At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin (β2m), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking. We identify non-covalent MHC-I FHC dimers mediated by the α3 domain as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single molecule co-localization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another FHC in a manner similar to the β2m light chain.

IL-15 is under clinical investigation toward the goal of curing HIV infection because of its abilities to reverse HIV latency and enhance immune effector function. However, increased potency through combination with other agents may be needed. 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhances IL-15–mediated latency reversal and NK cell function by increasing STAT5 activation. We hypothesized that HODHBt would also synergize with IL-15, via STAT5, to directly enhance HIV-specific cytotoxic T cell responses. We showed that ex vivo IL-15 + HODHBt treatment markedly enhanced HIV-specific granzyme B–releasing T cell responses in PBMCs from antiretroviral therapy–suppressed (ART-suppressed) donors. We also observed upregulation of antigen processing and presentation in CD4 + T cells and increased surface MHC-I. In ex vivo PBMCs, IL-15 + HODHBt was sufficient to reduce intact proviruses in 1 of 3 ART-suppressed donors. Our findings reveal the potential for second-generation IL-15 studies incorporating HODHBt-like therapeutics. Iterative studies layering on additional latency reversal or other agents are needed to achieve consistent ex vivo reservoir reductions.

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Previously, we demonstrated that major histocompatibility complex class I molecules (MHC-I) that lose the antigenic peptide and the light chain beta-2 microglobulin (β2m) to become free heavy chains (FHCs) associate to form dimers or higher-order oligomers in the plasma membrane (Dirscherl et al., 2018). Here, we investigate this homotypic interaction of MHC-I FHCs by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking to elucidate their molecular structure, abundance, and dynamics. We find that MHC-I FHC complexes are dimeric, transient, non-covalent, and mediated by the α3 domain. FHC interaction correlates with a decrease in the diffusion coefficient and an increase in the number of immobile molecules at the cell surface. Molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another FHC in a manner similar to the β2m light chain. We propose distinct functions of the MHC-I FHC dimers.

Major histocompatibility complex class I molecules (MHC I) that lose the antigenic peptide and the light chain beta-2 microglobulin (β2m) to become free heavy chains at the cell surface are known to associate, forming oligomers on the plasma membrane that are insufficiently understood. Here, we investigate the homotypic interaction of MHC I free heavy chains by combining a printed antibody micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking in order to elucidate their molecular structure, abundance, and dynamics. We find that MHC I free heavy chain complexes are dimeric, transient, non-covalent, and mediated by the α3 domain. Free heavy chain interaction correlates with a decrease in the diffusion coefficient and an increase in the number of immobile molecules at the cell surface. Molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another free heavy chain in a manner similar to the β2m light chain. We propose distinct functions of the MHC I free heavy chain dimers in signaling and in endocytic sorting.