Commensal bacteria maintain a Qa-1b-restricted unconventional CD8+ T population in gut epithelium.

Intestinal intraepithelial lymphocytes (IELs) are characterized by an unusual phenotype and developmental pathway, yet their specific ligands and functions remain largely unknown. Here by analysis of QFL T cells, a population of CD8+ T cells critical for monitoring the MHC I antigen processing pathway, we established that unconventional Qa-1b-restricted CD8+ T cells are abundant in intestinal epithelium. We found that QFL T cells showed a Qa-1b-dependent unconventional phenotype in the spleen and small intestine of naïve wild-type mice. The splenic QFL T cells showed innate-like functionality exemplified by rapid response to cytokines or antigens, while the gut population was refractory to stimuli. Microbiota was required for the maintenance, but not the initial gut homing of QFL T cells. Moreover, monocolonization with Pediococcus pentosaceus, which expresses a peptide that cross-activated QFL T cells, was sufficient to maintain QFL T cells in the intestine. Thus, microbiota is critical for shaping the Qa-1b-restricted IEL landscape.

Proper development of long-lived memory CD4 T cells requires HLA-DO function.

Introduction: HLA-DO (DO) is an accessory protein that binds DM for trafficking to MIIC and has peptide editing functions. DO is mainly expressed in thymic medulla and B cells. Using biochemical experiments, our lab has discovered that DO has differential effects on editing peptides of different sequences: DO increases binding of DM-resistant peptides and reduces the binding of DM-sensitive peptides to the HLA-DR1 molecules. In a separate line of work, we have established that appropriate densities of antigen presentation by B cells during the contraction phase of an infection, induces quiescence in antigen experienced CD4 T cells, as they differentiate into memory T cells. This quiescence phenotype helps memory CD4 T cell survival and promotes effective memory responses to secondary Ag challenge. Methods: Based on our mechanistic understanding of DO function, it would be expected that if the immunodominant epitope of antigen is DM-resistant, presentation of decreased densities of pMHCII by B cells would lead to faulty development of memory CD4 T cells in the absence of DO. We explored the effects of DO on development of memory CD4 T cells and B cells utilizing two model antigens, H5N1-Flu Ag bearing DM-resistant, and OVA protein, which has a DM-sensitive immunodominant epitope and four mouse strains including two DO-deficient Tg mice. Using Tetramers and multiple antibodies against markers of memory CD4 T cells and B cells, we tracked memory development. Results: We found that immunized DR1+DO-KO mice had fewer CD4 memory T cells and memory B cells as compared to the DR1+DO-WT counterpart and had compromised recall responses. Conversely, OVA specific memory responses elicited in HA immunized DR1+DO-KO mice were normal. Conclusion: These results demonstrate that in the absence of DO, the presentation of cognate foreign antigens in the DO-KO mice is altered and can impact the proper development of memory cells. These findings provide new insights on vaccination design leading to better immune memory responses.

H2-O deficiency promotes regulatory T cell differentiation and CD4 T cell hyperactivity.

Regulatory T cells (Treg) are crucial immune modulators, yet the exact mechanism of thymic Treg development remains controversial. Here, we present the first direct evidence for H2-O, an MHC class II peptide editing molecular chaperon, on selection of thymic Tregs. We provide evidence that lack of H2-O in the thymic medulla promotes thymic Treg development and leads to an increased peripheral Treg frequency. Single-cell RNA-sequencing (scRNA-seq) analysis of splenic CD4 T cells revealed not only of an enrichment of effector-like Tregs but also of activated CD4 T cells in the absence of H2-O. Our data support two concepts; a) lack of H2-O expression in the thymic medulla creates an environment permissive to Treg development and, b) that loss of H2-O drives increased basal auto-stimulation of CD4 T cells. These findings can help in better understanding of predispositions to autoimmunity and design of therapeutics for treatment of autoimmune diseases.

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.

Commensal Bacteria Maintain a Qa-1 b -restricted Unconventional CD8 + T Population in Gut Epithelium.

Intestinal intraepithelial lymphocytes (IELs) are characterized by an unusual phenotype and developmental pathway, yet their specific ligands and functions remain largely unknown. Here by analysis of QFL T cells, a population of CD8 + T cells critical for monitoring the MHC I antigen processing pathway, we established that unconventional Qa-1 b -restricted CD8 + T cells are abundant in intestinal epithelium. We found that QFL T cells showed a Qa-1 b -dependent unconventional phenotype in the spleen and small intestine of naïve wild-type mice. The splenic QFL T cells showed innate-like functionality exemplified by rapid response to cytokines or antigen, while the gut population was refractory to stimuli. Microbiota was required for the maintenance, but not the initial gut homing of QFL T cells. Moreover, monocolonization with Pediococcus pentosaceus, which expresses a peptide that cross-activated QFL T cells, was sufficient to maintain QFL T cells in the intestine. Thus, microbiota is critical for shaping the Qa-1 b -restricted IEL landscape.

H2-O deficiency promotes regulatory T cell differentiation and CD4 T cell hyperactivity.

Regulatory T cells (Treg) are crucial immune modulators, yet the exact mechanism of thymic Treg development remains controversial. Here, we present the first direct evidence for H2-O, an MHC class II peptide editing molecular chaperon, on selection of thymic Tregs. We identified that lack of H2-O in the thymic medulla promotes thymic Treg development and leads to an increased peripheral Treg frequency. Single-cell RNA-sequencing (scRNA-seq) analysis of splenic CD4 T cells revealed not only an enrichment of effector-like Tregs, but also activated CD4 T cells in the absence of H2-O. Our data support two concepts; a) lack of H2-O expression in the thymic medulla creates an environment permissive to Treg development and, b) that loss of H2-O drives increased basal auto-stimulation of CD4 T cells. These findings can help in better understanding of predispositions to autoimmunity and design of therapeutics for treatment of autoimmune diseases.

The ER Aminopeptidases, ERAP1 and ERAP2, synergize to self-modulate their respective activities.

Introduction: Critical steps in Major Histocompatibility Complex Class I (MHC-I) antigen presentation occur in the endoplasmic reticulum (ER). In general, peptides that enter the ER are longer than the optimal length for MHC-I binding. The final trimming of MHC-I epitopes is performed by two related aminopeptidases, ERAP1 and ERAP2 in humans that possess unique and complementary substrate trimming specificities. While ERAP1 efficiently trims peptides longer than 9 residues, ERAP2 preferentially trims peptides shorter than 9 residues. Materials and Methods: Using a combination of biochemical and proteomic studies followed by biological verification. Results: We demonstrate that the optimal ligands for either enzyme act as inhibitors of the other enzyme. Specifically, the presence of octamers reduced the trimming of long peptides by ERAP1, while peptides longer than nonomers inhibit ERAP2 activity. Discussion: We propose a mechanism for how ERAP1 and ERAP2 synergize to modulate their respective activities and shape the MHC-I peptidome by generating optimal peptides for presentation.

TCR-mimic bispecific antibodies to target the HIV-1 reservoir.

HIV-1 infection is incurable due to the persistence of the virus in a latent reservoir of resting memory CD4+ T cells. "Shock-and-kill" approaches that seek to induce HIV-1 gene expression, protein production, and subsequent targeting by the host immune system have been unsuccessful due to a lack of effective latency-reversing agents (LRAs) and kill strategies. In an effort to develop reagents that could be used to promote killing of infected cells, we constructed T cell receptor (TCR)-mimic antibodies to HIV-1 peptide-major histocompatibility complexes (pMHC). Using phage display, we panned for phages expressing antibody-like variable sequences that bound HIV-1 pMHC generated using the common HLA-A*02:01 allele. We targeted three epitopes in Gag and reverse transcriptase identified and quantified via Poisson detection mass spectrometry from cells infected in vitro with a pseudotyped HIV-1 reporter virus (NL4.3 dEnv). Sequences isolated from phages that bound these pMHC were cloned into a single-chain diabody backbone (scDb) sequence, such that one fragment is specific for an HIV-1 pMHC and the other fragment binds to CD3ε, an essential signal transduction subunit of the TCR. Thus, these antibodies utilize the sensitivity of T cell signaling as readouts for antigen processing and as agents to promote killing of infected cells. Notably, these scDbs are exquisitely sensitive and specific for the peptide portion of the pMHC. Most importantly, one scDb caused killing of infected cells presenting a naturally processed target pMHC. This work lays the foundation for a novel therapeutic killing strategy toward elimination of the HIV-1 reservoir.

How Does B Cell Antigen Presentation Affect Memory CD4 T Cell Differentiation and Longevity?

Dendritic cells are the antigen presenting cells that process antigens effectively and prime the immune system, a characteristic that have gained them the spotlights in recent years. B cell antigen presentation, although less prominent, deserves equal attention. B cells select antigen experienced CD4 T cells to become memory and initiate an orchestrated genetic program that maintains memory CD4 T cells for life of the individual. Over years of research, we have demonstrated that low levels of antigens captured by B cells during the resolution of an infection render antigen experienced CD4 T cells into a quiescent/resting state. Our studies suggest that in the absence of antigen, the resting state associated with low-energy utilization and proliferation can help memory CD4 T cells to survive nearly throughout the lifetime of mice. In this review we would discuss the primary findings from our lab as well as others that highlight our understanding of B cell antigen presentation and the contributions of the MHC Class II accessory molecules to this outcome. We propose that the quiescence induced by the low levels of antigen presentation might be a mechanism necessary to regulate long-term survival of CD4 memory T cells and to prevent cross-reactivity to autoantigens, hence autoimmunity.

Partnering for the major histocompatibility complex class II and antigenic determinant requires flexibility and chaperons.

Cytotoxic, or helper T cells recognize antigen via T cell receptors (TCRs) that can see their target antigen as short sequences of peptides bound to the groove of proteins of major histocompatibility complex (MHC) class I, and class II respectively. For MHC class II epitope selection from exogenous pathogens or self-antigens, participation of several accessory proteins, molecular chaperons, processing enzymes within multiple vesicular compartments is necessary. A major contributing factor is the MHC class II structure itself that uniquely offers a dynamic and flexible groove essential for epitope selection. In this review, I have taken a historical perspective focusing on the flexibility of the MHC II molecules as the driving force in determinant selection and interactions with the accessory molecules in antigen processing, HLA-DM and HLA-DO.

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.

The love and hate relationship of HLA-DM/DO in the selection of immunodominant epitopes.

Successful activation of CD4 T cells is centered around the ability of antigen presenting cells to successfully process, select Class II immunodominant epitopes from exogenous antigens and to present it to cognate T cells. To achieve this, newly synthesized MHC-II molecules are transferred to a specialized compartment which contain both exogenous antigens and the Class II processing machinery. Here in a process known as 'editing,' the Class II accessory molecule DM (HLA-DM human; murine H2-M) facilitates the loading and selection of exogenous peptides to MHC class II molecules thereby assuring proper selection of immunodominant epitopes. A second Class II accessory molecule, DO (HLA-DO human; murine H2-O), mainly present in B cells and thymic epithelium also contributes to the selection of immunodominant epitopes. Yet, despite a wealth of mechanistic insights into how DM functions, understanding the contributions of DO to epitope selection has proven to be highly challenging. In this review, we have attempted to discuss published in vitro and in vivo data during the past three years with insights into the biology of DO.

Lack of the MHC class II chaperone H2-O causes susceptibility to autoimmune diseases.

DO (HLA-DO, in human; murine H2-O) is a highly conserved nonclassical major histocompatibility complex class II (MHC II) accessory molecule mainly expressed in the thymic medulla and B cells. Previous reports have suggested possible links between DO and autoimmunity, Hepatitis C (HCV) infection, and cancer, but the mechanism of how DO contributes to these diseases remains unclear. Here, using a combination of various in vivo approaches, including peptide elution, mixed lymphocyte reaction, T-cell receptor (TCR) deep sequencing, tetramer-guided naïve CD4 T-cell precursor enumeration, and whole-body imaging, we report that DO affects the repertoire of presented self-peptides by B cells and thymic epithelium. DO induces differential effects on epitope presentation and thymic selection, thereby altering CD4 T-cell precursor frequencies. Our findings were validated in two autoimmune disease models by demonstrating that lack of DO increases autoreactivity and susceptibility to autoimmune disease development.

How a Proposed Hypothesis during My PhD Training Shaped My Career.

In this memoir-style essay, I have narrated the evolution of my scientific career, as deeply influenced by my PhD training and the mentorship of Professor Eli Sercarz. Starting in his lab, and continuing to my own laboratory, many of the questions we have pursued link in some way to Eli's ideas. In this essay, I have summarized the path that I followed after graduating from his lab and highlight findings along the way. I apologize to my colleagues whose work was not discussed here due to the nature of this review and space limitations.

A Public BCR Present in a Unique Dual-Receptor-Expressing Lymphocyte from Type 1 Diabetes Patients Encodes a Potent T Cell Autoantigen.

T and B cells are the two known lineages of adaptive immune cells. Here, we describe a previously unknown lymphocyte that is a dual expresser (DE) of TCR and BCR and key lineage markers of both B and T cells. In type 1 diabetes (T1D), DEs are predominated by one clonotype that encodes a potent CD4 T cell autoantigen in its antigen binding site. Molecular dynamics simulations revealed that this peptide has an optimal binding register for diabetogenic HLA-DQ8. In concordance, a synthetic version of the peptide forms stable DQ8 complexes and potently stimulates autoreactive CD4 T cells from T1D patients, but not healthy controls. Moreover, mAbs bearing this clonotype are autoreactive against CD4 T cells and inhibit insulin tetramer binding to CD4 T cells. Thus, compartmentalization of adaptive immune cells into T and B cells is not absolute, and violators of this paradigm are likely key drivers of autoimmune diseases.

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.

What to do with HLA-DO/H-2O two decades later?

The main objective of antigen processing is to orchestrate the selection of immunodominant epitopes for recognition by CD4 T cells. To achieve this, MHC class II molecules have evolved with a flexible peptide-binding groove in need of a bound peptide. Newly synthesized MHC-II molecules bind a class II invariant chain (Ii) upon synthesis and are shuttled to a specialized compartment, where they encounter exogenous antigens. Ii serves multiple functions, one of which is to maintain the shape of the MHC-II groove so that it can readily bind exogenous antigens upon dissociation of the Ii peptide in MHC- II compartment. MIIC contains processing enzymes, one or both accessory molecules, HLA-DM/H2-M (DM) and HLA-DO/H2-O (DO), and optimal denaturing conditions. In a process known as "editing," DM facilitates the dissociation of the invariant chain peptide, CLIP, for exchange with exogenous antigens. Despite the availability of mechanistic insights into DM functions, understanding how DO contributes to epitope selection has proven to be more challenging. The current dogma assumes that DO inhibits DM, whereas an opposing model suggests that DO fine-tunes the epitope selection process. Understanding which of these, or potentially other models of DO function is important, as DO variants have been linked to autoimmunity, cancer, and the generation of broadly neutralizing antibodies to viruses. This review therefore attempts to evaluate experimental evidence in support of these hypotheses, with an emphasis on the less discussed model, and to explore intriguing questions about the importance of DO in biology.

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.

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.