Development of effective vaccines for old mice in a tumor model.

Vaccines are often inefficient in old people and old mice. Few studies have focused on testing vaccines in old populations. Here we used DNA tumor antigen vaccines against melanoma and showed that old mice were not protected. Vaccines incorporating fusions of the tumor antigen with microbial adjuvant proteins OmpA (E. Coli) or Vp22 (Herpes simplex virus-1) dramatically improved protection of old mice. The mechanisms by which these adjuvant proteins act are distinct. TLR2 was not required for either OmpA or Vp22. Antigen processing and presentation were not boosted by these fusion constructs. However, fusion constructs with Vp22 gave a strong CD4 response to B16 melanoma and the OmpA response is MHC-II dependent. Both adjuvant fusion constructs stimulated CD4 and CD8 responses otherwise diminished in old mice.

Herpesviruses and autoimmunity.

Herpesvirus infection, in particular EBV infection, has been implicated in several major autoimmune diseases, including systemic lupus erythematosus (SLE), multiple sclerosis (MS) and rheumatoid arthritis (RA). Herpesvirus infection has potential roles in both initiating the autoimmune process and exacerbating disease progression. In particular, EBV has a proposed role in initiating the anti-nucleoprotein antibodies that are characteristic of SLE through molecular mimicry. There is also evidence to suggest that there is productive infection with EBV in the brain lesions of MS patients and in the synovium of RA patients. Research has been conducted in a mouse gamma-herpesvirus model, as it serves as a useful model for productive infection within autoimmune target tissues. The novel mechanisms by which EBV could contribute bystander effects by amplification of innate immune responses, along with preclinical and epidemiological studies into the role of herpesviruses in SLE, MS and RA, and clinical studies into the potential benefit of antiviral therapy, are discussed in this review.

Mechanisms of immunization against cancer using chimeric antigens.

Successful approaches to tumor immunotherapy must overcome the physiological state of tolerance of the immune system to self-tumor antigens. Immunization with appropriate variants of syngeneic antigens can achieve this. However, improvements in vaccine design are needed for efficient cancer immunotherapy. Here we explore nine different chimeric vaccine designs, in which the antigen of interest is expressed as an in-frame fusion with polypeptides that impact antigen processing or presentation. In DNA immunization experiments in mice, three of nine fusions elevated relevant CD8(+) T-cell responses and tumor protection relative to an unfused melanoma antigen. These fusions were: Escherichia coli outer membrane protein A (OmpA), Pseudomonas aeruginosa exotoxin A, and VP22 protein of herpes simplex virus-1. The gains of immunogenicity conferred by the latter two are independent of epitope presentation by major histocompatibility complex class II (MHC II). This finding has positive implications for immunotherapy in individuals with CD4(+) T-cell deficiencies. We present evidence that antigen instability is not a sine qua non condition for immunogenicity. Experiments using two additional melanoma antigens identified different optimal fusion partners, thereby indicating that the benefits of fusion vectors remain antigen specific. Therefore large fusion vector panels such as those presented here can provide information to promote the successful advancement of gene-based vaccines.

Apoptosis of T cells in peripheral blood and cerebrospinal fluid is associated with disease activity of multiple sclerosis.

Apoptotic elimination of pathogenic T cells is considered to be one of regulatory mechanisms in multiple sclerosis (MS). To explore the potential relationship between Fas-mediated apoptosis and the disease course of MS, we examined apoptosis, defined by annexin V (AV) binding, and Fas (CD95) expression in CD4+ and in CD8+ T cells in MS patients by using five-color flow cytometry. The percentage of AV+CD4+CD3+ cells and CD95+AV+CD4+CD3+ cells in peripheral blood and cerebrospinal fluid (CSF) were significantly decreased in active MS patients compared with inactive MS patients. A significantly lower proportion of CD95+AV+CD8+CD3+ cells in CSF was observed in active MS patients compared with inactive MS patients, but not in peripheral blood. These results indicate that the resistance of T cells to Fas-mediated apoptosis is involved in exacerbation of MS and/or that Fas-mediated apoptosis of T cells is associated with remission of MS.

Antiviral T cell responses: phalanx or multipronged attack?

Around 700 BCE, a new military formation called the phalanx was established in ancient Greece: a tight column of heavy infantry carrying long spears, or pikes, used in a single prong of attack. Later, in the battle of Marathon described by Herodotus, the Greeks learned the advantages of multipronged attacks, a strategy still used in modern warfare. Is the immune system similar in its approach to combatting pathogens or tumors?

The activation of memory CD4(+) T cells and CD8(+) T cells in patients with multiple sclerosis.

To reevaluate whether an association exists between the clinical course of multiple sclerosis (MS) and the activation of memory T cells, we investigated the phenotype of T cells in peripheral blood and cerebrospinal fluid (CSF) of patients with MS using five-color flow cytometry. A cross-sectional study with 39 relapsing-remitting MS patients demonstrated that the percentage of CD25(+)CD45RO(+)CD4(+)CD3(+) cells was significantly increased in peripheral blood as well as in CSF of active MS patients compared with inactive MS patients. A longitudinal study with 11 relapsing-remitting MS patients also showed a higher percentage of CD25(+)CD45RO(+)CD4(+)CD3(+) cells in peripheral blood at the phase of exacerbation than during remission. On the other hand, regardless of the disease activity, the percentage of CD25(+)CD45RO(+)CD8(+)CD3(+) cells in peripheral blood was significantly higher in patients with MS than in healthy control subjects. A lower percentage of CD25(+)CD45RO(+)CD8(+)CD3(+) cells in CSF was observed in active MS patients compared with inactive MS patients. These results suggest that the activation of memory CD4(+) T cells is associated with the exacerbation of MS and activation of memory CD8(+) T cells reflects systemic immunological dysregulation in MS patients. Transient as well as continuous activation of T cells by recall antigens may be involved in the disease course of MS.

Amplification of autoimmune disease by infection.

Reports of infection with certain chronic persistent microbes (herpesviruses or Chlamydiae) in human autoimmune diseases are consistent with the hypothesis that these microbes are reactivated in the setting of immunodeficiency and often target the site of autoimmune inflammation. New experimental animal models demonstrate the principle. A herpesvirus or Chlamydia species can be used to infect mice with induced transient autoimmune diseases. This results in increased disease severity and even relapse. The evidence suggests that the organisms are specifically imported to the inflammatory sites and cause further tissue destruction, especially when the host is immunosuppressed. We review the evidence for the amplification of autoimmune inflammatory disease by microbial infection, which may be a general mechanism applicable to many human diseases. We suggest that patients with autoimmune disorders receiving immunosuppressing drugs should benefit from preventive antiviral therapy.

A mouse herpesvirus induces relapse of experimental autoimmune arthritis by infection of the inflammatory target tissue.

It is not known what is required for successive relapses in autoimmune diseases or evolution to a progressive chronic disease. Autoimmune arthritis caused by passive transfer of autoantibodies against glucose 6-phosphate isomerase is transient and therefore lends itself well to test for what might extend the disease. Herpesviruses have long been suspected of contributing to human autoimmune disease. We infected mice with a murine gamma-herpesvirus (MHV-68). In immunodeficient mice, transient arthritis was followed by a relapse. This was due to lytic viral infection of synovial tissues demonstrated by PCR, immunohistochemistry, and electron microscopy. Latent infection could be reactivated in the synovium of normal mice when treated with Cytoxan and this was associated with increased clinical arthritis. We conclude that herpesviruses may play an ancillary pathogenic role in autoimmune arthritis by infection of the inflammatory target tissue.

Genomic absence of the gene encoding T cell receptor Vbeta7.2 is linked to the presence of autoantibodies in Sjögren's syndrome.

OBJECTIVE: It is not yet known whether the absence of certain T cell receptor V(beta) (TCRBV) genes (e.g., due to genomic deletion) has functional significance. We examined this question in relation to a known 21.6-kb insertion/deletion-related polymorphism (IDRP) in the human BV locus. METHODS: New polymerase chain reaction (PCR) genotyping methods were used. Monoclonal antibodies to TCRBV gene products were used to confirm the absence of the relevant proteins. Patients with Sjögren's syndrome (SS) or systemic lupus erythematosus (SLE) were compared with normal controls with regard to TCR genotypes and serologic profiles. RESULTS: There are 3 known haplotypes (I, D1, D2) and 6 possible genotypes related to the 21.6-kb IDRP. Novel PCR-based methods were used to define these genotypes. In subjects with deleted/deleted (D/D) genotypes, T cells could not express V(beta)7.2 TCRs, as assayed with a new antibody specific for V(beta)7.2. This was the sole significant difference between subjects without the insertion and those with either 1 or 2 copies. Surprisingly, we found that the D/D genotype was associated with primary SS, but only when pathogenic autoantibodies were present. CONCLUSION: These results suggest that T cells expressing TCRs with V(beta)7.2 are protective against a pathogenic immune response in SS. Thus, genomic polymorphism of TCR genes (along with the correct HLA alleles) determines whether T cells can direct a pathogenic autoimmune response.

Oligoclonal expansions of antigen-specific CD8+ T cells in aged mice.

Oligoclonal T cell expansions (TCE) are common in old humans and mice. It is not known whether an Ag-specific response becomes more oligoclonal with age, and, if so, how this might alter biological responses or compromise the immune response, thus contributing to the immunodeficiency of aging. We used a tumor antigen response to study these questions. Early on, antigen reactive T cell numbers at the site of tumor injection were lower and clonally more restricted in old mice. Subsequently, long-term oligoclonal TCE emerged in the blood and spleen of old mice. IL-15 was not necessary for development of TCE in the blood. Overall, the data pointed to a dysregulated immune response in old mice, perhaps due to lack of optimal IL-2 and CD4 help at the earliest stages and a lack of an efficient local peritoneal CTL response. This was associated with a deficient humoral response and, likely, persistence of tumor cells or tumor antigens. Perhaps the spleen is the site of persistence which explains clonal TCE observed primarily in PBL and spleen. The TCE appear to be inefficient as they are often anergic. As a result an occasional peritoneal or splenic tumor may arise in old mice.

Staphylococcus aureus express unique superantigens depending on the tissue source.

A comprehensive analysis of Staphylococcus aureus superantigen (SAG) genes was undertaken in isolates from a major hospital and compared with isolates from patients with toxic shock syndrome (TSS). Polymerase chain reaction (PCR) analysis included recently discovered SAGs. Staphylococcal enterotoxin (SE) G and SEI were uniquely expressed in genital isolates. Genital isolates were similar to TSS isolates, although the latter frequently expressed TSS toxin 1. Both had a high frequency of SEG/SEI and a high number of SAG genes per bacterium. Detection of an SAG gene by PCR correlated with positive results in functional assays for SAG activity. Levels of serum antibodies to SEG and SEI, but not to other superantigens, were higher in healthy women than in men and served as an independent measure of the higher frequency of exposure to SEG/SEI among women. Together, the data suggest a role for SEG/SEI or closely linked genes in the adaptation of S. aureus to the genital mucosa environment.

Tumor antigen drives a persistent oligoclonal expansion of CD8+ T cells in aged mice.

Oligoclonal T cell expansions (TCE) are common in old humans and mice, but it is not known whether the T cell response to a specific antigen is more restricted in old vs. young animals. Herein, we describe an enhanced and prolonged response of tumor antigen-specific CD8 cells in old mice identified by K(d)/peptide tetramers and Vbeta10 staining. At the onset of the response CD8 T cell numbers and Vbeta10+CD8+ cells at the site of tumor injection were lower in old mice, hinting that control of initial tumor growth may not be optimal. As further evidence of a dysregulated response in old mice, antibody titers to the tumor were deficient and the CD8 tumor antigen-specific response was greater and more prolonged in the blood and spleen. Old mice selected a more oligoclonal TCR repertoire based on TCRbeta chain CDR3 length analysis and sequences. Persistent expansions of Vbeta10+CD8+ cells in old mice had memory/activation phenotypes. This induced tumor antigen-specific response may represent a model for the spontaneous TCE observed with aging and demonstrates that the CD8 response to a defined peptide/MHC antigen is indeed more oligoclonal in old mice.