Direct Inhibition of T-Cell Responses by the Cryptococcus Capsular Polysaccharide Glucuronoxylomannan

The major virulence factor of the pathogenic fungi Cryptococcus neoformans and C. gattii is the capsule. Glucuronoxylomannan (GXM), the major component of the capsule, is a high-molecular-weight polysaccharide that is shed during cryptococcosis and can persist in patients after successful antifungal therapy. Due to the importance of T cells in the anticryptococcal response, we studied the effect of GXM on the ability of dendritic cells (DCs) to initiate a T-cell response. GXM inhibited the activation of cryptococcal mannoprotein-specific hybridoma T cells and the proliferation of OVA-specific OT-II T cells when murine bone marrow-derived DCs were used as antigen-presenting cells. Inhibition of OT-II T-cell proliferation was observed when either OVA protein or OVA323-339 peptide was used as antigen, indicating GXM did not merely prevent antigen uptake or processing. We found that DCs internalize GXM progressively over time; however, the suppressive effect did not require DCs, as GXM directly inhibited T-cell proliferation induced by anti-CD3 antibody, concanavalin A, or phorbol-12-myristate-13-acetate/ionomycin. Analysis of T-cell viability revealed that the reduced proliferation in the presence of GXM was not the result of increased cell death. GXM isolated from each of the four major cryptococcal serotypes inhibited the proliferation of human peripheral blood mononuclear cells stimulated with tetanus toxoid. Thus, we have defined a new mechanism by which GXM can impart virulence: direct inhibition of T-cell proliferation. In patients with cryptococcosis, this could impair optimal cell-mediated immune responses, thereby contributing to the persistence of cryptococcal infections. SynopsisInfections due to the pathogenic yeast Cryptococcus are a significant cause of morbidity and mortality in persons with impaired T-cell functions, particularly those with AIDS. The major virulence factor of Cryptococcus is its capsule, which is composed primarily of the polysaccharide glucuronoxylomannan (GXM). The capsule not only surrounds the organism but also is shed during cryptococcosis. GXM is taken up by macrophages in vitro and in vivo; however, little is known about the interaction between GXM and dendritic cells, which are the most potent cells capable of activating T cells. Because of the importance of T cells in the anticryptococcal response, the authors investigated the effect of GXM on the ability of dendritic cells to initiate a T-cell response. They found the polysaccharide was internalized by dendritic cells and inhibited antigen-specific T-cell responses. Furthermore, GXM had a direct, inhibitory effect on T-cell proliferation, independent of the effect on dendritic cells. These findings may help explain the persistence of cryptococcal infections and suggest that GXM could be therapeutic in situations where suppression of T-cell responses is desired.National Institutes of Health (R01 AI25780, R01 AI066087, R01 AI37532

Insights into HLA-Restricted T Cell Responses in a Novel Mouse Model of Dengue Virus Infection Point toward New Implications for Vaccine Design

The frequency of dengue virus (DENV) infection has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. To date, a convenient murine system to study human T cell responses to DENV has not been available. Mice transgenic for human leukocyte antigens (HLA) are widely used to model human immune responses and it has been shown that mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR−/− mice. To cover a wide range of HLA phenotypes, we backcrossed IFN-α/βR−/− mice with HLA A*0201, A*0101, A*1101, B*0702 and DRB1*0101 transgenic mice. A DENV proteome-wide screen identified a total of 42 epitopes across all HLA-transgenic IFN-α/βR−/− strains tested. In contrast only 8 of these elicited responses in the corresponding IFN-α/βR+/+ mice. We were able to identify T cell epitopes from 9 out of the 10 DENV proteins. However, the majority of responses were derived from the highly conserved nonstructural proteins NS3 and NS5. The relevance of this model is further demonstrated by the fact that most of the epitopes identified in our murine system are also recognized by PBMC from DENV exposed human donors, and a dominance of HLA B*0702 restricted responses has been detected in both systems. Our results provide new insights into HLA-restricted T cell responses against DENV, and we herein describe a novel murine model, which allows the investigation of T cell-mediated immune mechanisms relevant to vaccine design

Receptor-Mediated Clearance of Cryptococcus neoformans Capsular Polysaccharide In Vivo

Cryptococcus neoformans capsular glucuronoxylomannan (GXM) is shed during cryptococcosis and taken up by macrophages. The roles of the putative GXM receptors CD14, CD18, Toll-like receptor 2 (TLR2), and TLR4 in GXM clearance from serum and deposition in the liver and spleen in receptor-deficient mice were studied. While alterations in the kinetics of GXM redistribution were seen in the mutant mice, none of the receptors was absolutely required for serum clearance or hepatosplenic accumulation

Opsonic Requirements for Dendritic Cell-Mediated Responses to Cryptococcus neoformans

The encapsulated pathogenic yeast Cryptococcus neoformans is poorly recognized by phagocytic cells in the absence of opsonins. Macrophages will bind and internalize complement- or antibody-opsonized C. neoformans; however, less is known about the role of opsonins in dendritic cell (DC)-mediated recognition of the organism. Thus, we studied the opsonic requirements for binding to C. neoformans by cultured human monocyte-derived and murine bone marrow-derived DCs and whether binding leads to antifungal activity and cytokine release. Binding of unopsonized C. neoformans to human and murine DCs was negligible. Opsonization with pooled human serum (PHS) increased binding, while heat treatment of PHS virtually abolished this binding, thus suggesting a role for heat-labile complement components. PHS plus a monoclonal anticapsular antibody, 3C2, had an additive effect on binding for most cryptococcal strains. Human and murine DCs exhibited pronounced anticryptococcal activity in the presence of the antibody at early (2-h) and late (24-h) time points; however, PHS opsonization did not supplement this anticryptococcal activity. Antifungal activity against C. neoformans opsonized in PHS and/or antibody was partially reduced in the presence of inhibitors of the respiratory burst response. Human, but not murine, DCs released modest amounts of tumor necrosis factor alpha when stimulated with C. neoformans opsonized in PHS and/or antibody. However, opsonized C. neoformans failed to stimulate detectable release of interleukin 10 (IL-10) or IL-12p70 from either DC population. Thus, human and murine DCs show maximal binding to and antifungal activity against C. neoformans via a process highly dependent on opsonization

GXM from Four Cryptococcal Serotypes Inhibits Proliferation of Human PBMCs

<p>PBMCs were activated with tetanus toxoid (TT) in the presence or absence of GXM (100, 300, or 1,000 μg/ml) isolated from serotype A strain Cn6, serotype A strain H99, serotype B strain R265, serotype C strain Cn18, or serotype D strain B3501. Proliferation was measured by [³H]thymidine incorporation 6 d later. Data are expressed as the percent proliferation compared with TT alone (−) (set at 100%) and are expressed as the mean ± SEM of four donors (or two donors for strain Cn6 GXM) assayed in triplicate. The mean CPM for TT in the absence of GXM was 63,550. *<i>p</i> ≤ 0.005; **<i>p</i> < 0.0001.</p

GXM Inhibits the Activation of Cryptococcal MP-Specific T Cells

<p>BMDCs were pretreated with varying concentrations of GXM (30, 100, or 300 μg/ml) before the sequential addition of cryptococcal MP and MP-specific hybridoma T cells. Following 24-h incubation, IL-2 levels in the supernatants, a measure of T-cell activation, were measured by bioassay. The data are expressed as percent of IL-2 produced compared with MP alone (set at 100%) and are the average ± SEM of three to five independent experiments done in triplicate. Mean IL-2 in the MP-alone group was 68 U/ml. *<i>p</i> < 0.001.</p

Effect of GXM on T-Cell Proliferation Induced by Anti-CD3, Con A, or PMA/Ionomycin

<p>Purified CD4⁺ murine T cells were activated with plate-bound anti-CD3ɛ antibody, Con A, or PMA/ionomycin, in the presence or absence of GXM (100, 300, or 1,000 μg/ml). The data are expressed as the percent proliferation compared with the stimuli alone (−) (set at 100%) ±SEM of two to five independent experiments done in triplicate. The mean CPM for anti-CD3, Con A, and PMA/ionomycin were 224,440, 41,820, and 118,875, respectively. *<i>p</i> < 0.01.</p

GXM Inhibits the Proliferation of OVA-Specific T Cells

<p>BMDCs were pretreated with GXM (30, 100, or 300 μg/ml) before the addition of egg white or OVA_323–339 peptide. Purified CD4⁺ T cells from OT-II mice were then added and proliferation was measured by [³H]thymidine incorporation. The data are expressed as the percent proliferation compared with antigen alone (−) (set at 100%) ±SEM of three independent experiments done in triplicate. Mean CPM for egg white and OVA_323–339 peptide were 116,830 and 220,560, respectively. *<i>p</i> < 0.01; **<i>p</i> ≤ 0.001.</p

Effect of GXM on T-Cell Viability

<p>Purified CD4⁺ T cells from OT-II mice were activated with anti-CD3ɛ antibody or BMDC plus egg white. Cells were left untreated (−) or treated with GXM (300 μg/ml) or staurosporine (STS). At 48 h, T cells were stained with Annexin V/FITC and PI and analyzed by flow cytometry. Annexin−PI− cells are defined as live, Annexin+PI− as early apoptotic, and Annexin+PI+ as late apoptotic or dead. Data are expressed as mean ± SEM of one experiment (representative of three) done in duplicate (except STS).</p