HLA class I polymorphism creates diversity in epitope specificity and T cell repertoire. We show that HLA polymorphism also controls the choice of Ag presentation pathway. A single amino acid polymorphism that distinguishes HLA-B*4402 (Asp116) from B*4405 (Tyr116) permits B*4405 to constitutively acquire peptides without any detectable incorporation into the transporter associated with Ag presentation (TAP)-associated peptide loading complex even under conditions of extreme peptide starvation. This mode of peptide capture is less susceptible to viral interference than the conventional loading pathway used by HLA-B*4402 that involves assembly of class I molecules within the peptide loading complex. Thus, B*4402 and B*4405 are at opposite extremes of a natural spectrum in HLA class I dependence on the PLC for Ag presentation. These findings unveil a new layer of MHC polymorphism that affects the generic pathway of Ag loading, revealing an unsuspected evolutionary trade-off in selection for optimal HLA class I loading versus effective pathogen evasion.

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and β2-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

The retroviral Gag precursor plays an important role in the assembly of virion particles. The capsid (CA) protein of the Gag molecule makes a major contribution to this process. In the crystal structure of the free CA protein of the human immunodeficiency virus type 1 (HIV-1), 11 residues of the C terminus were found to be unstructured, and to date no information exists on the structure of these residues in the context of the Gag precursor molecule. We performed phylogenetic analysis and demonstrated a high degree of conservation of these 11 amino acids. Deletion of this cluster or introduction of various point mutations into these residues resulted in significant impairment of particle infectivity. In this cluster, two putative structural regions were identified, residues that form a hinge region (353-VGGP-356) and those that contribute to an ␣-helix (357-GHKARVL-363). Overall, mutations in these regions resulted in inhibition of virion production, but mutations in the hinge region demonstrated the most significant reduction. Although all the Gag mutants appeared to have normal Gag-Gag and Gag-RNA interactions, the hinge mutants were characterized by abnormal formation of cytoplasmic Gag complexes. Gag proteins with mutations in the hinge region demonstrated normal membrane association but aberrant rod-like membrane structures. More detailed analysis of these structures in one of the mutants demonstrated abnormal trapped Gag assemblies. These data suggest that the conserved CA C terminus is important for HIV-1 virion assembly and release and define a putative target for drug design geared to inhibit the HIV-1 assembly process.

Peptide assembly with class I molecules is orchestrated by multiple chaperones including tapasin, which bridges class I molecules with the TAP and is critical for efficient Ag presentation. In this paper, we show that, although constitutive levels of endogenous murine tapasin apparently are sufficient to form stable and long-lived complexes between the human HLA-B*4402 (B*4402) and mouse TAP proteins, this does not result in normal peptide loading and surface expression of B*4402 molecules on mouse APC. However, increased expression of murine tapasin, but not of the human TAP proteins, does restore normal cell surface expression of B*4402 and efficient presentation of viral Ags to CTL. High levels of soluble murine tapasin, which do not bridge TAP and class I molecules, still restore normal surface expression of B*4402 in the tapasin-deficient human cell line 721.220. These findings indicate distinct roles for tapasin in class I peptide loading. First, tapasin-mediated bridging of TAP-class I complexes, which despite being conserved across the human-mouse species barrier, is not necessarily sufficient for peptide loading. Second, tapasin mediates a function which probably involves stabilization of empty class I molecules and which is sensitive to structural compatibility of components within the loading complex. These discrete functions of tapasin predict limitations to the study of HLA molecules across some polymorphic and species barriers.

Crystallographic analysis of human Hfe has documented an overall structure similar to classical (class Ia) MHC molecules with a peptide binding groove deprived of ligand. Thus, to address the question of whether ␣␤ T cells could recognize MHC molecules independently of bound ligands, we studied human and mouse Hfe interactions with T lymphocytes. We provide formal evidence of direct cytolytic recognition of human Hfe by mouse ␣␤ T cell receptors (TCR) in HLA-A*0201 transgenic mice and that this interaction results in ZAP-70 phosphorylation. Furthermore, direct recognition of mouse Hfe molecules by cytotoxic T lymphocytes (CTLs) was demonstrated in DBA͞2 Hfe knockout mice. These CTLs express predominantly two T cell antigen receptor ␣ variable gene segments (AV6.1 and AV6.6). Interestingly, in wild-type mice we identified a subset of CD8 ؉ T cells positively selected by Hfe that expresses the AV6.1͞AV6.6 gene segments. T cell antigen receptor recognition of MHC molecules independently of bound ligand has potential general implications in alloreactivity and identifies in the Hfe case a cognitive link supporting the concept that the immune system could be involved in the control of iron metabolism. T cell receptor M ost histocompatibility class I molecules have an opengroove in their ␣1␣2-domains in which they bind peptides (HLA and H-2 class Ia molecules) less often than glycolipids (CD1 molecules) (1). The composite structures are recognized through their T cell antigen receptors (TCRs) by subsets of T lymphocytes [CD8 T lymphocytes, natural killer T cells (2), and mucosal associated invariant T cells (3)], resulting in cytolysis and͞or cytokine secretion, depending on the subset activated. Several TCR͞peptide͞MHC crystal structures have shown that the ␣1-and ␣2-helices of the MHC molecules are contacted by the TCR ␣-and ␤-chain complementarity-determining regions (CDR)1͞CDR2, the bound peptides interacting essentially with the hypervariable CDR3s (4). The dual selectivity of some alloreactive CTL clones for a given allogeneic molecule and a given peptide presented by this molecule might suggest that effective TCR͞MHC molecule interaction always requires a direct participation of the bound ligand (5, 6). However, less stringent peptide selectivity has been documented for other CTL clones (7,8), suggesting in those cases predominant TCR interaction with the ␣1␣2-helices of their allogeneic MHC molecular targets.Hfe is a nonclassical MHC class Ib molecule sharing 37% amino acid identity with HLA-A*0201. Hfe is formed, like classical MHC class I, by the noncovalent association of a heavy ␣ and a ␤2-microglobulin (␤2m) light chain (9). The 2.8-Å crystallographic structure of the human Hfe (hHfe) molecule has been published (10). Whereas its overall structure is similar to MHC class Ia molecules, Hfe has a narrow and empty version of the classical MHC class I peptide groove. To determine whether cytotoxic ␣␤ T cells are able to directly recognize the hHfe or mouse Hfe (mHfe), which are MHC class I molecules without an...