Angela Meier

Sex differences in the Toll-like receptor–mediated response of plasmacytoid dendritic cells to HIV-1

Manifestations of viral infections can differ between women and men, and marked sex differences have been described in the course of HIV-1 disease. HIV-1–infected women tend to have lower viral loads early in HIV-1 infection but progress faster to AIDS for a given viral load than men. Here we show substantial sex differences in the response of plasmacytoid dendritic cells (pDCs) to HIV-1. pDCs derived from women produce markedly more interferon-α (IFN-α) in response to HIV-1–encoded Toll-like receptor 7 (TLR7) ligands than pDCs derived from men, resulting in stronger secondary activation of CD8+ T cells. In line with these in vitro studies, treatment-naive women chronically infected with HIV-1 had considerably higher levels of CD8+ T cell activation than men after adjusting for viral load. These data show that sex differences in TLR-mediated activation of pDCs may account for higher immune activation in women compared to men at a given HIV-1 viral load and provide a mechanism by which the same level of viral replication might result in faster HIV-1 disease progression in women compared to men. Modulation of the TLR7 pathway in pDCs may therefore represent a new approach to reduce HIV-1–associated pathology.

HLA Alleles Associated with Delayed Progression to AIDS Contribute Strongly to the Initial CD8+ T Cell Response against HIV-1

Background Very little is known about the immunodominance patterns of HIV-1-specific T cell responses during primary HIV-1 infection and the reasons for human lymphocyte antigen (HLA) modulation of disease progression. Methods and Findings In a cohort of 104 individuals with primary HIV-1 infection, we demonstrate that a subset of CD8+ T cell epitopes within HIV-1 are consistently targeted early after infection, while other epitopes subsequently targeted through the same HLA class I alleles are rarely recognized. Certain HLA alleles consistently contributed more than others to the total virus-specific CD8+ T cell response during primary infection, and also reduced the absolute magnitude of responses restricted by other alleles if coexpressed in the same individual, consistent with immunodomination. Furthermore, individual HLA class I alleles that have been associated with slower HIV-1 disease progression contributed strongly to the total HIV-1-specific CD8+ T cell response during primary infection. Conclusions These data demonstrate consistent immunodominance patterns of HIV-1-specific CD8+ T cell responses during primary infection and provide a mechanistic explanation for the protective effect of specific HLA class I alleles on HIV-1 disease progression.

Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus -induced activation of murine macrophages

Aspergillus fumigatius is a ubiquitous saprophytic fungus that has become the most prevalent airborne fungal pathogen for immunocompromised patients during the last two decades. In this report we have analysed how macrophages recognize this microorganism. Using transfected human HEK 293 cells we demonstrate that NF‐κB‐dependent promoter activation triggered by A. fumigatus is mediated by Toll‐like receptors TLR2 and TLR4, whereas no activation was observed in cells overexpressing other distinct TLR proteins (TLR1, TLR3, TLR5–10). Using macrophages derived from mice lacking TLR2 expression, expressing defective TLR4 or both we found that A. fumigatus conidia and hyphae induce NF‐κB translocation, release of pro‐inflammatory molecules, like TNFα, and the chemoattractant MIP‐2 in a TLR2‐ and TLR4‐dependent manner. Recognition of A. niger and A. fumigatus , was similar in terms of the parameters analysed, suggesting that pathogenic and non‐pathogenic aspergilli are sensed by macrophages in a similar fashion. Finally, we found that recruitment of neutrophils is severely impaired in mice lacking both functional TLR2 and TLR4, but is less impaired in single TLR2‐ or TLR4‐deficient mice, providing evidence that both receptors are required for an optimal immune response to Aspergillus in vivo.

MyD88-Dependent Immune Activation Mediated by Human Immunodeficiency Virus Type 1-Encoded Toll-Like Receptor Ligands

ABSTRACT Immune activation is a major characteristic of human immunodeficiency virus type 1 (HIV-1) infection and a strong prognostic factor for HIV-1 disease progression. The underlying mechanisms leading to immune activation in viremic HIV-1 infection, however, are not fully understood. Here we show that, following the initiation of highly active antiretroviral therapy, the immediate decline of immune activation is closely associated with the reduction of HIV-1 viremia, which suggests a direct contribution of HIV-1 itself to immune activation. To propose a mechanism, we demonstrate that the single-stranded RNA of HIV-1 encodes multiple uridine-rich Toll-like receptor 7/8 (TLR7/8) ligands that induce strong MyD88-dependent plasmacytoid dendritic cell and monocyte activation, as well as accessory cell-dependent T-cell activation. HIV-1-encoded TLR ligands may, therefore, directly contribute to the immune activation observed during viremic HIV-1 infection. These data provide an initial rationale for inhibiting the TLR pathway to directly reduce the chronic immune activation induced by HIV-1 and the associated immune pathogenesis.

Antigen Load and Viral Sequence Diversification Determine the Functional Profile of HIV-1–Specific CD8+ T Cells

Background Virus-specific CD8+ T lymphocytes play a key role in the initial reduction of peak viremia during acute viral infections, but display signs of increasing dysfunction and exhaustion under conditions of chronic antigen persistence. It has been suggested that virus-specific CD8+ T cells with a “polyfunctional” profile, defined by the capacity to secrete multiple cytokines or chemokines, are most competent in controlling viral replication in chronic HIV-1 infection. We used HIV-1 infection as a model of chronic persistent viral infection to investigate the process of exhaustion and dysfunction of virus-specific CD8+ T cell responses on the single-epitope level over time, starting in primary HIV-1 infection. Methods and Findings We longitudinally analyzed the polyfunctional epitope-specific CD8+ T cell responses of 18 patients during primary HIV-1 infection before and after therapy initiation or sequence variation in the targeted epitope. Epitope-specific CD8+ T cells responded with multiple effector functions to antigenic stimulation during primary HIV-1 infection, but lost their polyfunctional capacity in response to antigen and up-regulated programmed death 1 (PD-1) expression with persistent viremic infection. This exhausted phenotype significantly decreased upon removal of stimulation by antigen, either in response to antiretroviral therapy or by reduction of epitope-specific antigen load in the presence of ongoing viral replication, as a consequence of in vivo selection of cytotoxic T lymphocyte escape mutations in the respective epitopes. Monofunctionality increased in CD8+ T cell responses directed against conserved epitopes from 49% (95% confidence interval 27%–72%) to 76% (56%–95%) (standard deviation [SD] of the effect size 0.71), while monofunctionality remained stable or slightly decreased for responses directed against escaped epitopes from 61% (47%–75%) to 56% (42%–70%) (SD of the effect size 0.18) (p < 0.05). Conclusion These data suggest that persistence of antigen can be the cause, rather than the consequence, of the functional impairment of virus-specific T cell responses observed during chronic HIV-1 infection, and underscore the importance of evaluating autologous viral sequences in studies aimed at investigating the relationship between virus-specific immunity and associated pathogenesis.

Evolution of Innate and Adaptive Effector Cell Functions during Acute HIV-1 Infection

Early events during acute human immunodeficiency virus type 1 (HIV-1) infection are critical in determining the course of disease progression. Cells of the innate and adaptive immune responses are involved in this acute response to infection; however, little is known about the coevolution of innate and adaptive effector cell populations during the initial phase of HIV-1 infection. Here, we have characterized the development of innate natural killer (NK) cell and adaptive HIV-1-specific CD8(+) T cell function during acute HIV-1 infection. Although NK cell populations were significantly expanded during acute infection before HIV-1 seroconversion, HIV-1-specific CD8(+) T cell responses were absent or weak and were inversely correlated with the level of NK cell activity. NK cell activity was directly correlated with the level of viral replication during acute HIV-1 infection and declined rapidly in subjects who initiated highly active antiretroviral therapy, whereas NK cell activity remained elevated in subjects who did not initiate therapy. Yet, reexposure to HIV-1 antigen during treatment discontinuation in chronic infection resulted in a synchronous increase in NK and CD8(+) T cell activity. Overall, these data demonstrate that expansion of the NK cell population precedes the development of adaptive HIV-1-specific CD8(+) T cells during acute infection but that both effector cell subsets respond with similar kinetics during chronic HIV-1 infection.

Recognition of a Defined Region within p24 Gag by CD8+ T Cells during Primary Human Immunodeficiency Virus Type 1 Infection in Individuals Expressing Protective HLA Class I Alleles

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-specific immune responses during primary HIV-1 infection appear to play a critical role in determining the ultimate speed of disease progression, but little is known about the specificity of the initial HIV-1-specific CD8+ T-cell responses in individuals expressing protective HLA class I alleles. Here we compared HIV-1-specific T-cell responses between subjects expressing the protective allele HLA-B27 or -B57 and subjects expressing nonprotective HLA alleles using a cohort of over 290 subjects identified during primary HIV-1 infection. CD8+ T cells of individuals expressing HLA-B27 or -B57 targeted a defined region within HIV-1 p24 Gag (amino acids 240 to 272) early in infection, and responses against this region contributed over 35% to the total HIV-1-specific T-cell responses in these individuals. In contrast, this region was rarely recognized in individuals expressing HLA-B35, an HLA allele associated with rapid disease progression, or in subjects expressing neither HLA-B57/B27 nor HLA-B35 (P < 0.0001). The identification of this highly conserved region in p24 Gag targeted in primary infection specifically in individuals expressing HLA class I alleles associated with slower HIV-1 disease progression provides a rationale for vaccine design aimed at inducing responses to this region restricted by other, more common HLA class I alleles.

Single-Stranded RNA Derived from HIV-1 Serves as a Potent Activator of NK Cells

Persistent immune activation is a hallmark of chronic viremic HIV-1 infection. Activation of cells of the innate immune system, such as NK cells, occurs rapidly upon infection, and is sustained throughout the course of the disease. However, the precise underlying mechanism accounting for the persistent HIV-1-induced activation of NK cells is poorly understood. In this study, we assessed the role of uridine-rich ssRNA derived from the HIV-1 long terminal repeat (ssRNA40) on activation of NK cells via TLR7/8. Although dramatic activation of NK cells was observed following stimulation of PBMC with ssRNA40, negligible activation was observed following stimulation of purified NK cells despite their expression of TLR8 mRNA and protein. The functional activation of NK cells by this HIV-1-encoded TLR7/8 ligand could not be reconstituted with exogenous IL-12, IFN-α, or TNF-α, but was critically dependent on the direct contact of NK cells with plasmacytoid dendritic cells or CD14+ monocytes, indicating an important level of NK cell cross-talk and regulation by accessory cells during TLR-mediated activation. Coincubation of monocyte/plasmacytoid dendritic cells, NK cells, and ssRNA40 potentiated NK cell IFN-γ secretion in response to MHC-devoid target cells. Studies using NK cells derived from individuals with chronic HIV-1 infection demonstrated a reduction of NK cell responsiveness following stimulation with TLR ligands in viremic HIV-1 infection. These data demonstrate that HIV-1-derived TLR ligands can contribute to the immune activation of NK cells and may play an important role in HIV-1-associated immunopathogenesis and NK cell dysfunction observed during acute and chronic viremic HIV-1 infection.

Upregulation of Pd-l1 on monocytes and dendritic cells by Hiv-1 derived Tlr ligands

Increased PD-L1 expression has been reported in HIV-1-infected individuals, but the mechanisms leading to PD-L1 upregulation remain to be elucidated. Here we demonstrate that HIV-1-derived Toll-like receptor (TLR)7/8 ligands can induce MyD88-dependent upregulation of PD-L1 on plasmacytoid dendritic cells, myeloidic dendritic cells and monocytes. These data suggest a mechanism through which HIV-1-derived TLR ligands might contribute to the functional impairment of virus-specific PD-1-positive T cells by inducing the upregulation of PD-L1 on antigen-presenting cells.

RAPID EX VIVO ISOLATION AND LONG-TERM CULTURE OF HUMAN TH17 CELLS

T helper (Th) 17 cells are a distinct lineage of CD4+ T cells mediating tissue inflammation through the secretion of IL-17. In addition, it has been shown that the expression of the transcriptional factor RORgammat is responsible for the induction and maintenance of this cell line. Th17 cells are believed to be involved in a variety of autoimmune disorders, but may also play an important role in host defense. Here we describe a novel technique to reproducibly isolate viable Th17 cells based on their IL-17 secreting ability. We confirmed Th17 cell enrichment by quantitative PCR analysis and demonstrate that positively selected cells using this technique express significantly increased mRNA levels of RORgammat, IL-23 receptor and CCR4 when compared to negatively selected cells. Furthermore, we show that purified Th17 cells can be maintained in long-term culture and expand in vitro. In conclusion, this technique will allow for the first time the direct, ex vivo analysis of phenotypic and functional properties of Th17 cells.