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NK cells recognize MHC class I (MHC-I) Ags via stochastically expressed MHC-I–specific inhibitory receptors that prevent NK cell activation via cytoplasmic ITIM. We have identified a pan anti–MHC-I mAb that blocks NK cell inhibitory receptor binding at a site distinct from the TCR binding site. Treatment of unmanipulated mice with this mAb disrupted immune homeostasis, markedly activated NK and memory phenotype T cells, enhanced immune responses against transplanted tumors, and augmented responses to acute and chronic viral infection. mAbs of this type represent novel checkpoint inhibitors in tumor immunity, potent tools for the eradication of chronic infection, and may function as adjuvants for the augmentation of the immune response to weak vaccines.

Unlike cytosolic processing and presentation of viral antigens by virus-infected cells, antigens first expressed in an infected non-professional antigen-presenting cell, such as CD4(+) T cells in the case of HIV, and then taken up by dendritic cells are cross-presented. This generally requires entry through the endocytic pathway, where endosomal proteases have first access for processing. Thus, understanding virus escape during cross-presentation requires an understanding of resistance to endosomal proteases such as cathepsin S. We have modified HIV-1(MN) gp120 (gp120(MN)) by mutating a key cathepsin S cleavage site T(322)T(323) in the V3 loop of the immunodominant epitope IGPGRAFYTT to IGPGRAFYVV to prevent digestion. We found this mutation to facilitate cross-presentation, and provide evidence from both MHC-binding and X-ray crystallographic structural studies that this results from preservation of the epitope rather than an increased epitope affinity for the class I MHC molecule. In contrast, when the protein is expressed by a vaccinia virus in the cytosol, the wild type protein is immunogenic without this mutation. These results demonstrate proof-of-concept that a virus like HIV, infecting predominantly non-professional presenting cells, can escape T cell recognition by incorporating a cathepsin S cleavage site that leads to destruction of an immunodominant epitope when the antigen undergoes endosomal cross-presentation.

Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8+ cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8+ T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2Dd better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8+ T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

We have recently described a mAb, KP15, directed against the MHC-I/peptide molecular complex consisting of H-2Dd and a decamer peptide corresponding to residues 311–320 of the HIV IIIB envelope glycoprotein gp160. When administered at the time of primary immunization with a vaccinia virus vector encoding gp160, the mAb blocks the subsequent appearance of CD8+ CTL with specificity for the immunodominant Ag, P18-I10, presented by H-2Dd. This inhibition is specific for this particular peptide Ag; another H-2Dd-restricted gp160 encoded epitope from a different HIV strain is not affected, and an H-2Ld-restricted epitope encoded by the viral vector is also not affected. Using functional assays and specific immunofluorescent staining with multivalent, labeled H-2Dd/P18-I10 complexes (tetramers), we have enumerated the effects of blocking of priming on the subsequent appearance, avidity, and TCR Vβ usage of Ag-specific CTL. Ab blocking skews the proportion of high avidity cells emerging from immunization. Surprisingly, Vβ7-bearing Ag-specific TCR are predominantly inhibited, while TCR of several other families studied are not affected. The ability of a specific MHC/peptide mAb to inhibit and divert the CD8+ T cell response holds implications for vaccine design and approaches to modulate the immune response in autoimmunity.