Kari R. Irvine

Tumor-specific Th17-polarized cells eradicate large established melanoma

CD4+ T cells can differentiate into multiple effector subsets, but the potential roles of these subsets in anti-tumor immunity have not been fully explored. Seeking to study the impact of CD4+ T cell polarization on tumor rejection in a model mimicking human disease, we generated a new MHC class II-restricted, T-cell receptor (TCR) transgenic mouse model in which CD4+ T cells recognize a novel epitope in tyrosinase-related protein 1 (TRP-1), an antigen expressed by normal melanocytes and B16 murine melanoma. Cells could be robustly polarized into Th0, Th1, and Th17 subtypes in vitro, as evidenced by cytokine, chemokine, and adhesion molecule profiles and by surface markers, suggesting the potential for differential effector function in vivo. Contrary to the current view that Th1 cells are most important in tumor rejection, we found that Th17-polarized cells better mediated destruction of advanced B16 melanoma. Their therapeutic effect was critically dependent on interferon-gamma (IFN-gamma) production, whereas depletion of interleukin (IL)-17A and IL-23 had little impact. Taken together, these data indicate that the appropriate in vitro polarization of effector CD4+ T cells is decisive for successful tumor eradication. This principle should be considered in designing clinical trials involving adoptive transfer-based immunotherapy of human malignancies.

Identification of poxvirus CD8 T cell determinants to enable rational design and characterization of smallpox vaccines

The large size of poxvirus genomes has stymied attempts to identify determinants recognized by CD8+ T cells and greatly impeded development of mouse smallpox vaccination models. Here, we use a vaccinia virus (VACV) expression library containing each of the predicted 258 open reading frames to identify five peptide determinants that account for approximately half of the VACV-specific CD8+ T cell response in C57BL/6 mice. We show that the primary immunodominance hierarchy is greatly affected by the route of VACV infection and the poxvirus strain used. Modified vaccinia virus ankara (MVA), a candidate replacement smallpox vaccine, failed to induce responses to two of the defined determinants. This could not be predicted by genomic comparison of viruses and is not due strictly to limited MVA replication in mice. Several determinants are immunogenic in cowpox and ectromelia (mousepox) virus infections, and immunization with the immunodominant determinant provided significant protection against lethal mousepox. These findings have important implications for understanding poxvirus immunity in animal models and bench-marking immune responses to poxvirus vaccines in humans.

HLA-A*0201, HLA-A*1101, and HLA-B*0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products.

In virus models explored in detail in mice, CTL typically focus on a few immunodominant determinants. In this study we use a multipronged approach to understand the diversity of CTL responses to vaccinia virus, a prototypic poxvirus with a genome ∼20-fold larger than that of the model RNA viruses typically studied in mice. Based on predictive computational algorithms for peptide binding to HLA supertypes, we synthesized a panel of 2889 peptides to begin to create an immunomic map of human CTL responses to poxviruses. Using this panel in conjunction with CTLs from vaccinia virus-infected HLA transgenic mice, we identified 14 HLA-A*0201-, 4 HLA-A*1101-, and 3 HLA-B*0702-restricted CD8+ T cell determinants distributed over 20 distinct proteins. These peptides were capable of binding one or multiple A2, A3, and B7 supertype molecules with affinities typical of viral determinants. Surprisingly, many of the viral proteins recognized are predicted to be late gene products, in addition to the early intermediate gene products expected. Nearly all of the determinants identified have identical counterparts encoded by modified vaccinia virus Ankara as well as variola virus, the agent of smallpox. These findings have implications for the design of new smallpox vaccines and the understanding of immune responses to large DNA viruses in general.

Sympathetic nervous system control of anti-influenza CD8+ T cell responses

Despite the longstanding appreciation of communication between the nervous and the immune systems, the nature and significance of these interactions to immunity remain enigmatic. Here, we show that 6-hydroxydopamine-mediated ablation of the mouse peripheral sympathetic nervous system increases primary CD8+ T cell responses to viral and cellular antigens presented by direct priming or cross-priming. The sympathetic nervous system also suppresses antiviral CD4+ T cell responses, but this is not required for suppressing CD8+ T cell responses. Adoptive transfer experiments indicate that enhanced CD8+ responses do not result from permanent alterations in CD8+ T cell function in sympathectomized mice. Rather, additional findings suggest that the sympathetic nervous system tempers the capacity of antigen-presenting cells to activate naïve CD8+ T cells. We also show that antiviral CD8+ T cell responses are enhanced by administration of a β2 (but not β1 or α) adrenergic antagonist. These findings demonstrate a critical role for the sympathetic nervous system in limiting CD8+ T cell responses and indicate that CD8+ T cell responses may be altered in patients using β-blockers, one of the most widely prescribed classes of drugs.

GILT Accelerates Autoimmunity to the Melanoma Antigen Tyrosinase-Related Protein 1

Melanocyte differentiation Ags, including tyrosinase-related protein (TRP) 1, are relevant to both autoimmune skin depigmentation (vitiligo) and tumor immunity, because they are expressed by both benign melanocytes and many malignant melanomas. Melanoma patients generate CD4+ T cells that specifically recognize these proteins. TRP1 contains internal disulfide bonds and is presented by MHC class II molecules. γ-IFN–inducible lysosomal thiol reductase (GILT) facilitates the generation of class II-binding peptides by the endocytic reduction of protein disulfide bonds. We show in this study that GILT is required for efficient MHC class II-restricted processing of a TRP1 epitope in vitro and accelerates the onset of vitiligo in TRP1-specific TCR transgenic mice. The presence of GILT confers a small increase in the percentage of autoreactive T cells with an effector memory phenotype that may contribute to earlier disease onset. The onset of vitiligo is associated with a greater increase in the percentage of autoreactive T cells with an effector memory phenotype. Given that many self and tumor Ags have disulfide bonds and are presented on MHC class II, GILT is likely to be important in the pathogenesis of other CD4+ T cell-mediated autoimmune diseases and for the development of effective cancer immunotherapy.

Terminal Deoxynucleotidyl Transferase Establishes and Broadens Antiviral CD8+ T Cell Immunodominance Hierarchies

The action of TdT on mouse TCR genes accounts for ∼90% of T cell repertoire diversity. We report that in TdT−/− mice, total TCD8+ responses to influenza and vaccinia viruses are reduced by ∼30% relative to wild-type mice. We find that TCD8+ responses to three subdominant influenza virus determinants are reduced to background values in TdT−/− mice while responses to three immunodominant determinants undergo a major reshuffling. A similar reshuffling occurs in TCD8+ responses to immunodominant vaccinia virus determinants, and is clearly based on broad differences in TCR family usage and CDR3 length between wild-type and TdT−/− mice. These findings demonstrate that TdT plays a critical role in the magnitude and breadth of anti-viral TCD8+ responses toward individual determinants and suggests that germline TCR repertoire bias toward the most dominant determinants is a major factor in establishing immunodominance hierarchies.

The next wave of recombinant and synthetic anticancer vaccines

The identification of tumor-associated antigens (TAA) recognized by T lymphocytes makes the development of antigen-specific synthetic and recombinant vaccines possible. The expression of TAA within a recombinant vector increases control over the kinetics and quantity, the molecular form, and the subcellular location of the immunogen delivered. The next generation of antitumor vaccines employs cytokines and costimulatory molecules expressed in concert with TAA that are capable of augmenting the activation and proliferation of antitumor immune responses. The ultimate goal of these new strategies, the treatment of established cancer, is now being realized in animal models.

Engineering the vaccinia virus L1 protein for increased neutralizing antibody response after DNA immunization

BackgroundThe licensed smallpox vaccine, comprised of infectious vaccinia virus, has associated adverse effects, particularly for immunocompromised individuals. Therefore, safer DNA and protein vaccines are being investigated. The L1 protein, a component of the mature virion membrane that is conserved in all sequenced poxviruses, is required for vaccinia virus entry into host cells and is a target for neutralizing antibody. When expressed by vaccinia virus, the unglycosylated, myristoylated L1 protein attaches to the viral membrane via a C-terminal transmembrane anchor without traversing the secretory pathway. The purpose of the present study was to investigate modifications of the gene expressing the L1 protein that would increase immunogenicity in mice when delivered by a gene gun.ResultsThe L1 gene was codon modified for optimal expression in mammalian cells and potential N-glycosylation sites removed. Addition of a signal sequence to the N-terminus of L1 increased cell surface expression as shown by confocal microscopy and flow cytometry of transfected cells. Removal of the transmembrane domain led to secretion of L1 into the medium. Induction of binding and neutralizing antibodies in mice was enhanced by gene gun delivery of L1 containing the signal sequence with or without the transmembrane domain. Each L1 construct partially protected mice against weight loss caused by intranasal administration of vaccinia virus.ConclusionModifications of the vaccinia virus L1 gene including codon optimization and addition of a signal sequence with or without deletion of the transmembrane domain can enhance the neutralizing antibody response of a DNA vaccine.

Factors influencing immunodominance hierarchies in TCD8+-mediated antiviral responses

CD8+ T-lymphocytes (TCD8+) perform a critical role in immunity against tumors and virus infections. A central feature of TCD8+ immune responses is immunodominance: the observation that TCD8+ responses consist of a limited collection of specificities with a structured hierarchy. These immunodominance hierarchies result from a complex combination of factors. Major roles are played by peptide binding affinity, T-cell repertoire, and antigen processing and presentation. While the bulk of our information comes from mouse model systems, an increasing number of human studies suggest that immunodominance will be even more complicated. This review outlines current knowledge of TCD8+ immunodominance to viral antigens and discusses the relevance and importance of a thorough understanding for the rational design of vaccines that elicit effective TCD8+ responses.