TLR Agonists Downregulate H2-O in CD8α− Dendritic Cells

Abstract

Peptide loading of MHC class II (MHCII) molecules is catalyzed by the non-classical MHCII-related molecule, H2-M. H2-O, another MHCII-like molecule, associates with H2-M and modulates H2-M function. The MHCII presentation pathway is tightly regulated in dendritic cells (DCs); yet how the key modulators of MHCII presentation, H2-M and H2-O, are affected in different DC subsets in response to maturation is unknown. Here we show that H2-O is markedly downregulated in vivo in mouse CD8α− DCs in response to a broad array of TLR agonists. In contrast, CD8α+ DCs only modestly downregulated H2-O in response to TLR-agonists. H2-M levels were slightly down-modulated in both CD8α− and CD8α+ DCs. As a consequence, H2-M:H2-O ratios significantly increased for CD8α− but not CD8α+ DCs. The TLR-mediated downregulation was DC-specific, as B cells did not show significant H2-O and H2-M downregulation. TLR4 signaling was required to mediate DC H2-O downregulation in response to LPS. Finally, our studies showed that the mechanism of H2-O downregulation was likely due to direct protein degradation of H2-O as well as down regulation of H2-O mRNA levels. The differential H2-O and H2-M modulation after DC maturation support the proposed roles of CD8α− dendritic cells in initiating CD4-restricted immune responses by optimal MHCII presentation and CD8α+ DCs in promoting immune tolerance via presentation of low levels of MHCII-peptide.

HLA-DO and Its Role in MHC Class II Antigen Presentation

“…While the model that describes DO solely as an inhibitor of DM by preventing its major role in the selection of immunodominant epitopes (44, 52) fits some of the observed effects of DO (53), it does not provide an adequate explanation for why DO has been evolutionary so conserved nor does it explain the variable expression of DO among different subsets of professional APCs, and at different stages of cellular differentiation (8, 54). A more likely explanation is that DO has some critical role in fine tuning, or improving the antigenic peptide repertoire selection in collaboration with DM: the expression of DO in B cells could easily be explained by the need to limit the number of long-lived memory T cells developed against different antigens (55–57), and in the thymus, DO contributes to the presentation of a repertoire that is most contrived yet effective permitting deletion of all possible self-reactive T cells (58).…”

pH-susceptibility of HLA-DO tunes DO/DM ratios to regulate HLA-DM catalytic activity

“…This is the first evidence to suggest possible in vivo consequences of acid exposure of DM-DO complexes and is consistent with the mechanism that acidic pH down-regulates DO/DM ratios in favor of free DM. DO levels are known to be reduced in association with activation of B cells and DO-expressing dendritic cells (DCs), influencing the spectrum of antigens that these APC present to CD4+ T cells 10 18 19 20 . Our finding suggests that, in addition to the transcriptional down-regulation of DO expression induced by cell activation stimuli 18 20 , the acidification of late-endosomes driven by receptor-activated signaling pathways 33 34 will promote down-regulation of DO/DM ratios (as a result of DO destruction), leading to increased free DM level and activity.…”

“…DO levels are known to be reduced in association with activation of B cells and DO-expressing dendritic cells (DCs), influencing the spectrum of antigens that these APC present to CD4+ T cells 10 18 19 20 . Our finding suggests that, in addition to the transcriptional down-regulation of DO expression induced by cell activation stimuli 18 20 , the acidification of late-endosomes driven by receptor-activated signaling pathways 33 34 will promote down-regulation of DO/DM ratios (as a result of DO destruction), leading to increased free DM level and activity. Conversely, interference with late-endosomal acidification by chloroquine would be expected to abrogate free DM generation and reduce antigen presentation, especially in high DO-expressing cells 35 .…”

“…Free DO has not been observed in cells. This suggests that DO/DM ratios are always ≤1 in endosomal compartments despite differential expression of DO among APC types 9 11 and among maturation/activation states of DO-expressing cells 10 18 19 20 . Both DM and DO are residents of the very acidic lysosomes 16 21 22 , which significantly complicates the investigation of the pH-regulation of DM-DO function in cells.…”

Pathways of Antigen Processing

“…DO is expressed in B cells and thymic epithelium and at low levels in select DC subsets, where there is evidence that it is regulated by Toll-like receptor (TLR) agonists (91-94). DO αβ dimers associate tightly with DM molecules, and are retained in the ER in the absence of DM, suggesting that in DO-positive cells DM and DO move in concert to endosomes (Figure 4) (95).…”