying concentrations of individual SCFAs or of their mixtures of acetate, propionate and butyrate. The productions of interleukin (IL) IL-1β, IL-2, IL-6, IL-10, IL-17, IL-21, IL-23 and transforming growth factor beta 1 (TGF-β1) were assessed. T cell differentiation after exposure to SCFAs was also examined. Compared with lipopolysaccharide (LPS)-stimulated cells (controls), SCFAs slightly decreased TGF-β1 production and reduced IL-6 production; butyrate was more effective than acetate or propionate. SCFAs particularly butyrate caused the induction of CD4+CD25+ regulatory T cells (Treg) rather than Th17 cells. SCFAs may up-regulate the production of anti-inflammatory cytokines in PBMCs, resulting in the induction of CD4+CD25+ Treg cells.     

Effect of Cholesterol Depletion on Interleukin-8 Production in Human Respiratory Epithelial Cells

Purpose

The lipid entities of cell membranes are components of the immune system and important mediators of inflammation. Despite increasing interest in the function of epithelial cells in inflammation, the role of cholesterol in this process has not been described. Here, we investigated the effect of cholesterol depletion on the inflammatory process in airway epithelial cells via the expression of interleukin (IL)-8 as a marker of inflammation.

Methods

A 549 cells were treated with 0.5% methyl-β-cyclodextrin as a selective cholesterol extractor. The IL-8 level was assessed by enzyme-linked immunosorbent assay and reassessed after cholesterol repletion. Mitogen-activated protein kinase (MAPK) inhibitors were used to determine the upstream signaling pathway for IL-8 production in cholesterol-depleted cells.

Results

We found a relationship between the amount of cholesterol in A 549 cells and inflammation of the airway. IL-8 production was increased in cholesterol-depleted A 549 cells and restored by cholesterol repletion. IL-8 production was decreased by pretreatment with the extracellular signal-regulated kinase (ERK) inhibitor U0126 but not with JNK inhibitor II or the p38 MAPK inhibitor SB202190.

Conclusions

Our findings suggest that inflammatory responses are increased in cholesterol-depleted epithelial cells via the MAPK signaling system, predominantly by the ERK pathway. We conclude that the lipid components of airwayepithelial cells may play a role in the inflammatory process.     

肺炎克雷伯杆菌分泌因子及活菌诱导人肺上皮细胞表达分泌IL-8的研究

目的 :探讨肺炎克雷伯杆菌 (Klebsiellapneumoniae ,Kp)分泌因子及活菌诱导肺上皮细胞株表达和分泌IL 8的状况。方法 :用临床分离株Kp0 3 1 1 6、Kp0 3 1 83的细菌培养上清或活菌刺激肺上皮细胞株A5 49和SPC A 1 ,酶联免疫吸附实验 (ELISA)检测细胞IL 8表达量。结果 :①两株Kp培养上清分别刺激A5 49或SPC A 1后 ,IL 8表达量均有显著增高 (P <0 .0 1 ) ,且随上清刺激浓度增加而上升。②两株Kp及DH5 (活菌分别与SPC A 1共孵育 2h ,用庆大霉素杀死胞外菌 ,继续孵育 2 4h后两株Kp诱导细胞IL 8表达量均显著高于DH5α(P <0 .0 1 ) ,且随细菌数 /细胞数比例增大而上升。结论 :Kp分泌因子及活菌都能够诱导肺上皮细胞IL 8表达 ,而活菌上调IL 8的效应较培育上清分泌因子更显著 ,提示肺上皮细胞在Kp感染刺激的肺部炎症反应中起重要作用。     

Generation of MHC Class I-Restricted Cytotoxic T Cell Lines and Clones Against Colonic Epithelial Cells from Ulcerative Colitis

We established CTL lines and clones against colonic epithelial cells from PBLs of patients with ulcerative colitis by continuous stimulation with HLA-A locus-matched colonic epithelial cell lines. We developed a nonradioactive europium release cytotoxicity assay to detect CTLs. PBLs from 3 of 12 patients but not from any of 14 normal controls who shared at least one haplotype of HLA-A locus with two colonic epithelial cell lines, CW2 and ACM, showed increased cytotoxicity against these lines. Three CTL lines established from the PBLs of patients showed increased cytotoxicity against HLA-A locus-matched CW2 or ACM but not against matched lung or esophagus cell lines. The phenotypes of CTL lines were -TCR⁺CD3⁺CD8⁺CD16^–. The CTL line MS showed increased cytotoxicity against freshly isolated colonic epithelial cells but not against cells with a different HLA-A locus. Two CTL clones were generated from MS and clone 3-2, expressing CD3⁺CD8⁺CD4^–CD56^–, showed high MHC class I-restricted cytotoxicity against the colonic epithelial cells. These results indicated that CTLs against colonic epithelial cells may contribute to epithelial cell damage in ulcerative colitis.     

游离脂肪酸诱导3T3-L1细胞胰岛素抵抗模型的建立

目的观察不同种类和浓度游离脂肪酸(FFA)对3T3-L1细胞葡萄糖摄取及胰岛素敏感性的影响,建立FFA诱导的胰岛素抵抗细胞模型。方法3T3-L1细胞体外诱导分化为脂肪细胞,油红O染色鉴定,不同浓度软脂酸(PA)和油酸(OA)诱导,测定基础状态和胰岛素刺激下葡萄糖特异性转运情况。结果3T3-L1脂肪细胞诱导率为98%±1.3%,PA和OA各组胰岛素刺激后的葡萄糖特异性转运呈时间和浓度依赖性下降趋势,均显著低于对照组(p<0.01);0.5mM PA作用24h后基础葡萄糖特异性转运明显低于对照组和PA 0.25mM作用24h组(p<0.05)。结论3T3-L1细胞在体外可稳定分化为成熟脂肪细胞,经FFA诱导成为胰岛素抵抗细胞模型;随FFA作用时间延长和浓度增加,胰岛素抵抗程度加重,并以时间和浓度依赖性方式抑制3T3-L1脂肪细胞胰岛素刺激后的葡萄糖特异性转运,高浓度时抑制基础状态下的葡萄糖特异性转运。     

人IL—6在CHO细胞表达及其实验室生产工艺的建立

本文以PKCR6质粒为载体,构建了人IL-6的次级克隆PKCR6-IL-6,并成功地转染了CHO细胞,获得高表达、稳定分泌人IL-6的转基因细胞,由此建立了实验室生产IL-6的基本工艺,即从方瓶过渡到转瓶,经转瓶高密度培养,CHO上清中IL-6含量可以提高近6倍,这为扩大规模生产奠定了一定的基础。     

Regulation and production of IL-8 by human proximal tubular epithelial cells in vitro

A number of inflammatory kidney diseases are associated with interstitial nephritis and influx of leucocytes in the renal interstitium. Potentially the influx of neutrophils in the interstitium may be induced by the chemotactic cytokine IL-8. In the present study we have analysed the production of IL-8 by cultured human proximal tubular epithelial cells (PTEC) in response to a number of proinflammatory cytokines. Primary cell lines of proximal tubular epithelium obtained from ten different kidneys, and cultured under serum-free conditions, were found to produce IL-8 to different degrees from not detectable levels up to 10·8±1·5 ng IL-8 per 1×10⁵ cells in 72 h. Gel filtration chromatography of PTEC supernatant indicated that the size of IL-8 of PTEC is 15·1 and 8·1 kD, and is chemotactically active for polymorphonuclear neutrophils (PMN). Addition of 0·5 ng/ml rIL-1α or 1000 U/ml recombinant tumour necrosis factor-alpha (TNF-α) to the culture media of PTEC induced an up-regulation of IL-8 production up to 6·3-fold and 3·0-fold, respectively. The up-regulation by IL-1α and TNF-α was dose- and time-dependent. In contrast, 500 U/ml recombinant interferon-gamma (rIFN-γ) down-regulated the production of IL-8 3·4-fold. Northern blot analysis showed that IL-1α and TNF-α increased the expression of IL-8 mRNA, whereas IFN-γ reduced IL-8 mRNA expression. Taken together, these experiments indicate that human PTEC are a potential source of IL-8 in the kidney, and that IL-8 produced in the proximal tubule can be induced by various proinflammatory cytokines.     

子宫内膜异位症在位内膜腺上皮细胞及基质细胞的分离培养和鉴定

目的分离培养子宫内膜异位症在位内膜组织中子宫内膜腺上皮细胞及基质细胞,建立研究子宫内膜异位症的细胞模型。方法对分泌期子宫内膜异位症在位内膜组织用混合酶消化,滤网过滤和差速梯度离心的方法分离,体外培养后通过光镜观察及免疫细胞化学、免疫荧光化学方法对分离细胞鉴定。结果分离的细胞角质蛋白阳性子宫内膜腺细胞百分率约90～95%;分离的骨架蛋白形成蛋白阳性的基质细胞百分率达90%以上。结论本研究获得较高纯度的子宫内膜腺细胞和基质细胞,成功建立了研究子宫内膜异位症的细胞模型。     

子宮内膜异位症在位内膜腺上皮细胞及基质细胞的分离培养和鉴定

目的 分离培养子宫内膜异位症在位内膜组织中子宫内膜腺上皮细胞及基质细胞,建立研究子宫内膜异位症的细胞模型.方法 对分泌期子宫内膜异位症在位内膜组织用混合酶消化,滤网过滤和差速梯度离心的方法分离,体外培养后通过光镜观察及免疫细胞化学、免疫荧光化学方法对分离细胞鉴定.结果 分离的细胞角质蛋白阳性子宫内膜腺细胞百分率约90～95%;分离的骨架蛋白形成蛋白阳性的基质细胞百分率达90%以上.结论 本研究获得较高纯度的子宫内膜腺细胞和基质细胞,成功建立了研究子宫内膜异位症的细胞模型.     

IL-24对非小细胞肺癌患者CD8+T细胞体外功能的影响

目的观察IL-24在体外对非小细胞肺癌(non-small cell lung cancer,NSCLC)患者CD8⁺T细胞功能的影响。方法本研究入组28例NSCLC患者和17例健康对照者,收集外周血单个核细胞(peripheral blood mononuclear cells,PBMC)和支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF),分选CD8⁺T细胞,反转录实时定量PCR检测CD8⁺T细胞中IL-24受体(IL-20R1、IL-20R2和IL-22R1)mRNA的相对表达量。不同浓度重组人IL-24(10 ng/ml和100 ng/ml)刺激纯化CD8⁺T细胞后,流式细胞术检测穿孔素和颗粒酶B的表达变化。建立CD8⁺T细胞和NSCLC细胞系NCI-H1882细胞的直接接触和间接接触体外共培养系统,观察IL-24刺激后CD8⁺T细胞诱导靶细胞死亡比例,以及IFN-γ和TNF-α的表达变化。组间比较采用t检验或LSD-t检验。结果CD8⁺T细胞中未检测到IL-22R1 mRNA表达,CD8⁺T细胞中IL-20R1和IL-20R2 mRNA相对表达量在健康对照者和NSCLC患者之间以及在非肿瘤部位和肿瘤部位之间的差异均无统计学意义(P>0.05)。NSCLC患者外周血和肿瘤部位CD8⁺T细胞中穿孔素和颗粒酶B水平显著低于健康对照者和非肿瘤部位(P<0.05),低浓度IL-24(10 ng/ml)刺激不影响CD8⁺T细胞中穿孔素和颗粒酶B水平(P>0.05),而高浓度IL-24(100 ng/ml)则显著提升NSCLC患者CD8⁺T细胞中穿孔素和颗粒酶B水平(P<0.05)。直接接触共培养系统中,高浓度IL-24(100 ng/ml)刺激NSCLC患者肿瘤部位CD8⁺T细胞后可诱导靶细胞死亡比例升高,以及IFN-γ和TNF-α表达升高,而低浓度IL-24(10 ng/ml)刺激对CD8⁺T细胞诱导的靶细胞死亡和细胞因子分泌无显著影响。间接接触共培养系统中,IL-24刺激对CD8⁺T细胞诱导的靶细胞死亡和细胞因子分泌均无显著影响。结论高浓度IL-24在体外可增强NSCLC患者CD8⁺T细胞的直接细胞杀伤功能,但在体内IL-24可能并不影响CD8⁺T细胞的功能。     

A subset of CD8+ T cells from allergic patients produce IL-4 and stimulate IgE production in vitro

Background and Objective A subset of IL-4 producing CD8⁺ T cells was recently identified in HIV patients. Based on these findings we examined whether IL-4 producing CD8⁺ T cells would also be present in allergic patients and what would be the functional relevance of this T-cell population. Methods We investigated the role of CD8⁺ T cells in IgE production of allergic diseases by analysing the cytokine profile of individual CD4⁺ and CD8⁺ T cells. Results In allergic patients about twice as many CD4⁺ T cells and six times as many CD8⁺ T cells produced IL-4 as in non-allergic controls. In contrast the frequency of IFNγ⁺ T-cell subsets did not significantly differ between the allergic and non-allergic individuals. The frequency of 1L4⁺CD8⁺ T cells correlated with the level of serum IgE. Coculture experiments with T cells or purified CD8⁺ T cells together with autologous B cells indicated that CD8⁺ T cells enhanced IgE in vitro, but not IgM production, even when they were physically separated from B cells. This effect could be partially blocked by addition of an IL-4 binding protein, a soluble IL-4 receptor indicating that lL-4 is involved in CD8⁺ T-cell mediated IgE production. Conclusions These data indicate a positive role of IL-4 secreting CD8⁺ T cells in IgE regulation in allergic patients.     

Evaluation of functional heterogeneity in the CD8 subset with T cells from T cell receptor-transgenic mice

The question of functional differentiation within the CD8 subset has been addressed in a model of TcR-transgenic (TcR-tg) mice expressing a TcR specific for H-2K^b (Ti). CD8⁺ Ti⁺ T cells present in the periphery of these mice have no cytotoxic T lymphocyte (CTL) activity unless they are stimulated with H-2K^b-expressing cells. In contrast to T cells from normal H-2^k littermates, alloantigen induction of CTL from TcR-tg mice is independent of CD4⁺ T helper (T_h) cells and is accompanied by high level secretion of interleukin-(IL)-2 by Ti⁺ CD8⁺ T cells. Precursor frequency analysis performed on CD8⁺ cells from TcR-tg mice revealed a high frequency of T_h as compared to CTL precursors. This raised the possibility of the existence of distinct subpopulations within CD8⁺ precursors with different requirements for differentiation to functional CTL. FACS analyses (performed on resting and on in vitro stimulated T cells from normal and TcR-tg mice) demonstrated a heterogeneous expression of Ly-6C on CD8⁺ cells with a large enrichment of Ly-6C^? cells among the Ti⁺ cells which persisted after stimulation with H-2^b cells in conditions that led to a homogeneous expression of the activation markers pgp-1 and CD69. The possibility that Ly-6C expression could mark functionally different subpopulations in CD8⁺ T cells was investigated. Stimulation of sorted populations of Ly-6C^? and Ly-6C⁺ cells allowed detection of CTL precursors in both these subsets and the majority of limiting dilution wells containing one pCTL also scored positive for IL-2 secretion. Thus, for CD8⁺ T cells expressing the same TcR, differentiation led to acquisition of both IL-2 secretion and CTL function and there was no evidence for the existence of a distinct population of helper-dependent CTL precursors.     

Neoplastic thymic epithelial cells of human thymoma support T cell development from CD4−CD8− cells to CD4+CD8+ cells in vitro

Human thymoma is a thymic epithelial cell tumour which often contains a large number of immature T cells and is frequently associated with autoimmune diseases. Since thymic epithelial cells play key roles in the development and selection of T cells in the normal thymus, we hypothesized that the neoplastic thymic epithelial cells of thymoma may support T cell differentiation in the tumour. We characterized CD4^?CD8^? cells in thymoma and applied an in vitro reconstitution culture system using the CD4^?CD8^? cells and the neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on 29.9 ± 12.2% of CD4^?CD8^? cells in thymoma. TCRγδ was expressed on 27.4 ± 15.1% of CD4^?CD8^? cells and CD19, a B cell marker, was expressed on 14.1 ± 23.1% of CD4^?CD8^? cells. CD4^?CD8^? cells expressed both IL-7R α-chain and common γ-chain. Purified CD4^?CD8^? cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4⁺CD8⁺ cells via CD4 single-positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. Furthermore, we examined the reconstitution culture using CD34⁺CD4^?CD8^? cells purified from normal infant thymus. The CD34⁺CD4^?CD8^? cells in normal thymus also differentiated to CD4⁺CD8⁺ cells in the allogeneic co-culture with the neoplastic epithelial cells of thymoma. These results indicate that the tumour cells of thymoma retain the function of thymic epithelial cells and can induce differentiation of T cells in thymoma.     

Human intestinal epithelial cells down-regulate IL-8 expression in human intestinal microvascular endothelial cells; role of transforming growth factor-beta 1 (TGF-β1)

Cytokines produced from intestinal epithelial cells may function as signals to neighbouring immune cells. In the present study we analysed the effects of colonic epithelial cell lines (HT-29, Caco-2, HCT-116, Colo-320) and freshly isolated intestinal epithelial cells on IL-8 expression in the SV-40T transfected human microvascular endothelial cell line (HMEC-1). Epithelial cell-conditioned media and transwells preventing physical contact between epithelial and endothelial cells were used. TGF-β1 and IL-8 levels were determined by ELISA and Northern blot analysis. Increasing concentrations of IL-1β led to increasing production of IL-8. The addition of epithelial cell-conditioned medium or epithelial cells to HMEC-1 cells in a two-compartment co-culture system resulted in a strong decrease in IL-8 at the protein and mRNA level. Decrease of IL-8 was markedly stronger when epithelial cells were co-cultured in contact with HMEC-1 cells, indicating that not only soluble factor(s) play a role in the induction of IL-8 suppression in HMEC-1 cells. MoAbs against TGF-β1 partially inhibited down-regulation of endothelial IL-8 expression. In further studies, IL-8 expression in freshly isolated human intestinal microvascular endothelial cells (HIMEC) was also down-regulated by intestinal epithelial cells. Our results demonstrate that intestinal epithelial cells down-regulate IL-8 expression in HMEC-1 cells. TGF-β1 is a candidate factor of epithelial–endothelial communication in the colonic mucosa.     

Human mast cells express receptors for IL-3, IL-5 and GM-CSF; a partial map of receptors on human mast cells cultured in vitro

BACKGROUND: Mast cells have long been recognized as the principal cell type that initiates the inflammatory response characteristic of acute allergic type 1 reactions. Our goal has been to further characterize maturation of progenitors to mast cells. METHODS: Mast cells were cultured from human cord blood derived CD133(+) progenitors. Mast cell function was tested using histamine release. During differentiation mast cells surface marker expression was monitored by flow cytometry. RESULTS: CD133(+) progenitors expressed the early haematopoietic and myeloid lineage markers CD34, CD117, CD13 and CD33. Mature mast cells expressed CD117, CD13 and CD33, and expression of the high affinity immunoglobulin E receptor FcepsilonRI increased during culture. Cytokine receptors interleukin (IL)-5R, IL-3R, granulocyte-macrophage-colony stimulating factor (GM-CSF)R and IL-18R were expressed at high levels during maturation. Chemokine receptors CXCR4 and CXCR2 were highly expressed on both newly purified CD133(+) cells and mature cells. CONCLUSION: Human mast cells can be cultured from a CD34(+)/CD117(+)/CD13(+)/CD33(+) progenitor cell population in cord blood that is tryptase and chymase negative. Developing and mature mast cells express a wide range of chemokine and cytokine receptors. We found high levels of expression of CD123, IL-5R and GM-CSF receptors, also found on eosinophils and basophils, and high levels of expression of the receptor for the inflammatory cytokine IL-18.     

CD4+ T cell-independent maintenance and expansion of memory CD8+ T cells derived from in vitro dendritic cell activation

CD4+ T cells are essential for the maintenance of CD8+ memory T (Tm) cells following acute infection, but the importance of CD4+ T cells for the maintenance and expansion of CD8+ Tm cells to non-infectious antigens remains mostly unknown. Here, we showed that ovalbumin (OVA)-specific CD8+ Tm cell precursors derived from in vitro stimulation of TCR transgenic OT I CD8+ T cells with OVA protein-pulsed bone marrow-derived dendritic cells (DCOVA) can give rise to functional CD8+ Tm cells after adoptively transferred into mice. These CD8+ Tm cells can be maintained and remain fully functional in CD4+ T cell-absent environments in vivo. Furthermore, CD4+ T cells are not essential for the expansion of these CD8+ Tm cells. Finally, these in vitro DCOVA-activated CD8+ Tm cells maintained in CD4-deficient mice are also able to confer fully protective immunity against a later challenge of OVA-expressing tumor cells. Collectively, these findings demonstrate that in contrast to acute infections, maintenance and expansion of CD8+ Tm cells after priming with OVA protein-pulsed dendritic cells are independent of CD4+ T cells.     

Expansion of CD3+CD4-CD8- T cell population expressing high levels of IL-5 in Omenn's syndrome.

Omenn's syndrome is a fatal, autosomal-recessive combined immune deficiency characterized by several erythematous exfoliative eruptions, lymphadenopathy, hepatosplenomegaly, and elevated eosinophil count. In some of these patients an expansion of CD3+CD4-CD8- double negative (DN) T cell population was observed. We show here that the DN population represents a clonal expansion of T cells which preferentially use V beta 14 in their T cell receptor complex. Using polymerase chain reaction, we show that patient's DN cells express spontaneously high levels of IL-5, thus possibly explaining the abundance of eosinophils in this disorder. The increase of IgE observed in patients with Omenn's syndrome is unlikely to be related to IL-4 production, as IL-4 levels in patient samples were low. However, patient's low expression of interferon-gamma (IFN-gamma), which has been reported to inhibit IgE production, may explain the elevated levels of IgE in this patient. The results thus highlight the importance of the inhibitory effect of IFN-gamma on regulation of IgE production.     

The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4+, CD8+, and CD19+ cell populations derived from allergic and healthy donors

The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4⁺ and CD8⁺ T cells and CD19⁺ B cells, positively selected by immunomagnetic separation, was investigated. The cells were obtained from allergic patients with moderate serum IgE levels and mild to moderate atopic dermatitis, and healthy controls. The different cell subfractions were stimulated with mitogens or specific allergens, as well as cell supernatants from the lymphoblastoid B- (RPMI 8866) and T-hybridoma (166 A₂) cell lines. Proliferative responses of T- and B-cell subsets stimulated with mitogens together with recombinant interleukin-2 (rIL-2) or accessory cells (AC) could be inhibited by DSCG. In allergic individuals, significant allergen-specific stimulation could be observed in the CD8-depleted peripheral blood mononuclear cell (PBMC) fractions. Isolated CD4⁺ T cells, without AC or IL-2, could also be stimulated with specific allergen, but the responses were rather low. DSCG inhibited, concentration dependently, all allergen-induced responses. Interestingly, only atopic derived CD4⁺ and CD8⁺ T cells were stimulated by soluble low-affinity IgE receptor (Fc?RII/sCD23) and IgE binding factor (IgEBF), including IgE enhancing factor, present in culture supernatants from RPMI 8866 and 166 A₂, respectively. These responses were also inhibited by DSCG. This was in contrast to the amplifying effect of DSCG on spontaneously proliferating RPMI 8866 and 166 A₂ cells, cultured in fresh cRPMI 1640 medium without sCD23 and IgE enhancing factor. Our results show that DSCG delivers an inhibitory signal or signals to PBMC subpopulations expressing Fc?RII/sCD23, either upregulated by phytohemagglutinin in normal and atopic cells, or by allergens or sCD23 in atopic cells. The findings suggest that sCD23 in supernatants or in serum may reverse the general inhibitory mode of DSCG.     

Epitope-specific in vivo protection against cytomegalovirus disease by CD8 T cells in the murine model of preemptive immunotherapy

Preclinical research in murine models as well as subsequent clinical trials have concordantly revealed a high protective potential of antiviral CD8 T cells, of donor-derived ex vivo memory CD8 T cells in particular, in the immunotherapy of cytomegalovirus (CMV) infection in immunocompromised recipients. Although it is generally held view that the observed beneficial effect of the transferred cells is viral epitope-specific, involving the recognition of MHC class-I presented peptides by cognate T cell receptors, this assumption awaits formal proof, at least with regard to the in vivo function of the CD8 T cells. This question is particularly evident for CMV, since the function of viral immune evasion proteins interferes with the MHC class-I pathway of peptide presentation. Alternatively, therefore, one has to consider the possibility that the requirement for epitope recognition may be bypassed by other ligand–receptor interactions between CD8 T cells and infected cells, which may trigger the signaling for effector functions. Clearly, such a mechanism might explain why CD8 T cells are so efficient in controlling CMV infection despite the expression of viral immune evasion proteins. Here we provide direct evidence for epitope-specificity of antiviral protection by employing a recombinant murine CMV (mCMV), namely the mutant virus mCMV-IE1-L176A, in which an immunodominant viral epitope of the regulatory immediate-early protein IE1 is functionally deleted by a point mutation replacing leucine with alanine at the C-terminal MHC anchor position of the antigenic peptide.