CD8alpha(-) and CD8alpha(+) subclasses of dendritic cells undergo phenotypic and functional maturation in vitro and in vivo.

Dendritic cells (DCs) are essential for the priming of immune responses. This antigen-presenting function of DCs develops in sequence in a process called maturation, during which they become potent sensitizers of naïve T cells but reduce their ability to capture and process antigens. Some heterogeneity exists in mouse-DC populations, and two distinct subsets of DCs expressing high levels of CD11c can be identified on the basis of CD8alpha expression. We have studied the phenotype and maturation state of mouse splenic CD8alpha(-) and CD8alpha(+) DCs. Both subsets were found to reside in the spleen as immature cells and to undergo a phenotypic maturation upon culture in vitro in GM-CSF-containing medium or in vivo in response to lipopolysaccharide. In vitro and in vivo analyses showed that this maturation process is an absolute requisite for DCs to acquire their T-cell priming capacity, transforming CD8alpha(-) and CD8alpha(+) DCs into potent and equally efficient activators of naïve CD4(+) and CD8(+) T cells. Furthermore, these results highlight the importance that environmental factors may have on the ability of DC subsets to influence Th responses qualitatively; i.e., the ability to drive Th1 versus Th2 differentiation may not be fixed immutably for each DC subset.

Induction of polyomavirus-specific CD8(+) T lymphocytes by distinct dendritic cell subpopulations.

Dendritic cells are pivotal antigen-presenting cells for generating adaptive T-cell responses. Here, we show that dendritic cells belonging to either the myeloid-related or lymphoid-related subset are permissive for infection by mouse polyomavirus and, when loaded with a peptide corresponding to the immunodominant anti-polyomavirus CD8(+) T-cell epitope or infected by polyomavirus, are each capable of driving expansion of primary polyomavirus-specific CD8(+) T-cell responses in vivo.

Requirements for bone marrow-derived antigen-presenting cells in priming cytotoxic T cell responses to intracellular pathogens.

Bone marrow (BM)-derived antigen-presenting cells (APCs) are potent stimulators of T cell immune responses. We investigated the requirements for antigen presentation by these cells in priming cytotoxic T lymphocyte (CTL) responses to intracellular bacterial and viral pathogens. [Parent-->F(1)] radiation BM chimeras were constructed using C57BL/6 donors and (C57BL/6 x BALB/c)F(1) recipients. Infection of chimeric mice with either Listeria monocytogenes or vaccinia virus expressing the nucleoprotein (NP) antigen from lymphocytic choriomeningitis virus (LCMV) primed H2-D(b)-restricted, but not H2-K(d)-restricted CTL responses, demonstrating the requirement for BM-derived APCs for successful priming of CTL responses to these pathogens. Surprisingly, this did not hold true for chimeric mice infected with LCMV itself. LCMV-infected animals developed strong CTL responses specific for both H2-D(b)- and H2-L(d)-restricted NP epitopes. These findings indicate that in vivo priming of CTL responses to LCMV is remarkably insensitive to deficiencies in antigen presentation by professional BM-derived APCs.

Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice.

C57BL/6 mice genetically deficient in interleukin 15 (IL-15(-/-) mice) were generated by gene targeting. IL-15(-/-) mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8(+) T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these populations in IL-15(-/-) mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15(-/-) mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15 in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15(-/-) mice remained healthy when maintained under specific pathogen-free conditions. However, IL-15(-/-) mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid lineages. Moreover, the ability to rescue lymphoid defects in IL-15(-/-) mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.

Polyomavirus-infected dendritic cells induce antiviral CD8(+) T lymphocytes.

CD8(+) T cells are critical for the clearance of acute polyomavirus infection and the prevention of polyomavirus-induced tumors, but the antigen-presenting cell(s) involved in generating polyomavirus-specific CD8(+) T cells have not been defined. We investigated whether dendritic cells and macrophages are permissive for polyomavirus infection and examined their potential for inducing antiviral CD8(+) T cells. Although dendritic cells and macrophages both supported productive polyomavirus infection, dendritic cells were markedly more efficient at presenting the immunodominant viral epitope to CD8(+) T cells. Additionally, infected dendritic cells, but not infected macrophages, primed anti-polyomavirus CD8(+) T cells in vivo. Treatment with Flt3 ligand, a hematopoietic growth factor that dramatically expands the number of dendritic cells, markedly enhanced the magnitude of virus-specific CD8(+) T-cell responses during acute infection and the pool of memory anti-polyomavirus CD8(+) T cells. These findings suggest that virus-infected dendritic cells induce polyomavirus-specific CD8(+) T cells in vivo and raise the potential for their use as cellular adjuvants to promote CD8(+) T cell surveillance against polyomavirus-induced tumors.

Massive expansion of antigen-specific CD8+ T cells during an acute virus infection.

During LCMV infection, CD8+ T cells expand greatly. Bystander activation has been thought to play a role because few cells score as LCMV specific in limiting dilution analysis. In contrast, we find that at least a quarter of the CD8+ cells secrete IFNgamma specifically in response to LCMV peptides at the peak of the response. Moreover, by analyzing the expansion of adoptively transferred LCMV-specific, TCR-transgenic CD8+ T cells in congenic hosts, we have determined that most of the CD8+ cell expansion is virus specific. Analysis of the effect of the monospecific TCR-transgenic T cells on the host response to three LCMV epitopes suggests that CTL precursors compete for sites on the APC in an epitope-specific fashion and that this competition determines the specificity of the response.

Differential presentation of the same MHC class I epitopes by fibroblasts and dendritic cells.

Ag is presented to CTL as peptide associated with MHC class I molecules, which are present on most types of cells. We have investigated the presentation of Db-restricted lymphocytic choriomeningitis virus (LCMV) peptides by a fibroblast line (MC57) and a dendritic cell line (JawsII) to splenocytes from LCMV-immune C57BL/6 mice. We found that when LCMV-infected MC57 were used to restimulate the spleen cells, the resulting CTL line lost its ability to respond to the two dominant epitopes of the immune response to LCMV glycoprotein (gp)33 and nucleoprotein (np)396 but remained strongly lytic for targets coated with the subdominant gp276 epitope. In contrast, when LCMV-infected JawsII cells were used to restimulate the splenocytes, the resulting line continued to target gp33 and np396 but lost reactivity to gp276. When uninfected JawsII or MC57 cells were coated with peptides and used as stimulators, the resulting CTL lines continued to recognize all three epitopes, indicating that costimulatory or other potential innate differences in Ag presentation between the two cell lines are unlikely to account for the selective expansion of CTL specificities. When infected, both cell types produce similar levels of infectious LCMV, have similar levels of the NP and GP proteins from which np396 and gp33 are derived, and can be recognized by CTL specific for each of the three epitopes. These data indicate that in the generation of peptides for MHC-I binding and presentation to CTL, MC57 and JawsII process the same set of virus proteins in quantitatively different ways.

Virus-mediated delivery of antigenic epitopes into dendritic cells as a means to induce CTL.

Dendritic cells (DC) are potent inducers of CD8+ T cells and can stimulate protective antitumor immunity when pulsed with an antigenic peptide or protein. We used a replication-deficient adenovirus containing a Kb-restricted antigenic peptide of chicken OVA to study CTL induction in vitro and in vivo after adenovirus-mediated gene transfer into DC. The efficiency of adenovirus-infected DC in eliciting a specific CTL response was compared with immunizations with a recombinant vaccinia virus and DC pulsed with peptide or protein. An immortalized DC line derived from a C57BL/6 mouse and freshly isolated splenic DC from C57BL/6 mice were used in CTL induction. Virus-infected DC elicited the strongest Ag-specific CTL response in vitro and in vivo and induced protective antitumor immunity to a challenge with EG.7 tumors (EL-4 cell line expressing OVA). Direct immunization of mice with recombinant adenovirus resulted in the induction of high titers of neutralizing Abs, which precluded a boost of a CTL response after repeated inoculations. However, repeated injections of virus-infected DC induced only low titers of neutralizing Abs. Furthermore, the presence of neutralizing Abs specific for the virus did not affect the usefulness of infected DC as repeated applications of virus-infected DC boosted the CTL response even in mice previously infected with the recombinant vector. The use of DC infected with a recombinant virus has advantages over other forms of immunization and could provide an alternative approach for designing vaccination therapies.

Differential immunological cross-reactions with antisera against the V-ATPase of Kalanchoë daigremontiana reveal structural differences of V-ATPase subunits of different plant species.

Two antisera (ATP88 and ATP95) raised against the V-ATPase holoenzyme of Kalanchoë daigremontiana were tested for their cross-reactivity with subunits of V-ATPases from other plant species. V-ATPases from Kalanchoë blossfeldiana, Mesembryanthemum crystallinum, Nicotiana tabacum, Lycopersicon esculentum, Citrus limon, Lemna gibba, Hordeum vulgare and Zea mays were immunoprecipitated with an antiserum against the catalytic V-ATPase subunit A of M. crystallinum. As shown by silver staining and Western blot analysis with ATP88, subunits A, B, C, D and c were present in all immunoprecipitated V-ATPases. In contrast, ATP95 recognized the whole set of subunits only in K. blossfeldiana, M. crystallinum, H. vulgare and Z. mays. This differential cross reactivity of ATP95 indicates the presence of structural differences of certain V-ATPase subunits. Based on the Bafilomycin A1-sensitive ATPase activity of tonoplast enriched vesicles, and on the amount of V-ATPase solubilized and immunoprecipitated, the specific ATP-hydrolysis activity of the V-ATPases under test was determined. The structural differences correlate with the ability of V-ATPases from different species to hydrolyze ATP at one given assay condition for ATP-hydrolysis measurements. Interestingly V-ATPases showing cross-reactivity of subunits A, B, C, D and c with ATP95 showed higher rates of specific ATP hydrolysis compared to V-ATPases containing subunits which were not labeled by ATP95. Thus, V-ATPases with high turnover rates in our assay conditions may show common structural characteristics which separate them from ATPases with low turnover rates.

Lymphocytic choriomeningitis virus-induced immune dysfunction: induction of and recovery from T-cell anergy in acutely infected mice.

Acute infection of immunocompetent mice by lymphocytic choriomeningitis virus induces a potent cytotoxic T-lymphocyte response that eliminates infectious virus. Concurrently and paradoxically, there is a general suppression of lymphocyte responses to mitogens and to other infectious agents. Splenocytes from infected mice released significant amounts of gamma interferon in response to mitogenic stimulation in vitro, but neither interleukin 2 nor interleukin 4 was similarly elevated relative to the amounts released by control cells. Early T-cell receptor-proximal signaling events were found to be intact, confirming that the cells were viable and had received the mitogenic stimuli in an appropriate manner. Acutely infected adult thymectomized mice regained concanavalin A responsiveness in parallel with euthymic mice, if T cells were left unmanipulated for several weeks after clearance of virus from the mice. Therefore, although acute lymphocytic choriomeningitis virus infection has the effect of disrupting proliferation when the T-cell receptor is ligated, this state is only temporary. In contrast, T cells from persistently infected adult mice reveal long-lasting alterations in concanavalin A responsiveness.

Nitric oxide production by splenic macrophages is not responsible for T cell suppression during acute infection with lactate dehydrogenase-elevating virus.

Cellular immune responses of mice are transiently suppressed during acute infection with lactate dehydrogenase-elevating virus (LDV). Immunosuppression of mice correlated with a greatly impaired in vitro proliferative response of the majority of the T cells to Con A or anti-CD3 Abs, which could not be reversed by the addition of rIL-2. We have examined whether the T cell suppression is caused by nitric oxide (NO) produced by activated macrophages, which are observed in acutely infected mice. Spleen macrophages from 3-day LDV-infected mice exhibited a 6- to 10-fold increased potential for producing NO, measured as nitrite or nitrite plus nitrate in the culture fluid, but produced significant amounts of NO in vitro only when incubated with IFN-gamma produced by Con A-stimulated T cells in the spleen cell population. Furthermore, we found that the concentrations of NO produced by macrophages in cultures of spleen cells from LDV-infected mice in the presence of IFN-gamma were insufficient to cause a reduction in the proliferative response of T cells in the spleen cell population. An excess of activated macrophages had to be added to achieve T cell suppression. NO inhibition of Con A-induced T cell proliferation exhibited a very sharp dose-response curve. In one experiment little suppression was observed at NO concentrations equivalent to 12 microM nitrite and below, whereas almost complete inhibition was observed at twice the NO concentration. We conclude that NO is not responsible for T cell suppression in LDV-infected mice.

Macrophages in mice acutely infected with lymphocytic choriomeningitis virus are primed for nitric oxide synthesis.

Following infection of adult mice with lymphocytic choriomeningitis virus (LCMV) there is a well documented suppression of T-cell and B-cell functions concurrent with the strong anti-LCMV immune response. Macrophages have been shown to be infected and activated during acute LCMV infection and there is some evidence to indicate that there is altered antigen presentation in acutely infected mice. We have examined nitric oxide (NO) production by splenic macrophages during acute infection of adult mice. Our results show that these macrophages are primed for production of NO, that the inducible production of NO parallels the immune suppression, and that NO production is dependent on the presence of IFN gamma. However, neither in vivo nor in vitro treatment with N-monomethyl-L-arginine (NMA), a specific inhibitor of nitric oxide synthase, altered the induction or maintenance of virus-induced immune suppression in mice acutely infected with LCMV.

Evidence for compartmentalized adenylate kinase catalysis serving a high energy phosphoryl transfer function in rat skeletal muscle.

The first characterization of the kinetics and subcellular compartmentation of adenylate kinase activity in intact muscle has been accomplished using rat diaphragm equilibrated with [18O]water. Rates of adenylate kinase-catalyzed phosphoryl transfer were measured by appearance of 18O-labeled beta-phosphoryls in ADP and ATP resulting from the transfer to AMP of newly synthesized 18O-labeled gamma-ATP. Unique features of adenylate kinase catalysis were uncovered in the intact cell not predictable from cell free analysis. This enzyme activity, which in non-contracting muscle is limited to 1/1000 of the estimated Vmax (cell free) apparently because of restricted ADP availability, is localized in subcellular compartments that increase in size and/or number with contractile frequency. Contraction also causes frequency-dependent increments in adenylate kinase velocity (22-fold at 4 Hz) as does oxygen deprivation (35-fold). These enhanced rates of adenylate kinase activity, equivalent to processing all the cellular ATP and ADP in approximately 1 min, occur when levels of ATP, ADP, and AMP are maintained very near their basal steady state. These characteristics of the dynamics of adenylate kinase catalysis in the intact cell demonstrate that rapid rates of AMP production from ADP are balanced by equally rapid rates of AMP phosphorylation with no net synthesis or accumulation of any adenine nucleotide. This rapid processing of nucleotide phosphoryls conforms to a proposed scheme whereby the adenylate kinase system provides the unique function of transferring, as beta-ADP, high energy phosphoryls generated by glycolytic metabolism to ATP-utilizing components in muscle.

Transient potentials in dendritic systems of arbitrary geometry.

A simple graphical calculus is developed that generates analytic solutions for membrane potential transforms at any point on the dendritic tree of neurons with arbitrary dendritic geometries, in response to synaptic "current" inputs. Such solutions permit the computation of transients in neurons with arbitrary geometry and may facilitate analysis of the role of dendrites in such cells.