Dendritic Cells from Lupus-Prone Mice Are Defective in Repressing Immunoglobulin Secretion

Autoimmunity results from a breakdown in tolerance mechanisms that regulate autoreactive lymphocytes. We recently showed that during innate immune responses, secretion of IL-6 by dendritic cells (DCs) maintained autoreactive B cells in an unresponsive state. In this study, we describe that TLR4-activated DCs from lupus-prone mice are defective in repressing autoantibody secretion, coincident with diminished IL-6 secretion. Reduced secretion of IL-6 by MRL/lpr DCs reflected diminished synthesis and failure to sustain IL-6 mRNA production. This occurred coincident with lack of NF-κB and AP-1 DNA binding and failure to sustain IκBα phosphorylation. Analysis of individual mice showed that some animals partially repressed Ig secretion despite reduced levels of IL-6. This suggests that in addition to IL-6, DCs secrete other soluble factor(s) that regulate autoreactive B cells. Collectively, the data show that MRL/lpr mice are defective in DC/IL-6-mediated tolerance, but that some individuals maintain the ability to repress autoantibody secretion by an alternative mechanism. The Journal of Immunology, 2007, 178: 4803–4810

Vaccination with Ad5 Vectors Expands Ad5-Specific CD8+ T Cells without Altering Memory Phenotype or Functionality

Adenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself. To what degree Ad-specific CD8(+) T cells are restimulated by Ad vector vaccination is unclear, although such knowledge would be important as vector-specific CD8(+) T cell expansion could potentially further limit Ad vaccine efficacy beyond Ad-specific neutralizing antibody alone.Here we addressed this issue by measuring human Adenovirus serotype 5 (Ad5)-specific CD8(+) T cells in recipients of the Merck Ad5 HIV-1 vaccine vector before, during, and after vaccination by multicolor flow cytometry. Ad5-specific CD8(+) T-cells were detectable in 95% of subjects prior to vaccination, and displayed primarily an effector-type functional profile and phenotype. Peripheral blood Ad5-specific CD8(+) T-cell numbers expanded after Ad5-HIV vaccination in all subjects, but differential expansion kinetics were noted in some baseline Ad5-neutralizing antibody (Ad5 nAb) seronegative subjects compared to baseline Ad5 nAb seropositive subjects. However, in neither group did vaccination alter polyfunctionality, mucosal targeting marker expression, or memory phenotype of Ad5-specific CD8(+) T-cells.These data indicate that repeat Ad5-vector administration in humans expands Ad5-specific CD8(+) T-cells without overtly affecting their functional capacity or phenotypic properties. This is a secondary analysis of samples collected during the 016 trial. Results of the Merck 016 trial safety and immunogenicity have been previously published in the journal of clinical infectious diseases [1].ClinicalTrials.gov NCT00849680[http://www.clinicaltrials.gov/show/NCT00849680]

Harnessing activin A adjuvanticity to promote antibody responses to BG505 HIV envelope trimers

T follicular helper (

Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells

Human immunodeficiency virus (HIV) persists indefinitely in individuals with HIV who receive antiretroviral therapy (ART) owing to a reservoir of latently infected cells that contain replication-competent virus1–4. Here, to better understand the mechanisms responsible for latency persistence and reversal, we used the interleukin-15 superagonist N-803 in conjunction with the depletion of CD8+ lymphocytes in ART-treated macaques infected with simian immunodeficiency virus (SIV). Although N-803 alone did not reactivate virus production, its administration after the depletion of CD8+ lymphocytes in conjunction with ART treatment induced robust and persistent reactivation of the virus in vivo. We found viraemia of more than 60 copies per ml in all macaques (n = 14; 100%) and in 41 out of a total of 56 samples (73.2%) that were collected each week after N-803 administration. Notably, concordant results were obtained in ART-treated HIV-infected humanized mice. In addition, we observed that co-culture with CD8+ T cells blocked the in vitro latency-reversing effect of N-803 on primary human CD4+ T cells that were latently infected with HIV. These results advance our understanding of the mechanisms responsible for latency reversal and lentivirus reactivation during ART-suppressed infection

Collapse of Cytolytic Potential in SIV-Specific CD8+ T Cells Following Acute SIV Infection in Rhesus Macaques

<div><p>Poor maintenance of cytotoxic factor expression among HIV-specific CD8+ T cells, in part caused by dysregulated expression of the transcription factor T-bet, is associated with HIV disease progression. However, the precise evolution and context in which CD8+ T cell cytotoxic functions become dysregulated in HIV infection remain unclear. Using the rhesus macaque (RM) SIV infection model, we evaluated the kinetics of SIV-specific CD8+ T cell cytolytic factor expression in peripheral blood, lymph node, spleen, and gut mucosa from early acute infection through chronic infection. We identified rapid acquisition of perforin and granzyme B expression in SIV-specific CD8+ T cells in blood, secondary lymphoid tissues and gut mucosa that collapsed rapidly during the transition to chronic infection. The evolution of this expression profile was linked to low expression of T-bet and occurred independent of epitope specificity, viral escape patterns and tissue origin. Importantly, during acute infection SIV-specific CD8+ T cells that maintained T-bet expression retained the ability to express granzyme B after stimulation, but this relationship was lost in chronic infection. Together, these data demonstrate the loss of cytolytic machinery in SIV-specific CD8+ T cells in blood and at tissue sites of viral reservoir and active replication during the transition from acute to chronic infection. This phenomenon occurs despite persistent high levels of viremia suggesting that an inability to maintain properly regulated cytotoxic T cell responses in all tissue sites enables HIV/SIV to avoid immune clearance, establish persistent viral reservoirs in lymphoid tissues and gut mucosa, and lead ultimately to immunopathogenesis and death.</p></div

Kinetics of T-bet expression in SIV-specific CTL during acute SIV infection.

<p>A) Flow cytometry plots of T-bet expression in TL8 and CM9 tetramer+ SIV-specific CD8+ T cells (TL8 tetramer+ in red, and CM9 tetramer+ in blue) overlayed on total CD8+ T cells (black) in blood, sLN, mLN, and spleen at the indicated time points from representative animals. Frequencies of T-bet+ tetramer+ events are shown. B) The frequencies of T-bet expression among tetramer+ SIV-specific CD8+ T cells in mLN, sLN, blood and spleen at the indicated time points; Linear regression ANOVA for samples collected between 13dpi and 90dpi. Lines connecting data points represent longitudinally collected data from sLN and blood of individual animals. No significant differences “ns”.</p

Evolution of CTL in peripheral blood during acute SIV infection.

<p>A) Experimental design including SIV infection, tissue and blood sample collection (TC), and necropsy (Nec) time points. B) Flow cytometry plots of Tat-TL8 and Gag-CM9 Mamu A*01 tetramer staining in CD8+ T cells at pre-infection, acute, and chronic infection from a representative animal. Frequencies of single positive tetramer+ events are shown. C) The frequency of TL8 and CM9 tetramer+ SIV-specific CD8+ T cells in peripheral blood CD8+ T cells throughout infection. D) Flow cytometry plots of perforin and granzyme B expression in TL8 and CM9 tetramer+ SIV-specific CD8+ T cells (TL8 tetramer+ in red, and CM9 tetramer+ in blue) overlayed on total CD8+ T cells (black) at pre-infection, 13dpi, and 90dpi from representative animals. Frequencies of gated populations are shown of TL8 or CM9 tetramer+ cells. E) The frequency of perforin and granzyme B (Perf+GrzB+) co-expression among TL8 or CM9 tetramer+ SIV-specific CD8+ T cells throughout infection. F) The median fluorescence intensity (MFI) of perforin and granzyme B in Perf+GrzB+, tetramer+ SIV-specific CD8+ T cells throughout infection. Lines connecting data points represent longitudinally collected data from individual animals.</p

Evolution of CTL in blood, SLT and gut mucosa during acute SIV infection.

<p>A) The frequencies of TL8 and CM9 tetramer+ SIV-specific CD8+ T cells among CD8+ T cells in mLN, sLN, spleen, gut mucosa and blood at the indicated time points. B) Flow cytometry plots of perforin and granzyme B expression in TL8 and CM9 tetramer+ SIV-specific CD8+ T cells (TL8 tetramer+ in red, and CM9 tetramer+ in blue) overlayed on total CD8+ T cells (black) in blood, sLN, mLN and spleen at pre- infection, 13dpi, and 90dpi from representative animals. Frequencies of gated populations are shown of TL8 or CM9 tetramer+ cells. C) The frequencies of Perf+GrzB+ co-expression in TL8 and CM9 tetramer+ SIV-specific CD8+ T cells from blood, sLN, mLN and spleen at the indicated time points throughout infection; Linear regression ANOVA of samples collected between 13dpi and 90dpi. D) Kinetics of perforin median fluorescence intensity (MFI) in Perf+GrzB+ TL8 and CM9 tetramer+ SIV-specific CD8+ T cells at the indicated time points throughout infection; Linear regression ANOVA of samples collected between 13dpi and 90dpi. E) Perforin MFI in Perf+GrzB+ tetramer+ cells in sLN and blood at 13dpi or 90dpi; Paired, one-tailed Student’s t-test; 13dpi (n = 12 pairs) or 90dpi (n = 3 pairs). Lines connecting data points represent longitudinally collected data from sLN and blood of individual animals. * p≤0.05, ** p<0.01. No significant differences “ns”.</p

Target cell availability, rather than breast milk factors, dictates mother-to-infant transmission of SIV in sooty mangabeys and rhesus macaques.

Mother-to-infant transmission (MTIT) of HIV is a serious global health concern, with over 300,000 children newly infected in 2011. SIV infection of rhesus macaques (RMs) results in similar rates of MTIT to that of HIV in humans. In contrast, SIV infection of sooty mangabeys (SMs) rarely results in MTIT. The mechanisms underlying protection from MTIT in SMs are unknown. In this study we tested the hypotheses that breast milk factors and/or target cell availability dictate the rate of MTIT in RMs (transmitters) and SMs (non-transmitters). We measured viral loads (cell-free and cell-associated), levels of immune mediators, and the ability to inhibit SIV infection in vitro in milk obtained from lactating RMs and SMs. In addition, we assessed the levels of target cells (CD4+CCR5+ T cells) in gastrointestinal and lymphoid tissues, including those relevant to breastfeeding transmission, as well as peripheral blood from uninfected RM and SM infants. We found that frequently-transmitting RMs did not have higher levels of cell-free or cell-associated viral loads in milk compared to rarely-transmitting SMs. Milk from both RMs and SMs moderately inhibited in vitro SIV infection, and presence of the examined immune mediators in these two species did not readily explain the differential rates of transmission. Importantly, we found that the percentage of CD4+CCR5+ T cells was significantly lower in all tissues in infant SMs as compared to infant RMs despite robust levels of CD4+ T cell proliferation in both species. The difference between the frequently-transmitting RMs and rarely-transmitting SMs was most pronounced in CD4+ memory T cells in the spleen, jejunum, and colon as well as in central and effector memory CD4+ T cells in the peripheral blood. We propose that limited availability of SIV target cells in infant SMs represents a key evolutionary adaptation to reduce the risk of MTIT in SIV-infected SMs

Analysis of the In Vivo Turnover of CD4+ T-Cell Subsets in Chronically SIV-Infected Sooty Mangabeys

Aberrant turnover of memory CD4+ T-cells is central to Acquired Immunodeficiency Syndrome (AIDS) progression. Understanding the relationship between the turnover of CD4+ subsets and immunological homeostasis during simian immunodeficiency virus (SIV) infection in natural hosts may provide insight into mechanisms of immune regulation that may serve as models for therapeutic intervention in Human Immunodeficiency Virus (HIV)-infected persons. Sooty mangabeys (SMs) have naturally evolved with SIV to avoid AIDS progression while maintaining healthy peripheral CD4+ T-cell counts and thus represent a model by which therapeutic interventions for AIDS progression might be elucidated. To assess the relationship between the turnover of CD4+ subsets and immunological homeostasis during SIV infection in non-progressive hosts, we treated 6 SIV-uninfected and 9 SIV-infected SMs with 2'-bromo-5'-deoxyuridine (BrdU) for 14 days and longitudinally assessed CD4+ T-cell subset turnover by polychromatic flow cytometry. We observed that, in SIV-infected SMs, turnover of CD4+ T-cell naïve and central, transitional, and effector memory subsets is comparable to that in uninfected animals. Comparable turnover of CD4+ T-cell subsets irrespective of SIV-infection status likely contributes to the lack of aberrant immune activation and disease progression observed after infection in non-progressive hosts