Spatial Separation of HLA-DM/HLA-DR Interactions within MIIC and Phagosome-Induced Immune Escape

SummaryMajor Histocompatibility Complex (MHC) class II molecules, including Human Leukocyte Antigen (HLA)-DR, present peptide fragments from proteins degraded in the endocytic pathway. HLA-DR is targeted to late-endocytic structures named MHC class II-containing Compartments (MIIC), where it interacts with HLA-DM. This chaperone stabilizes HLA-DR during peptide exchange and is critical for successful peptide loading. To follow this process in living cells, we have generated cells containing HLA-DR3/Cyan Fluorescent Protein (CFP), HLA-DM/Yellow Fluorescent Protein (YFP), and invariant chain. HLA-DR/DM interactions were observed by Fluorescence Resonance Energy Transfer (FRET). These interactions were pH insensitive, yet occurred only in internal structures and not at the limiting membrane of MIIC. In a cellular model of infection, phagosomes formed a limiting membrane surrounding internalized Salmonella. HLA-DR and HLA-DM did not interact in Salmonella-induced vacuoles, and HLA-DR was not loaded with antigens. The absence of HLA-DR/DM interactions at the limiting membrane prevents local loading of MHC class II molecules in phagosomes. This may allow these bacteria to successfully evade the immune system

Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to γ-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy

B Cell Receptor-Mediated Internalization of Salmonella: A Novel Pathway for Autonomous B Cell Activation and Antibody Production

The present paradigm is that primary B cells are nonphagocytosing cells. In this study, we demonstrate that human primary B cells are able to internalize bacteria when the bacteria are recognized by the BCR. BCR-mediated internalization of Salmonella typhimurium results in B cell differentiation and secretion or anti-Salmonella Ab by the Salmonella-specific B cells. In addition, BCR-mediated internalization leads to efficient Ag delivery to the MHC class II Ag-loading compartments', even though Salmonella remains vital intracellularly in primary B cells. Consequently, BCR-mediated bacterial uptake induces efficient CD4(+) T cell help, which boosts Salmonella-specific Ab production. BCR-mediated internalization of Salmonella by B cells is superior over extracellular Ag extraction to induce rapid and specific Immoral immune responses and efficiently combat infection. The Journal of Immunology, 2009, 182: 7473-748

Spatial separation of HLA-DM/HLA-DR interactions within MIIC and phagosome-induced immune escape

Major Histocompatibility Complex (MHC) class II molecules, including Human Leukocyte Antigen (HLA)-DR, present peptide fragments from proteins degraded in the endocytic pathway. HLA-DR is targeted to late-endocytic structures named MHC class II-containing Compartments (MIIC), where it interacts with HLA-DM. This chaperone stabilizes HLA-DR during peptide exchange and is critical for successful peptide loading. To follow this process in living cells, we have generated cells containing HLA-DR3/Cyan Fluorescent Protein (CFP), HLA-DM/Yellow Fluorescent Protein (YFP), and invariant chain. HLA-DR/DM interactions were observed by Fluorescence Resonance Energy Transfer (FRET). These interactions were pH insensitive, yet occurred only in internal structures and not at the limiting membrane of MIIC. In a cellular model of infection, phagosomes formed a limiting membrane surrounding internalized Salmonella. HLA-DR and HLA-DM did not interact in Salmonella-induced vacuoles, and HLA-DR was not loaded with antigens. The absence of HLA-DR/DM interactions at the limiting membrane prevents local loading of MHC class II molecules in phagosomes. This may allow these bacteria to successfully evade the immune syste

<i>Salmonella</i>-specific B cells form a survival niche supporting in vivo <i>Salmonella</i> spreading to systemic sites.

<p>C57BL/6 mice were adoptively transferred with 0, 2*10⁵ or 10⁶ HEL-specific CD43- naïve B cells labeled with CFSE, as indicated. Mice were orally infected with surface HEL-expressing <i>Salmonella</i> one day after B cell transfer. (A) Distribution of CFSE-labeled HEL-specific B cells in the mesenteric lymph nodes (mLN) and spleen before infection (BI), and 24 or 72 hours post-infection, as indicated. One representative example from 3 experiments with 4 mice for each experimental setting is shown. (B) Recovery of viable bacteria 72 hours post-infection from mesenteric lymph nodes (mLN) spleen (SP) and blood (BL) in infected mice transferred with 0, 2*10⁵ or 10⁶ HEL-specific B cells. Depicted are colony-forming units (CFU)/10⁵ eukaryotic cells. One representative example from 3 experiments with 4 mice for each experimental setting is shown.</p

Quantification of the fate of the GFP-expressing <i>Salmonella</i> in infected B cells.

<p>(A) B cells were infected with anti-BCR coated GFP-expressing <i>Salmonella</i> (green).The plasma membrane of the B cells (red) was stained using an anti-CD20 mAb to discriminate between intracellular and extracellular <i>Salmonella</i>. (B) The relative amount of intracellular <i>Salmonella</i> were measured immediately after infection (0 h), or 4 h and 18 h post-infection. Error bars represent SD from two independent experiments. (C) The number of <i>Salmonella</i> per B cell was measured immediately after infection and 18h post-infection. A representative experiment of two individual experiments is shown.</p

<i>Salmonella</i> is actively excreted by B cells.

<p>(A) Primary B cells having phagocytosed anti-BCR coated GFP-<i>Salmonella</i> on a monolayer of 3T3-CD40L fibroblasts were imaged using widefield fluorescence microscopy. Depicted is the GFP signal projected on the transmission image with images taken every 30 min. Scalebar = 10 µm. Arrows indicate the B cell, white arrow: B cells moves op top of the monolayer, black arrow: B cells moves below the monolayer. Images are frames from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050667#pone.0050667.s005" target="_blank">Video S4</a>. (B) Primary B cells having phagocytosed anti-BCR coated GFP-<i>Salmonella</i> on a monolayer of 3T3-CD40L fibroblasts were imaged using widefield fluorescence microscopy in the presence of TexasRed labeled anti-LPS mAbs. Depicted are GFP and Texas-Red signals projected on the transmission image. Scalebar = 10µm. Images are frames from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050667#pone.0050667.s006" target="_blank">Video S5</a>. (C) Quantification of <i>Salmonella</i> secretion from B cells. Primary B cells were incubated with live uncoated GFP-<i>Salmonella</i>. Cells were stained with antibodies against LPS, fixed and analyzed using FACS. Left panel: increase in cell surface exposed LPS from bacteria exposed at the cell surface after initial uptake by B cells. Middle panel: percentage of B cells having excreted <i>Salmonella</i> as calculated from the percentage of B cells containing GFP-<i>Salmonella</i> followed in time. Right panel: left and middle panels are projected to illustrate that both processes show similar kinetics. Error bars represent SD from three independent experiments. (D) Primary B cells were incubated with live uncoated GFP-expressing <i>Salmonella</i> and followed for the time points indicated. The fraction of living B cells is plotted to demonstrate that loss of GFP-<i>Salmonella</i> positive B cells is not correlated with cell death.</p