Udayasankar Kumaraguru

Bystander Activation Involving T Lymphocytes in Herpetic Stromal Keratitis

Herpes simplex virus infection of mouse corneas can lead to the development of an immunopathological lesion, termed herpetic stromal keratitis (HSK). Such lesions also occur in TCR-transgenic mice backcrossed to SCID (TgSCID) that are unable to mount detectable HSV-specific immune responses. The present study demonstrates that lesion expression in such mice depends on continuous viral replication, whereas in immunocompetent mice, lesions occurred even if virus replication was terminated at 4 days after infection. The continuous replication in TgSCID mice was considered necessary to produce an activating stimulus to CD4+ T cells that invade the cornea. Lesions in TgSCID were resistant to control by cyclosporin A, but were inhibited by treatment with rapamycin. This result was interpreted to indicate that T cell activation involved a non-TCR-mediated cytokine-driven bystander mechanism. Bystander activation was also shown to play a role in HSK lesions in immunocompetent mice. Accordingly, in immunocompetent DO11.10 mice, lesions were dominated by KJ1.26+ OVA-specific CD4+ T cells that were unreactive with HSV. In addition, KJ1.26+ HSV nonimmune cells parked in ocularly infected BALB/c mice were demonstrable in HSK lesions. These results provide insight for the choice of new strategies to manage HSK, an important cause of human blindness.

Lymphotoxin α−/− Mice Develop Functionally Impaired CD8+ T Cell Responses and Fail to Contain Virus Infection of the Central Nervous System

Recent observations have indicated that viral persistence and tumor spreading could occur because of effector function-defective CD8+ T cells. Although chronic exposure to Ag, lack of CD4 help, and epitope dominance are suggested to interfere with CTL differentiation, mechanisms underlying the defective effector function remain obscure. We demonstrate in this report that lymphotoxin α-deficient mice develop CD8+ T cells at normal frequencies when infected with HSV or immunized with OVA Ag but show impaired cytotoxic and cytokine-mediated effector functions resulting in enhanced susceptibility to HSV-induced encephalitis. Although these cells display near normal levels of perforin and Fas ligand, they remain largely at a naive state as judged by high expression of CD62 ligand and failure to up-regulate activation or memory markers. In particular, these CD8+ T cells revealed inadequate expression of the IL-12 receptor, thus establishing a link between CTL differentiation and LTα possibly through regulation of IL-12 receptor. Viruses and tumors could evade immunity by targeting the same pathway.

Immunopotentiation of DNA vaccine against herpes simplex virus via co-delivery of plasmid DNA expressing CCR7 ligands

The CCR7 ligands, secondary lymphoid tissue chemokine (SLC) and Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC), were recently recognized as key molecules in establishing functional microenvironments for the initiation of immune responses in secondary lymphoid tissue. Here, we investigated the effect of CCR7 ligands-DNA administration on systemic and mucosal immune responses to plasmid DNA encoding gB of herpes simplex virus (HSV). Systemic co-transfer of both CCR7 ligands enhanced serum gB-specific IgG Ab but failed to elicit enhancement of distal mucosal IgA responses. In contrast, mucosal co-transfer provided significant increases of distal mucosal IgA responses. CCR7 ligands also enhanced T cell-mediated immunity as measured by CD4+ T helper cell proliferation and CD8+ T cell-mediated CTL activity. Of particular interest, is the observation that SLC significantly increased the production of Th1-type cytokines (IL-2 and IFN-gamma) (P<0.05), whereas ELC increased the production of both Th1-type and Th2-type (IL-4) cytokines (P<0.05). Moreover, co-vaccination of CCR7 ligands increased the number of dendritic cells in secondary lymphoid tissue. These data indicate that CCR7 ligands may prove to be useful adjuvants for genetic vaccination against intracellular infection as well as cancer.

Immunization with Chaperone-Peptide Complex Induces Low-Avidity Cytotoxic T Lymphocytes Providing Transient Protection against Herpes Simplex Virus Infection

ABSTRACT Heat shock proteins loaded with viral peptides were shown to induce a CD8+ T cell response and confer protective immunity against challenge with herpes simplex virus (HSV). The delivery system consisted of recombinant human hsp70 coupled to the peptide SSIEFARL, which is the immunodominant peptide epitope, recognized by HSV specific T cells in C57BL/6 mice. Immunization resulted in CD8+ T-cell responses, measured by peptide-specific tetramers and peptide-induced intracellular gamma interferon expression and cytotoxicity, similar to responses resulting from immunization with a recombinant vaccinia virus that expressed SSIEFARL as a minigene (VvgB) and UV-inactivated HSV. However, the durability of the hsp70-SSIEFARL response was less than that resulting from VvgB and HSV immunization and in addition the CD8+ T-cell responses in the memory phase were functionally less effective. Mice challenged soon after immunization showed excellent immunity, but by 90 days postimmunization this had waned to be significantly less than the level of immunity in both VvgB- and HSV-immunized mice.

Involvement of an ATP-Dependent Peptide Chaperone in Cross-Presentation After DNA Immunization

Immunization with plasmid DNA holds promise as a vaccination strategy perhaps useful in situations that currently lack vaccines, since the major means of immune induction may differ from more conventional approach. In the present study, we demonstrate that exposure of macrophages to plasmid DNA encoding viral proteins or OVA generates Ag-specific material that, when presented in vitro by dendritic cells to naive T cells, induces primary CTL response or elicits IL-2 production from an OVA peptide-specific T-T hybridoma. The immunogenic material released was proteinaceous in nature, free of apoptotic bodies, and had an apparent m.w. much larger than a 9–11-aa CTL-recognizable peptide. The macrophage-released factor(s) specifically required a hydrolyzable ATP substrate and was inhibited by procedures that removed or hydrolyzed ATP; in addition, anti-heat-shock protein 70 antiserum abrogated the activity to a large extent. These results indicate the possible involvement of a heat-shock protein 70-linked peptide chaperone in a cross-priming method of immune induction by DNA vaccination. Such a cross-priming process may represent a principal mechanism by which plasmid DNA delivered to cells such as myocytes effectively shuttle Ag to DC or other APC to achieve CTL induction in vivo.

Herpetic stromal keratitis in the absence of viral antigen recognition

Herpetic stromal keratitis (HSK), resulting from ocular infection with herpes simplex virus (HSV), is thought to represent a T cell mediated immunopathologic lesion. Antigens recognized by the inflammatory T cells remain unresolved and non-TCR mediated activation of T cells (bystander activation) is considered as also involved. This report documents further evidence for the bystander activation mechanisms using three T cell transgenic RAG-/- mouse strains. Accordingly HSK occurred in PCC RAG-/-, P14 RAG-/-, and OT-1 RAG-/- mice. In none of the models could HSV specific T cell reactivity be demonstrated and animals were unprotected from lesion development by immunization prior to HSV ocular infection. The results support the role of bystander activation as a mechanism of T cell mediated immunopathology and show that CD8(+) as well as CD4(+) T cells can participate in HSK lesion development.

Chemokines and ocular pathology caused by corneal infection with herpes simplex virus.

The role played by chemokines in disease process is an active area of research that continues to uncover new players. In this report we discuss the likely role of selected chemokines in the disease herpetic stromal keratitis (HSK). This lesion occurs as a sequel to herpes simplex virus infection and is currently accepted as an immunopathological process which primarily involves CD4+ T lymphocytes. In this review we discuss the events involved in HSK, the chemokine profile associated with this disease, and speculate on cellular activities and molecular events which characterize HSK as an immunopathological disease.

Why do we lack an effective vaccine against herpes simplex virus infections

This review discusses the possible causes for the lack of an effective antiherpes vaccine. Future prospects of vaccines based on the current knowledge of immune responses to herpes viruses are discussed. It is argued that vaccines capable of expanding CD8 T-cell memory responses should be the focus of future anti-herpes simplex virus research.

Concomitant Helper Response Rescues Otherwise Low Avidity CD8+ Memory CTLs to Become Efficient Effectors In Vivo

This report seeks a means of maximizing memory CD8 T cell responses to peptide immunization. Delivery of the CD8 peptide epitope by stress protein, heat shock protein (hsp)70, results in excellent immunogenicity at the acute phase but memory responses were poor both in terms of the number of responding cells as well as their functional avidity. We demonstrate for the first time that hsp70 can also be used as a vehicle to achieve CD4 T cell responses to loaded peptide epitopes and that coimmunization with hsp70 loaded with both CD8 and CD4 peptide epitopes may increase memory up to 3-fold. Furthermore, CD8+ T cell memory responses were of higher avidity measured both by in vitro cytotoxicity assays and a new methodology that measures the avidity of CTL activity in vivo in mice. Our results emphasize that peptide immunization remains a viable approach to induce long-term CD8+ T cell function, providing steps are taken to assure appropriate stimulation of Th cell responses.

Toll-like receptor ligand links innate and adaptive immune responses by the production of heat-shock proteins.

The report shows that CpG can exert additional adjuvant effects by inducing cells that are normally inferior antigen (Ag)‐presenting cells to participate in immune induction by cross‐priming. Macrophages (Mφ) exposed to protein Ag in the presence of bioactive CpG DNA released material that induced primary CD8+ T cell responses in DC‐naïve T cell cultures. This cross‐priming event was accompanied by up‐regulation of the stress protein response as well as inflammatory cytokine expression in treated Mφ. The material released was indicated to contain inducible heat shock protein‐70 and epitope peptide, which in turn, were presented by dendritic cells (DCs) to responder T cells. Such an adjuvant effect by CpG may serve to salvage immunogenic material from otherwise inert depot cellular sites and additionally stimulate DCs to effectively cross‐prime. The cross‐priming, shown also to occur in vivo, may be particularly useful when Ag doses are low and have minimal opportunity for delivery to DCs for consequent direct priming.