Antibody sequence determinants of viral antigen specificity.

Throughout life, humans experience repeated exposure to viral antigens through infection and vaccination, resulting in the generation of diverse, antigen-specific antibody repertoires. A paramount feature of antibodies that enables their critical contributions in counteracting recurrent and novel pathogens, and consequently fostering their utility as valuable targets for therapeutic and vaccine development, is the exquisite specificity displayed against their target antigens. Yet, there is still limited understanding of the determinants of antibody-antigen specificity, particularly as a function of antibody sequence. In recent years, experimental characterization of antibody repertoires has led to novel insights into fundamental properties of antibody sequences but has been largely decoupled from at-scale antigen specificity analysis. Here, using the LIBRA-seq technology, we generated a large data set mapping antibody sequence to antigen specificity for thousands of B cells, by screening the repertoires of a set of healthy individuals against 20 viral antigens representing diverse pathogens of biomedical significance. Analysis uncovered virus-specific patterns in variable gene usage, gene pairing, somatic hypermutation, as well as the presence of convergent antiviral signatures across multiple individuals, including the presence of public antibody clonotypes. Notably, our results showed that, for B-cell receptors originating from different individuals but leveraging an identical combination of heavy and light chain variable genes, there is a specific CDRH3 identity threshold above which B cells appear to exclusively share the same antigen specificity. This finding provides a quantifiable measure of the relationship between antibody sequence and antigen specificity and further defines experimentally grounded criteria for defining public antibody clonality.IMPORTANCEThe B-cell compartment of the humoral immune system plays a critical role in the generation of antibodies upon new and repeated pathogen exposure. This study provides an unprecedented level of detail on the molecular characteristics of antibody repertoires that are specific to each of the different target pathogens studied here and provides empirical evidence in support of a 70% CDRH3 amino acid identity threshold in pairs of B cells encoded by identical IGHV:IGL(K)V genes, as a means of defining public clonality and therefore predicting B-cell antigen specificity in different individuals. This is of exceptional importance when leveraging public clonality as a method to annotate B-cell receptor data otherwise lacking antigen specificity information. Understanding the fundamental rules of antibody-antigen interactions can lead to transformative new approaches for the development of antibody therapeutics and vaccines against current and emerging viruses.

Computational detection of antigen-specific B cell receptors following immunization.

B cell receptors (BCRs) play a crucial role in recognizing and fighting foreign antigens. High-throughput sequencing enables in-depth sampling of the BCRs repertoire after immunization. However, only a minor fraction of BCRs actively participate in any given infection. To what extent can we accurately identify antigen-specific sequences directly from BCRs repertoires? We present a computational method grounded on sequence similarity, aimed at identifying statistically significant responsive BCRs. This method leverages well-known characteristics of affinity maturation and expected diversity. We validate its effectiveness using longitudinally sampled human immune repertoire data following influenza vaccination and SARS-CoV-2 infections. We show that different lineages converge to the same responding Complementarity Determining Region 3, demonstrating convergent selection within an individual. The outcomes of this method hold promise for application in vaccine development, personalized medicine, and antibody-derived therapeutics.

Refined analytical pipeline for the pharmacodynamic assessment of T-cell responses to vaccine antigens.

Pharmacodynamic assessment of T-cell-based cancer immunotherapies often focus on detecting rare circulating T-cell populations. The therapy-induced immune cells in blood-derived clinical samples are often present in very low frequencies and with the currently available T-cell analytical assays, amplification of the cells of interest prior to analysis is often required. Current approaches aiming to enrich antigen-specific T cells from human Peripheral Blood Mononuclear Cells (PBMCs) depend on in vitro culturing in presence of their cognate peptides and cytokines. In the present work, we improved a standard, publicly available protocol for T-cell immune analyses based on the in vitro expansion of T cells. We used PBMCs from healthy subjects and well-described viral antigens as a model system for optimizing the experimental procedures and conditions. Using the standard protocol, we first demonstrated significant enrichment of antigen-specific T cells, even when their starting frequency ex vivo was low. Importantly, this amplification occurred with high specificity, with no or neglectable enrichment of irrelevant T-cell clones being observed in the cultures. Testing of modified culturing timelines suggested that the protocol can be adjusted accordingly to allow for greater cell yield with strong preservation of the functionality of antigen-specific T cells. Overall, our work has led to the refinement of a standard protocol for in vitro stimulation of antigen-specific T cells and highlighted its reliability and reproducibility. We envision that the optimized protocol could be applied for longitudinal monitoring of rare blood-circulating T cells in scenarios with limited sample material.

A comparison of clustering models for inference of T cell receptor antigen specificity.

The vast potential sequence diversity of TCRs and their ligands has presented an historic barrier to computational prediction of TCR epitope specificity, a holy grail of quantitative immunology. One common approach is to cluster sequences together, on the assumption that similar receptors bind similar epitopes. Here, we provide the first independent evaluation of widely used clustering algorithms for TCR specificity inference, observing some variability in predictive performance between models, and marked differences in scalability. Despite these differences, we find that different algorithms produce clusters with high degrees of similarity for receptors recognising the same epitope. Our analysis strengthens the case for use of clustering models to identify signals of common specificity from large repertoires, whilst highlighting scope for improvement of complex models over simple comparators.

Broad-spectrum defenders: γδ T cells take on a multitude of immune challenges.

A recent article in Proceedings of the National Academy of Sciences investigated γδ T cell antigen specificity in mice and humans, in which the authors show that γδ T cell antigen specificity is not constrained to one epitope. Rather, γδ T cells recognize a broad range of diverse antigens containing similar chemical structures or properties. In this News and Views, the importance of γδ T cell antigen polyspecificity during immune responses is highlighted.

Human CD4+CD25+ T cells expressing a chimeric antigen receptor against aberrant superoxide dismutase 1 trigger antigen-specific immunomodulation.

BACKGROUND AIMS: Amyotrophic lateral sclerosis (ALS) is a fatal disease associated with motor neuron degeneration, accumulation of aggregated misfolded proteins and neuroinflammation in motor regions of the central nervous system (CNS). Clinical trials using regulatory T cells (Tregs) are ongoing because of Tregs' immunomodulatory function, ability to traffic to the CNS, high numbers correlating with slower disease in ALS and disease-modifying activity in ALS mouse models. In the current study, a chimeric antigen receptor (CAR) was developed and characterized in human Tregs to enhance their immunomodulatory activity when in contact with an ALS protein aggregate. METHODS: A CAR (DG05-28-3z) consisting of a human superoxide dismutase 1 (hSOD1)-binding single-chain variable fragment, CD28 hinge, transmembrane and co-stimulatory domain and CD3ζ signaling domain was created and expressed in human Tregs. Human Tregs were isolated by either magnetic enrichment for CD4+CD25hi cells (Enr-Tregs) or cell sorting for CD4+CD25hiCD127lo cells (FP-Tregs), transduced and expanded for 17 days. RESULTS: The CAR bound preferentially to the ALS mutant G93A-hSOD1 protein relative to the wild-type hSOD1 protein. The CAR Tregs produced IL-10 when cultured with aggregated G93A-hSOD1 proteins or spinal cord explants from G93A-hSOD1 transgenic mice. Co-culturing DG05-28-3z CAR Tregs with human monocytes/macrophages inhibited production of tumor necrosis factor alpha and reactive oxygen species. Expanded FP-Tregs resulted in more robust Tregs compared with Enr-Tregs. FP-Tregs produced similar IL-10 and less interferon gamma, had lower Treg-specific demethylated region methylation and expressed higher FoxP3 and CD39. CONCLUSIONS: Taken together, this study demonstrates that gene-modified Tregs can be developed to target an aggregated ALS-relevant protein to elicit CAR-mediated Treg effector functions and provides an approach for generating Treg therapies for ALS with the goal of enhanced disease site-specific immunomodulation.

Utilizing immunogenomic approaches to prioritize targetable neoantigens for personalized cancer immunotherapy.

Advancements in sequencing technologies and bioinformatics algorithms have expanded our ability to identify tumor-specific somatic mutation-derived antigens (neoantigens). While recent studies have shown neoantigens to be compelling targets for cancer immunotherapy due to their foreign nature and high immunogenicity, the need for increasingly accurate and cost-effective approaches to rapidly identify neoantigens remains a challenging task, but essential for successful cancer immunotherapy. Currently, gene expression analysis and algorithms for variant calling can be used to generate lists of mutational profiles across patients, but more care is needed to curate these lists and prioritize the candidate neoantigens most capable of inducing an immune response. A growing amount of evidence suggests that only a handful of somatic mutations predicted by mutational profiling approaches act as immunogenic neoantigens. Hence, unbiased screening of all candidate neoantigens predicted by Whole Genome Sequencing/Whole Exome Sequencing may be necessary to more comprehensively access the full spectrum of immunogenic neoepitopes. Once putative cancer neoantigens are identified, one of the largest bottlenecks in translating these neoantigens into actionable targets for cell-based therapies is identifying the cognate T cell receptors (TCRs) capable of recognizing these neoantigens. While many TCR-directed screening and validation assays have utilized bulk samples in the past, there has been a recent surge in the number of single-cell assays that provide a more granular understanding of the factors governing TCR-pMHC interactions. The goal of this review is to provide an overview of existing strategies to identify candidate neoantigens using genomics-based approaches and methods for assessing neoantigen immunogenicity. Additionally, applications, prospects, and limitations of some of the current single-cell technologies will be discussed. Finally, we will briefly summarize some of the recent models that have been used to predict TCR antigen specificity and analyze the TCR receptor repertoire.

Novel antibody competition binding assay identifies distinct serological profiles associated with protection.

Introduction: Pre-erythrocytic malaria vaccines hold the promise of inducing sterile protection thereby preventing the morbidity and mortality associated with Plasmodium infection. The main surface antigen of P. falciparum sporozoites, i.e., the circumsporozoite protein (CSP), has been extensively explored as a target of such vaccines with significant success in recent years. Systematic adjuvant selection, refinements of the immunization regimen, and physical properties of the antigen may all contribute to the potential of increasing the efficacy of CSP-based vaccines. Protection appears to be dependent in large part on CSP antibodies. However due to a knowledge gap related to the exact correlates of immunity, there is a critical need to improve our ability to down select candidates preclinically before entering clinical trials including with controlled human malaria infections (CHMI). Methods: We developed a novel multiplex competition assay based on well-characterized monoclonal antibodies (mAbs) that target crucial epitopes across the CSP molecule. This new tool assesses both, quality and epitope-specific concentrations of vaccine-induced antibodies by measuring their equivalency with a panel of well-characterized, CSP-epitope-specific mAbs. Results: Applying this method to RTS,S-immune sera from a CHMI trial demonstrated a quantitative epitope-specificity profile of antibody responses that can differentiate between protected vs. nonprotected individuals. Aligning vaccine efficacy with quantitation of the epitope fine specificity results of this equivalency assay reveals the importance of epitope specificity. Discussion: The newly developed serological equivalence assay will inform future vaccine design and possibly even adjuvant selection. This methodology can be adapted to other antigens and disease models, when a panel of relevant mAbs exists, and could offer a unique tool for comparing and down-selecting vaccine formulations.

Maternally Derived Antibodies to Foot-and-Mouth Disease Virus Modulate the Antigenic Specificity of Humoral Responses in Vaccinated Cattle.

Vaccination is widely used to control foot-and-mouth disease (FMD), but maternal antibodies may interfere with the response to vaccination in calves. This study, conducted on a regularly vaccinated Malaysian dairy farm, aimed to optimise the vaccination regime by measuring the in vitro neutralising virus antibody responses of 51 calves before and after vaccination with a one or two dose vaccination regime starting at 2-7 months old. The presence of maternal antibodies was associated with poor post-vaccination antibody responses after a single dose of vaccine in calves less than 6 months old. However, a second dose of vaccine given three weeks later, improved the antibody responses in all ages of calves. This confirms the view that in regularly vaccinated farms, some combination of delay and revaccination is needed to achieve effective immunization of calves. Sera from cows and pre-vaccinated calves neutralised homologous serotype A vaccine virus more strongly than a heterologous serotype A field virus, but this pattern was reversed in some calves after vaccination. The strength of heterologous responses in calves 49 days after first vaccination correlated to the amount of transferred maternal antibody, suggesting that pre-existing antibodies could have modulated the specificity of these active antibody responses. If confirmed, such an effect by pre-existing antibodies could have wider implications for broadening the coverage of FMD vaccine responses.

Adoptive cell immunotherapy for breast cancer: harnessing the power of immune cells.

Breast cancer is the most prevalent malignant neoplasm worldwide, necessitating the development of novel therapeutic strategies owing to the limitations posed by conventional treatment modalities. Immunotherapy is an innovative approach that has demonstrated significant efficacy in modulating a patient's innate immune system to combat tumor cells. In the era of precision medicine, adoptive immunotherapy for breast cancer has garnered widespread attention as an emerging treatment strategy, primarily encompassing cellular therapies such as tumor-infiltrating lymphocyte therapy, chimeric antigen receptor T/NK/M cell therapy, T-cell receptor gene-engineered T-cell therapy, lymphokine-activated killer cell therapy, cytokine-induced killer cell therapy, natural killer cell therapy, and γδ T cell therapy, among others. This treatment paradigm is based on the principles of immune memory and antigen specificity, involving the collection, processing, and expansion of the patient's immune cells, followed by their reintroduction into the patient's body to activate the immune system and prevent tumor recurrence and metastasis. Currently, multiple clinical trials are assessing the feasibility, effectiveness, and safety of adoptive immunotherapy in breast cancer. However, this therapeutic approach faces challenges associated with tumor heterogeneity, immune evasion, and treatment safety. This review comprehensively summarizes the latest advancements in adoptive immunotherapy for breast cancer and discusses future research directions and prospects, offering valuable guidance and insights into breast cancer immunotherapy.

In-depth analysis of human virus-specific CD8+ T cells delineates unique phenotypic signatures for T cell specificity prediction.

Following viral infection, the human immune system generates CD8+ T cell responses to virus antigens that differ in specificity, abundance, and phenotype. A characterization of virus-specific T cell responses allows one to assess infection history and to understand its contribution to protective immunity. Here, we perform in-depth profiling of CD8+ T cells binding to CMV-, EBV-, influenza-, and SARS-CoV-2-derived antigens in peripheral blood samples from 114 healthy donors and 55 cancer patients using high-dimensional mass cytometry and single-cell RNA sequencing. We analyze over 500 antigen-specific T cell responses across six different HLA alleles and observed unique phenotypes of T cells specific for antigens from different virus categories. Using machine learning, we extract phenotypic signatures of antigen-specific T cells, predict virus specificity for bulk CD8+ T cells, and validate these predictions, suggesting that machine learning can be used to accurately predict antigen specificity from T cell phenotypes.

Harnessing autoimmunity with dominant self-peptide: Modulating the sustainability of tissue-preferential antigen-specific Tregs by governing the binding stability via peptide flanking residues.

Sensitization to self-peptides induces various immunological responses, from autoimmunity to tumor immunity, depending on the peptide sequence; however, the underlying mechanisms remain unclear, and thus, curative therapeutic options considering immunity balance are limited. Herein, two overlapping dominant peptides of myelin proteolipid protein, PLP136-150 and PLP139-151, which induce different forms of experimental autoimmune encephalomyelitis (EAE), monophasic and relapsing EAE, respectively, were investigated. Mice with monophasic EAE exhibited highly resistant to EAE re-induction with any encephalitogenic peptides, whereas mice with relapsing EAE were susceptible, and progressed, to EAE re-induction. This resistance to relapse and re-induction in monophasic EAE mice was associated with the maintenance of potent CD69+CD103+CD4+CD25high regulatory T-cells (Tregs) enriched with antigen specificity, which expanded preferentially in the central nervous system with sustained suppressive activity. This tissue-preferential sustainability of potent antigen-specific Tregs was correlated with the antigenicity of PLP136-150, depending on its flanking residues. That is, the flanking residues of PLP136-150 enable to form pivotally arranged strong hydrogen bonds that secured its binding stability to MHC-class II. These potent Tregs acting tissue-preferentially were induced only by sensitization of PLP136-150, not by its tolerance induction, independent of EAE development. These findings suggest that, for optimal therapy, "benign autoimmunity" can be critically achieved through inverse vaccination with self-peptides by manipulating their flanking residues.

Antigen specificities of HIV-infected cells: A role in infection and persistence?

Antigen-experienced memory CD4+ T cells are the major target of HIV infection and support both productive and latent infections, thus playing a key role in HIV dissemination and persistence, respectively. Here, we reviewed studies that have shown direct association between HIV infection and antigen specificity. During untreated infection, some HIV-specific cells host productive infection, while other pathogen-specific cells such as cytomegalovirus (CMV) and Mycobacterium tuberculosis also contribute to viral persistence on antiretroviral therapy (ART). These patterns could be explained by phenotypic features differing between these pathogen-specific cells. Mechanisms involved in these preferential infection and selection processes include HIV entry and restriction, cell exhaustion, survival, self-renewal and immune escape. For instance, MIP-1ß expressing cells such as CMV-specific memory cells were shown to resist infection by HIV CCR5 coreceptor downregulation/inhibition. Conversely, HIV-infected CMV-specific cells undergo clonal expansion during ART. We have identified several research areas that need further focus such as the role of other pathogens, viral genome intactness, inducibility and phenotypic features. However, given the sheer diversity of both the CD4+ T cell repertoire and antigenic history of each individual, studying HIV-infected, antigen-experienced cells still imposes numerous challenges.

Dissecting regulatory T cell expansion using polymer microparticles presenting defined ratios of self-antigen and regulatory cues.

Biomaterials allow for the precision control over the combination and release of cargo needed to engineer cell outcomes. These capabilities are particularly attractive as new candidate therapies to treat autoimmune diseases, conditions where dysfunctional immune cells create pathogenic tissue environments during attack of self-molecules termed self-antigens. Here we extend past studies showing combinations of a small molecule immunomodulator co-delivered with self-antigen induces antigen-specific regulatory T cells. In particular, we sought to elucidate how different ratios of these components loaded in degradable polymer particles shape the antigen presenting cell (APC) -T cell interactions that drive differentiation of T cells toward either inflammatory or regulatory phenotypes. Using rapamycin (rapa) as a modulatory cue and myelin self-peptide (myelin oligodendrocyte glycoprotein- MOG) - self-antigen attacked during multiple sclerosis (MS), we integrate these components into polymer particles over a range of ratios and concentrations without altering the physicochemical properties of the particles. Using primary cell co-cultures, we show that while all ratios of rapa:MOG significantly decreased expression of co-stimulation molecules on dendritic cells (DCs), these levels were insensitive to the specific ratio. During co-culture with primary T cell receptor transgenic T cells, we demonstrate that the ratio of rapa:MOG controls the expansion and differentiation of these cells. In particular, at shorter time points, higher ratios induce regulatory T cells most efficiently, while at longer time points the processes are not sensitive to the specific ratio. We also found corresponding changes in gene expression and inflammatory cytokine secretion during these times. The in vitro results in this study contribute to in vitro regulatory T cell expansion techniques, as well as provide insight into future studies to explore other modulatory effects of rapa such as induction of maintenance or survival cues.

Antigen-specific T cell balance reveals Why patients with atopic dermatitis fail to achieve immune tolerance.

The number of regulatory T cells (Tregs) and how they behave in the pathogenesis of atopic dermatitis (AD) are still controversial. We identified and quantified Tregs, mite-specific Tregs, and mite-specific effector T cells (Teffs) in patients with AD and healthy controls (HCs). We collected peripheral blood and analyzed the cells using flow cytometry after stimulation with mite antigens. Mite-specific Tregs and mite-specific Teffs were recognized by the expression of CD137 and CD154, respectively. Patients with AD had more Tregs than HCs; however, when focusing on a single antigen, the ratio of mite-specific Tregs/Teffs was lower in patients with AD than in HCs. Furthermore, the mite-specific Teffs in patients with AD were more likely to produce proinflammatory cytokines interleukin (IL)-4 and IL-13. This Teff-dominant imbalance is thought to be the cause of development of atopic status in patients with AD without immune tolerance.

Antigen-specificity measurements are the key to understanding T cell responses.

Antigen-specific T cells play a central role in the adaptive immune response and come in a wide range of phenotypes. T cell receptors (TCRs) mediate the antigen-specificities found in T cells. Importantly, high-throughput TCR sequencing provides a fingerprint which allows tracking of specific T cells and their clonal expansion in response to particular antigens. As a result, many studies have leveraged TCR sequencing in an attempt to elucidate the role of antigen-specific T cells in various contexts. Here, we discuss the published approaches to studying antigen-specific T cells and their specific TCR repertoire. Further, we discuss how these methods have been applied to study the TCR repertoire in various diseases in order to characterize the antigen-specific T cells involved in the immune control of disease.

The Influence of Adjuvant Type on the Immunogenicity of RBD/N Cocktail Antigens as a Vaccine Candidate against SARS-CoV-2 Virus.

The emerging virus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2 virus), agent of COVID-19, appeared in December 2019 in Wuhan, China, and became a serious threat to global health and public safety. Many COVID-19 vaccines have been approved and licensed around the world. Most of the developed vaccines include S protein and induce an antibody-based immune response. Additionally, T-cell response to the SARS-CoV-2 antigens could be beneficial for combating the infection. The type of immune response is greatly dependent not only on the antigen, but also on adjuvants used in vaccine formulation. Here, we compared the effect of four different adjuvants (AddaS03, Alhydrogel/MPLA, Alhydrogel/ODN2395, Quil A) on the immunogenicity of a mixture of recombinant RBD and N SARS-CoV-2 proteins. We have analyzed the antibody and T-cell response specific to RBD and N proteins and assessed the impact of adjuvants on virus neutralization. Our results clearly indicated that Alhydrogel/MPLA and Alhydrogel/ODN2395 adjuvants elicited the higher titers of specific and cross-reactive antibodies to S protein variants from various SARS-CoV-2 and SARS-CoV-1 strains. Moreover, Alhydrogel/ODN2395 stimulated high cellular response to both antigens, as assessed by IFN-γ production. Importantly, sera collected from mice immunized with RBD/N cocktail in combination with these adjuvants exhibited neutralizing activity against the authentic SARS-CoV-2 virus as well as particles pseudotyped with S protein from various virus variants. The results from our study demonstrate the immunogenic potential of RBD and N antigens and point out the importance of adjuvants selection in vaccine formulation in order to enhance the immunological response. IMPORTANCE Although several COVID-19 vaccines have been approved worldwide, continuous emergence of new SARS-CoV-2 variants calls for new efficient vaccines against them, providing long-lasting immunity. As the immune response after vaccination is dependent not only on antigen used, but also on other vaccine components, e.g., adjuvants, the purpose of this work was to study the effect of different adjuvants on the immunogenicity of RBD/N SARS-CoV-2 cocktail proteins. In this work, it has been shown that immunization with both antigens plus the different adjuvants studied elicited higher Th1 and Th2 responses against RBD and N, which contributed to higher neutralization of the virus. The obtained results can be used for design of new vaccines, not only against SARS-CoV-2, but also against other important viral pathogens.

Simultaneous targeting of PD-1 and IL-2Rßγ with radiation therapy inhibits pancreatic cancer growth and metastasis.

In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to radiation therapy (RT) is characterized by increased IL-2Rß and IL-2Rγ along with decreased IL-2Rα expression. The bispecific PD1-IL2v is a PD-1-targeted IL-2 variant (IL-2v) immunocytokine with engineered IL-2 cis targeted to PD-1 and abolished IL-2Rα binding, which enhances tumor-antigen-specific T cell activation while reducing regulatory T cell (Treg) suppression. Using PD1-IL2v in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local and metastatic survival, along with a profound increase in tumor-infiltrating CD8+ T cell subsets with a transcriptionally and metabolically active phenotype and preferential activation of antigen-specific CD8+ T cells. In combination with single-dose RT, PD1-IL2v treatment results in a robust, durable expansion of polyfunctional CD8+ T cells, T cell stemness, tumor-specific memory immune response, natural killer (NK) cell activation, and decreased Tregs. These data show that PD1-IL2v leads to profound local and distant response in PDAC.

Translational immunology: Applying fundamental discoveries to human health and autoimmune diseases.

Studying the human immune system is challenging. These challenges stem from the complexity of the immune system itself, the heterogeneity of the immune system between individuals, and the many factors that lead to this heterogeneity including the influence of genetics, environment, and immune experience. Studies of the human immune system in the context of disease are increased in complexity as multiple combinations and variations in immune pathways can lead to a single disease. Thus, although individuals with a disease may share clinical features, the underlying disease mechanisms and resulting pathophysiology can be diverse among individuals with the same disease diagnosis. This has consequences for the treatment of diseases, as no single therapy will work for everyone, therapeutic efficacy varies among patients, and targeting a single immune pathway is rarely 100% effective. This review discusses how to address these challenges by identifying and managing the sources of variation, improving access to high-quality, well-curated biological samples by building cohorts, applying new technologies such as single-cell omics and imaging technologies to interrogate samples, and bringing to bear computational expertise in conjunction with immunologists and clinicians to interpret those results. The review has a focus on autoimmune diseases, including rheumatoid arthritis, MS, systemic lupus erythematosus, and type 1 diabetes, but its recommendations are also applicable to studies of other immune-mediated diseases.

T-Helper 22 Cell Type Responses to Nickel in Contact Allergic Subjects Are Associated with T-Helper 1, T-Helper 2, and T-Helper 17 Cell Cytokine Profile Responses and Patch Test Reactivity.

INTRODUCTION: Contact allergy to nickel (Ni) is a delayed-type hypersensitivity reaction mediated by Ni-reactive T cells producing the hallmark cytokines of several T-helper cell (Th) populations including IFN-γ (Th1), IL-4, IL-5 and IL-13 (Th2), and IL-17A (Th17). IL-22-expressing CD4+ cells, which could be either Th17 co-expressing IL-22 or Th22, expressing IL-22 in the absence of IL-17A, have also been found in Ni-provoked skin of allergic subjects. It has been unclear if Ni-reactive T cells consist of distinct Th cell type populations or if they secrete a mix of Th cell hallmark cytokines. The aim herein was to assess if cellular cytokine responses to Ni, in ex vivo-stimulated peripheral blood mononuclear cells (PBMCs) from Ni-allergic subjects, include not only Th1, Th2, and Th17 but also Th22 hallmark cytokines and to define if the cytokines are produced by distinct cell populations representing different Th profiles. METHODS: PBMC from Ni-allergic subjects (n = 15) with different degrees of patch test reactivity and non-allergic controls (n = 5) were in vitro stimulated with Ni. Cytokine levels in PBMC supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) (IFN-γ, IL-2, IL-3, IL-5, IL-6, IL-13, IL-17A, IL-22, and IL-31). FluoroSpot was used to assess if individual Ni-reactive cells produced single, or combinations of, cytokines representing different Th profiles. Cytokine combinations analyzed were IL-17A/IL-22/IFN-γ, IL-5/IL-17A/IFN-γ, IL-13/IL-22/IFN-γ, and IL-5/IL-13. RESULTS: IL-22 as well as all other cytokines measured by ELISA were induced by Ni at higher levels in PBMC from allergic versus non-allergic subjects, with higher levels being associated with stronger patch test reactivity. The levels of most Ni-induced cytokines were positively correlated with each other; IL-2 displayed the highest correlation with other cytokines and IL-6 the lowest. FluoroSpot analysis showed that Th signature cytokines, IFN-γ (Th1), IL-5 and IL-13 (Th2), IL-17A (Th17), and IL-22 (Th22), were almost exclusively produced by distinct cell populations. CONCLUSION: Distinct Th cell populations, including Ni-reactive cells displaying Th1, Th2, Th17, and Th22 cytokine profiles, are all increased in PBMC from Ni-allergic subjects and positively associated with patch test reactivity. The relevance of these different Th profile populations for the up- or down-regulation of inflammatory reactions in the skin of Ni-allergic subjects remains to be clarified.