Stephen R. Jennings

CD4-positive T lymphocytes are required for the generation of the primary but not the secondary CD8-positive cytolytic T lymphocyte response to herpes simplex virus in C57BL/6 mice

To understand the cellular basis for recovery from HSV infection, it is critical to identify functional interactions between HSV-specific T lymphocyte subpopulations involved in the generation of the optimal response. To this end, the requirement for CD4+ (L3T4+) T lymphocytes in the development of the primary and secondary CD8+ (Lyt-2+) cytolytic T lymphocyte (CTL) response following HSV infection in C57BL/6 mice was investigated. It was found that chronic depletion of CD4+ cells in vivo by treatment with the mAb GK1.5, which resulted in greater than 95% depletion of peripheral CD4+ T lymphocytes in treated animals, caused a profound decrease in the levels of cytolytic activity obtained during the primary response in the draining popliteal lymph nodes of mice responding to infection in the hind footpads. However, treatment did not affect the levels of in vivo secondary CTL activity in the popliteal lymph nodes, nor the in vitro secondary response in the spleen. The decreased CTL activity observed during the primary response was not due to an inability to prime HSV-specific CTL precursors (CTLp), as full cytolytic activity was obtained following culture of lymphocytes in the presence of exogenous IL-2 and antigen, and the response could be reconstituted by treatment with recombinant IL-2 in vivo. Analysis of the secondary CTL response in the spleen indicated that CD4+ cells were not required for either the generation or maintenance of this aspect of the response. However, blockade of IL-2 utilization by CTL using anti-IL-2R antibodies indicated that this lymphokine was absolutely essential for secondary CTL expansion in vitro. Finally, mice that had been infected 12 months previously exhibited a decreased ability to generate secondary HSV-specific CTL in vitro following CD4-depletion in vivo. Taken together, these results suggest two distinct stages of CTL development during the response: an early primary stage dependent upon the presence of CD4+ cells, and a later, CD4-independent stage operative during the secondary response, which decays with time postinfection.

In Vivo Ablation of CD11c-Positive Dendritic Cells Increases Susceptibility to Herpes Simplex Virus Type 1 Infection and Diminishes NK and T-Cell Responses

ABSTRACT The precise role of each of the seven individual CD11c+ dendritic cell subsets (DCs) identified to date in the response to viral infections is not known. DCs serve as critical links between the innate and adaptive immune responses against many pathogens, including herpes simplex virus type 1 (HSV-1). The role of DCs as mediators of resistance to HSV-1 infection was investigated using CD11c-diphtheria toxin (DT) receptor-green fluorescent protein transgenic mice, in which DCs can be transiently depleted in vivo by treatment with low doses of DT. We show that ablation of DCs led to enhanced susceptibility to HSV-1 infection in the highly resistant C57BL/6 mouse strain. Specifically, we showed that the depletion of DCs led to increased viral spread into the nervous system, resulting in an increased rate of morbidity and mortality. Furthermore, we showed that ablation of DCs impaired the optimal activation of NK cells and CD4+ and CD8+ T cells in response to HSV-1. These data demonstrated that DCs were essential not only in the optimal activation of the acquired T-cell response to HSV-1 but also that DCs were crucial for innate resistance to HSV-1 infection.

Neutrophil-derived oxidants promote leukocyte adherence in postcapillary venules

The objective of this study was to determine whether hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and monochloramine (NH2Cl), at concentrations produced by activated neutrophils, promote leukocyte adherence to microvascular endothelium in post-capillary venules. Cat mesenteric venules (30-45 microns diameter) were examined using intravital video microscopy. Red blood cell velocity (VRBC), venular diameter (DV), and the number of adherent leukocytes (NWBC) were measured in postcapillary venules. Venular blood flow and wall shear rate (tau) were calculated from the measured values of VRBC and DV. Different concentrations (0.01-1.0 mM) of H2O2, HOCl, or NH2Cl were superfused on the mesentery. In some experiments, the contributions of the leukocyte adhesive glycoprotein CD11/CD18 and platelet-activating factor (PAF) in the oxidant-induced leukocyte adherence were determined using a CD18-specific antibody (IB4) and a PAF-receptor antagonist (WEB 2086), respectively. The results of our in vivo experiments indicate that H2O2 and NH2Cl, but not HOCl, promote leukocyte adhesion to venular endothelium. Incubation of isolated cat neutrophils with either NH2Cl or H2O2 resulted in activation of CD11/CD18, as assessed by flow cytometry. Although the leukocyte adhesion induced by both H2O2 and NH2Cl was associated with a reduction in venular wall shear rate, corresponding decrements in shear rate induced by partial occlusion of the mesenteric artery did not lead to similar levels of leukocyte adherence. The leukocyte adherence induced by H2O2 and NH2Cl was largely prevented by monoclonal antibody IB4, indicating that both oxidants promote leukocyte adherence via activation of CD11/CD18. The H2O2-induced, CD18-mediated leukocyte adherence appears to be elicited by PAF and by a direct effect of the oxidant on CD11/CD18 expression. The mechanism underlying the NH2Cl-induced leukocyte adherence remain unclear.

Memory CD8+ T Cells Require CD28 Costimulation

CD8+ T cells are a critical component of the adaptive immune response against infections and tumors. A current paradigm in immunology is that naive CD8+ T cells require CD28 costimulation, whereas memory CD8+ T cells do not. We show here, however, that during viral infections of mice, costimulation is required in vivo for the reactivation of memory CD8+ T cells. In the absence of CD28 costimulation, secondary CD8+ T cell responses are greatly reduced and this impairs viral clearance. The failure of CD8+ T cells to expand in the absence of CD28 costimulation is CD4+ T cell help independent and is accompanied by a failure to down-regulate Bcl-2 and by cell cycle arrest. This requirement for CD28 costimulation was shown in both influenza A and HSV infections. Thus, contrary to current dogma, memory CD8+ T cells require CD28 costimulation to generate maximal secondary responses against pathogens. Importantly, this CD28 requirement was shown in the context of real infections were multiple other cytokines and costimulators may be up-regulated. Our findings have important implications for pathogens, such as HIV and measles virus, and tumors that evade the immune response by failing to provide CD28 costimulation. These findings also raise questions about the efficacy of CD8+ T cell-based vaccines against such pathogens and tumors.

Characterization of age-related changes in natural killer cells during primary influenza infection in mice.

The current investigation examined the importance of natural killer (NK) cells during the innate immune response to primary influenza infection in young and aged mice. Young (6-8 weeks) and aged (22 months) C57BL/6 mice were infected intranasally with influenza A virus, and NK cell-mediated cytotoxicity was determined in lung and spleen during the first 4 days of infection. Aged mice demonstrated both a decrease in influenza-inducible NK activity and a reduction in the percentage and number of NK1.1+ cells in response to primary influenza infection, relative to young mice. In order to further establish a role for NK cells in controlling influenza infection, young mice were depleted of NK cells in vivo by injecting rabbit anit-NK1.1 antibody 2 days and 1 day prior to influenza infection. Young mice depleted of NK cells exhibited increased weight loss and lung virus titers during the course of infection, compared to young mice infected with influenza virus. These data indicate that NK cell function is impaired in response to primary influenza infection in aged mice. More importantly, these results underscore the essential role of NK cells in controlling virus titers in lung during the early course of influenza infection, regardless of age.

Immunological control of herpes simplex virus infections.

Herpes simplex virus type 1 (HSV-1) is capable of causing a latent infection in sensory neurons that lasts for the lifetime of the host. The primary infection is resolved following the induction of the innate immune response that controls replication of the virus until the adaptive immune response can clear the active infection. HSV-1-specific CD8+ T cells survey the ganglionic regions containing latently infected neurons and participate in preventing reactivation of HSV from latency. The long-term residence and migration dynamics of the T cells in the trigeminal ganglia appear to distinguish them from the traditional memory T cell subsets. Recently described tissue resident memory (TRM) T cells establish residence and survive for long periods in peripheral tissue compartments following antigen exposure. This review focuses on the immune system response to HSV-1 infection. Particular emphasis is placed on the evidence pointing to the HSV-1-specific CD8+ T cells in the trigeminal belonging to the TRM class of memory T cells and the role of TRM cells in virus infection, pathogenesis, latency, and disease.

T-lymphocytes contribute to hepatic leukostasis and hypoxic stress induced by gut ischemia/reperfusion

Although neutrophils have been implicated in the hepatic injury elicited by gut ischemia/reperfusion (I/R), the contribution of other leukocyte populations to this injury process remains unclear. The objective of this study was to determine whether lymphocytes contribute to gut I/R‐induced microvascular dysfunction and inflammatory responses in the liver. Intravital videomicroscopy was used to monitor leukocyte recruitment, the number of nonperfused sinusoids and pyridine nucleotide (NADH) autofluorescence in livers of wild‐type, SCID, and interferon‐γ (IFN‐γ) knockout mice exposed to 15 min of gut ischemia and 1 h of reperfusion. In wild‐type mice, gut I/R elicited significant increases in the number of stationary leukocytes, nonperfused sinusoids, NADH autofluorescence (indicating hypoxia), and elevated plasma alanine aminotransferase (ALT) and TNF‐α levels. All of these responses were profoundly attenuated in SCID mice, while only some of the responses (in the midzonal region) were blunted in IFN‐γ knockout mice. Reconstitution (24 h before ischemia) of the circulating lymphocyte pool with T‐cell enriched splenocytes, but not T‐cell deficient (from nude mice), CD4+ T‐cell depleted splenocytes or splenocytes derived from IFN‐γ knockout mice, allowed the SCID mice to respond to gut I/R in a manner similar to wild‐type mice. Some of the responses were restored following reconstitution with CD8+ T‐cell depleted splenocytes. These findings implicate CD4+ Tlymphocytes and IFN‐γ in the hepatic microvascular dysfunction and inflammatory cell accumulation elicited by gut I/R.

CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1

ABSTRACT The role of CD4+ helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4+ T cells are needed for the generation of the protective HSV-1-specific CD8+-T-cell response. This study examined the contribution of CD4+ T cells in the generation of the primary CD8+-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8+-T-cell response generated in the draining lymph nodes of CD4+-T-cell-depleted C57BL/6 mice and B6-MHC-II−/− mice is quantitatively and qualitatively distinct from the CD8+ T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8+ T cells express comparable levels of the activation marker CD44 in mice lacking CD4+ T cells and normal mice. In contrast, CD8+ T cells generated in the absence of CD4+ T cells express the interleukin 2 receptor α-chain (CD25) at lower levels. Importantly, the CD8+ T cells in the CD4+-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8+ T cells is diminished in the absence of CD4+-T-cell help. These results suggest that CD4+-T-cell help is essential for the generation of fully functional CD8+ T cells during the primary response to HSV-1 infection.

Cytokine and adhesion molecule expression in SCID mice reconstituted with CD4+ T cells.

Summary: The objectives of this study were to quantify colonic cytokine and endothelial cell adhesion molecule (ECAM) expression in the colons of severe combined immunodeficient (SCID) mice reconstituted with different subsets of CD4+ T lymphocytes. We found that animals injected with CD45RBhigh but not CD45RBlow T cells or phosphate‐buffered saline (PBS) developed clinical evidence of colitis at 6‐8 weeks following reconstitution, as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of a variety of Thl and macrophage‐derived cytokines including interferon &ggr;, tumor necrosis factor‐&agr;, interleukin (IL)‐1&bgr;, IL‐6, IL‐12, and IL‐18 lymphotoxin‐&bgr;. In addition, message levels and vascular surface expression of ICAM‐1, VCAM‐1, and MAdCAM‐1 were all significantly enhanced in the colitic SCID mice reconstituted with CD45RBhigh T cells compared with SCID mice reconstituted with PBS or CD45RBlow T cells that did not develop disease. Significant increases in some of these ECAMs were also noted in the cecum and stomach and to a lesser degree in the small bowel. Our data confirm that reconstitution of SCID mice with CD45RBhigh but not CD45RBlow T cells induces chronic colitis, and that the colonic inflammation is associated with enhanced expression of proinflammatory cytokines and different ECAMs in the colon. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RBhigh T cells enhances ECAM expression in tissues distant from the site of active inflammation.

Protective immunity against equine herpesvirus type-1 (EHV-1) infection in mice induced by recombinant EHV-1 gD

The ability of recombinant preparations of equine herpesvirus type 1 (EHV-1) glycoprotein D (gD) to elicit specific antibody and T lymphocyte responses in the BALB/c mouse model of respiratory infection was investigated. Recombinant gD (rgD) expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli elicited both high titer neutralizing antibody (nAb) and CD4 T cell proliferative responses following subcutaneous or intranasal immunization, but elicited only a weak antibody response after intraperitoneal immunization. Protection against respiratory tract infection with pathogenic EHV-1 RacL11 was observed in mice immunized subcutaneously with GST-gD. Furthermore, the degree of protection correlated to the titer of nAb and the T cell response observed. Finally, GST-gD was more effective in protecting against respiratory RacL11 infection if delivered intranasally. These results confirm that gD plays an important role in eliciting the protective immune response against EHV-1 infection, and indicate that subunit vaccines containing preparations of gD may be very effective if delivered directly to the upper respiratory tract.