Tammy Rickabaugh

Homeostasis of the Naive CD4+ T Cell Compartment during Aging

Despite thymic involution, the number of naive CD4+ T cells diminishes slowly during aging, suggesting considerable peripheral homeostatic expansion of these cells. To investigate the mechanisms behind, and consequences of, naive CD4+ T cell homeostasis, we evaluated the age-dependent dynamics of the naive CD4+ T cell subsets CD45RA+CD31+ and CD45RA+CD31−. Using both a cross-sectional and longitudinal study design, we measured the relative proportion of both subsets in individuals ranging from 22 to 73 years of age and quantified TCR excision circle content within those subsets as an indicator of proliferative history. Our findings demonstrate that waning thymic output results in a decrease in CD45RA+CD31+ naive CD4+ T cells over time, although we noted considerable individual variability in the kinetics of this change. In contrast, there was no significant decline in the CD45RA+CD31− naive CD4+ T cell subset due to extensive peripheral proliferation. Our longitudinal data are the first to demonstrate that the CD45RA+CD31+CD4+ subset also undergoes some in vivo proliferation without immediate loss of CD31, resulting in an accumulation of CD45RA+CD31+ proliferative offspring. Aging was associated with telomere shortening within both subsets, raising the possibility that accumulation of proliferative offspring contributes to senescence of the naive CD4+ T cell compartment in the elderly. In contrast, we observed retention of clonal TCR diversity despite peripheral expansion, although this analysis did not include individuals over 65 years of age. Our results provide insight into naive CD4+ T cell homeostasis during aging that can be used to better understand the mechanisms that may contribute to immunosenescence within this compartment.

Transcription Program of Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (MHV-68 [also referred to as γHV68]) is phylogenetically related to Kaposis sarcoma-associated herpesvirus (KSHV [also referred to as HHV-8]) and Epstein-Barr virus (EBV). However, unlike KSHV or EBV, MHV-68 readily infects fibroblast and epithelial cell lines derived from several mammalian species, providing a system to study productive and latent infections as well as reactivation of gammaherpesviruses in vivo and in vitro. To carry out rapid genome-wide analysis of MHV-68 gene expression, we made DNA arrays containing nearly all of the known and predicted open reading frames (ORFs) of the virus. RNA obtained from an MHV-68 latently infected cell line, from cells lytically infected with MHV-68 in culture, and from the lung tissue of infected mice was used to probe the MHV-68 arrays. Using a tightly latent B-cell line (S11E), the MHV-68 latent transcription program was quantitatively described. Using BHK-21 cells and infected mice, we demonstrated that latent genes are transcribed during lytic replication and are relatively independent of de novo protein synthesis. We determined that the transcription profiles at the peak of lytic gene expression are similar in cultured fibroblast and in the lung of infected mice. Finally, the MHV-68 DNA arrays were used to examine the gene expression profile of a recombinant virus that overexpresses replication and transcription activator (RTA), C-RTA/MHV-68, during lytic replication in cell culture. The recombinant virus replicates faster then the parental strain and the DNA arrays revealed that nearly every MHV-68 ORF examined was activated by RTA overexpression. Examination of the gene expression patterns of C-RTA/MHV-68 over a time course led to the finding that the M3 promoter is RTA responsive in the absence of other viral factors.

An epigenetic clock analysis of race/ethnicity, sex, and coronary heart disease.

BackgroundEpigenetic biomarkers of aging (the “epigenetic clock”) have the potential to address puzzling findings surrounding mortality rates and incidence of cardio-metabolic disease such as: (1) women consistently exhibiting lower mortality than men despite having higher levels of morbidity; (2) racial/ethnic groups having different mortality rates even after adjusting for socioeconomic differences; (3) the black/white mortality cross-over effect in late adulthood; and (4) Hispanics in the United States having a longer life expectancy than Caucasians despite having a higher burden of traditional cardio-metabolic risk factors.ResultsWe analyzed blood, saliva, and brain samples from seven different racial/ethnic groups. We assessed the intrinsic epigenetic age acceleration of blood (independent of blood cell counts) and the extrinsic epigenetic aging rates of blood (dependent on blood cell counts and tracks the age of the immune system). In blood, Hispanics and Tsimane Amerindians have lower intrinsic but higher extrinsic epigenetic aging rates than Caucasians. African-Americans have lower extrinsic epigenetic aging rates than Caucasians and Hispanics but no differences were found for the intrinsic measure. Men have higher epigenetic aging rates than women in blood, saliva, and brain tissue.ConclusionsEpigenetic aging rates are significantly associated with sex, race/ethnicity, and to a lesser extent with CHD risk factors, but not with incident CHD outcomes. These results may help elucidate lower than expected mortality rates observed in Hispanics, older African-Americans, and women.

Function of Rta Is Essential for Lytic Replication of Murine Gammaherpesvirus 68

ABSTRACT Rta, encoded primarily by open reading frame 50, is well conserved among gammaherpesviruses. It has been shown that the Rta proteins of Epstein Barr virus (EBV), Kaposis sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV-68; also referred to as γHV68) play an important role in viral reactivation from latency. However, the role of Rta during productive de novo infection has not been characterized in gammaherpesviruses. Since there are cell lines that can support efficient productive de novo infection by MHV-68 but not EBV or KSHV, we examined whether MHV-68 Rta plays a role in initiating viral lytic replication in productively infected cells. Rta, functioning as a transcriptional activator, can activate the viral promoter of early lytic genes. The amino acid sequence alignments of the Rta homologues suggest that the organizations of their functional domains are similar, with the DNA binding and dimerization domains at the N terminus and the trans-activation domain at the C terminus. We constructed two mutants of MHV-68 Rta, Rd1 and Rd2, with deletions of 112 and 243 amino acids from the C terminus, respectively. Rd1 and Rd2 could no longer trans-activate the promoter of MHV-68 gene 57, consistent with the deletions of theirtrans-activation domains at the C terminus. Furthermore, Rd1 and Rd2 were able to function as dominant-negative mutants, inhibiting trans-activation of wild-type Rta. To study whether Rd1 and Rd2 blocked viral lytic replication, purified virion DNA was cotransfected with Rd1 or Rd2 into fibroblasts. Expression of viral lytic proteins was greatly suppressed, and the yield of infectious viruses was reduced up to 104-fold. Stable cell lines constitutively expressing Rd2 were established and infected with MHV-68. Transcription of the immediate-early gene, rta, and the early gene, tk, of the virus was reduced in these cell lines. The presence of Rd2 also led to attenuation of viral lytic protein expression and virion production. The ability of Rta dominant-negative mutants to inhibit productive infection suggests that the trans-activation function of Rta is essential for MHV-68 lytic replication. We propose that a single viral protein, Rta, governs the initiation of MHV-68 lytic replication during both reactivation and productive de novo infection.

The dual impact of HIV-1 infection and aging on naive CD4 T-cells: additive and distinct patterns of impairment.

HIV-1-infected adults over the age of 50 years progress to AIDS more rapidly than adults in their twenties or thirties. In addition, HIV-1-infected individuals receiving antiretroviral therapy (ART) present with clinical diseases, such as various cancers and liver disease, more commonly seen in older uninfected adults. These observations suggest that HIV-1 infection in older persons can have detrimental immunological effects that are not completely reversed by ART. As naïve T-cells are critically important in responses to neoantigens, we first analyzed two subsets (CD45RA+CD31+ and CD45RA+CD31-) within the naïve CD4+ T-cell compartment in young (20–32 years old) and older (39–58 years old), ART-naïve, HIV-1 seropositive individuals within 1–3 years of infection and in age-matched seronegative controls. HIV-1 infection in the young cohort was associated with lower absolute numbers of, and shorter telomere lengths within, both CD45RA+CD31+CD4+ and CD45RA+CD31-CD4+ T-cell subsets in comparison to age-matched seronegative controls, changes that resembled seronegative individuals who were decades older. Longitudinal analysis provided evidence of thymic emigration and reconstitution of CD45RA+CD31+CD4+ T-cells two years post-ART, but minimal reconstitution of the CD45RA+CD31-CD4+ subset, which could impair de novo immune responses. For both ART-naïve and ART-treated HIV-1-infected adults, a renewable pool of thymic emigrants is necessary to maintain CD4+ T-cell homeostasis. Overall, these results offer a partial explanation both for the faster disease progression of older adults and the observation that viral responders to ART present with clinical diseases associated with older adults.

Acceleration of Age-Associated Methylation Patterns in HIV-1-Infected Adults

Patients with treated HIV-1-infection experience earlier occurrence of aging-associated diseases, raising speculation that HIV-1-infection, or antiretroviral treatment, may accelerate aging. We recently described an age-related co-methylation module comprised of hundreds of CpGs; however, it is unknown whether aging and HIV-1-infection exert negative health effects through similar, or disparate, mechanisms. We investigated whether HIV-1-infection would induce age-associated methylation changes. We evaluated DNA methylation levels at >450,000 CpG sites in peripheral blood mononuclear cells (PBMC) of young (20-35) and older (36-56) adults in two separate groups of participants. Each age group for each data set consisted of 12 HIV-1-infected and 12 age-matched HIV-1-uninfected samples for a total of 96 samples. The effects of age and HIV-1 infection on methylation at each CpG revealed a strong correlation of 0.49, p<1 x10-200 and 0.47, p<1x10-200. Weighted gene correlation network analysis (WGCNA) identified 17 co-methylation modules; module 3 (ME3) was significantly correlated with age (cor=0.70) and HIV-1 status (cor=0.31). Older HIV-1+ individuals had a greater number of hypermethylated CpGs across ME3 (p=0.015). In a multivariate model, ME3 was significantly associated with age and HIV status (Data set 1: βage= 0.007088, p=2.08 x 10-9; βHIV= 0.099574, p=0.0011; Data set 2: βage= 0.008762, p=1.27x 10-5; βHIV= 0.128649, p= 0.0001). Using this model, we estimate that HIV-1 infection accelerates age-related methylation by approximately 13.7 years in data set 1 and 14.7 years in data set 2. The genes related to CpGs in ME3 are enriched for polycomb group target genes known to be involved in cell renewal and aging. The overlap between ME3 and an aging methylation module found in solid tissues is also highly significant (Fisher-exact p=5.6 x 10-6, odds ratio=1.91). These data demonstrate that HIV-1 infection is associated with methylation patterns that are similar to age-associated patterns and suggest that general aging and HIV-1 related aging work through some common cellular and molecular mechanisms. These results are an important first step for finding potential therapeutic targets and novel clinical approaches to mitigate the detrimental effects of both HIV-1-infection and aging.

Generation of a Latency-Deficient Gammaherpesvirus That Is Protective against Secondary Infection

ABSTRACT Kaposis sarcoma-associated herpesvirus and murine gammaherpesvirus-68 (MHV-68) establish latent infections and are associated with various types of malignancies. They are members of the gamma-2 herpesvirus subfamily and encode a replication and transcriptional activator, RTA, which is necessary and sufficient to disrupt latency and initiate the viral lytic cycle in vitro. We have constructed a recombinant MHV-68 virus that overexpresses RTA. This virus has faster replication kinetics in vitro and in vivo, is deficient in establishing latency, exhibits a reduction in the development of a mononucleosis-like disease in mice, and can protect mice against challenge by wild-type MHV-68. The present study, by using MHV-68 as an in vivo model system, demonstrated that RTA plays a critical role in the control of viral latency and suggests that latency is a determinant of viral pathogenesis in vivo.

Induction of Protective Immunity against Murine Gammaherpesvirus 68 Infection in the Absence of Viral Latency

ABSTRACT Human gammaherpesviruses, Epstein-Barr virus, and human herpesvirus 8/Kaposis sarcoma-associated herpesvirus are important pathogens associated with diseases, including lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) is used as an experimental model system to study the host immune control of infection and explore novel vaccine strategies based on latency-deficient live viruses. We studied the properties and the potential of a recombinant MHV-68 (AC-RTA) in which the genes required for persistent infection were replaced by a constitutively expressed viral transcription activator, RTA, which dictates the virus to lytic replication. After intranasal infection of mice, replication of AC-RTA in the lung was attenuated, and no AC-RTA virus or viral DNA was detected in the isolated splenocytes, indicating a lack of latency in the spleen. Infection of the AC-RTA virus elicited both cellular immune responses and virus-specific IgG at a level comparable to that elicited by infection of the wild-type virus. Importantly, vaccination of AC-RTA was able to protect mice against subsequent challenge by the wild-type MHV-68. AC-RTA provides a vaccine strategy for preventing infection of human gammaherpesviruses. Furthermore, our results suggest that immunity to the major latent antigens is not required for protection.

Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 RTA Reactivates Murine Gammaherpesvirus 68 from Latency

ABSTRACT Murine gammaherpesvirus 68 (MHV-68), Kaposis sarcoma-associated herpesvirus (HHV-8), and Epstein-Barr virus (EBV) are all members of the gammaherpesvirus family, characterized by their ability to establish latency in lymphocytes. The RTA protein, conserved in all gammaherpesviruses, is known to play a critical role in reactivation from latency. Here we report that HHV-8 RTA, not EBV RTA, was able to induce MHV-68 lytic viral proteins and DNA replication and processing and produce viable MHV-68 virions from latently infected cells at levels similar to those for MHV-68 RTA. HHV-8 RTA was also able to activate two MHV-68 lytic promoters, whereas EBV RTA was not. In order to define the domains of RTA responsible for their functional differences in viral promoter activation and initiation of the MHV-68 lytic cycle, chimeric RTA proteins were constructed by exchanging the N-terminal and C-terminal domains of the RTA proteins. Our data suggest that the species specificity of MHV-68 RTA resides in the N-terminal DNA binding domain.

Response to Comment on “Homeostasis of the Naive CD4+ T Cell Compartment during Aging”

Despite thymic involution, the number of naive CD4(+) T cells diminishes slowly during aging, suggesting considerable peripheral homeostatic expansion of these cells. To investigate the mechanisms behind, and consequences of, naive CD4+ T cell homeostasis, we evaluated the age-dependent dynamics of the naive CD4+ T cell subsets CD45RA+CD31+ and CD45RA+CD31-. Using both a cross-sectional and longitudinal study design, we measured the relative proportion of both subsets in individuals ranging from 22 to 73 years of age and quantified TCR excision circle content within those subsets as an indicator of proliferative history. Our findings demonstrate that waning thymic output results in a decrease in CD45RA+CD31+ naive CD4+ T cells over time, although we noted considerable individual variability in the kinetics of this change. In contrast, there was no significant decline in the CD45RA+CD31- naive CD4+ T cell subset due to extensive peripheral proliferation. Our longitudinal data are the first to demonstrate that the CD45RA+CD31+CD4+ subset also undergoes some in vivo proliferation without immediate loss of CD31, resulting in an accumulation of CD45RA+CD31+ proliferative offspring. Aging was associated with telomere shortening within both subsets, raising the possibility that accumulation of proliferative offspring contributes to senescence of the naive CD4+ T cell compartment in the elderly. In contrast, we observed retention of clonal TCR diversity despite peripheral expansion, although this analysis did not include individuals over 65 years of age. Our results provide insight into naive CD4+ T cell homeostasis during aging that can be used to better understand the mechanisms that may contribute to immunosenescence within this compartment.