Correction: Early Loss of Splenic Tfh Cells in SIV-Infected Rhesus Macaques.

[This corrects the article DOI: 10.1371/journal.ppat.1005287.].

Early Loss of Splenic Tfh Cells in SIV-Infected Rhesus Macaques.

Follicular T helper cells (Tfh), a subset of CD4 T lymphocytes, provide crucial help to B cells in the production of antigen-specific antibodies. Although several studies have analyzed the dynamics of Tfh cells in peripheral blood and lymph nodes (LNs) during Aids, none has yet addressed the impact of SIV infection on the dynamics of Tfh cells in the spleen, the primary organ of B cell activation. We show here a significant decrease in splenic Tfh cells in SIVmac251-infected rhesus macaques (RMs) during the acute phase of infection, which persists thereafter. This profound loss is associated with lack of sustained expression of the Tfh-defining transcription factors, Bcl-6 and c-Maf but with higher expression of the repressors KLF2 and Foxo1. In this context of Tfh abortive differentiation and loss, we found decreased percentages of memory B cell subsets and lower titers of SIV-specific IgG. We further demonstrate a drastic remodeling of the lymphoid architecture of the spleen and LNs, which disrupts the crucial cell-cell interactions necessary to maintain memory B cells and Tfh cells. Finally, our data demonstrated the early infection of Tfh cells. Paradoxically, the frequencies of SIV DNA were higher in splenic Tfh cells of RMs progressing more slowly suggesting sanctuaries for SIV in the spleen. Our findings provide important information regarding the impact of HIV/SIV infection on Tfh cells, and provide new clues for future vaccine strategies.

A novel life cycle modeling system for Ebola virus shows a genome length-dependent role of VP24 in virus infectivity.

UNLABELLED: Work with infectious Ebola viruses is restricted to biosafety level 4 (BSL4) laboratories, presenting a significant barrier for studying these viruses. Life cycle modeling systems, including minigenome systems and transcription- and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus life cycle under BSL2 conditions; however, all current systems model only certain aspects of the virus life cycle, rely on plasmid-based viral protein expression, and have been used to model only single infectious cycles. We have developed a novel life cycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP1,2. This system is ideally suited for studying morphogenesis, budding, and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ∼ 500-fold higher than that in previous systems. Using this system for functional studies of VP24, we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings, we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription. IMPORTANCE: Ebola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently being available, and must be studied in maximum-containment laboratories. Only a few of these laboratories exist worldwide, limiting our ability to study Ebola viruses and develop countermeasures. Here we report the development of a novel reverse genetics-based system that allows the study of Ebola viruses without maximum-containment laboratories. We used this system to investigate the Ebola virus protein VP24, showing that, contrary to previous reports, it only modestly inhibits virus genome replication and transcription but is important for packaging of genomes into virus particles, which constitutes a previously unknown function of VP24 and a potential antiviral target. We further propose a comprehensive model for the function of VP24 in nucleocapsid assembly. Importantly, on the basis of this approach, it should easily be possible to develop similar experimental systems for other viruses that are currently restricted to maximum-containment laboratories.

Despite early antiretroviral therapy effector memory and follicular helper CD4 T cells are major reservoirs in visceral lymphoid tissues of SIV-infected macaques.

Whereas antiretroviral therapy (ART) suppresses viral replication, ART discontinuation results in viral rebound, indicating the presence of viral reservoirs (VRs) established within lymphoid tissues. Herein, by sorting CD4 T-cell subsets from the spleen, mesenteric and peripheral lymph nodes (LNs) of SIVmac251-infected rhesus macaques (RMs), we demonstrate that effector memory (TEM) and follicular helper (TFH) CD4+ T cells harbor the highest frequency of viral DNA and RNA, as well of early R-U5 transcripts in ART-naïve RMs. Furthermore, our results highlight that these two CD4 T cells subsets harbor viral DNA and early R-U5 transcripts in the spleen and mesenteric LNs (but not in peripheral LN) of RMs treated with ART at day 4 post infection suggesting that these two anatomical sites are important for viral persistence. Finally, after ART interruption, we demonstrate the rapid and, compared to peripheral LNs, earlier seeding of SIV in spleen and mesenteric LNs, thereby emphasizing the importance of these two anatomical sites for viral replication dynamics. Altogether our results advance understanding of early viral seeding in which visceral lymphoid tissues are crucial in maintaining TEM and TFH VRs.

Mucosal T follicular helper cells in SIV-infected rhesus macaques: contributing role of IL-27.

Mesenteric lymph nodes (MLNs), that drain the large and small intestine, are critical sites for the induction of oral tolerance. Although depletion of CD4 T cells in the intestinal lamina propria is a hallmark of HIV infection, CD4 T cell dynamics in MLNs is less known due to the lack of accessibility to these LNs. We demonstrate the early loss of memory CD4 T cells, including T follicular helper cells (Tfh) and a remodeling of MLN architecture in SIV-infected rhesus macaques (RMs). Along with the loss of Tfh cells, we observe the loss of memory B cells and of germinal center B cells. Tfh cells display a Th1 profile with increased levels of the transcription factors that negatively impact on Tfh differentiation and of Stat5 phosphorylation. MLNs of SIV-infected RMs display lower mRNA transcripts encoding for IL-12, IL-23, and IL-35, whereas those coding for IL-27 are not impaired in MLNs. In vitro, IL-27 negatively impacts on Tfh cells and recapitulates the profile observed in SIV-infected RMs. Therefore, early defects of memory CD4 T cells, as well of Tfh cells in MLNs, which play a central role in regulating the mucosal immune response, may have major implications for Aids.

CD4 T Follicular Helper Cells and HIV Infection: Friends or Enemies?

Follicular T helper (Tfh) cells, a subset of CD4 T lymphocytes, are essential for memory B cell activation, survival, and differentiation and assist B cells in the production of antigen-specific antibodies. Work performed in recent years pointed out the importance of Tfh cells in the context of HIV and SIV infections. The importance of tissue distribution of Tfh is also an important point since their frequency differs between peripheral blood and lymph nodes compared to the spleen, the primary organ for B cell activation, and differentiation. Our recent observations indicated an early and profound loss of splenic Tfh cells. The role of transcriptional activator and repressor factors that control Tfh differentiation is also discussed in the context of HIV/SIV infection. Because Tfh cells are important for B cell differentiation and antibody production, accelerating the Tfh responses early during HIV/SIV infection could be promising as novel immunotherapeutic approach or alternative vaccine strategies. However, because Tfh cells are infected during the HIV/SIV infection and represent a reservoir, this may interfere with HIV vaccine strategy. Thus, Tfh represent the good and bad guys during HIV infection.

Maturation and Mip-1ß Production of Cytomegalovirus-Specific T Cell Responses in Tanzanian Children, Adolescents and Adults: Impact by HIV and Mycobacterium tuberculosis Co-Infections.

It is well accepted that aging and HIV infection are associated with quantitative and functional changes of CMV-specific T cell responses. We studied here the expression of Mip-1ß and the T cell maturation marker CD27 within CMVpp65-specific CD4(+) and CD8(+) T cells in relation to age, HIV and active Tuberculosis (TB) co-infection in a cohort of Tanzanian volunteers (≤ 16 years of age, n = 108 and ≥ 18 years, n = 79). Independent of HIV co-infection, IFNγ(+) CMVpp65-specific CD4(+) T cell frequencies increased with age. In adults, HIV co-infection further increased the frequencies of these cells. A high capacity for Mip-1ß production together with a CD27(low) phenotype was characteristic for these cells in children and adults. Interestingly, in addition to HIV co-infection active TB disease was linked to further down regulation of CD27 and increased capacity of Mip-1ß production in CMVpp65-specific CD4+ T cells. These phenotypic and functional changes of CMVpp65-specific CD4 T cells observed during HIV infection and active TB could be associated with increased CMV reactivation rates.

Assessment of the novel T-cell activation marker-tuberculosis assay for diagnosis of active tuberculosis in children: a prospective proof-of-concept study.

BACKGROUND: The diagnosis of paediatric tuberculosis is complicated by non-specific symptoms, difficult specimen collection, and the paucibacillary nature of the disease. We assessed the accuracy of a novel immunodiagnostic T-cell activation marker-tuberculosis (TAM-TB) assay in a proof-of-concept study to identify children with active tuberculosis. METHODS: Children with symptoms that suggested tuberculosis were prospectively recruited at the NIMR-Mbeya Medical Research Center in Mbeya, and the Ifakara Health Institute in Bagamoyo, Tanzania, between May 10, 2011, and Sept 4, 2012. Sputum and peripheral blood mononuclear cells were obtained for Mycobacterium tuberculosis culture and performance assessment of the TAM-TB assay. The children were assigned to standardised clinical case classifications based on microbiological and clinical findings. FINDINGS: Among 290 children screened, we selected a subgroup of 130 to ensure testing of at least 20 with culture-confirmed tuberculosis. 17 of 130 children were excluded because of inconclusive TAM-TB assay results. The TAM-TB assay enabled detection of 15 of 18 culture-confirmed cases (sensitivity 83·3%, 95% CI 58·6-96·4). Specificity was 96·8% (95% CI 89·0-99·6) in the cases that were classified as not tuberculosis (n=63), with little effect from latent tuberculosis infection. The TAM-TB assay identified five additional patients with highly probable or probable tuberculosis, in whom M tuberculosis was not isolated. The median time to diagnosis was 19·5 days (IQR 14-45) for culture. INTERPRETATION: The sputum-independent TAM-TB assay is a rapid and accurate blood test that has the potential to improve the diagnosis of active tuberculosis in children. FUNDING: European and Developing Countries Clinical Trials Partnership, German Federal Ministry of Education and Research, and Swiss National Science Foundation.