Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

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.

Multiple genetic programs contribute to CD4 T cell memory differentiation and longevity by maintaining T cell quiescence.

While memory T-cells represent a hallmark of adaptive immunity, little is known about the genetic mechanisms regulating the longevity of memory CD4 T cells. Here, we studied the dynamics of gene expression in antigen specific CD4 T cells during infection, memory differentiation, and long-term survival up to nearly a year in mice. We observed that differentiation into long lived memory cells is associated with increased expression of genes inhibiting cell proliferation and apoptosis as well as genes promoting DNA repair response, lipid metabolism, and insulin resistance. We identified several transmembrane proteins in long-lived murine memory CD4 T cells, which co-localized exclusively within the responding antigen-specific memory CD4 T cells in human. The unique gene signatures of long-lived memory CD4 T cells, along with the new markers that we have defined, will enable a deeper understanding of memory CD4 T cell biology and allow for designing novel vaccines and therapeutics.

Proteolysis by Granzyme B Enhances Presentation of Autoantigenic Peptidylarginine Deiminase 4 Epitopes in Rheumatoid Arthritis.

Proteolysis of autoantigens can alter normal MHC class II antigen processing and has been implicated in the induction of autoimmune diseases. Many autoantigens are substrates for the protease granzyme B (GrB), but the mechanistic significance of this association is unknown. Peptidylarginine deiminase 4 (PAD4) is a frequent target of autoantibodies in patients with rheumatoid arthritis (RA) and a substrate for GrB. RA is strongly associated with specific MHC class II alleles, and elevated levels of GrB and PAD4 are found in the joints of RA patients, suggesting that GrB may alter the presentation of PAD4 by RA-associated class II alleles. In this study, complementary proteomic and immunologic approaches were utilized to define the effects of GrB cleavage on the structure, processing, and immunogenicity of PAD4. Hydrogen-deuterium exchange and a cell-free MHC class II antigen processing system revealed that proteolysis of PAD4 by GrB induced discrete structural changes in PAD4 that promoted enhanced presentation of several immunogenic peptides capable of stimulating PAD4-specific CD4+ T cells from patients with RA. This work demonstrates the existence of PAD4-specific T cells in patients with RA and supports a mechanistic role for GrB in enhancing the presentation of autoantigenic CD4+ T cell epitopes.

Oscarellin, an Anthranilic Acid Derivative from a Philippine Sponge, Oscarella stillans, as an Inhibitor of Inflammatory Cytokines in Macrophages.

A new anthranilic acid derivative (1) was isolated from a Philippine sponge, Oscarella stillans (Bergquist and Kelly). The structure of compound 1, named oscarellin, was determined as 2-amino-3-(3'-aminopropoxy)benzoic acid from spectroscopic data and confirmed by synthesis. We examined the immunomodulating effect of compound 1 and its mechanism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Our data indicated that the expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were significantly reduced by the pretreatment of 1 (0.1-10 µM) for 2 h. In addition, compound 1 suppressed activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-termimal kinase (JNK), but not p38 mitogen-activated protein kinase (MAPK) in LPS-stimulated RAW 264.7 cells. Compound 1 abrogated LPS-induced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activities, whereas the induction of activating transcription factor-3 (ATF-3) was increased. Taken together, our results suggest that compound 1 attenuates pro-inflammatory cytokines via the suppression of JNK, ERK, AP-1, and NF-κB and the activation of the ATF-3 signaling pathway.

A cell-free antigen processing system informs HIV-1 epitope selection and vaccine design.

Distinct CD4+ T cell epitopes have been associated with spontaneous control of HIV-1 replication, but analysis of antigen-dependent factors that influence epitope selection is lacking. To examine these factors, we used a cell-free antigen processing system that incorporates soluble HLA-DR (DR1), HLA-DM (DM), cathepsins, and full-length protein antigens for epitope identification by LC-MS/MS. HIV-1 Gag, Pol, Env, Vif, Tat, Rev, and Nef were examined using this system. We identified 35 novel epitopes, including glycopeptides. Epitopes from smaller HIV-1 proteins mapped to regions of low protein stability and higher solvent accessibility. HIV-1 antigens associated with limited CD4+ T cell responses were processed efficiently, while some protective epitopes were inefficiently processed. 55% of epitopes obtained from cell-free processing induced memory CD4+ T cell responses in HIV-1+ donors, including eight of 19 novel epitopes tested. Thus, an in vitro processing system utilizing the components of Class II processing reveals factors influencing epitope selection of HIV-1 and represents an approach to understanding epitope selection from non-HIV-1 antigens.

Autonomous IL-36R signaling in neutrophils activates potent antitumor effector functions.

While the rapid advancement of immunotherapies has revolutionized cancer treatment, only a small fraction of patients derive clinical benefit. Eradication of large, established tumors appears to depend on engaging and activating both innate and adaptive immune system components to mount a rigorous and comprehensive immune response. Identifying such agents is a high unmet medical need, because they are sparse in the therapeutic landscape of cancer treatment. Here, we report that IL-36 cytokine can engage both innate and adaptive immunity to remodel an immune-suppressive tumor microenvironment (TME) and mediate potent antitumor immune responses via signaling in host hematopoietic cells. Mechanistically, IL-36 signaling modulates neutrophils in a cell-intrinsic manner to greatly enhance not only their ability to directly kill tumor cells but also promote T and NK cell responses. Thus, while poor prognostic outcomes are typically associated with neutrophil enrichment in the TME, our results highlight the pleiotropic effects of IL-36 and its therapeutic potential to modify tumor-infiltrating neutrophils into potent effector cells and engage both the innate and adaptive immune system to achieve durable antitumor responses in solid tumors.

Short peptide sequences mimic HLA-DM functions.

HLA-DM (DM) plays a critical role in Ag presentation to CD4 T cells by catalyzing the exchange of peptides bound to MHC class II molecules. It is known that DM interaction with MHC II involves conformational changes in the MHC II molecule leading to the disturbance of H-bonds formed between the bound peptide and the MHC II groove leading to peptide dissociation. The specific region of the DM molecule that induces this peptide dissociation is not defined. In this study, we describe three short peptides (helper peptides) that accelerate DM-catalyzed peptide exchange. Kinetic studies presented here demonstrate that these peptides act similarly to DM in; (a) enhancing peptide binding to HLA-DR1; (b) dissociation of complexes of peptide-DR1; and (c) maintaining a receptive conformation of empty DR1. We further report that helper peptides are effective in increasing peptide binding to DR1 expressed on B cells in vitro, and, when mixed with peptide and adjuvant, cause enhanced T cell priming in HLA-DR1 Tg mice. We suggest that helper peptides might interact with the same critical residues on MHC class II that is targeted by DM.

Selection of immunodominant epitopes during antigen processing is hierarchical.

MHC II proteins present processed antigens to CD4 + T cells through a complex set of events and players that include chaperons and accessory molecules. Antigen processing machinery is optimized for the selection of the best fitting peptides, called 'immunodominant epitopes', in the MHC II groove to which, specific CD4 + T cells respond and differentiate into memory T cells. However, due to the complexity of antigen processing, understanding the parameters that lead to immunodominance has proved difficult. Moreover, immunodominance of epitopes vary, depending on multiple factors that include; simultaneous processing of multiple proteins, involvement of multiple alleles of MHC II that can bind to the same antigen, or competition among several suitable epitopes on a single protein antigen. The current dogma assumes that once an antigenic determinant is selected under a specific condition, it would emerge immunodominant wherever it is placed. Here we will discuss some established parameters that contribute to immunodominance as well as some new findings, which demonstrate that slight changes to antigen structure can cause a complete shift in epitope selection during antigen processing and distort the natural immunodominant epitope.

Studying MHC Class II Peptide Loading and Editing In Vitro.

HLA-DM is now known to have a major contribution to the selection of immunodominant epitopes. A better understanding of the mechanisms controlling epitope selection can be achieved by examination of the biophysical behavior of MHC class II molecules upon binding of antigenic peptides and of the effect of DM on the interactions. Using purified soluble molecules, in this chapter we describe several in vitro methods for measuring peptide binding to HLA-DR molecules and the effects of HLA-DM on this interaction. A simple qualitative method, Gentle SDS-PAGE Assay assesses the ability of peptides to form tight complexes with MHC class II molecules. Measuring binding kinetics is among the most informative approaches to understanding molecular mechanisms, and here we describe two different methods for measuring binding kinetics of peptide-MHC complexes. In one method, rates of association and dissociation of fluorescently labeled peptides to soluble MHC class II molecules can be determined using G50 spin columns to separate unbound peptides from those in complex with MHC molecules. In another method, association and dissociation of unlabeled peptides and MHC class II molecules can be determined in real time using BIAcore Surface Plasmon Resonance (SPR). We also describe an intrinsic tryptophan fluorescence assay for studying transient interactions of DM and MHC class II molecules.

Distorted Immunodominance by Linker Sequences or other Epitopes from a Second Protein Antigen During Antigen-Processing.

The immune system focuses on and responds to very few representative immunodominant epitopes from pathogenic insults. However, due to the complexity of the antigen processing, understanding the parameters that lead to immunodominance has proved difficult. In an attempt to uncover the determinants of immunodominance among several dominant epitopes, we utilized a cell free antigen processing system and allowed the system to identify the hierarchies among potential determinants. We then tested the results in vivo; in mice and in human. We report here, that immunodominance of known sequences in a given protein can change if two or more proteins are being processed and presented simultaneously. Surprisingly, we find that new spacer/tag sequences commonly added to proteins for purification purposes can distort the capture of the physiological immunodominant epitopes. We warn against adding tags and spacers to candidate vaccines, or recommend cleaving it off before using for vaccination.

Determinants of immunodominance for CD4 T cells.

The term immunodominance was originally defined as a restricted T cell response to a short peptide sequence derived from a given protein. The question of what determines immunodominance has been a longstanding battle for the past two decades. Hundreds of papers have been written on different aspects of epitope selection during antigen processing documenting the complexity of the process. Antigen processing machinery involves several accessory molecules and chaperons coevolved with proteins of Major Histocompatibility Complex (MHC) molecules that each plays its part in epitope selection. These molecules are targeted to specialized vesicular compartments that also accommodate antigen processing enzymes called cathepsins. Within the antigen processing compartments, highly regulated pH gradient and reducing conditions and enzymes necessary for denaturation of the antigens are available and function to optimize processing of antigen and selection of the fittest for transport to the cell membrane and presentation to T cells. Despite the complexity, a cell free reductionist antigen processing system was recently reported that included only few purified proteins, but was shown to process and select physiologically relevant epitopes from full length protein antigens. Due to its minimalist nature the system has been quite helpful in dissecting the factors that contribute to epitope selection during antigen processing. In this review, we would summarize and highlight models that may explain how the dominant epitope may be selected for presentation to CD4(+) helper T cells.

MHC Class II Auto-Antigen Presentation is Unconventional.

Antigen presentation is highly critical in adoptive immunity. Only by interacting with antigens presented by major histocompatibility complex class II molecules, helper T cells can be stimulated to fight infections or diseases. The degradation of a full protein into small peptide fragments bound to class II molecules is a dynamic, lengthy process consisting of many steps and chaperons. Deregulation in any step of antigen processing could lead to the development of self-reactive T cells or defective immune response to pathogens. Indeed, human leukocyte antigens class II genes are the predominant contributors to susceptibility to autoimmune diseases. Conventional antigen-processing calls for internalization of extracellular antigens followed by processing and epitope selection within antigen-processing subcellular compartments, enriched with all necessary accessory molecules, processing enzymes, and proper pH and denaturing conditions. However, recent data examining the temporal relationship between antigen uptakes, processing, and epitope selection revealed unexpected characteristics for auto-antigenic epitopes, which were not shared with antigenic epitopes from pathogens. This review provides a discussion of the relevance of these findings to the mechanisms of autoimmunity.

Exogenous antigens bind MHC class II first, and are processed by cathepsins later.

The field of antigen processing and presentation is likely one of the most well defined areas in immunology based on decades of intense molecular and structural studies. Many molecules contributing to antigen processing and presentation have been discovered and their mechanisms of action been largely defined, yet a major question, which lies at the very core of the field has remained hard to pin down. The question is what determines immunodominance? Immunodominance is defined as a few specific epitopes being selected to represent an antigen to the immune system and provide targets for T cells. Many studies have aimed at understanding how epitopes are selected. A range of hypotheses related to the structural features of antigens, sensitivity to proteases, epitope affinity for MHC II, T cell precursor frequency, and T cell receptor affinity for peptide/MHC II have been considered. However, because of the variety of proteins and factors involved in antigen processing and enormous complexity, finding an answer has been challenging. Here we make an effort to tease out the sequence of events in antigen processing that promote selection of immunodominant epitopes for exogenous antigens.

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) before 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 and Its Role in MHC Class II Antigen Presentation.

Helper T cells are stimulated to fight infections or diseases upon recognition of peptides from antigens that are processed and presented by the proteins of Major Histocompatibility Complex (MHC) Class II molecules. Degradation of a full protein into small peptide fragments is a lengthy process consisting of many steps and chaperones. Malfunctions during any step of antigen processing could lead to the development of self-reactive T cells or defective immune response to pathogens. Although much has been accomplished regarding how antigens are processed and presented to T cells, many questions still remain unanswered, preventing the design of therapeutics for direct intervention with antigen processing. Here, we review published work on the discovery and function of a MHC class II molecular chaperone, HLA-DO, in human, and its mouse analog H2-O, herein called DO. While DO was originally discovered decades ago, elucidating its function has proven challenging. DO was discovered in association with another chaperone HLA-DM (DM) but unlike DM, its distribution is more tissue specific, and its function more subtle.

Studying MHC class II peptide loading and editing in vitro.

HLA-DM is now known to have a major contribution to the selection of immunodominant epitopes. A better understanding of the mechanisms controlling epitope selection can be achieved by examination of the biophysical behavior of major histocompatibility complex (MHC) class II molecules upon binding of antigenic peptides and the effect of DM on the interactions. Using purified soluble molecules, in this chapter, we describe several in vitro methods for measuring peptide binding to HLA-DR molecules and the effects of HLA-DM on the interactions. A simple qualitative method, Gentle SDS-PAGE Assay, would assess the ability of peptides to form tight complexes with MHC class II molecules. Measuring binding kinetics is among the most informative approaches to understanding molecular mechanisms, and here we describe two different methods for measuring binding kinetics of peptide-MHC complexes. In one method, rates of association and dissociation of fluorescently labeled peptides to soluble MHC class II molecules can be determined using G50 spin columns to separate unbound peptides from those in complex with MHC molecules. In another method, association and dissociation of unlabeled peptides and MHC class II molecules can be determined in real time using BIAcore surface plasmon resonance (SPR). We also have described an Intrinsic Tryptophan Fluorescence Assay for studying transient interactions of DM and MHC class II molecules.

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.