Aire mediates tolerance to insulin through thymic trimming of high-affinity T cell clones.

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.

The Receptor Binding Domain of SARS-CoV-2 Lambda Variant Has a Better Chance Than the Delta Variant in Evading BNT162b2 COVID-19 mRNA Vaccine-Induced Humoral Immunity.

The SARS-CoV-2 Delta and Lambda variants had been named variants of concern (VOC) and variants of interest (VOI), respectively, by the World Health Organization (WHO). Both variants have two mutations in the spike receptor binding domain (RBD) region, with L452R and T478K mutations in the Delta variant, and L452Q and F490S mutations in the Lambda variant. We used surface plasmon resonance (SPR)-based technology to evaluate the effect of these mutations on human angiotensin-converting enzyme 2 (ACE2) and Bamlanivimab binding. The affinity for the RBD ligand, ACE2, of the Delta RBD is approximately twice as strong as that of the wild type RBD, an increase that accounts for the increased infectivity of the Delta variant. On the other hand, in spite of its amino acid changes, the Lambda RBD has similar affinity to ACE2 as the wild type RBD. The protective anti-wild type RBD antibody Bamlanivimab binds very poorly to the Delta RBD and not at all to the Lambda RBD. Nevertheless, serum antibodies from individuals immunized with the BNT162b2 vaccine were found to bind well to the Delta RBD, but less efficiently to the Lambda RBD in contrast. As a result, the blocking ability of ACE2 binding by serum antibodies was decreased more by the Lambda than the Delta RBD. Titers of sera from BNT162b2 mRNA vaccinated individuals dropped 3-fold within six months of vaccination regardless of whether the target RBD was wild type, Delta or Lambda. This may account partially for the fall off with time in the protective effect of vaccines against any variant.

SARS-CoV-2 Variants of Concern and Variants of Interest Receptor Binding Domain Mutations and Virus Infectivity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has lasted more than 2 years with over 260 million infections and 5 million deaths worldwide as of November 2021. To combat the virus, monoclonal antibodies blocking the virus binding to human receptor, the angiotensin converting enzyme 2 (ACE2), have been approved to treat the infected patients. Inactivated whole virus or the full-length virus spike encoding adenovirus or mRNA vaccines are being used to immunize the public. However, SARS-CoV-2 variants are emerging. These, to some extent, escape neutralization by the therapeutic antibodies and vaccine-induced immunity. Thus, breakthrough infections by SARS-CoV-2 variants have been reported in previously virus-infected or fully vaccinated individuals. The receptor binding domain (RBD) of the virus spike protein reacts with host ACE2, leading to the entry of the virus into the cell. It is also the major antigenic site of the virus, with more than 90% of broadly neutralizing antibodies from either infected patients or vaccinated individuals targeting the spike RBD. Therefore, mutations in the RBD region are effective ways for SARS-CoV-2 variants to gain infectivity and escape the immunity built up by the original vaccines or infections. In this review, we focus on the impact of RBD mutations in SARS-CoV-2 variants of concern (VOC) and variants of interest (VOI) on ACE2 binding affinity and escape of serum antibody neutralization. We also provide protein structure models to show how the VOC and VOI RBD mutations affect ACE2 binding and allow escape of the virus from the therapeutic antibody, bamlanivimab.

Structures suggest an approach for converting weak self-peptide tumor antigens into superagonists for CD8 T cells in cancer.

Tumors frequently express unmutated self-tumor-associated antigens (self-TAAs). However, trial results using self-TAAs as vaccine targets against cancer are mixed, often attributed to deletion of T cells with high-affinity receptors (TCRs) for self-TAAs during T cell development. Mutating these weak self-TAAs to produce higher affinity, effective vaccines is challenging, since the mutations may not benefit all members of the broad self-TAA-specific T cell repertoire. We previously identified a common weak murine self-TAA that we converted to a highly effective antitumor vaccine by a single amino acid substitution. In this case the modified and natural self-TAAs still raised very similar sets of CD8 T cells. Our structural studies herein show that the modification of the self-TAA resulted in a subtle change in the major histocompatibility complex I-TAA structure. This amino acid substitution allowed a dramatic conformational change in the peptide during subsequent TCR engagement, creating a large increase in TCR affinity and accounting for the efficacy of the modified self-TAA as a vaccine. These results show that carefully selected, well-characterized modifications to a poorly immunogenic self-TAA can rescue the immune response of the large repertoire of weakly responding natural self-TAA-specific CD8 T cells, driving them to proliferate and differentiate into functional effectors. Subsequently, the unmodified self-TAA on the tumor cells, while unable to drive this response, is nevertheless a sufficient target for the CD8 cytotoxic effectors. Our results suggest a pathway for more efficiently identifying variants of common self-TAAs, which could be useful in vaccine development, complementing other current nonantigen-specific immunotherapies.

Hidden in Plain View: Discovery of Chimeric Diabetogenic CD4 T Cell Neo-Epitopes.

The T cell antigens driving autoimmune Type 1 Diabetes (T1D) have been pursued for more than three decades. When diabetogenic CD4 T cell clones and their relevant MHCII antigen presenting alleles were first identified in rodents and humans, the path to discovering the peptide epitopes within pancreatic beta cell proteins seemed straightforward. However, as experimental results accumulated, definitive data were often absent or controversial. Work within the last decade has helped to clear up some of the controversy by demonstrating that a number of the important MHCII presented epitopes are not encoded in the natural beta cell proteins, but in fact are fusions between peptide fragments derived from the same or different proteins. Recently, the mechanism for generating these MHCII diabetogenic chimeric epitopes has been attributed to a form of reverse proteolysis, called transpeptidation, a process that has been well-documented in the production of MHCI presented epitopes. In this mini-review we summarize these data and their implications for T1D and other autoimmune responses.

Rational discovery of a cancer neoepitope harboring the KRAS G12D driver mutation.

Cytotoxic T cells targeting cancer neoantigens harboring driver mutations can lead to durable tumor regression in an HLAI-dependent manner. However, it is difficult to extend the population of patients who are eligible for neoantigen-based immunotherapy, as immunogenic neoantigen-HLA pairs are rarely shared across different patients. Thus, a way to find other human leukocyte antigen (HLA) alleles that can also present a clinically effective neoantigen is needed. Recently, neoantigen-based immunotherapy targeting the KRAS G12D mutation in patients with HLA-C*08:02 has shown effectiveness. In a proof-of-concept study, we proposed a combinatorial strategy (the combination of phylogenetic and structural analyses) to find potential HLA alleles that could also present KRAS G12D neoantigen. Compared to in silico binding prediction, this strategy avoids the uneven accuracy across different HLA alleles. Our findings extend the population of patients who are potentially eligible for immunotherapy targeting the KRAS G12D mutation. Additionally, we provide an alternative way to predict neoantigen-HLA pairs, which maximizes the clinical usage of shared neoantigens.

Immune-based mutation classification enables neoantigen prioritization and immune feature discovery in cancer immunotherapy.

Genetic mutations lead to the production of mutated proteins from which peptides are presented to T cells as cancer neoantigens. Evidence suggests that T cells that target neoantigens are the main mediators of effective cancer immunotherapies. Although algorithms have been used to predict neoantigens, only a minority are immunogenic. The factors that influence neoantigen immunogenicity are not completely understood. Here, we classified human neoantigen/neopeptide data into three categories based on their TCR-pMHC binding events. We observed a conservative mutant orientation of the anchor residue from immunogenic neoantigens which we termed the "NP" rule. By integrating this rule with an existing prediction algorithm, we found improved performance in neoantigen prioritization. To better understand this rule, we solved several neoantigen/MHC structures. These structures showed that neoantigens that follow this rule not only increase peptide-MHC binding affinity but also create new TCR-binding features. These molecular insights highlight the value of immune-based classification in neoantigen studies and may enable the design of more effective cancer immunotherapies.

Beryllium-specific CD4+ T cells induced by chemokine neoantigens perpetuate inflammation.

Discovering dominant epitopes for T cells, particularly CD4+ T cells, in human immune-mediated diseases remains a significant challenge. Here, we used bronchoalveolar lavage (BAL) cells from HLA-DP2-expressing patients with chronic beryllium disease (CBD), a debilitating granulomatous lung disorder characterized by accumulations of beryllium-specific (Be-specific) CD4+ T cells in the lung. We discovered lung-resident CD4+ T cells that expressed a disease-specific public CDR3ß T cell receptor motif and were specific to Be-modified self-peptides derived from C-C motif ligand 4 (CCL4) and CCL3. HLA-DP2-CCL/Be tetramer staining confirmed that these chemokine-derived peptides represented major antigenic targets in CBD. Furthermore, Be induced CCL3 and CCL4 secretion in the lungs of mice and humans. In a murine model of CBD, the addition of LPS to Be oxide exposure enhanced CCL4 and CCL3 secretion in the lung and significantly increased the number and percentage of CD4+ T cells specific for the HLA-DP2-CCL/Be epitope. Thus, we demonstrate a direct link between Be-induced innate production of chemokines and the development of a robust adaptive immune response to those same chemokines presented as Be-modified self-peptides, creating a cycle of innate and adaptive immune activation.

Tolerance induction in memory CD4 T cells is partial and reversible.

Memory T cells respond rapidly in part because they are less reliant on a heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen-specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of reactivating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggests they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re-infection. These data indicate that antigen-specific tolerogenic strategies must examine multiple parameters of Tcell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells.

Lysosomal cathepsin creates chimeric epitopes for diabetogenic CD4 T cells via transpeptidation.

The identification of the peptide epitopes presented by major histocompatibility complex class II (MHCII) molecules that drive the CD4 T cell component of autoimmune diseases has presented a formidable challenge over several decades. In type 1 diabetes (T1D), recent insight into this problem has come from the realization that several of the important epitopes are not directly processed from a protein source, but rather pieced together by fusion of different peptide fragments of secretory granule proteins to create new chimeric epitopes. We have proposed that this fusion is performed by a reverse proteolysis reaction called transpeptidation, occurring during the catabolic turnover of pancreatic proteins when secretory granules fuse with lysosomes (crinophagy). Here, we demonstrate several highly antigenic chimeric epitopes for diabetogenic CD4 T cells that are produced by digestion of the appropriate inactive fragments of the granule proteins with the lysosomal protease cathepsin L (Cat-L). This pathway has implications for how self-tolerance can be broken peripherally in T1D and other autoimmune diseases.

A monoclonal antibody with broad specificity for the ligands of insulin B:9-23 reactive T cells prevents spontaneous type 1 diabetes in mice.

Activation of T cells specific for insulin B chain amino acids 9 to 23 (B:9-23) is essential for the initiation of type 1 diabetes (T1D) in non-obese diabetic mice. We previously reported that peptide/MHC complexes containing optimized B:9-23 mimotopes can activate most insulin-reactive pathogenic T cells. A monoclonal antibody (mAb287) targeting these complexes prevented disease in 30-50% of treated animals (compared to 10% of animals given an isotype control). The incomplete protection is likely due to the relatively low affinity of the antibody for its ligand and limited specificity. Here, we report an enhanced reagent, mAb757, with improved specificity, affinity, and efficacy in modulating T1D. Importantly, mAb757 bound with nanomolar affinity to agonists of both "type A" and "type B" cells and suppressed "type B" cells more efficiently than mAb287. When given weekly starting at 4 weeks of age, mAb757 protected ~70% of treated mice from developing T1D for at least 35 weeks, while mAb287 only delayed disease in 25% of animals under the same conditions. Consistent with its higher affinity, mAb757 was also able to stain antigen-presenting cells loaded with B:9-23 mimotopes in vivo. We conclude that monoclonal antibodies that can block the presentation of pathogenic T cell receptor epitopes are viable candidates for antigen-specific immunotherapy for T1D.

Age-associated B Cells Appear in Patients with Granulomatous Lung Diseases.

Rationale: A subpopulation of B cells (age-associated B cells [ABCs]) is increased in mice and humans with infections or autoimmune diseases. Because depletion of these cells might be valuable in patients with certain lung diseases, the goal was to find out if ABC-like cells were at elevated levels in such patients.Objectives: To measure ABC-like cell percentages in patients with lung granulomatous diseases.Methods: Peripheral blood and BAL cells from patients with sarcoidosis, beryllium sensitivity, or hypersensitivity pneumonitis and healthy subjects were analyzed for the percentage of B cells that were ABC-like, defined by expression of CD11c, low levels of CD21, FcRL 1-5 (Fc receptor-like protein 1-5) expression, and, in some cases, T-bet.Measurements and Main Results: ABC-like cells in blood were at low percentages in healthy subjects and higher percentages in patients with sarcoidosis as well as at high percentages among BAL cells of patients with sarcoidosis, beryllium disease, and hypersensitivity pneumonitis. Treatment of patients with sarcoidosis led to reduced percentages of ABC-like cells in blood.Conclusions: Increased levels of ABC-like cells in patients with sarcoidosis may be useful in diagnosis. The increase in percentage of ABC-like cells in patients with lung granulomatous diseases and decrease in treated patients suggests that depletion of these cells may be valuable.

Inherent reactivity of unselected TCR repertoires to peptide-MHC molecules.

The repertoire of αß T cell antigen receptors (TCRs) on mature T cells is selected in the thymus where it is rendered both self-tolerant and restricted to the recognition of major histocompatibility complex molecules presenting peptide antigens (pMHC). It remains unclear whether germline TCR sequences exhibit an inherent bias to interact with pMHC prior to selection. Here, we isolated TCR libraries from unselected thymocytes and upon reexpression of these random TCR repertoires in recipient T cell hybridomas, interrogated their reactivities to antigen-presenting cell lines. While these random TCR combinations could potentially have reacted with any surface molecule on the cell lines, the hybridomas were stimulated most frequently by pMHC ligands. The nature and CDR3 loop composition of the TCRß chain played a dominant role in determining pMHC-reactivity. Replacing the germline regions of mouse TCRß chains with those of other jawed vertebrates preserved reactivity to mouse pMHC. Finally, introducing the CD4 coreceptor into the hybridomas increased the proportion of cells that could respond to pMHC ligands. Thus, αß TCRs display an intrinsic and evolutionary conserved bias for pMHC molecules in the absence of any selective pressure, which is further strengthened in the presence of coreceptors.

Structural characteristics of lipocalin allergens: Crystal structure of the immunogenic dog allergen Can f 6.

Lipocalins represent the most important protein family of the mammalian respiratory allergens. Four of the seven named dog allergens are lipocalins: Can f 1, Can f 2, Can f 4, and Can f 6. We present the structure of Can f 6 along with data on the biophysical and biological activity of this protein in comparison with other animal lipocalins. The Can f 6 structure displays the classic lipocalin calyx-shaped ligand binding cavity within a central ß-barrel similar to other lipocalins. Despite low sequence identity between the different dog lipocalin proteins, there is a high degree of structural similarity. On the other hand, Can f 6 has a similar primary sequence to cat, horse, mouse lipocalins as well as a structure that may underlie their cross reactivity. Interestingly, the entrance to the ligand binding pocket is capped by a His instead of the usually seen Tyr that may help select its natural ligand binding partner. Our highly pure recombinant Can f 6 is able to bind to human IgE (hIgE) demonstrating biological antigenicity.

How C-terminal additions to insulin B-chain fragments create superagonists for T cells in mouse and human type 1 diabetes.

In type 1 diabetes (T1D), proinsulin is a major autoantigen and the insulin B:9-23 peptide contains epitopes for CD4+ T cells in both mice and humans. This peptide requires carboxyl-terminal mutations for uniform binding in the proper position within the mouse IAg7 or human DQ8 major histocompatibility complex (MHC) class II (MHCII) peptide grooves and for strong CD4+ T cell stimulation. Here, we present crystal structures showing how these mutations control CD4+ T cell receptor (TCR) binding to these MHCII-peptide complexes. Our data reveal stricking similarities between mouse and human CD4+ TCRs in their interactions with these ligands. We also show how fusions between fragments of B:9-23 and of proinsulin C-peptide create chimeric peptides with activities as strong or stronger than the mutated insulin peptides. We propose transpeptidation in the lysosome as a mechanism that could accomplish these fusions in vivo, similar to the creation of fused peptide epitopes for MHCI presentation shown to occur by transpeptidation in the proteasome. Were this mechanism limited to the pancreas and absent in the thymus, it could provide an explanation for how diabetogenic T cells escape negative selection during development but find their modified target antigens in the pancreas to cause T1D.

Development of T cell lines sensitive to antigen stimulation.

Immortalized T cells such as T cell hybridomas, transfectomas, and transductants are useful tools to study tri-molecular complexes consisting of peptide, MHC, and T cell receptor (TCR) molecules. These cells have been utilized for antigen discovery studies for decades due to simplicity and rapidness of growing cells. However, responsiveness to antigen stimulation is typically less sensitive compared to primary T cells, resulting in occasional false negative outcomes especially for TCRs having low affinity to a peptide-MHC complex (pMHC). To overcome this obstacle, we genetically engineered T cell hybridomas to express additional CD3 molecules as well as CD4 with two amino acid substitutions that increase affinity to MHC class II molecules. The manipulated T cell hybridomas that were further transduced with retroviral vectors encoding TCRs of interest responded to cognate antigens more robustly than non-manipulated cells without evoking non-antigen specific reactivity. Of importance, the manipulation with CD3 and mutated human CD4 expression was effective in increasing responsiveness of T cell hybridomas to a wide variety of TCR, peptide, and MHC combinations across class II genetic loci (i.e. HLA-DR, HLA-DQ, HLA-DP, and murine H2-IA) and species (i.e. both humans and mice), and thus will be useful to identify antigen specificity of T cells.

MHC class I loaded ligands from breast cancer cell lines: A potential HLA-I-typed antigen collection.

To build a catalog of peptides presented by breast cancer cells, we undertook systematic MHC class I immunoprecipitation followed by elution of MHC class I-loaded peptides in breast cancer cells. We determined the sequence of 3196 MHC class I ligands representing 1921 proteins from a panel of 20 breast cancer cell lines. After removing duplicate peptides, i.e., the same peptide eluted from more than one cell line, the total number of unique peptides was 2740. Of the unique peptides eluted, more than 1750 had been previously identified, and of these, sixteen have been shown to be immunogenic. Importantly, half of these immunogenic peptides were shared between different breast cancer cell lines. MHC class I binding probability was used to plot the distribution of the eluted peptides in accordance with the binding score for each breast cancer cell line. We also determined that the tested breast cancer cells presented 89 mutation-containing peptides and peptides derived from aberrantly translated genes, 7 of which were shared between four or two different cell lines. Overall, the high throughput identification of MHC class I-loaded peptides is an effective strategy for systematic characterization of cancer peptides, and could be employed for design of multi-peptide anticancer vaccines. SIGNIFICANCE: By employing proteomic analyses of eluted peptides from breast cancer cells, the current study has built an initial HLA-I-typed antigen collection for breast cancer research. It was also determined that immunogenic epitopes can be identified using established cell lines and that shared immunogenic peptides can be found in different cancer types such as breast cancer and leukemia. Importantly, out of 3196 eluted peptides that included duplicate peptides in different cells 89 peptides either contained mutation in their sequence or were derived from aberrant translation suggesting that mutation-containing epitopes are on the order of 2-3% in breast cancer cells. Finally, our results suggest that interfering with MHC class I function is one of the mechanisms of how tumor cells escape immune system attack.

C-terminal modification of the insulin B:11-23 peptide creates superagonists in mouse and human type 1 diabetes.

A polymorphism at ß57 in some major histocompatibility complex class II (MHCII) alleles of rodents and humans is associated with a high risk for developing type 1 diabetes (T1D). However, a highly diabetogenic insulin B chain epitope within the B:9-23 peptide is presented poorly by these alleles to a variety of mouse and human CD4 T cells isolated from either nonobese diabetic (NOD) mice or humans with T1D. We have shown for both species that mutations at the C-terminal end of this epitope dramatically improve presentation to these T cells. Here we present the crystal structures of these mutated peptides bound to mouse IAg7 and human HLA-DQ8 that show how the mutations function to improve T-cell activation. In both peptide binding grooves, the mutation of B:22R to E in the peptide changes a highly unfavorable side chain for the p9 pocket to an optimal one that is dependent on the ß57 polymorphism, accounting for why these peptides bind much better to these MHCIIs. Furthermore, a second mutation of the adjacent B:21 (E to G) removes a side chain from the surface of the complex that is highly unfavorable for a subset of NOD mouse CD4 cells, thereby greatly enhancing their response to the complex. These results point out the similarities between the mouse and human responses to this B chain epitope in T1D and suggest there may be common posttranslational modifications at the C terminus of the peptide in vivo to create the pathogenic epitopes in both species.

Clipping of arginine-methylated histone tails by JMJD5 and JMJD7.

Two of the unsolved, important questions about epigenetics are: do histone arginine demethylases exist, and is the removal of histone tails by proteolysis a major epigenetic modification process? Here, we report that two orphan Jumonji C domain (JmjC)-containing proteins, JMJD5 and JMJD7, have divalent cation-dependent protease activities that preferentially cleave the tails of histones 2, 3, or 4 containing methylated arginines. After the initial specific cleavage, JMJD5 and JMJD7, acting as aminopeptidases, progressively digest the C-terminal products. JMJD5-deficient fibroblasts exhibit dramatically increased levels of methylated arginines and histones. Furthermore, depletion of JMJD7 in breast cancer cells greatly decreases cell proliferation. The protease activities of JMJD5 and JMJD7 represent a mechanism for removal of histone tails bearing methylated arginine residues and define a potential mechanism of transcription regulation.

The domestic cat antibody response to feline herpesvirus-1 increases with age.

Herpesviruses establish lifelong infections, normally characterized by prolonged periods of latency with intermittent episodes of viral reactivation. Feline herpesvirus-1 (FHV-1) infects domestic cats, and epidemiological studies indicate that many or most domestic cats are exposed to FHV-1, but the strength and longevity of the antibody response to FHV-1 is not fully characterized. Here we describe development of an ELISA, using lysates of cat cells infected with FHV-1, that measure feline antibodies against FHV-1. The assay is sensitive, quantitative and has a large dynamic range. We found that serum anti-FHV-1 antibodies primarily recognize FHV-1 proteins of the Late (L) class and are primarily of the IgG isotype. We then analyzed serum from a cross-sectional cohort of 100 client-owned cats that differed in age, sex and vaccination history. While there was no difference in FHV-1 antibody responses between females and males, antibody levels were significantly increased in older cats in comparison with younger animals (p=0.01). Surprisingly, as the length of time since the most recent vaccination increased, there was no corresponding drop in serum anti-FHV-1 antibody. These data suggest that FHV-1 immunity is very long-lived and support the current recommendation that many cats do not require revaccination against FHV-1 annually.