Glycolipid-peptide conjugate vaccines enhance CD8+ T cell responses against human viral proteins.

An important goal of vaccination against viruses and virus-driven cancers is to elicit cytotoxic CD8+ T cells specific for virus-derived peptides. CD8+ T cell responses can be enhanced by engaging help from natural killer T (NKT) cells. We have produced synthetic vaccines that induce strong peptide-specific CD8+ T cell responses in vivo by incorporating an NKT cell-activating glycolipid. Here we examine the effect of a glycolipid-peptide conjugate vaccine incorporating an NKT cell-activating glycolipid linked to an MHC class I-restricted peptide from a viral antigen in human peripheral blood mononuclear cells. The vaccine induces CD1d-dependent activation of human NKT cells following enzymatic cleavage, activates human dendritic cells in an NKT-cell dependent manner, and generates a pool of activated antigen-specific CD8+ T cells with cytotoxic potential. Compared to unconjugated peptide, the vaccine upregulates expression of genes encoding interferon-γ, CD137 and granzyme B. A similar vaccine incorporating a peptide from the clinically-relevant human papilloma virus (HPV) 16 E7 oncoprotein induces cytotoxicity against peptide-expressing targets in vivo, and elicits a better antitumor response in a model of E7-expressing lung cancer than its unconjugated components. Glycolipid-peptide conjugate vaccines may prove useful for the prevention or treatment of viral infections and tumors that express viral antigens.

Sputum basophils are increased in eosinophilic asthma compared with non-eosinophilic asthma phenotypes.

Sputum basophil numbers are increased in allergic asthmatics, but it is unclear what role airway basophils play in "TH2-low" asthma phenotypes. Using flow cytometry, we found that basophils were significantly increased in all asthmatics (n=26) compared with healthy controls (n=8) (P=0.007) with highest levels observed in eosinophilic asthma (EA); median 0.22%, IQR 0.11%-0.47%; n=14) compared with non-EA (NEA) (0.06%, 0.00%-0.20%; n=12; P<0.05). In asthmatics, basophils were positively correlated with sputum eosinophils (r=0.54; P<0.005) and inversely with sputum neutrophils (r=-0.46: P<0.05), but not with FEV1 (% predicted), FEV1 /FVC or bronchodilator reversibility. In a subgroup initially identified as inadequately controlled asthma (n=7), there was a trend (P=0.08) towards a reduction in sputum basophils following increased inhaled corticosteroid (ICS) treatment. Our findings suggest that basophils may be particularly important in eosinophilic asthma and that sputum basophil assessment could be a useful additional indicator of "TH2-high" asthma.

B-Lymphocytes activated by CD40 ligand induce an antigen-specific anti-tumour immune response by direct and indirect activation of CD8(+) T-cells.

In this report, we describe the ability of CD40-ligand (CD40L)-activated, antigen-loaded B-cells to initiate antigen-specific anti-tumour immune responses in vivo. Mice immunized by means of intravenous administration of CD40L-activated B-cells loaded with an MHC class-I-binding peptide, and challenged with a tumour cell line expressing the same class-I epitope, showed a marked delay in tumour growth, compared to non-immunized controls or to mice receiving either freshly isolated B-cells or B-cells activated with lipopolysaccharide or interleukin-4. The ability of CD40L-activated B-cells to induce antigen-specific T-cell activation appeared to be through a combination of cross-presentation of antigen from activated B-cells to resident antigen-presenting cells and direct T-cell activation by the administered B-cells themselves. Immunization with CD40L-activated B-cells may, therefore, represent a means by which to stimulate anti-tumour CD8(+) T-cell responses in vivo.

Dendritic cell elimination as an assay of cytotoxic T lymphocyte activity in vivo.

We show in this paper that the survival of antigen-loaded dendritic cells in vivo may be used as a sensitive readout of CTL activity. We have previously shown that dendritic cells labeled with the fluorescent dye CFSE and injected sub-cutaneously into mice migrate spontaneously to the draining lymph node where they persist for several days. In the presence of effector CTL responses, dendritic cells loaded with specific antigen rapidly disappear from the draining lymph node. In this paper we extend the above observations and set up a simple and sensitive method to reveal CTL activity in individual mice in vivo. Dendritic cells were labeled with two different fluorochromes, loaded with antigen or left untreated, and mixed together before injection into mice. We show that only the dendritic cells loaded with specific antigen were cleared from the draining lymph node, while dendritic cells not loaded with antigen remained unaffected. Cytotoxic responses generated by immunization with peptide-loaded dendritic cells, or by infection with influenza virus, could be revealed using this method. Comparison of the differential survival of dendritic cells populations mixed together also allowed us to accurately evaluate the disappearance of dendritic cells, irrespective of variability in the injection site and other parameters. Given the ability of dendritic cells to efficiently take up and present complex antigens, nucleic acids and apoptotic bodies, this method may also allow the evaluation of cytotoxic activity against antigens that are not characterized in terms of peptide epitopes.

Immunotherapy with dendritic cells and tumor major histocompatibility complex class I-derived peptides requires a high density of antigen on tumor cells.

Immunization with dendritic cells and unfractionated MHC class I-binding peptides derived from autologous tumor cells has been shown to induce effective antitumor immunity. However, the importance of the relative abundance of tumor peptides on the surface of tumor cells is not known. We have addressed this question using peptides isolated from three tumor cell lines transfected with a minigene encoding amino acids 33-41 of the lymphocytic choriomeningitis virus glycoprotein (LCMV(33-41)). The three cell lines expressed different levels of MHC class I molecules and had different abilities to stimulate proliferation of LCMV(33-41)-specific T cells in vitro. We found that antitumor immune responses were best elicited by immunizing mice with dendritic cells and synthetic LCMV(33-41) peptide. Peptide preparations from a given tumor cell line conferred protection against challenge with the same tumor cell line. However, protective immunity to a different tumor could be induced only if the cell line used for peptide preparation presented a high relative proportion of LCMV(33-41) in association with MHC class I. Our results suggest that multiple peptide epitopes are required for the induction of an effective antitumor immune response using MHC class I-binding peptides from tumor cells. Also, the ability to induce an antitumor immune response appears to correlate with the proportion, rather than the absolute amount, of tumor-specific peptide in the mixture used for immunization.

CD8+ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity.

The fate of dendritic cells (DC) after they have initiated a T cell immune response is still undefined. We have monitored the migration of DC labeled with a fluorescent tracer and injected s.c. into naive mice or into mice with an ongoing immune response. DC not loaded with Ag were detected in the draining lymph node in excess of 7 days after injection with maximum numbers detectable approximately 40 h after transfer. In contrast, DC that had been loaded with an MHC class I-binding peptide disappeared from the lymph node with kinetics that parallel the known kinetics of activation of CD8+ T cells to effector function. In the presence of high numbers of specific CTL precursors, as in TCR transgenic mice, DC numbers were significantly decreased by 72 h after injection. The rate of DC disappearance was extremely rapid and efficient in recently immunized mice and was slower in "memory" mice in which memory CD8+ cells needed to reacquire effector function before mediating DC elimination. We also show that CTL-mediated clearance of Ag-loaded DC has a notable effect on immune responses in vivo. Ag-specific CD8+ T cells failed to divide in response to Ag presented on a DC if the DC were targets of a pre-existing CTL response. The induction of antitumor immunity by tumor Ag-loaded DC was also impaired. Therefore, CTL-mediated clearance of Ag-loaded DC may serve as a negative feedback mechanism to limit the activity of DC within the lymph node.

Impaired ability of MHC class II-/- dendritic cells to provide tumor protection is rescued by CD40 ligation.

The contribution of CD4+ T cells to dendritic cell (DC) activation and to the induction of CD8+ T cell responses in vivo was investigated using a model of antitumor immune responses. Immunization with peptide-loaded MHC class II-deficient (MHC class II-/-) DC induced the activation of Ag-specific CD8+ T cells and their accumulation in the lymph nodes and spleens of immunized mice. The accumulation induced by MHC class II-/- DC immunization was lower than the accumulation observed after immunization with MHC class II+/+ DC. Similarly, immunization with peptide-loaded, MHC class II-/- DC induced some degree of protection against tumor challenge, but this protection was lower than the protection achieved after immunization with MHC class II+/+ DC. Incubation with a membrane-associated form of CD40 ligand resulted in the up-regulation of costimulatory molecules on MHC class II-/- DC and fully rescued their ability to induce antitumor immunity. We conclude that CD4+ T cells play a critical role in the generation of antitumor immune responses through their capacity to induce the activation of DC via CD40/CD40 ligand interaction, and thus maximize CD8+ T cell responses.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) can regulate dendritic cell-induced activation and cytotoxicity of CD8(+) T cells independently of CD4(+) T cell help.

The mechanisms that regulate the strength and duration of CD8(+) cytotoxic T cell activity determine the effectiveness of an antitumor immune response. To better understand the antitumor effects of anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibody treatment, we analyzed the effect of CTLA-4 signaling on CD8(+) T cells in vitro and in vivo. In vitro, cross-linking of CTLA-4 on purified CD8(+) T cells caused decreased proliferative responses to anti-CD3 stimulation and rapid loss of activation marker expression. In vivo, blockade of CTLA-4 by neutralizing anti-CTLA-4 mAb greatly enhanced the accumulation, activation, and cytotoxic activity of CD8(+) T cells induced by immunization with Ag on dendritic cells (DC). This enhanced response did not require the expression of MHC class II molecules on DC or the presence of CD4(+) T cells. These results demonstrate that CTLA-4 blockade is able to directly enhance the proliferation and activation of specific CD8(+) T cells, indicating its potential for tumor immunotherapy even in situations in which CD4(+) T cell help is limited or absent.

Antigen expressed on tumor cells fails to elicit an immune response, even in the presence of increased numbers of tumor-specific cytotoxic T lymphocyte precursors.

We have used T-cell receptor (TCR) transgenic mice to analyze the interaction of tumors with the immune system. We show that the tumor cell line Lewis lung-lymphocytic choriomeningitis virus (LL-LCMV), genetically manipulated to express an H-2 Db-restricted epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV33-41), can grow progressively in TCR transgenic mice, where approximately 50% of CD8+ T cells are specific for LCMV33-41. TCR transgenic T cells were not deleted in tumor-bearing mice, and their surface phenotype and cytokine secretion patterns remained typical of naive T cells. Also, TCR transgenic T cells from tumor-bearing mice had undiminished capacity to proliferate to antigen in vitro. Tumor-protective immune responses could be elicited in TCR transgenic mice by immunization with LCMV33-41 peptide-loaded dendritic cells. Tumor resistance correlated with the switch of TCR transgenic T cells from a CD44low to a CD44high phenotype and increased capacity to produce IFNgamma in vitro. Results similar to those obtained in TCR transgenic mice were also obtained using an adoptive transfer system, where small numbers of TCR transgenic T cells were injected into normal C57BL/6 hosts. These data indicate that even large tumors may not induce specific immunization, tolerance, or anergy to tumor antigens, and that high numbers of tumor-specific CTL precursors are not sufficient to provide tumor resistance.

The emerging role of the dendritic cell in novel cancer therapies.

Research over the last two years has explored a number of possible approaches to applying dendritic cell immunotherapy to the treatment of human cancers. The chosen strategy in clinical situations will vary for individual patients and will depend on the type of tumour, availability of tissue samples and potential source of dendritic cells. We believe that the isolation of fractionated tumour peptide from individual patients' tumours for use with autologous stem cell-derived dendritic cells may provide, in at least some cases, an effective and practical approach to cancer immunotherapy. This approach will provide a 'closed' system of tumour immunotherapy with all components (dendritic cells, antigen and cytotoxic T lymphocytes) being provided by the patient and applied in a tailor-made fashion to individual patients as an adjuvant to current anti-tumour therapies.

Tumor-peptide-pulsed dendritic cells isolated from spleen or cultured in vitro from bone marrow precursors can provide protection against tumor challenge.

Dendritic cells (DC) purified from murine spleen or generated in vitro from bone marrow precursors were compared for their respective abilities to stimulate T cell responses and provide tumor protection in vivo. In vitro incubation with synthetic tumor peptide conferred on both DC populations the ability to induce proliferation of tumor-peptide-specific T cells in vitro. Spleen DC were reproducibly about twofold more effective than bone-marrow-derived DC in this assay. Both DC populations could also induce cytotoxic activity in vivo. In vitro cytoxicity assays showed that, while cytotoxic activity induced by immunization with spleen DC was clearly peptide-specific, a high non-specific cytotoxic activity was consistently observed after immunization with bone-marrow-derived DC, whether peptide-pulsed or not. Regardless of such high non-specific activity in vitro, only tumor-peptide-pulsed DC could provide protection against subsequent inoculation of tumor cells. DC not pulsed with tumor peptide were ineffective. We conclude that DC isolated from spleen or generated in vitro from bone marrow precursors are suitable reagents for use in tumor vaccination studies.

Assessment of DNA fingerprinting for determining genetic diversity in sheep populations.

Genomic DNA, prepared from 12 animals from four sheep flocks, was digested with either HaeIII or HinfI and probed with three DNA fingerprinting probes. Mean DNA fingerprint band sharing and band frequency calculated for each flock were used to estimate genetic diversity. Each of the DNA fingerprinting systems showed the same trend in diversity within the sampled flocks, and greater diversity between the flocks than within the flocks. DNA fingerprinting therefore provides a useful measure of genetic diversity in sheep.

Three cases of disputed paternity in dogs resolved by the use of DNA fingerprinting.

DNA fingerprinting with the probes 33.15 and alpha-globin 3'HVR has been used to resolve three cases of disputed paternity in dogs. For each pedigree it was necessary to establish which bands in the DNA fingerprints of the offspring were of paternal origin, and then establish which putative sire carried all these bands. In the first case, a litter of Rhodesian Ridgebacks, twelve DNA bands were informative in establishing paternity. In the second case, a litter of Afghan hounds, five DNA bands established paternity, Lastly, in a litter of Border collies, five DNA bands established paternity. In each case a single dog only sired the entire litter.