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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Mix to validate: a facile, reversible PEGylation for fast screening of potential therapeutic proteins in vivo.

: To advance the rate of novel protein therapies entering the clinic, we provide researchers a facile tool for protein drug efficacy testing in animal models in a high throughput manner. Here, we utilize the concept of PEGylating proteins through complementary interactions between His-tag and Ni complex of NTA, a well-established practice in protein research, to improve blood half-life of therapeutic protein candidates after systemic administration .

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.

How HLA-DM works: recognition of MHC II conformational heterogeneity.

Helper T cells respond to peptide antigens derived from exogenous sources presented by MHC II on antigen presenting cells. Antigens from pathogens are internalized by professional antigen presenting cells (APC) and processed for presentation. Certain epitopes are selected during processing as the final peptides for stimulation of T cells and are termed "immunodominant".

A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes.

Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4(+) T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: human leukocyte antigen-DR1 (HLA-DR1), HLA-DM and cathepsins.

Conformational heterogeneity of MHC class II induced upon binding to different peptides is a key regulator in antigen presentation and epitope selection.

T cells bearing alphabeta receptors recognize antigenic peptides bound to class I and class II glycoproteins encoded in the major histocompatibility complex (MHC). Cytotoxic and helper T cells respond respectively to peptide antigens derived from endogenous sources presented by MHC class I, and exogenous sources presented by MHC II, on antigen presenting cells. Differences in the MHC class I and class II structures and their maturation pathways have evolved to optimize antigen presentation to their respective T cells.

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.

HLA-DM targets the hydrogen bond between the histidine at position beta81 and peptide to dissociate HLA-DR-peptide complexes.

The peptide editor HLA-DM (DM) mediates exchange of peptides bound to major histocompatibility (MHC) class II molecules during antigen processing; however, the mechanism by which DM displaces peptides remains unclear. Here we generated a soluble mutant HLA-DR1 with a histidine-to-asparagine substitution at position 81 of the beta-chain (DR1betaH81N) to perturb an important hydrogen bond between MHC class II and peptide. Peptide-DR1betaH81N complexes dissociated at rates similar to the dissociation rates of DM-induced peptide-wild-type DR1, and DM did not enhance the dissociation of peptide-DR1betaH81N complexes.