LILRB1 blockade enhances bispecific T cell engager antibody-induced tumor cell killing by effector CD8+ T cells

Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. In addition to using immune checkpoint blockade to reinvigorate existing but unresponsive tumor-specific T cells, alternative therapeutic approaches have been developed, including stimulation of polyclonal T cell cytolytic activity against tumors using bispecific T cell engager (BiTE) molecules that simultaneously engage the TCR complex and a tumor-associated Ag. BiTE molecules are efficacious against hematologic tumors and are currently being explored as an immunotherapy for solid tumors. To understand mechanisms regulating BiTE molecule­–mediated CD8+ T cell activity against solid tumors, we sought to define human CD8+ T cell populations that efficiently respond to BiTE molecule stimulation and identify factors regulating their cytolytic activity. We find that human CD45RA+CCR7− CD8+ T cells are highly responsive to BiTE molecule stimulation, are enriched in genes associated with cytolytic effector function, and express multiple unique inhibitory receptors, including leukocyte Ig-like receptor B1 (LILRB1). LILRB1 and programmed cell death protein 1 (PD1) were found to be expressed by distinct CD8+ T cell populations, suggesting different roles in regulating the antitumor response. Engaging LILRB1 with its ligand HLA-G on tumor cells significantly inhibited BiTE molecule–induced CD8+ T cell activation. Blockades of LILRB1 and PD1 induced greater CD8+ T cell activation than either treatment alone. Together, our data suggest that LILRB1 functions as a negative regulator of human CD8+ effector T cells and that blocking LILRB1 represents a unique strategy to enhance BiTE molecule therapeutic activity against solid tumors

Divergent paths for the selection of immunodominant epitopes from distinct antigenic sources

Immunodominant epitopes are few selected epitopes from complex antigens that initiate T cell responses. Here, to provide further insights into this process, we use a reductionist cell-free antigen processing system composed of defined components. We use the system to characterize steps in antigen processing of pathogen-derived proteins or autoantigens and we find distinct paths for peptide processing and selection. Autoantigen-derived immunodominant epitopes are resistant to digestion by cathepsins, whereas pathogen-derived epitopes are sensitive. Sensitivity to cathepsins enforces capture of pathogen-derived epitopes by Major Histocompatibility Complex class II (MHC class II) prior to processing, and resistance to HLA-DM-mediated-dissociation preserves the longevity of those epitopes. We show that immunodominance is established by higher relative abundance of the selected epitopes, which survive cathepsin digestion either by binding to MHC class II and resisting DM-mediated-dissociation, or being chemically resistant to cathepsins degradation. Non-dominant epitopes are sensitive to both DM and cathepsins and are destroyed

HLA-DO as the optimizer of epitope selection for MHC class II antigen presentation.

Processing of antigens for presentation to helper T cells by MHC class II involves HLA-DM (DM) and HLA-DO (DO) accessory molecules. A mechanistic understanding of DO in this process has been missing. The leading model on its function proposes that DO inhibits the effects of DM. To directly study DO functions, we designed a recombinant soluble DO and expressed it in insect cells. The kinetics of binding and dissociation of several peptides to HLA-DR1 (DR1) molecules in the presence of DM and DO were measured. We found that DO reduced binding of DR1 to some peptides, and enhanced the binding of some other peptides to DR1. Interestingly, these enhancing and reducing effects were observed in the presence, or absence, of DM. We found that peptides that were negatively affected by DO were DM-sensitive, whereas peptides that were enhanced by DO were DM-resistant. The positive and negative effects of DO could only be measured on binding kinetics as peptide dissociation kinetics were not affected by DO. Using Surface Plasmon Resonance, we demonstrate direct binding of DO to a peptide-receptive, but not a closed conformation of DR1. We propose that DO imposes another layer of control on epitope selection during antigen processing

A model for the effects of HLA-DO on antigen presentation.

<p>DO likely interacts with peptide-receptive DR molecules and may stabilize an overly receptive conformation. This conformation lends itself to a more efficient release of the poorly binding peptides while helping the formation of compact complexes with the well-fitting peptides. In the pool of available peptides those with weak anchoring residues that tend to be more DM-sensitive may not get a chance to stabilize in the groove, and therefore are outcompeted by DM-resistant peptides with bulkier hydrophobic P1 pocket residues. We theorize that DO interacts primarily with DR molecules in receptive conformation mostly generated by the effector function of DM.</p

The observed effect of DO on peptide presentation is DO specific and can manifest in complex with DM.

<p>(A) Association of HA(Y308A) peptide to DR1 molecules in the presence of coinfected DM/DO complex (Ni-NTA purified). The peptide binding experiment was performed with no accessory molecules (black squares), with DM (red dots), DM/DO (green triangles), or both DM/DO and DM (blue triangles) over the course of 10 hours. The fluorescence signals (Arbitrary Fluorescence Units) associated with the control samples incubated >10 hours in the absence of DR1 were measured: HA(Y308A) peptide alone, 1140; HA(Y308A)+DM, 894; HA(Y308A)+DM/DO, 1404; HA(Y308A)+DM+DM/DO, 1944. (B) DO was depleted from a DO stock by immunoprecipitation via Ni-NTA followed by Mags.DO5 resin. The depleted sample was used instead of DO in reactions measuring HA(anchorless) peptide/DR complex formation in the presence or absence of DM after 5 hours of incubation. The fluorescence intensity of peptide/DR1 complexes formed in the DO depleted reaction was compared to a reaction containing no DO (left bar in each set of three), and a reaction that contained DO that did not undergo depletion (right bar in each set of three). The experiment is representative of three separate trials. (C) A DO-depleted sample was used instead of DO in a reaction measuring of HA(306–318) peptide/DR complex formation in the presence or absence of DM after 5 hours of incubation. The fluorescence intensity of peptide/DR1 complexes formed in the DO depleted reaction was compared to a reaction containing no DO (left bar in each set of three), and a reaction that contained DO that did not undergo depletion (right bar in each set of three). The experiment is representative of three separate trials.</p

HLA-DO interacts with DR1 in a receptive conformation but not a peptide-loaded, compact form.

<p>(A) SPR sensograms of constitutively receptive DR1βG86Y (4µM) binding to DO. Ni-NTA purified DO was immobilized on anti-His antibody coupled chip (blue trace). After a brief wash, DR1βG86Y was injected over the captured DO surface (green trace). An injection of unloaded DR1 over the anti-His antibody surface (red trace) was performed to control for potential nonspecific binding DR1 to the chip surface. (B) SPR sensograms of closed compact DR1/HA(306–318) complex (4µM) binding to DO. Ni-NTA purified DO was immobilized on anti-His antibody coupled chip (blue trace). After a brief wash, DR1/HA(306–318) was injected over the captured DO surface (green trace). An injection of unloaded DR1 over the anti-His antibody surface (red trace) was performed to control for potential nonspecific binding DR1 to the chip surface. (C) DR1βG86Y binding to DM/DO complex molecules. Mags.DO5 purified DM/DO was immobilized on anti-His antibody coupled chip surface to a level of 2000–3000 RU. After a brief wash, DR1βG86Y was injected over the captured DM/DO at concentrations of 0.5 µM (blue trace), 1 µM (red trace), 2 µM (green trace). Before every injection of DR1βG86Y, the DM/DO molecules captured on the surface were regenerated to insure that the surface was not saturated by bound DR1 molecules. The signal of the resulting binding ∼200–300 after the end of the injection is marked on the graph. (D) Binding controls of DR1βG86Y and DR1/HA(306–318) with anti-His antibody, DM/DO and DM surfaces. Following the immobilization of anti-His antibody, 4 µM DR1βG86Y (red trace) and 4 µM DR1/HA(306–318) (black trace) were injected over the immobilized antibody. Upon capturing 2000–3000 RU of DM/DO by the anti-His antibody, 4 µM DR1/HA(306–318) was injected over the DM/DO (green trace). In a separate control, 3000 RU of DM was captured by immobilized anti-FLAG antibody. 4 µM DR1βG86Y (cyan trace), or 4 µM DR1/HA(306–318) (blue trace) was injected over the captured DM. The magnitude of binding was measured at the <i>stability point</i> ∼200–300 seconds after the end of the injection. Data shown are representative of two independent experiments.</p

Soluble recombinant DO recognizes soluble recombinant DM.

<p>(A) DO (Ni-NTA purified) was immobilized on an anti-His antibody surface (blue trace). After a brief wash, DM was injected over the captured DO (green trace). A control injection of DM over anti-His antibody surface (red trace) showed no non-specific binding. Data shown are representative of six independent experiments. (B) DO (Ni-NTA purified) binding to DM immobilized by anti-FLAG antibody surface in concentrations ranging from 0.01 to 10 µM in separate experiments.</p

DO can increase the binding of peptides to DR1 molecules.

<p>(A) Association (left) and dissociation (right) of HA(306–318) peptide to DR1 molecules with no accessory molecules (black squares), with DM (red dots), DO (green triangles) or both DO and DM (blue triangles) over the course of 10 hours. The fluorescence signals (Arbitrary Fluorescence Units) associated with the control samples incubated >10 hours in the absence of DR1 were measured: HA(306–318) peptide alone, 1390; HA(306–318)+DM, 1376; HA(306–318)+DO, 3316; HA(306–318)+DM+DO, 9236. (B) Association (left) and dissociation (right) of H5N1-HA1(259–274) flu peptide to DR1 molecules with no accessory molecules (black squares), with DM (red dots), DO (green triangles) or both DO and DM (blue triangles) over the course of 10 hours. The fluorescence signals (Arbitrary Fluorescence Units) associated with the control samples incubated >10 hours in the absence of DR1 were measured: H5N1-HA1(259–274) peptide alone, 1312; H5N1-HA1(259–274)+DM, 1250; H5N1-HA1(259–274)+DM+DO, 9012. (C) Prolonged 96 hour dissociation experiment of HA(306–318) peptide from DR1 molecules with DM (red dots) or both DO and DM (blue triangles). Data shown are representative of at least three independent experiments.</p

DO diminishes binding of peptides to DR1 molecules.

<p>(A) Association (left) and dissociation (right) of CII(259–273) peptide to DR1 molecules with no accessory molecules (black squares), with DM (red dots), with DO (green triangles), or both DO and DM (blue triangles) over the course of 10 hours. The fluorescence signals (Arbitrary Fluorescence Units) associated with the control samples incubated >10 hours in the absence of DR1 were measured: CII(259–273) peptide alone, 3996; CII(259–273)+DM, 1026; CII(259–273)+DO, 3326; CII(259–273)+DM+DO, 8278. (B) Association (left) and dissociation (right) of HA(anchorless) peptide to DR1 molecules with no accessory molecules (black squares), with DM (red dots), DO (green triangles) or both DO and DM (blue triangles) over the course of 10 hours. The fluorescence signals (Arbitrary Fluorescence Units) associated with the control samples incubated >10 hours in the absence of DR1 were measured: HA(anchorless) peptide alone, 3364; HA(anchorless)+DM, 1334; HA(anchorless)+DO, 1558; HA(anchorless)+DM+DO, 1726. Data shown are representative of at least three independent experiments.</p

The initial rates of peptide/DR1 complex formation for tested peptides with and without accessory molecules.

#<p>Initial Rates were determined for 1 hour (Response; Arbitrary Fluorescence Units min⁻¹).</p>*<p>The effect of DO on HA(Y308A) was determined by using co-expressed DM/DO complex.</p>¶<p>Determined in a separate experiment.</p