Sarah G. Crist

IL-15-Independent Proliferative Renewal of Memory CD8 + T Cells in Latent Gammaherpesvirus Infection

IL-15 is known to be critical in the homeostasis of Ag-specific memory CD8+ T cells following acute viral infection. However, little is known about the homeostatic requirements of memory CD8+ T cells during a latent viral infection. We have used the murine gammaherpesvirus-68 (MHV-68) model system to investigate whether IL-15 is necessary for the maintenance of memory CD8+ T cells during a latent viral infection. IL-15 is not essential either for the initial control of MHV-68 infection or for the maintenance of MHV-68-specific memory CD8+ T cells. Even at 140 days postinfection, the proportion of CD8+ T cells recognizing the MHV-68 epitopes were the same as in control mice. The maintenance of these memory CD8+ T cells was attributable to their ability to turn over in vivo, probably in response to the presence of low levels of Ag. IL-15−/− mice had a significantly higher turnover rate within the virus-specific memory CD8+ T cell population, which was the result of increased levels of viral gene expression rather than an increase in viral load. These cells did not accumulate in the spleens of the IL-15−/− mice due to an increased sensitivity to apoptosis as a result of decreased Bcl-2 levels. Intriguingly, memory CD8+ T cells from latently infected mice failed to undergo homeostatic proliferation in a naive secondary host. These data highlight fundamental differences between memory CD8+ T cells engaged in active immune surveillance of latent viral infections vs memory CD8+ T cells found after acute viral infections.

Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection

Gammaherpesviruses can persist in the host in the face of an aggressive immune response. T cells recognize Ags expressed in both the productive and latent phases of the virus life cycle, however little is known about their relative roles in the long-term control of the infection. In this study we used the murine gammaherpesvirus 68 model system to investigate the relative properties of CD8 T cells recognizing lytic and latent viral Ags. We report that the CD8 T cell response to lytic phase epitopes is maximal in the lungs of infected mice at ∼10 days postinfection, and is of progressively lesser magnitude in the mediastinal lymph nodes and spleen. In contrast, the CD8 T cell response to the latent M2 protein is maximal at ∼19 days postinfection and is most prominent in the spleen, then progressively less in the mediastinal lymph node and the lung. Latent and lytic Ag-specific CD8 T cells had markedly different cell surface phenotypes during chronic infection, with latent Ag-specific cells being predominantly CD62Lhigh or CD43 (1B11)high. Lytic Ag-specific T cells had significantly lower expression of these markers. Importantly, latent but not lytic Ag-specific T cells could kill target cells rapidly in vivo during the chronic infection. These two different sets of CD8 T cells also responded differentially to IL-7, a cytokine involved in T cell homeostasis and the maintenance of T cell memory. These data have important implications for our understanding of immunological control during chronic gammaherpesvirus infections.

Phenotype and susceptibility to HIV infection of CD4+ Th17 cells in the human female reproductive tract.

Prevention of sexual acquisition of HIV in women requires a substantial increase in our knowledge about HIV-target cell availability and regulation in the female reproductive tract (FRT). In this study, we analyzed the phenotype and susceptibility to HIV infection of CD4+ T cell in the endometrium (EM), endocervix (END), and ectocervix (ECT) of the FRT. We found that T helper type 17 (Th17) cells represent a major subset in FRT tissues analyzed and that Th17 cells were the main CD4+ T-cell population expressing C-C motif chemokine receptor 5 (CCR5) and CD90. In premenopausal women, CD4+ T cells and Th17 cells, in particular, were significantly lower in EM relative to END and ECT. Th17 cells were elevated in EM from postmenopausal women relative to premenopausal tissues but not changed in END and ECT. Susceptibility of CD4+ T cells to HIV infection measured as intracellular p24 was lowest in the EM and highest in the ECT. Additionally, we found that Th17 cells co-expressing CCR5 and CD90 were the most susceptible to HIV infection. Our results provide valuable information for designing preventive strategies directed at targeting highly susceptible target cells in the FRT.

Estradiol Reduces Susceptibility of CD4 + T Cells and Macrophages to HIV-Infection

The magnitude of the HIV epidemic in women requires urgent efforts to find effective preventive methods. Even though sex hormones have been described to influence HIV infection in epidemiological studies and regulate different immune responses that may affect HIV infection, the direct role that female sex hormones play in altering the susceptibility of target cells to HIV-infection is largely unknown. Here we evaluated the direct effect of 17-β-estradiol (E2) and ethinyl estradiol (EE) in HIV-infection of CD4+ T-cells and macrophages. Purified CD4+ T-cells and monocyte-derived macrophages were generated in vitro from peripheral blood and infected with R5 and X4 viruses. Treatment of CD4+ T-cells and macrophages with E2 prior to viral challenge reduced their susceptibility to HIV infection in a dose-dependent manner. Addition of E2 2 h after viral challenge however did not result in reduced infection. In contrast, EE reduced infection in macrophages to a lesser extent than E2 and had no effect on CD4+ T-cell infection. Reduction of HIV-infection induced by E2 in CD4+ T-cells was not due to CCR5 down-regulation, but was an entry-mediated mechanism since infection with VSV-G pseudotyped HIV was not modified by E2. In macrophages, despite the lack of an effect of E2 on CCR5 expression, E2–treatment reduced viral entry 2 h after challenge and increased MIP-1β secretion. These results demonstrate the direct effect of E2 on susceptibility of HIV-target cells to infection and indicate that inhibition of target cell infection involves cell-entry related mechanisms.

Control of murine gammaherpesvirus infection is independent of NK cells.

Numerous studies have shown that NK cells are important in controlling the early stages of infection with alpha‐ or betaherpesviruses. In contrast, little is known about the impact of NK cells on gammaherpesvirus infections. We tested mice with defects in NK cells for their ability to resist murine gammaherpesvirus (MHV‐68) infection. The depletion of NK cells had no effect on the control of the acute or latent stages of the infection. In addition, transgenic mice deficient in NK cells controlled the infection in a comparable manner to wild‐type mice. We also showed that the antiviral CD8 T cell response was unaffected by the presence or absence NK cells. We conclude that NK cells contribute little to the control of MHV‐68 infection, and therefore, NK cells are not essential for controlling all herpesvirus infections.

Pathogen Recognition in the Human Female Reproductive Tract: Expression of Intracellular Cytosolic Sensors NOD1, NOD2, RIG-1, and MDA5 and response to HIV-1 and Neisseria gonorrhea

Expression patterns and regulation of cytosolic pattern recognition receptors (PRR) NOD‐1, NOD‐2, RIG‐1, and MDA5 have not been elucidated in the human female reproductive tract (FRT).

Modulation of hepatocyte growth factor secretion in human female reproductive tract stromal fibroblasts by poly (I:C) and estradiol.

Citation Coleman KD, Ghosh M, Crist SG, Wright JA, Rossoll RM, Wira CR, Fahey JV. Modulation of Hepatocyte Growth Factor secretion in human female reproductive tract stromal fibroblasts by poly (I:C) and estradiol. Am J Reprod Immunol 2012; 67: 44–53

Estradiol regulation of nucleotidases in female reproductive tract epithelial cells and fibroblasts.

The use of topical and oral adenosine derivatives in HIV prevention that need to be maintained in tissues and cells at effective levels to prevent transmission prompted us to ask whether estradiol could influence the regulation of catabolic nucleotidase enzymes in epithelial cells and fibroblasts from the upper and lower female reproductive tract (FRT) as these might affect cellular TFV-DP levels. Epithelial cells and fibroblasts were isolated from endometrium (EM), endocervix (CX) and ectocervix (ECX) tissues from hysterectomy patients, grown to confluence and treated with or without estradiol prior to RNA isolation. The expression of nucleotidase (NT) genes was measurable by RT-PCR in epithelial cells and fibroblasts from all FRT tissues. To determine if sex hormones have the potential to regulate NT, we evaluated NT gene expression and NT biological activity in FRT cells following hormone treatment. Estradiol increased expression of Cytosolic 5′-nucleotidase after 2 or 4 h in endometrial epithelial cells but not epithelial cells or fibroblasts from other sites. In studies using a modified 5′-Nucleotidase biological assay for nucleotidases, estradiol increased NT activity in epithelial cells and fibroblasts from the EM, CX and ECX at 24 and 48 h. In related studies, HUVEC primary cells and a HUVEC cell line were unresponsive to estradiol in terms of nucleotidase expression or biological activity. Our findings of an increase in nucleotidase expression and biological activity induced by estradiol do not directly assess changes in microbicide metabolism. However, they do suggest that when estradiol levels are elevated during the menstrual cycle, FRT epithelial cells and fibroblasts from the EM, CX and ECX have the potential to influence microbicide levels that could enhance protection of HIV-target cells (CD4+T cells, macrophages and dendritic cells) throughout the FRT.

Effect of tenofovir on nucleotidases and cytokines in HIV-1 target cells.

Tenofovir (TFV) has been widely used for pre-exposure prophylaxis of HIV-1 infection with mixed results. While the use of TFV in uninfected individuals for prevention of HIV-1 acquisition is actively being investigated, the possible consequences of TFV exposure for the HIV-target cells and the mucosal microenvironment are unknown. In the current study, we evaluated the effects of TFV treatment on blood-derived CD4+ T cells, monocyte-derived macrophages and dendritic cells (DC). Purified HIV-target cells were treated with different concentrations of TFV (0.001-1.0 mg/ml) for 2 to 24hr. RNA was isolated and RT-PCR was performed to compare the levels of mRNA expression of nucleotidases and pro-inflammatory cytokine genes (MIP3α, IL-8 and TNFα) in the presence or absence of TFV. We found that TFV increases 5’-ecto-nucleotidase (NT5E) and inhibits mitochondrial nucleotidase (NT5M) gene expression and increases 5’ nucleotidase activity in macrophages. We also observed that TFV stimulates the expression and secretion of IL-8 by macrophages, DC, and activated CD4+ T cells and increases the expression and secretion of MIP3α by macrophages. In contrast, TFV had no effect on TNFα secretion from macrophages, DC and CD4+ T cells. Our results demonstrate that TFV alters innate immune responses in HIV-target cells with potential implications for increased inflammation at mucosal surfaces. As new preventive trials are designed, these findings should provide a foundation for understanding the effects of TFV on HIV-target cells in microbicide trials.

T-Cell Responses to the M3 Immune Evasion Protein of Murid Gammaherpesvirus 68 Are Partially Protective and Induced with Lytic Antigen Kinetics

ABSTRACT DNA vaccination with the M3 gene, encoding an immune evasion molecule expressed during both the acute lytic and persistent phases of murid gammaherpesvirus 68 infection, yielded a significantly lower titer of virus in the lung than controls. The protection seen was dependent on T cells, and we mapped an epitope recognized by CD8 T cells. The immune response to this epitope follows the same kinetics as lytic cycle antigens, despite the fact that this gene is expressed in both lytic and persistent stages of infection. This has important implications for our understanding of T-cell responses to putative latency-associated gammaherpesvirus proteins and how vaccination may improve control of these viruses.