Naturally occurring CD4+CD25+ regulatory T cells appear important to prevent activation of autoreactive T cells. This article demonstrates that the magnitude of a CD8+ T cell–mediated immune response to an acute viral infection is also subject to control by CD4+CD25+ T regulatory cells (Treg). Accordingly, if natural Treg were depleted with specific anti-CD25 antibody before infection with HSV, the resultant CD8+ T cell response to the immunodominant peptide SSIEFARL was significantly enhanced. This was shown by several in vitro measures of CD8+ T cell reactivity and by assays that directly determine CD8+ T cell function, such as proliferation and cytotoxicity in vivo. The enhanced responsiveness in CD25-depleted animals was between three- and fourfold with the effect evident both in the acute and memory phases of the immune response. Surprisingly, HSV infection resulted in enhanced Treg function with such cells able to suppress CD8+ T cell responses to both viral and unrelated antigens. Our results are discussed both in term of how viral infection might temporarily diminish immunity to other infectious agents and their application to vaccines. Thus, controlling suppressor effects at the time of vaccination could result in more effective immunity.

MicroRNAs are small regulatory RNAs that post-transcriptionally regulate gene expression and can be encoded by viral as well as cellular genomes. The functions of most viral miRNAs are unknown and few have been studied in an in vivo context. Here we show that the murine polyomavirus (PyV) encodes a precursor microRNA that is processed into two mature microRNAs, both of which are active at directing the cleavage of the early PyV mRNAs. Furthermore, we identify a deletion mutant of polyomavirus that is defective in encoding the microRNAs. This mutant replicates normally and transforms cultured cells with efficiencies comparable to wildtype PyV. The miRNA mutant is competent to establish a transient infection of mice following parenteral inoculation, and is cleared post infection at approximately the same rate as the wildtype virus. In addition, under these laboratory conditions, we observe no differences in anti-viral CD8 T cell responses. These results indicate that PyV miRNA expression is not essential for infection of cultured cells or experimentally inoculated mice, and raise the possibility that its role in natural infection might involve aspects of acquisition or spread that are not recapitulated by experimental inoculation.

Induction of neonatal T cell tolerance to soluble antigens requires the use of incomplete Freund's adjuvant (IFA). The side effects that could be associated with IFA and the ill-defined mechanism underlying neonatal tolerance are setbacks for this otherwise attractive strategy for prevention of T cell–mediated autoimmune diseases. Presumably, IFA contributes a slow antigen release and induction of cytokines influential in T cell differentiation. Immunoglobulins (Igs) have long half-lives and could induce cytokine secretion by binding to Fc receptors on target cells. Our hypothesis was that peptide delivery by Igs may circumvent the use of IFA and induce neonatal tolerance that could confer resistance to autoimmunity. To address this issue we used the proteolipid protein (PLP) sequence 139–151 (hereafter referred to as PLP1), which is encephalitogenic and induces experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. PLP1 was expressed on an Ig, and the resulting Ig–PLP1 chimera when injected in saline into newborn mice confers resistance to EAE induction later in life. Mice injected with Ig–PLP1 at birth and challenged as adults with PLP1 developed T cell proliferation in the lymph node but not in the spleen, whereas control mice injected with Ig–W, the parental Ig not including PLP1, developed T cell responses in both lymphoid organs. The lymph node T cells from Ig–PLP1 recipient mice were deviated and produced interleukin (IL)-4 instead of IL-2, whereas the spleen cells, although nonproliferative, produced IL-2 but not interferon (IFN)-γ. Exogenous IFN-γ, as well as IL-12, restored splenic proliferation in an antigen specific manner. IL-12–rescued T cells continued to secrete IL-2 and regained the ability to produce IFN-γ. In vivo, administration of anti–IL-4 antibody or IL-12 restored disease severity. Therefore, adjuvant-free induced neonatal tolerance prevents autoimmunity by an organ-specific regulation of T cells that involves both immune deviation and a new form of cytokine- dependent T cell anergy.

Although many studies have investigated the requirement for CD4+ T cell help for CD8+ T cell responses to acute viral infections that are fully resolved, less is known about the role of CD4+ T cells in maintaining ongoing CD8+ T cell responses to persistently infecting viruses. Using mouse polyoma virus (PyV), we asked whether CD4+ T cell help is required to maintain antiviral CD8+ T cell and humoral responses during acute and persistent phases of infection. Though fully intact during acute infection, the PyV-specific CD8+ T cell response declined numerically during persistent infection in MHC class II-deficient mice, leaving a small antiviral CD8+ T cell population that was maintained long term. These unhelped PyV-specific CD8+ T cells were functionally unimpaired; they retained the potential for robust expansion and cytokine production in response to Ag rechallenge. In addition, although a strong antiviral IgG response was initially elicited by MHC class II-deficient mice, these Ab titers fell, and long-lived PyV-specific Ab-secreting cells were not detected in the bone marrow. Finally, using a minimally myeloablative mixed bone marrow chimerism approach, we demonstrate that recruitment and/or maintenance of new virus-specific CD8+ T cells during persistent infection is impaired in the absence of MHC class II-restricted T cells. In summary, these studies show that CD4+ T cells differentially affect CD8+ T cell responses over the course of a persistent virus infection.

Although immunity against intracellular pathogens is primarily provided by CD8 T lymphocytes that recognize pathogen-derived peptides presented by major histocompatibility complex (MHC) class Ia molecules, MHC class Ib–restricted CD8 T cells have been implicated in antiviral immunity. Using mouse polyoma virus (PyV), we found that MHC class Ia–deficient (Kb−/−Db−/−) mice efficiently control this persistently infecting mouse pathogen. CD8 T cell depletion mitigates clearance of PyV in Kb−/−Db−/− mice. We identified the ligand for PyV-specific CD8 T cells in Kb−/−Db−/− mice as a nonamer peptide from the VP2 capsid protein presented by Q9, a member of the β2 microglobulin–associated Qa-2 family. Using Q9-VP2 tetramers, we monitored delayed but progressive expansion of these antigen-specific CD8αβ T cells in Kb−/−Db−/− mice. Importantly, we demonstrate that Q9-VP2–specific CD8 T cells more effectively clear wild-type PyV than a VP2 epitopenull mutant PyV. Finally, we show that wild-type mice also generate Q9-restricted VP2 epitope–specific CD8 T cells to PyV infection. To our knowledge, this is the first evidence for a defined MHC class Ib–restricted antiviral CD8 T cell response that contributes to host defense. This study motivates efforts to uncover MHC class Ib–restricted CD8 T cell responses in other viral infections, and given the limited polymorphism of MHC class Ib molecules, it raises the possibility of developing peptide-based viral vaccines having broad coverage across MHC haplotypes.

Mucosal surfaces represent the entry route of a multitude of viral pathogens. For many of these viruses, such as the herpes simplex viruses and human immunodeficiency virus, no effective vaccine exists. Hence, it is important that prospective vaccines engender maximal immunity at these susceptible sites. Genetic vaccines encoding adjuvant molecules represent one approach to optimize mucosal as well as systemic immunity. Promising candidates include various inflammatory cytokines and chemokines that might be used to enhance the primary response to a level sufficient for protection. Encouraging studies involving cytokines such as granulocyte/macrophage colony-stimulating factor, interleukin-2 (IL-2), IL-12, IL-18, and many others are examined. Notable chemokines that may offer hope in such efforts include IL-8, RANTES, CCL19, CCL21, and a few others. Combinatorial approaches utilizing several cytokines and chemokines will most likely yield the greatest success. In addition, as more is discovered regarding the requirements for memory development of T cells, boosters involving key cytokines such as IL-15 and IL-23 may prove beneficial to long-term maintenance of the memory pool. This review summarizes the progress in the use of genetic vaccines to achieve mucosal immunity and discusses the needed strategies to maximize long-term prospective immunity at this vulnerable entry site.

Antigen delivered within particulate materials leads to enhanced antigen-specific immunity compared to soluble administration of antigen. However, current delivery approaches for antigen encapsulated in synthetic particulate materials are limited by the complexity of particle production that affects stability and immunogenicity of the antigen. Herein, we describe a protein delivery system that utilizes plasma membrane vesicles (PMVs) derived from biological materials such as cultured cells or isolated tissues and a simple protein transfer technology. We show that these particulate PMVs can be easily modified within 4 h by a protein transfer process to stably incorporate a glycosylphosphatidylinositol (GPI)-anchored form of the breast cancer antigen HER-2 onto the PMV surface. Immunization of mice with GPI-HER-2-modified-PMVs induced strong HER-2-specific antibody responses and protection from tumor challenge in two different breast cancer models. Further incorporation of the immunostimulatory molecules IL-12 and B7-1 onto the PMVs by protein transfer enhanced tumor protection and induced beneficial Th1 and Th2-type HER-2-specific immune responses. Since protein antigens can be easily converted to GPI-anchored forms, these results demonstrate that isolated plasma membrane vesicles can be modified with desired antigens along with immunostimulatory molecules by protein transfer and used as a vaccine delivery vehicle to elicit potent antigen-specific immunity.

The CD8 coreceptor is important for positive selection of major histocompatibility complex I (MHC-I)-restricted thymocytes and in the generation of pathogen-specific T cells. However, the requirement for CD8 in these processes may not be essential. We previously showed that mice lacking ␤ 2 -microglobulin are highly susceptible to tumors induced by mouse polyoma virus (PyV), but CD8-deficient mice are resistant to these tumors. In this study, we show that CD8-deficient mice also control persistent PyV infection as efficiently as wild-type mice and generate a substantial virus-specific, MHC-I-restricted, T-cell response. Infection with vesicular stomatitis virus (VSV), which is acutely cleared, also recruited antigen-specific, MHC-I-restricted T cells in CD8-deficient mice. Yet, unlike in VSV infection, the antiviral MHC-I-restricted T-cell response to PyV has a prolonged expansion phase, indicating a requirement for persistent infection in driving T-cell inflation in CD8-deficient mice. Finally, we show that the PyV-specific, MHC-I-restricted T cells in CD8-deficient mice, while maintained long term at near-wild-type levels, are short lived in vivo and have extremely narrow T-cell receptor repertoires. These findings provide a possible explanation for the resistance of CD8-deficient mice to PyV-induced tumors and have implications for the maintenance of virus-specific MHC-I-restricted T cells during persistent infection.