Our investigations into the biological function of HLA-F support a novel role for the interaction between HLA-F and OC of MHC-I in the uptake of extracellular Ag for cross-presentation. Several lines of evidence suggest an active role for OC of MHC-I in the uptake of Ag as whole or partial protein that contains epitopes specific to receptive MHC-I expressed on target cells. Exogenous Ag was internalized together with MHC-I, and downmodulation of surface MHC-I resulted in reduced binding, uptake, and presentation of Ag. Differential binding of polypeptides containing low- and high-affinity MHC-I–binding epitopes to MHC-I and HLA-F suggested that exogenous Ag binds to the surface of activated cells to a structure that includes MHC-I OC and HLA-F and that is in contact with the MHC-I–specific epitope sequence found within the extended polypeptide. The requirement of this model for exposure of linear epitopes is consistent with the observation that in our experiments protein required denaturation to enter this pathway, as it did in previously described experiments for cross-presentation of BZLF-1 and pp65 (28
). We speculate that MHC-I OC, possibly stabilized by HLA-F, retains rudimentary peptide–binding specificity conferred by the peptide-binding cleft and can bind epitopes in an open-ended fashion without size limitations, possibly similar to MHC class II.
It is well known, based on many crystal structures and considerable binding and T cell recognition data, that a folded class I MHC molecule binds 8- to 10-mer peptides, depending on the allele and epitope, and that, in contrast to MHC-II, the ends of the pocket are closed (4
). There are few reported exceptions to this rule, but, whereas the suggestion that extended polypeptide chains are capable of binding to MHC-I OC may be considered controversial, peptide length specificity of some HLA class I alleles has been shown to be very broad and includes peptides of up to 25 aa in length (47
). The structure of the MHC-I OC is unknown and is most certainly distinct from the classical structure consisting of H chain, β2
m, and peptide. Solving the MHC-I OC structure could address possibilities for an alternative Ag-binding mechanism related to our proposed model for interaction of the MHC-I–binding cleft with epitopes within extended polypeptides.
In addition to MHC-I OC, several experiments directly implicated HLA-F in this pathway. First, the observed overlapping internalization and localization of Ag and HLA-F were coincident with the same observation with MHC-I OCs. Secondly, HLA-F was coprecipitated with MHC-I by polypeptide containing MHC-I–binding epitopes at relative levels approximately paralleling those of MHC-I. Third, downmodulation of HLA-F resulted in interference with Ag binding, uptake, and processing for presentation, and, although downmodulation was coincident with downmodulation of MHC-I, blocking with HLA-F–specific mAb alone interfered with Ag cross-presentation. Indeed, the fact that HLA-F and MHC-I OC, and not MHC-I complex, physically interact in cells relates the findings of MHC-I OC function directly to HLA-F (15
). One possible role for HLA-F could be in the stabilization and transport of MHC-I OC to, on, and from the surface. The physical interaction between HLA-F and classical MHC-I OC, their coincident surface expression on activated lymphocytes, and their coincident downmodulation in HLA-F knockdowns combine to suggest that they are interdependent for surface transport. HLA-F may stabilize MHC-I OC as it is formed and the two proteins, possibly as a heterodimer, transit to the surface. Other not mutually exclusive roles include cooperation in the internalization of Ag and MHC-I.
The evidence presented in this work suggests that Ag, MHC-I OC, and possibly HLA-F transit from the surface through the endosomal pathway into lysosomes or lysosome-like structures, where protein is degradated to produce target peptide independently of TAP or tapasin. After complex formation, MHC-I–containing specific peptide derived from the exogenous Ag source is transported to the surface. This model has evident similarities with the MHC-II Ag-presenting pathway in particular, and there is already good evidence that MHC-I molecules visit phagolysosomal compartments to acquire peptides prior to surface expression (48
). MHC-I proteins have been shown to reside in endosomes and lysosomes of dendritic cells, and exchange of MHC-I between the cell membrane and endosomal compartments has been demonstrated in both T cells and macrophages (50
). Furthermore, in the mouse, exogenous MHC-I Ag loading has been associated with endosomal and lysosomal trafficking in dendritic cells (53
Although previous studies have provided good evidence in support of a pathway for class I loading that is shared with class II molecules (49
), our work presents at least three major new findings. First, no evidence implicating the participation of MHC-I OC nor any including HLA-F has been reported. Second, no studies or proposed models have suggested an allelic dependence on MHC-I–specific epitopes for Ag uptake. Third, most cross-presentation pathways studied were operating in professional APCs, primarily dendritic cells (40
). In that regard, evidence that other cell types can take up and process Ags has been accumulating. Voeten et al. (56
) showed that exogenous recombinant influenza A virus nucleoprotein is processed and presented via MHC-I by EBV-transformed B-LCLs. Also, not only dendritic cells and monocytes, but also B cells can cross-present uBZLF1 in vitro (28
As noted above, the experimental responses reported in this study were dependent on the use of denatured proteins or polypeptides as opposed to intact proteins, which generally did not evoke similar responses. The likelihood that denatured polypeptides arise in vivo would appear to be high, considering the plethora of proteases active during apoptosis and in cells subjected to cytolysis, thereby producing fragmented proteins containing class I MHC epitopes (57
). Because the requirement for HLA-F in this pathway implies it is operating primarily when cells are activated (17
) and thus during an inflammatory response, cell lysis at the site of activation arising either from the lytic effects of virus or from the cytolytic activity of innate or adaptive cells would expose the local environment to resultant degradation products. Activated cells exposed to such degradation products could benefit from the ability to take up and process extracellular Ag for a number of different reasons. For example, the possibility that monocyte-derived cells can participate critically in processing Ag for cross-presentation has been suggested, even if they do not present that Ag to T cells themselves (55
). Also, cross-presentation by nonprofessional APCs has been demonstrated for HLA class I epitopes from exogenous NY-ESO-1 polypeptides (58
). Membrane transfer of Ags from activated B cells to bystander B cells was recently demonstrated (59
). Transfer from other activated cells could also be explained by the process of intercellular communication of biomolecules through exosomes (60
). This points to the possibility of transfer of lipid rafts containing processed Ag, presented by MHC-I from activated NK or T cells to monocytes, dendritic cells, or B cells (60
). Other more speculative possibilities include NK cells acquiring Ag from target cells for subsequent stimulation of T cells (in this case memory T cells), particularly when a target cell is lacking in MHC expression. Very little detail has been reported regarding how memory CD8+
T cells are activated, including their potential activation by amateur APCs (64
Whereas the details of cross-presentation for MHC-I have not been well elucidated, several lines of evidence suggest this immune pathway could play a fundamental role in protection from pathogens (40
). If MHC-I OC and HLA-F function in MHC-I Ag cross-presentation, then uncovering the biochemical rules for Ag uptake, processing, and presentation particular to this pathway could provide new strategies for the design and optimization of novel immunogens.