The Abl-1 Kinase is Dispensable for NK Cell Inhibitory Signalling and is not Involved in Murine NK Cell Education.

Natural killer (NK) cell responsiveness in the mouse is determined in an education process guided by inhibitory Ly49 and NKG2A receptors binding to MHC class I molecules. It has been proposed that inhibitory signalling in human NK cells involves Abl-1 (c-Abl)-mediated phosphorylation of Crk, lowering NK cell function via disruption of a signalling complex including C3G and c-Cbl, suggesting that NK cell education might involve c-Abl. Mice deficient in c-Abl expression specifically in murine NK cells displayed normal inhibitory and activating receptor repertoires. Furthermore, c-Abl-deficient NK cells fluxed Ca2+ normally after triggering of ITAM receptors, killed YAC-1 tumour cells efficiently and showed normal, or even slightly elevated, capacity to produce IFN-γ after activating receptor stimulation. Consistent with these results, c-Abl deficiency in NK cells did not affect NK cell inhibition via the receptors Ly49G2, Ly49A and NKG2A. We conclude that signalling downstream of murine inhibitory receptors does not involve c-Abl and that c-Abl plays no major role in NK cell education in the mouse.

TLR-Stimulated Eosinophils Mediate Recruitment and Activation of NK Cells In Vivo.

Eosinophils like many myeloid innate immune cells can provide cytokines and chemokines for the activation of other immune cells upon TLR stimulation. When TLR-stimulated eosinophils were inoculated i.p. into wild-type mice, and NK cells were rapidly recruited and exhibited antitumour cytotoxicity. However, when mice depleted of CD11c+ cells were used, a marked decrease in the number of recruited NK cells was observed. We postulated that CpG or LPS from the injected eosinophils could be transferred to host cells, which in turn could recruit NK cells. However, by inoculating mice deficient in TLR4 or TLR9 with LPS or CpG-stimulated eosinophils respectively, NK cell recruitment was still observed alongside cytotoxicity and IFNγ production. CpG stimulation of eosinophils produced the pro-inflammatory cytokine IL-12 and the chemokine CXCL10, which are important for NK cell activation and recruitment in vivo. To demonstrate the importance of CXCL10 in NK cell recruitment, we found that CpG-stimulated eosinophils pretreated with the gut microbial metabolite butyrate had reduced expression and production of CXCL10 and IL-12 and concomitantly were poor at recruitment of NK cells and inducing IFNγ in NK cells. Therefore, eosinophils like other innate immune cells of myeloid origin can conceivably stimulate NK cell activity. In addition, products of the gut microbiota can be potential inhibitors of NK cell.

The effects of hepatitis C virus core protein on functional responses in the NK cell line YTS.

Hepatitis C virus infection affects more than 170 million people worldwide. More than 80% of the patients are not able to eliminate the virus and progress to a chronic infection that usually culminates in complications such as cirrhosis and/or hepatocellular carcinoma. Although the adaptive immune response has been widely shown to be essential for viral clearance, the role of natural killer (NK) cells is not clearly understood. In this study, the effect of HCV core protein is examined on NK cell function, i.e., cytotoxicity and cytokine secretion. The expression of core protein in the YTS NK cell line led to an increase in the percentage of apoptotic cells soon after transduction. The surviving cells exhibited decreased cytotoxicity associated with decreases in perforin and granzyme B expression. Furthermore, the HCV core protein-transduced YTS NK cells had reduced IFNγ production as well as an altered surface receptor expression pattern. These features may correspond to a state of functional anergy similar to that seen in T cells transduced with HCV core protein. Together, these data suggest that HCV core protein may alter NK cell function.

Microbial water pollution: a screening tool for initial catchment-scale assessment and source apportionment.

The European Union Water Framework Directive requires that Management Plans are developed for individual River Basin Districts. From the point of view of faecal indicator organisms (FIOs), there is a critical need for screening tools that can provide a rapid assessment of the likely FIO concentrations and fluxes within catchments under base- and high-flow conditions, and of the balance ('source apportionment') between agriculture- and sewage-derived sources. Accordingly, the present paper reports on: (1) the development of preliminary generic models, using water quality and land cover data from previous UK catchment studies for assessing FIO concentrations, fluxes and source apportionment within catchments during the summer bathing season; (2) the calibration of national land use data, against data previously used in the models; and (3) provisional FIO concentration and source-apportionment assessments for England and Wales. The models clearly highlighted the crucial importance of high-flow conditions for the flux of FIOs within catchments. At high flow, improved grassland (and associated livestock) was the key FIO source; FIO loadings derived from catchments with high proportions of improved grassland were shown to be as high as from urbanized catchments; and in many rural catchments, especially in NW and SW England and Wales, which are important areas of lowland livestock (especially dairy) farming, ≥ 40% of FIOs was assessed to be derived from agricultural sources. In contrast, under base-flow conditions, when there was little or no runoff from agricultural land, urban (i.e. sewerage-related) sources were assessed to dominate, and even in rural areas the majority of FIOs were attributed to urban sources. The results of the study demonstrate the potential of this type of approach, particularly in light of climate change and the likelihood of more high-flow events, in underpinning informed policy development and prioritization of investment.

Critical role of Qa1b in the protection of mature dendritic cells from NK cell-mediated killing.

Molecular interactions in natural killer (NK) cell-mediated killing of dendritic cells (DC) have under recent years come under scrutiny. Upon stimulation with IFN-gamma or lipopolysaccharide, DC become relatively resistant to NK cell-mediated lysis. In the present study, we investigated the role of Qa1(b) on DC and its receptor NKG2A on NK cells in the protection of mature DC from NK cells. We demonstrate that while both NKG2A+ and NKG2A- NK cells can efficiently lyse unstimulated DC, NKG2A+ NK cells but not NKG2A- NK cells are largely impaired in their ability to lyse mature DC. Similarly, mature DC from mice expressing H-2D(b), whose leader peptide sequence binds and stabilizes Qa1(b), were resistant to NK cell-mediated killing, suggesting that stable Qa1(b) expression contributes to the protection of mature DC. This finding was further validated by the demonstration that addition of the Qdm leader peptide could protect TAP1-/- DC from NK cell-mediated lysis both in vitro and in vivo. The present data suggest that stable expression of Qa1 on the surface of mature DC contributes to the protection of DC from NK cell-mediated lysis.

Measuring ammonia emissions from land applied manure: an intercomparison of commonly used samplers and techniques.

A number of techniques have been developed to quantify ammonia (NH(3)) emissions following land application of manure or fertiliser. In this study, coefficients of variation were determined for three commonly used field techniques (mass balance integrated horizontal flux, wind tunnels and the equilibrium concentration technique) for measuring emissions from a range of manure types. Coefficients of variation (CV) for absorption flasks, passive flux samplers and passive diffusion samplers were 21, 10 and 14%, respectively. In comparative measurements, concentrations measured using passive flux samplers and absorption flasks did not differ significantly, but those measured using passive diffusion samplers were on average 1.8 times greater. The mass balance technique and wind tunnels gave broadly similar results in two out of four field tests. Overexposure of passive diffusion samplers for some sampling periods meant that estimation of cumulative NH(3) emission using the equilibrium concentration technique in the field tests could not be made. For cumulative NH(3) emissions, CVs were in the range of 23-52, 46-74 and 21-39% for the mass balance, wind tunnel and equilibrium concentration techniques, respectively. Lower CVs were associated with measurements following slurry compared with solid manure applications. Our conclusions from this study are that for the measurement of absolute emissions the mass balance technique is to be preferred, and for small-plot comparative measurements the wind tunnel system is preferred to the equilibrium concentration technique.

Predicting ammonia losses following the application of livestock manure to land.

A series of experiments was conducted using small wind tunnels to assess the influence of a range of environmental, manure and management variables on ammonia emissions following application of different manure types to grassland and arable land. Wind speed and dry matter content (for cattle slurry in particular) were identified as the parameters with greatest influence on ammonia emissions from slurries. For solid manures, rainfall was identified as the parameter with most influence on ammonia emissions. A Michaelis-Menten function was used to describe emission rates following manure application. Linear regression was then used to develop statistical models relating the Michaelis-Menten function parameters to the experimental variables for each manure type/land use combination. The fitted models accounted for between 62% and 94% of the variation in the data. Validation of the models for cattle slurry to grassland and pig slurry to arable land against independent data sets obtained from experiments using the micrometeorological mass balance measurement technique showed that the models overestimated losses, which was most probably due to inherent differences between the wind tunnel and the micrometerological mass balance measurement techniques.

Factors affecting the concentrations of lead in British wheat and barley grain.

The entry of Pb into the food chain is of concern as it can cause chronic health problems. The concentration of Pb was determined in cereal grain samples collected representatively from British Cereal Quality Surveys in 1982 and 1998 (n = 176, 250 and 233 for wheat collected in 1982 and 1998, and barley in 1998, respectively). In addition, paired soil and grain samples were collected from 377 sites harvested across Britain in 1998-2000. Wheat grain Pb ranged from below the analytical detection limit (0.02 mg kg(-1) dry weight, DW) to 1.63 mg kg(-1) DW, and barley grain Pb from <0.02 to 0.48 mg kg(-1) DW. The vast majority of samples (>99% for both wheat and barley, excluding Scottish barley samples collected in 2000) were well below the newly introduced EU limit for the maximum permissible concentration of Pb in cereals (0.2 mg kg(-1) fresh weight, equivalent to 0.235 mg kg(-1) DW). There was a significant reduction in wheat grain Pb in the 1998 survey compared with the 1982 survey. However, 40 barley samples collected from Scotland in 2000 in the paired soil and crop survey showed anomalously high concentrations of Pb, with 10 samples exceeding the EU limit. Washing experiments demonstrated that surface contamination, introduced during grain harvest and/or storage, was the main reason for the high concentrations in these samples. In the paired soil and crop surveys, there were no significant correlations between grain Pb concentrations with total soil Pb and other soil properties, indicating low bioavailability of Pb in the soils and limited uptake and transport of Pb to grain. The Pb in cereal grain is likely to originate mainly from atmospheric deposition and other routes of surface contamination during harvest and storage.

Predicting cadmium concentrations in wheat and barley grain using soil properties.

The entry of Cd into the food chain is of concern as it can cause chronic health problems. To investigate the relationship between soil properties and the concentration of Cd in wheat (Triticum aestivum L.) and harley (Hordeum vulgare L.) grain, we analyzed 162 wheat and 215 barley grain samples collected from paired soil and crop surveys in Britain, and wheat and barley samples from two long-term sewage sludge experiments. Cadmium concentrations were much lower in barley grain than in wheat grain under comparable soil conditions. Multiple regression analysis showed that soil total Cd and pH were the significant factors influencing grain Cd concentrations. Significant cultivar differences in Cd uptake were observed for both wheat and barley. Wheat grain Cd concentrations could be predicted reasonably well from soil total Cd and pH using the following model: log(grain Cd) = a + b log(soil Cd) - c(soil pH), with 53% of the variance being accounted for. The coefficients obtained from the data sets of the paired soil and crop surveys and from long-term sewage sludge experiments were similar, suggesting similar controlling factors of Cd bioavailability in sludge-amended or unamended soils. For barley, the model was less satisfactory for predicting grain Cd concentration (22% of variance accounted for). The model can be used to predict the likelihood of wheat grain Cd exceeding the new European Union (EU) foodstuff regulations on the maximum permissible concentration of Cd under different soil conditions, particularly in relation to the existing Directive and the proposed new Directive on land applications of sewage sludge.

Nitric oxide produced by murine dendritic cells is cytotoxic for intracellular Salmonella enterica sv. Typhimurium.

The pathogenicity of Salmonella enterica serovar Typhimurium has traditionally been correlated with its ability to survive and grow in macrophages. Macrophage-derived production of nitric oxide (NO) has been implicated as a major innate defence, restricting bacterial proliferation both in macrophage cultures and in mice. In the present study, we show that the ability of primary murine dendritic cells (DCs) to ingest Salmonella is low, but greatly enhanced by serum complement. Ingestion of bacteria was followed by the expression of inducible nitric oxide synthase (iNOS), as well as by NO production. iNOS mRNA was detected as early as 6 h post infection and production of NO 12 h post infection, rising further at 16 h post infection. Inhibition of the iNOS activity with the inhibitor N-monomethyl-l-arginine or using DCs from iNOS-/- mice resulted in increased intracellular bacterial yields. To further define the potential defensive role of DC-derived NO, the actual intracellular replication rate of S. Typhimurium in DCs was measured. DC-derived NO was shown to exert a bactericidal effect, whereas the effect of NO in macrophage-like J774-A.1 cells was found to be bacteriostatic. These results identified an important role for NO in restricting S. Typhimurium survival in DCs, indicating that DCs may actively participate in the innate defence against intracellular pathogens.

Expansion of natural killer (NK) and natural killer-like T (NKT)-cell populations derived from patients with B-chronic lymphocytic leukemia (B-CLL): a potential source for cellular immunotherapy.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world. It is currently an incurable disease, making new treatment options such as immunotherapy desirable. Monoclonal antibodies (Mabs) to surface antigens of the tumor cell is one option. Administration of cytotoxic cells such as natural killer (NK) and natural killer-like T (NKT) cells expanded in vitro might be a useful treatment modality alone or in combination with MAbs. A limiting step in the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Here, we report the feasibility of expanding populations of the human killer cells, CD3-CD56+ NK and CD3+CD56+ NKT cells, from peripheral blood mononuclear cells (PBMCs) of B-CLL patients. The influence of tumor B cells on the in vitro expansion of killer cells was assessed by depleting B cells from PBMCs by microbead separation before culture. The 21-day cultures from both B-cell- and non-B-cell-depleted PBMC showed a marked expansion of NK cells, and also of T cells, among which almost half had the NKT phenotype. Depletion of B cells before culture did not change the expansion rates of NK and NKT cells significantly. In patients with progressive B-CLL, NK cell expansion capacity was improved after fludarabine treatment when compared to samples obtained before treatment. Repeated samples of PBMCs from individual untreated patients with both indolent and progressive disease cultured under identical conditions gave similar NK cell expansion rates. Expanded killer cell populations had cytotoxic function against the NK-sensitive target K562 cell line and expressed high levels of Granzyme B. From our studies, we conclude that NK cells as well as NKT cells from the peripheral blood of B-CLL patients can be expanded, and that these cells have cytotoxic capacity.

An inventory of heavy metals inputs to agricultural soils in England and Wales.

An inventory of heavy metal inputs (Zn, Cu, Ni, Pb, Cd, Cr, As and Hg) to agricultural soils in England and Wales in 2000 is presented, accounting for major sources including atmospheric deposition, sewage sludge, livestock manures, inorganic fertilisers and lime, agrochemicals, irrigation water, industrial by-product 'wastes' and composts. Across the whole agricultural land area, atmospheric deposition was the main source of most metals, ranging from 25 to 85% of total inputs. Livestock manures and sewage sludge were also important sources, responsible for an estimated 37-40 and 8-17% of total Zn and Cu inputs, respectively. However, at the individual field scale sewage sludge, livestock manures and industrial wastes could be the major source of many metals where these materials are applied. This work will assist in developing strategies for reducing heavy metal inputs to agricultural land and effectively targeting policies to protect soils from long-term heavy metal accumulation.

Effects of past sewage sludge additions on heavy metal availability in light textured soils: implications for crop yields and metal uptakes.

The effect of heavy metal additions in past sewage sludge applications on soil metal availability and the growth and yield of crops was evaluated at two sites in the UK. At Gleadthorpe, sewage sludges enriched with salts of zinc (Zn), copper (Cu) and nickel (Ni) had been applied to a loamy sand in 1982 and additionally naturally contaminated Zn and Cu sludge cakes in 1986. At Rosemaund, sewage sludges naturally contaminated with Zn, Cu, Ni and chromium (Cr) had been applied in 1968-1971 to a sandy loam. From 1994 to 1997, the yields of both cereals and legumes at Gleadthorpe were up to 3 t/ha lower than the no-sludge control where total topsoil Zn and Cu concentrations exceeded 200 and 120 mg/kg, respectively, but only when topsoil ammonium nitrate extractable metal levels also exceeded 40 mg/kg Zn and 0.9 mg/kg Cu. At Rosemaund, yields were only decreased where total topsoil Cu concentrations exceeded 220 mg/kg or 0.7 mg/kg ammonium nitrate extractable Cu. These results demonstrate the importance of measuring extractable as well as total heavy metal concentrations in topsoils when assessing likely effects on plant yields and metal uptakes, and setting soil quality criteria.

IL-2 down-regulates the expression of TCR and TCR-associated surface molecules on CD8(+) T cells.

CD8(+) T cells are known to down-regulate the TCR complex upon ligation with its cognate MHC class I-peptide complex. In the present report, we demonstrate that stimulation of CD8(+) T cells with cytokines also leads to down-regulation of the TCR complex and TCR-associated surface molecules. A significant reduction of TCRalpha beta, CD3, CD8alpha and CD8beta surface expression was observed when CD8(+) T cells were cultured in IL-2 and to a lesser extent in IL-4 or IL-15. The down-regulation was apparent after 2 days of culture and was observed at IL-2 concentrations as low as 10 U/ml. Using TCR transgenic mice, we found that the down-regulation was associated with a decreased affinity of CD8(+) T cells to MHC class I-peptide complexes, as determined by MHC class I tetramer staining. Furthermore, the antigen-specific proliferation of IL-2-pre-activated CD8(+) T cells was significantly reduced compared to naive CD8(+) T cells or to CD8(+) T cells previously stimulated with peptide-pulsed dendritic cells. Moreover, only CD8alpha(high) but not CD8alpha(low) cells sorted from IL-2-activated CD8(+) T cells proliferated in response to specific antigen, although both subsets proliferated equally well to IL-2. Taken together, these data suggest that the down-regulation of TCR components and a subsequent decrease in affinity towards MHC class I-peptide complexes may be a mechanism by which TCR-dependent proliferation of non-specifically activated CD8(+) T cells is avoided.

Cutting edge: Regulation of CD8(+) T cell proliferation by 2B4/CD48 interactions.

The biological function of 2B4, a CD48-binding molecule expressed on T cells with an activation/memory phenotype, is not clear. In this report, we demonstrate that proliferation of CD8(+) T cells is regulated by 2B4. Proliferative responses of CD8(+) T cells were significantly reduced by anti-2B4 Ab. The effects were not potentiated by anti-CD48 Ab, suggesting that the observed responses were driven by 2B4/CD48 interactions. Surprisingly, the 2B4/CD48-dependent proliferative responses were also observed in the absence of APCs. This suggests that 2B4/CD48 interactions can occur directly between T cells. Furthermore, when activated 2B4(+)CD8(+) T cells were mixed with 2B4(-)CD8(+) TCR-transgenic T cells and specific peptide-loaded APC, the proliferation of the latter T cells was inhibited by anti-2B4 Ab. Taken together, this suggests that 2B4 on activated/memory T cells serves as a ligand for CD48, and by its ability to interact with CD48 provides costimulatory-like function for neighboring T cells.

Cadmium content of wheat grain from a long-term field experiment with sewage sludge.

Grain Cd concentrations were determined in the wheat (Triticum aestivum L.) cultivars Soissons, Brigadier, and Hereward grown in 1994,1996, and 1999, respectively, in soils of a long-term field experiment to which sewage sludges contaminated with Zn, Cu, Ni, or Cr had previously been added. Soil pore water soluble Cd and free Cd2+ increased linearly with increasing total soil Cd (R2=0.82 and 0.84, respectively; P<0.001). Similarly, soil pore water free Cd2+ increased linearly with increasing soil pore water soluble Cd (R2=0.98; P<0.001). There was no evidence of a plateau in soil pore water Cd concentrations with increasing soil Cd concentrations. Grain Cd concentrations were significantly correlated with total soil Cd (P<0.001), soil pore water Cd (P<0.001), and free Cd2+ (P<0.001). A slight curvilinear relationship between grain Cd and soil Cd was apparent, but there was no plateau, even at the maximum soil Cd concentration of about 2.7 mg kg(-1). The relationship between soil pore water Cd and grain Cd was linear for all three cultivars. The slopes were in the order 1994 > 1996 > 1999, with more Cd being taken up into the grain by Soissons grown in 1994, and least by Hereward grown in 1999. For Soissons, Cd concentration in the grain greater than the EU limit (0.24 mg kg(-1) dry wt.) occurred at soil Cd less than the current UK limit of 3 mg kg(-1) for soils receiving sewage sludge. In contrast, for Brigadier and Hereward, grain Cd concentrations were near to and less than the EU limit, respectively, at soil Cd concentrations of 3 mg kg(-1).

A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells.

Adoptive transfer of immunocompetent cells may induce anti-tumor effects in vivo. However, a significant obstacle to the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Among the immunologic effector cells that are considered mediators of anti-tumor effects, those with the highest per-cell cytotoxic capacity express a natural killer (NK) cell phenotype, i.e., CD56(+)CD3(-). However, such cells are normally present only in low numbers in peripheral blood mononuclear cells (PBMCs), lymphokine activated killer (LAK), and cytokine induced killer (CIK) cell preparations. To optimize the expansion of human NK cells, PBMCs were cultured in different serum free medium supplemented with monoclonal anti-CD3 antibodies and interleukin (IL)-2 at varying concentrations. By using Cellgro stem cell growth medium supplemented with 5% human serum and IL-2 (500 U/ml) cells expanded 193-fold (median, range 21-277) after 21 days, and contained 55% (median, range 7-92) CD3(-)CD56(+) cells. The remaining cells were CD3(+) T cells, 22% (median, range 2-68) of which co-expressed CD56. The expanded cell population lysed 26 to 45% of K562 targets in a 1:1 effector to target ratio, signifying substantial cytotoxic efficacy. The described method is a simple and efficient way of expanding and enriching human NK cells. We have termed these high-yield CD3(-)CD56(+) cells cytokine-induced natural killer (CINK) cells.

In vivo activation of dendritic cells and T cells during Salmonella enterica serovar Typhimurium infection.

The present study was initiated to gain insight into the interaction between splenic dendritic cells (DC) and Salmonella enterica serovar Typhimurium in vivo. Splenic phagocytic cell populations associated with green fluorescent protein (GFP)-expressing bacteria and the bacterium-specific T-cell response were evaluated in mice given S. enterica serovar Typhimurium expressing GFP and ovalbumin. Flow cytometry analysis revealed that GFP-positive splenic DC (CD11c+ major histocompatibility complex class II-positive [MHC-II+] cells) were present following bacterial administration, and confocal microscopy showed that GFP-expressing bacteria were contained within CD11c+ MHC-II+ splenocytes. Furthermore, splenic DC and T cells were activated following Salmonella infection. This was shown by increased surface expression of CD86 and CD40 on CD11c+ MHC-II+ cells and increased CD44 and CD69 expression on CD4+ and CD8+ T cells. Salmonella-specific gamma interferon (IFN-gamma)-producing cells in both of these T-cell subsets, as well as cytolytic effector cells, were also generated in mice given live bacteria. The frequency of Salmonella-specific CD4+ T cells producing IFN-gamma was greater than that of specific CD8+ T cells producing IFN-gamma in the same infected animal. This supports the argument that the predominant source of IFN-gamma production by cells of the specific immune response is CD4+ T cells. Finally, DC that phagocytosed live or heat-killed Salmonella in vitro primed bacterium-specific IFN-gamma-producing CD4+ and CD8+ T cells as well as cytolytic effector cells following administration into naïve mice. Together these data suggest that DC are involved in priming naïve T cells to Salmonella in vivo.

Expression of the DX5 antigen on CD8+ T cells is associated with activation and subsequent cell death or memory during influenza virus infection.

The antigen recognized by the DX5 antibody (DX5 antigen) is expressed on all murine NK cells. In the present study we found that a proportion of CD8+ T cells (approximately 5%) also express the DX5 antigen in uninfected mice, and that numbers of CD8+ T cells expressing DX5 are significantly higher in the lungs of influenza virus-infected mice representing up to 50% of all CD8+ T cells on day 10 post infection. The expression of the DX5 antigen on CD8+ T cells was associated with a memory phenotype in uninfected C57BL/6 mice and with an activation phenotype during influenza virus infection. Interestingly, when lymphocytes were isolated from lungs of influenza virus-infected mice on day 10 post infection and adoptively transferred into recombination activating gene-1 (RAG1)-deficient mice, CD8+DX5+ cells could not be recovered from the recipient mice 2 days later. Moreover, CD8+DX5+ cells were not detected when lung cells were removed from day 10 influenza virus-infected mice and cultured in vitro for 2 days. However, CD8+DX5+ cells could be detected when apoptosis inhibitors were added to these cultures, suggesting that the CD8+DX5+ cells underwent apoptosis during cell culture. Furthermore, almost all DX5 expressing CD8+ cells from lungs of mice on day 10 post influenza virus infection stained positively with Annexin-V. Taken together, the data suggest that CD8+ T cells expressing DX5 are associated with an activation/memory phenotype and are biased towards apoptosis.

Anti-CTLA-4 antibody treatment triggers determinant spreading and enhances murine myasthenia gravis.

CTLA-4 appears to be a negative regulator of T cell activation and is implicated in T cell-mediated autoimmune diseases. Experimental autoimmune myasthenia gravis (EAMG), induced by immunization of C57BL/6 mice with acetylcholine receptor (AChR) in adjuvant, is an autoantibody-mediated disease model for human myasthenia gravis (MG). The production of anti-AChR Abs in MG and EAMG is T cell dependent. In the present study, we demonstrate that anti-CTLA-4 Ab treatment enhances T cell responses to AChR, increases anti-AChR Ab production, and provokes a rapid onset and severe EAMG. To address possible mechanisms underlying the enhanced autoreactive T cell responses after anti-CTLA-4 Ab treatment, mice were immunized with the immunodominant peptide alpha(146-162) representing an extracellular sequence of the ACHR: Anti-CTLA-4 Ab, but not control Ab, treatment subsequent to peptide immunization results in clinical EAMG with diversification of the autoantibody repertoire as well as enhanced T cell proliferation against not only the immunizing alpha(146-162) peptide, but also against other subdominant epitopes. Thus, treatment with anti-CTLA-4 Ab appears to induce determinant spreading, diversify the autoantibody repertoire, and enhance B cell-mediated autoimmune disease in this murine model of MG.