Upstream toward the “DRiP”-ing Source of the MHC Class I Pathway

MHC class I binding peptides are generated via cytosolic degradation of a previously undefined substrate. In this issue of Immunity, Kunisawa and Shastri (2006) isolate pre-degradation polypeptide intermediates bound to a cytosolic chaperone

Viral Sequestration of Antigen Subverts Cross Presentation to CD8+ T Cells

Virus-specific CD8+ T cells (TCD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector TCD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated TCD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the TCD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines

Tolerogenic Properties of Lymphatic Endothelial Cells Are Controlled by the Lymph Node Microenvironment

Peripheral self-tolerance eliminates lymphocytes specific for tissue-specific antigens not encountered in the thymus. Recently, we demonstrated that lymphatic endothelial cells in mice directly express peripheral tissue antigens, including tyrosinase, and induce deletion of specific CD8 T cells via Programmed Death Ligand-1 (PD-L1). Here, we demonstrate that high-level expression of peripheral tissue antigens and PD-L1 is confined to lymphatic endothelial cells in lymph nodes, as opposed to tissue (diaphragm and colon) lymphatics. Lymphatic endothelial cells in the lymph node medullary sinus express the highest levels of peripheral tissue antigens and PD-L1, and are the only subpopulation that expresses tyrosinase epitope. The representation of lymphatic endothelial cells in the medullary sinus expressing high-level PD-L1, which is necessary for normal CD8 T cell deletion kinetics, is controlled by lymphotoxin-β receptor signaling and B cells. Lymphatic endothelial cells from neonatal mice do not express high-level PD-L1 or present tyrosinase epitope. This work uncovers a critical role for the lymph node microenvironment in endowing lymphatic endothelial cells with potent tolerogenic properties.</p

Tolerogenic Properties of Lymphatic Endothelial Cells Are Controlled by the Lymph Node Microenvironment

<div><p>Peripheral self-tolerance eliminates lymphocytes specific for tissue-specific antigens not encountered in the thymus. Recently, we demonstrated that lymphatic endothelial cells in mice directly express peripheral tissue antigens, including tyrosinase, and induce deletion of specific CD8 T cells via Programmed Death Ligand-1 (PD-L1). Here, we demonstrate that high-level expression of peripheral tissue antigens and PD-L1 is confined to lymphatic endothelial cells in lymph nodes, as opposed to tissue (diaphragm and colon) lymphatics. Lymphatic endothelial cells in the lymph node medullary sinus express the highest levels of peripheral tissue antigens and PD-L1, and are the only subpopulation that expresses tyrosinase epitope. The representation of lymphatic endothelial cells in the medullary sinus expressing high-level PD-L1, which is necessary for normal CD8 T cell deletion kinetics, is controlled by lymphotoxin-β receptor signaling and B cells. Lymphatic endothelial cells from neonatal mice do not express high-level PD-L1 or present tyrosinase epitope. This work uncovers a critical role for the lymph node microenvironment in endowing lymphatic endothelial cells with potent tolerogenic properties.</p></div

LtβR-Ig signaling and B cells do not control tyrosinase expression but influence the kinetics of FH T cell deletion.

<p><b><i>a.</i></b> LEC were purified from the LN of mice treated with PBS or LtβR-Ig for 4 weeks, or from WT (C57BL/6) or µMT^−/− mice. RNA was purified and 40-cycle qPCR was performed for tyrosinase. Graphs represent results from 2 independent experiments. Data are represented as mean +/− SEM. <b><i>b.</i></b> LNSC were purified by enzymatic digestion of pooled LN from the indicated mice, obtained via magnetic bead separation, and were analyzed by flow cytometry. Plots are gated on CD45^neg gp38⁺ CD31⁺ cells. <b><i>c.</i></b> 1 × 10⁶ CD8-enriched FH T cells were labeled with cell trace violet (CTV)-labeled and adoptively transferred into WT, µMT^−/−, or Tyr^neg (albino) recipients. At day 3 post-transfer, pooled peripheral LN were harvested and stained for CD8 and Tyr₃₆₉-tetramer, and assessed for CTV dilution. <i>Left panel</i>, representative histogram plot gated on CD8⁺ Tet⁺ cells. <i>Right panel,</i> summary graph of percent FH T cells divided per division representative of 4 independent experiments. Data are represented as mean +/− SEM. *p<0.05, **p<0.01, ^#p = 0.06 ns = not significant. <b><i>d.</i></b> Experiment performed as in (c), except FH T cells were identified using CD45.1 and LN were harvested at 7 days post transfer.</p

LN-LEC do not mediate tolerance to tyrosinase in the neonatal period.

<p><b><i>a.</i></b> Brachial LN were purified from mice at the indicated age and enzymatically digested. For postnatal day 7 and 6 week old mice, LNSC were purified by CD45 magnetic bead separation. Single cell suspensions were stained with antibodies specific for CD45, gp38 and CD31. Numbers in quadrants to the right of plots represent the percentage out of CD45^neg cells. <b><i>b.</i></b> Frozen brachial LN sections from mice of the indicated age were stained with antibodies specific for Lyve-1, CD31, B220, and CD3ε. Scale bar = 200 µm. <b><i>c.</i></b> LNSC from the brachial LN of the indicated mice were stained with antibodies specific for CD45, gp38, CD31, PD-L1. Plots are gated on CD45^neg gp38⁺ CD31⁺ cells. <b><i>d.</i></b> Proliferation of CFSE-labeled naïve Thy1.2⁺ FH T cells co-cultured with the indicated Tyr₃₆₉ pulsed, or un-pulsed LN-LEC population after 86hr. Numbers indicate the percentage of divided FH T cells of total FH T cells. <i>Left panels</i>, representative experiment; <i>right panel</i>, summary data for 2 independent experiments. **p<0.01.</p